High-affinity Thrombin Receptor Research Articles

Acetaminophen Overdose Reveals PAR4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium in Mice.

The protease thrombin, which elicits multiple physiological and pathological effects on vascular endothelial cells (ECs), can signal through PARs (protease-activated receptors) 1 and 4. PAR1 is a high-affinity thrombin receptor known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generation and hepatic vascular dysfunction that occur during acetaminophen (APAP) overdose to determine (1) whether hepatic endothelial PAR4 is a functional receptor, and (2) the endothelial-specific functions for PAR1 and PAR4 in a high thrombin and pathological setting. We generated mice with conditional deletion of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte accumulation in the liver, thrombin generation, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial translating ribosome affinity purification followed by next-generation sequencing. We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability, although mice deficient for both receptors had lower vascular permeability at an earlier timepoint after APAP overdose than either of the single mutants. Additionally, mice with loss of both endothelial Par1 and Par4 had reduced thrombin generation after APAP overdose, suggesting decreased hypercoagulability. Last, we found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

Acetaminophen Overdose Reveals Protease-Activated Receptor 4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium.

Hepatic endothelial cell (EC) dysfunction and centrilobular hepatocyte necrosis occur with acetaminophen (APAP) overdose. The protease thrombin, which is acutely generated during APAP overdose, can signal through protease-activated receptors 1 and 4 (PAR1/PAR4). PAR1 is a high-affinity thrombin receptor that is known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generated during APAP overdose to determine (1) if hepatic endothelial PAR4 is a functional receptor, and (2) endothelial-specific functions for PAR1 and PAR4 in a high thrombin setting. We generated mice with conditional deletion(s) of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte congestion/bleeding, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial Translating Ribosome Affinity Purification followed by next-generation sequencing (TRAPseq). We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability but no effect on hepatocyte necrosis. Additionally, mice with loss of endothelial Par1 and Par4 had reduced permeability at an earlier time point after APAP overdose when compared to mice singly deficient in either receptor in ECs. We also found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

Thrombomodulin Regulation of Mitogen-Activated Protein Kinases.

The multifaceted role of mitogen-activated protein kinases (MAPKs) in modulating signal transduction pathways in inflammatory conditions such as infection, cardiovascular disease, and cancer has been well established. Recently, coagulation factors have also emerged as key players in regulating intracellular signaling pathways during inflammation. Among coagulation factors, thrombomodulin, as a high affinity receptor for thrombin on vascular endothelial cells, has been discovered to be a potent anti-inflammatory and anti-tumorigenic signaling molecule. The protective signaling function of thrombomodulin is separate from its well-recognized role in the clotting cascade, which is to function as an anti-coagulant receptor in order to switch the specificity of thrombin from a procoagulant to an anti-coagulant protease. The underlying protective signaling mechanism of thrombomodulin remains largely unknown, though a few published reports link the receptor to the regulation of MAPKs under different (patho)physiological conditions. The goal of this review is to summarize what is known about the regulatory relationship between thrombomodulin and MAPKs.

The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

Thrombin is frequently increased in the CNS after injury yet little is known regarding its effects on neural stem cells. Here we show that the subventricular zone (SVZ) of adult mice lacking the high affinity receptor for thrombin, proteinase activated receptor 1 (PAR1), show increased numbers of Sox2+ and Ki-67+ self-renewing neural stem cells (NSCs) and Olig2+ oligodendrocyte progenitors. SVZ NSCs derived from PAR1-knockout mice, or treated with a PAR1 small molecule inhibitor (SCH79797), exhibited enhanced capacity for self-renewal in vitro, including increases in neurosphere formation and BrdU incorporation. PAR1-knockout SVZ monolayer cultures contained more Nestin, NG2+ and Olig2+ cells indicative of enhancements in expansion and differentiation towards the oligodendrocyte lineage. Cultures of NSCs lacking PAR1 also expressed higher levels of myelin basic protein, proteolipid protein and glial fibrillary acidic protein upon differentiation. Complementing these findings, the corpus callosum and anterior commissure of adult PAR1-knockout mice contained greater numbers of Olig2+ progenitors and CC1+ mature oligodendrocytes. Together these findings highlight PAR1 inhibition as a means to expand adult SVZ NSCs and to promote an increased number of mature myelinating oligodendrocytes in vivo that may be of particular benefit in the context of neural injury where PAR1 agonists such as thrombin are deregulated.

TGFβ upregulates PAR-1 expression and signalling responses in A549 lung adenocarcinoma cells.

The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an αvβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2- and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin αv and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer.

Regulation of neutrophilic inflammation by proteinase-activated receptor 1 during bacterial pulmonary infection.

Neutrophils are key effector cells of the innate immune response to pathogenic bacteria, but excessive neutrophilic inflammation can be associated with bystander tissue damage. The mechanisms responsible for neutrophil recruitment to the lungs during bacterial pneumonia are poorly defined. In this study, we focus on the potential role of the major high-affinity thrombin receptor, proteinase-activated receptor 1 (PAR-1), during the development of pneumonia to the common lung pathogen Streptococcus pneumoniae. Our studies demonstrate that neutrophils were indispensable for controlling S. pneumoniae outgrowth but contributed to alveolar barrier disruption. We further report that intra-alveolar coagulation (bronchoalveolar lavage fluid thrombin–antithrombin complex levels) and PAR-1 immunostaining were increased in this model of bacterial lung infection. Functional studies using the most clinically advanced PAR-1 antagonist, SCH530348, revealed a key contribution for PAR-1 signaling in influencing neutrophil recruitment to lung airspaces in response to both an invasive and noninvasive strain of S. pneumoniae (D39 and EF3030) but that PAR-1 antagonism did not impair the ability of the host to control bacterial outgrowth. PAR-1 antagonist treatment significantly decreased pulmonary levels of IL-1β, CXCL1, CCL2, and CCL7 and attenuated alveolar leak. Ab neutralization studies further demonstrated a nonredundant role for IL-1β, CXCL1, and CCL7 in mediating neutrophil recruitment in response to S. pneumoniae infection. Taken together, these data demonstrate a key role for PAR-1 during S. pneumoniae lung infection that is mediated, at least in part, by influencing multiple downstream inflammatory mediators.

Regulation of neutrophilic inflammation in lung injury induced by community-acquired pneumonia

BackgroundCommunity-acquired pneumonia is commonly caused by Streptococcus pneumoniae, which is associated with excessive neutrophilic inflammation. The high-affinity thrombin receptor, proteinase-activated receptor 1 (PAR1), has been implicated in mediating the interplay between coagulation and inflammation. However, its role during S pneumoniae-induced neutrophilic inflammation, and the mechanisms for neutrophil recruitment in this context are poorly understood. We aimed to investigate the role of neutrophilic inflammation and PAR1 in S pneumoniae-induced pneumonia. MethodsWe used the most clinically advanced PAR-1 antagonist, SCH530348, and performed neutrophil depletion and chemokine neutralisation studies in two murine models. We also did translational studies to examine CXC and CC chemokine receptor expression by flow cytometry on neutrophils in blood and bronchoalveolar lavage fluid (BALF) from mechanically ventilated patients with acute respiratory distress syndrome induced by community-acquired pneumonia. FindingsS pneumoniae infection led to activation of coagulation, increased neutrophil recruitment, and increased PAR-1 expression. By contrast with neutrophil depletion, PAR1 antagonist treatment significantly reduced neutrophil recruitment (mean difference 26·7 × 103 cells per mL [SE 4·9] at 4 h, p=0·0002; and 149·3 [41·4] at 24 h, p=0·0032) without being detrimental to host defence. Markers of alveolar leak, coagulation activation, and proinflammatory cytokines and chemokines (interleukin 1β, CXCL1, CCL2, and CCL7) were attenuated. Neutralisation studies demonstrated that interleukin 1β and CCL7, but not CXCL1 and CCL2, had a key role in neutrophil recruitment in this model. In patients with acute respiratory distress syndrome induced by community-acquired pneumonia (n=10), CXCR1 and CXCR2 expression on BALF neutrophils was higher than in controls (n=3) (median difference in mean fluorescence intensity [MFI] 703 arbitrary units [p=0·0699] for CXCR1 and 658·7 [p=0·0280] for CXCR2). The expression of CXCR1 was decreased on neutrophils from BALF compared with blood (median difference in MFI 1337, p=0·0020) and that of CXCR2, CCR1, CCR2, and CCR3 was increased (125·5, p=0·0020; 335·1, p=0·0020; 116, p=0·0068; and 275, p=0·0020; respectively). InterpretationThese findings suggest that clinically available PAR1 antagonists might offer a novel therapeutic approach for prevention and management of excessive neutrophilic inflammation and alveolar barrier dysfunction in pneumococcal pneumonia without compromising host defence. Furthermore, these data highlight a role for chemokine receptor switching in acute respiratory distress syndrome induced by community-acquired pneumonia. FundingWellcome Trust.

S100 Proteinase-activated Receptor 1 Signalling Contributes To Neutrophilic Inflammation And Alveolar Barrier Disruption In Streptococcus Pneumoniae Pneumonia

Introduction Streptococcus pneumoniae is the most common cause of community-acquired pneumonia (CAP) and is associated with excessive neutrophilic inflammation. The high-affinity thrombin receptor, proteinase-activated receptor (PAR)-1, has been implicated in mediating the interplay between coagulation and inflammation. However, its role during S. pneumoniae -induced neutrophilic inflammation and the mechanisms for neutrophil recruitment in this context are poorly understood. Methods and Results The role of neutrophilic inflammation and PAR-1 was investigated in two models of murine pneumococcal pneumonia (serotype 2 (D39) and serotype 19F (EF3030)) by using the most clinically advanced PAR-1 antagonist, SCH530348. Neutrophil depletion and chemokine neutralisation studies were also performed. Samples were analysed by immunohistochemistry, cytology, flow cytometry, ELISA and microbiological techniques. Our models were characterised by evidence of intra-alveolar coagulation, increased neutrophil recruitment to areas of bacterial infection and increased PAR-1 expression (demonstrated by quantitative image-analysis). Neutrophil depletion protected mice against barrier disruption but resulted in compromised host defence. In contrast, PAR-1 antagonist treatment significantly reduced neutrophil recruitment to the bronchoalveolar space without being detrimental to host defence. Markers of alveolar leak, coagulation activation and pro-inflammatory cytokines and chemokines (IL-1β, CXCL1, CCL2 and CCL7) were also attenuated. Neutralisation studies demonstrated that IL-1β and CCL7, but not CXCL1 and CCL2, played a key role in neutrophil recruitment to the airspaces in this model. Translational studies were performed to examine by flow cytometry the CXC and CC chemokine receptor expression on neutrophils from blood and BALF of mechanically ventilated CAP-induced ARDS patients and controls. CXCR1 and CXCR2 expression on BALF neutrophils was higher in CAP-ARDS patients compared to controls. Additionally, chemokine expression patterns on neutrophils from CAP-ARDS patients changed within different compartments, evidenced by decreased expression of CXCR1 and increased expression of CXCR2, CCR1, CCR2 and CCR3 on neutrophils from BALF compared with blood. Conclusion These data provide preclinical proof-of-concept that recently developed PAR-1 antagonists may offer a novel therapeutic approach for controlling or preventing alveolar barrier dysfunction and excessive neutrophilic inflammation in pneumococcal pneumonia without compromising host defence. Furthermore, they highlight a role for chemokine receptor switching in CAP-ARDS with important implications for future targeting of these chemokine receptors.

Thrombin promotes proliferation of human lung fibroblasts via protease activated receptor‐1‐dependent and NF‐κB‐independent pathways

Acute and chronic respiratory diseases are associated with abnormal coagulation regulation and fibrolysis. However, the detailed mechanism by which coagulation regulation and fibrolysis affect the occurrence and development of lung diseases remain to be elucidated. Protease activated receptor-1 (PAR-1), a major high-affinity thrombin receptor, and nuclear factor kappa B (NF-κB), a transcription factor, are involved in cell survival, differentiation, and proliferation. We have investigated the potential mechanism of thrombin-induced fibroblast proliferation and roles of PAR-1 and NF-κB signalling in this process. The effect of thrombin on proliferation of human pulmonary fibroblasts (HPF) was assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. The expression of PAR1 and NF-κB subunit p65 protein was detected by Western blot. Nuclear translocation of p65 was examined by laser scanning confocal microscopy. We show that thrombin significantly increased proliferation of HPF as determined by induction of BrdU-positive incorporation ratio. Induced PAR1 protein expression was also seen in HPF cells treated with thrombin. However, thrombin had no significant effect on expression and translocation of NF-κB p65 in HPF cells. The results indicate that, by increasing protein expression and interacting with PAR1, thrombin promotes HPF proliferation. NF-κB signalling appears to play no role in this process.

PL-25 Mechanisms coupling thrombomodulin to tumor dissemination

Thrombomodulin (TM) is a primarily endothelial associated high-affinity thrombin receptor crucial for thrombin-dependent protein C activation. The activated protein C/protein S complex proteolyzes coagulation factors V and VIII, rendering them nonfunctional [1]. The TM-thrombin complex also activates thrombin activatable fibrinolysis inhibitor (TAFI), which renders clot more resistant to lysis [2]. In addition to biological functions mediated through regulation of hemostatic function, TM has been shown to regulate multiple functions important in inflammation, including cytokine production, cell adhesion, and complement activation, through mechanisms involving the lectin-like domain [3]. A potential role for TM in tumor biology is suggested by studies showing that TM expression by tumor tissues correlates with a less advanced stage at diagnosis and a better prognosis for multiple cancers, including tumors of neural [4] and epithelial origin [5–8]. For example, studies by Ogawa and colleagues of tumor tissue from patients with squamous cell carcinoma (SCC) of the lung revealed decreased TM expression in tumor tissue from metastatic foci relative to primary tumors. Furthermore, patients with TM-negative tumors had a significantly poorer survival rate than those with TMpositive tumors [6]. Consistent with these observations, tumor cell lines subcloned from patients with malignant melanoma displayed a negative correlation between TM expression and cell proliferation in vitro and in vivo [9]. This effect appeared to be thrombin and thrombin receptor independent, and possibly related to lectin like domain mediated signal transduction pathways. TM expression by tumor cells has also been implicated in limiting tumor cell invasive potential. For example, a human esophageal SCC cell line with high TM expression had significantly lower invasive potential than the same cell line with low TM expression [8].

Serine and metalloprotease signaling through PAR1 in arterial thrombosis and vascular injury

Thrombin-dependent platelet activation has been shown to be important in the setting of angioplasty and stenting, which may cause ischemic complications including acute myocardial infarction and death. Inhibitors of the high-affinity thrombin receptor, protease-activated receptor 1 (PAR1), are now being evaluated in clinical trials for safety and efficacy in patients with atherothrombotic disease. However, it is unknown whether chronic inhibition of PAR1 in these large patient populations will have beneficial or possibly adverse effects on other biologic processes involved in blood vessel homeostasis and the response to vascular injury. Most recently, PAR1 was found to be cleaved at a distinct site by matrix metalloprotease-1 (MMP-1) to create a longer tethered ligand, which activates a distinct spectrum of G protein pathways in platelets. The differential activation by serine proteases such as thrombin and the metalloprotease MMP-1, places the protease receptor PAR1 at the junction of two major protease classes critically involved in thrombosis, matrix remodeling, and the response to vascular injury.

Challenges and Promises of Developing Thrombin Receptor Antagonists

Despite the availability of dual antiplatelet therapy comprised of aspirin and clopidogrel, there is still significant unmet medical need for treating and preventing arterial thrombotic diseases. To achieve further reduction of cardiovascular events without exceeding bleeding tolerability and safety limits, novel antiplatelet strategies might need to trade in antiplatelet efficacy by partial inhibition of an important platelet activation pathway or by differentially targeting pathological versus physiological thrombogenesis pathways. Thrombin, the central enzyme in coagulation and the most potent platelet agonist tested in vitro, is one of the key factors driving the formation of occlusive thrombi. Platelet thrombin receptors, namely protease-activated receptor 1 (PAR-1) and protease-activated receptor 4 (PAR-4), act in concert to elicit robust platelet responses to thrombin. PAR-1 is the high affinity thrombin receptor and represents a novel antithrombotic target. PAR-4 is a low affinity thrombin receptor with less understood function. This review discusses the genetic and pharmacological evidence for PAR-1 target validation and highlights the progresses and challenges in developing oral PAR-1 antagonists, especially SCH 530348 from Merck/Schering-Plough and E-5555 from Eisai Co. Recent patents disclosing several novel chemical series of PAR-1 antagonists from Sanofi-Aventis and Pierre Fabre are also presented.

SCH 602539, a Protease-Activated Receptor-1 Antagonist, Inhibits Thrombosis Alone and in Combination With Cangrelor in a Folts Model of Arterial Thrombosis in Cynomolgus Monkeys

To determine the antithrombotic effects of SCH 602539, an analog of the selective protease-activated receptor (PAR)-1 antagonist vorapaxar (formerly SCH 530348) currently in advanced clinical development, and the P2Y(12) ADP receptor antagonist cangrelor, alone and in combination. Multiple platelet activation pathways contribute to thrombosis. The effects of SCH 602539 and cangrelor alone and in combination on cyclic flow reductions were evaluated in a Folts model of thrombosis in cynomolgus monkeys. The effects of these treatments on ex vivo platelet aggregation and coagulation parameters were also monitored. Dose-dependent inhibition of cyclic flow reductions was observed after treatment with SCH 602539 alone and cangrelor alone (P<0.05 versus vehicle for the 2 highest concentrations of each agent). The combination of SCH 602539 and cangrelor was associated with synergistic antithrombotic effects (P<0.05 versus vehicle for all combinations tested). The 2 highest doses of SCH 602539 inhibited platelet aggregation in response to PAR-1-selective high-affinity thrombin receptor agonist peptide by greater than 80% but did not affect platelet aggregation induced by other agonists; also, they did not affect any coagulation parameters. The combined inhibition of the PAR-1 and the P2Y(12) ADP platelet activation pathways had synergistic antithrombotic and antiplatelet effects. The addition of a PAR-1 antagonist to a P2Y(12) ADP receptor antagonist may provide incremental clinical benefits in patients with atherothrombotic disease, both in short- and long-term settings. These hypotheses need to be tested clinically.

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.

Why Thrombin PAR1 Receptors Are Important to the Cardiac Surgical Patient

Targeting of the high-affinity thrombin receptor protease-activated receptor-1 (PAR1) on platelets represents an exciting strategy to curb the pro-thrombotic complications of cardiac surgery without interfering with the hemostatic benefits of thrombin in the coagulation cascade. The first dedicated PAR1 antagonist to complete safety trials this year has justified expectations, showing no increased risk of bleeding when added to standard anti-platelet therapy but halving major adverse cardiovascular events after percutaneous coronary intervention. In the setting of cardiothoracic surgery with cardiopulmonary bypass, an FDA-approved drug already exists with anti-PAR1 properties: aprotinin has been shown to inhibit thrombin-induced platelet activation in vitro and clinically, through sparing of PAR1 receptor cleavage and activation. Because aprotinin also exerts anti-fibrinolytic effects through blockade of plasmin, this indicates a subtle clinical mechanism of action that is simultaneously anti-thrombotic yet hemostatic. PAR1 antagonists would also be expected to exert anti-inflammatory properties through targeting of PAR1 on endothelium, and this principle has been validated in vitro for aprotinin and newer peptidomimetric antagonists. PAR1 antagonism is likely to remain an active and exciting area of research in cardiac surgery, with newer generations of PAR1 antagonists and recombinant aprotinin variants entering clinical development.

Protease-Activated Receptor-1 Contributes to Cardiac Remodeling and Hypertrophy

Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Aprotinin and the Protease-Activated Receptor 1 Thrombin Receptor: Antithrombosis, Inflammation, and Stroke Reduction

Cardiopulmonary bypass, although remaining an indispensable asset in cardiac surgery, especially in more complex and repeat operations, is associated with significant thrombin generation in the bypass circuit, leading to the activation of platelets, the coagulation system, an inflammatory response, and perioperative stroke. Recent clinical studies and meta-analyses of clinical trials in coronary artery bypass grafting surgery have confirmed that aprotinin not only reduces transfusion requirements in cardiac surgery but also confers significant protection against platelet dysfunction, activation of the systemic inflammatory response, and perioperative stroke when administered at the full (or "Hammersmith") dose. This article reviews research from several independent groups to propose a novel mechanism through which the antithrombotic, anti-inflammatory, and neuroprotective mechanism might be mediated, via protection of the high-affinity thrombin receptor protease-activated receptor 1 (PAR1).

Effect of Aprotinin and Recombinant Variants on Platelet Protease-Activated Receptor 1 Activation

Thrombin generated during cardiopulmonary bypass activates the high-affinity thrombin receptor, protease-activated receptor 1 (PAR1), causing platelet dysfunction and excessive bleeding. The serine protease inhibitor aprotinin protects platelets against thrombin-mediated PAR1 activation in vitro and in vivo. Here we have investigated three novel recombinant aprotinin variants with specific modifications to the active site lysine at amino acid position 15 (arginine-15, arginine-15-alanine-17, and valine-15-leucine-17) for their effect on PAR1-mediated platelet aggregation in vitro. Aggregation studies were carried out using washed human platelets (n = 9) or platelet rich plasma (n = 7) from healthy volunteers activated with 1 or 5 nM thrombin. Recombinant aprotinin variants were used at the molar equivalent to 50 KIU/mL of the parent compound. The PAR1-specific antagonist peptide, FLLRN, was used at 500 microM. Platelet aggregation at low concentrations of thrombin (1 nM) was mediated exclusively through PAR1, as shown by inhibition of aggregation in the presence of FLLRN. At 1 nM thrombin, the mean percentage +/- SD aggregation of washed platelets was 68.6% +/- 12.3%. This was suppressed by each aprotinin variant at the 50 KIU/mL equivalent dose: arginine-15 (23.0% +/- 17.5%, p < 0.001); arginine-15-alanine-17 (33.3% +/- 22.9%, p < 0.01); aprotinin (37.5% +/- 19.4%, p < 0.05); valine-15-leucine-17 (50.0% +/- 16.1%, not significant)). At 5 nM thrombin, which activates both high (PAR1) and low-affinity (PAR4) thrombin receptors on platelets, FLLRN and aprotinin failed to block aggregation: this finding indicates that aprotinin selectively targeted PAR1. In platelet-rich plasma, aggregation at 1 nM thrombin was 77.1% +/- 10.0%, and this was inhibited in the following order: arginine-15 (30.1% +/- 9.6%, p < 0.001); arginine-15-alanine-17 (52.3% +/- 9.7%, p > 0.001); aprotinin (55.9% +/- 6.2%, p > 0.001); valine-15-leucine-17 (73.7% +/- 7.1%, not significant). Aprotinin variants differentially inhibit PAR1-mediated platelet aggregation. With more understanding of the mechanisms of action of aprotinin and its derivatives, safer and more efficacious aprotinin variants may become available for clinical use.

Aprotinin inhibits proinflammatory activation of endothelial cells by thrombin through the protease-activated receptor 1

Thrombin is generated in significant quantities during cardiopulmonary bypass and mediates adverse events, such as platelet aggregation and proinflammatory responses, through activation of the high-affinity thrombin receptor protease-activated receptor 1, which is expressed on platelets and endothelium. Thus antagonism of protease-activated receptor 1 might have broad therapeutic significance. Aprotinin, used clinically to reduce transfusion requirements and the inflammatory response to bypass, has been shown to inhibit protease-activated receptor 1 on platelets in vitro and in vivo. Here we have examined whether aprotinin inhibits endothelial protease-activated receptor 1 activation and resulting proinflammatory responses induced by thrombin. Protease-activated receptor 1 expression and function were examined in cultured human umbilical vein endothelial cells after treatment with alpha-thrombin at 0.02 to 0.15 U/mL in the presence or absence of aprotinin (200-1600 kallikrein inhibitory units/mL). Protease-activated receptor 1 activation was assessed by using an antibody, SPAN-12, which detects only the unactivated receptor, and thrombin-mediated calcium fluxes. Other thrombin-dependent inflammatory pathways investigated were phosphorylation of the p42/44 mitogen-activated protein kinase, upregulation of the early growth response 1 transcription factor, and production of the proinflammatory cytokine interleukin 6. Pretreatment of cultured endothelial cells with aprotinin significantly spared protease-activated receptor 1 receptor cleavage (P < .0001) and abrogated calcium fluxes caused by thrombin. Aprotinin inhibited intracellular signaling through p42/44 mitogen-activated protein kinase (P < .05) and early growth response 1 transcription factor (P < .05), as well as interleukin 6 secretion caused by thrombin (P < .005). This study demonstrates that endothelial cell activation by thrombin and downstream inflammatory responses can be inhibited by aprotinin in vitro through blockade of protease-activated receptor 1. Our results provide a new molecular basis to help explain the anti-inflammatory properties of aprotinin reported clinically.

Endothelial Thrombomodulin Induces Ca2+ Signals and Nitric Oxide Synthesis through Epidermal Growth Factor Receptor Kinase and Calmodulin Kinase II

Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.