Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay.
Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg506-->Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 micromol/L), and APC. APC, at concentrations < or = 5.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a clot was observed in plasma from APC-resistant individuals. Gel electrophoresis analyses of factor V fragments showed that membrane-bound factor V is primarily cleaved at Arg306 in both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast degradation after cleavage at Arg506/Arg306, in the APC-resistant individuals' plasma, early generation and accumulation of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg306. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed in either plasma from normal or APC-resistant individuals. Our data show that resistance to APC in patients with the Arg506-->Gln mutation is due to the inefficient degradation (inactivation) of factor Va heavy chain by APC.
Kalafatis
M
M
Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405-0068, USA.
Haley
P E
PE
Lu
D
D
Bertina
R M
RM
Long
G L
GL
Mann
K G
KG
D000595:Amino Acid Sequence; D001777:Blood Coagulation; D005165:Factor V / Q000235:genetics*; D005166:Factor V Deficiency / Q000097:blood*; D015943:Factor Va / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010314:Partial Thromboplastin Time; D017354:Point Mutation; D011486:Protein C / Q000378:metabolism*; D015203:Reproducibility of Results; D013917:Thrombin / Q000378:metabolism
D011486:Protein C; C095381:factor V Leiden; D015943:Factor Va; D005165:Factor V; D013917:Thrombin