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

Abstract

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.

Inhibition of angiogenesis by antibody blocking the action of proangiogenic high-molecular-weight kininogen

“…The epitope was mapped to D5, H441‐502 [10] and the cell‐binding peptides are included in the epitope. A second MAb, B38, a neutralizing antibody directed to the light chain domain of factor V [21], was produced in ascites and purified in a similar manner. A third monoclonal antibody to α v β 3 (LM 609) was donated by Dr David A. Cheresh (Scripps Foundation, LaJolla, CA, USA) [22].…”

Thrombin-stimulated Human Platelets Express Molecular Forms of α-Granule Factor V (FV), which Differ from FVa

“…Human plasma containing inhibitory activity against FV was a kind gift of Dr. A. Koneti Rao, Philadelphia, PA and was purified as described by Annamalai et al (6). Hz is IgGIK, neutralizes FV activity and is directed specifically to the FVa heavy chain (D).…”

“…FVa binds with high affinity to platelet surfaces (4) through its light chain (5), while the other cleavage products, the ac-tivation peptides do not. Once bound to the platelet surface, FVa functions as an essential non-enzymatic cofactor of the prothrombinase complex by combining with factor Xa to allow a much more efficient catalytic conversion of prothrombin to thrombin than by factor Xa alone (6)(7)(8).…”

“…Purification of Human Plasma FV FV was purified using a murine Mab BIO (against FV activation peptide C 1 ) coupled to Sepharose 4B as previously described (6). The purified FV was a major single band of molecular weight 330 kDa when subjected to electrophoresis under reduced conditions.…”

Coagulation factor Va Glu-96-Asp-111: a chelator-sensitive site involved in function and subunit association

“…Excision of the unique B-domain by thrombin or FXa generates FVa, a heterodimer consisting of the FVaH (FVa heavy subunit) and FVaL (FVa ligh subunit) derived from A 1 -A 2 and A 3 -C 1 -C 2 , respectively. Both FVaH and FVaL are required for prothrombinase function [3][4][5] and consequently their Ca 2+ -dependent non-covalent association [6,7] is fundamental for haemostasis. Despite the importance of the intrasubunit association, little is known about the points of contact or how divalent cations facilitate the interaction.…”