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.
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.
Norton
K J
KJ
Division of Hematology, State University of New York at Stony Brook 11794-8151.
Scarborough
R M
RM
Kutok
J L
JL
Escobedo
M A
MA
Nannizzi
L
L
Coller
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011817:Rabbits / Q000276:immunology; D018179:Receptors, Thrombin / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D010455:Peptides; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D013917:Thrombin