Loss of membrane-dependent factor Va cleavage: a mechanistic interpretation
of the pathology of protein CVermont
D Lu, M Kalafatis, KG Mann and GL Long
Department of Biochemistry, University of Vermont, College of Medicine,
Burlington.
Clinical manifestations of arterial and venous thrombosis in a family with
protein C deficiency was associated with two mutations in the light chain
of protein C: Glu20-->Ala and Val34-->Met. Further studies showed
that the mutation Glu20-->Ala which eliminated a gamma- carboxylation
site was exclusively responsible for the anticoagulant defect of activated
protein C (APC). Membrane-bound human factor Va is inactivated by APC after
two sequential cleavages of the heavy chain at Arg506 and Arg306. Human
factor Va inactivation by human recombinant APC (rAPC) and a mutant
molecule with an alanine instead of a glutamic acid at position 20
(rAPC(gamma 20A)) was investigated in the presence and absence of
phospholipid vesicles. During a 2-hour incubation period of the cofactor
with either rAPC or rAPC(gamma 20A). In the absence of a membrane surface,
factor Va is cleaved quantitatively at Arg506 and retains approximately 60%
of its initial cofactor activity. After a 2- hour incubation period with
rAPC membrane-bound factor Va has no cofactor activity, whereas in the
presence of a membrane surface and rAPC(gamma 20A) factor Va retains 60% of
its initial cofactor activity. The completed loss in factor Va cofactor
activity upon incubation of the membrane-bound cofactor with phospholipid
vesicles and rAPC is associated with cleavages at Arg506 and Arg306,
whereas membrane-bound factor Va cleavage at Arg306 by rAPC(gamma 20A) is
impaired, resulting in a cofactor that is cleaved at Arg506. Slow cleavage
at Arg306 occurs when membrane-bound factor Va is incubated with rAPC(gamma
20A) and only small amounts of fragments of M(r) = 45,000 and 30,000 are
noticed. Our data show that the genetic defect which leads to the absence
of a gamma-carboxylation site at Glu20 impairs membrane binding of human
APC, which in turn is required for cleavage of factor Va at Arg306 and
inactivation of the cofactor. The consequence of impaired
membrane-dependent cleavage at Arg306 is manifested in vivo by venous and
arterial thrombosis.
Volume 84,
Issue 3,
pp. 687-690,
08/01/1994
Copyright © 1994 by The American Society of Hematology
