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J. Biol. Chem., Vol. 279, Issue 4, 2383-2393, January 23, 2004

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

CHARACTERIZATION OF PURIFIED PLATELET-DERIVED FACTOR V/Va^*

Weston R. Gould, Jay R. Silveira¶, and Paula B. Tracy||

From the Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405-0086

Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from 40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2–3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2–3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction (10–15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser⁶⁹² catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr⁴⁰² with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser⁶⁹² remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr¹⁵⁴³ rather than Arg¹⁵⁴⁵ accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.

Received for publication, August 5, 2003 , and in revised form, October 31, 2003.

^* This work was supported by Grant P01 HL47603, Project 4 (to P. B. T.), and the Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT. Parts of this work were presented in abstract form (71, 72). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

Present address: Pfizer Global Research and Development, 2800 Plymouth Rd., Ann Arbor, MI 48105.

^¶ Present address: NIAID Rocky Mountain Laboratories, Department of Health and Human Services, 903 South 4th St., Hamilton, MT 59840.

^|| To whom correspondence should be addressed: Dept. of Biochemistry, University of Vermont College of Medicine, Given Bldg., Rm. C409, 89 Beaumont Ave., Burlington, VT 05405-0086. Tel.: 802-656-1995; Fax: 802-862-8229; E-mail: paula.tracy{at}uvm.edu.

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