Homocysteine inhibits inactivation of factor Va by activated protein C.
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).
Undas
A
A
Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405-0068, USA.
Williams
E B
EB
Butenas
S
S
Orfeo
T
T
Mann
K G
KG
D015153:Blotting, Western; D015943:Factor Va / Q000037:antagonists & inhibitors*; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D010743:Phospholipids / Q000378:metabolism; D011486:Protein C / Q000037:antagonists & inhibitors* / Q000378:metabolism
D010743:Phospholipids; D011486:Protein C; D006710:Homocysteine; D015943:Factor Va