Abstract Number: PB0730
Meeting: ISTH 2021 Congress
Theme: Role of Hemostatic System in Cancer, Inflammation and Immunity » Coagulation Proteins Beyond Hemostasis
Background: Thrombin, factor VIIa (FVIIa), FXa, and activated protein C (APC) affect multiple cellular functions via protease activated receptors (PAR).
Aims: To help understand differences between these proteases, their relative potencies for cleaving PAR1, PAR2, and PAR3 were determined.
Methods: PAR sensitivities to cleavage were determined using HEK-293 cells expressing EPCR and secreted embryonic alkaline phosphatase (SEAP)-PAR1, SEAP-PAR2, and SEAP-PAR3.
Results: In the presence of EPCR, dose-response curves for APC and FXa cleavage of PAR1 and PAR3 were similar. Each required a 100-fold higher concentration for 50% cleavage (EC50) compared to thrombin for PAR1 cleavage (EC50= 36.0, 49.8 and 0.3 nmol/L for APC, FXa and thrombin, respectively) and a 16-fold higher concentration for PAR3 cleavage (EC50=20.1, 19.2 and 1.22 nM for APC, FXa and thrombin, respectively). Notably, thrombin and FXa cleave PAR1 at Arg41 whereas APC cleaves at Arg46; APC and FXa cleave PAR3 at Arg41 whereas thrombin cleaves at K38. FVIIa showed minor cleavage of PAR1 at >100 nM and no cleavage of PAR3. Dose-response curves for APC and FVIIa PAR2 cleavage at Arg36 in the presence of EPCR were similar but they required a 20-fold higher concentration compared to FXa (EC50=15.5, 349 and 369 nM for FXa, APC and FVIIa, respectively). Mutation of positively charged residues in the PAR2 N-terminus (R31Q, K34Q, K41Q and K51Q) revealed strikingly different effects for FXa, APC and FVIIa. Notably, K34Q improved PAR2 cleavage by FXa 4-fold (EC50=3.7 nM) but reduced cleavage by APC or FVIIa 2 to 4-fold (EC50=1431 and 779 nM for APC and FVIIa, respectively).
Conclusions: Cleavages of PAR1, PAR2 and PAR3 by thrombin, APC, FXa and FVIIa vary greatly with respect to relative sensitivities based on dose-responses, EPCR-dependency, and cleavage sites. These remarkable variabilities help explain how these proteases use the same receptor systems to modulate diverse cellular functions.
To cite this abstract in AMA style:
Yamashita A, Taki M, Griffin JH, Mosnier LO. Comparison of the Relative PAR1, PAR2 and PAR3 Cleavage Profiles by Thrombin, APC, Factor Xa and Factor VIIa [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/comparison-of-the-relative-par1-par2-and-par3-cleavage-profiles-by-thrombin-apc-factor-xa-and-factor-viia/. Accessed November 29, 2023.« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/comparison-of-the-relative-par1-par2-and-par3-cleavage-profiles-by-thrombin-apc-factor-xa-and-factor-viia/