The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.     

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.     

Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30–100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3^−/− mice had increased G_q signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca²⁺) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca²⁺ release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3^+/−) had an intermediate increase in maximum Ca²⁺ mobilization. Treatment of PAR3^−/− mice platelets with P2Y₁₂ antagonist (2MeSAMP) did not affect Ca²⁺ mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G_12/13 signaling in response to thrombin was not significantly different between wild type and PAR3^−/− mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca²⁺ mobilization and PKC activation in mouse platelets by physical interaction.     

Proteinase-activated receptor 4 (PAR4): activation and inhibition of rat platelet aggregation by PAR4-derived peptides

We studied the actions of receptor-activating peptide analogues (PAR4APs), modeled on the proteolytically-revealed tethered ligand sequence of murine proteinase-activated receptor-4 (PAR4), in a rat platelet aggregation assay. The PAR4APs GYPGKF-NH2 (GY-NH2) and AYPGKF-NH2 (AY-NH2) were able to cause aggregation with EC50 values of about 40 microM and 15 microM, respectively. The reverse human PAR4 sequence (VQGPYG-NH2, YG-NH2) and the PAR1AP SFLLR-NH2, did not cause aggregation. In contrast, trans-cinnamoyl-YPGKF-NH2 (tcY-NH2) did not cause aggregation but blocked aggregation caused by GY-NH2, AY-NH2, and thrombin without affecting ADP-mediated aggregation. We conclude that in contrast to the PAR1AP, the PAR4APs GY-NH2 and AY-NH2 activate rat platelets via a PAR4-related receptor and that peptide analogues modeled on the PAR4 tethered activating sequence can serve as useful agonist and antagonist probes for assessing the consequence of activating PAR4 either by PAR4APs or thrombin in rat tissue preparations.     

Akt activation in platelets depends on Gi signaling pathways

The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr308 and Ser473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental Gi or Gz signaling. Unlike wild-type mouse platelets, platelets from Galphaq-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y1 receptor. However, ADP caused Akt phosphorylation in Galphaq- and P2Y1-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.     

Interaction of viper venom serine peptidases with thrombin receptors on human platelets

The serine peptidases, thrombocytin and PA-BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA-BJ, and thrombocytin on the degradation of the soluble N-terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41-Ser42. In addition, a rapid cleavage of the peptide bond at Arg46-Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA-BJ and thrombocytin both cleaved at 41-42 and 46-47 peptide bonds, and fragment 42-103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.     

Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: evidence for a novel mechanism for preventing or terminating signaling by PAR1's tethered ligand

The thrombin receptor PAR1 is activated when thrombin cleaves the receptor's amino-terminal exodomain to reveal the new N-terminal sequence SFLLRN which then acts as a tethered peptide ligand. Free SFLLRN activates PAR1 independent of receptor cleavage and has been used to probe PAR1 function in various cells and tissues. PAR1-expressing cells desensitized to thrombin retain responsiveness to SFLLRN. Toward determining the mechanism of such responses, we utilized fibroblasts derived from a PAR1-deficient mouse. These cells were unresponsive to thrombin and SFLLRN and became sensitive to both ligands after transfection with human PAR1 cDNA. Moreover, PAR1-transfected cells responded to SFLLRN after thrombin-desensitization, indicating that signaling of thrombin-desensitized cells to SFLLRN was mediated by PAR1 itself. SFLLRN caused signaling in thrombin-desensitized cells when no uncleaved PAR1 was detectable on the cell surface; however, cleaved PAR1 was present. To determine whether the cleaved receptors could still signal, fibroblasts were transfected with a PAR1 mutant containing a trypsin site/SFLLRN sequence carboxyl terminal to the native thrombin site. These cells retained responsiveness to trypsin after thrombin-desensitization. Conversely, fibroblasts expressing a PAR1 mutant with the trypsin site/SFLLRN sequence amino terminal to the native thrombin site retained responsiveness to thrombin after trypsin-desensitization. This suggests that a population of thrombin-cleaved PAR1 can respond both to exogenous SFLLRN and to a second tethered ligand. In this population, the tethered ligand unmasked by thrombin cleavage must not be functional, suggesting the possibility of a novel mechanism of receptor shutoff involving sequestration or modification of the tethered ligand to prevent or terminate its function.     

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibα of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca²⁺ mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbα by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbα binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbα proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbα cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbα.     

Coordinate activation of human platelet protease-activated receptor-1 and -4 in response to subnanomolar alpha-thrombin

We previously demonstrated that human platelets activated with SFLLRN release PAR-1 activation peptide, PAR-1-(1-41), even in the presence of hirudin. This observation suggests that during their activation, platelets generate a protease that activates PAR-1. In this study, PAR-1 and -4 activation peptides were detected 10 s after 相似文献   

Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin. Human umbilical vein endothelial cells (HUVEC) are known to express PAR1 and the trypsin/tryptase receptor, PAR2. Northern blots detected both of those receptors and, to a lesser extent, PAR3, but PAR4 message was undetectable and there was no response to PAR4 agonist peptides. To determine whether PAR3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cleavage was blocked with two selective antibodies and PAR1 activation was inhibited with the antagonist, BMS200261. The antibodies completely inhibited HUVEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibits PAR1 signaling at the concentrations used. Since peptides mimicking the PAR1 tethered ligand domain can also activate PAR2, we asked whether the remaining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1. Evidence that transactivation can occur was obtained in COS-7 cells co-expressing PAR2 and a variant of PAR1 that can be cleaved, but not signal. There was a substantial response to thrombin only in cells expressing both receptors. Conversely, in HUVEC, complete blockade of the thrombin response by the PAR1 antagonist occurred only when signaling through PAR2 was also blocked. From these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelial cell responses to thrombin; 2) although PAR3 may be expressed, there is still no evidence that it mediates thrombin responses; 3) PAR4 is not expressed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling through PAR1 is blocked. Such transactivation may limit the effectiveness of PAR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage.     

Proteinase-activated receptors (PARs)--the PAR3 Neo-N-terminal peptide TFRGAP interacts with PAR1

Thrombin activates proteinase-activated receptor (PAR)1, PAR3 and PAR4 by a unique mechanism that involves cleavage of the receptor and exposure of a new N-terminal domain acting as a tethered ligand. Synthetic peptides based on the proteolytically revealed receptor sequence can selectively activate PAR1 or PAR4 independently of receptor cleavage. However, corresponding peptides for PAR3 have not been identified thus far. Here, we demonstrate that the synthetic peptide TFRGAP representing the 1st six residues of the new amino terminus of PAR3 induced ERK activation in human A-498 carcinoma cells endogeneously expressing PAR1 and PAR3. This effect was completely abolished by single alanine substitution at positions 3, 4 and 6 in the peptide. Since the specific PAR1 antagonist RWJ 56110 completely abolished TFRGAP-induced ERK activation in A-498 cells we speculate that TFRGAP does signal MAPK via interaction with PAR1. This was underlined by experiments on PAR1-/- mouse lung fibroblasts (KOLF cells) that stably overexpress human PAR1 and PAR3, respectively. While TFRGAP was without effect on ERK activation in PAR3+ KOLF cells, it induced MAPK activation in KOLF cells transfected with PAR1. These studies provide evidence that analogues of the PAR3 tethered ligand can mediate cell signaling by interaction with PAR1-type thrombin receptors.     

Conserved structure and adjacent location of the thrombin receptor and protease-activated receptor 2 genes define a protease-activated receptor gene cluster.

BACKGROUND: Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism. Recently, a second protease-activated receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of receptor cleavage. Thus, functionally, the thrombin receptor and PAR2 constitute a fledgling receptor family that shares a novel proteolytic activation mechanism. To further explore the relatedness of the two known protease-activated receptors and to examine the possibility that a protease-activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin receptor and PAR2 genes. MATERIALS AND METHODS: The genomic structures of the two protease-activated receptor genes were determined by analysis of lambda phage, P1 bacteriophage, and bacterial artificial chromosome (BAC) genomic clones. Chromosomal location was determined with fluorescent in situ hybridization (FISH) on metaphase chromosomes, and the relative distance separating the two genes was evaluated both by means of two-color FISH and analysis of YACs and BACs containing both genes. RESULTS: Analysis of genomic clones revealed that the two protease-activated receptor genes share a two-exon genomic structure in which the first exon encodes 5'-untranslated sequence and signal peptide, and the second exon encodes the mature receptor protein and 3'-untranslated sequence. The two receptor genes also share a common locus with the two human genes located at 5q13 and the two mouse genes at 13D2, a syntenic region of the mouse genome. These techniques also suggest that the physical distance separating these two genes is less than 100 kb. CONCLUSIONS: The fact that the thrombin receptor and PAR2 genes share an identical structure and are located within approximately 100 kb of each other in the genome demonstrates that these genes arose from a gene duplication event. These results define a new protease-activated receptor gene cluster in which new family members may be found.     

蛋白酶激活受体

蛋白酶激活受体(protease-activated receptor,PAR)属于G蛋白偶联受体家族,包括4个成员,除PAR2为胰蛋白酶受体外,其他三个都是凝血酶受体,PAR通过形成或暴露新的N末端被激活。PAR广泛表达于全身各组织,尤其在消化系统表现出多种功能;通过促进细胞增殖、迁移、浸润、血管生成以及组织重构(通过促进细胞增殖、迁移、浸润和血管生成),同时抑制细胞的分化和凋亡等因素参与肿瘤的发生和发展,这为临床诊治和预后评判提供了有力的手段。     

Protease activated receptors in cardiovascular function and disease

Recent studies have shown that a novel class of protease activated receptors (PARs), which are composed of seven transmembrane G protein-coupled domains, are activated by serine proteases such as thrombin, trypsin and tryptase. Although four types (PAR 1, PAR 2, PAR 3 and PAR 4) of this class of receptors have been identified, their discrete physiological and pathological roles are still being unraveled. Extracellular proteolytic activation of PARs results in the cleavage of specific sites in the extracellular domain and formation of a new N-terminus which functions as a tethered ligand. The newly formed tethered ligand binds intramolecularly to an exposed site in the second transmembrane loop and triggers G-protein binding and intracellular signaling. Recent studies have shown that PAR-1, PAR-2 and PAR-4 have been involved in vascular development and a variety of other biological processes including apoptosis and remodeling. The use of animal model systems, mainly transgenic mice and synthetic tethered ligand domains, have contributed enormously to our knowledge of molecular signaling and the regulatory properties of various PARs in cardiomyocytes. This review focuses on the role of PARs in cardiovascular function and disease.     

Thrombin Regulation of Cell Function through Protease-Activated Receptors: Implications for Therapeutic Intervention

The serine protease thrombin is well recognized as being pivotal to the maintenance of hemostasis under both normal and pathological conditions. Its cellular actions are mediated through a unique family of protease-activated receptors (PARs). These receptors represent a novel family of G protein-coupled receptors that undergo proteolytic cleavage of their amino terminus and subsequent autoactivation by a tethered peptide ligand. This paper reviews the consequences of PAR activation in thrombosis, vascular injury, inflammation, tissue injury, and within the tumor microenvironment.     

Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity

Proteinase-activated receptor 2 (PAR(2)), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR(2), often measured by Ca(2+) release. PAR(2) contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR(2) in HEK293T cells resulted in concentration-dependent, PAR(2) AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple G(i) proteins or sequester intracellular Ca(2+). Because previous studies described synergistic PAR(2)- and TLR4-mediated cytokine production, we hypothesized that PAR(2) and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR(2)-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR(2) in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR(2) and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR (-/-)(2) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.     

Cathepsin G activates protease-activated receptor-4 in human platelets

Of the four known protease-activated receptors (PARs), PAR1 and PAR4 are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressing Xenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophil-dependent platelet activation was blocked by the PAR4 antibody. These data show that PAR4 mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.     

Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy

It has been hypothesized that protease-activated receptors may be activated and attenuated by more than one protease. Here, we explore a desensitization mechanism of the PAR1 thrombin receptor by anticoagulant proteases and provide an explanation to the enigma of why plasmin/tissue plasminogen activator (t-PA) can both activate and deactivate platelets prior to thrombin treatment. By using a soluble N-terminal exodomain (TR78) as a model for the full-length receptor, we were able to unambiguously compare cleavage rates and specificities among the serum proteases. Thrombin cleaves TR78 at the R41-S42 peptide bond with a kcat of 120 s-1 and a KM of 16 microM to produce TR62 (residues 42-103). We found that, of the anticoagulant proteases, only plasmin can rapidly truncate the soluble exodomain at the R70/K76/K82 sites located on a linker region that tethers the ligand to the body of the receptor. Plasmin cleavage of the TR78 exodomain is nearly equivalent to that of thrombin cleavage at R41 with similar rates (kcat = 30 s-1) and affinity (KM = 18 microM). Specificity was demonstrated since there is no observed cleavage at the five other potential plasmin-cleavage sites. Plasmin also cleaves the TR78 exodomain at the R41 thrombin-cleavage site generating transiently activated exodomain. We directly demonstrated that plasmin cleaves these same sites in full-length membrane-embedded receptor expressed in yeast and COS7 fibroblasts. The rate of plasmin truncation is similar between the extensively glycosylated COS7-expressed receptor and the nonglycosylated yeast-produced receptor. Mutation of the R70/K76/K82 sites to A70/A76/A82 eliminates plasmin truncation and desensitization of thrombin-dependent Ca2+ signaling and converts PAR1 into a plasmin-activated receptor with full agonist activity for plasmin. Plasmin does not desensitize the Ca2+ response of platelets or COS7 cells to SFLLRN consistent with intermolecular ligand-binding sites being located to the C-terminal side of K82. Truncation of the wild-type receptor at the C-terminal plasmin-cleavage sites removes the N-terminal tethered ligand or preligand, thereby providing an effective pathway for PAR1 desensitization in vivo.     

Protease activated receptors in cardiovascular function and disease

Recent studies have shown that a novel class of protease activated receptors (PARs), which are composed of seven transmembrane G protein-coupled domains, are activated by serine proteases such as thrombin, trypsin and tryptase. Although four types (PAR 1, PAR 2, PAR 3 and PAR 4) of this class of receptors have been identified, their discrete physiological and pathological roles are still being unraveled. Extracellular proteolytic activation of PARs results in the cleavage of specific sites in the extracellular domain and formation of a new N-terminus which functions as a tethered ligand. The newly formed tethered ligand binds intramolecularly to an exposed site in the second transmembrane loop and triggers G-protein binding and intracellular signaling. Recent studies have shown that PAR-1, PAR-2 and PAR-4 have been involved in vascular development and a variety of other biological processes including apoptosis and remodeling. The use of animal model systems, mainly transgenic mice and synthetic tethered ligand domains, have contributed enormously to our knowledge of molecular signaling and the regulatory properties of various PARs in cardiomyocytes. This review focuses on the role of PARs in cardiovascular function and disease. (Mol Cell Biochem 263: 227–239, 2004)     

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2? mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y?? receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.