Kornecki E, Ehrlich Y H, Egbring R, Gramse M, Seitz R, Eckardt A, Lukasiewicz H, Niewiarowski S
Department of Psychiatry, University of Vermont, Burlington 05405.
Am J Physiol. 1988 Sep;255(3 Pt 2):H651-8. doi: 10.1152/ajpheart.1988.255.3.H651.
We have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase (0.5-1 microgram/ml). Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. Experiments performed by immunoblotting with use of polyclonal and monoclonal antibodies directed to GPIIIa showed that pretreatment of human platelets with granulocyte elastase resulted in the appearance of an additional proteolytic derivative of GPIIIa migrating with an apparent molecular mass of 120 kDa in a nonreduced system. GPIIIa appears to be the preferred substrate of elastase, since GPIIb was not degraded by human granulocyte elastase. We conclude that 1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and 2) human granulocyte elastase exposes active fibrinogen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen.
我们研究了人粒细胞弹性蛋白酶与人血小板之间的相互作用。用人粒细胞弹性蛋白酶孵育人血小板后，125I标记的纤维蛋白原结合及纤维蛋白原诱导的自发血小板聚集均证明，其暴露了活性纤维蛋白原结合位点。纤维蛋白原诱导血小板聚集发生在低浓度人粒细胞弹性蛋白酶（0.5 - 1微克/毫升）的情况下。用人粒细胞弹性蛋白酶预处理的血小板，每个血小板平均暴露10,500个纤维蛋白原结合位点，即约为最佳浓度ADP所暴露结合位点数目的三分之一。使用多克隆抗血小板膜抗体，从用浓度递增的人粒细胞弹性蛋白酶预处理的125I放射性标记人血小板的去污剂提取物表面免疫沉淀出糖蛋白IIb（GPIIb）、IIIa（GPIIIa）以及源自GPIIIa的一种60,000道尔顿（60 kDa）的蛋白质（在还原系统中为66 kDa）。使用针对GPIIIa的多克隆和单克隆抗体进行免疫印迹实验表明，用人粒细胞弹性蛋白酶预处理人血小板会导致在非还原系统中出现一种表观分子量为120 kDa的GPIIIa额外蛋白水解衍生物。GPIIIa似乎是弹性蛋白酶的首选底物，因为人粒细胞弹性蛋白酶不会降解GPIIb。我们得出结论：1）人粒细胞弹性蛋白酶对血小板GPIIIa的蛋白水解作用导致形成两种主要水解产物；2）人粒细胞弹性蛋白酶暴露与GPIIb/GPIIIa复合物相关的活性纤维蛋白原结合位点，从而导致纤维蛋白原直接诱导血小板聚集。
