Characterization of PAR-mediated signaling induced by activated coagulation factor X mutants

Coagulation Factor X (FX) is a serine protease which plays a crucial role in blood coagulation. In addition FXa has been involved in cell signaling through activation of protease activated receptors (PARs). We compared FX mutants, found in FX deficient patients, for their activity toward the synthetic substrate Spectrofluor FXa (amidolytic activity) and prothrombin, and for their cell signaling capacity. The study of dysfunctional variants might help to identify FXa exosites responsible for the specific interaction with PARs. Methods. Missense changes in the catalytic domain (V342A, G381D, R386C) not impairing FX biosynthesis but responsible for mild to severe reduction of coagulant activity were selected. The recombinant FX wild type (rFX-wt) and mutants were stably expressed in eukaryotic cells and immuno-purified. FX procoagulant function was determined by measuring prothrombin time, thrombin generation and amidolytic activity. Murine myoblast C2C12 were stimulated with the rFXa variants (after activation by Russell’s viper venom) and PAR activation was evaluated through Western blot analysis of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2). Results and discussion. The rFXa-381D mutant (chymotrypsin numbering, c197), affecting the active site cleft of FX, showed low amidolytic (5%) and thrombin generation (<1%) activity, as compared to the rFXa-wt, and did not induce signal transduction, thus behaving like the active serine mutant rFX-379A, produced as negative control. The rFXa-386C mutant (c202), which affects a variable surface-exposed region of the serine protease domain, presented a reduced amidolytic (50%) and thrombin generation (40%) activitiy, and a reduced transduction activity. In particular, the ERK1/2 phosphorilation induced by 18 nM rFXa-wt was observed at 130nM concentration. The rFXa-342A mutant (c160), in a conserved superficial region of the catalytic domain, showed a reduced amidolytic (30%) and thrombin generation (25%) activitiy, and was able to induce ERK1/2 phosphorilation with a similar intensity to the rFXa-wt (18 nM) at 44-88 nM concentration. In time-dependent assays both mutants showed, at the highest concentration (174 nM), a signaling capacity comparable to that of rFXa-wt. Conclusion. Catalytic activity of FXa is required to induce activation of signal transduction pathways and the signaling capacity of recombinant mutants was found to be correlated with their activity toward synthetic or natural substrates in the coagulation pathway.