Adhesive properties of the isolated amino-terminal domain of platelet glycoprotein Ibα in a flow field

Abstract

We have examined the interaction between the amino-terminal domain of platelet glycoprotein (GP) Ib␣ and immobilized von Willebrand Factor (vWF) under f low conditions in the absence of other components of the GP Ib-IX-V complex. Latex beads were coated with a recombinant fragment containing GP Ib␣ residues 1-302, either with normal sequence or with the single G233V substitution that causes enhanced affinity for plasma vWF in platelet-type pseudo-von-Willebrand disease. Beads coated with native fragment adhered to vWF in a manner comparable to platelets, showing surface translocation that ref lected the transient nature of the bonds formed. Thus, the GP Ib␣ extracellular domain is necessary and sufficient for interacting with vWF under high shear stress. Beads coated with the mutated fragment became tethered to vWF in greater number and had lower velocity of translocation than beads coated with the normal counterpart, suggesting that the G233V mutation lowers the rate of bond dissociation. Our findings define an approach for studying the biomechanical properties of the GP Ib␣-vWF bond and suggest that this interaction is tightly regulated to allow rapid binding at sites of vascular injury, while permitting the concurrent presence of receptor and ligand in the circulation.Platelet adhesion to von Willebrand factor (vWF) immobilized at sites of vascular injury initiates thrombus formation in areas of rapid blood flow (1) and represents a critical mechanism in hemostasis and thrombosis. The process is mediated by the glycoprotein (GP) Ib-IX-V complex, in which the aminoterminal domain of the GP Ib␣-chain (2, 3) contains the binding site for the vWF A1 domain (4, 5). This interaction supports platelet tethering to surfaces even at extremely high shear rates but without irreversible attachment. If no other bonds are formed, tethered platelets translocate in the direction of flow, albeit at a markedly lower velocity than freely flowing blood cells (6). On reactive substrates, however, initial contact allows the rapid establishment of additional bonds typically mediated by receptors of the integrin superfamily and results in essentially instantaneous irreversible adhesion, followed by events leading to subsequent thrombus development (1). At present, it is unknown whether any single domain of the GP Ib-IX-V complex can exhibit the vWF binding function of the intact receptor expressed on the platelet surface. Information in this regard could indicate whether linkage to the cytoskeleton (7,8) and͞or interactions between components of the complex (9, 10) contribute to vWF binding.We have addressed these questions by using a recombinant fragment comprising GP Ib␣ residues 1-302 (11). The isolated domain has been shown to undergo tyrosine sulfation and support modulator-dependent vWF binding as the native receptor (12). In the present studies, plastic beads were coated with the fragment, and its activity was tested in a flow field to mimic the function of a surface-expressed cell membrane receptor during vascular in...

Interaction of von Willebrand Factor Domain A1 with Platelet Glycoprotein Ibα-(1–289)

“…Platelet adhesion mediated by VWF is rapid and efficient, and the evaluation of the process by video microscopy suggests the rate constants for the binding of A1 to GPIb␣ could be correspondingly fast (19,21,40). However, these studies do not provide direct information about intrinsic dissociation constants or reaction rates, and the results are compatible with other models.…”

“…Placed in context with the results of flow studies performed by Ruggeri and colleagues (37,38,40), the intrinsic binding parameters of VWF domain A1 and GPIb␣-(1-289) may dictate a reformulation of the role of shear stress in VWF function. The isolated VWF A1 domain and GPIb␣-(1-289) are too small to react directly to any physiological fluid shear stress but are sufficient to mediate particle adhesion that exhibits a physiological dependence on wall shear rate.…”

“…The specific binding of NMC-4 to the microtiter wells was determined as described under "Experimental Procedures." ilar shear-dependent adhesion to immobilized VWF (40). Inclusion of the mutation G233V, which causes platelet-type pseudo-VWD, increases the affinity of GPIb␣-(1-302) binding to VWF and increases the adhesion of GPIb␣-(1-302)-coated latex beads to VWF (40).…”

“…ilar shear-dependent adhesion to immobilized VWF (40). Inclusion of the mutation G233V, which causes platelet-type pseudo-VWD, increases the affinity of GPIb␣-(1-302) binding to VWF and increases the adhesion of GPIb␣-(1-302)-coated latex beads to VWF (40). Thus, the adhesive properties of VWF domain A1 and its binding site on the N terminus of GPIb␣ do not appear to depend on interactions with more remote structures in either protein.…”

Influence of Anatomical Location on Arterial Thrombosis

“…[25][26][27][28][29] Both selectin-and glycoprotein Ib␣-binding have been shown to have a shear threshold dependence whereby resistance to detachment increases proportional to increasing shear. These shear requirements are important not only for adhesion promotion but also maintenance.…”