Destabilization of the A1 Domain in von Willebrand Factor Dissociates the A1A2A3 Tri-domain and Provokes Spontaneous Binding to Glycoprotein Ibα and Platelet Activation under Shear Stress

Abstract

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 Ib␣ (GPIb␣). 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 GPIb␣ 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 GPIb␣ and ␣IIb␤3 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.Platelet adhesion at sites of vascular injury contributes to the arrest of bleeding as well as to the pathologic occlusion of diseased vessels under elevated shear stress. Under this high shear stress, the platelet-von Willebrand factor (VWF) 3 interaction is essential for platelet adhesion. The interaction between VWF and the exposed subendothelium permits the VWF to interact with circulating platelets via the receptor glycoprotein (GP)Ib/ IX/V complex (1). Mature VWF consists of a 2050-residue subunit that contains domains that are arranged in the order DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (cystine knot) (2-4). The VWF contains a triplicate repeat sequence or A domains in the central portion of the subunit. Binding sites for platelet GPIb␣, heparin, sulfatides, and collagen (5-9) are within the A1 domain, whereas its homologous A3 domain only binds to collagen, and the A2 domain contains the cleavage site for the metalloprotease ADAMTS-13 (10 -12). The functions of these A domains relevant to the biology of VWF have been characterized by individual recombinant expression of each of the A domains (9, 10, 13).The interaction between VWF and GPIb␣ occurs when the binding site for GPIb␣ in the A1 domain of VWF has been exposed by the influence of high fluid forces or when the multimeric protein has been immobilized (14). This activation can also be induced by nat...

N-terminal Flanking Region of A1 Domain in von Willebrand Factor Stabilizes Structure of A1A2A3 Complex and Modulates Platelet Activation under Shear Stress

“…Previously, we described the functional similarities between a monomeric A1A2A3 protein and full-length vWF and suggested that the recombinant tri-domain fragment (amino acids 1238 -1874) of vWF contains regulatory structural elements that modulate activation of vWF in solution (4). On the other hand, another recombinant A1A2A3 protein (amino acids 1261-1874) from our laboratory apparently resulted in a GPIb␣ binding site that was less cryptic in the A1 domain.…”

“…However, the binding site for GPIb␣ in the A1 domain remained cryptic in our A1A2A3 protein, which excludes the DЈD3 domains (4). This outcome may indicate that the amino acid sequence 1238 -1247 from the C terminus of the D3 domain forms part of the mechanism that masks the binding site for GPIb␣.…”

“…One ml of citrated whole blood containing either the A1A2A3 protein or buffer (TBS) was perfused over the analyte surface-coated plate followed by TBS or PBS. Tethered platelets were observed with phase contrast objectives, recorded by video microscopy, and analyzed as described previously (4). Experiments were performed in duplicate using different blood donors.…”

“…The recombinant A1A2A3 variants were expressed in mammalian (HEK293) cells, purified from the conditioned medium, and subjected to gel electrophoresis and gel filtration chromatography to verify its purity and monomeric state as we described previously (4,25). A monospecific polyclonal antibody against the A1 domain, A108 (against sequence 1444 -1452), was commercially prepared using synthetic peptides in rabbits.…”

The N-terminal Flanking Region of the A1 Domain Regulates the Force-dependent Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

“…Circulating platelets do not bind VWF unless putative conformational changes occur around its A1 domain that exposes the GPIb␣ binding site (VWF activation) as a result of high hemodynamic force (8). The present view of the force-dependent VWF activation includes two steps: 1) relief of an autoinhibitory mechanism involving interdomain associations within the A1A2A3 tri-domain (9,10) and between A1 and DЈD3 (11) and 2) upregulation of A1 binding affinity to GPIb␣ (6,(12)(13)(14). However, the biophysical and structural basis of how the VWF-GPIb␣ interaction is regulated when its hemostatic adhesive function is most needed remains unclear, in part, because of discrepant data.…”