Development and characterization of monoclonal antibodies against Protease Activated Receptor 4 (PAR4)

Background: Protease activated receptor 4 (PAR4) is a G protein coupled receptor (GPCR) which is activated by proteolytic cleavage of its N-terminal exodomain. This generates a tethered ligand that activates the receptor and triggers downstream signaling events. With the current focus in the development of anti-platelet therapies shifted towards PARs, new reagents are needed for expanding the field's knowledge on PAR4. Currently, there are no PAR4 reagents which are able to detect activation of the receptor. Methods: Monoclonal PAR4 antibodies were purified from hybridomas producing antibody that were generated by fusing splenocytes with NS-1 cells. Immunoblotting, immunofluorescence, and flow cytometry were utilized to detect the epitope for each antibody and to evaluate the interaction of the antibodies with cells. Results: Here, we report the successful generation of three monoclonal antibodies to the N-terminal extracellular domain of PAR4: 14H6, 5 F10, and 2D6. We mapped the epitope on PAR4 of 14H6, 5 F10, and 2D6 antibodies to residues (48-53), (41-47), and (73-78), respectively. Two of the antibodies (14H6 and 5 F10) interacted close to the thrombin cleavage and were sensitive to alpha-thrombin cleavage of PAR4. In addition, 5 F10 was able to partially inhibit the cleavage of PAR4 expressed in HEK293 cells by alpha-thrombin. Conclusions: These new antibodies provide a means to monitor endogenous PAR4 expression and activation by proteases on cells. (C) 2015 Elsevier Ltd. All rights reserved.