Contribution of the Heavy and Light Chains of Factor Va to the Interaction with Factor Xa

Abstract

The interactions of the isolated heavy and light chains of factor Va with factor Xa were evaluated using active-site-modified factor Xa [(carboxytetramethyl)rhodamine-Glu-Gly- Arg-factor Xa (ctr-EGR-Xa)]. The Kd for the factor Va heavy-chain interaction with ctr-EGR-Xa was 60 microM. A series of monoclonal antibodies directed against bovine factor Va were tested for their ability to inhibit thrombin formation in an assay using the fluorescent thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Monoclonal antibody alpha BFV-4, which recognizes the light chain of the cofactor, was found to inhibit the formation of thrombin. Similarly, monoclonal antibody alpha BFV-5, which is directed against the heavy chain of the cofactor, was found to inhibit thrombin formation. In contrast, monoclonal antibody alpha BFV-1, also directed against the heavy chain of the cofactor, did not inhibit thrombin generation by the prothrombinase complex. Monoclonal antibodies alpha BFV-4 and alpha BFV-5 inhibited the interaction of active-site-modified radiolabeled factor Xa (125I-Xa-EGR) with factor Va bound to PC/PS-coated microtiter wells, whereas nonimmune mouse IgG did not have any effect on the 125I-Xa-EGR.membrane-bound factor Va interaction. The antibodies effect upon the phospholipid-independent interaction between the cofactor and ctr-EGR-Xa was evaluated by analytical ultracentrifugation. Both alpha BFV-4 and alpha BFV-5 inhibited the phospholipid-independent interaction between factor Va and ctr-EGR-Xa.(ABSTRACT TRUNCATED AT 250 WORDS)

Defining the Factor Xa-binding Site on Factor Va by Site-directed Glycosylation

“…There is no consensus at present as to whether FXa interacts with the light chain of FVa. The observation, that a monoclonal antibody directed against the A3 domain of FVa inhibits the FVa⅐FXa interaction, supports this direct interaction (43). However, the membrane-bound light chain of FVa has been proposed to not interact with FXa (44).…”

Factor V: a combination of Dr Jekyll and Mr Hyde

“…85 When coupled with the interactions of both proteins with the membrane, both the light and heavy chains of factor Va interact with factor Xa; each contributing to dissociation constants in the millimolar range, resulting in a tight association of the 2 proteins (K d ϳ 1 nM) when coupled with membrane binding. 81,82,86 The membrane-bound factor Va-factor Xa complex cleaves membrane-bound prothrombin to produce ␣-thrombin only in the local environment of membrane-bound complex (principally the immobilized activated platelet membrane surface). As a consequence of the common membrane binding of both substrate and enzyme, the effective K m for prothrombin activation is decreased by 2 orders of magnitude.…”

Factor Va Residues 311–325 Represent an Activated Protein C Binding Region

“…fVa is a two-chain protein with a heavy chain (1-709 residues) consisting of the A1 (residues 1-303) and A2 (residues 317-656) domains noncovalently bound via divalent metal ions to the light chain (residues 1546 -2196, A3-C1-C2 domains) (3). Both the light chain and the heavy chain of fVa have been shown to interact with fXa (4).…”