Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus.
Based upon its recently cloned nucleotide sequence, the human platelet thrombin receptor is thought to be formed by a single polypeptide chain with seven transmembrane domains and an extracellular N terminus that can be cleaved by thrombin. As yet, however, little is known from studies of the receptor protein itself. To obtain such information, we have prepared monoclonal antibodies against a peptide corresponding to receptor residues Ser42 through Phe55, the domain immediately distal to the site of cleavage by thrombin. By flow cytometry, all of the antibodies reacted with the thrombin-responsive megakaryoblastic CHRF-288 and HEL cell lines, but not with the T-lymphoid Sup-T1 cell line. Functionally, the antibodies inhibited platelet responses to alpha-thrombin, gamma-thrombin, and trypsin, but had no effect on platelet activation by ADP, epinephrine, or the thromboxane analog U46619. Radioiodinated antibody bound to approximately 1,800 sites/platelet, a value similar to the reported number of moderate affinity thrombin binding sites per platelet. On Western blots, the antibodies recognized a 66-kDa protein in platelet, HEL, and CHRF-288 membranes. The discrepancy between this apparent size and the predicted mass of the receptor suggests that, as with other G protein-coupled receptors, one or more of the potential sites for N-linked glycosylation have been utilized. Therefore, these results suggest that: 1) the cloned thrombin receptor is involved in a broad range of platelet responses to thrombin, as well as gamma-thrombin and trypsin; 2) as predicted, the N terminus of the receptor is accessible on the platelet surface; 3) the moderate affinity thrombin binding site noted in earlier studies may be the receptor; 4) potentially as much as one third of the mass of the receptor is carbohydrate.
Brass
L F
LF
Department of Medicine, University of Pennsylvania, Philadelphia 19104.
Vassallo
R R
RR
Belmonte
E
E
Ahuja
M
M
Cichowski
K
K
Hoxie
J A
JA
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D004837:Epinephrine / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D015539:Platelet Activation / Q000187:drug effects; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*; D014357:Trypsin / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D010455:Peptides; D011450:Prostaglandin Endoperoxides, Synthetic; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D014662:Vasoconstrictor Agents; D000244:Adenosine Diphosphate; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D014357:Trypsin; D013917:Thrombin; D004837:Epinephrine