Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 11, Pages 7539-7546Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.11.7539
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- NHLBI NIH HHS [HL60617] Funding Source: Medline
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We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ib alpha. Recombinant VWF A1 domain (residues Glu(497)-Pro(705) of VWF) bound stoichiometrically to a GrPIb alpha-calmodulin fusion protein (residues His(1)-Val(289) of GPIb alpha; GPIb alpha-CaM) immobilized on W-7-agarose with a K-d of 3.3 mu M. The variant VWF A1(R545A) bound to GPIb alpha-CaM 20-fold more tightly, mainly because the association rate constant k(on) increased from 1,100 to 8,800 M-1 s(-1). The GPIb alpha mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIb alpha(G233V)-CaM and GPIb alpha(M239V)-CaM with a K-d of 1.0 and 0.63 mu M, respectively. The increased affinity of VWF A1 for GPIb alpha(M239V)-CaM was explained by an increase in k(on) to 4,580 M-1 s(-1) GPIb alpha-CaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu(480)/Val(481)-Gly(718)). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIb alpha-CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIb alpha-CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIb alpha, and the increased binding affinity caused by mutations in VWF or GPIb alpha, are reproduced by isolated structural domains. The substantial increase in k(on) caused by mutations in either A1 or GPIb alpha suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k(on) and k(off) provide important new constraints on models for rapid platelet tethering at high wall shear rates.
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