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 antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:10(6) 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 NaCl 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.
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- Bajaj S. P., Butkowski R. J., Mann K. G. Prothrombin fragments. Ca2+ binding and activation kinetics. J Biol Chem. 1975 Mar 25;250(6):2150–2156. [PubMed] [Google Scholar]
- Barton P. G., Hanahan D. J. The preparation and properties of a stable factor V from bovine plasma. Biochim Biophys Acta. 1967 Apr 11;133(3):506–518. doi: 10.1016/0005-2795(67)90555-7. [DOI] [PubMed] [Google Scholar]
- Bloom J. W., Nesheim M. E., Mann K. G. A rapid technique for the preparation of factor V deficient plasma. Thromb Res. 1979;15(5-6):595–599. doi: 10.1016/0049-3848(79)90169-5. [DOI] [PubMed] [Google Scholar]
- Bloom J. W., Nesheim M. E., Mann K. G. Phospholipid-binding properties of bovine factor V and factor Va. Biochemistry. 1979 Oct 2;18(20):4419–4425. doi: 10.1021/bi00587a023. [DOI] [PubMed] [Google Scholar]
- Colman R. W. The effect of proteolytic enzymes on bovine factor V. I. Kinetics of activation and inactivation by bovine thrombin. Biochemistry. 1969 Apr;8(4):1438–1445. doi: 10.1021/bi00832a019. [DOI] [PubMed] [Google Scholar]
- Colman R. W., Weinberg R. M. Factor V. Methods Enzymol. 1976;45:107–122. doi: 10.1016/s0076-6879(76)45015-2. [DOI] [PubMed] [Google Scholar]
- Esmon C. T., Owen W. G., Jackson C. M. A plausible mechanism for prothrombin activation by factor Xa, factor Va, phospholipid, and calcium ions. J Biol Chem. 1974 Dec 25;249(24):8045–8047. [PubMed] [Google Scholar]
- Esmon C. T. The subunit structure of thrombin-activated factor V. Isolation of activated factor V, separation of subunits, and reconstitution of biological activity. J Biol Chem. 1979 Feb 10;254(3):964–973. [PubMed] [Google Scholar]
- Esnouf M. P., Jobin F. The isolation of factor V from bovine plasma. Biochem J. 1967 Mar;102(3):660–665. doi: 10.1042/bj1020660. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fass D. N., Knutson G. J., Bowie E. J. Porcine Willebrand factor: a population of multimers. J Lab Clin Med. 1978 Feb;91(2):307–320. [PubMed] [Google Scholar]
- Hibbard L. S., Mann K. G. The calcium-binding properties of bovine factor V. J Biol Chem. 1980 Jan 25;255(2):638–645. [PubMed] [Google Scholar]
- Jobin F., Esnouf M. P. Studies on the formation of the prothrombin-converting complex. Biochem J. 1967 Mar;102(3):666–674. doi: 10.1042/bj1020666. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kandall C. L., Rosenberg R., Colman R. W. Molecular changes associated with proteolysis of bovine factor V by thrombin. Eur J Biochem. 1975 Oct 1;58(1):203–211. doi: 10.1111/j.1432-1033.1975.tb02365.x. [DOI] [PubMed] [Google Scholar]
- Kane W. H., Lindhout M. J., Jackson C. M., Majerus P. W. Factor Va-dependent binding of factor Xa to human platelets. J Biol Chem. 1980 Feb 10;255(3):1170–1174. [PubMed] [Google Scholar]
- Mann K. G., Heldebrant C. M., Fass D. N. Multiple active forms of thrombin. I. Partial resolution, differential activities, and sequential formation. J Biol Chem. 1971 Oct 10;246(19):5994–6001. [PubMed] [Google Scholar]
- March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
- Nesheim M. E., Mann K. G. Thrombin-catalyzed activation of single chain bovine factor V. J Biol Chem. 1979 Feb 25;254(4):1326–1334. [PubMed] [Google Scholar]
- Nesheim M. E., Myrmel K. H., Hibbard L., Mann K. G. Isolation and characterization of single chain bovine factor V. J Biol Chem. 1979 Jan 25;254(2):508–517. [PubMed] [Google Scholar]
- Nesheim M. E., Prendergast F. G., Mann K. G. Interactions of a fluorescent active-site-directed inhibitor of thrombin: dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. Biochemistry. 1979 Mar 20;18(6):996–1003. doi: 10.1021/bi00573a010. [DOI] [PubMed] [Google Scholar]
- Nesheim M. E., Taswell J. B., Mann K. G. The contribution of bovine Factor V and Factor Va to the activity of prothrombinase. J Biol Chem. 1979 Nov 10;254(21):10952–10962. [PubMed] [Google Scholar]
- PAPAHADJOPOULOS D., HOUGIE C., HANAHAN D. J. PURIFICATION AND PROPERTIES OF BOVINE FACTOR V: A CHANGE OF MOLECULAR SIZE DURING BLOOD COAGULATION. Biochemistry. 1964 Feb;3:264–270. doi: 10.1021/bi00890a021. [DOI] [PubMed] [Google Scholar]
- Rosing J., Tans G., Govers-Riemslag J. W., Zwaal R. F., Hemker H. C. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem. 1980 Jan 10;255(1):274–283. [PubMed] [Google Scholar]
- Saraswathi S., Rawala R., Colman R. W. Subunit structure of bovine factor V. Influence of proteolysis during blood collection. J Biol Chem. 1978 Feb 25;253(4):1024–1029. [PubMed] [Google Scholar]
- Smith C. M., Hanahan D. J. The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure. Biochemistry. 1976 May 4;15(9):1830–1838. doi: 10.1021/bi00654a007. [DOI] [PubMed] [Google Scholar]
- Tracy P. B., Peterson J. M., Nesheim M. E., McDuffie F. C., Mann K. G. Interaction of coagulation factor V and factor Va with platelets. J Biol Chem. 1979 Oct 25;254(20):10354–10361. [PubMed] [Google Scholar]
- Travis J., Bowen J., Tewksbury D., Johnson D., Pannell R. Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma. Biochem J. 1976 Aug 1;157(2):301–306. doi: 10.1042/bj1570301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]