Receptor-specific binding of von Willebrand factor to platelets in response to shear stress
McCrary, Jeffrey Kirk
Hellums, J. David
Doctor of Philosophy
Physical and biochemical forces convert platelets in human blood in vivo from the quiescent state as freely moving, discoid, singlets to aggregates adhering to subendothelial surfaces and secreting a wide variety of bioactive substances. Platelet aggregation, one of the essential components of hemostasis, can be caused in vitro by several chemical agonists, and by shear stress, as is found in flowing blood. Platelet aggregation at high shear stress is a receptor-mediated process requiring the binding of the plasma protein von Willebrand factor (vWf) to platelets. This study was undertaken to detect and characterize by direct measurement the binding of vWf to platelets under shear stress. Three different binding assays were developed. Two utilize radioassays for detection of vWf-platelet binding, one using radioiodinated, plasma-derived vWf multimers, the other using a novel technique which produces radiolabeled, unusually large vWf multimers from human umbilical vein cell cultures. The third assay utilizes an immunoassay technique to measure extracellular vWf concentration. Each experimental technique was verified using the platelet agonist, ristocetin. The measurements reported here constitute the first direct determination of vWf multimer binding to platelets in response to shear stress. Binding of exogenous vWf multimers to platelets is detected over the entire ranges of shear stresses and times of exposure tested (15-180 dynes/cm$\sp2$ and 15-300 seconds, respectively). It is receptor-specific, involving both platelet receptors GPIb and GPIIb/IIIa without apparent interdependence. Binding of the exogenous forms persists after long periods of shear, where platelet secretion of vWf can exceed the extent of binding of exogenous vWf, resulting in a net increase in extracellular vWf concentration. Shear stress-induced binding requires large vWf multimers, and involves the secretion of adenosine diphosphate from intraplatelet granules. These results demonstrate that vWf binds to platelet receptors GPIb and GPIIb/IIIa under levels of shear stress which cause platelet aggregation in vitro, and which correspond to arterial thrombosis occurrence in vivo. The findings discussed herein thus contribute to our understanding of the role of platelets in thrombotic disorders, and may be useful in development of methods for therapy and/or prophylaxis.
Biomedical engineering; Chemical engineering; Cell biology; Biology; Engineering