Discussion

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 .