Destabilization of the A1 domain in von Willebrand factor dissociates the A1A2A3 tri-domain and provokes spontaneous binding to glycoprotein Ibalpha and platelet activation under shear stress.
This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ibalpha (GPIbalpha). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIbalpha without ristocetin with an apparent K(D) of 85 +/- 34 nm, comparable with that of WT (36 +/- 12 nm) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s(-1) shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s(-1), platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIbalpha and alphaIIbbeta3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.
Auton
Matthew
M
Section of Cardiovascular Research, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
Sowa
Katie E
KE
Smith
Scott M
SM
Sedlák
Erik
E
Vijayan
K Vinod
KV
Cruz
Miguel A
MA
D001696:Biomechanical Phenomena; D002460:Cell Line; D006801:Humans; D009154:Mutation; D015539:Platelet Activation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012996:Solutions; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019038:Platelet Glycoprotein GPIb-IX Complex; D012996:Solutions; D014841:von Willebrand Factor