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 im portant role in reocclusion following thrombolytic therapy of angiopla sty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cl eavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the exter nal membrane. High-molecular-mass kininogen (HK) contains, in its heav y chain, domain 2, which is responsible for its action as a potent inh ibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic st udies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserv ed sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary pr ecursors, the cystatins, is thought to be involved in the binding of c ysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P 1, corresponding to the thiol-protease-binding sequence in HK and cont aining a ligand, Npys, that can react with the free sulfhydryl group i n the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induc ed aggregation of washed platelets and platelets in plasma, but did no t inhibit the aggregatory effects of other platelet agonists. P1 did n ot 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 neithe r raised intracellular levels of cAMP nor did it interfere with the ab ility of thrombin to antagonize the rise in intracellular levels of cA MP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhib itors that selectively block thrombin-induced platelet aggregation whi le sparing other functions of this pathophysiological protease and wit hout inhibiting the action of other platelet agonists.