Coagulation, Protease-Activated Receptors, and Diabetic Kidney Disease: Lessons from eNOS-Deficient Mice.

Endothelial nitric oxide synthase (eNOS) dysfunction is known to exacerbate the progression and prognosis of diabetic kidney disease (DKD). One of the mechanisms through which this is achieved is that low eNOS levels are associated with hypercoagulability, which promotes kidney injury. In the extrinsic coagulation cascade, the tissue factor (factor III) and downstream coagulation factors, such as active factor X (FXa), exacerbate inflammation through activation of the protease-activated receptors (PARs). Recently, it has been shown that the lack of or reduced eNOS expression in diabetic mice, as a model of advanced DKD, increases renal tissue factor levels and PAR1 and 2 expression in their kidneys. Furthermore, pharmaceutical inhibition or genetic deletion of coagulation factors or PARs ameliorated inflammation in DKD in mice lacking eNOS. In this review, we summarize the relationship between eNOS, coagulation, and PARs and propose a novel therapeutic option for the management of patients with DKD.

Kallikrein-related peptidase 6 orchestrates astrocyte form and function through proteinase activated receptor-dependent mechanisms.

Kallikrein-related peptidase 6 (Klk6) is the most abundant serine proteinase in the adult central nervous system (CNS), yet we know little regarding its physiological roles or mechanisms of action. Levels of Klk6 in the extracellular environment are dynamically regulated in CNS injury and disease positioning this secreted enzyme to affect cell behavior by potential receptor dependent and independent mechanisms. Here we show that recombinant Klk6 evokes increases in intracellular Ca2+ in primary astrocyte monolayer cultures through activation of proteinase activated receptor 1 (PAR1). In addition, Klk6 promoted a condensation of astrocyte cortical actin leading to an elongated stellate shape and multicellular aggregation in a manner that was dependent on the presence of either PAR1 or PAR2. Klk6-evoked changes in astrocyte shape were accompanied by translocation of ß-catenin from the plasma membrane to the cytoplasm. These data are exciting because they demonstrate that Klk6 can influence astrocyte plasticity through receptor-dependent mechanisms. Furthermore, this study expands our understanding of the mechanisms by which kallikreins can contribute to neural homeostasis and remodeling and point to both PAR1 and PAR2 as new therapeutic targets to modulate astrocyte form and function.

Silencing of protease-activated receptors attenuates synovitis and cartilage damage following a joint bleed in haemophilic mice.

INTRODUCTION AND AIM: Joint bleeding results in blood-induced arthropathy. We investigate whether a joint bleed alters protease-activated receptor (PAR) expression, and whether treatment with small interfering RNA (siRNA) targeted against PAR1-4 attenuates synovitis and cartilage damage. METHODS: Protease-activated receptor expression was evaluated upon a joint bleed in haemophilic mice and in humans. In addition, mice with a joint bleed were randomized between treatment with PAR1-4 siRNA or control and evaluated for the presence of synovitis and cartilage damage. Also, human cartilage was transfected with PAR1-4 siRNA or control, and evaluated for plasmin-induced cartilage damage. RESULTS: Following a joint bleed, we observed an increase in synovial PAR1, -2 and -4 expression, and an increase in chondrocyte PAR2 and -3 expression in mice (all P < 0.05). Also an increase in synovial PAR1 and chondrocyte PAR4 expression in patients was observed (both P < 0.05). Treatment of a joint bleed in haemophilic mice with PAR1-4 siRNA attenuates synovitis and cartilage damage (both P < 0.01). Treatment of human cartilage tissue explants with PAR1-4 siRNA reduced plasmin-induced cartilage damage (P < 0.01). CONCLUSION: This study demonstrates that synovial and chondrocyte PAR expression is altered upon a joint bleed, and that treatment with PAR1-4 siRNA attenuates synovitis and plasmin-induced cartilage damage.

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

OBJECTIVE: 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. APPROACH AND RESULTS: 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. CONCLUSIONS: 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.

Roles and interactions among protease-activated receptors and P2ry12 in hemostasis and thrombosis.

Toward understanding their redundancies and interactions in hemostasis and thrombosis, we examined the roles of thrombin receptors (protease-activated receptors, PARs) and the ADP receptor P2RY12 (purinergic receptor P2Y G protein-coupled 12) in human and mouse platelets ex vivo and in mouse models. Par3(-/-) and Par4(+/-) mouse platelets showed partially decreased responses to thrombin, resembling those in PAR1 antagonist-treated human platelets. P2ry12(+/-) mouse platelets showed partially decreased responses to ADP, resembling those in clopidogrel-treated human platelets. Par3(-/-) mice showed nearly complete protection against carotid artery thrombosis caused by low FeCl(3) injury. Par4(+/-) and P2ry12(+/-) mice showed partial protection. Increasing FeCl(3) injury abolished such protection; combining partial attenuation of thrombin and ADP signaling, as in Par3(-/-):P2ry12(+/-) mice, restored it. Par4(-/-) mice, which lack platelet thrombin responses, showed still better protection. Our data suggest that (i) the level of thrombin driving platelet activation and carotid thrombosis was low at low levels of arterial injury and increased along with the contribution of thrombin-independent pathways of platelet activation with increasing levels of injury; (ii) although P2ry12 acts downstream of PARs to amplify platelet responses to thrombin ex vivo, P2ry12 functioned in thrombin/PAR-independent pathways in our in vivo models; and (iii) P2ry12 signaling was more important than PAR signaling in hemostasis models; the converse was noted for arterial thrombosis models. These results make predictions being tested by ongoing human trials and suggest hypotheses for new antithrombotic strategies.

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

Absence 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.

Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth.

A fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.

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.