THE 2ND KRINGLE DOMAIN OF PROTHROMBIN PROMOTES FACTOR VA-MEDIATED PROTHROMBIN ACTIVATION BY PROTHROMBINASE

The incorporation of factor Xa into the prothrombinase complex, factor Xa-factor Va-phospholipid-Ca(II), results in an approximately 10(5)-f old higher rate of substrate activation than that of the enzyme alone, To examine the role of the prothrombin kringle domains in the interac tion with prothrombinase we have employed site-directed mutagenesis to produce prothrombin species that lack either the first kringle domain , PT/Delta K1, or the second kringle domain, PT/Delta K2. Previously, we have shown that these proteins are fully carboxylated and that they bind to phospholipid vesicles. In this investigation we demonstrate t hat cleavage at Arg(271). Thr(272) and Arg(320)-Ile(321) peptide bonds occurs upon activation with prothrombinase to yield normal thrombin f rom both PT/Delta K1 and PT/Delta K2. In the absence of factor Va, the K-m(app) for the activation of PT/Delta K1, PT/Delta K2, or plasma-de rived prothrombin by factor Xa-phospholipid-Ca(II) are equivalent, The K-m(app) for the activation of PT/Delta K2, by prothrombinase is appr oximately 4-5 fold higher than that obtained for plasma derived prothr ombin or PT/Delta K1. These data demonstrate that the prothrombin krin gle domains do not contribute significantly to the binding affinity of the substrate-enzyme interaction, In the absence of factor Va, equiva lent k(cat) values were obtained for all of the prothrombin species wh en they were activated by factor Xa-Ca(II)-phospholipid, In contrast, a 7-fold lower k(cat) value was obtained for the activation of PT/Delt a K2 by prothrombinase as compared with that obtained for plasma proth rombin or PT/Delta K1. Collectively, these data suggest that determina nts within the second prothrombin kringle domain interact with factor Va to elicit a significant acceleration in the catalytic rate of subst rate turnover, Indeed, in contrast to plasma derived prothrombin, no d irect binding of PT/Delta K2 to factor Va to form the PT/Delta K2-fact or Va complex could be demonstrated by 90 degrees Light scattering.