Monoclonal antibodies to human coagulation factor V and factor Va

Abstract

BALB/c mice were immunized with human factor V. The immunogen was a mixture of procofactor (factor V) and thrombin-activated cofactor (factor Va). Spleen cells were obtained from an immunized animal and fused with NS-1 murine myeloma cells. Hybrid cell cultures were assayed for the production of antibodies to human factor V and factor Va by a solid-phase radioimmunoassay. Factor V and/or factor-Va-specific antibodies were detected in 38 of the 96 cultures assayed. The cells from 10 of these positive cultures were subcloned by limiting dilution and grown as ascites tumors in BALB/c mice. Ascitic fluids were obtained and characterized with respect to their binding interaction with human factor V and factor Va. Three hybridoma cell lines produce monoclonal antibodies that react equally well with factor V and factor Va. Another antibody reacts with both antigens, but the reactivity with factor V is better than with factor Va. An additional two antibodies react with factor Va better than factor V in the radioimmunoassay (RIA). The remaining four antibodies react exclusively with factor V. A previously described murine monoclonal antibody to human factor V (alpha HFV-1) has been used to study the peptides produced during the thrombin-catalyzed activation of human factor V. This antibody binds both factor V and factor Va, releases them at high ionic strength, and has an apparent dissociation constant for factor Va of 3 x 10(-9)M. When human factor V (mol wt 330,000) is activated by thrombin and passed over an alpha HFV-1-Sepharose affinity resin, factor Va binds and subsequently can be eluted. The eluate in 1.2 M NaCl contains two fragments of apparent mol wt 93,000 and 70,000. EDTA, which inactivates factor Va, promotes release of the mol wt 93,000 fragment from factor Va bound to the antibody. Subsequent elution with 1.2 M NaCl releases the mol wt 70,000 fragment. These observations indicate that human factor Va is a two subunit protein and that the epitope for alpha HFV-1 is on the mol wt 70,000 fragment.

Platelet concentrates prepared and stored under currently optimal conditions: minor impact on platelet adhesive and cohesive functions after storage

“…Platelet‐bound von Willebrand factor (vWF), fibrinogen, and fibronectin were quantified with polyclonal antibodies (Dako, Glostrup, Denmark) conjugated to FITC by standard techniques 5 . The amount of coagulation factor Va bound to platelets was measured with a murine MoAb (American Diagnostica, Greenwich, CT, clone V237) conjugated with FITC 6 . For all the studies, the negative control was an IgG 1 ‐FITC MoAb (clone 679.1Mc7).…”

Unfractionated heparin inhibits thrombin-catalysed amplification reactions of coagulation more efficiently than those catalysed by factor Xa

“…In experiments that employed exogenous Factor Xa (Fig. 1, procedure A), Factor Xa was introduced simultaneously with the CaCl2 and heparin, since Factor Xa can activate Factor VIII and Factor V only when CaC12 and coagulant phospholipids are present (Smith & Hanahan, 1976;Foster et al, 1983). As no cofactor is required when thrombin activates Factor V and Factor VIII (Nesheim & Mann, 1979;Vehar & Davie, 1980), thrombin could be added to CAP, either simultaneously with heparin and CaCl2 (Fig. 1, procedure B) or before (Fig.…”

“…We have proposed that during coagulation the efficient catalytic action of heparin on thrombin inhibition by antithrombin III results in the inhibition of thrombinmediated activation of Factor VIII and Factor V, thereby delaying prothrombinase formation (Ofosu et al, 1987a). Factor Xa can also activate Factor VIII and Factor V (Smith & Hanahan, 1976;Vehar & Davie, 1980;Foster et al, 1983). Secondly, Factor Xa is less sensitive than thrombin to inhibition by antithrombin III, with and without heparin (Jordan et al, 1980;Ofosu et al, 1984).…”

The factor V light chain mediates the binding and endocytosis of plasma‐derived factor V by megakaryocytes

“…Inhibition of 125 I-FV endocytosis by various anti-FV antibodies provided and characterized by Dr Kenneth Mann (University of Vermont, Burlington, VT, USA) was determined in parallel. Antibodies directed against the FV light chain (E9, #2 and #9) or FV heavy chain (#17) [15] were preincubated with 125 I-FV prior to the addition of CMK cells. Consistent with the results described above, anti-FV light chain antibodies, E9 and anti-FV#2 inhibited FV endocytosis by megakaryocytes by greater than 80% (Fig.…”