Endothelial Protein C Receptor and 3K3A-Activated Protein C Protect Mice from Allergic Contact Dermatitis in a Contact Hypersensitivity Model.

Endothelial protein C receptor (EPCR) is a receptor for the natural anti-coagulant activated protein C (aPC). It mediates the anti-inflammatory and barrier-protective functions of aPC through the cleavage of protease-activated receptor (PAR)1/2. Allergic contact dermatitis is a common skin disease characterized by inflammation and defective skin barrier. This study investigated the effect of EPCR and 3K3A-aPC on allergic contact dermatitis using a contact hypersensitivity (CHS) model. CHS was induced using 1-Fluoro-2,4-dinitrobenzene in EPCR-deficient (KO) and matched wild-type mice and mice treated with 3K3A-aPC, a mutant form of aPC with diminished anti-coagulant activity. Changes in clinical and histological features, cytokines, and immune cells were examined. EPCRKO mice displayed more severe CHS, with increased immune cell infiltration in the skin and higher levels of inflammatory cytokines and IgE than wild-type mice. EPCR, aPC, and PAR1/2 were expressed by the skin epidermis, with EPCR presenting almost exclusively in the basal layer. EPCRKO increased the epidermal expression of aPC and PAR1, whereas in CHS, their expression was reduced compared to wild-type mice. 3K3A-aPC reduced CHS severity in wild-type and EPCRKO mice by suppressing immune cell infiltration/activation and inflammatory cytokines. In summary, EPCRKO exacerbated CHS, whereas 3K3A-aPC could reduce the severity of CHS in both EPCRKO and wild-type mice.

Procoagulant platelets: Laboratory detection and clinical significance.

Platelets play a critical role in both haemostasis and thrombosis, and it is now evident that not all platelets behave the same when they are called to action. A functionally distinct subpopulation of platelets forms in response to maximal agonist stimulation: the procoagulant platelet. This platelet subpopulation is defined by its ability to expose phosphatidylserine on its surface, allowing for coagulation factor complexes to form and generate bursts of thrombin and fibrin to stabilize platelet clots. Reduced levels of procoagulant platelets have been linked to bleeding in Scott's syndrome and haemophilia A patients, and elevated levels have been demonstrated in many thrombotic disorders, including identifying patients at higher risk for stroke recurrence. One obstacle for incorporating an assay for measuring procoagulant platelets into clinical management algorithms is the lack of consensus on the exact definition and markers for this subpopulation. This review will outline the biological characteristics of procoagulant platelets and the laboratory assays currently used to identify them in research settings. It will summarize the findings of clinical research demonstrating the relevance of measuring the procoagulant platelet levels in patients and will discuss how an appropriate assay can be used to elucidate the mechanism behind the formation of this subpopulation, facilitating novel drug discovery to improve upon current outcomes in cardiovascular and other thrombotic disorders.

Remote ischemic preconditioning inhibits platelet αIIb ß3 activation in coronary artery disease patients receiving dual antiplatelet therapy: A randomized trial.

OBJECTIVES: We investigated whether remote ischemic preconditioning (RIPC) inhibits agonist-induced conformational activation of platelet αIIb ß3 in patients with coronary artery disease already receiving conventional antiplatelet therapy. PATIENTS/METHODS: Consecutive patients with angiographically confirmed coronary artery disease were randomized to RIPC or sham treatment. Venous blood was collected before and immediately after RIPC/sham. Platelet aggregometry (ADP, arachidonic acid) and whole blood platelet flow cytometry was performed for CD62P, CD63, active αIIb ß3 (PAC-1 binding) before and after stimulation with ADP, thrombin ± collagen, or PAR-1 thrombin receptor agonist. RESULTS: Patients (25 RIPC, 23 sham) were well matched, 83% male, age (mean ± standard deviation) 63.3 ± 13.2 years, 95% aspirin, 81% P2Y12 inhibitor. RIPC did not affect platelet aggregation, nor agonist-induced expression of CD62P, but selectively and significantly decreased αIIb ß3 activation after stimulation with either PAR-1 agonist peptide or the combination of thrombin + collagen, but not after ADP nor thrombin alone. The effect of RIPC on platelet αIIb ß3 activation was evident in patients receiving both aspirin and P2Y12 inhibitor, and was not associated with an increase in vasodilator-stimulated phosphoprotein phosphorylation. CONCLUSIONS: Remote ischemic preconditioning inhibits conformational activation of platelet αIIb ß3 in response to exposure to thrombin and collagen in patients with coronary artery disease receiving dual antiplatelet therapy. These findings indicate agonist-specific inhibition of platelet activation by RIPC in coronary artery disease that is not obviated by the prior use of P2Y12 inhibitors.

Variable phenotypic penetrance of thrombosis in adult mice after tissue-selective and temporally controlled Thbd gene inactivation.

Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd-/-) mice to term. Half of the Thbd-/- offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd-/- animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd-/- mice. However, Thbd-/- females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy.

Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice.

Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow-derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

Pharmacological targeting of the thrombomodulin-activated protein C pathway mitigates radiation toxicity.

Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)-activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.