Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity.     

Role of protease-activated receptors in the vascular system

Thrombin is one of the key molecules involved in the development of vascular diseases. Thrombin does not only serve as a coagulation factor, but it also exerts cellular effects by activating protease (proteinase)-activated receptors (PARs), a family of seven-transmembrane G protein-coupled receptors. This study focused on the role of PARs in the vascular system. Among the four members so far identified, PAR-1 and PAR-2 were found to play an important role in the vascular system, while the functional roles of PAR-3 and PAR-4 appear to be mostly limited to platelets. The endothelial cells play a primary role in mediating the vascular effects of PARs under physiological conditions, while PARs of the smooth muscle cells can be induced under pathological conditions, and therefore play a more pathophysiological role. PAR-1 and PAR-2 mediate various vascular effects including regulation of vascular tone, proliferation and hypertrophy of smooth muscle and angiogenesis. Since proteases are activated under pathological conditions such as hemorrhage, tissue damage, and inflammation, PARs are suggested to play a critical role in the development of functional and structural abnormality in the vascular lesion. Understanding the functional role of PARs in the vascular system can thus help in the development of new strategies for the prevention and therapy of vascular diseases.     

Extracellular mediators in atherosclerosis and thrombosis: lessons from thrombin receptor knockout mice

It is well appreciated that thrombin as well as other proteases can act as signaling molecules that specifically regulate cells by cleaving and activating members of a novel class of protease-activated receptors (PARs). The utility of gene knockout strategies to define and better comprehend the physiological role of specific proteins is perhaps best exemplified in the field of thrombin receptors. The development of PAR knockout mice has provided the unique opportunity to identify and characterize new members of this novel family of GPCRs, evaluate the interaction of PARs jointly expressed in common cells and tissues, and better understand the role of PARs in thrombosis, restenosis, vascular remodeling, angiogenesis, and inflammation. Presently, 4 members of the PAR family have been cloned and identified. In this review, we examine experimental evidence gleaned from PAR-/- mouse models as well as how the use of PAR-/- mice has provided insights toward understanding the physiological role of thrombin in cells of the vascular system and vascular pathology.     

How the protease thrombin talks to cells.

How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The irreversibility of PAR1's proteolytic activation mechanism stands in contrast to the reversible ligand binding that activates classical G protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G protein-coupled receptors. This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with na?ve receptors, thereby maintaining thrombin responsiveness. Thus cells have evolved a trafficking solution to the signaling problem presented by PARs. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Recent studies with knockout mice, receptor-activating peptides, and blocking antibodies are beginning to define the role of these receptors in vivo.     

Roles of protease-activated receptors in a mouse model of endotoxemia

Endotoxemia is often associated with extreme inflammatory responses and disseminated intravascular coagulation. Protease-activated receptors (PARs) mediate cellular responses to coagulation proteases, including platelet activation and endothelial cell reactions predicted to promote inflammation. These observations suggested that PAR activation by coagulation proteases generated in the setting of endotoxemia might promote platelet activation, leukocyte-mediated endothelial injury, tissue damage, and death. Toward testing these hypotheses, we examined the effect of PAR deficiencies that ablate platelet and endothelial activation by coagulation proteases in a mouse endotoxemia model. Although coagulation was activated as measured by thrombin-antithrombin (TAT) production and antithrombin III (ATIII) depletion, Par1(-/-), Par2(-/-), Par4(-/-), Par2(-/-):Par4(-/-), and Par1(-/-):Par2(-/-) mice all failed to show improved survival or decreased cytokine responses after endotoxin challenge compared with wild type. Thus, our results fail to support a necessary role for PARs in linking coagulation to inflammation or death in this model. Interestingly, endotoxin-induced thrombocytopenia was not diminished in Par4(-/-) mice. Thus, a mechanism independent of platelet activation by thrombin was sufficient to cause thrombocytopenia in our model. These results raise the possibility that decreases in platelet count in the setting of sepsis may not be caused by disseminated intravascular coagulation but instead report on a sometimes parallel but independent process.     

Progress in the understanding of protease-activated receptors

Thrombin results from the activation of the blood coagulation system. It is a multifunctional protein that has, besides its function in hemostasis and thrombosis, several cellular effects that link the coagulation system with the inflammatory response. Many years of investigations were necessary for the discovery of the first functional thrombin receptor, which was found to have a unique mechanism of activation. The receptor was named protease-activated receptor 1 (PAR-1) because proteolysis is necessary for its activation. Subsequent studies led to the identification of the other PARs, PAR-2, PAR-3, and PAR-4. PAR-2 is activated by trypsin, tryptase, factor Xa, or factor VIIa, but it cannot be activated by thrombin, PAR-3 and PAR-4 can also be activated by thrombin. Activation of PARs by protease involves proteolytic cleavage and unmasking of an amino-terminal receptor sequence, which acts as a tethered ligand by binding to the second extracellular loop of the receptor to initiate transmembrane signaling. Sequence analysis has shown that all PARs are members of the 7-transmembrane domain receptor superfamily. Expression of PARs has been detected in most tissues and in numerous cells, and thus these molecules have been implicated in several physiological processes and in the pathogenesis of several diseases.     

Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)--factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF--VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF--VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF--VIIa--X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TF--VIIa--Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF--VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.     

Protease-activated receptor-4 inhibition protects from multiorgan failure in a murine model of systemic inflammation

Coagulation proteases may act as cell signaling molecules via protease-activated receptor (PAR) cleavage, subsequently affecting cellular and inflammatory responses. Activation of PARs in the setting of systemic inflammation and disseminated intravascular coagulation (DIC) might thus exacerbate the inflammatory response contributing to tissue and organ damage. To investigate the role of PAR-4 in these processes, we subjected mice to a model of systemic inflammation and DIC (Shwartzman reaction) in the absence or presence of a cell-penetrating pepducin antagonist of PAR-4 (P4pal-10). P4pal-10 dose-dependently diminished the severity of endotoxemia and preserved liver, kidney, as well as lung function. Moreover, systemic inflammation and local (neutrophilic) inflammatory responses were attenuated. In vitro migration assays and P4pal-10 treatment in neutropenic mice suggest an essential role for neutrophils in PAR-4-mediated pathology. P4pal-10 treatment of thrombocytopenic mice excluded the involvement of platelets in this phenomenon. These results uncover an important role for PAR-4 in the Shwartzman reaction and suggest that inhibition of PAR-4 signaling in neutrophils could be protective in systemic inflammation and DIC.     

Signaling properties and functions of two distinct cardiomyocyte protease-activated receptors

Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal-regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal-regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH(2)-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by alpha(1)-adrenergic receptor activation. These studies provide novel evidence that cardiomyocytes coexpress 2 functional PARs, which link to a common set of signals that culminate in changes in contractile function and hypertrophic growth. PAR actions may assume clinical importance in the border zone surrounding an infarction, where local proteolysis of PARs by serine proteases generated during inflammatory or thrombogenic pathways would elevate calcium concentration (setting the stage for arrhythmias), promote hypertrophic growth, and/or influence cardiomyocyte survival.     

Tissue factor and glycoprotein C on herpes simplex virus type 1 are protease-activated receptor 2 cofactors that enhance infection

The coagulation system provides physiologic host defense, but it can also be exploited by pathogens for infection. On the HSV1 surface, host-cell-derived tissue factor (TF) and virus-encoded glycoprotein C (gC) can stimulate protease activated receptor 1 (PAR1)-enhanced infection by triggering thrombin production. Using novel engineered HSV1 variants deficient in either TF and/or gC, in the present study, we show that activated coagulation factors X (FXa) or VII (FVIIa) directly affect HSV1 infection of human umbilical vein endothelial cells in a manner that is dependent on viral TF and gC. The combination of FXa and FVIIa maximally enhanced infection for TF(+)/gC(+) HSV1 and receptor desensitization and Ab inhibition demonstrated that both proteases act on PAR2. Inhibitory TF Abs showed that the required TF source was viral. Individually, TF or gC partly enhanced the effect of FXa, but not FVIIa, revealing gC as a novel PAR2 cofactor for FVIIa. In sharp contrast, thrombin enhanced infection via PAR1 independently of viral TF and gC. Thrombin combined with FXa/FVIIa enhanced infection, suggesting that PAR1 and PAR2 are independently involved in virus propagation. These results show that HSV1 surface cofactors promote cellular PAR2-mediated infection, indicating a novel mode by which pathogens exploit the initiation phase of the host hemostatic system.     

蛋白酶激活受体在免疫炎症性疾病中的研究进展

蛋白酶激活受体(protease activated receptors, PARs)是G蛋白偶联受体的一个亚家族,最早发现其与血栓形成、组织止血、癌症相关,随着研究的深入,发现PARs在感染、免疫炎症性疾病、纤维化疾病中发挥重要作用。目前已发现PARs有4种亚型,分别为PAR1~4,研究较多的为PAR1和PAR2,现就PAR1、PAR2在免疫炎症性疾病中的作用作一概述。     

Protease-activated receptors: how proteases signal to cells to cause inflammation and pain

Certain serine proteases that originate from the circulation (coagulation factors), inflammatory cells (mast cell tryptase, neutrophil granzyme A, and proteinase 3), and epithelial and neuronal tissues (trypsins) can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors. Proteases cleave PARs on multiple cell types to reveal tethered ligand domains that bind to and activate the cleaved receptors. The proteases that activate PARs are often generated and secreted during injury and inflammation, and PARs orchestrate tissue responses to these insults, including hemostasis, inflammation, nociception, and repair mechanisms. Agonists of PARs, notably PAR2, induce inflammation in many tissues that is characterized by hyperemia, extravasation of plasma proteins, granulocyte infiltration, and alterations in epithelial permeability. These effects are mediated in part by the release of neuropeptides substance P and calcitonin gene-related peptide from sensory nerve fibers in peripheral tissues. Proteases that activate PAR2 also induce the release of neuropeptides from the central projections of these nerves in the dorsal horn of the spinal cord, where they participate in pain transmission. Accumulating evidence from PAR-deficient mice indicates that these mechanisms may contribute to experimental models of disease and raise the possibility that protease inhibitors and PAR antagonists may be useful therapies for a variety of inflammatory and painful conditions.     

Protease-activated receptors mediate apamin-sensitive relaxation of mouse and guinea pig gastrointestinal smooth muscle

BACKGROUND & AIMS: Protease-activated receptor (PAR)-1 and PAR-2 are expressed on gastrointestinal smooth muscle, but knowledge of their functionality is limited. The aim of this study was to determine if PAR-1 and PAR-2 mediate gastrointestinal smooth muscle relaxation and to clarify the underlying mechanisms. METHODS: Responses to PAR activation using the serine proteases thrombin and trypsin and the peptide agonists for PAR-1 and PAR-2, SFLLRN-NH2 and SLIGRL-NH2, respectively, were investigated in submaximally contracted longitudinal strips of mouse gastric fundus and guinea pig taenia coli. RESULTS: In mouse gastric fundus, both thrombin and trypsin caused relaxations followed by contractions. SFLLRN-NH2 and SLIGRL-NH2 caused similar biphasic responses, the relaxation components of which were eliminated by apamin or ryanodine. For SFLLRN-NH2, apamin and ryanodine revealed contractions. Nifedipine inhibited both relaxations and contractions to each peptide. In guinea-pig taenia coli, thrombin but not trypsin caused relaxation, whereas SFLLRN-NH2 and SLIGRL-NH2 caused concentration-dependent relaxations that were eliminated by apamin but were unaffected by ryanodine. CONCLUSIONS: The mouse gastric fundus and guinea pig taenia coli contain functional PAR-1 and PAR-2 that mediate relaxations via ryanodine-sensitive and -insensitive activation of small-conductance, Ca2+-activated K+ channels. We propose that smooth muscle PARs act as sensors for inflammatory signals in gut and respond by inhibiting gut motility during peritoneal infections or tissue damage.     

Orchestration of coagulation protease signaling by tissue factor

Protease-activated receptors (PARs) are vascular sensors for signaling of the trypsinlike coagulation serine proteases that play key roles in cardiovascular medicine. In the initiation phase of coagulation, tissue factor (TF) orchestrates the assembly of VIIa with substrate X, forming a ternary complex in which product Xa is generated. The resulting TF-VIIa-Xa complex is an efficient activator of PAR1 and PAR2. TF initiation of the coagulation cascade is thus intimately linked to inflammatory cell signaling. Inflammation is an increasingly appreciated component of the vulnerable atherosclerotic plaque. Targeting inflammatory cell signaling events of the coagulation system may become an important aspect of efforts to improve antithrombotic therapy.     

Protease-activated receptor 2 promotes experimental liver fibrosis in mice and activates human hepatic stellate cells

Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated after proteolytic cleavage by serine proteases, including mast cell tryptase and activated coagulation factors. PAR-2 activation augments inflammatory and profibrotic pathways through the induction of genes encoding proinflammatory cytokines and extracellular matrix proteins. Thus, PAR-2 represents an important interface linking coagulation and inflammation. PAR-2 is widely expressed in cells of the gastrointestinal tract, including hepatic stellate cells (HSCs), endothelial cells, and hepatic macrophages; however, its role in liver fibrosis has not been previously examined. We studied the development of CCl(4) -induced liver fibrosis in PAR-2 knockout mice, and showed that PAR-2 deficiency reduced the progression of liver fibrosis, hepatic collagen gene expression, and hydroxyproline content. Reduced fibrosis was associated with decreased transforming growth factor beta (TGFβ) gene and protein expression and decreased matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinase 1 gene expression. In addition, PAR-2 stimulated activation, proliferation, collagen production, and TGFβ protein production by human stellate cells, indicating that hepatic PAR-2 activation increases profibrogenic cytokines and collagen production both in vivo and in vitro. CONCLUSION: Our findings demonstrate the capacity of PAR-2 activation to augment TGFβ production and promote hepatic fibrosis in mice and to induce a profibrogenic phenotype in human HSCs. PAR-2 antagonists have recently been developed and may represent a novel therapeutic approach in preventing fibrosis in patients with chronic liver disease.     

Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia

Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.     

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH(2), RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.     

Peptide-derived protease-activated receptor-1 (PAR-1) antagonists

Protease activated receptor-1 (PAR-1) is a G-coupled receptor cleaved by thrombin and other proteases to expose a new N-terminus, a "tethered ligand", that activates the receptor. Independently of proteolytic cleavage, peptides similar to the new N-terminus also activate the receptor, and structure activity relationships for the activating peptides have been extensively studied. Modification of activating peptides led to rationally designed peptide antagonists. The more potent peptide antagonists were N-terminal and 3-position modifications of the agonist peptides. The resulting PAR-1 antagonists have proved useful in pharmacological studies resolving the contribution of PAR-1 signaling mechanisms relative to other PARs in platelets, vascular endothelial and other cell types. High affinity peptide agonists and antagonists have been radiolabled and proven useful in binding assays. Screening of combinatorial libraries and compound collections using the radioligands have identified non-peptide antagonists of several different chemotypes. When the "thrombin receptor" (PAR-1) was first cloned and its mechanism of activation elucidated, there was great enthusiasm for the receptor as a drug target. The use of peptide agonists and antagonists has made possible much progress in our understanding of the role of this receptor.     

Protease-activated receptors in cardiovascular diseases

Thrombosis associated with the pathophysiological activation of platelets and vascular cells has brought thrombin and its receptors to the forefront of cardiovascular medicine. Thrombin signaling through the protease-activated receptors (PARs) has been shown to influence a wide range of physiological responses including platelet activation, intimal hyperplasia, inflammation, and maintenance of vascular tone and barrier function. The thrombin receptors PAR1 and PAR4 can be effectively targeted in animals in which acute or prolonged exposure to thrombin leads to thrombosis and/or restenosis. In the present study, we describe the molecular and pharmacological basis of small-molecule inhibitors that target PAR1. In addition, we discuss a new class of cell-penetrating inhibitors, termed pepducins, that provide insight into previously unidentified roles of PAR1 and PAR4 in protease signaling.     

Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1

Cell signaling by coagulation factor Xa (Xa) contributes to pro-inflammatory responses in vivo. This study characterizes the signaling mechanism of Xa in a HeLa cell line that expresses protease-activated receptor 1 (PAR-1) but not PAR-2, -3, or -4. Xa induced NF-kappaB in HeLa cells efficiently but with delayed kinetics compared to thrombin. This delay caused no difference in gene expression patterns, as determined by high-density microarray analysis. Both proteases prominently induced the angiogenesis-promoting gene Cyr61 and connective tissue growth factor. Inhibition of PAR-1 cleavage abolished MAP kinase phosphorylation and gene induction by Xa, demonstrating that Xa signals through PAR-1 and not through a novel member of the PAR family. Activation of cell surface prothrombin with the snake venom enzyme Ecarin also produced PAR-1-dependent signaling. However, though the response to Ecarin was completely blocked by the thrombin inhibitor hirudin, the response to Xa was not. This suggests that the Xa response is not mediated by locally generated thrombin. The concentration dependence of Xa for PAR-1 activation is consistent with previously characterized Xa-mediated PAR-2 signaling, suggesting that local concentration of Xa on the cell surface, rather than sequence-specific recognition of the PAR scissile bond, determines receptor cleavage. This study demonstrates that PAR-1 cleavage by Xa can elicit the same cellular response as thrombin, but mechanistic differences in receptor recognition may be crucial for specific roles for Xa in signaling during spatial or temporal separation from thrombin generation.