Molino M, Di Lallo M, Martelli N, de Gaetano G, Cerletti C
Instituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy.
Blood. 1993 Oct 15;82(8):2442-51.
Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC-1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA-fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb-IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.
组织蛋白酶G是一种丝氨酸类、类胰凝乳蛋白酶的蛋白酶，由活化的多形核白细胞（PMN）释放，可能作为血小板激动剂。通过细胞荧光分析，使用针对糖蛋白（Gp）Ibα链（SZ2）、Gp IX或Gp IIb-IIIa复合物（P2）的异硫氰酸荧光素标记单克隆抗体（MoAb）以及纤维蛋白原受体特异性MoAb PAC-1，评估了这种酶对血小板表面糖蛋白Ib和IIb-IIIa的影响。在人洗涤血小板中，组织蛋白酶G增加了P2和PAC-1的结合，减少了SZ2的结合，但仅轻微影响抗Gp IX的结合。与凝血酶刺激的血小板相比，组织蛋白酶G刺激的血小板中SZ2结合减少更快，而P2和PAC-1结合的增加在两种激动剂作用下程度相当。在多聚甲醛（PFA）固定且能量耗尽的血小板中，凝血酶对Gp Ib或Gp IIb-IIIa复合物均无影响。相反，组织蛋白酶G仍能减少SZ2的结合，而未观察到P2或PAC-1抗体结合增加。与凝血酶不同，用Triton X-100通透处理组织蛋白酶G处理过的PFA固定血小板并不能恢复SZ2结合。此外，血小板与组织蛋白酶G孵育导致在存在血管性血友病因子的情况下瑞斯托霉素诱导的凝集丧失，并在上清液中出现Gp Ib衍生的蛋白水解产物。在通过EDTA预处理使表面Gp IIb-IIIa复合物解离后，组织蛋白酶G仍诱导P2结合增加。阿司匹林和二磷酸腺苷清除系统对组织蛋白酶G诱导的抗体结合变化仅有轻微但不显著的影响。所有这些数据表明，组织蛋白酶G与凝血酶一样，与血小板表面Gp相互作用，诱导Gp IIb-IIIa复合物细胞内池的暴露以及功能性纤维蛋白原受体的伴随表达。此外，它还诱导Gp Ib上抗原位点的丧失，但其涉及的机制，即Gp Ib的蛋白水解裂解，与凝血酶的机制有很大不同。由活化的PMN产物诱导的这些变化可能会降低血小板对内皮下层的反应性，同时增加其发生聚集和释放反应的能力。
