Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine.
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.
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
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Puri
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Matsueda
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Umeyama
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Bradford
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Colman
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D000242:Cyclic AMP / Q000097:blood; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007704:Kininogens / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D016415:Sequence Alignment; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*
D007704:Kininogens; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; C055422:aggregin; C081604:phenylalanyl-glutaminyl-valyl-valyl-S-(3-nitro-2-pyridinesulfenyl)cysteinyl-glycinamide; D005340:Fibrinogen; D000242:Cyclic AMP; D013917:Thrombin; D002154:Calpain; D003546:Cysteine Endopeptidases