PAR-4 Deficiency Research Articles

Genetic deletion of platelet PAR4 results in reduced thrombosis and impaired hemostatic plug stability

BackgroundProtease‐activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis. ObjectiveWe aimed to generate a mouse line with a megakaryocyte/platelet‐specific deletion of PAR4 (PAR4fl/fl;PF4Cre+) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice. MethodsPlatelets from PAR4fl/fl;PF4Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet‐specific deletion of PAR4 or wild‐type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors. ResultsPAR4fl/fl;PF4Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4−/− and PAR4fl/fl;PF4Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4−/− mice and PAR4fl/fl;PF4Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild‐type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability. ConclusionWe generated a novel PAR4fl/fl;PF4Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability.

Loss of Protease-Activated Receptor 4 Prevents Inflammation Resolution and Predisposes the Heart to Cardiac Rupture After Myocardial Infarction.

Cardiac rupture is a major lethal complication of acute myocardial infarction (MI). Despite significant advances in reperfusion strategies, mortality from cardiac rupture remains high. Studies suggest that cardiac rupture can be accelerated by thrombolytic therapy, but the relevance of this risk factor remains controversial. We analyzed protease-activated receptor 4 (Par4) expression in mouse hearts with MI and investigated the effects of Par4 deletion on cardiac remodeling and function after MI by echocardiography, quantitative immunohistochemistry, and flow cytometry. Par4 mRNA and protein levels were increased in mouse hearts after MI and in isolated cardiomyocytes in response to hypertrophic and inflammatory stimuli. Par4-deficient mice showed less myocyte apoptosis, reduced infarct size, and improved functional recovery after acute MI relative to wild-type (WT). Conversely, Par4-/- mice showed impaired cardiac function, greater rates of myocardial rupture, and increased mortality after chronic MI relative to WT. Pathological evaluation of hearts from Par4-/- mice demonstrated a greater infarct expansion, increased cardiac hemorrhage, and delayed neutrophil accumulation, which resulted in impaired post-MI healing compared with WT. Par4 deficiency also attenuated neutrophil apoptosis in vitro and after MI in vivo and impaired inflammation resolution in infarcted myocardium. Transfer of Par4-/- neutrophils, but not of Par4-/- platelets, in WT recipient mice delayed inflammation resolution, increased cardiac hemorrhage, and enhanced cardiac dysfunction. In parallel, adoptive transfer of WT neutrophils into Par4-/- mice restored inflammation resolution, reduced cardiac rupture incidence, and improved cardiac function after MI. These findings reveal essential roles of Par4 in neutrophil apoptosis and inflammation resolution during myocardial healing and point to Par4 inhibition as a potential therapy that should be limited to the acute phases of ischemic insult and avoided for long-term treatment after MI.

Abstract 53: Ablation of Thrombin Signaling in Platelets but Not in Endothelial Cells Impairs Hemostasis in a Mouse Model of Spontaneous Gastrointestinal Bleeding

Human PAR1 is expressed in endothelial cells as well as in platelets where it facilitates the response to thrombin and platelet activation. Vorapaxar, a PAR1 antagonist, prevents myocardial infarction and stroke in patients with prior MI or peripheral arterial disease at a cost of increased bleeding risk. Par1 is also highly expressed in endothelial cells in mice, and Par1-deficiency is associated with bleeding in the mouse embryo at midgestation. Additionally, known actions of endothelial PAR1 activation suggest pro-hemostatic functions. This raises the question of whether inhibition of PAR1 function in endothelial cells (in addition to PAR1 inhibition in platelets) contributes to the bleeding risk associated with Vorapaxar treatment. Our previous work demonstrated that Par1 deficiency results in loss of thrombin signaling in mouse endothelial cells but not mouse platelets, while Par4 deficiency ablated thrombin-induced platelet activation in mice. Thus, mice allow us to separate loss of thrombin signaling in platelets from loss of thrombin signaling in endothelial cells. Accordingly, we used Apc min/+ mice, which develop intestinal polyposis and spontaneous GI bleeding, as a model to determine whether loss of thrombin signaling in platelets (Par4 KO) or endothelial and other cells (Par1 KO) exacerbates spontaneous bleeding. Hematocrit and other hematologic parameters were measured biweekly from 7 weeks through 15 weeks of age. Hematocrits in mice wild-type for Apc were stable over this period (41.48 ± 0.48 at 7 weeks; 40.48 ± 0.37 at 15 weeks, n=15). Hematocrits in Apc min/+ mice fell approximately linearly from 37.06 ± 0.82 at 7 weeks to 14.39 ± 1.12 at 15 weeks (n=15). Hematocrits in Par1-deficient Apc min/+ mice were indistinguishable from those in Apc min/+ without Par deficiency (14.39 ± 1.12 vs 14.47 ± 1.66 at 15 weeks; n=6-15). By contrast, Par4-deficient Apc min/+ mice were already severely anemic at 7 weeks compared to Apc min/+ mice (19 ± 2.0 vs 39 ± 3.6; p<0.01, n=4). Par-dependent differences in polyp count and size were not detected. Taken together, our results suggest that loss of thrombin signaling in platelets promotes spontaneous GI bleeding in the Apc min model while loss of thrombin signaling in endothelial cells is without effect in this system.

A combined deficiency of tissue factor and PAR-4 is associated with fatal pulmonary hemorrhage in mice

IntroductionMice with a complete absence of tissue factor (TF) die during embryonic development whereas mice with low levels of TF (Low-TF mice) survive to adulthood. Low-TF mice exhibit spontaneous hemorrhage in various organs, including the lung. In contrast, mice can survive without protease-activated receptor (PAR)-4, which is the major thrombin receptor on mouse platelets. We determined the effect of combining a deficiency PAR-4 (primary hemostasis) with a deficiency in TF (secondary hemostasis) on embryonic development and survival of adult mice. Materials and methodsLow-TF mice (mTF−/−, hTF+/+) were crossed with PAR-4−/− mice to generate heterozygous mice (mTF+/−, hTF+/−, PAR-4+/−). These mice were intercrossed to generate Low-TF mice lacking PAR-4. Mice surviving to wean were genotyped and survival was monitored for 6months. ResultsWe observed the expected number of Low-TF,PAR-4−/− mice at wean indicating survival in utero and after birth. However, an absence of PAR-4 was associated with premature death of all Low-TF,PAR-4−/− mice in the 6month observational period. This compares with 40% death of the Low-TF,PAR-4+/+ mice (p=0.003). Low-TF,PAR-4+/− mice had an intermediate phenotype with 55% of the mice dying within 6months. The primary cause of mortality of Low-TF,PAR-4−/− mice was pulmonary hemorrhage. ConclusionsLow-TF,PAR-4−/− mice survive into adulthood, but combining a deficiency of primary hemostasis (PAR-4 deficiency) with secondary hemostasis (low levels of TF) leads to premature death primarily due to pulmonary hemorrhage.

Protease-activated receptor 4 deficiency offers cardioprotection after acute ischemia reperfusion injury

Protease-activated receptor (PAR)4 is a low affinity thrombin receptor with less understood function relative to PAR1. PAR4 is involved in platelet activation and hemostasis, but its specific actions on myocyte growth and cardiac function remain unknown. This study examined the role of PAR4 deficiency on cardioprotection after myocardial ischemia–reperfusion (IR) injury in mice. When challenged by in vivo or ex vivo IR, PAR4 knockout (KO) mice exhibited increased tolerance to injury, which was manifest as reduced infarct size and a more robust functional recovery compared to wild-type mice. PAR4 KO mice also showed reduced cardiomyocyte apoptosis and putative signaling shifts in survival pathways in response to IR. Inhibition of PAR4 expression in isolated cardiomyocytes by shRNA offered protection against thrombin and PAR4-agonist peptide-induced apoptosis, while overexpression of wild-type PAR4 significantly enhanced the susceptibility of cardiomyocytes to apoptosis, even under low thrombin concentrations. Further studies implicate Src- and epidermal growth factor receptor-dependent activation of JNK on the proapoptotic effect of PAR4 in cardiomyocytes. These findings reveal a pivotal role for PAR4 as a regulator of cardiomyocyte survival and point to PAR4 inhibition as a therapeutic target offering cardioprotection after acute IR injury.

Abstract 15433: Thrombin Activation of Platelet Protease-activated Receptor 4 Augments Atherosclerosis

Objective: Platelet activation has been shown to play a critical role in both formation and propagation of atherosclerosis. Protease-activated receptors (PARs) 1 and 4 mediate signaling by the coagulation protease thrombin. While PAR1 and 4 mediate thrombin activation on human platelets, mouse platelets contain PAR4 with PAR3 acting as a cofactor. Recent studies have demonstrated direct thrombin inhibitors (DTIs) reduced atherosclerosis in hypercholesterolemic mice. In this study, we examined the effect of PAR1 and 4 deficient on atherosclerosis using low-density lipoprotein receptor deficient (Ldlr -/- ) mice. Methods and Results: Ldlr -/- mice that were PAR1 +/+ , PAR1 -/- , PAR4 +/+ , or PAR4 -/- (n = 5-20 each group) were fed a fat and cholesterol-enriched diet for 12 weeks. PAR4 deficiency attenuated aortic root (65% decrease; P = 0.001) and aortic arch (71% decrease; P = 0.001) atherosclerosis with no effects on total plasma cholesterol concentrations or lipoprotein distributions. These reductions were attributable to hematopoietic-derived PAR4 from analysis of bone marrow experiments. To determine if PAR4 mediated all effects of thrombin signaling, Ldlr -/- /PAR4 +/+ or Ldlr -/- /PAR4 -/- mice were fed a fat and cholesterol-enriched diet, supplemented with placebo or the DTI dabigatran etexilate (30 g/kg diet) for 12 weeks. Dabigatran etexilate administration reduced atherosclerosis in PAR4 +/+ mice but not in PAR4 -/- mice. PAR1 deficiency had no effect on atherosclerotic burden. Conclusion: We demonstrated whole body and hematopoietic PAR4 deficiency resulted in decreased aortic root and sinus atherosclerosis. PAR1 deficiency had no effects on atherosclerosis. Thrombin inhibition did not alter atherosclerosis in PAR4 deficient mice. Together, these results suggest that thrombin activation of PAR4 on platelets contributes to atherosclerosis.

Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα

BackgroundPlatelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated. ObjectiveTo investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation. Methods and resultsHighly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins. ConclusionsBoth GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.

Platelets and protease-activated receptor-4 contribute to acetaminophen-induced liver injury in mice

AbstractAcetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity.

Coagulation‐driven platelet activation reduces cholestatic liver injury and fibrosis in mice

The coagulation cascade has been shown to participate in chronic liver injury and fibrosis, but the contribution of various thrombin targets, such as protease activated receptors (PARs) and fibrin(ogen), has not been fully described. Emerging evidence suggests that in some experimental settings of chronic liver injury, platelets can promote liver repair and inhibit liver fibrosis. However, the precise mechanisms linking coagulation and platelet function to hepatic tissue changes following injury remain poorly defined. To determine the role of PAR-4, a key thrombin receptor on mouse platelets, and fibrin(ogen) engagement of the platelet αII b β3 integrin (αIIb β3 ) in a model of cholestatic liver injury and fibrosis. Biliary and hepatic injury was characterized following 4 week administration of the bile duct toxicant α-naphthylisothiocyanate (ANIT) (0.025%) in PAR-4-deficient mice, mice expressing a mutant form of fibrin(ogen) incapable of binding integrin αII b β3 (Fibγ(Δ5) ), and wild-type mice. Elevated plasma thrombin-antithrombin and serotonin levels, hepatic fibrin deposition, and platelet accumulation in liver accompanied hepatocellular injury and fibrosis in ANIT-treated wild-type mice. PAR-4 deficiency reduced plasma serotonin levels, increased serum bile acid concentration, and exacerbated ANIT-induced hepatocellular injury and peribiliary fibrosis. Compared with PAR-4-deficient mice, ANIT-treated Fibγ(Δ5) mice displayed more widespread hepatocellular necrosis accompanied by marked inflammation, robust fibroblast activation, and extensive liver fibrosis. Collectively, the results indicate that PAR-4 and fibrin-αII b β3 integrin engagement, pathways coupling coagulation to platelet activation, each exert hepatoprotective effects during chronic cholestasis.

Redundancy and interaction of thrombin- and collagen-mediated platelet activation in tail bleeding and carotid thrombosis in mice.

Current antiplatelet strategies to prevent myocardial infarction and stroke are limited by bleeding risk. A better understanding of the roles of distinct platelet-activating pathways is needed. We determined whether platelet activation by 2 key primary activators, thrombin and collagen, plays distinct, redundant, or interacting roles in tail bleeding and carotid thrombosis in mice. Platelets from mice deficient for the thrombin receptor protease-activated receptor-4 (Par4) and the collagen receptor glycoprotein VI protein (GPVI) lack responses to thrombin and collagen, respectively. We examined tail bleeding and FeCl3-induced carotid artery occlusion in mice lacking Par4, GPVI, or both. We also examined a series of Par mutants with increasing impairment of thrombin signaling in platelets. Ablation of thrombin signaling alone by Par4 deficiency increased blood loss in the tail bleeding assay and impaired occlusive thrombus formation in the carotid occlusion assay. GPVI deficiency alone had no effect. Superimposing GPVI deficiency on Par4 deficiency markedly increased effect size in both assays. In contrast to complete ablation of thrombin signaling, 9- and 19-fold increases in EC50 for thrombin-induced platelet activation had only modest effects. The observation that loss of Par4 uncovered large effects of GPVI deficiency implies that Par4 and GPVI made independent, partially redundant contributions to occlusive thrombus formation in the carotid and to hemostatic clot formation in the tail under the experimental conditions examined. At face value, these results suggest that thrombin- and collagen-induced platelet activation can play partially redundant roles, despite important differences in how these agonists are made available to platelets.

PAR-4 Deficiency Rescues The Embryonic Lethal Phenotype Of TFPI Null Mice

BackgroundTissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor that dampens tissue factor (TF) initiated blood coagulation. It is produced by endothelial cells and megakaryocytes. Mice lacking TFPI activity die in utero from yolk sac hemorrhage or a presumptive consumptive coagulopathy. The embryonic lethal phenotype of TFPI null mice can be rescued by TF or factor VII deficiency. Our laboratory has shown that platelet TFPI limits platelet accumulation, but not fibrin formation, in an in vivo vascular injury model. Interestingly, mice lacking PAR4, the major thrombin receptor in mouse platelets, have decreased platelet accumulation but not fibrin formation following vascular injury. Therefore, we hypothesized that PAR-4 deficiency would counterbalance the absence of TFPI in platelets and rescue the embryonic lethal phenotype of TFPI null mice. Methods/ResultsTFPI heterozygous mice (TFPI+/-; C57/Bl6) were bred with PAR4 null mice (PAR4-/-; C57/Bl6) to generate TFPI+/-/PAR4-/- mice. These mice were bred to assess rescue of TFPI lethality. Of 76 pups closely monitored following birth, 12 (15.8%) were TFPI-/-/PAR4-/-, with 8 (10.5%) surviving to wean. TFPI-/-/PAR4-/- male mice were bred with TFPI+/-/PAR4-/- females for subsequent studies. Of 447 pups generated, 101 TFPI-/-/PAR4-/- mice (22.6%) were produced. Thus, TFPI-/-/PAR4-/- pups survive to wean at approximately one-half the expected frequency. TFPI-/-/PAR4-/- mice surviving to wean appeared healthy with no overt signs of thrombosis, and have apparently normal survival to over 12 months of age. Compared to TFPI+/-/PAR4-/- littermates, TFPI-/-/PAR4-/- have reduced body weight at 8 weeks. Female TFPI-/-/PAR4-/- mice can reproduce successfully. Approximately one-third of the TFPI-/-/PAR4-/- mice have short or totally absent tails. The short tail phenotype develops in utero and changes in tail:body proportion do not occur following birth. There was no difference in blood loss following tail clip between TFPI+/+/PAR4-/-, TFPI+/-/PAR4-/- or TFPI-/-/PAR4-/- mice. There also was no difference in fibrin or platelet accumulation between TFPI+/+/PAR4-/- or TFPI-/-/PAR4-/-mice following venous electrolytic injury. ConclusionsThis is the first reported rescue of the TFPI null embryonic lethal phenotype by a platelet activation defect. Almost one-half of the TFPI-/-/PAR4-/- mice survive through adulthood despite having normal tissue factor function not regulated by TFPI. This, and our inability, thus far, to identify a procoagulant state in the adult mice, suggests that an essential TFPI anticoagulant function is to modulate PAR-4 mediated platelet function; likely by limiting thrombin generation. TFPI may do this through inhibition of TF-fVIIa, or via inhibition of early forms of prothrombinase on the platelet surface, a very recently recognized inhibitory function of TFPI. The long term survival of these mice also demonstrates that unfettered TF-fVlla-fXa mediated signaling events through PAR-2 are not solely responsible for the embryonic lethality of TFPI null mice. However, the short or absent tails that arise during development of TFPI deficient mice suggests that TF and/or TFPI may have roles in notochord development that is separate from the coagulation system. Disclosures:Mast:Novo Nordisk: Honoraria, Research Funding.

Abstract 283: Platelet Inhibition Increases Angiotensin II-induced Abdominal Aortic Aneurysm Incidence and Rupture in Mice

Objective Platelets play a central role in both hemostasis and thrombosis. The coagulation protease thrombin activates platelets by cleavage of protease-activated receptors (PAR1 and PAR4 in humans, and PAR3 and PAR4 in mice). Circulating thrombin is increased in patients with abdominal aortic aneurysms (AAAs). We recently demonstrated that PAR4 deficiency in mice increased the incidence of aneurysm (P = 0.001) and rupture-induced death (P = 0.003) in an angiotensin II (AngII) infusion model of AAA. Furthermore, platelet depletion significantly increased rupture in this model (P = 0.048). The purpose of this study was to examine clinically used anti-platelet drugs in this mouse model of AAA. Methods and Results Male Ldlr -/- mice (8-12 weeks in age) were fed a fat and cholesterol-enriched diet (21% milk fat, 0.2% cholesterol). Groups of mice received either aspirin (30 mg/L via drinking water [ASA]), or diet supplemented with the direct thrombin inhibitor dabigatran etexilate (10 g/kg chow [DE]) or the P2Y 12 inhibitor clopidogrel (50 mg/kg/day [Plavix]) 1 week prior to and throughout AngII (1,000 ng/kg/min) infusion for 28 days. Drug bioavailability was confirmed with all treatments. Medial diameters in the suprarenal aortic region were increased significantly from baseline to day 28 in all groups infused with AngII, as measured by in vivo ultrasound. Medial diameters were not different in any of the treatment groups compared with placebo controls. However, DE (87% vs. 47%) and Plavix (82% vs. 40%) significantly increased the incidence of AAA versus placebo groups (P < 0.05). ASA also increased the incidence of AAA (93% vs. 70% P = NS). Importantly, all treatments had a significant increase in aortic rupture-induced death versus placebo groups (P < 0.05; DE [67% vs. 7%]; Plavix [41% vs. 0%]; and ASA [64% vs. 10%]). None of the treatments affected total plasma cholesterol, lipoprotein-cholesterol distributions, or AngII-induced increases in systolic blood pressure. Conclusion This study indicates that the presence of functional platelets reduces the formation and rupture of AAA in this mouse model. This suggests that inhibition of platelet function may be detrimental to patients with existing AAAs, a conclusion that will be addressed in future mouse studies.

Introduction to mini‐theme issue on “protease‐activated receptor signaling”

Introduction to mini‐theme issue on “protease‐activated receptor signaling”

Protease‐activated receptors and myocardial infarction

Protease-activated receptors (PARs) are widely expressed within the heart. They are activated by a myriad of proteases, including coagulation proteases. In vitro studies showed that activation of PAR-1 and PAR-2 on cardiomyocytes induced hypertrophy. In addition, PAR-1 stimulation on cardiac fibroblasts induced proliferation. Genetic and pharmacologic approaches have been used to investigate the role of the different PARs in cardiac ischemia/reperfusion (I/R) injury. In mice and rats, PAR-1 is reported to play a role in inflammation, infarct size, and remodeling after cardiac I/R injury. However, there are notable differences between the effect of a deficiency in PAR-1 and inhibition of PAR-1. For instance, inhibition of PAR-1 reduced infarct size whereas there was no effect of a deficiency of PAR-1. These differences maybe due to off-target effects of the inhibitor or PAR-4 compensation of PAR-1 deficiency. Similarly, a deficiency of PAR-2 was associated with reduced cardiac inflammation and improved heart function after I/R injury, whereas pharmacologic activation of PAR-2 was found to be protective due to increased vasodilatation. These differences maybe due to different signaling responses induced by an endogenous protease versus an exogenous agonist peptide. Surprisingly, PAR-4 deficiency resulted in increased cardiac injury and increased mortality after I/R injury. In contrast, a pharmacological study indicated that inhibition of PAR-4 was cardioprotective. It is possible that the major cellular target of the PAR-4 inhibitor is platelets, which have been shown to contribute to inflammation in the injured heart, whereas PAR-4 signaling in cardiomyocytes may be protective. These discrepant results between genetic and pharmacological approaches indicate that further studies are needed to determine the role of different PARs in the injured heart.

Maternal Par4 and platelets contribute to defective placenta formation in mouse embryos lacking thrombomodulin

AbstractAbsence of the blood coagulation inhibitor thrombomodulin (Thbd) from trophoblast cells of the mouse placenta causes a fatal arrest of placental morphogenesis. The pathogenesis of placental failure requires tissue factor, yet is not associated with increased thrombosis and persists in the absence of fibrinogen. Here, we examine the role of alternative targets of coagulation that might contribute to the placental failure and death of Thbd−/− embryos. We demonstrate that genetic deficiency of the protease-activated receptors, Par1 or Par2, in the embryo and trophoblast cells does not prevent the death of Thbd−/− embryos. Similarly, genetic ablation of the complement pathway or of maternal immune cell function does not decrease fetal loss. In contrast, Par4 deficiency of the mother, or the absence of maternal platelets, restores normal development in one-third of Thbd-null embryos. This finding generates new evidence implicating increased procoagulant activity and thrombin generation in the demise of thrombomodulin-null embryos, and suggests that platelets play a more prominent role in placental malfunction associated with the absence of thrombomodulin than fibrin formation. Our findings demonstrate that fetal prothrombotic mutations can cause localized activation of maternal platelets at the feto-maternal interface in a mother with normal hemostatic function.

The Caenorhabditis elegans AMP-activated Protein Kinase AAK-2 Is Phosphorylated by LKB1 and Is Required for Resistance to Oxidative Stress and for Normal Motility and Foraging Behavior

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.