Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand

We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by its natural ligand activated protein C (APC)/protein C switches the protease activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin from a disruptive to a protective effect in cultured human umbilical vein endothelial cells. Given the phenotypic differences between endothelial cells in venular and arterial beds, in this study we evaluated the signaling function of thrombin in human pulmonary artery endothelial cells (HPAECs) before and after treating them with PC-S195A which lacks catalytic activity but exhibits a normal affinity for EPCR. As expected, both thrombin and thrombin receptor agonist peptide (TRAP) enhanced the permeability barrier of HPAECs; however, both PAR-1 agonists exhibited a potent barrier protective effect when the cells were treated with PC-S195A prior to stimulation by the agonists. Interestingly, similar to APC, thrombin exhibited a potent cytoprotective activity in the LPS-induced permeability and TNF-alpha-induced apoptosis and adhesion assays in the PC-S195A treated HPAECs. Treatment of HPAECs with the cholesterol depleting molecule methyl-beta-cyclodextrin eliminated the protective effect of both APC and thrombin. These results suggest that the occupancy of EPCR by its natural ligand recruits PAR-1 to a protective signaling pathway within lipid rafts of HPAECs. Based on these results we conclude that the activation of PAR-1 by thrombin would initiate a protective response in intact arterial vascular cells expressing EPCR. These findings may have important ramifications for understanding the mechanism of the participation of the vascular PAR-1 in pathophysiology of the inflammatory disorders.     

Thrombin and activated protein C inhibit the expression of secretory group IIA phospholipase A2 in the TNF-α-activated endothelial cells by EPCR and PAR-1 dependent mechanisms

Introduction

Thrombin and tumor necrosis factor (TNF)-α up-regulate the expression of proinflammatory molecules in human umbilical vein endothelial cells (HUVECs). However, activated protein C (APC) down-regulates the expression of the same molecules. The expression level of secretory group IIA phospholipase A₂ (sPLA₂-IIA) is known to be elevated in inflammatory disorders including in sepsis. Here, we investigated the effects of APC and thrombin on the expression of sPLA₂-IIA and extracellular signal-regulated kinase (ERK) in HUVECs.

Materials and methods

The expression level of sPLA₂-IIA was quantitatively measured by an enzyme-linked-immunosorbent-assay following stimulation of HUVECs with either thrombin or TNF-α in the absence and presence of the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 and the cholesterol-depleting drug methyl-β-cyclodextrin (MβCD).

Results and conclusions

Thrombin had no effect on the expression of sPLA₂-IIA in HUVECs, however, TNF-α potently induced its expression. The prior treatment of cells with APC inhibited expression of sPLA₂-IIA through the EPCR-dependent cleavage of PAR-1. Further studies revealed that if HUVECs were pretreated with the zymogen protein C to occupy EPCR, thrombin also inhibited the TNF-α-mediated expression of sPLA₂-IIA through the cleavage of PAR-1. The EPCR-dependent cleavage of PAR-1 by both APC and thrombin increased the phosphorylation of ERK 1/2. Pretreatment of cells with either LY294002 or MβCD abolished the inhibitory activity of both APC and thrombin against sPLA₂-IIA expression, suggesting that the protein C occupancy of EPCR confers a PI3-kinase dependent protective activity for thrombin such that its cleavage of the lipid-raft localized PAR-1 inhibits the TNF-α-mediated expression of sPLA₂-IIA in HUVECs.     

Inflammation and coagulation. An overview

Inflammation and coagulation are two main host-defence systems that interact with each other. Inflammation activates coagulation and coagulation modulates the inflammatory activity in many ways. The contributing molecular pathways are reviewed. Thrombin and activated protein C (APC) and its receptor EPCR constitute a major physiological regulatory system to control vascular wall permeability during sepsis. Pro-inflammatory cellular effects of coagulation proteases as well as the anti-inflammatory effects of APC/EPCR are mediated by signaling via protease activated receptors PAR on mononuclear cells, endothelial cells, platelets, fibroblast, and smooth muscle cells. The beneficial effects of APC in sepsis are mainly dependent on the PAR-mediated cell-protective properties rather than the anticoagulant protease function on coagulation cofactors FV/Va and FVIII/VIIIa. Animal experiments with signaling selective APC-variants show promise in improving the therapeutic efficacy and safety of APC in sepsis.     

The structure-function relationship of activated protein C. Lessons from natural and engineered mutations

Protein C is the central enzyme of the natural anticoagulant pathway and its activated form APC (activated protein C) is able to proteolyse non-active as well as active coagulation factors V and VIII. Proteolysis renders these cofactors inactive, resulting in an attenuation of thrombin formation and overall down-regulation of coagulation. Presences of the APC cofactor, protein S, thrombomodulin, endothelial protein C receptor and a phospholipid surface are important for the expression of anticoagulant APC activity. Notably, APC also has direct cytoprotective effects on cells: APC is able to protect the endothelial barrier function and expresses anti-inflammatory and anti-apoptotic activities. Exact molecular mechanisms have thus far not been completely described but it has been shown that both the protease activated receptor 1 and EPCR are essential for the cytoprotective activity of APC. Recently it was shown that also other receptors like sphingosine 1 phosphate receptor 1, Cd11b/CD18 and tyrosine kinase with immunoglobulin-like and EGF-like domains 2 are likewise important for APC signalling. Mutagenesis studies are being performed to map the various APC functions and interactions onto its 3D structure and to dissect anticoagulant and cytoprotective properties. The results of these studies have provided a wealth of structure-function information. With this review we describe the state-of-the-art of the intricate structure-function relationships of APC, a protein that harbours several important functions for the maintenance of both humoral and tissue homeostasis.     

Polymorphisms in the endothelial protein C receptor gene and thrombophilia

The protein C anticoagulant pathway plays a crucial role as a regulator of the blood clotting cascade. Protein C is activated on the vascular endothelial cell membrane by the thrombin-thrombomodulin complex. Once formed, activated protein C (APC) down-regulates thrombin formation by inactivating factors (F)Va and FVIIIa. Endothelial protein C receptor (EPCR) is able to bind protein C and increase the rate of protein C activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of thrombin, thrombomodulin, protein C and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced/increased APC generation and modify the risk of thrombosis. This review highlights the different mutations/polymorphisms reported in the EPCR gene and their association with the risk of thrombosis.     

Thrombomodulin-mediated catabolism of protein C by pleural mesothelial and vascular endothelial cells

Pleural mesothelial and vascular endothelial cells express protein C (PC) pathway components including thrombomodulin (TM) and endothelial protein C receptor (EPCR) and activate PC by the thrombin-TM dependent mechanism. We used these cells as model systems to identify molecules involved in endocytosis and degradation of PC. We find that mesothelial and endothelial cells can bind, internalize and degrade PC. Addition of thrombin markedly induced degradation of PC by these cells in a TM-dependent fashion, implicating the involvement of the thrombin-TM complex in internalization and degradation of PC. This observation defines a novel function for the thrombin-TM complex as a degradation receptor for PC and suggests that PC is degraded concurrent with its activation. A PC Gla-domain mutant, which is unable to bind to the EPCR, was degraded by the cells to a lesser extent than wild-type PC, implicating the PC degradation concurrent with its activation. Consistent with the role of thrombin-TM complex as a degradation receptor, the catalytically inactive thrombin-S195A also induced PC degradation though to a lesser extent than wild-type thrombin. This suggests that generation of activated PC (APC) can contribute to accumulation of degradation products, but is not essential for the thrombin-induced degradation of PC. The thrombin-TM-mediated degradation of PC by both cell types suggest a previously unrecognized mechanism, which can contribute to PC consumption. This mechanism may be pathophysiologically relevant and can contribute to an acquired PC deficiency in conditions characterized by sustained thrombin generation.     

Protective effects of activated protein C in sepsis

Sepsis remains a complex syndrome associated with significant morbidity and mortality. It is now widely accepted that the pathways of inflammation, coagulation, apoptosis, and endothelial permeability are intimately linked in sepsis pathophysiology. The clinical success of activated protein C (APC), a natural anticoagulant, in reducing mortality in patients with severe sepsis has fuelled basic and preclinical research on the protective effects of this molecule. Over the past 15 years, impressive research advances have provided novel insights into the multifunctional activities of APC. APC is now viewed not only as an anticoagulant, but also as a cell signaling molecule that dampens the excessive or insufficiently controlled host response during sepsis. This review attempts to summarize the pleiotropic activities of APC with focus on its ability to inhibit coagulation, inflammation, apoptosis, and endothelial barrier breakdown. A comprehensive PUBMED literature review up to May 2008 was conducted.     

Control of thrombin mediated cleavage of protein S

Thrombin has been shown to cleave the vitamin K dependent cofactor protein S with subsequent loss of its cofactor activity. This study examines the control mechanisms for thrombin cleavage of protein S. The anticoagulant activity of activated protein C (APC) is enhanced fourteen fold by the addition of protein S. Thrombin cleaved protein S is seven fold less efficient than the native protein, and this loss of activity is due to reduced affinity of cleaved protein S for APC or the lipid surface compared to the intact protein. In the absence of Ca++, protein S is very sensitive to minimal concentrations of thrombin. As little as 1.5 nM thrombin results in complete cleavage of 20 nM protein S in 10 min and loss of cofactor activity. Ca++, in concentrations greater than 0.5 mM, will inhibit this cleavage and in the presence of physiological Ca++ concentrations, no cleavage of protein S could be demonstrated in spite of high concentrations of thrombin (up to 1 microM) and prolonged incubations (up to two hours). The endothelial surface protein thrombomodulin is very efficient in inhibiting the cleavage of protein S by thrombin suggesting that any thrombin formed on the endothelial cell surface is unlikely to cleave protein S, thus allowing the intact protein to act as a cofactor to APC. We conclude that the inhibitory effects of Ca++ and thrombomodulin on thrombin mediated cleavage of protein S imply that this event, by itself, is unlikely to represent a physiological control of the activity of protein S.     

Activated protein C stimulates osteoblast proliferation via endothelial protein C receptor

Introduction

Bone is continually remodeled by the action of osteoblasts, osteocytes, and osteoclasts. Resting osteoblasts are able to proliferate and differentiate into mature osteoblasts when physiologically required, as after tissue injury. Activated protein C (APC) is a serine protease that functions in anticoagulation, anti-inflammation, anti-apoptosis, cell proliferation, and wound repair. In this study, we examined the effect of APC on osteoblast proliferation and differentiation.

Materials and Methods

We examined the presence of protein C in human fracture hematoma by immunohistochemical staining. We then evaluated the effect of APC, diisopropyl fluorophosphate-inactivated APC (DIP-APC) or protein C zymogen on normal human osteoblast (NHOst) proliferation using tetrazolium salt assay in the presence or absence of aprotinin, hirudin, protein C, antibody against protein C, endothelial protein C receptor (EPCR) or protease-activated receptor (PAR)-1. Finally, activation of p44/42 MAP kinase was evaluated by Western blot analysis.

Results

Both APC and DIP-APC increased osteoblast proliferation in a dose-dependent manner, while protein C did not. The APC-induced increased proliferation of osteoblast was not affected by aprotinin, hirudin, and anti-protein C antibody which inhibits the protease activity of APC. Treatment with protein C or anti-EPCR antibody which inhibits APC binding to EPCR inhibited APC-mediated osteoblast proliferation, while treatment with anti-PAR-1 antibody did not. APC promoted the phosphorylation of p44/42 MAP kinase within osteoblasts; this effect was inhibited by the anti-EPCR antibody.

Conclusions

APC stimulates osteoblast proliferation by activating p44/42 MAP kinase through a mechanism that requires EPCR but not PAR-1 or the proteolytic activity of APC. APC generated at fracture sites may contribute to fracture healing by promoting osteoblast proliferation.     

Activated protein C, a natural anticoagulant protein, has antioxidant properties and inhibits lipid peroxidation and advanced glycation end products formation

INTRODUCTION: Activated protein C (APC) is an important natural anticoagulant that is proteolytically generated from protein C (PC) by the modulation of thrombin activity in the presence of thrombomodulin on an endothelial surface. Recent studies have demonstrated that, beyond its anticoagulant acitivities, APC had anti-inflammatory and cytoprotective properties. The mechanisms underlying APC's anti-inflammatory effects remain unknown. Our goal was to elucidate and confirm these mechanisms. METHODS: We first examined the effect of APC on reactive oxygen species (ROS) and inflammatory cytokine production in murine macrophage-like RAW264.7 cells. We further examined the effect of APC on chemically induced lipid peroxidation and advanced glycation end-products (AGE) formation. RESULTS AND CONCLUSIONS: APC in the range of 10-50 microg/mL could reduce lipopolysaccharide (LPS)-induced ROS generation, nuclear factor kappaB (NF-kappaB) activation and resultant proinflammatory cytokine production. Additional cell-free experiments revealed that APC (10-50 microg/mL) had inhibitory effects on lipid peroxidation and AGE formation. These findings suggest that APC, via its intrinsic anti-oxidant properties, may, in settings of oxidant stress, exert important cytoprotective and anti-inflammatory effects that are distinct from its anticoagulant activity as an antioxidant protein. If that is true, APC may contribute to ROS-related chronic disorders including atherosclerosis and diabetes as well as acute shock conditions.     

Evidence for functionally active protease-activated receptor-3 (PAR-3) in human vascular smooth muscle cells

The present study investigates whether vascular smooth muscle cells of the human saphenous vein (SMC) express a functionally active protease-activated receptor-3 (PAR-3). PAR-3 mRNA was detected by RT-PCR. In the presence of thrombin, a rapid and transient increase in PAR-3 mRNA was observed. Stimulation of SMC with thrombin or the synthetic PAR-3-activating peptide, TFRGAP, resulted in transient mobilization of intracellular calcium. After a preceding challenge with thrombin, the calcium signal to TFRGAP was abolished, suggesting cleavage and subsequent desensitization of PAR-3 by thrombin. Activation of PAR-3 by TFRGAP elicited a time-dependent activation of the extracellular-signal-regulated kinase (ERK)-1/2 with a maximum response 10-20 min after stimulation. At 200 microM, TFRGAP increased [3H]-thymidine incorporation into cellular DNA about two-fold. These data indicate that PAR-3 is expressed in human SMC and triggers intracellular signaling. Thus, in the SMC PAR-3 might contribute to thrombin-induced responses.     

Factor VIIa binding to endothelial cell protein C receptor

Recent studies have shown that factor VIIa (FVIIa) binds specifically to endothelial protein C receptor (EPCR), a known cellular receptor for protein C and activated protein C, on the endothelium. The formation of FVIIa:EPCR complexes neither supports the activation of coagulation nor modulates tissue factor-initiated coagulation. However, FVIIa interaction with EPCR, particularly at pharmacological concentrations of FVIIa, may impair EPCR-dependent protein C activation and activated protein C-mediated cell signaling by competing directly with them for binding to EPCR. FVIIa binding to EPCR may also contribute to FVIIa clearance. This review summarizes recent data on FVIIa interaction with EPCR and discusses potential physiological significance and consequences of the interaction.     

Calcium mobilization and protease-activated receptor cleavage after thrombin stimulation in motor neurons

Thrombin, the ultimate enzyme in the blood coagulation cascade, has prominent actions on various cells, including neurons. As in platelets, thrombin increases [Ca²⁺ _i mobilization in neurons, and also retracts neurites. Both these effects are mediated through a G protein-coupled, proteolytically activated receptor for thrombin (PAR-1). Prolonged exposure to thrombin kills neurons via apoptosis, that may also involve PAR-1 activation. Increased [Ca²⁺]ⁱ has been a unifying mechanism proposed for cell death in several neurodegenerative diseases. Thrombin-elevated calcium levels may activate intracellular cascades in neurons leading to cell death. Since thrombin mediates its diverse effects on cells through both heterotrimeric and monomeric G proteins, we also explored what effect altering differential G protein coupling would have on the neuronal response to thrombin. We studied calcium mobilization by thrombin in a model motor neuronal cell line, NSC19, using fluorescence image analysis. Confirming effects in other neuronal types, thrombin caused dramatic increases in [Ca²⁺]_i levels, both transiently and after prolonged exposure, which involved activation and cleavage of the PAR-1 receptor. Using enzyme linked immunosorbent assay (ELISA) and dot-blot analysis, we found that the N-terminal fragment of PAR-1 was released into the medium after exposure to thrombin. We confirmed that PAR-1 protein and mRNA expression occurred in motor neurons. We found that cholera toxin inhibited thrombin-mediated Ca²⁺ influx, pertussis toxin did not significantly alter thrombin action, and lovastatin, a small 21-kDa Ras GTPase (Rho) modulator, showed a tendency to reduce the thrombin effect. These data indicate that thrombin-increased [Ca²⁺]_i, sufficient to trigger cell death in motor neurons, might be approached in vivo by modulating thrombin signaling through PAR-1.     

Cytoprotective protein C pathways and implications for stroke and neurological disorders

Recent studies indicate that single-action-single-target agents are unlikely to cure CNS disorders sharing a pathogenic triad consisting of vascular damage, neuronal injury/neurodegeneration and neuroinflammation. Here we focus on a recent example of a multiple-action-multiple-target approach for CNS disorders based on newly discovered biological properties of activated protein C (APC), an endogenous plasma protease with antithrombotic, cytoprotective and anti-inflammatory activities in the CNS. We propose that APC-mediated signaling through the protease activated receptor-1 (PAR1) can favorably regulate multiple pathways within the neurovascular unit in non-neuronal cells and neurons during acute or chronic CNS insults, leading to stabilization of the blood-brain barrier (BBB), neuroprotection and control of neuroinflammation. Although much remains to be understood regarding the biology of APC, preclinical studies suggest that APC has promising applications as disease-modifying therapy for ischemic stroke and other neuropathologies whose underlying pathology involves deficits in the vasculo-neuronal-inflammatory triad.     

Activated protein C generation is greatly decreased in plasma from newborns compared to adults in the presence or absence of endothelium

Activated protein C (APC) generation strongly affects sepsis and thrombosis by inhibition of thrombin generation. However, it is unclear if there are age-related differences in effectiveness of protein C (PC). We studied age effects on plasma APC generation +/- endothelium. Defibrinated (Ancrod) plasma (from adults or newborns (umbilical cord)) was recalcified with buffer containing tissue factor +/- thrombomodulin (TM) on either plastic or endothelium (HUVEC) at 37 degrees C. Timed subsamples of reaction mixture were taken into either heparin-EDTA or FFRCMK-EDTA solutions and analyzed for APC-PC inhibitor (APC-PCI) or APC-alpha1 antitrypsin (APC-alpha1 AT) by ELISAs. Since heparin converts free APC to APC-PCI, the difference in APC-PCI measured in heparin-EDTA and FFRCMK-EDTA samples was equal to free active APC. APC-alpha2 macroglobulin (APC-alpha2M) was measured as remaining chromogenic activity in heparin-EDTA. Free APC, APC-PCI and APC-alpha1 AT were decreased in newborn compared to adult plasma on plastic. However, APC-alpha2M made up a larger fraction of inhibitor complexes in new-born plasma. On endothelium, significantly more APC, APC-PCI and APC-alpha1AT were generated in either plasma compared to that on plastic with excess added TM. APC, APC-PCI and APC-alpha1AT were also reduced and total APC-alpha2M increased in newborn plasma on HUVEC. Addition of PC to newborn plasma gave APC generation similar to adult plasma. Thus, free APC, APC-PCI and APC-alpha1AT generation is reduced in newborn compared to adult plasma with or without endothelium, likely due to reduced plasma PC levels. Endothelium enhances APC generation, regardless of plasma type, possibly because of cell surface factors such as TM, phospholipid and endothelial PC receptor.     

The effects of supra-normal protein C levels on markers of coagulation, fibrinolysis and inflammation in a human model of endotoxemia

The protein C pathway serves as a modulating system with both anti-inflammatory and anticoagulant properties and is intimately involved in the pathophysiology of inflammation and sepsis. Treatment with recombinant human activated protein C (rhAPC) can reduce the mortality of severe sepsis.We investigated whether an elevation of plasma protein C levels to supra-normal levels by infusion of a protein C zymogen concentrate has an effect on coagulation, protein C activation or inflammation in a human endotoxemia model. Eleven healthy male volunteers were enrolled in a double-blind, placebo-controlled two-way cross-over trial. Ten minutes after infusion of 2ng/kg endotoxin each volunteer received either placebo or a plasma-derived protein C zymogen concentrate (Ceprotin, Baxter) (150 U/kg as a slow bolus infusion followed by 30 U/kg/h continuous infusion until 4 hours after LPS-infusion). Protein C antigen and activity increased 4- to 5-fold after infusion of the concentrate. APC was generated during endotoxin-induced inflammation in the placebo (1.6 fold increase) and the protein C period (4.0-fold increase). The increase of APC levels correlated with the TNF-alpha and IL-6 release in both periods (r = 0.65-0.68; p < 0.05) and paralleled the protein C antigen and activity levels in the period with supranormal protein C levels. Supra normal protein C levels resulted in slightly, although non-significant, lower tissue factor mRNA expression and thrombin generation (TAT, F1+2). Systemic inflammation (TNF-alpha, IL-6) was not influenced by protein C zymogen concentrate administration. Infusion of protein C zymogen was safe and no adverse effects occurred. The increase of protein C levels several fold above the normal range resulted in a proportional increase of the APC levels, but had no major anticoagulant, anti-inflammatory or profibrinolytic effects. Low grade endotoxemia itself induces significant protein C activation, which correlates with the TNF release.     

TRYPSIN INDUCED VON WILLEBRAND FACTOR RELEASE FROM HUMAN ENDOTHELIAL CELLS IS MEDIATED BY PAR-2 ACTIVATION

Nystedt and co-workers cloned in 1994 a second protease activatable receptor (PAR-2) that could be activated by trypsin but not by thrombin [1]. In this study, we investigated whether trypsin induced stimulation of endothelial cells is linked to PAR-2 activation. We have found by mRNA analysis that endothelial cells of venous and arterial origin express both protease activatable receptors. The functional thrombin receptor and the protease activated receptor-2 (PAR-2) mediate apparently the same effects in human vascular endothelial cells. Both, the activation of the thrombin receptor with thrombin or SFLLRN and the activation of the PAR-2 with trypsin or SLIGRL induced intracellular calcium mobilisation and a subsequent release of von Willebrand factor (vWf) from Weibel-Palade bodies. As a consequence, it can be concluded that endothelial cells have two different receptors mediating the same cellular responses after activation. Copyright © 1996 Elsevier Science Ltd