Release of the Thrombin Receptor (PAR-1) N-terminus from the Surface of Human Platelets Activated by Thrombin

Summary

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 mega- karyoblastic 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 supemate of thrombin-treated human plate lets. The concentration of the fragment obtained with platelets from 15 normal donors was 4.8 ± 0.9 pmol per 10⁹ 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 supemates 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 supemate. 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.

• References

• 1 Vu T-KH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-1068
• 2 Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T, Coughlin SR. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 1997; 386: 502-508
• 3 Connolly AJ, Ishihara H, Kahn ML, Farese Jr VR, Coughlin SR. Role of the thrombin receptor In development and evidence for a second receptor. Nature 1996; 381: 516-519
• 4 Darrow AD, Fung-Leung W-P, Ye RD, Santulli RJ, Cheung W-I, Derian CK, Bums CL, Damiano DP, Zhou L, Keenan CM, Peterson PA, Andrade-Gordon P. Biological consequences of thrombin receptor deficiency in mice. Thromb Haemost 1996; 76: 860-866
• 5 Nystedt S, Emilsson K, Wahlestedt C, Sundelin J. Molecular Cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 1994; 91: 9208-9212
• 6 Hwa JJ, Ghibaudi L, Williams P, Chintala M, Zhang RM, Chatteijee M, Sybertz E. Evidence for the presence of a proteinase-activated receptor distinct from the thrombin receptor in vascular endothelial cells. Circ Res 1996; 78: 581-588
• 7 Blackhart BD, Emilsson K, Nguyen D, Teng W, Martelli AJ, Nystedt S, Sundelin J, Scarborough RM. Ligand cross-reactivity within the protease- activated receptor family. J Biol Chem 1996; 271: 16466-16471
• 8 Gerszten RE, Chen J, Ishii M, Ishii K, Wang L, Nanevicz T, Turck CW, Vu T-KH, Coughlin SR. Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 1994; 368: 648-651
• 9 Nanevicz T, Wang L, Chen M, Ishii M, Coughlin SR. Thrombin receptor activating mutations – Alteration of an extracellular agonist recognition domain causes constitutive signaling. J Biol Chem 1996; 271: 702-706
• 10 Furman MI, Liu L, Benoit SE, Becker RC, Barnard RC, Michelson AD. The cleaved peptide of the thrombin receptor is a strong platelet agonist. Circulation 1996; (Suppl. 094) 01: 1-580 (abstr).
• 11 Hoxie JA, Ahuja M, Belmonte E, Pizarro S, Parton RG, Brass LF. Internal ization and recycling of activated thrombin receptors. J Biol Chem 1993; 268: 13756-13763
• 12 Brass LF, Pizarro S, Ahuja M, Belmonte E, Stadel J, Hoxie JA. Changes in the structure and function of the human thrombin receptor during activa tion, internalization and recycling. J Biol Chem 1994; 269: 2943-2952
• 13 Brass LF, Vassallo Jr RR, Belmonte E, Ahuja M, Cichowski K, Hoxie JA. Structure and function of the human platelet thrombin receptor: studies using monoclonal antibodies against a defined epitope within the receptor N-terminus. J Biol Chem 1992; 267: 13795-13798
• 14 Molino M, Bainton DF, Hoxie JA, Coughlin SR, Brass LF. Thrombin receptors on human platelets: initial localization and subsequent redistribution during platelet activation. J Biol Chem 1997; 272: 6011-6017
• 15 Molino M, Blanchard N, Belmonte E, Tarver AP, Abrams C, Hoxie JA, Cerletti C, Brass LF. Proteolysis of the human platelet and endothelial cell thrombin receptor by neutrophil-derived cathepsin G. J Biol Chem 1995; 270: 11168-11175
• 16 Harmon JT, Jamieson GA. Activation of platelets by a-thrombin is a recep tor-mediated event. J Biol Chem 1986; 261: 15928-15933
• 17 Norton KJ, Scarborough RM, Kutok JL, Escobedo M-A, Nannizzi L, Coller BS. 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. Blood 1993; 82: 2125-2136
• 18 Mirza H, Yatsula V, Bahou WF. The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells: molecular characterization and evidence for functional coupling to the thrombin receptor. J Clin Invest 1996; 97: 1705-1714
• 19 Molino M, Bamathan ES, Numerof R, Clark J, Dreyer M, Cumashi A, Hoxie J, Schechter N, Woolkalis MJ, Brass LF. Interactions of mast cell tryptase with thrombin receptors and PAR-2. J Biol Chem 1997; 272: 4043-4049
• 20 Molino M, Woolkalis MJ, Reavey-Cantwell J, Pratico D, Andrade-Gordon P, Bamathan ES, Brass LF. Endothelial cell thrombin receptors and PAR-2: two protease-activated receptors located in a single cellular environment. J Biol Chem 1997; 272: 11133-11141