Activated protein C up-regulates procoagulant tissue factor activity on endothelial cells by shedding the TFPI Kunitz 1 domain.

Thrombin and activated protein C (APC) signaling can mediate opposite biologic responses in endothelial cells. Given that thrombin induces procoagulant tissue factor (TF), we examined how TF activity is affected by APC. Exogenous or endogenously generated APC led to increased TF-dependent factor Xa activity. Induction required APC's proteolytic activity and binding to endothelial cell protein C receptor but not protease activated receptors. APC did not affect total TF antigen expression or the availability of anionic phospholipids on the apical cell membrane. Western blotting and cell surface immunoassays demonstrated that APC sheds the Kunitz 1 domain from tissue factor pathway inhibitor (TFPI). A TFPI Lys86Ala mutation between the Kunitz 1 and 2 domains eliminated both cleavage and the enhanced TF activity in response to APC in overexpression studies, indicating that APC up-regulates TF activity by endothelial cell protein C receptor-dependent shedding of the Kunitz 1 domain from membrane-associated TFPI. Our results demonstrate an unexpected procoagulant role of the protein C pathway that may have important implications for the regulation of TF- and TFPI-dependent biologic responses and for fine tuning of the hemostatic balance in the vascular system.

Protection of vascular barrier integrity by activated protein C in murine models depends on protease-activated receptor-1.

Protease activated receptor-1 (PAR1) mediates barrier protective signalling of activated protein C (APC) in human endothelial cells in vitro and may contribute to APC's beneficial effects in patients with severe sepsis. Mouse models are of key importance for translational research but species differences may limit conclusions for the human system. We analysed whether mouse APC can cleave, activate and induce signalling through murine PAR1 and tested in newly established mouse models if long-term infusion of APC prevents from vascular leakage. Cell surface immunoassays demonstrated efficient cleavage of endogenous murine endothelial PAR1 by either murine or human APC. Pharmacological concentrations of APC of either species had powerful barrier protective effects on cultured murine endothelial cells that required PAR1 cleavage. Vascular endothelial growth factor-mediated hyperpermeability in the skin was reduced by either endogenously generated as well as directly infused recombinant mouse APC in wild-type mice. However APC did not significantly alter the vascular barrier function in PAR1-deficient mice. In endotoxin-challenged mice, infused APC significantly prevented from pulmonary fluid accumulation in the wild-type mice but not in mice lacking PAR1. Our results directly show that murine APC cleaves and signals through PAR1 in mouse endothelial cells. APC reduces vascular permeability in mouse models and PAR1 plays a major role in mediating these effects. Our data in vitro and in vivo support the paradigm that PAR1 contributes to protective effects of APC on vascular barrier integrity in sepsis.

Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant.

Activated protein C (APC) reduces mortality in severe sepsis patients. APC exerts anticoagulant activities via inactivation of factors Va and VIIIa and cytoprotective activities via endothelial protein C receptor and protease-activated receptor-1. APC mutants with selectively altered and opposite activity profiles, that is, greatly reduced anticoagulant activity or greatly reduced cytoprotective activities, are compared here. Glu149Ala-APC exhibited enhanced in vitro anticoagulant and in vivo antithrombotic activity, but greatly diminished in vitro cytoprotective effects and in vivo reduction of endotoxin-induced murine mortality. Thus, residue Glu149 and the C-terminal region of APC's light chain are identified as functionally important for expression of multiple APC activities. In contrast to Glu149Ala-APC, 5A-APC (Lys191-193Ala + Arg229/230Ala) with protease domain mutations lacked in vivo antithrombotic activity, although it was potent in reducing endotoxin-induced mortality, as previously shown. These data imply that APC molecular species with potent antithrombotic activity, but without robust cytoprotective activity, are not sufficient to reduce mortality in endotoxemia, emphasizing the need for APC's cytoprotective actions, but not anticoagulant actions, to reduce endotoxin-induced mortality. Protein engineering can provide APC mutants that permit definitive mechanism of action studies for APC's multiple activities, and may also provide safer and more effective second-generation APC mutants with reduced bleeding risk.

Activated protein C-cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin.

Activated protein C (APC) signals in endothelial cells ex vivo through protease activated receptor-1 (PAR1). However, it is controversial whether PAR1 can mediate APC's protective effects in sepsis because the inflammatory response results in thrombin generation and thrombin proteolytically activates PAR1 much more efficiently than APC. Here we show that APC can induce powerful barrier protective responses in an endothelial cell monolayer in the presence of thrombin. Using cell surface immunoassays with conformation sensitive monoclonal anti-PAR1 antibodies we analyzed cleavage of endogenous PAR1 on the endothelial cell surface by APC in the absence and presence of thrombin. Incubation with APC caused efficient PAR1 cleavage and upon coincubation with thrombin APC supported additional PAR1 cleavage. Thrombin-cleaved PAR1 rapidly disappeared from the cell surface whereas, unexpectedly, the APC-cleaved PAR1 remained and could be detected on the cell surface, even when thrombin at concentrations of up to 1 nM was also present. Our findings demonstrate for the first time directly that APC can generate a distinct PAR1 population on endothelial cells in the presence of thrombin. The data suggest that different trafficking of activated PAR1 might explain how PAR1 signaling by APC can be relevant when thrombin is present.

Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells.

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.

Endothelial protein C receptor-dependent inhibition of migration of human lymphocytes by protein C involves epidermal growth factor receptor.

The protein C pathway is an important regulator of the blood coagulation system. Protein C may also play a role in inflammatory and immunomodulatory processes. Whether protein C or activated protein C affects lymphocyte migration and possible mechanisms involved was tested. Lymphocyte migration was studied by micropore filter assays. Lymphocytes that were pretreated with protein C (Ceprotin) or activated protein C (Xigris) significantly reduced their migration toward IL-8, RANTES, MCP-1, and substance P, but not toward sphingosine-1-phosphate. The inhibitory effects of protein C or activated protein C were reversed by Abs against endothelial protein C receptor and epidermal growth factor receptor. Evidence for the synthesis of endothelial protein C receptor by lymphocytes is shown by demonstration of receptor mRNA expression and detection of endothelial protein C receptor immunoreactivity on the cells' surface. Data suggest that an endothelial protein C receptor is expressed by lymphocytes whose activation with protein C or activated protein C arrests directed migration. Exposure of lymphocytes to protein C or activated protein C stimulates phosphorylation of Tyr845 of epidermal growth factor receptor, which may be relevant for cytoprotective effects of the protein C pathway.

Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling.

Activated protein C (APC) has anti-inflammatory and vascular protective effects independent of anticoagulation. We previously identified the prototypical thrombin receptor, protease-activated receptor-1 (PAR1), as part of a novel APC-endothelial cell protein C receptor (EPCR) signaling pathway in endothelial cells. Experiments in wild-type and PAR1(-/-) mice demonstrated that intravenous injection of APC leads to PAR1-dependent gene induction in the lung. The vascular endothelium undergoes profound changes in severe sepsis, the approved therapeutic indication for APC. Similar to PAR1, APC activated PAR2 through canonical cleavage. Although PAR2 was up-regulated in cytokine-stimulated endothelial cells, APC signaling remained PAR1-dependent. Large scale gene expression profiling documented marked differences in both up- and down-regulated genes between APC and thrombin signaling in cytokine-stimulated cells. APC down-regulated transcripts for proapoptotic proteins including p53 and thrombospondin-1, but p53 was unchanged, and thrombospondin was even up-regulated by thrombin. Concordant PAR1-dependent effects on protein levels were found. Thus, by signaling through the same receptor PAR1, APC, and thrombin can exert distinct biological effects in perturbed endothelium. These data may explain how APC can be therapeutically protective through the EPCR-PAR1 signaling despite ongoing thrombin generation due to disseminated intravascular coagulopathy.

Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation.

Endothelial cells normally form a dynamically regulated barrier at the blood-tissue interface, and breakdown of this barrier is a key pathogenic factor in inflammatory disorders such as sepsis. Pro-inflammatory signaling by the blood coagulation protease thrombin through protease activated receptor-1 (PAR1) can disrupt endothelial barrier integrity, whereas the bioactive lipid sphingosine 1-phosphate (S1P) recently has been demonstrated to have potent barrier protective effects. Activated protein C (APC) inhibits thrombin generation and has potent anti-inflammatory effects. Here, we show that APC enhanced endothelial barrier integrity in a dual-chamber system dependent on binding to endothelial protein C receptor, activation of PAR1, and activity of cellular sphingosine kinase. Small interfering RNA that targets sphingosine kinase-1 or S1P receptor-1 blocked this protective signaling by APC. Incubation of cells with PAR1 agonist peptide or low concentrations of thrombin (approximately 40 pM) had a similar barrier-enhancing effect. These results demonstrate that PAR1 activation on endothelial cells can have opposite biologic effects, reveal a role for cross-communication between the prototypical barrier-protective S1P and barrier-disruptive PAR1 pathway, and suggest that S1P receptor-1 mediates protective effects of APC in systemic inflammation.

Thrombin affects eosinophil migration via protease-activated receptor-1.

BACKGROUND: Protease-activated receptors (PARs) are a unique class of G-protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. Although expression of the PAR1, which is typically activated by thrombin, on human eosinophils has been demonstrated, no effect of thrombin on eosinophil function has been shown yet. Thus we investigated whether thrombin affects eosinophil migration in vitro. METHODS: Eosinophils were obtained from venous blood of healthy donors. Cell migration was studied by micropore filter assays. Involvement of PARs in thrombin-dependent migration was tested functionally using selective agonist peptides for PARs and a cleavage blocking PAR1 antibody. RESULTS: Thrombin significantly stimulated eosinophil chemotaxis in a dose-dependent manner. This effect was mimicked by the PAR1 but not the PAR2 agonist and was reversed by the cleavage blocking PAR1 antibody. Checkerboard experiments indicated that eosinophil migration depends on the presence of thrombin in a concentration gradient. CONCLUSIONS: Data suggest that activation of PAR1 by thrombin stimulates directed migration of human eosinophils and thereby may affect eosinophils in tissue and allergic inflammation.

Activated protein C signals through the thrombin receptor PAR1 in endothelial cells.

The anti-inflammatory effects of activated protein C (APC) have lead to its recent approval for the treatment of sepsis. Although the endothelial cell protein C receptor (EPCR) plays a crucial role in APC's protective roles in septicemia, the precise signaling mechanism of the protease APC remains unclear. In fibroblast overexpression systems, we find that APC activates protease activated receptors (PAR) 1 and 2 in an EPCR-dependent manner. Human endothelial cells (HUVECs) express PAR1, PAR2 and EPCR. Stimulation of HUVECs with either APC, or specific receptor activating peptides for PAR1 or PAR2, show that all three agonists induce a very similar set of early response genes as assessed by high density microarray analysis. Only the transcript for monocyte chemo-attractant protein-1 (MCP-1) was selectively induced by APC and the PAR1 agonist, but not by the PAR2 agonist. APC-mediated MAP kinase phosphorylation and gene induction were inhibited by cleavage blocking antibodies to PAR1, demonstrating that APC signals exclusively through PAR1 in endothelial cells. MCP-1 is protective in animal models of endotoxemia, suggesting that APC may prevent lethality in sepsis by inducing MCP-1 expression through EPCR-dependent activation of endothelial cell PAR1. These data demonstrate unexpected protective functions of the major thrombin receptor PAR1 in endothelial cells.

Science review: role of coagulation protease cascades in sepsis.

Cellular signaling by proteases of the blood coagulation cascade through members of the protease-activated receptor (PAR) family can profoundly impact on the inflammatory balance in sepsis. The coagulation initiation reaction on tissue factor expressing cells signals through PAR1 and PAR2, leading to enhanced inflammation. The anticoagulant protein C pathway has potent anti-inflammatory effects, and activated protein C signals through PAR1 upon binding to the endothelial protein C receptor. Activation of the coagulation cascade and the downstream endothelial cell localized anticoagulant pathway thus have opposing effects on systemic inflammation. This dichotomy is of relevance for the interpretation of preclinical and clinical data that document nonuniform responses to anticoagulant strategies in sepsis therapy.

Tissue factor-dependent coagulation protease signaling in acute lung injury.

OBJECTIVES: To review the role of tissue factor-dependent coagulation in acute lung injury. To interpret preclinical and clinical data on therapeutic intervention of the coagulation cascade, focusing on the principles of proteolytic cell signaling of the coagulant and anticoagulant pathways. DATA EXTRACTION AND SYNTHESIS: This review is based on published original research and relevant review articles on cell signaling by coagulation proteases and on experimental models that implicate the tissue factor-initiated coagulation cascade in acute lung injury and systemic inflammation. CONCLUSIONS: The coagulation cascade signals via protease activated receptors in the tissue factor-initiation phase and downstream via the effector protease, thrombin. Bleomycin-induced acute lung injury is an example of thrombin signaling-dependent pathology. Frequently, thrombin signaling is a major contributor to inflammation in the extravascular space but intravascular thrombin signaling is a threshold-regulated event. At low concentrations, intravascular thrombin activates the protein C pathway by converting protein C (bound to endothelial cell protein C receptor) to activated protein C and this generates antiinflammatory signals along the activated protein C-endothelial cell protein C receptor-protease activated receptor 1 pathway on endothelial cells. Direct thrombin signaling only occurs when intravascular thrombin concentrations exceed a coagulant threshold. In systemic bacterial toxin-mediated inflammation, inhibition of thrombin is not sufficient to limit inflammation, whereas tissue factor inhibition interrupts a self-sustaining inflammatory escalation in acute lung injury. Therefore, in the vasculature, inflammatory signaling by the tissue factor initiation complex is favored over thrombin signaling.

Activation of endothelial cell protease activated receptor 1 by the protein C pathway.

The coagulant and inflammatory exacerbation in sepsis is counterbalanced by the protective protein C (PC) pathway. Activated PC (APC) was shown to use the endothelial cell PC receptor (EPCR) as a coreceptor for cleavage of protease activated receptor 1 (PAR1) on endothelial cells. Gene profiling demonstrated that PAR1 signaling could account for all APC-induced protective genes, including the immunomodulatory monocyte chemoattractant protein-1 (MCP-1), which was selectively induced by activation of PAR1, but not PAR2. Thus, the prototypical thrombin receptor is the target for EPCR-dependent APC signaling, suggesting a role for this receptor cascade in protection from sepsis.

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.