Thrombin-induced platelet aggregation is inhibited by the heptapeptide Leu(271)-ALa(277) of domain 3 in the heavy chain of high molecular weight kininogen

The ability of kininogens to modulate thrombin-induced aggregation of human platelets has been assigned to domain 3 (D3) in the common heavy chain coded for by exons 7, 8, and 9 of kininogen gene. We expressed each of the exons 7, 8, and 9, and various combinations as glutathione S-transferase fusion proteins in Escherichia coli. Each of the exon products 7 (Lys(236)-Gln(292)), 8 (Va1(293)-Gly(328)), and 9 (Gln(329)-Met(357)), and their combinations were evaluated for the ability to inhibit thrombin induced platelet aggregation. Only products containing exon 7 inhibited platelet aggregation induced by thrombin with an IC50 of >20 mu M. A deletion mutant of exon 7 product, polypeptide 7A product (Lys(236)-Lys(270)) did not block thrombin-induced platelet aggregation, while 7B product (Thr-(255)-Gln(292)) and 7C product (Leu(271)-Gln(292)) inhibited aggregation. These findings indicated that the inhibitory activity is localized to residues Leu(271)-Gln(292). Peptides Phe(279)-Ile(283) and Phe(281)-Gln(292) did not block thrombin, and Asn(275)-Phe(279) had only minimal inhibitory activity. A heptapeptide Leu(271)-Ala(277) inhibited thrombin-induced aggregation of platelets with an IC50 of 65 mu M. The effect is specific for the. activation of platelets by thrombin but not ADP or collagen. No evidence for a thrombin-kininogen complex was found, and neither HK nor its derivatives directly inhibited thrombin activity. Knowledge of the critical sequence of kininogen should allow design of compounds that can modulate thrombin activation of platelets.