Summary
The formation of platelet-rich thrombi under high shear rates requires both fibrinogen
and von Willebrand factor (VWF) as molecular adhesives between platelets. We attempted
to describe the role of VWF as a potential substrate and modulator of the fibrinolytic
system using binding assays, as well as kinetic measurements on the cleavage of fibrin(ogen)
and a synthetic plasmin substrate (Spectrozyme-PL). The similar dissociation constants
for the binding of plasminogen, plasmin, and active site-blocked plasmin onto immobilised
VWF suggest that the primary binding site in plasmin(ogen) is not the active site.
The progressive loss of clottability and generation of degradation products during
fibrinogen digestion with plasmin were delayed in the presence of VWF at physiological
concentrations, while VWF cleavage was not detectable. Determination of kinetic parameters
for fibrinogen degradation by plasmin, miniplasmin and microplasmin showed that VWF
did not modify the Km, whereas kcat values decreased with increasing VWF concentrations
following the kinetic model of non-competitive inhibition. Inhibitory constants calculated
for VWF were in the range of its physiological plasma concentration (5.4 μg/ml, 5.7
μg/ml and 10.0 μg/ ml for plasmin, miniplasmin and microplasmin, respectively) and
their values suggested a modulating role of the kringle 5 domain in the interaction
between VWF and (mini)plasmin. VWF had no effect on the amidolytic activity of plasmin
on Spectrozyme-PL, or on fibrin dissolution by (mini)plasmin. Our data suggest that
VWF, while a poor plasmin substrate relative to fibrinogen, protects fibrinogen against
degradation by plasmin preserving its clottability in plasma and its adhesive role
in platelet-rich thrombi.
Keywords
von Willebrand factor - fibrinogen - plasmin - fibrinolysis - thrombolysis
