Platelet Tissue Factor: Fast and Specific Clotting Activation Pathway Mediated by VWF-GPIba Interaction and Platelet Membrane FVII. Human Platelets Contain All the Components for Assembling the Prothrombinase Complex and Their Procoagulant Function Is Independent of Aggregation/Secretion and GPVI Function.

Abstract 3000

Poster Board II-977

Human platelets synthesize and store functionally silent tissue factor (TF) which expresses procoagulant activity (PCA) after platelet activation. Fast activation of TF was elicited by VWF-Ristocetin (VWF-R) through GPIbαa activation and Src-Lyn transduction pathway (Blood, Nov 2008; 112:113). Given that GPVI, along with GPIb and TF have been found in “lipid rafts”, and the activated form of GPVI signals through Fyn, another member of the Src family, we tested if GPVI was involved in TF-initiated PCA. We also studied the time-course and pathway specificity of TF activation and the role of platelet FVII in PCA. Weak TF immunofluorescence and co-localization with GPIba were observed in non stimulated washed platelets. A mild increase of TF fluorescence was detected 2 min after TRAP activation, which augmented when the stimulus was VWF-R. Furthermore, striking enhancement of TF fluorescence occurred 2 min after depositing platelets over a VWF-coated surface, but not over fibrinogen or albumin. Platelets adherent to VWF matrix showed GPIb clustering and loss of co-localization with TF. Externalization of TF was confirmed by immunoprecipitation (Ip) of biotinylated membranes before and after platelet activation. Concomitantly, TF-dependent FXa generation increased 5-10-fold shortly after VWF stimulus. Washed platelets stimulated with VWF-R agglutinated normally when stirred in an aggregometer, and the fraction of platelets exposing anionic phospholipids (annexin V binding) was similar to parallel samples stimulated with TRAP. However, VWF-R induced null ¹⁴C-serotonin secretion and P-selectin exposure (flow cytometry) in washed platelets. In contrast, TRAP, collagen, ADP and convulxin induced full platelet aggregation, ¹⁴C-serotonin and P-selectin secretion at 2-5 min, but with no increase in FXa generation. Platelet PCA was inhibited by antibodies against TF, GPIba, FVIIa, as well as by SU6656 and PP2 (Src pathway inhibitors), but not by Gö6850 (a PKC inhibitor) or exogenous TFPI. p85, a subunit of PI-3K constitutively associated with GPIb complex, becomes strongly associated with TF after stimulation with VWF-R, though only weakly after TRAP activation, confirming the coordinate activation of GPIb and TF.

FVII and FX were revealed in platelet membrane fractions by immunoblotting and both co-precipitate with TF in non-stimulated platelets. Two min after activation with VWF-R striking co-precipitations of TF with FVII and FX light chains were evidenced, denoting activation of platelet FVII and FX. When exogenous FX was added to the assay, the amount of FXa generated after 1 and 2 min stimulation was similar whether or not exogenous FVIIa was added.

Platelets from four non-related patients with bleeding related to hereditary defect of GPVI had null aggregation and secretion with convulxin and collagen, less than 7% labeling of GPVI by flow cytometry and an immunoreactive membrane GPVI of Mr≈40kDa (native GPVI Mr=62kDa). All of them had normal agglutination with VWF-R and normal FXa generation.

In summary, GPIb activation by VWF constitutes a unique and fast inducer of platelet TF-dependent PCA. This process requires anionic phospholipid exposure, but is independent of platelet GPIIb/IIIa and GPVI function. Platelet FVII can initiate FXa generation without need of plasma FVII. The associations of platelet FVII and FX with TF on membrane fractions, together with the large amount of FV in platelets, indicate that human platelets provide not just TF and a PCA phospholipid platform, but also all the components of the prothrombinase complex to trigger the clotting process. Taken together, our results underline the central role of platelets in the whole hemostatic process, unifying primary and secondary hemostasis and circumscribing thrombin generation and fibrin deposition where platelet plug is being formed. Platelet PCA should become a pharmacological target for preventing or managing bleeding and thrombotic disorders.