Recombinant vWF type 2A mutants R834Q and R834W show a defect in mediatingplatelet adhesion to collagen, independent of enhanced sensitivity to a plasma protease
H. Lankhof et al., Recombinant vWF type 2A mutants R834Q and R834W show a defect in mediatingplatelet adhesion to collagen, independent of enhanced sensitivity to a plasma protease, THROMB HAEM, 81(6), 1999, pp. 976-983
Type 2A von Willebrand Disease (VWD) is characterized by the absence of hig
h molecular weight von Willebrand factor (VWF) multimers in plasma which is
caused by enhanced extracellular proteolysis or defective intracellular tr
ansport. We identified in vWD type 2A patients two mutations in the A2 doma
in at position 834 in which arginine (R) was substituted for glutamine. (R8
34Q) or tryptophan (R834W). We reproduced these mutations in VWF cDNA and e
xpressed the recombinant proteins in furin cDNA containing baby hamster kid
ney (fur-BHK) cells. The subunit composition and the multimeric structure o
f both mutants was similar to wild-type (WT) vWF. Characterization of mutan
t R834Q by ristocetin or botrocetin induced platelet binding, and by bindin
g to heparin showed no abnormality. R834W had normal botrocetin induced pla
telet binding, but ristocetin induced platelet binding and binding to hepar
in were decreased. Under static conditions R834Q and R834W, at 10 mu g/ml,
bound equally well to collagen type III as WT-VWF. At high shear rate condi
tions both mutants supported platelet adhesion normally when coated to a gl
ass surface or preincubated on collagen. When R834Q or R834W was added to t
he perfusate, adhesion to collagen type III was 50% of the WT-VWF value, wh
ich was not due to a decreased collagen binding under flow. A divalent cati
on dependent protease, purified from plasma; degraded the 2A mutants rapidl
y while WT-VWF was not affected. In conclusion; the mutations present in th
e A2 domain of VWF result in an enhanced proteolytic sensitivity to a dival
ent ion-dependent protease. When present in the perfusate, R834Q and R834W
show a decrease in platelet adhesion to collagen type III under flow condit
ions, which is not caused by decreased binding of the mutant VWF to collage
n or enhanced proteolysis.