Correction of thrombin generation in hemophilia plasma by recombinant factor V mutants

Congenital or acquired deficiency of factor VIII (commonly referred to as
hemophilia A) leads to a serious bleeding tendency.1 Affected individuals may
present with spontaneous bleeding, most frequently bleeding in joints and
muscle but also gastro-intestinal bleeding or intracerebral hemorrhage. Also,
invasive procedures may be complicated by severe bleeding. Prophylactic or
therapeutic administration of (recombinant) coagulation factor concentrates has
markedly improved the management of hemophilia patients in recent decades. One
of the most challenging complications of this treatment nowadays is the
formation of antibodies towards factor VIII, which may hamper adequate recovery
of exogenously administered factor VIII and cause significant bleeding
complications. Affected patients are commonly treated with activated
prothrombin complex concentrates (such as FEIBA) or recombinant human activated
factor VII (NovoSeven).2,3 However, these options are associated with
complications themselves, such as thrombosis.
Pro-hemostatic proteins interfering with the coagulation system have been found
to be able to induce generation of thrombin even in the absence of sufficient
concentrations of factor VIII. Previous in vitro studies point to a potential
role of factor V in the prothrombinase complex. In particular, factor V that
has mutations which render this protease less susceptible to proteolytic
cleavage by activated protein C may potentially have pro-hemostatic properties.
This notion is underlined by the fact that a commonly occurring factor V
mutation (Factor V Leiden) leads to a mild pro-thrombotic phenotype and may
reduce the risk of bleeding. Other factor V mutations (factor V Cambridge and
factor V Hong Kong) have similar effects.

What is the effect of ex vivo incubation of plasma from patients with
hemophilia A with or without inhibiting anti-factor VIII antibodies with
recombinant human factor V mutants on activation of coagulation?

Laboratory experiments in plasma derived from patients with hemophilia A.
Patients will have to provide informed consent. From participating patients 10
ml blood will be drawn in standard citrated vacuum tubes. The collection of
blood will be combined with blood sampling for other (routine) reasons. Plasma
will be obtained by centrifuging the tubes for 30 min at 2500 RPM. The
supernatant plasma will be aliquoted and frozen at *80oC until assayed.
Citrated blood will be thawed and incubated with increasing concentrations of
various recombinant human factor V mutants.

donation of 10 ml blood, during venipuncture that will be peformed for routine
patientcare.