Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Abstract

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

Thrombin binding to platelets and their activation in plasma

“…Our results demonstrate the cloned thrombin receptor to be an important site to which athrombin binds platelets suspended in plasma. Whereas it was not directly measured, our results are consistent with athrombin-mediated activation of platelets in PRP involving the cleavage of the cloned thrombin receptor (Norton et al 1993). However, a GPIb-dependent pathway for thrombininduced platelet activation (Harmon & Jamieson, 1988: Yamamoto et al, 1991 was not ruled out by this study.…”

“…Platelet activation by a-thrombin involving this receptor requires the cleavage of a scissile bond within the receptor, a reaction probably facilitated by the interactions of the fibrin(ogen) recognition exosite of a-thrombin with the hirudin tail-like domain of the thrombin receptor (Vu et al, 1991b: Hung et al, 1991: Brass et al, 1992. Antibodies to parts of the hirudin-like domain and to portions of the activation peptide cleaved from the receptor by a-thrombin inhibit the aggregation of washed platelets induced by low concentrations (,< 1.0 m) of a-thrombin (Brass et al, 1992: Norton et al, 1993. ATAP 138, a monoclonal antibody to part of the hirudin-like domain of the cloned thrombin receptor, also abrogated for 60 s the binding of up to 10 n M a-thrombin to platelets suspended in plasma.…”

“…The 6rst 2 20 residues of the cleaved receptor have elements structurally similar to the carboxyl tail of hirudin, and therefore have the potential to bind thrombin (Hung et d, 1991;Liu et d, 1991). Antisera to the hirudm-like domain and to residues encompassing the thrombin cleavage site of the receptor inhibit the a-thrombin induced aggregation of washed platelets (Brass et al, 1992;Norton et al, 1993).…”

Agonists of proteinase‐activated receptor 1 induce plasma extravasation by a neurogenic mechanism

“…Sections and cultures were incubated with primary antibodies in PBS with 2% normal goat serum and detergent for 24 ± 48 h at 48C. The following primary antibodies were used: mouse PAR1 antibody 61-1 (raised to residues 29 ± 43 human PAR1; 1 : 100 ± 200) (Norton et al, 1993); rabbit PAR1 antibody 4274 (raised to the residues 1 ± 42 of human PAR1; 1 : 100); mouse antibody to the neuronal marker PGP9.5 (Accurate Chemicals, Westbury, NY, U.S.A., 1 : 100); mouse antibody to calcitonin gene-related peptide (CGRP; Dr J.H. Walsh, UCLA, 1 : 20,000).…”

Platelet Membrane Receptor Proteolysis: Implications for Platelet Function

“…In human platelets, α-thrombin cleaves the N-terminal extracellular site of isoforms PAR1 and PAR4 ( 24 , 25 ). The proteolysis liberates a new N-terminus, which autocatalytically activates the receptor molecule ( 26 , 27 ). By ensuing signaling via G-proteins (in particular Gqα) ( 24 , 28 ), thrombin induces a wide range of platelet responses, including shape change, Ca 2+ fluxes, integrin activation, and secretion ( 23 ).…”