title: The second kringle domain of prothrombin promotes factor Va-mediated prothrombin activation by prothrombinase.
abstract: 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.
authors.affiliation: Division of Hematology-Oncology, New England Medical Center, Boston, Massachusetts.
authors.forename: K J
authors.initials: KJ
authors.lastname: Kotkow
authors.forename: S R
authors.initials: SR
authors.lastname: Deitcher
authors.forename: B
authors.initials: B
authors.lastname: Furie
authors.forename: B C
authors.initials: BC
authors.lastname: Furie
mesh_terms: D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015943:Factor Va / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018082:Kringles; D011485:Protein Binding; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000096:biosynthesis; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D015943:Factor Va; D011516:Prothrombin; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa