Human Platelet Tissue Factor Is Localized in Lipid Rafts and Is Associated with GPIb-IX-V Complex in the Membrane: Association between Adhesive and Procoagulant Activities of Platelets.

The binding of platelet membrane GPIb-IX-V to extracellular matrix proteins and von Willebrand factor initiates the adhesion, spreading and activation of platelets. On the other hand, tissue factor (TF) triggers the blood coagulation by binding FVII/VIIa. In resting platelets, TF is stored mainly as a non active (“encrypted”) form, possibly through a dimer- or heterodimer complex. The encryption seems not to affect the TF capacity to bind FVII and VIIa, in such form that the membrane-based complex would be “primed” to trigger the clot formation upon platelet activation. Accordingly, the platelet adhesion and activation would be tightly interwoven with thrombin generation. Given the described localization of GPIbα in platelet lipid rafts, which appears to modulate its activity, we investigated whether TF is also present in these cholesterol-rich domains of the platelet membrane. Continuous sucrose gradients generated by ultracentrifugation at 180.000g x 18h at 4°C were used to obtain cholesterol-rich fractions from platelet lysates (1% Triton X-100 at 4°C). Western blots of each gradient fraction using a polyclonal anti-TF antibody revealed a protein of ~60kDa only in the fractions rich in cholesterol (using flotillin-1 as a marker). TF-dependent procoagulant activity (PCA), assessed by FXa generation was measured in resting and 5μM TRAP-stimulated, leukocyte-free platelets. In both cases, PCA was reduced after membrane cholesterol depletion with methyl-β-cyclo-dextrin. We also found that PCA was significantly enhanced in washed platelets treated with VWF and ristocetin, suggesting some form of functional or structural relationship between GPIb-IX-V complex and TF. GPIb-IX was immunoprecipitated (IP) from human platelet membranes by anti-GPIbα MoAbs (AP-1 or SZ2), in both resting and stimulated platelets (TRAP or VWF-ristocetin). The IP product was revealed with MoAb anti-TF (American Diagnostica, clone 4509) or polyclonal anti-TF, detecting a protein of ~47kDa. When we used MoAb anti-TF for the IP and MoAb anti-GPIX (BL-H6, anti-CD42a) to reveal the western blot, two distinct bands of ~22 and ~47kDa were disclosed, probably corresponding to GPIX and a heterodimer of GPIX and TF, respectively. These observations indicate that, at least a fraction of the platelet TF is localized in lipid rafts and that TF is also in close association with the GPIb-IX-V complex. This co-localization probably has important functional implications as demonstrated by the defective thrombin generation in patients with Bernard-Soulier syndrome. Moreover, this functional association may be relevant in both normal hemostasis and thrombus formation and growth.