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Comparison of the Relative PAR1, PAR2 and PAR3 Cleavage Profiles by Thrombin, APC, Factor Xa and Factor VIIa

A. Yamashita1,2, M. Taki2, J.H. Griffin1, L.O. Mosnier1

1Department of Molecular Medicine, The Scripps Research Institute, La Jolla, United States, 2Department of Pediatrics, St. Marianna University School of Medicine, Kawasaki, Japan

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.

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