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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
AE Annamalai, AK Rao, HC Chiu, D Wang, AK Dutta-Roy, PN Walsh and RW Colman
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.
Volume 70,
Issue 1,
pp. 139-146,
07/01/1987
Copyright © 1987 by The American Society of Hematology
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