Puri R N, Matsueda R, Umeyama H, Bradford H N, Colman R W
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
Eur J Biochem. 1993 May 15;214(1):233-41. doi: 10.1111/j.1432-1033.1993.tb17916.x.
Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.
凝血酶诱导的血小板聚集被认为在心肌梗死血管成形术溶栓治疗后的再闭塞中起重要作用。我们之前证明，凝血酶诱导的洗涤血小板聚集伴随着聚集素（一种假定的ADP受体）的裂解，并且这些事件是由外膜表面表达的钙蛋白酶间接介导的。高分子量激肽原（HK）在其重链中含有结构域2，该结构域作为血小板钙蛋白酶的有效抑制剂发挥作用。HK重链的结构域3直接抑制凝血酶与血小板的结合，这使得使用整个分子进行的机制研究变得复杂。此外，HK是一种120 kDa的蛋白酶，不适合作为潜在的药物制剂。存在于HK及其进化前体胱抑素中的高度保守序列Gln-Val-Val-Ala-Gly，被认为参与半胱氨酸蛋白酶的结合，但本身没有抑制作用。合成了一种亲和类似物Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2（Npys，3-硝基-2-硫代吡啶），P1，它对应于HK中的硫醇蛋白酶结合序列，并含有一种可以与钙蛋白酶活性位点中的游离巯基反应的配体Npys。P1是血小板钙蛋白酶的不可逆抑制剂。P1选择性抑制凝血酶诱导的洗涤血小板和血浆中血小板的聚集，但不抑制其他血小板激动剂的聚集作用。P1不抑制凝血酶的酰胺水解活性和凝血活性。与HK不同，P1不抑制凝血酶与洗涤血小板的结合。P1不抑制凝血酶诱导的血小板形状改变。P1既不提高细胞内cAMP水平，也不干扰凝血酶拮抗前列腺素I2类似物伊洛前列素诱导的细胞内cAMP水平升高的能力。P1的设计和合成可能会促成一类新型抑制剂的开发，这类抑制剂可以选择性地阻断凝血酶诱导的血小板聚集，同时保留这种病理生理蛋白酶的其他功能，并且不抑制其他血小板激动剂的作用。
