Contribution of the heavy and light chains of factor Va to the interaction with factor Xa.
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)
Kalafatis
M
M
Department of Biochemistry, University of Vermont, College of Medicine, Burlington 05405-0068.
Xue
J
J
Lawler
C M
CM
Mann
K G
KG
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
D000911:Antibodies, Monoclonal; D010743:Phospholipids; D015943:Factor Va; D013917:Thrombin; D015951:Factor Xa