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• W2000263382 abstract "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 blocked by inhibiting plasmin with the protease inhibitor phenylmethylsulfonyl fluoride. It should be noted that neither plasminogen nor its activator streptokinase are able to stimulate platelet aggregation on its own (Fig. 1). To begin investigating the identity of receptors with which plasmin may interact, we considered the protease-activated receptors present on human platelets, PAR-1 and PAR-4. PARs use G proteins in their signaling pathways, rendering PAR signaling subject to the phenomenon of desensitization (i.e. a decrease in the ability of a receptor to respond to further agonist stimulation). Thus, in approaching the question of whether plasmin cleaves PAR-1 or PAR-4, we examined the intracellular calcium release profile generated by plasmin in platelets by reasoning that if plasmin cleaved either PAR, then it may desensitize calcium release to further agonist stimulation of that PAR. Using plasmin as an agonist, we observed a slow net increase in intracellular calcium to ∼100 nm, followed by an equally slow decrease over time (Fig. 2A, ii and iv, first peak); these results are consistent with what has been previously reported for plasmin-mediated intracellular calcium release (20Nakamura K. Kimura M. Fenton 2nd, J.W. Andersen T.T. Aviv A. Am. J. Physiol. 1995; 268: C958-C967Google Scholar, 21Turner J.S. Redpath G.T. Humphries J.E. Gonias S.L. Vandenberg S.R. Biochem. J. 1994; 297: 175-179Google Scholar). When SFLLRN was added subsequent to plasmin addition, the level of intracellular calcium increased to a comparable extent as seen with SFLLRN alone (compare Fig. 2A, i and ii); a similar situation was observed when AYPGKF was the secondary stimulator (Fig. 2A, iii and iv).Fig. 2Plasmin desensitizes PAR-1 or PAR-4 after long incubation periods. A, intracellular calcium release tracings upon stimulation with the PAR-1 agonist SFLLRN (50 μm) added alone (i) or within 4 min after the addition of 1 unit/ml plasmin (ii), whereas the PAR-4 agonist AYPGKF (500 μm) was added alone (iii) or within 4 min after the addition of plasmin (iv). B, washed human platelets were incubated at 37 °C for 45 min in the absence (control) or presence of 1 unit/ml plasmin, followed by stimulation with 1 unit/ml thrombin (arrows) to initiate aggregation (top) or intracellular calcium mobilization (bottom). Tracings are representative of at least three separate experiments.View Large Image Figure ViewerDownload (PPT) Platelet aggregation mediated by plasmin showed a greater extent and duration of shape change and a slower rate of aggregation onset compared with thrombin. If plasmin proteolysis proceeded at a slow rate relative to thrombin, then it could explain why a relatively short incubation time failed to desensitize further PAR stimulation. Therefore, in an effort to facilitate a greater extent of desensitization, platelets were incubated with plasmin for a prolonged period using a recently published protocol (19Dubois C. Steiner B. Kieffer N. Meyer Reigner S.C. Thromb. Haemost. 2003; 89: 853-865Google Scholar). Upon further stimulation with 1 unit/ml thrombin, a complete loss of both aggregation and calcium release occurred relative to control treatment (Fig. 2B). Role of PARs in Plasmin-induced Platelet Aggregation—As an alternate approach to our hypothesis, we proceeded to determine whether desensitization of either PAR with the respective activating peptide would have an effect on platelet aggregation and calcium release induced by plasmin. Fig. 3 shows that in comparison with nondesensitized platelets, desensitization of PAR-1 with SFLLRN has no real effect on plasmin-mediated aggregation; however, when PAR-4 is desensitized with AYPGKF, aggregation induced by plasmin is completely eliminated. To more directly ascertain whether there was a functional consequence to activation of either PAR-1 or PAR-4 by plasmin, we examined the effect of plasmin on platelet aggregation in conditions where PAR-1 was either inhibited by treatment of human platelets with the PAR-1-specific antagonist BMS-200261 (22Bernatowicz M.S. Klimas C.E. Hartl K.S. Peluso M. Allegretto N.J. Seiler S.M. J. Med. Chem. 1996; 39: 4879-4887Google Scholar) or absent as with mouse platelets. Using human platelets, thrombin-mediated platelet aggregation in the presence of BMS-200261 was abolished in reference to untreated platelet controls, but plasmin-mediated aggregation occurred without any apparent diminution (Fig. 4A). Plasmin stimulation of mouse platelets was also performed because mouse platelets contain PAR-3 and PAR-4 but not PAR-1. Our results show that mouse platelets aggregate in response to plasmin but not SFLLRN (Fig. 4B). PAR-4 Mediates Platelet Aggregation by Plasmin—To investigate the implicated involvement of PAR-4, we employed the use of a PAR-4 antagonist (tc-YPGKF) that was reported in the investigation of PAR-4-mediated activation of rat platelets (23Hollenberg M.D. Saifeddine M. Can. J. Physiol. Pharmacol. 2001; 79: 439-442Google Scholar). Upon testing of human platelets, a plot of aggregation response versus differing concentrations of AYPGKF showed a clear shift to the right in the presence of tc-YPGKF relative to the absence of the inhibitor (Fig. 5A). In response to plasmin, the presence of tc-YPGKF also inhibited platelet aggregation without inhibiting shape change (Fig. 5B). It should be noted that plasmin activity assays in the absence and presence of tc-YPGKF show that the antagonist itself does not inhibit plasmin activity directly (data not shown). To further identify PAR-4 as the receptor involved in plasmin-mediated platelet aggregation, we performed aggregation studies with mouse platelets under circumstances in which a functional absence of PAR-4 occurred. Platelets from mice were treated with tc-YPGKF at the same concentration that was found to inhibit plasmin-mediated aggregation in human platelets. The mouse platelets responded in the same manner as human platelets; i.e. aggregation was inhibited but shape change still occurred (Fig. 6A). In addition, we obtained PAR-4-null mice and tested their platelets' response to plasmin. Our results show that PAR-4-null mouse platelets did not display any measurable shape change or aggregatory response to plasmin but demonstrated aggregation in response to a non-PAR agonist, such as the thromboxane A2 mimetic U46619 (Fig. 6B). Plasmin Cleaves PAR-4 at the Thrombin Cleavage Site—In an effort to show conclusively that plasmin cleaves PAR-4 at its tethered ligand generation site, an expression plasmid for PAR-4 was transiently transfected into COS-7 cells in which the cleavage residue, arginine-47, was unchanged (wild-type) or was mutated to an alanine (R47A) to confer resistance to proteolytic cleavage. When challenged with either thrombin or plasmin, those cells expressing the wild-type PAR-4 showed an increased extent of intracellular calcium mobilization over basal levels (Fig. 7). Moreover, a difference in the rates of calcium release mediated by the two proteases was readily observed and was calculated that the thrombin-mediated calcium release was ∼10-fold higher than that mediated by plasmin (Fig. 7A). In contrast, the mutated PAR-4 showed no response to either thrombin or plasmin, indicating that plasmin cleaves PAR-4 at the same site, as does thrombin (i.e. R47). It should be noted that functional PAR-4 signaling was established in both wild-type and mutated forms of the receptor by showing that the PAR-4 agonist peptide AYPGKF was capable of activating PAR-4 in the respectively transfected cells (Fig. 7B). Thrombolytic drugs that catalyze the formation of active plasmin can cause the level of plasmin to reach up to 1 unit/ml of blood (24Pasche B. Ouimet H. Francis S. Loscalzo J. Blood. 1994; 83: 404-414Google Scholar), and the ability of plasmin to activate platelets involves proteolysis, because treatment with the protease inhibitor phenylmethylsulfonyl fluoride totally abolishes platelet shape change and aggregation (Fig. 1). Furthermore, the lack of a functional platelet response with inactivated plasmin rules out the idea of simple binding between plasmin and a platelet membrane protein to manifest an effect. In this study, we demonstrate that plasmin activates platelets via PAR-4 using multiple complementary approaches. It is given that human platelets express both PAR-1 and PAR-4 on their membrane surfaces and that our data show plasmin-mediated platelet aggregation continued during desensitization of PAR-1 (Fig. 3) and in the presence of a PAR-1 antagonist (BMS-200261). In addition, plasmin was unable to cause aggregation in PAR-4 desensitized platelets (Fig. 3), PAR-4-antagonized platelets (Fig. 5), or platelets from PAR-4-deficient mice (Fig. 6), and plasmin-mediated intracellular signaling ceased where PAR-4 had been rendered resistant to protease cleavage (Fig. 7). It has been reported that plasmin interacts with PAR-1 based on its activity toward a synthetic peptide designed to represent the thrombin cleavage site (25Parry M.A. Myles T. Tschopp J. Stone S.R. Biochem. J. 1996; 320: 335-341Google Scholar), albeit with an activity that is slower than thrombin by about 10-fold. Kuliopulos et al. (26Kuliopulos A. Covic L. Seeley S.K. Sheridan P.J. Helin J. Costello C.E. Biochemistry. 1999; 38: 4572-4585Google Scholar) showed with calcium mobilization studies that plasmin has a low affinity for the traditional thrombin cleavage site on PAR-1 and a higher affinity for a cleavage site that is further downstream; in effect, plasmin is more likely to make PAR-1 refractory to further thrombin stimulation than to stimulate calcium mobilization. In addition, platelets stimulated with plasmin after a short period failed to desensitize calcium mobilization to further simulation with SFLLRN (26Kuliopulos A. Covic L. Seeley S.K. Sheridan P.J. Helin J. Costello C.E. Biochemistry. 1999; 38: 4572-4585Google Scholar), and these observations were validated in our laboratory (data not shown). Furthermore, our results explain the contradiction of previous reports concerning the ability of plasmin to inhibit thrombin- and SFLLRN-induced calcium mobilization in platelets (20Nakamura K. Kimura M. Fenton 2nd, J.W. Andersen T.T. Aviv A. Am. J. Physiol. 1995; 268: C958-C967Google Scholar, 21Turner J.S. Redpath G.T. Humphries J.E. Gonias S.L. Vandenberg S.R. Biochem. J. 1994; 297: 175-179Google Scholar). The inability of past investigators to observe plasmin-mediated aggregation most likely reflects the fact that prolonged plasmin activity leads to desensitization of PARs and resulting in diminished responses. Concerning the inability of plasmin to initially desensitize PAR-4 (Fig. 2A), we considered three possibilities: 1) that plasmin cleaves only a finite subset of the total number of PAR-4 molecules on the platelet surface and thus leaves a majority of naive receptors to interact with the stronger agonists; 2) that plasmin cleaves PAR-4 at a site other than the thrombin cleavage site and results in aberrant G protein coupling; or 3) that plasmin cleaves PAR-4 at the thrombin cleavage site at a slower rate than thrombin. Considering the first possibility, if only a subset of PAR-4 molecules were being activated by plasmin, then a prolonged platelet exposure to plasmin should not show any difference from what was observed (i.e. thrombin-mediated aggregation would continue to occur after plasmin treatment); however, our results show that prolonged plasmin activity renders platelets refractory to additional thrombin-mediated aggregation and calcium mobilization (Fig. 2B). Plasmin activation of human platelets can be blocked by treatment with thrombin inactivators and anti-thrombin receptor antibodies (27Watabe A. Ohta M. Matsuyama N. Mizuno K. el Borai N. Tanimoto T. Kawanishi T. Hayakawa T. Res. Commun. Mol. Pathol. Pharmacol. 1997; 96: 341-352Google Scholar), reflecting the general similarity of action between the two proteases. Also, our data show that the PAR-4 antagonist tc-YPGKF, which was designed to compete with the PAR-4 tethered ligand, interfered with the ability of plasmin to mediate platelet aggregation (Fig. 5); this suggested that plasmin generates the same tethered ligand on PAR-4 as would thrombin. Indeed, our hypothesis was corroborated when a mutation of the thrombin cleavage site on PAR-4 prevented its activation by plasmin as well as by thrombin (Fig. 7). This allowed us to discount the second possibility because plasmin must recognize PAR-4 in the same tertiary conformation as would thrombin to cleave PAR-4; therefore, a similar extent of G protein coupling should be realized. Furthermore, a 10-fold decrease in the rate of intracellular calcium release was observed with plasmin relative to thrombin in the PAR-4-transfected cells (Fig. 7A). Because intracellular calcium release is a proximal signaling event to the cleavage of PAR-4, it represents a reliable assessment of the rate at which PAR-4 is activated by the two proteases. Taken together with the findings of a lengthy period of exposure to plasmin required to effect desensitization of PAR-4 as well as an increased shape change function and relatively slow rate of aggregation onset relative to thrombin, we surmise that plasmin cleaves PAR-4 at a reduced rate compared with thrombin. In conclusion, plasmin mediates platelet aggregation predominantly through proteolytic cleavage of protease-activated receptor 4 that ultimately leads to integrin αIIbβ3 activation. In addition, the functional absence of PAR-1 has no effect on plasmin-mediated platelet aggregation, whereas a functional absence of PAR-4 inhibits this response. We thank Drs. Steven Seiler and Shaun Coughlin for their kind gifts of the BMS-200261 and the PAR-4 null mice, respectively. We also thank Dr. James Daniel for his critical review of the manuscript." @default.
• W2000263382 created "2016-06-24" @default.
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• W2000263382 date "2004-04-01" @default.
• W2000263382 modified "2023-10-17" @default.
• W2000263382 title "Plasmin-mediated Activation of Platelets Occurs by Cleavage of Protease-activated Receptor 4" @default.
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• W2000263382 doi "https://doi.org/10.1074/jbc.m401431200" @default.
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