Immunomodulatory role of thrombin in cancer progression.

Coagulation proteases and the generation of thrombin are increased in tumors. In addition, chemotherapeutic agents commonly used to treat malignant cancers can exacerbate cancer-associated thromboses. Thrombin can modify tumor cell behavior directly through the activation of protease-activated receptors (PAR) or indirectly by generating fibrin matrices. In addition to its role in generating fibrin to promote hemostasis, thrombin acts directly on multiple effector cells of the immune system impacting both acute and chronic inflammatory processes. Thrombin-mediated release of interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 leads to the accumulation of multiple tumor-infiltrating immunosuppressive cell populations including myeloid derived suppresser cells, M2-like macrophages, and T regulatory cells. Ablation of PAR-1 from the tumor microenvironment, but not the tumor, has been shown to dramatically reduce tumor growth and metastasis in multiple tumor models. Thrombin-activated platelets release immunosuppressive cytokines including transforming growth factor-ß that can inhibit natural killer cell activity, helping tumor cells to evade host immunosurveillance. Taken together, there is strong evidence that thrombin influences cancer progression via multiple mechanisms, including the tumor immune response, with thrombin emerging as a target for novel therapeutic strategies for cancer.

Thrombin-Par1 signaling axis disrupts COP9 signalosome subunit 3-mediated ABCA1 stabilization in inducing foam cell formation and atherogenesis.

ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) play a vital role in promoting cholesterol efflux. Although, the dysregulation of these transporters was attributed as one of the mechanisms of atherogenesis, what renders their dysfunction is not well explored. Previously, we have reported that thrombin without having any effect on ABCG1 levels depletes ABCA1 levels affecting cholesterol efflux. In this study, we explored the mechanisms underlying thrombin-induced depletion of ABCA1 levels both in macrophages and smooth muscle cells. Under normal physiological conditions, COP9 signalosome subunit 3 (CSN3) was found to exist in complex with ABCA1 and in the presence of proatherogenic stimulants such as thrombin, ABCA1 was phosphorylated and dissociated from CSN3, leading to its degradation. Forced expression of CSN3 inhibited thrombin-induced ABCA1 ubiquitination and degradation, restored cholesterol efflux and suppressed foam cell formation. In Western diet (WD)-fed ApoE-/- mice, CSN3 was also disassociated from ABCA1 otherwise remained as a complex in Chow diet (CD)-fed ApoE-/- mice. Interestingly, depletion of CSN3 levels in WD-fed ApoE-/- mice significantly lowered ABCA1 levels, inhibited cholesterol efflux and intensified foam cell formation exacerbating the lipid laden atherosclerotic plaque formation. Mechanistic studies have revealed the involvement of Par1-Gα12-Pyk2-Gab1-PKCθ signaling in triggering phosphorylation of ABCA1 and its disassociation from CSN3 curtailing cholesterol efflux and amplifying foam cell formation. In addition, although both CSN3 and ABCA1 were found to be colocalized in human non-lesion coronary arteries, their levels were decreased as well as dissociated from each other in advanced atherosclerotic lesions. Together, these observations reveal for the first time an anti-atherogenic role of CSN3 and hence, designing therapeutic drugs protecting its interactions with ABCA1 could be beneficial against atherosclerosis.

Ankaferd hemostat (ABS) as a potential mucosal topical agent for the management of COVID-19 syndrome based on its PAR-1 inhibitory effect and oestrogen content.

COVID-19 due to the SARS-CoV-2 infection is a multi-systemic immune syndrome affecting mainly the lungs, oropharyngeal region, and other vascular endothelial beds. There are tremendous ongoing efforts for the aim of developing drugs against the COVID-19 syndrome-associated inflammation. However, currently no specific medicine is present for the absolute pharmacological cure of COVID-19 mucositis. The re-purposing/re-positioning of already existing drugs is a very important strategy for the management of ongoing pandemy since the development of a new drug needs decades. Apart from altering angiotensin signaling pathways, novel drug candidates for re-purposing comprise medications shall target COVID-19 pathobiology, including pharmaceutical formulations that antagonize proteinase-activated receptors (PARs), mainly PAR-1. Activation of the PAR-1, mediators and hormones impact on the hemostasis, endothelial activation, alveolar epithelial cells and mucosal inflammatory responses which are the essentials of the COVID-19 pathophysiology. In this context, Ankaferd hemostat (Ankaferd Blood Stopper, ABS) which is an already approved hemostatic agent affecting via vital erythroid aggregation and fibrinogen gamma could be a potential topical remedy for the mucosal management of COVID-19. ABS is a clinically safe and effective topical hemostatic agent of plant origin capable of exerting pleiotropic effects on the endothelial cells, angiogenesis, cell proliferation and vascular dynamics. ABS had been approved as a topically applied hemostatic agent for the management of post-surgical/dental bleedings and healing of infected inflammatory mucosal wounds. The anti-inflammatory and proteinase-activated receptor axis properties of ABS with a considerable amount of oestrogenic hormone presence highlight this unique topical hemostatic drug regarding the clinical re-positioning for COVID-19-associated mucositis. Topical ABS as a biological response modifier may lessen SARS-CoV-2 associated microthrombosis, endothelial dysfunction, oropharyngeal inflammation and mucosal lung damage. Moreover, PAR-1 inhibition ability of ABS might be helpful for reducing the initial virus propagation and mocasal spread of COVID-19.

Arrestin domain containing 3 promotes Helicobacter pylori-associated gastritis by regulating protease-activated receptor 1.

Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation, and cancer. Here, we demonstrate a proinflammation role of ARRDC3 in Helicobacter pylori-associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM-derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C-Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in Arrdc3-/- mice but increased in protease-activated receptor 1-/- (Par1-/-) mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori-associated gastritis. This study identifies a regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a proinflammatory effect within the gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may provide valuable strategies in treating of H. pylori-associated gastritis.

Thrombin Signaling Promotes Pancreatic Adenocarcinoma through PAR-1-Dependent Immune Evasion.

Pancreatic ductal adenocarcinoma (PDAC) is associated with robust activity of the coagulation system. To determine mechanisms by which clotting factors influence PDAC tumor progression, we generated and characterized C57Bl/6-derived KPC (KRasG12D, TRP53R172H ) cell lines. Tissue factor (TF) and protease-activated receptor-1 (PAR-1) were highly expressed in primary KPC pancreatic lesions and KPC cell lines similar to expression profiles observed in biopsies of patients with PDAC. In allograft studies, tumor growth and metastatic potential were significantly diminished by depletion of TF or Par-1 in cancer cells or by genetic or pharmacologic reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1-deleted KPC cells (KPC-Par-1KO) failed to generate sizable tumors, a phenotype completely rescued by restoration of Par-1 expression. Expression profiling of KPC and KPC-Par-1KO cells indicated that thrombin-PAR-1 signaling significantly altered immune regulation pathways. Accordingly, KPC-Par-1KO cells failed to form tumors in immune-competent mice but displayed robust tumor growth comparable to that observed with control KPC cells in immune-compromised NSG mice. Immune cell depletion studies indicated that CD8 T cells, but not CD4 cells or natural killer cells, mediated elimination of KPC-Par-1KO tumor cells in C57Bl/6 mice. These results demonstrate that PDAC is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1 and further indicate that one key mechanism of thrombin/PAR-1-mediated tumor growth is suppression of antitumor immunity in the tumor microenvironment. SIGNIFICANCE: The tissue factor-thrombin-PAR-1 signaling axis in tumor cells promotes PDAC growth and disease progression with one key mechanism being suppression of antitumor immunity in the microenvironment.

Coagulation management in patients undergoing neurosurgical procedures.

PURPOSE OF REVIEW: Management of coagulation in neurosurgical procedures is challenging. In this contest, it is imperative to avoid further intracranial bleeding. Perioperative bleeding can be associated with a number of factors, including anticoagulant drugs and coagulation status but is also linked to the characteristic and the site of the intracranial disorder. The aim of this review will be to focus primarily on the new evidence regarding the management of coagulation in patients undergoing craniotomy for neurosurgical procedures. RECENT FINDINGS: Antihemostatic and anticoagulant drugs have shown to be associated with perioperative bleeding. On the other hand, an increased risk of venous thromboembolism and hypercoagulative state after elective and emergency neurosurgery, in particular after brain tumor surgery, has been described in several patients. To balance the risk between thrombosis and bleeding, it is important to be familiar with the perioperative changes in coagulation and with the recent management guidelines for anticoagulated patients undergoing neurosurgical procedures, in particular for those taking new direct anticoagulants. We have considered the current clinical trials and literature regarding both safety and efficacy of deep venous thrombosis prophylaxis in the neurosurgical population. These were mainly trials concerning both elective surgical and intensive care patients with a poor grade intracranial bleed or multiple traumas with an associated severe traumatic brain injury (TBI). SUMMARY: Coagulation management remains a major issue in patients undergoing neurosurgical procedures. However, in this field of research, literature quality is poor and further studies are necessary to identify the best strategies to minimize risks in this group of patients.

Thrombin is a selective inducer of heparanase release from platelets and granulocytes via protease-activated receptor-1.

Heparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.

Dysregulated protease activated receptor 1 (PAR1) promotes metastatic phenotype in breast cancer through HMGA2.

As the majority of patients with basal-like breast carcinoma present with invasive, metastatic disease that do not respond to available therapies, it is essential to identify new therapeutic targets that impact invasion and metastasis. Protease-activated receptor 1 (PAR1), a G-protein coupled receptor has been shown to act as an oncogene, but underlying mechanisms are not well understood. Here, we show that ectopic expression of functionally active PAR1 in MCF-7 cells induced a hormone-refractory, invasive phenotype representative of advanced basal-like breast carcinoma that readily formed metastatic lesions in lungs of mice. PAR1 was found to globally upregulate mesenchymal markers, including vimentin, a direct target of PAR1, and downregulate the epithelial markers including E-cadherin, as well as estrogen receptor. In contrast, non-signaling PAR1 mutant receptor did not lead to an invasive, hormone refractory phenotype. PAR1 expression increased spheroid formation and the level of stemness markers and self-renewal capacity in human breast cancer cells. We identified HMGA2 (high mobility group A2) as an important regulator of PAR1-mediated invasion. Inhibition of PAR1 signaling suppresses HMGA2-driven invasion in breast cancer cells. HMGA2 gene and protein are highly expressed in metastatic breast cancer cells. Overall, our results show that PAR1/HMGA2 pathway may present a novel therapeutic target.

Colon Cancer Growth and Dissemination Relies upon Thrombin, Stromal PAR-1, and Fibrinogen.

Thrombin-mediated proteolysis is a major determinant of metastasis, but is not universally important for primary tumor growth. Here, we report that colorectal adenocarcinoma represents one important exception whereby thrombin-mediated functions support both primary tumor growth and metastasis. In contrast with studies of multiple nongastrointestinal cancers, we found that the growth of primary tumors formed by murine and human colon cancer cells was reduced in mice by genetic or pharmacologic reduction of circulating prothrombin. Reduced prothrombin expression was associated with lower mitotic indices and invasion of surrounding tissue. Mechanistic investigations revealed that thrombin-driven colonic adenocarcinoma growth relied upon at least two targets of thrombin-mediated proteolysis, protease-activated receptor-1 (PAR-1) expressed by stromal cells and the extracellular matrix protein, fibrinogen. Colonic adenocarcinoma growth was reduced in PAR-1-deficient mice, implicating stromal cell-associated PAR-1 as one thrombin target important for tumor outgrowth. Furthermore, tumor growth was dramatically impeded in fibrinogen-deficient mice, offering the first direct evidence of a critical functional role for fibrinogen in malignant tumor growth. Tumors harvested from fibrinogen-deficient mice displayed a relative reduction in cell proliferative indices, as well as increased tumor necrosis and decreased tumor vascular density. Collectively, our findings established a functional role for thrombin and its targets PAR-1 and fibrinogen in the pathogenesis of colonic adenocarcinoma, supporting tumor growth as well as local invasion and metastasis.

The inflammatory actions of coagulant and fibrinolytic proteases in disease.

Aside from their role in hemostasis, coagulant and fibrinolytic proteases are important mediators of inflammation in diseases such as asthma, atherosclerosis, rheumatoid arthritis, and cancer. The blood circulating zymogens of these proteases enter damaged tissue as a consequence of vascular leak or rupture to become activated and contribute to extravascular coagulation or fibrinolysis. The coagulants, factor Xa (FXa), factor VIIa (FVIIa), tissue factor, and thrombin, also evoke cell-mediated actions on structural cells (e.g., fibroblasts and smooth muscle cells) or inflammatory cells (e.g., macrophages) via the proteolytic activation of protease-activated receptors (PARs). Plasmin, the principle enzymatic mediator of fibrinolysis, also forms toll-like receptor-4 (TLR-4) activating fibrin degradation products (FDPs) and can release latent-matrix bound growth factors such as transforming growth factor-ß (TGF-ß). Furthermore, the proteases that convert plasminogen into plasmin (e.g., urokinase plasminogen activator) evoke plasmin-independent proinflammatory actions involving coreceptor activation. Selectively targeting the receptor-mediated actions of hemostatic proteases is a strategy that may be used to treat inflammatory disease without the bleeding complications of conventional anticoagulant therapies. The mechanisms by which proteases of the coagulant and fibrinolytic systems contribute to extravascular inflammation in disease will be considered in this review.

A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.

BACKGROUND: The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. OBJECTIVES: To determine the functional consequences of the R122C substitution for P2Y(12) function. PATIENT/METHODS: We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed. RESULTS: ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. CONCLUSIONS: Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait.

Differential contribution of FXa and thrombin to vascular inflammation in a mouse model of sickle cell disease.

Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.

Protease-activated receptor-1 drives pancreatic cancer progression and chemoresistance.

Protease activated receptor (PAR)-1 expression in tumor cells is associated with disease progression and overall survival in a variety of cancers of epithelial origin; however, the importance of PAR-1 in the tumor microenvironment remains unexplored. Utilizing an orthotopic pancreatic cancer model in which tumor cells are PAR-1 positive whereas stromal cells are PAR-1 negative, we show that PAR-1 expression in the microenvironment drives progression and induces chemoresistance of pancreatic cancer. PAR-1 enhances monocyte recruitment into the tumor microenvironment by regulating monocyte migration and fibroblast dependent chemokine production thereby inducing chemoresistance. Overall, our data identify a novel role of PAR-1 in the pancreatic tumor microenvironment and suggest that PAR-1 may be an attractive target to reduce drug resistance in pancreatic cancer.

Increased responsiveness to thrombin through protease-activated receptors (PAR)-1 and -4 in active Crohn's disease.

BACKGROUND AND AIMS: Platelets are essential in hemostasis and inflammation, thereby linking coagulation with inflammation. Abundant thrombin generation in association with inflammation is considered a major reason for the increased risk for thromboembolic events. We therefore investigated platelet responsiveness to thrombin. METHODS: In this case-control study 85 patients with Crohn's disease (active CD 42, remission 43) and 30 sex- and age-matched controls were enrolled. Clinical disease activity (Harvey-Bradshaw-Index) was assessed and CD-related data were determined by chart review. Platelets' response to protease activated receptor-1 and -4 (PAR-1, -4) was assessed by whole blood platelet aggregometry (MEA), levels of platelets adhering to monocytes (PMA), and platelet surface P-selectin. RESULTS: Platelets' aggregation after activation with the specific PAR-1 agonist (SFLLRN) and PAR-4 agonist (AYPGKF) was higher in patients with active CD compared to patients in remission and controls (p=0.0068 and p=0.0023 for SFLLRN, p=0.0019 and 0.0003 for AYPGKF). Likewise, levels of PMA after activation with PAR-1 and PAR-4 receptor agonists were higher in patients with active CD compared to patients in remission and controls (p=0.0001 and p<0.0001 for SFLLRN, p=0.0329 and p=0.0125 for AYPGKF). However, P-selectin expression on human platelets showed heterogeneous results. Only PAR-1 activation of platelets resulted in significant differences between CD patients and controls (p=0.0001 and p=0.0022 for active and inactive CD versus controls, respectively). CONCLUSIONS: Our data suggest a new mechanism of platelet activation which has the potential to increase risk for thromboembolism in patients with active CD which might be due to platelets poised for thrombin-inducible activation.

Inflammation, obesity, and thrombosis.

Clinical and epidemiological studies support a connection between obesity and thrombosis, involving elevated expression of the prothrombotic molecules plasminogen activator inhibitor-1 and tissue factor (TF) and increased platelet activation. Cardiovascular diseases and metabolic syndrome-associated disorders, including obesity, insulin resistance, type 2 diabetes, and hepatic steatosis, involve inflammation elicited by infiltration and activation of immune cells, particularly macrophages, into adipose tissue. Although TF has been clearly linked to a procoagulant state in obesity, emerging genetic and pharmacologic evidence indicate that TF signaling via G protein-coupled protease-activated receptors (PAR2, PAR1) additionally drives multiple aspects of the metabolic syndrome. TF-PAR2 signaling in adipocytes contributes to diet-induced obesity by decreasing metabolism and energy expenditure, whereas TF-PAR2 signaling in hematopoietic and myeloid cells drives adipose tissue inflammation, hepatic steatosis, and insulin resistance. TF-initiated coagulation leading to thrombin-PAR1 signaling also contributes to diet-induced hepatic steatosis and inflammation in certain models. Thus, in obese patients, clinical markers of a prothrombotic state may indicate a risk for the development of complications of the metabolic syndrome. Furthermore, TF-induced signaling could provide new therapeutic targets for drug development at the intersection between obesity, inflammation, and thrombosis.

A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure.

Analysis of hematopoietic stem cells (HSCs) in factor VIII knockout (FVIIIKO) mice revealed a novel regulatory role for the coagulation cascade in hematopoiesis. Thus, HSCs in FVIIIKO mice had reduced proportions of CD34(low) cells within Lin(-)Sca(+)Kit(+) progenitors, and exhibited reduced long-term repopulating capacity as well as hyper granulocyte-colony-stimulating factor (G-CSF)-induced mobilization. This disregulation of HSCs is likely caused by reduced levels of thrombin, and is associated with altered protease-activated receptor 1 (PAR1) signaling, as PAR1 KO mice also exhibited enhanced G-CSF-induced mobilization. Analysis of reciprocal bone marrow (BM) chimera (FVIIIKO BM into wild-type recipients and vice versa) and the detection of PAR1 expression on stromal elements indicates that this phenotype is likely controlled by stromal elements. Micro-computed tomography analysis of distal tibia metaphyses also revealed for the first time a major impact of the FVIII/thrombin/PAR1 axis on the dynamic bone structure, showing reduced bone:tissue volume ratio and trabecular number in FVIIIKO and PAR1KO mice. Taken together, these results show a critical and novel role for the coagulation cascade, mediated in part by thrombin-PAR1 interaction, and regulates HSC maintenance and a reciprocal interplay between HSCs and the dynamic bone structure.

Protease-activated receptor (PAR)2, but not PAR1, is involved in collateral formation and anti-inflammatory monocyte polarization in a mouse hind limb ischemia model.

AIMS: In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model. METHODS AND RESULTS: PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered. CONCLUSION: PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patients.

A critical role of thrombin/PAR-1 in ADP-induced platelet secretion and the second wave of aggregation.

BACKGROUND: The stable or second wave of platelet aggregation often observed in ADP-stimulated platelet-rich plasma (PRP) with an artificially lowered extracellular calcium level has been attributed to enhanced thromboxane A2 (TXA2 ) generation and inhibition of ectonucleotidase activity. However, the role of thrombin in ADP-induced platelet secretion and the second wave of aggregation is unknown. OBJECTIVES AND METHODS: We employed aggregometry, flow cytometry, immunoblotting and ELISA to determine whether and how thrombin participates in ADP-induced platelet secretion and the second wave of aggregation. RESULTS: ADP induces a phosphoinositide 3-kinase (PI3K) pathway-dependent thrombin generation, presumably resulting from the cleavage of αII b ß3 -associated prothrombin. Generated thrombin subsequently activates protease-activated receptor-1 (PAR-1) and mediates dense granule secretion and the second wave of platelet aggregation in ADP-stimulated citrated PRP. Thus, ADP-induced dense granule secretion and the second wave of platelet aggregation in PRP were similarly and non-additively blocked by thrombin inhibitor hirudin, PAR-1 antagonist SCH-79797 or PI3K inhibitor wortmannin. Moreover, ADP stimulation caused the dissociation of prothrombin from αII b ß3 and an increased plasma thrombin level; both were prevented by wortmannin. Furthermore, the wortmannin-inhibited second wave of platelet aggregation by ADP was restored by a subaggregation concentration of PAR-1 activating peptide SFLLRN. Blocking TXA2 production with indomethacin or restoring extracellular calcium to physiological concentration did not influence this thrombin/PAR-1 dependence. CONCLUSIONS: A PI3K-dependent thrombin generation and the resultant PAR-1 activation serve as an indispensable mechanism to relay the platelet activation process induced by ADP.

Thrombin promotes matrix metalloproteinase-13 expression through the PKCδ c-Src/EGFR/PI3K/Akt/AP-1 signaling pathway in human chondrocytes.

Thrombin is a key mediator of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of rheumatoid arthritis and osteoarthritis. Matrix metalloproteinase-13 (MMP-13) may contribute to the breakdown of articular cartilage during arthritis. However, the role of thrombin in MMP-13 production in chondrocytes is unknown. In this study, we investigated the intracellular signaling pathways involved in thrombin-induced MMP-13 expression in human chondrocytes. We found that stimulation with thrombin led to increased secretion of MMP-13 in cultured human chondrocytes. Further, this thrombin-induced MMP-13 production was reduced after transfection with siRNAs against protease activated receptors 1 and 3 (PAR1 and PAR3), but not with PAR4 siRNA. Treatment with specific inhibitors for PKCδ, c-Src, EGFR, PI3K, Akt, or AP-1 or with the corresponding siRNAs against these signaling proteins also abolished the thrombin-mediated increase in MMP-13 production in chondrocytes. Our results provide evidence that thrombin acts through the PAR1/PAR3 receptors and activates PKCδ and c-Src, resulting in EGFR transactivation and activation of PI3K, Akt, and finally AP-1 on the MMP-13 promoter, thereby contributing to cartilage destruction during arthritis.

Agonist stimulation, talin-1, and kindlin-3 are crucial for α(IIb)ß(3) activation in a human megakaryoblastic cell line, CMK.

Platelet integrin α(IIb)ß(3) activation is regulated by inside-out signaling via agonist stimulation. However, when α(IIb)ß(3) was exogenously expressed in cell lines such as Chinese hamster ovarian cells, physiological agonists hardly induced α(IIb)ß(3) activation. To overcome this disadvantage, we characterized the functional regulation of endogenously expressed α(IIb)ß(3) in a megakaryoblastic cell line, CMK, employing an initial velocity assay for PAC-1 binding. We firstly demonstrated that protease-activated receptor 1-activating peptide induced robust, but transient α(IIb)ß(3) activation in CMK cells with high glycoprotein-Ib expression. Stable talin-1 or kindlin-3 knockdown cells confirmed that the protease-activated receptor 1-activating peptide-induced α(IIb)ß(3) activation was dependent on talin-1 and kindlin-3 expression. In sharp contrast to exogenously expressed α(IIb)ß(3) in Chinese hamster ovarian cells, transient overexpression of full-length talin (FL-talin) or talin-head domain (THD) alone did not activate α(IIb)ß(3) in CMK cells, but required agonist stimulation. Similarly, kindlin-3 overexpression alone did not induce α(IIb)ß(3) activation, but it significantly augmented agonist-induced α(IIb)ß(3) activation. Several mutants of FL-talin and THD suggested that the head-rod interaction was critical for autoinhibition of talin-1, and the interaction between the THD and the membrane-proximal region of the ß(3) cytoplasmic tail was essential for talin-mediated α(IIb)ß(3) activation. In addition, THD and kindlin-3 cooperatively augmented protease-activated receptor 1-induced α(IIb)ß(3) activation. We proposed that the CMK cell line is an attractive platform for investigating agonist-, talin-1-, and kindlin-3- dependent α(IIb)ß(3) activation.