Isolation of functional human coagulation factor V by using a hybridoma antibody.

Abstract

Spleen cells obtained from mice immunized with partially purified human coagulation Factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibodypositive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:106 before losing reactivity in an anti-Factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed Factor V activity from human plasma. Factor V activity could be eluted with 1.2 M NaCI at pH 6.5. Homogeneous Factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated Factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent Mr comparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold -when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule.Blood coagulation Factor V as a functional entity was first described in 1947 by Owren (1). Studies by Owren (2), Ware and Seegers (3), and Murphy and Seegers (4) identified Factor V as one of the components essential for the rapid conversion of prothrombin to the blood clotting enzyme thrombin. Subsequent work indicated that Factor V functions as a cofactor, in concert with phospholipid (or platelets) and Ca2+, to promote the proteolytic activation of prothrombin by Factor Xa (5-12). In addition, several studies (2,6,8,(13)(14)(15)(16)(17) indicated that Factor V itselfis converted by thrombin to the more active form, Factor Va.Our knowledge concerning the function and molecular properties of Factor V expanded after the isolation of the bovine protein (18,19). Bovine Factor V is a high molecular weight (Mr = 330,000), single-chain precursor of Factor Va (18). Once activated by thrombin-catalyzed proteolytic cleavage (19,20), Factor Va enhances (in the presence of Ca2' and phospholipid) the activity ofFactor Xa toward prothrombin by at least 4 orders of magnitude (21,22). Studies of Factor V binding properties with regard to Ca2+ (23), phospholipid (24), and platelets (25) have indicated that Factor Va not only greatly augments rates of prothrombin activation but also can be viewed as a plateletbound receptor for Factor Xa in vivo (25,26).Although bovine Factor V has been characterized reasonably well, the properties of human Factor V have not been elucidated. In our hands, even when substantially modified, procedures that readily permit the isolation of the bovine protein do not yield electrophoretically homogeneous, undegraded human Factor V. Consequently, efforts were undertaken, using partially purified human Factor V, to generate a monoclonal hybridoma antibody for use as a tool in studying the human protein. An antibody was produce...

Unique in Vivo Modifications of Coagulation Factor V Produce a Physically and Functionally Distinct Platelet-derived Cofactor

“…Factor X and prothrombin were isolated as described (28,29). Plasmaderived FV was isolated by immunoaffinity chromatography as described (30,31). Thrombin was prepared by activation of prothrombin with Taipan snake venom by the method of Owen and Jackson (32).…”

“…1B) of purified platelet-derived FV/Va samples indicated that unlike plasma-derived FV, which is defined as a single chain 330-kDa procofactor (30,(43)(44)(45), platelet-derived FV/Va contained only a small fraction of the procofactor and several lower molecular weight peptides (Fig. 1B, lane 2).…”

“…Isolation of Platelet-derived FV/Va-Platelet-derived FV/Va was isolated by a modification of the procedure for the purification of plasmaderived FV (30). Washed platelets were diluted to a final concentration 4 ϫ 10 9 platelets/ml in TBS/Ca 2ϩ containing 5 M PGE 1 yielding ϳ250 -350 ml of platelet suspension.…”

“…The platelet lysate was then incubated at 0°C for 90 min to generate a cryoprecipitate, which was removed by centrifugation at 16,000 ϫ g for 40 min at 4°C with greater than 98% recovery of platelet-derived FV/Va. An anti-human FV monoclonal antibody (30) coupled to Sepharose CL-4B resin (50 ml resin; 3-4 mg antibody/ml resin) was added to the supernatant, and the suspension was slowly stirred at 4°C for 2 h to adsorb the FV onto the resin. The resin was then transferred to a sintered glass funnel, and the supernatant was removed by vacuum.…”

Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

“…Synthetic phospholipid vesicles composed of 75% L-palmitoyl-2-oleoyl phosphatidylcholine and 25% Lpalmitoyl 2-oleoyl phosphatidylserine (PCPS) and ␣-thrombin were prepared as described previously (38,39). Human factor V was purified and converted to the active form of the cofactor (Va) as described by Katzmann et al (40). Human recombinant protein S (rHPS) was produced in human kidney 293 cells, purified, and chemically characterized as described elsewhere (1).…”