HIGH-MOLECULAR-MASS AND LOW-MOLECULAR-MASS KININOGENS BLOCK PLASMIN-INDUCED PLATELET-AGGREGATION BY FORMING A COMPLEX WITH KRINGLE-5 OF PLASMINOGEN PLASMIN/

We have previously demonstrated a low-affinity (0.8 mu M, non-covalent complex formation between high-molecular-mass kininogen (HK) and plas minogen (Pig) which prevented Pl,a interaction with glioma and endothe lial cells. We have now extended our previous observations by explorin g the potential complex formation between Pig and low-molecular-mass k ininogen (LK) and between LK and HK with Pig cleaved with human neutro phil elastase (HNE). Plg cleavage by HNE (Plg(HNE)) yielded kringles 1 -3, kringle 4 and mini-plasminogen. Pig(HNE) was subjected to SDS/PAGE under non-reducing conditions, followed by western blotting, and incu bated with either I-125-HK or I-125-LK. Autoradiograms revealed that I -125-HK bound to miniplasminogen and to kringles 1-3 but not to kringl e 4 and the presence of 10 mM 6-aminohexanoic acid (Ahx) disrupted onl y the interaction with kringles 1-3, In contrast, I-125-LK bound to mi niplasminogen but not to kringles 1-3 or 4 and Ahx had no effect at al l. The complex formation of either HK (0.67 mu M) or LK (3 mu M) with Pig (1.5 mu M) did not affect its conversion to plasmin by tissue plas minogen activator (t-PA) (10 U/ml) in the presence of a tissue plasmin ogen stimulator (0.14 mu M). However, the rate of conversion of plasmi nogen to plasmin by t-PA was affected when platelets were added to the reaction mixture. Since HK (0.83 mu M) has been shown to inhibit plas min-induced platelet aggregation, we investigated whether this inhibit ory propel ty is found within the heavy chain shared by HK and LK. We found that LK inhibited plasmin-induced platelet aggregation, but a 4- fold molar excess was required when compared to HK. Compared to plasmi n, 3-5-fold molar excess of miniplasmin is required to induce platelet aggregation, indicating the important role of kringles 1-3 for plasmi n interactions with these cells. These results indicate that HK and LK -mediated inhibition of plasmin-induced platelet aggregation is likely due to complex formation with kringle 5 without interfering with plas min's active site. We found an additional interaction between HK and k ringles 1-3 enhancing the inhibitory effect, presumably by interfering with plasmin's interaction with platelets, This HK and LK-associated modulation of plasmin-induced platelet aggregation may serve as a temp late to develop synthetic peptides as novel therapeutic agents to prev ent some of the plasmin-associated thrombocytopenia seen during thromb olytic therapy.