Effect Of Chitosan/Calcium Alginatedressing Onthe Activity And Adhesion Of Platelets

Objective: Previously developed chitosan/calcium alginate composite dressings showed good hemostatic effect on femoral artery hemorrhage in general animals,and was expected to be an effective artery hemostatic dressing for clinical operation or Emergency rescue.But the mechanism of coagulation was not clear.Platelet adhesion is the initial step for physiological function of platelets,thus it is essential to promote platelet adhesion for hemostatic effect of bioactive materials.This study was to explore the effect of chitosan/calcium alginate composite dressing on platelet activity and adhesion and its mechanism,and to provide theoretical basis for its clinical application.Methods:1 The experiments were grouped as follows: blank control group（Control）;chitosan group（CS）;calcium alginate group（Alg-Ca）;chitosan/calcium alginate compound dressing group（CS/Alg-Ca）.2 Preparation of platelet-rich plasma by fractional centrifugation.3 Scanning electron microscope（SEM）and determination of lactate dehydrogenase（LDH）were used to observe the platelet adhesion.4 The expression of P-selectin（CD62p）and integrin（CD61）was detected by flow cytometry.5 The levels of thromboxane B₂（TXB₂）in plasma were measured by enzyme-linked immunosorbent assay（Elisa）.6 Real-time quantitative PCR（qPCR）was applied to test the expressions of genes related to platelet aggregation and adhesion: CD63、Yes1、Pafah1b2、Pecam1、Lamp1and CD40 Lg.7 Fluorescence spectrophotometry was used to detect the content of free calcium in plasma and plateletsResults:1 SEM and LDH test results showed: few platelets adhered to chitosan,but the pseudopod extension was activated in CS.While large quantity of platelets adhered to chitosan in Alg-Ca,and most of them were round and in inactivated state;most of platelets adhered to chitosan and were activated in Cs/Alg-Ca.2 The results of flow cytometryCompared with the Control group,there was no obvious change inthe expression level of CD62 p in CS,while high expression in Alg-Ca and CS/Alg-Ca.Compared with Control group,CD61 increased in CS group,decreased in Alg-Ca group,The change of CD61 level in CS / Alg-Ca group was consistent with that in CS group.3 ELISA showed that CS/Alg-Ca composite dressing could improve the level of TXB₂ in plasma（P<0.05）.4 qPCR detection resultsThe expression level of CD63 was increased in CS group（P<0.05）.The expression of Yes1,Pecam1,CD40 Lg was down-regulated by CS（P<0.05）,and level of Lamp1 was up-regulated（P<0.05）.CD63,Yes 1,Pecam 1 andCD40 Lggenes were down-regulated Alg-Ca（P<0.05）,but level of Pafah1b2 gene was upregulated（P<0.05）.Compared with Alg-Ca,CS/Alg-Ca complex could significantly up-regulated the expression of CD63（P<0.05）,and down-regulated the level of Yes 1、Pecam 1（P<0.05）.Compared with CS and Alg-Ca,the gene level of CD40 Lg was significantly up-regulated（P<0.05）.5 Fluorescence probe detection of plasma ATP and intracellular free calcium content showed that CS/Alg-Ca could not increase the plasma ATP and free calcium content.Conclusion:1 CS/Alg-Ca has better effect on platelet adhesion,which is due to overexpression ofAlg-Ca in early stage and CD61 in later stageinduced by Alg-Ca.2 CS/Alg-Ca composite dressing can increase level of TXB₂ in PRP.3Compared with Alg-Ca,CS/Alg-Ca complex can significantly up-regulated the expression of CD63 and down-regulated the level of Yes 1、Pecam 1（P<0.05）,Compared with CS and Alg-Ca,CS/Alg-Ca complex can significantly up-regulated the gene level of CD40Lg（P<0.05）.4 CS/Alg-Ca can not increase the level of ATP in plasma and the free calcium in platelets.