Role of membrane cholesterol in platelet calcium signalling in response to VWF and collagen under stasis and flow

Marjolijn van Lier; Sandra Verhoef; Sandra Cauwenberghs; Johan W. M. Heemskerk; Jan-Willem N. Akkerman; Harry F. G. Heijnen

doi:10.1160/TH07-08-0528

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2008; 99(06): 1068-1078
DOI: 10.1160/TH07-08-0528

Platelets and Blood Cells

Schattauer GmbH

Role of membrane cholesterol in platelet calcium signalling in response to VWF and collagen under stasis and flow

Marjolijn van Lier

¹Department of Clinical Chemistry and Hematology, UMCU; Institute for Biomembranes, Utrecht, the Netherlands

,

Sandra Verhoef

¹Department of Clinical Chemistry and Hematology, UMCU; Institute for Biomembranes, Utrecht, the Netherlands

,

Sandra Cauwenberghs

²Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, the Netherlands

,

Johan W. M. Heemskerk

²Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, Maastricht, the Netherlands

,

Jan-Willem N. Akkerman

¹Department of Clinical Chemistry and Hematology, UMCU; Institute for Biomembranes, Utrecht, the Netherlands

,

Harry F. G. Heijnen

¹Department of Clinical Chemistry and Hematology, UMCU; Institute for Biomembranes, Utrecht, the Netherlands

³Cell Microscopy Center, Department of Cell Biology, UMCU, Utrecht, the Netherlands

› Author Affiliations

Abstract

Summary

Several studies have highlighted a specific role for membrane cholesterol domains in platelet signalling. Upon adhesion to von Willebrand factor (VWF) or collagen, cholesterol-rich domains (CRDs) accumulate in filopodial extensions and selectively harbour counterpart receptors (GPIb and GPVI) and associated signalling molecules. In the present study we have addressed the role of membrane cholesterol in Ca²⁺ signalling and secretion during the interaction of platelets with VWF and collagen.VWF/ ristocetin-induced platelet aggregation was delayed after treatment with methyl β-cyclodextrin (mbCD), but the maximal aggregation response was not affected. Platelet spreading but not adhesion to immobilised VWF under flow was attenuated by cholesterol removal, and accompanied by moderate lowering in the spiking Ca²⁺ response. On the other hand, platelet interaction with collagen was quite sensitive to cholesterol depletion. Platelet aggregation decreased after treatment with mbCD, and Ca²⁺ responses were decreased, both under static and flow conditions. Cholesterol depletion affected the secondary feedback activation via release of thromboxane A₂ and ADP. The collagen-induced secretion of alpha granules and surface translocation of P-selectin and CD63 was also critically affected by cholesterol depletion. Confocal microscopy showed localization of p-Tyr at sites of contact with substrate and other platelets, where also CRDs accumulate. Our data thus reveal a more critical role for membrane cholesterol in collagen-induced than in VWF-induced Ca²⁺ signalling, and furthermore support the concept that secondary activation responses are dependent on intact CRDs.

Keywords

Collagen - von Willebrand factor - calcium - cholesterol-rich domains

Full Text

References

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