Plasmin-mediated Activation of Platelets Occurs by Cleavage of Protease-activated Receptor 4

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4. The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4. Platelet activation performs a significant function in hemostasis and thrombosis. Platelets mediate hemostasis by amplifying an initial stimulus and aggregating at a site of injury. The stimulus can range from the exposure of subendothelial proteins, as in the case of tissue injury, to the development of turbulent blood flow through a narrowed blood vessel as in atherosclerosis. In the treatment of stroke or deep vein thrombus formation, the systemic administration of thrombolytic drugs, such as tissue plasminogen activator or streptokinase, has been the most successful and widely used (1Schweizer J. Kirch W. Koch R. Elix H. Hellner G. Forkmann L. Graf A. J. Am. Coll. Cardiol. 2000; 36: 1336-1343Google Scholar). Thrombolytic drugs work by catalyzing the activation of plasmin (an enzyme that degrades fibrin clots) from its inactive circulating precursor plasminogen from plasma. An interesting paradox is that plasmin also has the ability to directly activate platelets (2Niewiarowski S. Senyi A.F. Gillies P. J. Clin. Investig. 1973; 52: 1647-1659Google Scholar, 3Ishii-Watabe A. Uchida E. Mizuguchi H. Hayakawa T. Biochem. Pharmacol. 2000; 59: 1345-1355Google Scholar) and potentially cause additional thrombus formation. An important agonist for platelet activation is thrombin, which is generated at sites of vascular injury by extrinsic and intrinsic coagulation cascades. Thrombin induces its platelet-activating effects mainly through a family of G protein-coupled protease-activated receptors (PARs) 1The abbreviation used is: PAR, protease-activated receptor. 1The abbreviation used is: PAR, protease-activated receptor.; these receptors are activated by a mechanism in which a protease creates a new amino terminus that functions as its own tethered ligand and thus results in intramolecular activation (4Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Google Scholar, 5Hollenberg M.D. Trends Pharmacol. Sci. 1999; 20: 271-273Google Scholar). Of the four known PARs, three (PAR-1, PAR-3 and PAR-4) are activated by thrombin. PAR-1 is present in human platelets and plays a chief role in platelet activation because of its high affinity for thrombin, but it is absent in mouse platelets (6Kahn M.L. Hammes S.R. Botka C. Coughlin S.R. J. Biol. Chem. 1998; 273: 23290-23296Google Scholar). PAR-2 functions as a receptor for the protease trypsin but not for thrombin (7Nystedt S. Emilsson K. Wahlestedt C. Sundelin J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9208-9212Google Scholar). PAR-4 is present in both human and mouse platelets, and it has a low affinity for thrombin (6Kahn M.L. Hammes S.R. Botka C. Coughlin S.R. J. Biol. Chem. 1998; 273: 23290-23296Google Scholar, 8Xu W.F. Andersen H. Whitmore T.E. Presnell S.R. Yee D.P. Ching A. Gilbert T. Davie E.W. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6642-6646Google Scholar). PAR-3 is present on mouse platelets and serves as a cofactor for PAR-4 activation at low thrombin concentrations but is absent from human platelets (9Ishihara H. Connolly A.J. Zeng D. Kahn M.L. Zheng Y.W. Timmons C. Tram T. Coughlin S.R. Nature. 1997; 386: 502-506Google Scholar, 10Nakanishi-Matsui M. Zheng Y.W. Sulciner D.J. Weiss E.J. Ludeman M.J. Coughlin S.R. Nature. 2000; 404: 609-613Google Scholar). Thus, PAR-1 and PAR-4 mediate thrombin activation of human platelets, whereas PAR-3 and PAR-4 perform the same function in mouse platelets. PARs can be activated without proteolysis by the use of specific agonist peptides that mimic the tethered ligand regions of the respective receptors. The hexapeptide SFLLRN specifically activates PAR-1 in human platelets to cause aggregation (11Hung D.T. Wong Y.H. Vu T.K. Coughlin S.R. J. Biol. Chem. 1992; 267: 20831-20834Google Scholar, 12Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Google Scholar), whereas the peptide AYPGKF activates PAR-4 in both human and mouse platelets (13Faruqi T.R. Weiss E.J. Shapiro M.J. Huang W. Coughlin S.R. J. Biol. Chem. 2000; 275: 19728-19734Google Scholar). Plasmin, as a protease, has an amino acid specificity similar to thrombin in that it cleaves its substrates at lysine and arginine residues. Because plasmin and thrombin are both plasma proteases and thrombin interacts with PAR-1 and PAR-4 in human platelets, the possibility was presented that plasmin might also activate platelets by interacting with PAR-1 and/or PAR-4. Because it is not known which receptor mediates platelet activation by plasmin, it should not be surprising that little is known about the intracellular signaling pathways directly downstream of receptor activation. Platelet degranulation and secretion seem to play a large part in plasmin-mediated aggregation, particularly the potentiation of the effects of plasmin by ADP and epinephrine (3Ishii-Watabe A. Uchida E. Mizuguchi H. Hayakawa T. Biochem. Pharmacol. 2000; 59: 1345-1355Google Scholar). Some investigators have shown that relatively lengthy incubations of washed platelets with low doses of plasmin can inhibit thrombin-mediated aggregation (14Schafer A.I. Zavoico G.B. Loscalzo J. Maas A.K. Blood. 1987; 69: 1504-1507Google Scholar), whereas others have demonstrated that prolonged incubation with low plasmin concentrations stimulates significant platelet aggregation (15Ervin A.L. Peerschke E.I. Blood Coagul. Fibrinolysis. 2001; 12: 415-425Google Scholar). In this study, we report that plasmin is capable of inducing platelet activation through cleavage of protease-activated receptor 4. Reagents—Plasminogen, streptokinase, and the chromogenic plasmin substrate S-2403 were purchased from DiaPharma (West Chester, OH). The PAR-1 antagonist BMS-200261 was obtained as a generous gift from Dr. Steven Seiler (Bristol-Myers Squibb). The PAR-1 activating peptide agonist SFLLRN, PAR-4 activating peptide agonist AYPGKF, and PAR-4 antagonist transcinnamoyl-YPGKF were synthesized by ResGen (Huntsville, AL). The thromboxane A2 analog 15(S)-hydroxy-9,11-epoxymethanoprosta-5Z,13E-dienoic acid (U44419) was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Wild-type 129P3/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). PAR-4 homozygous null mice have been previously characterized (16Sambrano G.R. Weiss E.J. Zheng Y.W. Huang W. Coughlin S.R. Nature. 2001; 413: 74-78Google Scholar) and were kindly provided by Dr. Shaun Coughlin (Cardiovascular Research Institute, University of California, San Francisco, CA). COS-7 cells were purchased from the American Type Culture Collection (Manassas, VA). SC-57101 was obtained from Searle Research and Development (Skokie, IL). Fura-2 was purchased from Molecular Probes (Eugene, OR). PCR amplification kit was purchased from Promega (Madison, WI). QuikChange site-directed mutagenesis kit was purchased from Stratagene (Cedar Creek, TX). Unless specifically mentioned, all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Platelet Isolation—Whole human blood was drawn from informed, healthy volunteers at the Sol Sherry Thrombosis Research Center of Temple University. Whole mouse blood was drawn from mice by the heart puncture method. Human and mouse platelets were treated with aspirin, suspended in platelet-rich plasma, then isolated and resuspended in Tyrode's buffer as described previously (17Paul B.Z. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Google Scholar). Plasmin Preparation—Plasmin was prepared by incubating 5 mg/ml plasminogen with 1.5 × 104 units/ml streptokinase for 3 min at 37 °C before addition as an agonist. The activities of plasmin preparations were assayed before each experiment; one plasmin activity unit was defined as the cleavage of 1 μmol of S-2403 per min. Intracellular Calcium Release—For platelet measurements, PRP was incubated with 2 μm fura-2 or an equal amount of Me2SO (vehicle) and incubated simultaneously with acetylsalicylic acid. Platelets were then isolated and washed as described above. The integrin αIIbβ3 antagonist SC-57101 (10 μm) was added before each assay to prevent agonist-mediated platelet aggregation from interfering with fluorescence measurement. Changes in fluorescence were measured using an Aminco-Bowman Series 2 luminescence spectrometer with a water-jacketed cuvette holder, equipped with a thermostat, at 37 °C and set at constant stirring. Sample volumes of 0.5 ml were analyzed with an excitation wavelength of 340 nm and an emission wavelength of 510 nm. Fluorescence measurements were converted to calcium concentrations using the equation reported by Grynkiewicz et al. (18Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar), where Fmin and Fmax were determined with each respective platelet preparation. Platelet Aggregation—Agonist-induced platelet aggregation was analyzed using a Chrono-Log model 440-VS aggregometer (Havertown, PA) with sample volumes of 0.5 ml in a cuvette holder, equipped with a thermostat, at 37 °C and set at constant stirring. Aggregometer output was recorded using a Kipp & Zonen type BD 12E flatbed chart recorder (SCI-TEC, Saskatoon, Canada) set at 0.2 mm/s. Platelet Desensitization—Platelet desensitization was performed according to the method of Dubois et al. (19Dubois C. Steiner B. Kieffer N. Meyer Reigner S.C. Thromb. Haemost. 2003; 89: 853-865Google Scholar), where washed platelets were incubated for 45 min at 37 °C without stirring in the absence or presence of either 50 μm SFLLRN, 500 μm AYPGKF, or 1 unit/ml plasmin. Immediately after incubation, platelets were assayed for their responses to various agonists. Construction of Wild-type and Mutant PAR-4 Expression Plasmids— PCR amplification of the wild-type PAR-4 coding sequence was carried out using forward and reverse primers specific for human PAR-4 cDNA (GenBank accession no. AF055917) (8Xu W.F. Andersen H. Whitmore T.E. Presnell S.R. Yee D.P. Ching A. Gilbert T. Davie E.W. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6642-6646Google Scholar). A sequence encoding a hemagglutinin epitope tag was inserted at the beginning of the translation initiation. The sense primer containing a HindIII restriction site and the hemagglutinin tag sequence is 5′-CGCGAAGCTTACCATGTACCCATACGATGTTCCAGATTACGCTTGGGGGCGACTGCTCCTGT-3′, and the antisense primer containing a XhoI restriction site is 5′-CGCTCGAGTCACTGGAGCAAAGAGGAGT-3′. The restriction enzyme sites are underlined, and the coding sequence of the hemagglutinin epitope is given in bold letters. After initial denaturation for 5 min at 95 °C, the PCR amplifications were carried out for 35 cycles using a 2× PCR mixture as follows: denaturation at 95 °C for 45 s, annealing at 55 °C for 45 s, and extension at 72 °C for 1 min. The final cycle was followed by an additional extension for 10 min at 72 °C. An expression plasmid (pcDNA3/PAR4) was constructed in the pcDNA3-Hygro (+) vector by digesting the reverse transcription-PCR product with Hind-IIII and XhoI and inserting into the vector digested with the same set of restriction enzymes. The nucleotide sequence of the wild-type PAR-4 cDNA in the expression plasmid was confirmed by DNA sequence analysis. For the mutant PAR-4, a site-directed mutation at arginine-47 (R47A) was introduced into pcDNA3/PAR4 using the QuikChange site-directed mutagenesis kit from Stratagene (Cedar Creek, TX) with changes in oligonucleotides from CGC to GCC. The sense primer sequence is 5′-GCCTGCCCCCGCCGGCTACCCAGGC-3′ and antisense primer is 5′-GCCTGGGTAGCCGGCGGGGGCAGGC-3′; the mutation was confirmed by DNA sequencing. Transient transfection of COS-7 cells was accomplished using the LipofectAMINE 2000 transfection reagent (Invitrogen). Cross-desensitization of PARs by Plasmin—Fig. 1 shows a typical human platelet aggregation tracing in response to 1 unit/ml plasmin, which can be totally by plasmin with the protease should be that plasminogen its activator streptokinase are to platelet aggregation on its own the of receptors with which plasmin we the protease-activated receptors present on human platelets, PAR-1 and PAR-4. PARs use G in their signaling PAR signaling to the of desensitization a in the ability of a receptor to to further agonist Thus, in the of whether plasmin cleaves PAR-1 or PAR-4, we the intracellular calcium profile generated by plasmin in platelets by that plasmin either PAR, then it desensitize calcium to further agonist stimulation of that plasmin as an we a slow in intracellular calcium to followed by an slow and these results are with has been previously reported for plasmin-mediated intracellular calcium K. M. Andersen A. Am. J. Scholar, S.R. Biochem. J. 1994; Scholar). SFLLRN was added to plasmin the of intracellular calcium to a as with SFLLRN and a similar was AYPGKF was the and PAR-1 or PAR-4 after incubation intracellular calcium stimulation with the PAR-1 agonist SFLLRN μm) added or min after the addition of 1 unit/ml plasmin whereas the PAR-4 agonist AYPGKF μm) was added or min after the addition of plasmin washed human platelets were incubated at 37 °C for 45 min in the absence or presence of 1 unit/ml plasmin, followed by stimulation with 1 unit/ml thrombin to aggregation or intracellular calcium are of at three Platelet aggregation by plasmin showed a and of and a of aggregation compared with thrombin. plasmin proteolysis at a slow to thrombin, then it a relatively incubation failed to desensitize further PAR in an to a of platelets were incubated with plasmin for a prolonged using a (19Dubois C. Steiner B. Kieffer N. Meyer Reigner S.C. Thromb. Haemost. 2003; 89: 853-865Google Scholar). further stimulation with 1 unit/ml thrombin, a of both aggregation and calcium to treatment of PARs in Platelet an to we to whether desensitization of either PAR with the respective activating peptide have an effect on platelet aggregation and calcium induced by plasmin. 3 shows that in comparison with platelets, desensitization of PAR-1 with SFLLRN has no effect on plasmin-mediated however, PAR-4 is desensitized with AYPGKF, aggregation induced by plasmin is completely directly whether was a to activation of either PAR-1 or PAR-4 by plasmin, we the effect of plasmin on platelet aggregation in where PAR-1 was either inhibited by treatment of human platelets with the PAR-1-specific antagonist BMS-200261 M. Seiler J. Chem. Scholar) or absent as with mouse platelets. human platelets, thrombin-mediated platelet aggregation in the presence of BMS-200261 was in to platelet but plasmin-mediated aggregation without Plasmin stimulation of mouse platelets was also performed because mouse platelets PAR-3 and PAR-4 but not results that mouse platelets aggregate in response to plasmin but not SFLLRN PAR-4 Platelet by the of PAR-4, we the use of a PAR-4 antagonist that was reported in the of activation of platelets M.D. M. J. Pharmacol. 2001; Scholar). of human platelets, a of aggregation response concentrations of AYPGKF showed a to the in the presence of to the absence of the In response to plasmin, the presence of also inhibited platelet aggregation without should be that plasmin activity in the absence and presence of that the antagonist not inhibit plasmin activity directly not further PAR-4 as the receptor in plasmin-mediated platelet aggregation, we performed aggregation with mouse platelets in which a absence of PAR-4 Platelets from mice were treated with at the same that was to inhibit plasmin-mediated aggregation in human platelets. The mouse platelets in the same as human aggregation was inhibited but In we obtained mice and their response to plasmin. results that mouse platelets not or response to plasmin but demonstrated aggregation in response to a such as the thromboxane A2 Plasmin PAR-4 at the Thrombin an to that plasmin cleaves PAR-4 at its tethered ligand an expression plasmid for PAR-4 was into COS-7 cells in which the cleavage was or was to an (R47A) to to with either thrombin or plasmin, cells the wild-type PAR-4 showed an of intracellular calcium a in the of calcium by the proteases was and was that the thrombin-mediated calcium was that by plasmin In the PAR-4 showed no response to either thrombin or plasmin, that plasmin cleaves PAR-4 at the same as thrombin should be that PAR-4 signaling was in both wild-type and of the receptor by that the PAR-4 agonist peptide AYPGKF was capable of activating PAR-4 in the cells Thrombolytic drugs that the of plasmin can cause the of plasmin to to 1 unit/ml of blood B. H. S. Loscalzo J. Blood. 1994; Scholar), and the ability of plasmin to activate platelets because treatment with the protease totally platelet and aggregation Furthermore, the of a platelet response with plasmin out the of plasmin and a platelet to an In this study, we that plasmin activates platelets via PAR-4 using multiple complementary approaches. is given that human platelets both PAR-1 and PAR-4 on their and that plasmin-mediated platelet aggregation desensitization of PAR-1 and in the presence of a PAR-1 antagonist In plasmin was to cause aggregation in PAR-4 desensitized platelets platelets or platelets from mice and plasmin-mediated intracellular signaling where PAR-4 had been to protease cleavage has been reported that plasmin interacts with PAR-1 on its activity a peptide to the thrombin cleavage site T. J. S.R. Biochem. J. Scholar), with an activity that is thrombin by about et al. A. L. J. 1999; Scholar) showed with calcium that plasmin has a low affinity for the thrombin cleavage site on PAR-1 and a affinity for a cleavage site that is further in plasmin is to PAR-1 refractory to further thrombin stimulation to calcium In platelets with plasmin after a failed to desensitize calcium to further with SFLLRN A. L. J. 1999; Scholar), and these were in not Furthermore, results the of the ability of plasmin to inhibit and calcium in platelets K. M. Andersen A. Am. J. Scholar, S.R. Biochem. J. 1994; Scholar). The of investigators to plasmin-mediated aggregation most the that prolonged plasmin activity to desensitization of PARs and in the of plasmin to initially desensitize PAR-4 we three that plasmin cleaves a of the of PAR-4 on the platelet and thus a of receptors to with the that plasmin cleaves PAR-4 at a site other the thrombin cleavage site and results in G or that plasmin cleaves PAR-4 at the thrombin cleavage site at a thrombin. the a of PAR-4 were activated by plasmin, then a prolonged platelet exposure to plasmin should not from was thrombin-mediated aggregation to after plasmin however, results that prolonged plasmin activity platelets refractory to additional thrombin-mediated aggregation and calcium Plasmin activation of human platelets can be by treatment with thrombin and receptor A. M. N. K. N. T. T. Hayakawa T. Pharmacol. 1997; 96: Scholar), the similarity of the that the PAR-4 antagonist which was to with the PAR-4 tethered with the ability of plasmin to mediate platelet aggregation this that plasmin the same tethered ligand on PAR-4 as thrombin. was a mutation of the thrombin cleavage site on PAR-4 its activation by plasmin as well as by thrombin to the possibility because plasmin PAR-4 in the same as thrombin to a similar of G should be Furthermore, a in the of intracellular calcium was with plasmin to thrombin in the cells Because intracellular calcium is a signaling to the cleavage of PAR-4, it a of the at which PAR-4 is activated by the with the of a lengthy of exposure to plasmin to effect desensitization of PAR-4 as well as an function and relatively slow of aggregation to thrombin, we that plasmin cleaves PAR-4 at a compared with thrombin. In plasmin mediates platelet aggregation through cleavage of protease-activated receptor that to integrin αIIbβ3 activation. In the absence of PAR-1 has no effect on plasmin-mediated platelet aggregation, whereas a absence of PAR-4 this We Steven Seiler and Shaun Coughlin for their of the BMS-200261 and the PAR-4 null mice, We also Dr. Daniel for of the

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