biomedical science

2009-12-17T10:11:00.001-08:00

Synthetic Platelets Put the Brakes on Blood Loss
In animals, they cut bleeding time in half, but use in humans is still far off

By Ed EdelsonHealthDay Reporter
WEDNESDAY, Dec. 16 (HealthDay News) -- Hoping to improve on nature, researchers have built and tested synthetic versions of the blood-clotting cells called platelets, to be used in trauma or other cases where blood just won't stop flowing.
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"We start by making a core, with material that is used in degradable stitches, which dissolve in the body," said Erin B. Lavik, a professor of biomedical engineering at Case Western Reserve University, and lead author of a report published Dec. 16 in Science Translational Medicine. "Then we attach a polymer that is soluble in water and is used in the pharmaceutical industry. Then we attach a molecule that interacts with activated platelets and helps them clot more quickly."
The hope is that the artificial platelets can replace or augment the activity of the currently used clotting medication, known as factor VIIa, Lavik explained.
Factor VIIa is a protein that plays a central role in blood clotting. A genetically engineered version of the protein is now available for medical use. It was introduced for use in people with hemophilia, a genetic condition in which normal clotting does not occur, and it is being increasingly used against uncontrollable hemorrhage.
But factor VIIa must be kept in refrigerated form and has a short shelf life, Lavik said. And it cannot be used for head or spinal cord injuries, for fear of complications.
"The reason we developed this synthetic platelet is that it is stable at all temperatures," Lavik said. "It is a fine powder that can be administered intravenously. The faster you can control bleeding, the better the outcome."
In animal tests, injured rats given injections of the artificial platelets stopped bleeding in half the time of those that went untreated. Rats that got injections 20 seconds after an injury stopped bleeding in 23 percent less time than untreated rats.
"We also did head-to-head comparisons with factor VIIa," Lavik said. "When the artificial platelets were introduced, bleeding was reduced even more." The artificial platelets induced clotting 25 percent faster than factor VIIa, the report said.
However, a long series of tests lie ahead before the artificial platelets can enter routine medical practice, Lavik said. "The next step would be an animal model that most closely mimics human injury," she said. "We have to move up to larger animals. Pigs are most commonly used."
Financial help is also needed. "We have applied to federal and non-federal groups for funding," Lavik said. She is hoping for support from a pharmaceutical company because "ultimately, you have to think about making it commercially viable."
"We have just started having those conversations," Lavik said. "Now that we have published this paper, we hope we can generate some interest."
A critical point is convincing the U.S. Food and Drug Administration that artificial platelets would have a useful medical application, Lavik said. Convincing the FDA would start with data from future animal studies. "Assuming that it replicates what we have seen so far, then we would talk with the FDA," she said. "There is no use estimating our chance of success until we see that data and talk with them."
"Anything new that would be safe to use with someone who has ongoing hemorrhage would be useful in a trauma center," added Dr. Michael Craun, trauma medical director at Scott and White Memorial Hospital in Temple, Texas. "We really have problems now with people who have major injuries."
Another expert agreed. "Any compound or device that can stem hemorrhage in patients can be helpful if the risk-benefit ratio is favorable," said Dr. Brian Harbrecht, director of trauma at the University of Louisville.
But he and Craun also stressed the early nature of the work.
"A lot more investigation needs to go into this particular product to see if it is clinically applicable or not," Harbrecht said. "That requires years and years of more precise work."
The new report described "preliminary experiments with rats only," Craun added, and there are questions about safety, cost and technology still to be answered

52 Facts About Blood Donation

2009-12-16T19:29:00.000-08:00

1. More than 4.5 million people need blood transfusions each year in the U.S. and Canada.
2. 43,000 pints: amount of donated blood used each day in the U.S. and Canada.
3. Someone needs blood every two seconds.
4. 37% of the U.S. population is eligible to donate blood – less than 10% do annually**.
5. About 1 in 7 people entering a hospital need blood.
6. One pint of blood can save up to three lives.
7. Healthy people who are at least 17 years old (16 with parental consent), and at least 110 pounds may donate whole blood every 56 days. Females receive 53% of blood transfusions; males receive 47%.
8. 94% of blood donors are registered voters.
9. In 1901, Dr. Karl Landsteiner first identified the major human blood groups: A, B, AB and O.
10. People with O- blood are universal donors of red blood cells.
11. People with AB+ blood are universal recipients of red blood cells, and universal donors of plasma.
12. One unit of whole blood can be separated into several components, including red blood cells, plasma, and platelets.
13. Red blood cells carry oxygen to the body's organs and tissues, and live for about 120 days in the circulatory system.
14. Platelets promote blood clotting and give those with leukemia and other cancers a chance to live.
15. Plasma is a pale yellow mixture of water, proteins and salts.
16. Plasma, which is 90% water, makes up 55% of blood volume.
17. Healthy bone marrow makes a constant supply of red cells, plasma and platelets.
18. Blood or plasma that comes from people who have been paid for it cannot be used for human transfusion.
19. Granulocytes, a type of white blood cell, roll along blood vessel walls in search of bacteria to engulf and destroy.
20. White cells are the body's primary defense against infection.
21. Apheresis is a special kind of blood donation that allows a donor to give specific blood components, such as platelets or red blood cells.
22. 42 days: how long most donated red blood cells can be stored.
23. Five days: how long most donated platelets can be stored.
24. One year: how long frozen plasma can be stored.
25. Much of today's medical care depends on a steady supply of blood from healthy donors.
26. 2.7 pints: the average whole blood and red blood cell transfusion.*
27. Children being treated for cancer, premature infants and children having heart surgery may receive blood and platelets during their treatments.
28. Anemic patients may need blood transfusions to increase their red blood cell levels.
29. Cancer, transplant and trauma patients, and patients undergoing open-heart surgery may require platelet transfusions to survive.
30. Sickle cell disease is an inherited disease that affects more than 80,000 people in the U.S., 98% of whom are of African descent.
31. Many patients with severe sickle cell disease receive blood transfusions every month.
32. Over 10 tests are performed on each unit of donated blood.
33. 17% of non-donors cite "never thought about it" as the main reason for not giving blood, while 15% say they're too busy.
34. The #1 reason blood donors say they give is because they "want to help others."
35. Blood centers often run short of types O and B red blood cells.
36. There is no substitute for human blood.
37. If all blood donors gave three times a year, blood shortages would be a rare event (The current average is about two).
38. 46.5 gallons: amount of blood you could donate if you begin at age 17 and donate every 56 days until you are 79 years old.
39. There are four easy steps to donate blood: medical history, a quick physical, donation and snacks.
40. The actual blood donation takes less than 15 minutes. The entire process – from the time you sign in until the time you leave – usually takes under an hour.
41. After donating blood, you replace the fluid in hours and the red blood cells within four weeks. It takes eight weeks to restore the iron lost after donating.
42. You cannot get AIDS or any other infectious disease by donating blood.
43. 10 pints: the amount of blood in the body of an average adult.
44. One unit of whole blood is roughly the equivalent of one pint.
45. Blood makes up about 7% of your body's weight.
46. Newborn babies have about one cup of blood in their bodies.
47. Giving blood will not decrease your strength.
48. Any company, community organization, place of worship or individual may contact their local community blood center to host a blood drive.
49. Roughly half of all blood donations across the U.S. are collected at blood drives.
50. People who donate blood are volunteers and are not paid for their donation.
51. 500,000 Americans donated blood in the days following the events of September 11.
52. Blood donation. It's about an hour of your time. It's About Life!

2009-12-15T17:22:00.000-08:00

Fake blood 2.0?
Posted by Bob Grant
[Entry posted at 14th December 2009 09:14 PM GMT]
View comment(1) Comment on this news story

Newly created synthetic particles that mimic red blood cells may one day carry drug molecules and/or oxygen through bloodstreams, according to researchers writing in this week's issue of the Proceedings of the National Academy of Sciences (PNAS). What's more, the team of scientists in Michigan and California say the particles could also be used to improve the resolution of magnetic resonance imaging.

The synthetic red blood cellsthat Mitragotri and his team developedImage: Nishit Doshi"It's a very nice paper and very exciting work," Krishnendo Roy, a biomedical engineer at the University of Texas at Austin who wasn't involved with the study, told The Scientist. "The beauty of their method is its simplicity." University of California, Santa Barbara, chemical engineer Samir Mitragotri led the team of scientists and told The Scientist that the blood cell-like particles could evolve into useful tools in the clinic. "What we got very excited about was making a structure with synthetic materials that begins to mimic a natural object," said Mitragotri. "If we can bridge the gap [between synthetic materials and living cells] it will open up tremendous opportunities for synthetic materials." Mitragotri said that he and his team tested the ability of the particles to carry oxygen, finding that they had a "comparable" oxygen-carrying capacity to actual red blood cells. He added that it may be possible in the future to link therapeutic agents destined for the vascular system, such as heparin, to the particles so that they can be easily distributed throughout the blood. The artificial blood cells, with attached iron oxide nanoparticles, could also one day improve MRI resolution by serving as contrast agents that provide a different imaging signal compared to the surrounding tissue, Mitragotri said. Mitragotri and his colleagues created the artificial red blood cells by first making tiny spheres out of a biodegradable polymer called poly(lactic acid-co-glycolide) (PLGA). They then exposed the spheres to isopropanol, which collapsed them into the discoid shape characteristic of red blood cells. The researchers then layered proteins -- either albumin or hemoglobin -- onto the doughnut-shaped disks, cross-linked the proteins to give them extra strength and stability, and finally dissolved away the PLGA template to leave only a strong but flexible shell of proteins in the shape and size (about 7 microns in diameter) of a red blood cell. Mitragotri and his team then tested the ability of the artificial cells to behave like real blood cells, passing them through glass capillary tubes that were narrower than the diameter of the particles and testing their oxygen-carrying capacity. They showed that the particles could carry about 90 percent of the oxygen real red blood cells can carry. They also showed that a drug-mimicking molecule could easily be loaded into and off of the artificial blood cells. "They conclusively demonstrated some stuff concerning oxygen-carrying capacity and the potential for drug release," Patrick Doyle, a chemical engineer at the Massachusetts Institute of Technology who was not involved with the study, told The Scientist. But years of continued testing lie between Mitragotri's synthetic red blood cells and clinical application. Several questions, including how long the particles will remain in circulation, how the immune system will react to the synthetic blood cells, and how efficiently they transport oxygen, remain to be answered. Mitragotri said that his lab plans on answering these questions by studying the particles in model organisms, research that is set to begin soon. "Whether this is applicable in an in vivo setting," said Roy, "we won't know that for 3, 4, or 5 years." "I don't think these [clinical applications] are far off ideas, but you have to go through all the usual regulatory hurdles," said Doyle, noting that the synthetic cells might also be used to study how cellular aberrations, such as tumor cells, behave in the body. "Ultimately they can also be model systems, by which you can understand disease states of cells," he added.

2009-10-23T18:25:00.000-07:00

Oswald Hope Robertson
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Oswald Hope Robertson (2 June 1886 – 23 March 1966) was an English-born medical scientist who pioneered the idea of blood banks in the "blood depots" he established in 1917 during service in France with the US Army Medical Corps.
Robertson was born in Woolwich in south-east London, but at the age of one-and-a-half he emigrated with his parents to California, settling in the San Joaquin Valley. He attended local schools in Dinuba, then graduated from the Polytechnic High School in San Francisco.
His initial plan to study basic biology was changed by a meeting with an American medical student while on holiday in Germany. After attending some lectures on anatomy, he decided to study medicine, being admitted to the University of California in 1906. He later studied at Harvard Medical School, the Massachusetts General Hospital and the Rockefeller Institute for Medical Research, but had to cut short his studies during World War I when he was called to join medical teams in France. Here he experimented with preserving human blood cells for use in blood transfusions, and became recognised as the inventor of the blood bank.
[edit] Commemoration

2009-04-23T17:22:00.001-07:00

project

2009-04-23T17:14:00.000-07:00

The PDF file of project is here.

Conslusion

2009-04-23T16:53:00.000-07:00

As already seen in the discussion part, the hypothesis which expressed there is no relationship between activation/inhibition of the platelets and externalization the phosphatidylserine would be, based on the results, ruled out.
Another hypothesis which expresses the relation ship is still on the table. It is not possible to declare:
-Activation of the platelets by Thrombin causes externalization of phosphatidylserine on the surface of the platelets.
-In this experiment the same results could be not applied to ADP as the test was performed for different concentrations of ADP but satisfactory results were not reached. Changing method in this regard in suggested.
-Inhibition of the platelets by Aspirin causes internalization of phosphatidylserine on the surface of the platelets. This statement has been approved by comparing the results of controls with and with out Aspirin.
In addition, having seen the papers, we would realize that COHEN Zoë and colleagues conducted a research in 2004 and their find out declare that if platelets become activated, they would express phosphatidylserine on the outer leaflet of the plasma membrane. They have working with ADP as they say ADP-induced PS exposure. They wanted to realise the role of Caspase in this process as well, while we were not. It can be a suggestion for further works as well to consider different enzymes in this process. Their findings could be useful to prove the activatory role of ADP as well.
Finally, the characteristics of Annexin V have been approved again. The property which they bind with negatively charged phospholipid on the surface of the cells, although it is not the main statement of this experiment but this viable facts could be used to be applied in other experiments as well.

6.1. Suggestions for further works
It should be realised how Thrombin/ADP or Aspirin apply their changes on the platelets. If we were working on cells which have nuclei the possibility of effect on the nuclei could be mentioned. But the platelets which are circulating in blood are not same cell. Therefore changing the expression of a gene which is highly possible for physiological reactions is ruled out. The other and simple way which comes to mind is acting by the aid of a second messenger. Further studies should be based on understanding the mechanism of this effect.
Repeating this experiment with different activators is suggested as well, in particular due to unclear results from ADP.
Further experiments should investigate about the role of Caspase and try to find out the mechanism of inhibition and activation with more details.
As stated about the receptors of ADP and Thrombin. The mechanism of action of stimulation (activation or inhibition) should be considered to find more relationship between the factor of change and response.

Discussion

2009-04-23T16:51:00.000-07:00

The hypothesis of this project as stated before is mainly about finding a relationship between activators of platelets (Thrombin and ADP) and Inhibitor of palettes (Aspirin) on externalisation the phosphatidylserine on the outer membrane of the platelets which would bind to Annexin V labelled with fluorescent. Therefore by the end of running the experiment for five times the data should be analysed to find logic relation ship between aforementioned parameters. By comparing the result of flowcytometry, few groups of analysis are reached. After ward in a separate graph they are compared. In all charts Y error bars represent Standard Deviations.

5.1. Thrombin and Aspirin
5.1.1. The first chart will demonstrate the externalisation of the Thrombin in different concentrations. It is with out Aspirin .This chart would reveal that what is the effect of Thrombin in externalisation of Phosphatidylserine with out Aspirin.
Graph 5.1.1.1. Externalising of Phosphatidylserine in different concentrations of Thrombin with out Aspirin.

The first graphs is indicating that , adding Thrombin on its own could rise up externalising phosphatidylserine on the outer membrane of the platelets. This is while Aspirin had not been added to the samples and Alcohol (ethanol) was used instead of Aspirin. As the chart suggests the general trend is increasing.

5.1.2. The next stage is to evaluate the role of Aspirin. The first eight tubes are with out Aspirin (Solution A had been added).The mean of fluorescent intensity for different concentration of Thrombin would be me calculated. For tubes from 8 to 16, solution B had been added which was including Aspirin. Then the mean of Fluorescent intensity for different concentration of Thrombin for these tubes would be taken as well. They should be compared with each other. The result comes below.
Chart 5.1.2.1. Compare the effect of Aspirin on different concentrations of Thrombin.

The concentration of each tube has been mentioned. The added Aspirin per each tube was 2µL. As the graph suggests, in most cases the ADP causes decreasing in externalisation of Thrombin although the change is not significant( P<0.05) however in this regard in a research conducted by Conehn Zoë and colleagues in 2004, revealed that when the plateletes become inhibited they express less phosphatidylserine on the outer membrane. This matches with the achievements of our experiment.

5.2. ADP and Aspirin
Before doing this experiment another method was used in order to get proper data for ADP. But as results were not satisfactory therefore only the double amount of final concentration of ADP is used in new method. In previous method the range of concentration of ADP was similar to Thrombin in current method and highest concentration of ADP was 10µL while in current method the concentration of ADP is 20 µL. Perhaps running this method will not reach a visible result about ADP. The role of ADP in activating the platelets has already been proved (Jianguo Jin et al, 1998) .The fact that activating by ADP cause measurable change is not something which could be found by this experiment. Maybe by applying different methods, better results are reached. Therefore the first chart (like Thrombin) will be excluded.

5.2.1. This stage is to evaluate the effect of Aspirin on platelets which have already been activated by ADP. The tube 8 is include 20µL ADP with out Aspirin (Solution A had been added) and tube 16 is include 20µL ADP with Aspirin. In five times of running the experiment the mean of fluorescent intensity of tubes 8 and 16 would be me calculated and the results are compared with each other. The concentration of added Aspirin as mentioned in method is 0.01 % V/V. The important factor in this regard is the amount of Aspirin used for each tube.
Chart 5.2.2.1. Aspirin has effect on platelets which had already been activated by ADP.
By this chart it is not quite clear to recognise what is the effect of Aspirin of the specimen which have already been activated with ADP. May be there are some interactions between the function of ADP and Aspirin in platelets cell membrane which can cause a visible change. But apart from specimen 4 which has a high SD (as the Y-error bars shows); generally it is logic to say that Aspirin cause internalisation the phosphatidylserine. We should not forget that we are looking for effect of Aspirin (either internalising or externalising). If platelets have already been activated with different activators although they may cause overlap, but the most important impact should not be forgotten. The next chart will reveals more valuable information in this regard.
Additionally, as stated before Aspirin Induces Apoptosis through -Release of Cytochrome c from Mitochondria (Katja C Zimmermann et al, 2000) and the inhibition of Proteasome Function (Priyanka Dikshit et al, 2006). It is logic and true. When a cell is inhibited for along time and is not in use, the final destination is not something apart from apoptosis. Although the main cause of apoptosis is not what was mentioned, but inhibition a cell.

5.3. Control and Aspirin
In order to prove the hypothesis, the sole effect of Aspirin on platelets which have not been activated by any factor would be evaluated as well. This is a compare between controls of samples which have been added Alcohol (from solution A) and the control of samples which have been added Aspirin (from solution B). Chart comes below manifest this compare.

Chart 5.2.2.1. Compare Controls with and with out Aspirin.
Here a significant change is observed (P<0.01). href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Casta%C3%B1o%20E%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract">Castaño E and colleagues in 1999 announced that Aspirin increases phosphatidylserine externalization when they were analysing HT-29 colon carcinoma cells .In this paper is said that Aspirin induces cell death and caspase-dependent phosphatidylserine externalization. Based on their paper one of two halls marks of apoptosis is: increase in phosphatidylserine externalization. : It does not correspond with other findings which discuss about inhibitory role of Aspirin. As mentioned before, when platelets become activated they express more phosphatidylserine on the outer leaflet, therefore when they become inhibited (for example by Aspirin) they logically must express less phosphatidylserine while bases on the aforementioned paper was not. But Pamela L in her book demonstrate that unlike observation about colorectal cell lines , if the method of measuring is based on Annexin V binding to phosphatidylserine, there was not dose dependent apoptotic increase .

2009-04-23T16:50:00.000-07:00

4. Experimental results
The results of flowcytometry of each tube come below. The experiment was performed for five times. Hence it is expected to see the five series of sixteen results.
In each group of results two diagrams and one table are seen. Details of each result come below (Guide line of the flowcytometry machine).

-Upper diagram
FSC Forward Scatter: Cell size
SSC Side Scatter: Granularity

-Lower diagram
X scatter: Fluorescent intensity emitted is proportional to the quantity of binding sites for the fluorescent compound on the cell/particle. The unit is MESF which is Molecules of Equivalent Soluble Fluorochrome. In this experiment the geometric mean of fluorescent is measured.
Y scatter: Events count

-Table
Geometric mean of all events
Geometric mean of M1 region (1% of the events)
FL2-H: 585/42 nm, yellow-green colour
Mean: Average X-axis channel number of linear value for events in the quadrant
Geometric Mean: average of the logarithm of the X-axis channel number or linear value for events in the quadrant expressed anti-log.

Picture 4.1.1. Flowcytometry machine.
BD FCSCalibur two laser channel.

4.1. Diagrams and table from flowcytometry.
Results of flowcytometry for each sample come next.

M&M

2009-04-23T16:49:00.000-07:00

3. Materials & Methods

3.1 Materials
Apart from ADP, Thrombin and Aspirin which have been explained before, further substances are required as well which would be described below.

3.1.1 HEPES buffer
HEPES buffer is a suitable alternative general-purpose zwitterionic buffer. HEPES buffer does not bind to Mg +2, Ca +2, Mn +2 and Cu +2. HEPES buffer is one from ten important biological buffers.
-HEPES buffer with CaCl2 is used in this experiment .It is a zwitterionic buffer does not bind Ca +2 in plasma. HEPES buffer can exhibit toxicity if the concentration is greater than 40 mM .20 mM HEPES is the most satisfactory concentration. HEPES buffer can maintain pH. That is why it is more used in cell culture.
HEPES used in this experiment is consisting of:
-10 mM NaCl, 10mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
,2.7 mM CaCl2 ,5 mM Glucose ,0.5 mg/mL BSA ,Distilled water up to 500mL ,Few drops of NaOH to reach the pH to 7.4 (Lepe-Zuniga JL et al , 1987)

3.1.2. Fixative solution
Fixative solution used in this experiment is formaldehyde also known as methanal, CH2O MW=30.03, which is a very good stabiliser and fixative in biological studies. Usually it is used in 4% w/v concentration. It does limit oxidation and polymerisation.

3.2. Method

3.2.1. General description
The idea if planning this method is based on similar study conducted by Jef L and colleagues in 2006.
In a nut shell the over view of all done in the method of this experiment is:

1-Preparing HEPES buffer
2-Pareparting PRP: Platelets Rich Plasma (Centrifuging in 200 g for 10 minutes)
3-Prepraing series of dilution of Thrombin and ADP.
4-Adding Alcohol (Ethanol) [tubes 1 to 8] and adding Aspirin [tubes 9 to 16]
5- Taking 10µL from indicated tubes +90µL fixative (formaldehyde)
6-Taineg 1ml from above and mixing with 1 ml distilled water.
7-Flowcytometry to plot a histogram and gather the data.
More details comes bellow.

3.2.2 Platelet isolation
First stage is platelets isolation.10 mL blood was collected in 2 mL anticoagulant ACD (anticoagulant citrate dextrose solution) (2.5% sodium citrate, 1.5% citric acid, and 2% glucose).The donor should not take Aspirin within last few days. Then the specimens are centrifuged for 10 minutes in 2000g to reach the Platelet Reach Plasma (PRP). Follow by order HEPES buffer is prepared to use in next stages as the main buffer of this experiment.

3.2.3 Solution A, Solution B
Two separate solutions called solution A and B are prepared as follow:
-Solution A: Solution A is consist of: 2mL buffer + 50 µL PRP + 2µL Alcohol (Ethanol) Concentration of Ethanol = 0.01 % (0.00009746) V/V
-Solution B: Solution B is consist of: 2mL buffer + 50 µL PRP + 2µL Aspirin
Concentration of Aspirin=0.01 % (0.00009746) V/V

3.2.4 Preparing preliminary concentration of Thrombin and ADP
A set of 16 tubes are required. The following concentration of Thrombin and ADP are made. By the end of this stage the concentration of Thrombin and ADP content of each tube will be as the following table.

Table 3.2.4.1. Preliminary concentrations of Thrombin and ADP.

Tube 1
10 µL buffer
Tube 2
10 µL buffer
Tube 3
0.10 Unit/mL Thrombin
Tube 4
0.30 Unit/mL Thrombin
Tube 5
1.00 Unit/mL Thrombin
Tube 6
3.00 Unit/mL Thrombin
Tube 7
10.00 Unit/mL Thrombin
Tube 8
200µM ADP
Tube 9
10µL buffer
Tube 10
10µL buffer
Tube 11
0.10 Unit/mL Thrombin
Tube 12
0.30 Unit/mL Thrombin
Tube 13
1.00 Unit/mL Thrombin
Tube 14
3.00 Unit/mL Thrombin
Tube 15
10.00 Unit/mL Thrombin
Tube 16
200µM ADP

By the end of this stage 16 tubes are ready. Now extra 16 tubes are required to carry on the experiment to prepare the final concentrations of Thrombin and ADP.

3.2.5. Preparing final concentrations of Thrombin and ADP
In these series of tubes we need to take 10 µL from the previous tubes and mix with 90 µL of solution A for tubes 1 to 8 and solution B for tubes 9 to 16. Hence we will see the new set of tubes in which the concentration of thrombin and ADP will become 10 fold less. By the end of this stage in new set of tubes the concentration of Thrombin or ADP and content of each tube will be as the following table.

Table 3.2.5.1. Final concentrations of Thrombin and ADP.

Tube 1
10mL buffer
Tube 2
10mL buffer
Tube 3
0.01 Unit/mL Thrombin
Tube 4
0.03 Unit/mL Thrombin
Tube 5
0.10 Unit/mL Thrombin
Tube 6
0.30 Unit/mL Thrombin
Tube 7
1.00 Unit/mL Thrombin
Tube 8
20µM ADP
Tube 9
10mL buffer
Tube 10
10mL buffer
Tube 11
0.01 Unit/mL Thrombin
Tube 12
0.03 Unit/mL Thrombin
Tube 13
0.10 Unit/mL Thrombin
Tube 14
0.30 Unit/mL Thrombin
Tube 15
1.00 Unit/mL Thrombin
Tube 16
20µM ADP

After this stage we need to wait for ten minutes for each tube .Then Annexin V will be added.

3.2.6. Adding Annexin V and preparing for flowcytometry
At this stage 5µL Annexin V is added to each tube. Annexin V is labelled with fluorescent substance (florin). Then after 50µL of each tube is taken and mixed with 100 µL fixative (formaldehyde) in another tube. We can now leave the tubes for a couple of hours. It is very important to do the flowcytometry at the same day. Although they are fixed with fixative but they should not be kept for more than some hours. Before final stage, 1 mL of each tube is taken and mixed with distilled water in another series of tube. This is to prepare for flowcytometry.

3.2.7. Flowcytometry
The final stage is flowcytometry. The flowcytometry machine will be set as described above and the results would be gathered. The results of Flowcytometry come in next part.

2009-04-23T16:46:00.000-07:00

.1. Annexin V (Endonexin II, Placental anticoagulant protein I, vascular anticoagulant-alpha, Lipocortin V, Placental protein 4; PP4, Anchorin CII) (Bohn H et al, 1979 ; Ernest Beutler et al, 2001 ;Reutelingsperger CP ,2001 ;Hendrikus H et al , 2005). Annexin V (Gene map locus 4q26-q28), a member of the Annexin family with an unknown function up to now , is a cellular protein which is known as a phosphatidylserine-binding protein (Ernest Beutler et al , 2001) . Annexin family has over 160 proteins. They all share the property of Ca 2+ dependent binding to negatively charged phospholipid surfaces (Gerke V et al, 2002). At late of 70s Annexin V for the first time was purified from an EDTA extract of human placenta (Bohn H et al, 1979; Hendrikus H et al, 2005). After ward, a protein (Fig. 2.1.1.) was discovered independently in blood vessels which was named vascular anticoagulant protein Alpha (VAC-Alpha) which inhibits blood coagulation (Reutelingsperger CP, 1985). The mechanism of anticoagulation is based on the high-affinity binding to Phospholipids (Fiedler K et al, 1995). This is the property which makes Annexin V quite effective in inhibiting the prothrombinase complex (Reutelingsperger CP, 1988; Homburg CH et al, 1995). In vitro, Annexin V is defined as a Phospholipid binding protein with a high affinity for PS (phosphatidylserine) (D’Arceuil H et al, 2000). One of the important functions of Annexin V is forming a shield around negatively-charged phospholipid molecules (Dumont EA et al, 2000; Dreier R et al, 1998). This formation of Annexin V shield would block the entry of phospholipid molecules into coagulation clotting reactions (Glaser M et al, 2003). An increased quantity of phospholipid molecules on cell membranes which speed up coagulation reactions is seen in the absence of the shield(Koopman G et al , 1994 ; Vermes I et al , 1995 ). Annexin V binds quite tightly to anionic phospholipids (Catherine .R et al, 1992). There are evidences suggesting Annexin V interacts with phospholipid vesicles (Hendrikus H et al, 2005). Annexin V can form a shield around phospholipid molecules which blocks their entry into coagulation (clotting) reactions (Ernest Beutler et al, 2001). The formation of this shield is disrupted by the abnormal antibodies in case of anti phospholipid antibody syndrome (Hayes MJ, 2004). In the absence of the shield, there is an increased quantity of phospholipid molecules on cell membranes (Gerke V et al, 2002). These molecules speed up coagulation reactions and cause the abnormal blood clotting which is characteristic of the anti phospholipid antibody syndrome (Ernest Beutler et al, 2001; Huber R et al , 1992).
Figure 2.1.1.X-ray analysis on Annexin V. Tertiary
Structure of Annexin V is revealed (Consists of 319
amino acids).Green spheres are indicating Ca 2+

2.2. Phosphatidylserine - C13H24NO10P (MW: 385.304)
Phosphatidylserine (Fig. 2.2.1.) is a phospholipid component essential to the functioning of all the cells of the body which is mainly known as a molecule which enables brain cells to metabolise glucose (Vermes I et al, 1995). It is mainly located entirely on the inner layer of the plasma membrane. Phosphatidylserine is mainly known as effective treatment for Alzheimer's disease . Phosphatidylserine is usually on the cytosolic side, of cell membranes (Catherine .R et al, 1992; Calderon F et al, 2008). By a flipase enzyme Phosphatidylserine become exposed on the surface when a cell undergoes apoptotic cell death (Boersma et al, 2003; Belhocine T et al, 2002). The normal distribution of Phosphatidylserine is altered during platelet activation and cellular apoptosis (Fadok VA et al, 1992) Phosphatidylserine modulates the activity of several enzymes involved in cellular signalling. Annexins (particularly Annexin V) bind to and polymerize through protein-protein interactions on membrane patches which are expressing phosphatidylserine. Annexin V specifically tends to bind to phosphatidylserine (Calderon F et al, 2008). As stated before Phosphatidylserine is almost exclusively located on the cytoplasmic side of the plasma membrane and after stimulation, Phosphatidylserine becomes exposed on the outer side of plasma membrane at a proportion depending on the type and the nature of the stimuli (Cohen Zoë et al , 2004 ;Kenis H et al ,2004).

Figure 2.2.1.Chemical structure
of Phosphatidylserine.
C13H24NO10P

2.3. Platelet Thrombocytes (Fig. 2.3.1.) are smallest (2-4µm in diameter) cytoplasmic bodies derived from megakaryocyte cytoplasm in bone marrow (White, GC, 1980). The development of megakaryocytes and consequently the production of platelets are unique processes in body (Dahlbäck B, 2000). Megakaryocyte maturation involves nuclear duplication without cell division (many sets of chromosomes in nucleus 8-64C) which resulting in giant cells. Over the process of cytoplasmic fragmentation of these giant cells, platelets are realised into blood stream .The production of platelet is controlled by thrombopoietin, IL-3, IL-6 and IL-11(Sixma JJ et al, 1977). Platelets have no nucleus. Platelets play a critical role in normal haemostasis .Their life span is between 7 to10 days with the normal count 1.5 - 4.0 x 105 ml -1 in blood (up to 1/3 of them are generally stored in spleen) (George.JG , 2000;Dahlbäck.B , 2000)
Figure 2.3.1. Platelets.
The main cells in the coagulation process.

There some types of granules in platelets which store different substances .Granules of platelets are including:
-α granules: fibrinogen, FV, FVIII, PDGF, PF4 VWF, b-thromboglobulin
-Dense bodies: Ca 2+, 5-HT, ADP
-Lysosomes: acid hydrolases

2.4. Activation of the platelets and activators
Platelets are normally circulating in blood in resting condition .They need to get activated to aggregate. They can become activated by some substances such as ADP and Thrombin. ADP (Adenosine diphosphate) and Thrombin can activate the platelets. Over the activation process their shape change. Platelets contract into a spheroid shape and throw out long protuberances “pseudopodia”. Platelets adhere to exposed structures in the sub endothelium .In this process, fibrinogen receptors (GPIIb/IIIa) on the platelet surface become activated and increase affinity for fibrinogen. Platelet cytoplasmic granules fuse with the plasma membrane and their contents are released into the blood (George.JG, 2000; Dahlbäck.B, 2000) .The platelets express more phosphatidylserine (PS) on the outer leaflet of the plasma membrane when they become activated (Cohen Zoë et al, 2004). Some substances could activate the platelets for instance ADP and Thrombin would be described.Thrombin needs to act on Protease-activated receptors (PRA) (a subfamily of related G protein-coupled receptors). Then it can activate the platelets. The mechanism is that after binding the receptors (PRA’s) become activated by cleavage of part of their extracellular domain. These receptors are presence on other cells such as endothelial cells, myocytes and neurons too. (Ayyanathan K et al, 1996) .Thrombin act on mainly PAR’s 1, 3 and 4 Thrombin cleaves the N-terminus of the receptor. The cleaved N-Terminus in turn would act as a tethered ligand. A part of the receptor itself acts as the agonist which can cause a physiological response , in the cleaved state (J Clin Invest , 2003) .ADP mainly binds to P2Y12.This receptors is mainly involved in platelet aggregation.P2Y12 is a potential target for the treatment of clotting disorders ( Murugappa S et al , 2006).
2.4.1. Activator ADP
Adenosine diphosphate is ester of pyrophosphoric acid with the nucleoside adenosine is a nucleotide. ADP (Fig. 2.4.1.1.) is more known as an energy transfer molecule. ADP is stored in dense granules of platelets (Born GV, 1962).ADP is know as activator of platelets activity. ADP induces platelet aggregation. It is by binding to specific receptor on the plasma membrane of platelet (Ernest Beutler et al, 2001). There is a molecule which inhibits this effect. It is Prostaglandin E1. Prostaglandin E1 interferes binding ADP to the receptors. The effect of ADP is enhanced by leptin (Nakata, M et al, 1999; Ozata M, et al, 2001). ADP is normally saved in dense granules of the platelets. ADP binds to GPIIb/GPIIIa (platelet membrane receptor) (Born GV et al, 1976).After initial activation the platelets undergo a change in shape. As stated before the disc shape cell would change into to a spherical form with protruding pseudopodia (Ernest Beutler et al, 2001). ADP stimulates the thromboxane A2 generation and release of arachidonic acid. ADP, inhibits adenylate cyclise activity, and therefore lowers cAMP activity. (Cohen Zoë et al, 2004). Inside the platelet When ADP binds to ADP leads to mobilization of intracellular calcium ions via activation of phospholipase C. This make a change in platelet shape, and consequently to platelet aggregation
Figure 2.4.1.1. Chemical structure of ADP

2.4.2. Activator Thrombin
Thrombin (Fig. 2.4.2.1.) with gene locus 11p11-q12 is a serine protease known as activated form of coagulation factor II. The main function of thrombin is converting soluble fibrininogen into insoluble strands of fibrin (Evans, I et al, 1992). It as well catalyses some other reactions in coagulation cascade. Thrombin is not known as a normal constituent of the circulating blood and would be generated by the catalytic cleavage of prootrombin(factor II) which is its plasma precursor (E.W.Davie et al , 2003).Thrombin is a glycoprotein formed by two peptides chains of 36 and 259 amino-acids linked by disulfure bonds (Francis CW et al , 1983).As already stated the earliest function of thrombin which has already been identified is the cleavage of fibrinogen into fibrin monomers and consequently the activation of the fibrin-stabilizing factor (factor XIII) and protein C (M.T. Stubbs et al , 1993).Therefore Thrombin would be more than a simple plasma enzyme because of its properties to stimulate platelets which cause them to expand aggregate and release components granules8alpha and dense) (Francis CW et al, 1983). LPS-induced liver injury is cause by thrombin as well which alters the synthesis, expression and release of proteins from endothelial cells (Liu CY et al, 1979). This process would results in increasing in production of factor VIII, tPA, PAI, platelet-derived growth factor (PDGF), factor XIII, platelet activating factor (PAF), and as well modifies the interactions between endothelial cells and the underlying matrix or even between endothelial cells and the expression of adhesion glycoproteins to the cell surface. That is a way to increase the binding of inflammatory cells to the endothelium (J.A. Huntington et al, 2005). Inducing chemotaxis in neutrophils and macrophages is performed by thrombin too (M.T. Stubbs et al, 1993) .The mechanism of activation of platelet by thrombin is based on receptor activation. Thrombin binds to its receptor then thrombin cleaves the amino-terminal extension to expose a new amino-acid sequence (Ernest Beutler et al, 2001). This new amino acid-sequence binds to the third extracellular loop of the receptor and causes activation. The similar mechanism exist on other ells as well (Francis CW et al, 1983).
Figure 2.4.2.1. Human Thrombin

2.5. Inhibition of the platelets and inhibitors
The inhibition process is the opposite of the activation. By definition inhibition the platelets means inhibiting clotting activity of platelets. Inhibitors of a platelets interfere with a chemical reaction, growth or other biological activity of the platelets where as an activator has the opposite effect (Siegle AM et al, 1982). For normal platelets keeping the equilibrium between activating and inhibiting processes is quite essential. It is important not only for platelets but also for vascular system as well .As it is linked with cardiovascular disease, Heparin and Aspirin are mow known as anti-platelet factors (Ernest Beutler et al, 2001). .The substances which eventually inhibit the platelets coagulation activity. Over the inhibiting the platelets, they tend to aggregate less (Joel S. Bennett, 2001). The molecules which were externalised over the activation would become less externalised. For instance some receptors such as GP IIb/IIIa and Glycoprotein IIb/IIIa (IIbß3). Prostacyclin (PG-I2), nitric oxide (NO) and Aspirin are some substances which can work as inhibit cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) are known, as two intracellular messengers, to mediate the effects of platelets inhibitors. Another instance is Aspirin which would be described (Gambaryan Stepan et al, 2004; Priyanka Dikshit et al, 2006).

2.5.1. Aspirin
Aspirin (acetyl salicylic acid) (Fig 2.5.1.1.) as a NSAID (non steroidal anti-inflammatory drug) inhibits platelet prostaglandin synthesis and the ADP- and collagen-induced platelet release reaction (Siegle AM et al, 1982). Although the whole mechanism of this irreversible inhibition of platelets is still unknown but may involve protein acetylation cyclo-oxygenase 1 (COX-1) which can permanently inactivates the enzyme activity Cyclooxygenase 1 enzyme catalyses the conversion of arachidonic acid to prostaglandin H2. (G J Roth et al, 1975; Barsom Aktas et al, 2005; Helgason, C.M, 2000). More importantly .Aspirin Induces Apoptosis through -Release of Cytochrome c from Mitochondria (Katja C Zimmermann et al , 2000) and -The inhibition of Proteasome Function (Priyanka Dikshit et al ,2006 ; Ernest Beutler et al, 2001).
Figure 2.5.1.1.
Chemical structure of Aspirin

2.6. Flowcytometry machine
Flowcytometry is a technique to counting, examine, and sort the microscopic particles which are suspended in a stream of fluid. Flowcytometry is one of the biggest advances in platelet function analysis (A.D Michelson et al, 1996; A.D. Michelson et al, 1999). The most commonly used routine tests of flowcytometry is the quantification of glycoprotein receptor density (R.J. Cohn et al , 1997; T.L. Lindahl et al , 1992) Only small quantities of blood are required for flowcytometry and platelets can be analysed in their circulating state. (A.D. Michelson et al ,1999 ; A.D. Michelson et al ,2002 .Platelet function in whole blood can be comprehensively evaluated by flowcytometry (Alice Longobardi Givan ,2001. Flowcytometry is a technique used for counting, examining, and sorting microscopic particles suspended in a fluid. Multiparametric analysis of the physical/chemical characteristics of single cell by an optical/electronic detection apparatus. Particles 0.2 to 150 micrometers. In this experiment flowcytometry is used to measure the phosphatidylserine exposed on the surface of platelets. They are bind with Annexin V which has been labelled with fluorescent substances (Florin). Light scatter and florescence channels were set at logarithmic gain (Alice Longobardi Givan, 2001). Forward scatter data are indicating fluorescence intensities were obtained from 10000 platelets and analysed. In this experiment flowcytometry is set up on FL2-H: which is set for 585/42 nm of light and yellow-green color. The result showing fluorescent intensity are express by a unit which is molecules of equivalent soluble fluorescein (MESF) (Alice Longobardi Givan ,2001).That indicates the number of molecules bind with Annexin V(in this experiment phosphatidylserine) revealed on the surface of platelet. In face in this experiment, phosphatidylserine is indirectly measured. Phosphatidylserine is bind with Annexin V and Annexin V was labelled with fluorescent. The fluorescent on the Annexin V is measured which indirectly is indicating the Annexin V and more importantly the phosphatidylserine express on the surface of the platelets (A.D Michelson et al , 1996 ; A.D. Michelson et al ,1999).
Figure 2.6.1. The mechanism of action of flowcytometry.

Abstract

2009-04-23T16:45:00.000-07:00

1 Abstract
Phosphatidylserine is well known protein .It is main function is in regard with memory and in brain cells. However it presence on the surface of the platelets as well. In this experiment , as a basic understanding , we are looking for to find a logic relationship between activation/inhibition of the platelets and manifesting phosphatidylserine .The hypothesis in this regard could be either no relationship or in case of relationship either increase or decreasing the amount of phosphatidylserine on the surface of the proteins. In a nutshell, in this experiment we will be involved with two processes. One is that first we activate the platelets by Thrombin and next time separately by ADP then we measure the amount of phosphatidylserine on the surface of the platelets. In second scenario we need to inhibit the platelets by Aspirin and then measure the phosphatidylserine. The measurement is performed by flowcytometry.
The problem was finding a way to measure the appeared molecules on the surface of the platelet. Annexin (a cellular protein with unknown functions up to now) has a property which is useful for this matter. Annexin family has over 160 members which share on a characteristic .They all share the property of Ca 2+ dependent binding to negatively charged phospholipid surfaces. The molecule which is preferred for this experiment is Annexin V. The mechanism of process is that Annexin V will be labelled with fluorescent materials. As it tends to bind to phospholipids on the surface of the platelet, hence it can bind to phosphatidylserine. Then by measuring the amount of Annexin V on surface of the platelets, indirectly we know the amount of phosphatidylserine on the surface of the platelet. As stated above the hypothesis is, expect for lack relationship scenario, activation the platelets by Thrombin/ADP could increase or decrease the externalisation of phosphatidylserine on the surface of the platelets .The second hypothesis could be: inhibition the platelets by Aspirin could increase or decrease the externalisation of phosphatidylserine on the surface of the platelets. In this experiment, plasma is taken from the blood of donors looks healthy and has not used Aspirin for at least a week ago. The findings could be used to have a basic understanding of their relationship which might be helpful in terms of producing a drug to suppress or stimulate this process.

Key Words: Annexin V, Phosphatidylserine, Flowcytometry, ADP, Thrombin, Aspirin, Externalization

British scientists to create 'synthetic' blood

2009-03-27T17:54:00.001-07:00

British scientists to create 'synthetic' blood
Human embryos will be used to make an unlimited supply for infection-free transfusions
By Steve Connor, Science Editor
Monday, 23 March 2009

Scientists in Britain plan to become the first in the world to produce unlimited amounts of synthetic human blood from embryonic stem cells for emergency infection-free transfusions.
Related articles
Steve Connor: A scientific dream for more than half a century
A major research project is to be announced this week that will culminate in three years with the first transfusions into human volunteers of "synthetic" blood made from the stem cells of spare IVF embryos. It could help to save the lives of anyone from victims of traffic accidents to soldiers on a battlefield by revolutionising the vital blood transfusion services, which have to rely on a network of human donors to provide a constant supply of fresh blood.
The multimillion-pound deal involving NHS Blood and Transplant, the Scottish National Blood Transfusion Service and the Wellcome Trust, the world's biggest medical research charity, means Britain will take centre stage in the global race to develop blood made from embryonic stem cells. The researchers will test human embryos left over from IVF treatment to find those that are genetically programmed to develop into the "O-negative" blood group, which is the universal donor group whose blood can be transfused into anyone without fear of tissue rejection.
This blood group is relatively rare, applicable to about 7 per cent of the population, but it could be produced in unlimited quantities from embryonic stem cells because of their ability to multiply indefinitely in the laboratory.
The aim is to stimulate embryonic stem cells to develop into mature, oxygen-carrying red blood cells for emergency transfusions. Such blood would have the benefit of not being at risk of being infected with viruses such as HIV and hepatitis, or the human form of "mad cow" disease. The military in particular needs a constant supply of fresh, universal donor blood for battlefield situations when normal supplies from donors can quickly run out.
But developing blood made from the cells of spare IVF embryos will raise difficult ethical issues for people not happy with the idea of destroying embryos to create stem cells. It also raises the intriguing philosophical question of whether the synthetic blood will have come from someone who never existed. In theory, just one embryo could meet the nation's needs.
The Wellcome Trust is believed to have promised £3m towards the cost of the project, with further funding coming from the blood transfusion services of Scotland, and England and Wales. The Irish government is also understood to be involved. A spokesman for the Wellcome Trust said complicated legal issues were still being ironed out between all the parties involved but that an announcement is likely to be made in the coming week.
The project will be led by Professor Marc Turner, of Edinburgh University, the director of the Scottish National Blood Transfusion Service. Professor Turner has been involved in studies investigating how to ensure donated blood is free of the infectious agent behind variant CJD, the human form of "mad cow" disease. Several vCJD patients are thought to have contracted the disease by blood transfusions.
Professor Turner was unavailable for comment but a spokeswoman for the National Blood Service for England and North Wales confirmed that negotiations on the joint research project were at an advanced stage and that legal, rather than scientific, issues were holding up the announcement.
The multi-centre collaboration is also understood to involve scientists at the Medical Research Council's Centre for Regenerative Medicine at the University of Edinburgh, and Roslin Cells, a spin-off company that has emerged out of the Roslin Institute, where Dolly the sheep was cloned in 1996.
Scientists in other countries, notably Sweden, France and Australia, are also known to be working on the development of synthetic blood from embryonic stem cells. And last year, a team from a US biotechnology company, Advanced Cell Technology, announced that it has been able to produce billions of functioning red blood cells from embryonic stem cells. But the US work had been held up because of funding problems dating back to the ban on embryonic stem cell work under the Bush administration. President Barack Obama has since reversed that policy.
In Britain, the project was held up because of the difficulty of finding funding for "translational" research that attempts to take scientific studies in the laboratory into the earliest stages of commercial development. This problem has now been overcome

2009-03-22T02:35:00.000-07:00

2009-03-22T02:34:00.000-07:00

2009-03-14T22:25:00.002-07:00

2009-03-14T22:25:00.001-07:00

2009-03-14T22:24:00.001-07:00

Taking blood

2008-11-26T19:51:00.001-08:00

My superviser,Dr Vickers is taking blood from one of my friends.

Flow cytometry

2008-11-23T20:31:00.000-08:00

Flow Cytometry machine.

If you surf the internet you can barley find a proper picture of a flow cytometry machine!

This is the machine which I am working with over doing my project.

Assignment 2

2008-09-29T17:46:00.001-07:00

I have handed in these two assignments as the start of the project .

Cytometric measurement of Annexin 5 by flowcytometry
Supervised by : Dr J Vickers.
Saman Yazdani

1- Describe the hypothesis to be tested in your project
This experiment is aimed for a basic understanding of the system. It is a quantitative measurement of Annexin5 (a family of calcium-binding proteins).
There are two hypotheses on the table:
-Activation of platelets increases A5 binding to platelets.
Or
-Activation of platelets has no effect on A5binding to platelets surface. (Conference abstract)

First, the amount of A5 on the surface of platelets is measured by flow cytometry (as a base line).A5 is bound to phospholipids inside the platelets include: phosphatidyl inositol 1, phostatidyl serine and ethanol amine (conference abstract).
This measurement is kept as a base line to be compared. Then some substances include: ADP and A23187( Calcimycin, Calcium Ionophore, Antibiotic A23187 ,Calcium Ionophore A23187), (both as activator) and PGE1 (prostaglandin E1-as inhibitor)
Are separately added to the platelets (conference abstract).
If the indicated substances (ADP, A23187 and PGE1) had any effect on the presence and exhibition of those phospholipids, then it can be displayed (it is assumed). The fact is the indicated phospholipids would bind to A5 and then they expo and present Anx5 to the surface of the platelets (it is expected). In flowcytometry a material (A5 binding) would bind to A5 out side the platelets and then A5 binding is measured, it does indirectly shows the amount of A5 out side the platelets.
As stated above, the hypothesis is base on the fact that, the ADP, A23187 & PGE1 (as activator and inhibitors) could cause a change in the manifestation the Anx5 on the surface of the platelets.

2. Briefly outline the type of experimental design you intend to employ, include appropriate control strategies.

Experimental Design.
Materials & Method:
Blood samples (up to 5) .ADP and A23187 (activator) PGE1 (inhibitor), Flow cytometry machine) either 2 or 4 laser channels) and general haematology laboratory equipments.
First of all, the blood is taken from donors (not necessarily patient).It would be centrifuged and the plasma is separated and then platelet is reached from the Buffy coat layer. Then the sample will be washed to have more pure platelets.
In order to have a base line for experiment, before doing the rest of process, the level of A5 is measured by flowcytometry .In this experiment the effect of some substances is measured in order to realise the activation or inhibition of platelets.
As mentioned before this experiment is aimed to either prove or disapprove the indicated hypothesis. Further explanations are about the assumptions coming part5.However it is essential to know that why this experiment is done and what should be focused in all part of the experiment.
It is required to remember that the Anx5 cellular protein which binds with some types of phospholipids (mainly phospholipids serine) and then appears on the surface side of the platelets. (Conference abstract).
Like every other experiment, the first time is a negative control, means measuring with out adding any substance and just measuring the Anx5 as a base line.

3. What type of statistical analysis will be used to analyse your data?
Using SPSS software or simple excel spread sheets could be used to evaluate and analyse the data. The main output of this experiment is some figures by flowcytometry (and graphs) which indicate the amount of A5 in different cases include before and after adding those activator or inhibitors.
A schematic diagram like follow is expected.
Blue: A5 on the surface of platelet before adding ADP
Red: A5 on the surface of platelet before adding A23187
Yellow: A5 on the surface of platelet before adding PGE1
This could be extend with different substances and materials as well .Up to know what ever is reached is this fact that those indicated substances have effect( either increase or decrease) on the level of A5 presenting on the surface of platelets (conference abstract). Although more specific and details are always expected over any experiment.
As stated above, by changing the amount of those indicated substances their effect will be changed as well, hence in t2 the more indicated substances would be added, t3 ant t4 and t5 as well.

4- Outline the number and type of experimental observations that are required to carry out this type of analysis.
As the out put results of flow cytometry is an average of a lot of measurements of doing the test, Longobardi Givan (2001); therefore even if in each of those conditions, there are measured once, should not cause technical errors.
Up to 10 donors blood could give satisfactory data to be analysed.

5-In the majority of experimental designs it is necessary to make a number of assumptions. Outline some of the assumptions you have made when designing experiments for your project. Give reasons for these decisions

ADP-induced platelet aggregation when added in vitro and increase aggregation, Carlo patrono et al 2001, means increasing the activity of platelets. In the opposite side PGE1 and A23187 decrease the activity of platelets (conference abstract).Another fact which has already been mention is the affinity of phosphatydil serine to bind with Anx5 and presenting on the surface of the platelets. Consequently the main assumption could be finding a logical link between the activation of platelets (or inhibition) and level of presenting of Anx5 on the surface of the platelets.
One of the assumptions could be null and void, means there is not any relation ship between these parameters.
Another assumption is, the more the platelets are activated, the more Anx5 they would expose. The third assumption is vice verse the second one, the more the platelets are activated the less Anx5 they would expose.
It is assumed that the assumption would be rejected, because lots of other researchers earlier have been proved that the phosphatydil molecules inside the platelet surface would be undergone due to the activity of the platelets, E.E Nishizawa et el (2008).If so, the level of manifesting the anx5 must logically change on the surface of the platelets (either increase or decrease). Base on those three indicated assumptions the experiment and following the hypothesis would be carried on.
There are other physiological activators which have similar effect on the platelets: thrombin, collagen, thromboxane A2, adrenaline, platelet activating factor, 5-HT, vasopressin, George, JG. (2000) and Dahlbäck, B (2000). Blood coagulation. It is assumed to use other substances over the experiment as well.

References:
-Alice Longobardi Givan , Flow Cytometry: First Principles, Second Edition.
Copyright 2001 by Wiley-Liss, Inc.
ISBNs 0-471-38224-8 (Paper); 0-471-22394-8 (Electronic)

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-Reutelingsperger CP, Kop JM, Hornstra G, Hemker HC. Purification and characterization of a novel protein from bovine aorta that inhibits coagulation:
inhibition of the phospholipid-dependent factor-Xa-catalyzed prothrombin activation, through a high-affinity binding of the anticoagulant to the phospholipids. Eur J Biochem. 1988;173:171–178

-Tait JF, Smith C, Blankenberg FG. Structural requirements for in vivo detection
of cell death with 99mTc-annexin V. J Nucl Med. 2005;46:807– 815

-Trotter,PJ Orchard, MA and Walker,J H . Relocation of annexin V to platelet membranes is a phosphorylation-dependent process. Biochem Journal 1997; 1 December , pp. 447–452. [Pubmed]

-Zimmermann KC, Green DR. How cells die: apoptosis pathways. J Allergy Clin
Immunol. 2001;108(suppl):S99 –S103

-Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid
asymmetry in blood cells. Blood. 1997;89:1121–1132.

Assignment 1

2008-09-29T17:44:00.000-07:00

I have handed in these two assignments as the start of the project .
Assignment one:
Cytometric measurement of Annexin V by flow cytometry
Supervised by: Dr James Vickers
Saman Yazdani

Annexin V is defined as a cellular protein with an unknown function, Ernest Beutler (2000). Therefore recognition of some functions of Annx5 could be the main object. The quantitative measurement of Annx5 is another way to get more information about this protein.Annx5 is called with other names as well like: Alternative titles; symbols Annexin V; ANX5 , endonexin II; ENX2, placental Anticoagulant protein I , Vascular anticoagulant-alpha lipocortin V placental protein 4; PP4 , anchorin CII and it gen map locus is: 4q26-q28 , Tait et al (1991). AnxA5 is a member of the Annexin family. This a multiprotein family of over 160 protein that share the property of Ca2+ dependent binding to negatively charged phospholipid surface Gerke V, et al (2002) . It is also indicated that Annexin V forms the voltage-dependent Ca (2+) channels in phospholipids bilayers and was the first ion channel to be structurally and functionally characterized. Demange et al. (1994) . At the end of the 1970s, anxA5 was first isolated from human placenta, Bohn H (1979). A few years later was discovered independently in blood vessels and named vascular anticoagulant protein alpha (VAC-alpha) because of its property to inhibit blood coagulation, Reutelingsperger CP (1985). The anticoagulant mechanism is based on the high-affinity binding to PS. This property makes Anx5 very effective in inhibiting the prothrombinase complex; Reutelingsperger CP et al (1988) .Elucidation of its primary structure revealed that VAC- alpha (vascular anticoagulant protein alpha) was a member of the Annexin family. It was therefore termed Annexin V. According to this system, human Annexin V was renamed anxA5. After its discovery, anxA5 was produced by the expression of its complementary DNA in the bacterium Escherichia coli, Maurer-Fogy I et al (1988), Iwasaki A et al (1987).Anx5 has an important role in Apoptosis as well, Ernest Beutler (2000) .Apoptosis, which is the major form of PCD, Leist M (2001), plays an important role in the development, homeostasis, and disease of an organism. In pathology, 2 opposing situations can arise concerning apoptosis: 1 situation in which there is abundant apoptosis, as in cases of transplanted organ rejections, AIDS, septic shock, and cardiovascular and neurodegenerative diseases, and 1 situation in which there is insufficient apoptosis, as in cases of cancer , Blankenberg FG et al (2001). Fadok et al (1992) revealed that PS is expressed on the cell surface of apoptotic cells, Fadok V et al (1992). Viable cells retain PS Predominantly located in the inner leaflet of the cell membrane, Zwaal RF et al (1997). This notion led, Koopman et al (1994) and others to design an apoptosis detection assay on the basis of fluorescence (fluorescin isothiocyanate)-labelled anxA5, Koopman (1994), Martin SJ (1995), Homburg (1995). To be able to discern between apoptotic cells and necrotic cells, which have compromised plasma membrane integrity, propidium iodide (PI) was added. By this means, viable, apoptotic, and necrotic cells can be discriminated by either fluorescence microscopy or flow cytometry. PCD is a rapidly changing topic. The current state of the art concerning apoptosis is described in several recent reviews, Leist M (2001), Kroemer G et al (2005), and Zimmermann KC et al (2001). A5 in involves in the following Biological Process: anti-apoptosis , Signal transduction , Negative regulation of coagulation , Ernest Beutler (2000) . A5 has a high affinity to bind to phosphatidylserine. Phosphatidylserine is translocated from the inner side of the membrane to the outer layer, if cell undergoes death by apoptosis (or necrosis) and serves as one of the several signals by which cell destined for death and also is recognized by phagocytes, Van England et al (1998). That is what is focused on in this experiment. The high affinity of anx5 to phosphatidylserine.The base of this experiment is ( Design Experiment) : First the sample is taken from the donors ( up to 5 ) there are centrifuged and washed and platelets are separated and then as a base line the amount of Anx5 on the surface of the platelets is measured by flow cytometry , conference abstract . Then in separate times some substances are added to the platelets.
ADP, A23187 ( Calcimycin, Calcium Ionophore, Antibiotic A23187 ,Calcium Ionophore A23187)and PGE1 (prostaglandin E1) .ADP is an activator of the platelets and A23187 & PGE2 will decrease the platelets activity, Carlo patrono et al 2001.
Prostaglandin E1 stimulated the formation of platelet radioactive 3′:5′-cyclic AMP in a dose-dependent manner, G. Ball et al (1970) .Which eventually causes inhibiting activity of the platelets. There are two hypotheses in this regard:
-Activation of the platelets will increase A5 binding to the platelets surface
Or
-Activation of the platelets has no effect on the Anx5 binding to the platelets surface.

The explanation of hypothesis is: Phosphatidylserine, which is one of the “eat me” signals at the surface of the apoptotic cell .A5, would bind to this molecule.
--In the case of increasing activity of the platelets: ADP will cause phosthatydil serine inside the platelets appears more on the surface of the platelets, E. E. Nishazawa (2008). As stated before Anx5 is bound to phosphatydil serine and therefore the appearance of Anx5 will be increased (it is assumed).
--In the case of inhibiting activity of the platelets:
A23187 and PGE1 will be added to the platelets separately .A23187 and PGE1 are inhibitor for platelet activates (conference abstract), the manifestation of phosphatydil serine will be reduces on the surface of the platelets consequently the Anx5 binding will decrease (it is assumed).
When the phsphatadyil serine appears on the surface of platelets ad stated before, Anx5 is bound and then by flowcytometry the level of Anx5 is measured.
, Alice Longobardi Givan (2001).The final part is getting conclusion .As stated above this experiment is designed to either prove or disapprove the two indicated hypothesis. The final conclusion will help to get a basic understating of the role of ADP, A23187 and PGE1 on the appearing of Anx5 on the surface of the platelets which might be helpful for future investigations.

References:
-Alice Longobardi Givan , Flow Cytometry: First Principles, Second Edition.
Copyright 2001 by Wiley-Liss, Inc.ISBNs 0-471-38224-8 (Paper); 0-471-22394-8 (Electronic)

- Blankenberg FG, Strauss HW. Will imaging of apoptosis play a role in clinical
care? A tale of mice and men. Apoptosis. 2001;6:117–123

- Bohn H, Kraus W. Isolation and characterization of a new placenta specific
protein (PP10) . Arch Gynecol. 1979; 227:125–134.

-Carlo Patrono, Barry Coller, James E. Dalen, Garret A. FitzGerald, Valentin Fuster, Michael Gent, Jack Hirsh and Gerald Roth . Platelet-Active Drugs: The Relationships among Dose, Effectiveness, and Side Effects .On line version of chest journal 2001.

-Dahlbäck, B. Blood coagulation. The Lancet 2000; 355: 1627-1632

-E. E. Nishizawa 1 J. F. Mustard 1 .The Effect of Synthetic Phosphatidyl Serines on Platelet Aggregation, Blood Coagulation and Haemostasis. British Journal of Hematology 2008 ; Volume 20 Issue 1, Pages 45 – 54.

-Ernest Beutler, Marshall A.Lichtman, Barry S.Coller, Thomas J.Kipps, Uri Seligsohn. Anti phospholipid syndrome.Williams Haematology 2000;(pp. 1715-1728).

-Fadok V, Voelker DR, Campbell PA, et al. Exposure of phosphatidylserine on the
surface of apoptotic lymphocytes triggers specific recognition and removal by
macrophages. J Immunol. 1992;148:2207–2216

-G. Ball, G. G. Brereton, Mary Fulwood, D. M. Ireland and Patricia Yates ,(1970) ,
Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3´:5´-cyclic monophosphate.Biochem. Journal. (1970) 120 (709–718)

- Gerke V, Moss SE. Annexins: from structure to function. Physiol Rev. 2002;82:
331–371

-George, JG. Platelets. The Lancet 2000; 355: 1531-1539

-Hendrikus H. Boersma, Bas L.J.H. Kietselaer,Leo M.L. Stolk, PharmD,
Abdelkader Bennaghmouch, Leonard Hofstra,Jagat Narula, . Past, Present, and Future of Annexin A5: From Protein Discovery to Clinical Applications. The Journal of Nuclear medicine. 2005; Vol. 46. No. 12

-Homburg CH, de Haas M, von dem Borne AE, et al. Human neutrophils lose their
Surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in
Vitro; Blood. 1995;85:532–540.

-Iwasaki A, Suda M, Nakao H, et al. Structure and expression of cDNA for an
inhibitor of blood coagulation isolated from human placenta: a new lipocortinlike
protein. J Biochem (Tokyo). 1987;102:1261–1273

-Koopman G, Reutelingsperger CP, Kuijten GA, et al. Annexin V for flow
cytometric detection of phosphatidylserine expression on B cells undergoing
apoptosis. Blood 1994;84:1415–1420

-Kroemer G, Martin SJ. Caspase-independent cell death. Nat Med. 2005;11:725–
730.

-Leist M, Jaattela M. Four deaths and a funeral: from caspases to alternative
mechanisms. Nat Rev Mol Cell Biol. 2001;2:589 –598
1985;151:625– 629

-Martin SJ, Reutelingsperger CP, McGahon AJ, et al. Early redistribution of
plasma membrane phosphatidylserine is a general feature of apoptosis regardless
of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp
Med. 1995;182:1545–1556.

-Maurer-Fogy I, Reutelingsperger CP, Pieters J, et al. Cloning and expression of
cDNA for human vascular anticoagulant, a Ca2_-dependent phospholipid-binding
protein. Eur J Biochem 1988;174:585–592.

- Reutelingsperger CP, Kop JM, Hornstra G, Hemker HC. Purification and characterization of a novel protein from bovine aorta that inhibits coagulation:
inhibition of the phospholipid-dependent factor-Xa-catalyzed prothrombin activation, through a high-affinity binding of the anticoagulant to the phospholipids. Eur J Biochem. 1988;173:171–178

-Tait JF, Smith C, Blankenberg FG. Structural requirements for in vivo detection
of cell death with 99mTc-annexin V. J Nucl Med. 2005;46:807– 815

-Trotter,PJ Orchard, MA and Walker,J H . Relocation of annexin V to platelet membranes is a phosphorylation-dependent process. Biochem Journal 1997; 1 December , pp. 447–452. [Pubmed]

-Zimmermann KC, Green DR. How cells die: apoptosis pathways. J Allergy Clin
Immunol. 2001;108(suppl):S99 –S103

-Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid
asymmetry in blood cells. Blood. 1997;89:1121–1132.

2008-08-28T12:10:00.001-07:00

Antiphospholipid Antibodies Accelerate Plasma Coagulation by Inhibiting Annexin-V Binding to Phospholipids: A "Lupus Procoagulant" Phenomenon

2008-08-19T12:33:00.001-07:00

Annexin A5
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Annexin A5

PDB rendering based on 1a8a.
Available structures: 1a8a, 1a8b, 1anw, 1anx, 1avh, 1avr, 1bc0, 1bc1, 1bc3, 1bcw, 1bcy, 1bcz, 1g5n, 1hak, 1hvd, 1hve, 1hvf, 1hvg, 1n41, 1n42, 1n44, 1sav, 2ie6, 2ie7, 2ran
Identifiers
Symbol(s)
ANXA5; ANX5; ENX2; PP4
External IDs
OMIM: 131230 MGI: 106008 HomoloGene: 20312
[show]Gene ontology
Molecular Function:
• phospholipase inhibitor activity• calcium ion binding• protein binding• calcium-dependent phospholipid binding
Cellular Component:
• intracellular• cytoplasm
Biological Process:
• anti-apoptosis• signal transduction• blood coagulation• negative regulation of coagulation
RNA expression pattern

More reference expression data
Orthologs
Human
Mouse
Entrez
308
11747
Ensembl
ENSG00000164111
ENSMUSG00000027712
Uniprot
P08758
Q3U5Q1
Refseq
NM_001154 (mRNA)NP_001145 (protein)
NM_009673 (mRNA)NP_033803 (protein)
Location
Chr 4: 122.81 - 122.84 Mb
Chr 3: 36.64 - 36.66 Mb
Pubmed search
[1]
[2]
Annexin A5 (or annexin V) is a cellular protein in the annexin group. The function of the protein is unknown, however annexin A5 has been proposed to play a role in the inhibition of blood coagulation by competing for phosphatidylserine binding sites with prothrombin and also to inhibit the activity of phospholipase A1 also by competing for phosphatidylserine binding sites. These properties have been found by in vitro experiments (in a test tube) and they have not been confirmed by experiments with laboratory animals.
Contents[hide]
1 Annexin A5 in pathology
2 Laboratory use of annexin A5
3 References
4 Further reading
5 External links
//

[edit] Annexin A5 in pathology
Antibodies directed against annexin A5 are the cause of a syndrome called the antiphospholipid syndrome.
Annexin A5 forms a shield around negatively-charged phospholipid molecules. The formation of an annexin A5 shield blocks the entry of phospholipids into coagulation (clotting) reactions. In the antiphospholipid antibody syndrome, the formation of the shield is disrupted by antibodies. Without the shield, there is an increased quantity of phospholipid molecules on cell membranes, speeding up coagulation reactions and causing the blood-clotting characteristic of the antiphospholipid antibody syndrome.

[edit] Laboratory use of annexin A5
Annexin A5 is used as a probe in the annexin A5 affinity assay to detect cells that have expressed phosphatidylserine on the cell surface, a feature found in apoptosis as well as other forms of cell death.[1][2]

[edit] References
^ Koopman G, Reutelingsperger CP, Kuijten GAM et al. (1994). "Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis". Blood 84 (5): 1415–20. PMID 8068938.
^ Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995). "A novel assay for apoptosis—flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V". J Immunol Methods 184 (1): 39. doi:10.1016/0022-1759(95)00072-I. PMID 7622868.

[edit] Further reading
Cederholm A, Frostegård J (2007). "Annexin A5 as a novel player in prevention of atherothrombosis in SLE and in the general population.". Ann. N. Y. Acad. Sci. 1108: 96–103. PMID 17893975.
Schlaepfer DD, Jones J, Haigler HT (1992). "Inhibition of protein kinase C by annexin V.". Biochemistry 31 (6): 1886–91. PMID 1310621.
Huber R, Berendes R, Burger A, et al. (1992). "Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.". J. Mol. Biol. 223 (3): 683–704. PMID 1311770.
Kirsch T, Pfäffle M (1992). "Selective binding of anchorin CII (annexin V) to type II and X collagen and to chondrocalcin (C-propeptide of type II collagen). Implications for anchoring function between matrix vesicles and matrix proteins.". FEBS Lett. 310 (2): 143–7. PMID 1397263.
Dawson SJ, White LA (1992). "Treatment of Haemophilus aphrophilus endocarditis with ciprofloxacin.". J. Infect. 24 (3): 317–20. PMID 1602151.
Tait JF, Frankenberry DA, Shiang R, et al. (1992). "Chromosomal localization of the human gene for annexin V (placental anticoagulant protein I) to 4q26----q28.". Cytogenet. Cell Genet. 57 (4): 187–92. PMID 1683830.
Huber R, Römisch J, Paques EP (1991). "The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.". EMBO J. 9 (12): 3867–74. PMID 2147412.
Huber R, Schneider M, Mayr I, et al. (1991). "The calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution. Implications for membrane binding and calcium channel activity.". FEBS Lett. 275 (1-2): 15–21. PMID 2148156.
Maurer-Fogy I, Reutelingsperger CP, Pieters J, et al. (1988). "Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein.". Eur. J. Biochem. 174 (4): 585–92. PMID 2455636.
Rothhut B, Coméra C, Cortial S, et al. (1990). "A 32 kDa lipocortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation.". Biochem. J. 263 (3): 929–35. PMID 2532007.
Schlaepfer DD, Mehlman T, Burgess WH, Haigler HT (1987). "Structural and functional characterization of endonexin II, a calcium- and phospholipid-binding protein.". Proc. Natl. Acad. Sci. U.S.A. 84 (17): 6078–82. PMID 2957692.
Funakoshi T, Heimark RL, Hendrickson LE, et al. (1987). "Human placental anticoagulant protein: isolation and characterization.". Biochemistry 26 (17): 5572–8. PMID 2960376.
Iwasaki A, Suda M, Nakao H, et al. (1988). "Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta: a new lipocortin-like protein.". J. Biochem. 102 (5): 1261–73. PMID 2963810.
Funakoshi T, Hendrickson LE, McMullen BA, Fujikawa K (1988). "Primary structure of human placental anticoagulant protein.". Biochemistry 26 (25): 8087–92. PMID 2964863.
Kaplan R, Jaye M, Burgess WH, et al. (1988). "Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein.". J. Biol. Chem. 263 (17): 8037–43. PMID 2967291.
Grundmann U, Abel KJ, Bohn H, et al. (1988). "Characterization of cDNA encoding human placental anticoagulant protein (PP4): homology with the lipocortin family.". Proc. Natl. Acad. Sci. U.S.A. 85 (11): 3708–12. PMID 2967495.
Pepinsky RB, Tizard R, Mattaliano RJ, et al. (1988). "Five distinct calcium and phospholipid binding proteins share homology with lipocortin I.". J. Biol. Chem. 263 (22): 10799–811. PMID 2968983.
Ahn NG, Teller DC, Bienkowski MJ, et al. (1989). "Sedimentation equilibrium analysis of five lipocortin-related phospholipase A2 inhibitors from human placenta. Evidence against a mechanistically relevant association between enzyme and inhibitor.". J. Biol. Chem. 263 (35): 18657–63. PMID 2974032.
Demange P, Voges D, Benz J, et al. (1994). "Annexin V: the key to understanding ion selectivity and voltage regulation?". Trends Biochem. Sci. 19 (7): 272–6. PMID 7519374.
Fernández MP, Morgan RO, Fernández MR, Carcedo MT (1994). "The gene encoding human annexin V has a TATA-less promoter with a high G+C content.". Gene 149 (2): 253–60. PMID 7958998.

[edit] External links
MeSH Annexin+A5
[show]
v • d • e Carrier protein: calcium-binding proteins
Intracellular calcium-sensing proteins
Calmodulin - Calnexin - Calreticulin - Gelsolin - neuronal (Hippocalcin, Neurocalcin, Recoverin)
Other
Annexin (A1, A2, A5) - Vitamin D-dependent calcium-binding protein/Calbindin - Calexcitin - Calsequestrin - Fibulin - Matrix gla protein - Osteocalcin - Osteonectin - S-100 - Synaptotagmin - Troponin C

This membrane protein-related article is a stub. You can help Wikipedia by expanding it.
Retrieved from "http://en.wikipedia.org/wiki/Annexin_A5"
Categories: Genes on chromosome 4 Human proteins Peripheral membrane proteins Membrane protein stubs

2008-08-19T11:44:00.001-07:00

Annexin-5 apoptosis assay
Annexin-5 is a member of a highly conserved protein family that bind acidic phospholipids in a calcium-dependent manner. Annexin-5 is known also as 35 kDa Calelectrin, 35-gamma Calcimedin, Anchorin C2, PAP-1 (placental anticoagulant protein-1), Calphobindin-1, Endonexin-2, Lipocortin V, VAC-alpha (vascular anticoagulant-alpha).
The protein has been shown to possess a high affinity for phosphatidylserine. Phosphatidylserine is translocated from the inner side of the plasma membrane to the outer layer when cells undergo death by apoptosis or cell necrosis and serves as one of several signals by which cell destined for death are recognized by phagocytes (see also: apoptotic bodies) (Van Engeland et al, 1998).
Cell death by apoptosis and necrosis differ from each other in that cell membranes maintain their integrity during the initial stages of apoptosis, while they become leaky during necrotic cell death. If Annexin-5 binds to the cell surface this indicates that cell death is imminent. In order to differentiate apoptosis from necrosis, a dye exclusion test with propidium iodide is performed to establish whether membrane integrity has been conserved or whether membranes have become leaky. A combination test measuring Annexin-5 binding and dye exclusion thus allows discrimination between intact cells, apoptotic cells, and necrotic cells. The test allows detection of apoptosis at early stages before gross morphological changes have occurred and has been adapted for use with flow cytometry, light and electron microscopy and can be applied to vital and fixed material (Miller, 2004; Pellicciari et al, 1997). Schellenberger et al (2002) have conjugated Annexin-5 to crosslinked iron oxide (CLIO) nanoparticles, a functionalized superparamagnetic preparation developed for target-specific magnetic resonance imaging (MRI). The resulting nanoparticles can be used to remove apoptotic cells from cell suspensions containing healthy and apoptotic cells by magnetic column chromatography and to visualize apoptotic cells by MRI.
Dillon et al (2000) have reported that detection of annexin-5 binding may not always be indicative of apotosis. Phosphatidylserine exposed on the majority of B-cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor-mediated signaling events.
For other assays allowing detection of apoptotic cells see also: apoptosis assays. Kawaminami et al (1998) have reported that the withdrawal of ovarian hormones induces huge castration cells in the anterior pituitary gland. These cells express annexin-5. Annexin-5 is detected also in follicular epithelial cells and parafollicular cells of the thyroid gland, adrenocortical cells of the zona fasciculata and zona reticularis, luteal cells, testicular interstitial cells, and Sertoli cells. It is not detected in the pineal gland, the parathyroid gland, the islet of Langerhans, the adrenal medulla, zona glomerulosa cells, and granulosa cells. The authors have suggested that annexin-5 contributes to secretory cell functions, which may be common to endocrine cells secreting chemically different hormones.

2008-08-19T11:44:00.000-07:00

2008-07-27T11:42:00.001-07:00

AnnexinV (endonexin II, lipocortin V, 35K calelectrin, 35-g-calcimedin, PP4, IBC, PAP-I, VAC-a, calphobindin I, anchorin CII, thromboblastin inhibitor)

Flow cytometry

2008-07-27T11:35:00.000-07:00

Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.

2008-07-26T20:28:00.001-07:00

The annexin V assay has been widely accepted as a marker of apoptosis
more info would be given in near future.

Platelets

2008-07-19T20:05:00.001-07:00

Platelets, or thrombocytes, are the cells circulating in the blood that are involved in the cellular mechanisms of primary hemostasis leading to the formation of blood clots. In strict sense, the term platelet should be reserved for anucleated thrombocytes of mammals. Nucleated thrombocytes of nonmammalian vertebrates differ from the mammalian plates not only in having a nucleus and thus resembling B lymphocytes, but also these nucleated thrombocytes do not aggregate in response to ADP, serotonin and adrenaline (they do aggregate with thrombin, of course.) [1]. Dysfunction or low levels of platelets predisposes to bleeding, while high levels, although usually asymptomatic, may increase the risk of thrombosis. An abnormality or disease of the platelets is called a thrombocytopathy[2] which could be either a decrease in number ie thrombocytopenia, decrease in function ie thrombasthenia or an increase in number ie thrombocytosis

Annexin 5

2008-07-10T19:52:00.000-07:00

To day we had a meeting with Dr Vickers , supervisior of project . We have been discussing about the project which is supposed to be done in this year.
At the end , while I did not want but I asked him, could we publish it? he said I have not already published the projects of students but it is good , perhaps!
now pray for me! if I can publish it it would be a real vistory!!!!!!!

2008-05-15T16:48:00.001-07:00

Definition of Annexin V
Annexin V: Annexin V is the cause of a syndrome called the antiphospholipid antibody syndrome with abnormal blood clotting.
The annexins are a family of proteins first described in 1990. All of the annexin proteins share the property of binding calcium and phospholipids.
Annexin V normally forms a shield around certain phospholipid molecules that blocks their entry into coagulation (clotting) reactions. In the antiphospholipid antibody syndrome, the formation of this shield is disrupted by the abnormal antibodies. Without the shield, there is an increased quantity of phospholipid molecules on cell membranes, speeding up coagulation reactions and causing the abnormal blood clotting characteristic of the antiphospholipid antibody syndrome

April 2008

2008-04-19T07:25:00.001-07:00

Blood Transfusion Journal -April 2008
please click here .

project

2008-04-11T12:33:00.000-07:00

Cytometric measurement of annexin V bind to platelet

project

2008-03-20T12:14:00.000-07:00

A couple of days ago, I change my project . The new topic is aabout Haematology . I will write more about it. waiting for new news.

Journal-March2008

2008-02-23T09:19:00.000-08:00

Journal-March2008

Transfusion
The Journal of AABB
March 2008 - Vol. 48 Issue 3 Page 405-571

You can download it for free, please click here .

Stroke

2008-02-20T05:44:00.000-08:00

Also known as a cerebrovascular accident (CVA), a stroke is a life-threatening event in which part of the brain is deprived of adequate oxygen. Strokes are extremely dangerous, accounting for more than 160,000 deaths each year in the United States, according to the Centers for Disease Control and Prevention (CDC). Stroke is the third leading cause of death in the United States, behind heart disease and cancer. It is also a leading cause of adult disability and institutionalization.
There are two kinds of strokes. An ischemic stroke occurs when the blood supply to the brain is interrupted, usually by a blood clot. The second kind of stroke is a hemorrhagic stroke, which occurs when there is bleeding in or around the brain. Some people may also experience a “mini-stroke” (also called a transient ischemic attack), where symptoms last for a short period of time. All strokes are considered medical emergencies.
Symptoms of a stroke may include numbness or weakness, confusion, dizziness, trouble speaking or understanding others and paralysis. After a stroke begins, it is imperative that people seek treatment as soon as possible to re-establish the flow of oxygen-rich blood to brain cells before permanent tissue damage or death occurs. Imaging tests may also be performed to confirm that a stroke has occurred, identify any potential causes and determine the extent of brain damage (if any).
People who survive a stroke should begin stroke rehabilitation as soon as possible to regain as many lost functions (e.g., lack of coordination, muscle strength) as possible.There are several risk factors associated with strokes. They include age, high blood pressure, heart disease, smoking and obesity. In general, prevention methods for stroke are aimed at eliminating or treating the risk factors. This can usually be accomplished by making certain lifestyle changes, such as eating a heart-healthy diet and taking medications. A physician might also recommend surgery for some patients.

A good linke in this regard:

Video , Text .

Stroke

2008-02-18T15:12:00.000-08:00

StrokeWhat causes a stroke?
Stroke is a disease that affects the blood vessels that supply blood to the brain.
A stroke occurs when a blood vessel that brings oxygen and nutrients to the brain either bursts (hemorrhagic stroke) or is clogged by a blood clot or some other mass (ischemic stroke). When a rupture or blockage occurs, parts of the brain don't get the blood and oxygen they need. Without oxygen, nerve cells in the affected area of the brain can't work properly, and die within minutes. And when nerve cells can't work, the part of the body they control can't work either. The devastating effects of a severe stroke are often permanent because dead brain cells aren't replaced.
There are two main types of stroke. One (ischemic stroke) is caused by blockage of a blood vessel; the other (hemorrhagic stroke) is caused by bleeding. Bleeding strokes have a much higher death rate than strokes caused by clots.
What is ischemic stroke?
Ischemic stroke is the most common type. It accounts for about 87 percent of all strokes. It occurs when a blood clot (thrombus) forms and blocks blood flow in an artery bringing blood to part of the brain. Blood clots usually form in arteries damaged by fatty buildups, called atherosclerosis.
When the blood clot forms within an artery of the brain, it's called a thrombotic stroke. These often occur at night or first thing in the morning. Another distinguishing feature is that very often they're preceded by a transient ischemic attack. This is also called a TIA or "warning stroke." TIAs have the same symptoms of stroke but only last a few minutes; stroke symptoms last much longer. If someones experiences a TIA, they should urgent medical care immediately.
What is a cerebral embolism?
A wandering clot (an embolus) or some other particle that forms away from the brain, usually in the heart, may also cause an ischemic stroke. This is called cerebral embolism. The clot is carried by the bloodstream until it lodges in an artery leading to or in the brain, blocking the flow of blood.
The most common cause of these emboli is blood clots that form during atrial fibrillation (AF). AF is a disorder found in about 2.2 million Americans. It's responsible for 15–20 percent of all strokes. In AF, the heart's two small upper chambers (the atria) quiver like a bowl of jello instead of beating strongly and effectively. Some blood isn't pumped completely out of them when the heart beats, so it pools and clots can form. When a blood clot enters the circulation and lodges in a narrowed artery of the brain, a stroke occurs. This is called a cardioembolic stroke, or a stroke that occurs because of a heart problem.
What is hemorrhagic stroke?
A subarachnoid hemorrhage occurs when a blood vessel on the brain's surface ruptures and bleeds into the space between the brain and the skull (but not into the brain itself).
A cerebral hemorrhage occurs when a defective artery in the brain bursts, flooding the surrounding tissue with blood.
Hemorrhage (or bleeding) from an artery in the brain can be caused by a head injury or a burst aneurysm. Aneurysms are blood-filled pouches that balloon out from weak spots in the artery wall. They're often caused or made worse by high blood pressure. Aneurysms aren't always dangerous, but if one bursts in the brain, they cause a hemorrhagic stroke.
When a cerebral or subarachnoid hemorrhage occurs, the loss of a constant blood supply means some brain cells no longer can work. Accumulated blood from the burst artery also may put pressure on surrounding brain tissue and interfere with how the brain works. Severe or mild symptoms can result, depending on the amount of pressure.
The amount of bleeding determines the severity of cerebral hemorrhages. In many cases, people with cerebral hemorrhages die of increased pressure on their brains. But those who live tend to recover much more than people who've had strokes caused by a clot. That's because when a blood vessel is blocked, part of the brain dies — and the brain doesn't regenerate itself; in other words, brain cells can't be replaced. But when a blood vessel in the brain bursts, pressure from the blood compresses part of the brain. If the person survives, gradually the pressure goes away. Then the brain may regain some of its former function.

Benefits of Mechanical Thrombectomy Catheter Devices.

2008-02-12T15:13:00.000-08:00

Benefits of Mechanical Thrombectomy Catheter Devices.
The benefits of mechanical thrombectomy techniques have mainly been demonstrated in short-term studies. In one such trial, the benefit of using thrombectomy devices alongside any adjuvant therapy was examined.16 With the exception of aspirin and thienopyridine, which are used to prevent stroke, combined usage of adjuvant therapy and thrombectomy provides no long-term improvements in left ventricular systolic function. This indicates that thrombectomy alone is sufficient to remove thrombolic occlusion in the coronary artery. The main improvement offered by mechanical thrombectomy over standard angioplasty is the reduction in risk of distal embolism. The balloon catheter has to pass directly through the thrombus in order to be inflated, which frequently leads to the breaking off of small fragments from the thrombus that can cause a blockage elsewhere. In a study examining the microcirculation between mechanical thrombectomy and angioplasty patients, the thrombectomy method resulted in higher flow rates due to the reduced risk of distal embolism.12 Occurrence of macroembolisation was also negligible with thrombectomy tested on both fresh and hardened thrombi.17 In addition, the procedure time of both thrombectomy and the standard balloon technique is extremely comparable. Hence, the thrombectomy technique fits into the tight time constraints in cardiac blockage. Thrombectomy also has advantages over thrombolytics. Administration of thrombolytic substances is time-consuming: the drugs take from one hour to 90 minutes to reduce the thrombus. This time delay exposes the patient to a 1% haemorrhagic stroke rate, which is an unacceptable risk.18

Mechanical Thrombectomy Devices Supplement Thrombolysis

2008-02-07T17:23:00.000-08:00

Managing thrombosis comprises a critical part of vascular patient care, because the development of thromboses poses considerable risk throughout the body, frequently precipitating stroke, myocardial infarction, compromised circulation and ischemia. Sites at particular risk for thromboses formation include dialysis access grafts, ischemic lower extremities with native artery or bypass occlusions, iliofemoral deep veins, cerebral arteries and coronary bypass grafts, which are prone restenose and to seed distal thromboemboli.
Percutaneous revascularization of thrombus-containing lesions has an increased incidence of adverse events such as abrupt vessel closure, periprocedural myocardial infarction and death. Various pharmacologic agents and mechanical approaches have demonstrated modest angiographic success with significant periprocedural complications.
The pharmacological treatment of thrombosis has advanced in recent years with the growing familiarity with tissue plasminogen activators (rt-PAs), the market withdrawal and return of urokinase, and the advent of newer agents such as tenecteplase and plasmin-based products. Despite these developments, many patients remain refractory to pharmacotherapy. Moreover, the collective costs of such therapy are daunting among those in whom thrombosis can be treated in-hospital by large doses of lytic agents.
In those patients non-responsive to pharmacotherapy, the application of mechanical trombectomy devices (MTDs) is expanding to encompass a greater range of thrombotic sites. These devices fall into two broad categories, those that fragment the thrombus, with or without clot removal and those that contact the vessel wall.
The advantages of MTDs can include faster reperfusion of ischemic limbs, potentially shorter hospital stays than those required for lytic drug administration alone, and lower rates of rethrombosis. MTD therapy also may provide comparative advantages of safety by leading to fewer bleeding complications, because they can reduce lytic drug doses, or eliminate them altogether. This theoretical advantage, however, must be balanced against device risks such as distal embolization, hemolysis, and vessel wall damage, the likelihood of which varies with each specific device and application.
Mechanical thrombolytic devices have become the mainstay of percutaneous treatment of clotted dialysis grafts. Properly used, they are faster, safer and equally or more effective than chemical lysis. This was one of the main findings presented at the recent conference of the Cardiovascular and Interventional Radiological Society of Europe (CIRSE 2003).
Surgical literature indicates that removing wall-adherent material (clot and pseudointima) is not only desirable but also essential for improved patency of these grafts. Currently, only two wall-contact devices are approved by the FDA for de-clotting synthetic dialysis grafts: the Arrow-Trerotola percutaneous thrombolytic device from Arrow International, Reading, Penna. and the Akonya Eliminator from IDev Technologies, Houston.
In a broader category, the AngioJet (Possis Medical, Inc., Minneapolis) is the only thrombectomy system currently approved for peripheral arterial applications; although many other MTDs have been used off-label for this purpose. The AngioJet is a dual lumen catheter designed to rapidly remove blood clots with minimal vascular trauma. In April 2000, March 1999 and December 1996, Possis received FDA clearances to commence United States marketing of the AngioJet System, for removal of blood clots in leg arteries, native coronary arteries and coronary bypass grafts and access grafts used by patients on kidney dialysis, respectively. The AngioJet System is typically used in conjunction with other medical devices, such as angioplasty balloons and stents (both bare metal and drug eluting), and drugs, such as thrombolytics and platelet inhibitors. Possis also markets the XMI, XVG, Xpeedior and AVX catheters. Each of these catheters features the company's proprietary Cross-Stream Technology.
When compared with urokinase in the randomized VeGAS 2 Trial, AngioJet therapy yields greater angiographic success with a lower incidence of 30-day major adverse events.
The Trellis, manufactured by Bacchus Vascular, Inc., Santa Clara, Calif., is a novel drug infusion device designed for isolated thrombolysis. A thrombus is isolated between two occlusion balloons while the thrombolytic is mechanically dispersed with an oscillating wire and then aspirated. Theoretically, isolated thrombolysis enables single-setting thrombolysis by delivering a high concentration of thrombolytic agent while preventing systemic dispersion. Adjunctive procedures may be performed in the same single setting. Data from a voluntary company registry for isolated thrombolysis have indicated lower hospital costs due to reduced ICU stay and lytic dosing, and no reported bleeding complications. Bacchus reports that a future registry is planned. The Trellis is approved for use in the peripheral vasculature.

Project

2008-02-06T07:18:00.000-08:00

16. An Investigation into clot removal times concerning artificial and real blood clots inserted in a straight plastic tube. Supervisor: GP DescriptionThe aim of this project is to investigate the times (together with pressures involved and volumes of fluid extracted) taken to removal artificial blood clots using the ‘GP’ MTD in straight plastic tubes compared to the same using porcine blood clots in porcine arteries. Different lengths of clot will be used.
GP’ MTD=’GP’ Mechanical Thrombectomy Device. Recently patented.

To be honest, do you understand any thing at all?
That is what I have to do as my final project. The supervisor says ,she is going to invent something new, who knows! May be she can. Let’s pray for her.

Who is Thomas Chang ?

2008-02-02T07:25:00.000-08:00

Thomas Chang, O.C. (born 1933) is a Canadian physician and scientist who invented the world’s first artificial cell in 1957.
Born in Shantou, China, he is the Director, Artificial Cells & Organs Research Centre and Professor of Physiology, Medicine & Biomedical Engineering in the Faculty of Medicine at McGill University.
In 1991, he was made an Officer of the Order of Canada.

Thomas Chang, Professor of Physiology
Like many driven young men, Thomas Chang would bring his work home with him. The difference with Chang was his "work" was that the near-impossible task of creating the world's first artificial blood cell. And as an undergraduate student in 1956, his "home" was his residence room in McGill's Douglas Hall.
Remarkably, Chang was successful. Working with improvised materials like perfume atomizers, Chang managed to create a permeable plastic sack that would effectively carry haemoglobin almost as effectively as a natural blood cell. In 1989, the New Scientist called Chang's student research project an "elegantly simple and intellectually ambitious" idea that "has grown into a dynamic field of biomedical research and development."
Chang's remarkable career continued as Director of the Artificial Cells and Organs Research Centre at McGill. In the late sixties he discovered enzymes carried by artificial cells could correct some metabolic disorders and also developed charcoal-filled cells to treat drug poisoning, a now widely used technique. His work on finding a safe blood substitute brought him to prominence in the 1980s and 1990s, earning him an Order of Canada.
At one point considered for a Nobel Prize, Chang has not lost focus on his primary motivation: "To me as a scientist, what is most important is what is most useful to the patient, not what is good for your reputation or what pays the most money. The sick patient should be the most important stimulus for our work."

For more please have look on the related website Univeristy of McGill .

Journal-Feb2008

2008-01-29T09:28:00.000-08:00

You can freely download :

Transfusion
The Journal of AABB
February 2008 - Vol. 48 Issue 2 Page 203-404

Click here.
( A *.rar file include pdf files)

Hb

2008-01-27T08:31:00.000-08:00

Hemoglobin is a protein that is carried by red cells. It picks up oxygen in the lungs and delivers it to the peripheral tissues to maintain the viability of cells. Hemoglobin is made from two similar proteins that "stick together". Both proteins must be present for the hemoglobin to pick up and release oxygen normally. One of the component proteins is called alpha, the other is beta. Before birth, the beta protein is not expressed. A hemoglobin protein found only during fetal development, called gamma, substitutes up until birth.

Artifical Blood & World

2008-01-27T07:59:00.000-08:00

Here are the name of 4 famous companies which are working on this issue in the world:

-Biopure Corporation, Cambridge, USA

Product : HemopureTM.

Source : Polymerised bovine Hb,Africa 2001.

Clinical trial level:Approved for use in South .
Application : Clinical use to treat acute anaemia and as alternative to donated blood in transfusion.

-Hemosol Incorporated, Toronto, Canada
Product :HemolinkTM.
Source : Phase III trials completed.
Clinical trial : Phase III trials completed.
Application : Application for use during cardiac surgery filed to regulatory authorities.

-Sangart Incorporated, San Diego, USA
Product : HemospanTM.
Source : Conjugated human Hb.
Clinical trial level: Phase Ib/II initiated in Sweden 2003.
Application : Trials underway in orthopaedic surgery.

-Baxter International,Deerfield, USA
Product : OptroTM.
Source : Cross-linked Hb from genetically modified E. coli bacteria.
Clinical trial level: Phase II trials completed.
Application : Trials completed in patients undergoing surgery in 1999.

First use

2008-01-27T07:35:00.000-08:00

The first successful use of the artificial cell in routine clinical applications is hemoperfusion . After initial clinical trials for poisoning, kidney failure, and liver failure , it is now in routine clinical use, especially for the treatment of suicidal or accidental poisoning from medications . It is also being used in combination with the hybrid artificial liver in clinical trials.

Blood Substitute

2008-01-27T07:24:00.001-08:00

The term 'blood substitute' is a little misleading since their development so far has concentrated primarily on only the blood's function to carry oxygen to our tissues. A more accurate term for blood substitutes being developed would be either:
Cell-free oxygen carriers
Oxygen therapeutics
Red cell substitutes
Essentially, blood substitutes are fluids which, when injected into the human blood stream, contribute significantly to the transport of oxygen around the body. What constitutes a suitable substitute?
Scientists have identified that an ideal blood substitute should have several key properties. These would ensure that a substitute would be free of all of the problems associated with donated human blood when it is used in transfusion. Such properties include:
Adequate oxygen uptake in the lungs
Adequate oxygen delivery to the tissues
Long circulation time
Non-toxic
Rapidly excreted without causing harm
Stable at room temperature and readily available for use
Easily sterelisable (i.e. easy to ensure absence of pathogens such as viruses)
Cheap to manufacture
Long shelf life and easy to store
Widely applicable (i.e. no need for crossmatching or compatibility testing)
Free of any side effects
There are 3 key advantages to a blood substitute that meets all of these criteria, and it is these that make the prospect of developing an effective substitute very exciting:
Unlike red blood cells, a blood substitute could be sterilised, this would mean the problems of disease spreading would be removed from transfusions.
Problems of human error, (mismatching of blood types) with donated blood would be removed since a substitute would not contain the properties of blood that cause this.
A blood substitute could be stored stably for a long time, which would allow for transport to third world countries or to a battlefield, or site of natural disaster without the need for refrigerators. Effectively there would be trouble free availability of blood.

Blood substitutes

2008-01-27T07:20:00.000-08:00

Blood substitutes, often called artificial blood, are used to fill fluid volume and/or carry oxygen and other blood gases in the cardiovascular system. Although commonly used, the term is not accurate since human blood performs many important functions. Red blood cells transport oxygen, white blood cells defend against disease, platelets promote clotting, and plasma proteins provide various functions. The preferred and more accurate terms are volume expanders for inert products, and oxygen therapeutics for oxygen-carrying products. Examples of these two "blood substitute" categories: Volume expanders: inert and merely increase blood volume. These may be crystalloid-based (Ringer's lactate, normal saline, D5W (dextrose 5% in water) or colloid-based (Haemaccel, Gelofusin). Oxygen therapeutics: mimic human blood's oxygen transport ability. Examples: Hemopure, Oxygent, PolyHeme. Oxygen therapeutics are in turn broken into two categories based on transport mechanism: perfluorocarbon based, and hemoglobin based. Volume expanders are widely available and are used in both hospitals and first response situations by paramedics and emergency medical technicians. Oxygen therapeutics are in clinical trials in the U.S. and Europe, however Hemopure is more widely available in South Africa.

Introduction

2008-01-25T10:13:00.000-08:00

I am planing to write something about my subject- biomedical science - and gather some info in this regard.Should you help me would be better.