GPIbα Activation Triggers Platelet Tissue Factor/FVIIa-Dependent Procoagulant Activity, Which Is Dampened By Platelet Secreted TFPI and Protein S. Novel Platelet-Based Model of Hemostasis

Human platelets contain Tissue Factor (TF) demonstrated by western blotting (WB), IP, confocal IF microscopy and flow cytometry (FC) using an array of different α-TF antibodies. Moreover, TF is synthesized by platelets and has procoagulant activity (PCA) (Panes et al. Blood 2007). Platelets also contain the full-length α-isoform of TFPI that is exposed on the plasma membrane of activated, coated-platelets. A new role of Protein S (PS) is to function as TFPI co-factor for TF/FVIIa/FXa inactivation. In fact, TFPI function, enhanced by protein S (PS), dampens thrombin generation on the platelet surface (Wood, JP et al, 2014). In endothelial cells TFPI appears associated with the cell surface through glycosylphosphatidyl-inositol-mediated anchorage, suggesting some type of association with cholesterol-rich domains in cell membranes (lipid rafts, LR). Platelets also contain PS, but its functional association with platelet TFPI to inhibit platelet TF-dependent PCA remains still unknown. Aim: to disclose the physiologic role of TFPI and PS on the TF-dependent PCA of human platelets. Results: Stimulation of isolated, washed platelets with VWF-Ristocetin (VWF-R) resulted in 10-fold increase of TF-dependent FXa generation within 2-5min, compared with non-stimulated (N-S) platelets: [median 17(10-37) to 182(43-847) nM FXa/2x10⁷ platelets, n=198]. VWF-R induced anionic phospholipid exposure, but no α-δ-granules release in washed platelets. FX activation could be triggered without external FVII, although this was required to sustain the reaction. FVII/FVIIa was demonstrated in washed platelets by WB, confocal IF microscopy and FC, and its membrane expression augmented after platelet activation. FXa induced by VWF-R was abolished by pre-incubation with TFPI or with several polyclonal or mAbs against to each TF, FVIIa or GPIbα. In contrast, TRAP stimulation (n=84) induced little or no FXa generation [Median 32(11-219) nM FXa/2x10⁷platelets], but FX activation was increased by 54% (range 13-109%, p=0.0039) in platelets pre-incubated with α-TFPI. This increase was more pronounced with pre-incubation with α-PS (90%, range 31-227%, p<0.004). Combination of α-TFPI and α-PS did not enhance further FX activation. The releasate fraction of TRAP-stimulated platelets inhibited the FXa produced by VWF-R-stimulated platelets, supporting that TRAP-induced secretion of TFPI and PS explained this effect. Membrane exposure of platelet TFPI measured by FC was decreased after TRAP stimulation, a paradox explained by the high content of TFPI and PS in microparticles (MP) contained in the TRAP releasates. In contrast, the smaller number of MP released by VWF-R activation contained neither TFPI nor PS (WB assay). IP assays in platelet membrane fractions showed co-precipitation of TF, FVII/FVIIa and GPIbα. All these proteins co-precipitated also with TFPI in lipid raft fractions. Importantly, TFPI was notably augmented in LR fractions after TRAP stimulation, as compared with VWF-R stimulation. Moreover, TRAP stimulation resulted in co-precipitation of TFPI and PS in cytosolic, membrane and released platelet fractions. Conclusions: 1. TF-dependent PCA of washed human platelets is specifically and rapidly induced by GPIbα activation and it´s not accompanied with α-δ-granules release, including TFPI and PS. 2. Platelets contain enough membrane FVII to trigger the FX activation. 3. TRAP induces granule release, including TFPI and PS, which block TF-dependent PCA. 3. This TFPI is predominantly localized in LR fractions and co-precipitates with PS. 4. Secreted protein S likely localizes TFPI to the platelet membrane rich in anionic-rich phospholipids. 5. These results suggest that TF is translocated to LR for its inactivation. 6.These findings lead us to propose a novel model in which clotting is triggered by platelet TF during the first stage of platelet adhesion through GPIbα-VWF interaction; and TF/FVIIa/FXa would be dampened by the secreted TFPI and PS during subsequent platelet activation. Human platelets TF, FVII and the anionic phospholipid surface become central players to assemble and localize the whole clotting process into and around the platelet plug for both hemostasis and atherothrombosis; and platelet TFPI and PS would modulate its growth inactivating TF/FVIIa/FXa on the platelet surface.