Characterization of the Unique Mechanism Mediating the Shear-dependent Binding of Soluble von Willebrand Factor to Plateletsстатья из журнала
Аннотация: 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 α-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) Ibα in the presence of ristocetin and with GP IIb-IIIa after stimulation with α-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 μl. Moreover, shear-induced vWF binding was blocked equally effectively by selected monoclonal antibodies against either GP Ibα 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 Ibα 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. 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 α-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) Ibα in the presence of ristocetin and with GP IIb-IIIa after stimulation with α-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 μl. Moreover, shear-induced vWF binding was blocked equally effectively by selected monoclonal antibodies against either GP Ibα 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 Ibα 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.
Год издания: 1995
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Platelet Disorders and Treatments, Blood properties and coagulation, Complement system in diseases
Другие ссылки: Journal of Biological Chemistry (PDF)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Открытый доступ: hybrid
Том: 270
Выпуск: 40
Страницы: 23352–23361