Abstract
We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by alpha-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min(-1) at 0, 0.03, 0.1, 1 mm homocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mm beta-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [(35)S]homocysteine (10-450 micrometer) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507-709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with beta-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s).
Highlights
Homocysteine is a nonprotein-forming sulfhydryl amino acid derived from the metabolism of methionine
We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments
Congenital defects in the enzymes involved in methionine metabolism, 5,10-methylenetetrahydrofolate reductase, N5-methyltetrahydrofolate methyltransferase, and cystathionine -synthase, along with deficiencies in the essential cofactors folic acid and
Summary
Homocysteine is a nonprotein-forming sulfhydryl amino acid derived from the metabolism of methionine. Homocysteine is metabolized in human cells through two pathways of remethylation to methionine and one pathway of transsulfuration to cysteine with cystathionine as an intermediate [1]. Congenital defects in the enzymes involved in methionine metabolism, 5,10-methylenetetrahydrofolate reductase, N5-methyltetrahydrofolate methyltransferase, and cystathionine -synthase, along with deficiencies in the essential cofactors folic acid and vitamins B6 or B12 can result in elevated levels of plasma homocysteine. Human factor V (Mr ϭ 330,000) is activated by ␣-thrombin cleavages at Arg-709, Arg-1018, and Arg-1545 [27,28]. Factor Va is proteolytically inactivated by activated protein C (APC) by three cleavages of the heavy chain at Arg-506, Arg-306, and Arg-679 [29, 30].
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