The Influence Of Phospholipid Metabolite S1P, LPA On Platelet Function

While performing physiological haemostatic function, platelets played a very important role in many important pathological processes like cardiovascular diseases, inflame/Latino, tumor formation, pulmonary fibrosis. Currently in clinical practice, platelet transfusion therapy was an important measure to prevent and alleviate thrombocytopenia and platelet dysfunction. But some patients appeared platelet transfusion refractoriness after repeating platelet transfusion. How to avoid the occurrence of platelet transfusion refractoriness and improve the treatment effect of platelet transfusion was an important issue currently in the medical profession of blood transfusion.In the part 1, in order to provide new ideas and methods for prevention and treatment of clinical platelet transfusion refractoriness. We detected and analyzed a series of morphological and function changes and the release rule of sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) of aphaeresis platelet during storage in vitro, to investigate the relation between SIP and LPA and platelet storage lesion. The results showed that platelet plasma S1P and LPA levels gradually increased with the extension of platelet storage time, but after increasing extracellular S1P concentrations, platelet preservation quality was better than the control group:we added 10.58 mol/L S1P to the plasma in 0 day,compared with the control group after storage for 5 days, the platelet apoptosis decreased from 15% to 5%, the activation decreased from 55% to 43%, the increasing of activation apoptosis was significantly inhibited (P<0.05), the aggregation rate increased from 5% to 23%, Hypotonic shock response (HSR) increased from 11% to 25%, Extent of shape change (ESC) increased from 3% to 7%, and the reeducation was significantly inhibited (P<0.05), but there was no significantly difference between the control group and the increasing the LPA content group (in 5 day, P>0.05). sphingosine kinase activity was effectively inhibited after we added N,N-methylsphingosine directly (DMS) in the plasma,the generation of S1P was reduced (in 5 day, DMS group vs. control group:1.06 mol/L vs.0.61 mol/L), the platelet storage lesion significantly increased:in 5 day, platelet apoptosis increased from 9% to 14%, activation increased from 43% to 60%, the activation and apoptosis significantly increased (P<0.05), aggregation rate decreased from 28% to 5%, HSR decreased from 30 to 18%, ESC decreased from 8% to 2%; the content of added S1P recovery of S1P in plasma after fifth days, platelet apoptosis decreased from 14% to 10%, the activation decreased from 51% to 42%, the activation and apoptosis of platelets were significantly inhibited (P<0.05), aggregation rate increased from 8% to 18%, HSR increased from 18% to 24%, ESC increased from 2% to 6%, the decrease was significantly inhibited (P<0.05). The above results show that SIP can effectively inhibit the occurrency of platelet storage lesion,N,N-Dimethylsphingosine (DMS) effectively inhibited sphingosine kinase activity, reduced the generation of S1P, and the platelet preserving quality was lower,compared with the control group. The above results showed that platelet S1P content increased gradually, this was probably because of the release of active molecules leading to more platelet during storage in vitro, resulted in the raise of S1P content in plasma. After the addition of S1P in vitro, the apoptosis pathway of platelet was inhibited (such as inhibit the activity of Caspase-3),reduced the release of active molecules, effectively inhibited the occurrence of platelet storage lesion, the mechanism needed further study.In addition, in Appendices 1 and 2 of this article, we had discussed the polymorphism of exon 10 of CD61 gene in Han and Uygur,and the frequency molecular mechanisms of CD36 deficiency in Shanghai populationWe used Bloodchip to the screen blood group gene type in Han population, and found that the type of HPA-6bw could not be determined in some individuals. Because the binding sites of the probe for HPA-6bw genotyping located in near the mutation site of HPA-6bw, there may be unknown mutations affecting the combination of primer and template, which resulted in no detection of signal. in order to study the polymorphism of platelet membrane glycoprotein CD61 coding gene of HPA-6bw blood group system, and explore the reasonable gene primers and probes in the part 2, Blood samples of 149 Han people and 96 Uighur people were collected randomly, The exon 10 of CD61 gene was amplified and sent for DNA sequencing. we found there were 3 new SNPs(1533A>G、 1545G>A、1529C>T) in the exon 10 of CD61 gene in both Han and Uygur, and the difference between Han and Uygur was no statistically significant, this showed the blood relationship between the two nations more intimate.Platelet membrane glycoprotein CD36 deficiency may produce anti-CD36 antibodies, resulting in thrombocytopenia, post transfusion purpura, platelet transfusion refractoriness and other transfusion reactions. CD36 deficiency phenotype was very rare in Caucasians (0.3%), and very high in the Asian population (3-10%) and Africa population (7.8%). In the third chapter, we screened 1022 healthy blood donors, analyzed the expression of CD36 on monocytes and platelets, and confirmed the types of CD36 deficiency, obtained D36 gene mutation types and frequencies in blood donors, discovered new gene mutations, and compared the differences of CD36 molecular background in China with other reports.We amplified CD36 gene exon 3-14 and on both sides of the relevant part of the intron, confirmed that some new CD36 gene mutations sequencing by direct sequencing. The results showed that in this study CD36 I deficiency and II deficiency frequency were 0.2% and 2% respectively. The population of Shanghai China CD36 deficiency frequency is higher than that in the European and American countries, slightly lower than that in the other countries in Asia. Our results showed that there were 13 types of mutations in our study,8 mutations have been reported, and other 5 kinds of mutation has not been reported, they were located in 3,12,13,14 exons respectively, caused a reading frame shift or amino acid change, resulting in changes the protein structure. the 371C>T in exon 5 mutation associated with CD36 I deficiency, the remaining mutations associated with CD36 type II deficiency. Most of the mutations in the CD36 gene caused the corresponding amino acid transformation (apart from synonymous mutation 1008G>T). In addition,1344insTCTT located in exon 14 leaded to the lack of terminator. However, the research on gene level was not enough to reveal the molecular behavior of protein level, to explore the molecular mechanism of CD36 deletion also needs more research.