融合蛋白TAP-SSL5对人血小板功能的影响

目的探讨抗炎抗凝双效融合蛋白TAP-SSL5对人血小板功能的影响。方法采用健康人富含血小板血浆以凝胶过滤法分离得到人血小板。流式细胞仪检测融合蛋白TAP-SSL5或小鼠抗人CD42b(glycoprotein Ibα,GPIbα)单克隆抗体(HIP1)与血小板的结合情况;以人血小板表面P-选择素的表达及PAC-1的结合反映血小板的激活程度;以全血电阻法定量分析TAP-SSL5对人血小板聚集功能的影响;为评价TAP-SSL5的出血风险,进一步观察了TAP-SSL5对小鼠尾部出血时间的影响。结果融合蛋白TAP-SSL5可与血小板结合,并竞争性抑制HIP1与血小板的结合,提示TAP-SSL5可与血小板表面的GPIbα结合,进而抑制GPIbα与vWF的相互作用。但高浓度的TAP-SSL5(终浓度30 mg/L)也能激活血小板,使人血小板表面P-选择素的表达增加(表达阳性率为90.4%)和PAC-1的结合量上升(阳性率为66.3%);终浓度为10、30 mg/L的TAP-SSL5可引起血小板的显著聚集。给小鼠单次尾静脉注射10 mg/kg的TAP-SSL5,可显著延长尾部出血时间,由对照组的(647.1±33.7)s延长至(753.6±127.3)s(P<0.01);而常规剂量组(3 mg/kg TAP-SSL5)尾部出血时间为(612.8±79.1)s,与对照组相比无显著差异(P>0.05)。结论融合蛋白TAP-SSL5保留了SSL5与血小板GPIbα结合的功能,有助于进一步提高TAP-SSL5的抗血栓作用,但同时也导致融合蛋白TAP-SSL5在高浓度情况下可延长出血时间和激活血小板。

Effect of TAP-SSL5 fusion protein on human platelet functions in vitro and in vivo

Objective To investigate the effect of anti-inflammatory and anticoagulant tick anticoagulant peptide(TAP)-staphylococcal superantigen-like protein 5(SSL5) fusion protein on human platelet functions.Methods Gel-filtered platelets(GFP) were prepared from human platelet-rich plasma(PRP).Flow cytometry(FCM) was applied for the binding assay of TAP-SSL5 fusion protein and mouse anti-human CD42b(GPIbα) monoclonal antibody(HIP1) to platelets.Both the expression of P-selectin on the platelet surface and the PAC-1 binding to the platelets were assayed by FCM.Platelet aggregation induced by TAP-SSL5 fusion protein was analyzed by whole blood impedance platelet aggregometry.The mouse tail bleeding time was recorded after venous injection of TAP-SSL5 fusion protein to evaluate the bleeding risk of TAP-SSL5 fusion protein.Results TAP-SSL5 fusion protein could bind to platelets and competitively inhibit HIP1 to bind to platelets,indicating that TAP-SSL5 fusion protein could bind to GPIbα on platelets and inhibit the interaction between GPIbα and von Willebrand Factor(vWF).A high concentration of TAP-SSL5 fusion protein could also activate platelets,and 30 mg/L TAP-SSL5 fusion protein resulted in 90.4% P-selectin positive platelets and 66.3% PAC-1 positive platelets.TAP-SSL5 fusion protein at high concentrations promoted platelets aggregation as well(10 and 30 mg/kg).TAP-SSL5 fusion protein(10 mg/kg) significantly prolonged the mouse tail bleeding time.The mouse tail bleeding time was prolonged to(753.6±127.3)s,which was significantly longer than that of the mice without the injection (647.1±33.7)s,P<0.01].However,the tail bleeding time of the conventional dose group(3 mg/kg) was(612.8±79.1)s,which was not significantly different from that of the control group(P>0.05).Conclusion TAP-SSL5 fusion protein keeps the ability of SSL5 binding to GPIbα on platelets,which may improve the anti-thrombosis function of TAP-SSL5 fusion protein and also lead to the prolongation of mouse tail bleeding time and platelets activation.