Preparation And Preliminary Investigation Of IPM

objective:Using outdated manual concentrated platelet to prepare Infusibie platelet membrane particle (IPM) and preliminary investigating the coagulation function of IPM. To development one hemostatic, by freezing and thawing and ultrasonic sonicationMethods:The outdated concentrated platelet provided by Blood Bank and separating Platelet concentrate from red blood cells, white blood cells and plasma, by differential centrifugation. Then the platelets precipitate was resuspended in physiological saline, and disrupted by repeated freezing at-70℃for5hours and thawing at25℃for2hours, three times. The frozen and thrawed suspension was centrifuged to remove intracellular residue components which were retained in supernatant. the lysed platelets then was homogenized by sonicating for200cycles (2second cycle,1.4s on,0.6s off). The sonicated platelets suspension was centrifuged to get the IPM which remain in the supernatant, and the supernatant was stored at4℃for the following experimental research. Include:the size of the IPM measured by Nano size detectim, getting particle profile by Scanning Electron Microscope, comparing IPM protein component with platelet draw support from SDS-PAGE, surface protein receptor of particles and aggregation function through Flow cytometry, and thrombin generation ability using chemiluminiscence. Results:The IPM particles size is about150nm、sphere and IPM is lack of protein in molecular weight200-85kDa. the particles inculding Gp Ⅰb a (CD42b), G p Ⅱ b-Ⅲ a (CD41/CD61), CD31, CD62p. in vitro, Ristocetin and Ⅲ-collagen could induce particles aggregation. Thrombin generation experiments show that IPM could provide catalytic surface for tenase complex and thrombin synthesis.Conclusion:By freezing and thawing and ultrasonic sonication, we could get IPM particles of uniform particle size and particle surface remains GpⅠ ba, G p Ⅱ b-Ⅲ a, CD31, CD62p, which were associated with hemostatic function, and under the stimulus of Ristocetin and Ⅲ-collagen, IPM particles can aggregate together. so, It is expected to become a new hemostatic agent.