Abstract
We have studied the mechanism of interaction between soluble von Willebrand factor (vWF), labeled with fluorescein isothiocyanate (FITC), and platelets exposed to shear in a cone-and-plate viscometer. A flow cytometer calibrated with fluorescent bead standards was used to calculate the number of molecules associated with each platelet in suspension. To validate the methods and reagents used, binding of the same labeled vWF was assessed in the presence of ristocetin or alpha-thrombin and found to be saturable, with a narrow and symmetric distribution on > 90% of the platelets. As expected, essentially all bound ligand interacted exclusively with platelet membrane glycoprotein (GP) Ib alpha in the presence of ristocetin and with GP IIb-IIIa after stimulation with alpha-thrombin. In contrast, only a minor proportion (< 20%) of the platelets exposed to shear were found to bind vWF, with no evidence for saturation and markedly decreased interaction when the platelet count was below 100,000 microliters. Moreover, shear-induced vWF binding was blocked equally effectively by selected monoclonal antibodies against either GP Ib alpha or GP IIb-IIIa or against the respective binding sites in vWF. Thus, both receptors are involved in the process, possibly through initial transient interactions mediated by GP Ib alpha that lead to platelet activation and subsequent irreversible binding supported by GP IIb-IIIa. While the levels of shear stress theoretically applied to platelets in these experiments are above those thought to occur in the normal circulation, our findings demonstrate a unique vWF binding mechanism that is not mimicked by other known modulators and correlates with platelet aggregation. Similar processes may occur in response to lower shear stress when platelets are exposed to thrombogenic surfaces and agonists generated at sites of vascular injury during thrombus formation.
Highlights
The role of von Willebrand factor1 in platelet thrombus formation, under conditions of high shear, is well established and supported by experimental evidence as well as clinical observations [1]
We have studied the mechanism of interaction between soluble von Willebrand factor, labeled with fluorescein isothiocyanate (FITC), and platelets exposed to shear in a cone-and-plate viscometer
Binding of soluble normal von Willebrand factor (vWF) to GP Ib␣ in the absence of flow requires the presence of exogenous modulators, like ristocetin [3] or botrocetin [4], while interaction with GP IIb-IIIa can only occur after platelet activation [5]
Summary
Preparation of Washed Platelets—Blood was drawn from an antecubital vein of normal donors with a 19-gauge needle into plastic syringes and immediately transferred into polypropylene tubes containing trisodium citrate, pH 7.5, as anticoagulant (final concentration 0.013 M) and the ADP scavenger, apyrase (Sigma), to prevent platelet activation (final concentration 10 units/ml). LJ-152B/6 (IgG1) reacts with the vWF site that includes the Arg-Gly-Asp sequence recognized by GP IIb-IIIa, but has no cross-reactivity with other ligands of this receptor containing the same sequence [28] This antibody selectively inhibits vWF binding to GP IIb-IIIa as well as platelet aggregation and thrombus formation under high shear conditions [9, 26]. It should be noted that all IgG against vWF interfered with shear-induced aggregation and binding, including those (LJ-229 and LJ-C3) not expected to block the interaction with platelet receptors This may be due to cross-linking effects between divalent antibody and multivalent vWF multimers since monovalent Fab fragments exhibited appropriate selectivity (i.e. the two control antibodies had no inhibitory effect). IgG and Fab fragments of anti-GP Ib␣ and anti-GP IIb-IIIa antibodies gave consistently similar results
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