Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1980 Sep;66(3):583–591. doi: 10.1172/JCI109890

Human coagluation factor V purification and thrombin-catalyzed activation.

B Dahlbäck
PMCID: PMC371687  PMID: 7400329

Abstract

Factor V was isolated from human plasma by barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B chromatography, ammonium sulfate fractionation, and gel chromatography on Ultrogel 22. Degradation of Factor V during purification was largely prevented by ample use of inhibitors of proteolytic enzyme. The purified Factor V was a stable, single-chain molecule with an apparent molecular weight of 330,000. Activation of human Factor V by thrombin resulted in a 10- to 15-fold increase in activity. The activation pattern as monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis was compared with that of bovine Factor V. Differences in the patterns of thrombin activation were noticed between the two species, whereas the final products were similar. The products of human Factor V activation are two closely spaced doublets, one with an apparent molecular weight of approximately 110,000, and the other, approximately 72,000. An antibody was raised against the purified protein. Crossed immunoelectrophoresis showed that the antibody recognized Factor V both before and after activation with thrombin.

Full text

PDF
583

Images in this article

586Image
on p.586
588Image
on p.588
589Image
on p.589
589Image
on p.589

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Bolhuis P. A., Hakvoort T. B., Breederveld K., Mochtar I. A., ten Cate J. W. Isolation and partial characterization of human factor V. Biochim Biophys Acta. 1979 May 23;578(1):23–30. doi: 10.1016/0005-2795(79)90108-9. [DOI] [PubMed] [Google Scholar]
  3. Breederveld K., Giddings J. C., ten Cate J. W., Bloom A. L. The localization of factor V within normal human platelets and the demonstration of a platelet-factor V antigen in congenital factor V deficiency. Br J Haematol. 1975 Mar;29(3):405–412. doi: 10.1111/j.1365-2141.1975.tb01838.x. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Dahlbäck B., Stenflo J. Binding of bovine coagulation factor Xa to platelets. Biochemistry. 1978 Nov 14;17(23):4938–4945. doi: 10.1021/bi00616a013. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Ganrot P. O. Crossed immunoelectrophoresis. Scand J Clin Lab Invest Suppl. 1972;124:39–47. doi: 10.3109/00365517209102749. [DOI] [PubMed] [Google Scholar]
  8. Johansson B. G. Agarose gel electrophoresis. Scand J Clin Lab Invest Suppl. 1972;124:7–19. doi: 10.3109/00365517209102747. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Lundblad R. L., Uhteg L. C., Vogel C. N., Kingdon H. S., Mann K. G. Preparation and partial characterization of two forms of bovine thrombin. Biochem Biophys Res Commun. 1975 Sep 16;66(2):482–489. doi: 10.1016/0006-291x(75)90536-7. [DOI] [PubMed] [Google Scholar]
  11. Miletich J. P., Jackson C. M., Majerus P. W. Interaction of coagulation factor Xa with human platelets. Proc Natl Acad Sci U S A. 1977 Sep;74(9):4033–4036. doi: 10.1073/pnas.74.9.4033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Miletich J. P., Jackson C. M., Majerus P. W. Properties of the factor Xa binding site on human platelets. J Biol Chem. 1978 Oct 10;253(19):6908–6916. [PubMed] [Google Scholar]
  13. Miletich J. P., Majerus D. W., Majerus P. W. Patients with congenital factor V deficiency have decreased factor Xa binding sites on their platelets. J Clin Invest. 1978 Oct;62(4):824–831. doi: 10.1172/JCI109194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Osterud B., Rapaport S. I., Lavine K. K. Factor V activity of platelets: evidence for an activated factor V molecule and for a platelet activator. Blood. 1977 May;49(5):819–834. [PubMed] [Google Scholar]
  17. Rosenberg J. S., Beeler D. L., Rosenberg R. D. Activation of human prothrombin by highly purified human factors V and X-a in presence of human antithrombin. J Biol Chem. 1975 Mar 10;250(5):1607–1617. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Suttie J. W., Jackson C. M. Prothrombin structure, activation, and biosynthesis. Physiol Rev. 1977 Jan;57(1):1–70. doi: 10.1152/physrev.1977.57.1.1. [DOI] [PubMed] [Google Scholar]
  21. 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]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES