| Objective: Aprotinin, a naturally occurring serine protease inhibitor derived from bovine lung, has been found be widespread used during cardiopulmonary bypass and involved in surgical procedures as a consequence to decrease blood loss and transfusion requirements. Aprotinin's biochemical effects on the fibrinolytic system have been well known, but its mechanisms on the protection of platelets have not been clear yet. Thrombin, a serine protease with multiple functions, is the key enzyme participating in the whole process of blood coagulation. The major thrombin receptor on platelets, protease-activated receptor 1 (PAR-1) requires proteolytic cleavage to transmit platelet-activating signals. Effects of aprotinin on platelet activation induced by different agonists were compared to observe the protective effect of aprotinin on human washed platelet in vitro and to explore its possible mechanism.Materials and Methods: Healthy male blood donors (age ranging from 25-45 years old) without use of any medicines two weeks prior to blood collection were randomly divided into 3 groups: saline control (0KIU/ml, A0), low-dose aprotinin group (100KIU/ml, A1) and high-dose aprotinin group (300KIU/ml, A2). Blood, collected from the cubital veins, was treated by ACD anti-coagulation (whole blood: ACD=9?1, v/v) and fabricated into washed platelet suspension of 2×108 platelets per milliliter by centrifugation. The expression of thrombin receptor PAR-1 on platelet membrane was measured by flow cytometric analysis. Platelet aggregation induced by proteolytic agonists (thrombin) and nonproteolytic agonists (ADP, PMA, adrenaline, SFLLRN and collagen) was recorded by light transmission and the inhibition rate of aprotinin on platelet aggregation was calculated, respectively. Then, the rise of cytosolic free calcium concentration in platelets stimulated by proteolytic agonists (thrombin) and nonproteolytic agonists (adrenaline and SFLLRN) and the effect of aprotinin on [Ca2+]i were also detected and calculated by fluorescence and imaging, respectively. Effects of aprotinin on the amounts of F-actin associated with cytoskeletal proteins induced by thrombin were observed by SDS-PAGE electrophoresis technique. Finally, effects of aprotinin on platelet ultrastructure induced by thrombin were observed under transmission electron microscopy. Results and Conclusions: In this study: ①Flow cytometry revealed: There was significant difference in expression of PAR1 receptor between washed platelets with first antibody or not. ② The platelet aggregation rates of A1 and A2 were remarkably lower than that of A0 when platelets were induced by thrombin, and there was significant difference between the groups. A2 had greater inhibitive effect on platelet aggregation with significant difference than A1. There was no significant difference between the aggregation rates of A1, A2 and that of A0 when platelets were induced by aggregating agents (ADP, PMA, adrenalin, SFLLRN and collagen). No significant difference in inhibitive degree was found between A1 and A2. ③In groups induced by thrombin, when the concentration of Ca2+ was 1mM, no significant difference in [Ca2+]i was found between A1 and A0 in static. Peak of [Ca2+]i after activation of A1 and A0 was remarkably higher than that in static with significant difference. However, peak of [Ca2+]i after activation of A1 was significantly lower than that of A0 with significant difference. In groups induced by SFLLRN and adrenalin, when the concentration of Ca2+ was 1mM, no significant difference in [Ca2+]i between A1 and A0 was found in static. Peak of [Ca2+]i after activation of A1 and A0 was remarkably higher than that in static with significant difference. However, peak of [Ca2+]i after activation of A1 was the same as that of A0 without significant difference. ④ At the band with molecular weight of 43kDa of SDS-PAGE, staining of A1, A2 and the positive control was darker than that of A0, but no significant difference in staining at the band with molecular weight of 43kDa was found among...
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