Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium.
We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.
D000469:Alkaline Phosphatase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001666:Binding Sites, Antibody / Q000187:drug effects*; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005165:Factor V / Q000276:immunology*; D015943:Factor Va; D005170:Factor X / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D015951:Factor Xa; D006801:Humans; D007124:Immunoenzyme Techniques; D007143:Immunoglobulin Heavy Chains / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D010664:Phenylmethylsulfonyl Fluoride / Q000031:analogs & derivatives / Q000494:pharmacology; D011516:Prothrombin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D010664:Phenylmethylsulfonyl Fluoride; D015943:Factor Va; C027226:(4-amidinophenyl)methanesulfonyl fluoride; D005165:Factor V; D011516:Prothrombin; D005170:Factor X; D000469:Alkaline Phosphatase; D015951:Factor Xa; D002118:Calcium