title: 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)
authors.affiliation: Department of Biochemistry, University of Vermont, College of Medicine, Burlington 05405-0068.
authors.forename: M
authors.initials: M
authors.lastname: Kalafatis
authors.forename: J
authors.initials: J
authors.lastname: Xue
authors.forename: C M
authors.initials: CM
authors.lastname: Lawler
authors.forename: K G
authors.initials: KG
authors.lastname: Mann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000918:Antibody Specificity; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D015943:Factor Va / Q000737:chemistry / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D000911:Antibodies, Monoclonal; D010743:Phospholipids; D015943:Factor Va; D013917:Thrombin; D015951:Factor Xa