Mapping the interaction of bradykinin 1–5 with the exodomain of human protease activated receptor 4

Abstract

The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1-5 (RPPGF), inhibits thrombininduced human or mouse platelet aggregation. RPPGF binds to the exodomain of human protease-activated receptor 1 (PAR1). Studies determined if RPPGF also binds to the exodomain of human PAR4. RPPGF binds to a peptide of the thrombin cleavage site on PAR4. Recombinant wild-type and mutated exodomain of human PAR4 was prepared. The N-terminal arginine on RPPGF binds to the P2 position or proline 46 on PAR4 to block thrombin cleavage. These data indicate that RPPGF influences thrombin activity by binding to the thrombin cleavage site on both PAR4 and PAR1.

Bradykinin B2 receptor knockout mice are protected from thrombosis by increased nitric oxide and prostacyclin

“…18 Further, RPPGF binds to the P4-P2 sites on protease activated receptor 1 and the P2 site on protease activated receptor 4 to prevent ␣-thrombin cleavage of these receptors. 33,36 Although these investigations indicate for the first time that elevation of RPPGF occurs in vivo in thromboprotected mice, our present studies do not show causality. Measurement of plasma levels of RPPGF may not reflect its site of action on the membranes of platelets and endothelium.…”

Mapping Human Protease-activated Receptor 4 (PAR4) Homodimer Interface to Transmembrane Helix 4

“…HEK293 cells in 6-well plates were transfected with 0.5 g of plasmid and were removed from plates 48 h post-transfection by rinsing with PBS. The cells were incubated with antibodies to PAR4 (32) or rhodopsin (monoclonal antibody B6 -30N (36), kindly provided by Dr. Krzysztof Palczewski (Case Western Reserve University)) followed by incubation with species specific antibodies conjugated to Alexa Fluor 647 (Invitrogen) to detect surface expression distinct from GFP expression. Mean fluorescence was determined by counting 10,000 cells on an LSRII (BD Biosciences) in the Case Comprehensive Cancer Center flow cytometry core.…”

“…The vectors pLUC-N2 and pGFP-N2 were purchased from PerkinElmer Life Sciences. The rhodopsin/PAR4 chimeras were generated using overlapping PCR as previously described (32). A detailed list of primers is available by request.…”

Bradykinin and Its Metabolite Bradykinin 1-5 Inhibit Thrombin-Induced Platelet Aggregation in Humans

“…The lack of an effect of BK1-5 on TRAP 1-6-induced platelet aggregation is consistent with the data of Hasan et al (2003), indicating that BK1-5 inhibits thrombin-induced platelet activation in vitro by preventing the cleavage of the proteaseactivated receptor (PAR-1) at its cleavage site, arginine 41, thereby preventing the exposure of the PAR-1 autoactivating tethered ligand. In addition, BK1-5 may inhibit thrombin by binding to the PAR-4 thrombin receptor (Nieman et al, 2005).…”