The second 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)-fold higher rate of substrate activation than that of the enzyme alone. To examine the role of the prothrombin kringle domains in the interaction 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 that cleavage at Arg271-Thr272 and Arg320-Ile321 peptide bonds occurs upon activation with prothrombinase to yield normal thrombin from both PT/delta K1 and PT/delta K2. In the absence of factor Va, the Km(app) for the activation of PT/delta K1, PT/delta K2, or plasma-derived prothrombin by factor Xa-phospholipid-Ca(II) are equivalent. The Km(app) for the activation of PT/delta K2 by prothrombinase is approximately 4-5-fold higher than that obtained for plasma-derived prothrombin or PT/delta K1. These data demonstrate that the prothrombin kringle domains do not contribute significantly to the binding affinity of the substrate-enzyme interaction. In the absence of factor Va, equivalent kcat values were obtained for all of the prothrombin species when they were activated by factor Xa-Ca(II)-phospholipid. In contrast, a 7-fold lower kcat value was obtained for the activation of PT/delta K2 by prothrombinase as compared with that obtained for plasma prothrombin or PT/delta K1. Collectively, these data suggest that determinants within the second prothrombin kringle domain interact with factor Va to elicit a significant acceleration in the catalytic rate of substrate turnover. Indeed, in contrast to plasma-derived prothrombin, no direct binding of PT/delta K2 to factor Va to form the PT/delta K2-factor Va complex could be demonstrated by 90 degrees light scattering.