Platelet-associated Tissue Factor: modulation of cell surface expression by platelet agonists

Abstract number: P1276

Camera^* M., Brambilla^* M., Frigerio† M., Cottell‡ D., Rossi† F., Maderna‡ P., Toschi§ V., Parolari† A., Biglioli† P., Tremoli^* E.

^*Department Pharm Sciences, Italy; †Monzino Cardiol Center, Italy; §San Carlo Hospital, Italy ‡University College, Dublin, Ireland;

Tissue Factor (TF) is one of the major determinants of the activation of coagulation cascade at site of atherosclerotic plaque rupture. It has been recently shown that human platelets contain TF which may derive from leukocytes. In this study we investigated whether intraplatelet TF can be exposed on the membrane by agonists of platelet activation. Flow cytometric analysis of unstimulated platelets showed a small amount of membrane-associated immunoreactive TF (irTF) in whole blood (WB), platelet-rich plasma (PRP) or washed platelets (WP) isolated from healthy subjects free of medication known to affect platelet function. After ADP stimulation a significant increase in irTF was observed on platelet membranes (WB: 3.4 ± 1.2 vs. 5.55 ± 1.37 MF, n= 20; PRP: 2.8 ± 0.6 vs. 4.8 ± 1.3 MF, n= 33, and WP: 4.2 ± 1.1 vs. 6.9 ± 1.9, n= 6, all P < 0.01). The increase in irTF paralleled that of P-selectin. The absence of leukocyte contamination in PRP and WP was confirmed by the lack of staining of platelet preparations for panspecific leukocyte CD45 antigen, as analyzed in the platelet as well as in the leukocyte-size ranges. ADP induces irTF expression in a concentration-dependent manner (0.1–20 µmol L^-1) peaking at 10 µmol L^-1. Time course experiments (15–240 min) performed in PRP showed that irTF specific fluorescence was maximal at 15–30 min. The presence of platelet membrane-associated TF was also confirmed by transmission mode immunoelectron microscopy. Experiments performed in PRP with human recombinant FVIIa-FITC labeled indicate that platelet membrane associated TF is able to bind to its physiological ligand and thus it might be functionally active. Thrombin receptor activating peptide (TRAP) and epinephrine also significantly increased membrane-associated irTF in WB, as well as in PRP and WP. Moreover, the concomitant addition of ADP and epinephrine to WB or PRP more markedly increased irTF compared with either agonist alone. Real Time PCR experiments showed detectable amounts of TF mRNA in unstimulated platelets, suggesting that the platelet itself has the machinery to potentially synthesize TF. These findings indicate that platelets may be a source of circulating irTF and that platelet agonists render this protein available for the interaction with blood or vessel wall components. Regulated TF expression establishes the potential for a previously unrecognized role for platelets in sustaining thrombus formation and growth via coagulation-mediated mechanisms.