Release of the thrombin receptor (PAR-1) N-terminus from the surface of human platelets activated by thrombin.
Platelet activation by thrombin is at least partially mediated by a G-protein-coupled receptor whose extended N-terminus is cleaved by thrombin. Theoretically, this should release a small fragment containing the original receptor N-terminus. However, the fate of this fragment is unknown, as is its biological role, if any. To begin to examine these issues, we have prepared monoclonal anti-receptor antibodies whose epitopes lie entirely N-terminal to the thrombin cleavage site. By flow cytometry and fluorescence microscopy of human platelets and megakaryoblastic CHRF-288 cells, these antibodies were found to recognize intact receptors and receptors activated by the agonist peptide, SFLLRN, but not receptors whose N-terminus had been cleaved by thrombin or cathepsin G. Incubating CHRF-288 cells with thrombin released pre-bound antibody from the cell surface. An assay based upon the antibodies was able to detect a fragment containing the original receptor N-terminus in the supernate of thrombin-treated human platelets. The concentration of the fragment obtained with platelets from 15 normal donors was 4.8 +/- 0.9 pmol per 10(9) platelets (mean +/- sem), which is similar to the value expected if all of the thrombin receptors present on human platelets have been cleaved. Taken together, these results demonstrate that 1) following receptor cleavage a fragment containing the original N-terminus of the receptor is released from the platelet surface, 2) based upon epitope mapping, this fragment is at least 15-20 residues long, 3) it is possible to quantitate the receptor fragment in the supernates of cells exposed to thrombin, and 4) the results of the quantitation suggest that on platelets all of the receptors have been cleaved and 100% of the fragment is present in the cell supernate. Depending on it survival time, measurements of the receptor fragment in blood or urine samples may eventually prove to be a useful marker for thrombin receptor activation in vivo.
Ramachandran
R
R
Department of Medicine of the University of Pennsylvania, Philadelphia, 19104, USA.
Klufas
A S
AS
Molino
M
M
Ahuja
M
M
Hoxie
J A
JA
Brass
L F
LF
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015539:Platelet Activation; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000737:chemistry / Q000378:metabolism*; D013499:Surface Properties; D013917:Thrombin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013917:Thrombin