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			PubMed Journals: Biochemistry

  Source:		PMID: 9538022


    		Biochemistry. 1998 Apr 7;37(14):5029-38.
     
			Structure/function analyses of recombinant
			variants of human factor Xa: factor Xa incorporation
			into prothrombinase on the thrombin-activated
			platelet surface is not mimicked by synthetic
			phospholipid vesicles.

			Larson PJ(1), Camire RM, Wong D, Fasano
			NC, Monroe DM, Tracy PB, High KA.

			Author Information
			(1) Department of Pediatrics,
			University of Pennsylvania School of Medicine,
			Philadelphia, Pennsylvania 19104, USA.

			This report describes the expression, purification,
			and characterization of a series of recombinant
			factor Xa variants bearing aspartate substitutions
			for each of the glutamate residues which
			normally undergo gamma-carboxylation. Factor
			X was expressed in human embryonic kidney
			cells and purified from conditioned media
			by immunoaffinity and hydroxylapatite
			chromatography. Factor X was activated with
			Russell's viper venom factor X activator,
			and single-chain unactivated factor X was
			removed from activated factor X by size-exclusion
			chromatography. Recombinant wild-type factor
			Xa had normal activity in a clotting assay,
			and mutants with aspartate substitutions
			for glas residues 16, 26, and 29 had no
			detectable clotting activity. In purified
			component assays, these gla variants had
			essentially no detectable activity in the
			prothrombinase complex assembled on synthetic
			phospholipid vesicles but had significant
			activity when the prothrombinase was assembled
			on thrombin-activated platelets. In addition,
			the gla 32 variant had normal activity in
			the platelet prothrombinase but diminished
			activity in prothrombinase assembled on
			synthetic PSPC vesicles. These differences
			were not accounted for by the total phospholipid
			composition of the thrombin-activated platelet
			membrane. We have produced fully active
			recombinant human factor Xa and demonstrated
			that gla residues 16, 26, and 29 are critical
			for normal activity of factor Xa. More importantly,
			this study provides an extensive characterization
			of macromolecular enzyme complex formation
			with gla variants of a vitamin K-dependent
			coagulation protein and provides evidence
			that prothrombinase complex assembly on
			thrombin-activated platelets is not equivalent
			to assembly on synthetic phospholipid vesicles.
			The data suggest that thrombin-activated
			platelets possess some element(s) (other
			than 30% phosphatidyl serine or factor Va),
			presumably either protein or phospholipid,
			that serves as a component of the factor
			Xa binding site.

			DOI: 10.1021/bi972428p PMID: 9538022 [Indexed
			for MEDLINE]

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