No abstract

Tissue factor (TF) circulates in plasma, largely on monocyte/macrophage-derived microvesicles that can bind activated platelets through a mechanism involving P-selectin glycoprotein ligand-1 (PSGL-1) on the microvesicles and P-selectin on the platelets. We found these mi-crovesicles to be selectively enriched in both TF and PSGL-1, and deficient in CD45, suggesting that they arise from distinct membrane microdomains. We investigated the possibility that mi-crovesicles arise from cholesterol-rich lipid rafts and found that both TF and PSGL-1, but not CD45, localize to lipid rafts in blood monocytes and in the mono-cytic cell line THP-1. Consistent with a raft origin of TF-bearing microvesicles, their shedding was significantly reduced with depletion of membrane cholesterol. We also evaluated the interaction between TF-bearing microvesicles and plate-lets. Microvesicles bound only activated platelets, and required PSGL-1 to do so. The microvesicles not only bound the activated platelets, they fused with them, transferring both proteins and lipid to the platelet membrane. Fusion was blocked by either annexin V or an antibody to PSGL-1 and had an important functional consequence: increasing the proteolytic activity of the TF-VIIa complex. These findings suggest a mechanism by which all of the membrane-bound reactions of the coagulation system can be localized to the surface of activated platelets.

The platelet glycoprotein (GP) Ib-IX-V complex mediates the attachment of platelets to the blood vessel wall by binding von Willebrand factor (VWF), an interaction that also transmits signals for platelet activation and aggregation. Because the complex is extensively palmitoylated, a modification known to target proteins to lipid rafts, we investigated the role of raft localization in GP Ib-IX-V functions. In unstimulated platelets, a minor portion of the complex localized to Triton-insoluble raft fractions; this portion increased three to sixfold with platelet activation by VWF. Raft-associated GP Ib-IX-V was selectively palmitoylated, with GP Ib-IX-V–associated palmitate increasing in the raft fraction on VWF-mediated activation. The raft fraction was also the site of association between GP Ib-IX-V and the Fc receptor FcγRIIA. The importance of this association was demonstrated by the ability of the FcγRIIA antibody IV.3 to inhibit shear-induced platelet aggregation. Disruption of rafts by depleting membrane cholesterol impaired several GP Ib-IX-V–dependent platelet fractions: aggregation to VWF under static conditions and under shear stress, tyrosine phosphorylation, and adhesion to a VWF surface. Partial restoration of membrane cholesterol content partially restored shear-induced platelet aggregation and tyrosine phosphorylation. Thus, localization of the GP Ib-IX-V complex within rafts is crucial for both platelet adhesion and postadhesion signaling.