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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
M Kalafatis, PE Haley, D Lu, RM Bertina, GL Long and KG Mann
Department of Biochemistry, University of Vermont College of Medicine,
Burlington, VT 05405-0068, USA.
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.
Volume 87,
Issue 11,
pp. 4695-4707,
06/01/1996
Copyright © 1996 by The American Society of Hematology
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