Group V secretory phospholipase A2 impairs endothelial protein C receptor-dependent protein C activation and accelerates thrombosis in vivo.

BACKGROUND: Endothelial protein C receptor (EPCR) must be bound to a molecule of phosphatidylcholine (PC) to be fully functional, i.e. to interact with protein C/activated protein C (APC) properly. PC can be replaced with other lipids, such as lysophosphatidylcholine or platelet-activating factor, by the action of group V secretory phospholipase A2 (sPLA2-V), an enzyme that is upregulated in a variety of inflammatory conditions. Studies in purified systems have demonstrated that the substitution of PC notably impairs EPCR function in a process called EPCR encryption. OBJECTIVES: To analyze whether sPLA2-V was able to regulate EPCR-dependent protein C activation in vivo, and its impact on thrombosis and the hemostatic system. METHODS: Mice were transfected with sPLA2-V by hydrodynamic gene delivery. The effects on thrombosis were studied with the laser carotid artery occlusion model, and APC generation capacity was measured with ELISA. Global hemostasis was analyzed with thromboelastometry. RESULTS: We found that sPLA2-V overexpression in mice significantly decreased their ability to generate APC. Furthermore, a murine carotid artery laser thrombosis model revealed that higher sPLA2-V levels were directly associated with faster artery thrombosis. CONCLUSIONS: sPLA2-V plays a thrombogenic role by impairing the ability of EPCR to promote protein C activation.

Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells.

BACKGROUND: Extracellular histones exert part of their prothrombotic activity through the stimulation of blood cells. Besides platelets, histones can bind to red blood cells (RBCs), which are important contributors to thrombogenesis, but little is known about the functional consequences of this interaction. OBJECTIVES: To evaluate the effect of histones on the procoagulant potential of human RBCs with particular regard to the expression of surface phosphatidylserine (PS). METHODS: PS exposure on human RBCs treated with a natural mixture of histones or recombinant individual histones was evaluated with fluorescein isothiocyanate-annexin-V binding and measured with flow cytometry. Calcium influx in RBCs loaded with the calcium-sensitive fluorophore Fluo-4 AM was assessed with flow cytometry. The procoagulant potential of histone-treated RBCs was evaluated with a purified prothrombinase assay and a one-stage plasma recalcification clotting test. RESULTS: Natural histones induced PS exposure on RBCs in a dose-dependent manner, and neutralization or cleavage of histones by heparin or activated protein C, respectively, abolished PS externalization. H4 was mainly responsible for the stimulating activity of histones, whereas the other subtypes were almost ineffective. Similarly, natural histones and H4 induced influx of calcium into RBCs, whereas the other individual histones did not. Histone-induced exposure of PS on RBCs translated into increased prothrombinase complex-mediated prothrombin activation and accelerated fibrin formation in plasma. CONCLUSIONS: Histones induce RBCs to express a procoagulant phenotype through the externalization of PS. This finding provides new insights into the prothrombotic activity of extracellular histones.

Factor VIIa binding to endothelial cell protein C receptor protects vascular barrier integrity in vivo.

BACKGROUND: Recent studies have shown that factor VIIa binds to endothelial cell protein C receptor(EPCR), a cellular receptor for protein C and activated protein C. At present, the physiologic significance of FVIIa interaction with EPCR in vivo remains unclear. OBJECTIVE: To investigate whether exogenously administered FVIIa, by binding to EPCR, induces a barrier protective effect in vivo. METHODS: Lipopolysaccharide(LPS)-induced vascular leakage in the lung and kidney,and vascular endothelial growth factor (VEGF)-induced vascular leakage in the skin, were used to evaluate the FVIIa-induced barrier protective effect. Wild-type, EPCR-deficient, EPCR-overexpressing and hemophilia A mice were used in the studies. RESULTS: Administration ofFVIIa reduced LPS-induced vascular leakage in the lung and kidney; the FVIIa-induced barrier protective effect was attenuated in EPCR-deficient mice. The extent of VEGF-induced vascular leakage in the skin was highly dependent on EPCR expression levels. Therapeutic concentrations of FVIIa attenuated VEGF-induced vascular leakage in control mice but not in EPCR-deficient mice.Blockade of FVIIa binding to EPCR with a blocking mAb completely attenuated the FVIIa-induced barrier protective effect. Similarly, administration of protease activated receptor 1 antagonist blocked the FVIIa induced barrier protective effect. Hemophilic mice showed increased vascular permeability, and administration of therapeutic concentrations of FVIIa improved barrier integrity in these mice. CONCLUSIONS: This is the first study to demonstrate that FVIIa binding to EPCR leads to a barrier protective effect in vivo. This finding may have clinical relevance, as it indicates additional advantages of using FVIIa in treating hemophilic patients.

Endogenous protein C has a protective role during Gram-negative pneumosepsis (melioidosis).

BACKGROUND: Activated protein C (APC) exerts anticoagulant effects via inactivation of factors Va and VIIIa and cytoprotective effects via protease activated receptor (PAR)1. Inhibition of endogenous APC in endotoxemia and sepsis results in exacerbation of coagulation and inflammation, with consequent enhanced lethality. OBJECTIVES: We here sought to dissect the distinct roles of the anticoagulant and cytoprotective functions of endogenous APC in severe Gram-negative pneumonia-derived sepsis (melioidosis). METHODS: We infected wild-type (WT) mice with Burkholderia pseudomallei, a common sepsis pathogen in southeast Asia, and treated them with antibodies inhibiting both the anticoagulant and cytoprotective functions of APC (MPC1609) or the anticoagulant functions of APC (MAPC1591) only. Additionally, we administered SEW2871 (stimulating the S1P1-pathway downstream from PAR1) to control and MPC1609-treated mice. RESULTS: MPC1609, but not MAPC1591, significantly worsened survival, increased coagulation activation, facilitated bacterial growth and dissemination and enhanced the inflammatory response. The effects of MPC1609 could not be reversed by SEW2871, suggesting that S1P1 does not play a major role in this model. CONCLUSIONS: These results suggest that the mere inhibition of the anticoagulant function of APC does not interfere with its protective role during Gram-negative pneumosepsis, suggesting a more prominent role for cytoprotective effects of APC .

Endothelial cell protein C receptor-mediated redistribution and tissue-level accumulation of factor VIIa.

BACKGROUND: Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown. OBJECTIVE: The aim of the present study was to investigate the role of the FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues. METHODS: Wild-type, EPCR-deficient or ECPR-over-expressing mice were injected with human recombinant (r)FVIIa (120 µg kg(-1) body weight) via the tail vein. At varying time intervals after rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR. RESULTS: The data reveal that, after intravenous (i.v.) injection, rFVIIa rapidly disappears from the blood and associates with the endothelium in an EPCR-dependent manner. Immunohistochemical analyses revealed that the association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. The FVIIa association with the endothelium was undetectable at time points exceeding 24 h post-FVIIa administration. The levels of rFVIIa accumulated in tissue correlate with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days. CONCLUSIONS: The observation that an EPCR-dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides.

Innate immunity and coagulation.

Infection frequently elicits a coagulation response. Endotoxin triggers the formation of tissue factor initiating coagulation, down regulates anticoagulant mechanisms including the protein C pathway and heparin-like proteoglycans and up regulates plasminogen activator inhibitor. The overall physiological result of this is to promote coagulation through enhancing initiation, suppressing negative regulation and impairing fibrin removal. The response to infection also leads to tissue destruction. Nucleosomes and histones released from the injured cells trigger further inflammation, protection from the pathogen but further tissue injury leading to multi-organ failure. Such a complex response to infection presumably arises due to the role of coagulation in the control and clearance of the infectious agent.

Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation.

BACKGROUND: Histones are basic proteins that contribute to cell injury and tissue damage when released into the extracellular space. They have been attributed a prothrombotic activity, because their injection into mice induces diffuse microvascular thrombosis. The protein C-thrombomodulin (TM) system is a fundamental regulator of coagulation, particularly in the microvasculature, and its activity can be differentially influenced by interaction with several cationic proteins. OBJECTIVE: To evaluate the effect of histones on the protein C-TM system in a plasma thrombin generation assay and in purified systems. METHODS: The effect of histones on plasma thrombin generation in the presence or absence of TM was analyzed by calibrated automated thrombinography. Protein C activation in purified systems was evaluated by chromogenic substrate cleavage. The binding of TM and protein C to histones was evaluated by solid-phase binding assay. RESULTS: Histones dose-dependently increased plasma thrombin generation in the presence of TM, independently of its chondroitin sulfate moiety. This effect was not caused by inhibition of activated protein C activity, but by the impairment of TM-mediated protein C activation. Histones were able to bind to both protein C and TM, but the carboxyglutamic acid domain of protein C was required for their effect. Histones H4 and H3 displayed the highest activity. Importantly, unlike heparin, DNA did not inhibit the potentiating effect of histones on thrombin generation. CONCLUSIONS: Histones enhance plasma thrombin generation by reducing TM-dependent protein C activation. This mechanism might contribute to microvascular thrombosis induced by histones in vivo at sites of organ failure or severe inflammation.

Endogenous activated protein C is essential for immune-mediated cancer cell elimination from the circulation.

Fibrinogen and platelets play an important role in cancer cell survival in the circulation by protecting cancer cells from the immune system. Moreover, endogenous activated protein C (APC) limits cancer cell extravasation due to sphingosine-1-phosphate receptor-1 (S(1)P(1)) and VE-cadherin-dependent vascular barrier enhancement. We aimed to study the relative contribution of these two mechanisms in secondary tumor formation in vivo. We show that fibrinogen depletion limits pulmonary tumor foci formation in an experimental metastasis model in C57Bl/6 mice but not in NOD-SCID mice lacking a functional immune system. Moreover, we show that in the absence of endogenous APC, fibrinogen depletion does not prevent cancer cell dissemination and secondary tumor formation in immune-competent mice. Overall, we thus show that endogenous APC is essential for immune-mediated cancer cell elimination.

Inflammation, innate immunity and blood coagulation.

Inflammation drives arterial, venous and microvascular thrombosis. Chronic inflammation contributes to arterial thrombotic complications, whereas acute inflammation drives venous thrombosis and microvascular thrombosis. Mechanistically, inflammation modulates thrombotic responses by upregulating procoagulants, downregulating anticoagulants and suppressing fibrinolysis. The inflammatory response can also result in cell apoptosis or necrosis. Products released from the dead cells, particularly histones, propagate further inflammation, tissue death and organ failure. Inhibition of histone mediated cytotoxicity appears to be a new mechanism for protecting against this deadly cascade.

Endogenous activated protein C signaling is critical to protection of mice from lipopolysaccaride-induced septic shock.

SUMMARY BACKGROUND: Activated protein C (APC) is known to protect animals from sepsis. Endogenous protein C is important in protection. It is unknown whether the cytoprotective or anticoagulant properties of protein C (PC) are responsible for the protective effect of endogenous PC. OBJECTIVE: To determine if signaling by endogenous activated protein C contributes to survival in sepsis. METHODS: We used an immunochemical approach to either block all of the known activities of protein C using mAb MPC1609 or, alternatively, selectively block the anticoagulant activity of activated protein C while sparing some of its cytoprotective activities using mAb MAPC1591. RESULTS: MPC1609 blocked APC binding to endothelium whereas MAPC1591 enhanced binding. MPC1609 prevented APC protection of endothelial barrier function whereas MAPC1591 did not. Injection of MPC1609, but not MAPC1591, with a sublethal dose of lipopolysaccharide (LPS) caused lethality. At 18 h, the mice injected with MPC1609 plus LPS had much higher interleukin-6 (IL-6) levels than mice injected with LPS alone or LPS plus MPC1591. In these mice treated with LPS plus MPC1609, higher blood urea nitrogen (BUN) and creatinine levels suggested that an acute renal failure might contribute to a slow clearance of IL-6. CONCLUSIONS: These studies demonstrate for the first time that cytoprotective activities of APC, and not the anticoagulant activity, is required for protection in this sepsis model. Similar anti-human antibodies may prove useful in clinical conditions such as trauma and hemophilia where cytoprotection is desirable, but the anticoagulant activity of endogenous activated protein C may contribute to bleeding.

Role of coagulation pathways and treatment with activated protein C in hyperoxic lung injury.

BACKGROUND: Activated protein C (APC) significantly decreases mortality in severe sepsis, but its role in acute lung injury from non-infectious aetiologies is unclear. The role of APC in hyperoxic acute lung injury was tested by studying the physiology of lung injury development, measurement of key coagulation proteins and treatment with murine APC (mAPC). METHODS: Mice were continuously exposed to >95% oxygen and lung injury was assessed by extravascular lung water, lung vascular protein permeability and alveolar fluid clearance. Coagulation proteins were measured in bronchoalveolar lavage (BAL) fluid and plasma. Recombinant mAPC was administered in preventive and treatment strategies. RESULTS: Hyperoxia produced dramatic increases in lung vascular permeability and extravascular lung water between 72 and 96 h. Lung fluid balance was also adversely affected by progressive decreases in basal and cAMP-stimulated alveolar fluid clearance. Plasma levels of APC decreased at 72 h and were 90% depleted at 96 h. There were significant increases in BAL fluid levels of thrombomodulin, thrombin-antithrombin complexes and plasminogen activator inhibitor-1 at later time points of hyperoxia. Lung thrombomodulin expression was severely decreased during late hyperoxia and plasma levels of APC were not restored by excess thrombin administration. Administration of recombinant mAPC failed to improve indices of lung injury. CONCLUSIONS: Hyperoxic acute lung injury produces procoagulant changes in the lung with a decrease in plasma levels of APC due to significant endothelial dysfunction. Replacement of mAPC failed to improve lung injury.

Non-hematopoietic EPCR regulates the coagulation and inflammatory responses during endotoxemia.

BACKGROUND: Activated protein C (APC) protects the host from severe sepsis. Endothelial protein C receptor (EPCR) is expressed on both hematopoietic leukocytes and non-hematopoietic endothelium, and plays a key role in protein C activation. OBJECTIVES: We explore the influence of EPCR deletion on the responses to lipopolysaccharide (LPS) and then determine whether the observed differences are due to loss of hematopoietic or non-hematopoietic EPCR. METHODS AND RESULTS: After LPS challenge, EPCR null (Procr(-/-)) mice exhibited more thrombin and cytokine generation, neutrophil sequestration in the lung and a higher mortality rate than Procr(+/-) mice. Procr(+/-) BM/Procr(-/-) (non-hematopoietic Procr(-/-)) and Procr(-/-) BM/Procr(+/-) (hematopoietic Procr(-/-)) chimeric mice were generated by bone marrow (BM) transplantation. Compared with control Procr(+/-) mice, non-hematopoietic Procr(-/-) mice exhibited reduced protein C activation by thrombin and exaggerated responses to LPS challenge, whereas Procr(+/-) mice and hematopoietic Procr(-/-) mice exhibited similar protein C activation by thrombin and similar responses to LPS challenge. CONCLUSIONS: EPCR deletion exaggerates the host responses to LPS primarily due to deficiency of EPCR on the non-hematopoietic cells.

Regulated endothelial protein C receptor shedding is mediated by tumor necrosis factor-alpha converting enzyme/ADAM17.

Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway. Previously, we have reported that EPCR can be shed from the cell surface, and that this is mediated by an unidentified metalloproteinase. In this study, we demonstrate that tumor necrosis factor-alpha converting enzyme/ADAM17 (TACE) is responsible for EPCR shedding. Phorbol-12-myristate 13-acetate (PMA)-stimulated EPCR shedding is reduced by approximately 50% in HEK293 cells transfected with human EPCR cDNA and by 60% in human umbilical vein endothelial cells after transfection of TACE small interfering RNA (siRNA) into these cells. PMA-stimulated EPCR shedding is completely blocked in fibroblasts from TACE-deficient mice transfected with human EPCR cDNA, and restored by transfection of TACE cDNA into this cell line. To characterize the EPCR sequence requirement for shedding, we generated several mutants of EPCR. Replacing amino acids from residue 193 to residue 200 with the FLAG sequence (DYKDDDDK) completely blocks EPCR shedding, whereas a single amino acid substitution in this region has less effect on EPCR shedding.

The Ser219-->Gly dimorphism of the endothelial protein C receptor contributes to the higher soluble protein levels observed in individuals with the A3 haplotype.

The endothelial cell protein C receptor (EPCR) plays an important role in regulating blood coagulation and in activated protein C-mediated anti-inflammatory and antiapoptotic processes. Recent studies reported that there are polymorphisms in the human EPCR gene. One of the polymorphisms (haplotype A3) results in substitution of the Ser at residue 219 with Gly in the transmembrane domain. This haplotype is associated with increased plasma levels of soluble EPCR and is a candidate risk factor for thrombosis. We established stable cell lines expressing either the EPCR A1 (Ser at residue 219) or A3 (Gly at residue 219) haplotype. Both constitutive and PMA-stimulated shedding are five- to sevenfold higher in the A3 cell line than the A1 cell line. We also isolated human umbilical vein endothelial cells (HUVEC) from A1/A1 or A1/A3 origins. PMA-stimulated shedding is fourfold higher in HUVEC derived from A1/A3 origin than from A1/A1 origin. After PMA treatment, the rate of human protein C activation decreased 36% in HUVEC derived from A1/A3 origin, while it only decreased 18% in HUVEC derived from A1/A1 origin. These results indicate that the A3 haplotype does promote cellular shedding in either 293 or endothelial cells and therefore is likely directly contributory to the higher soluble EPCR levels seen in patients carrying this haplotype.

Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin.

Previous studies have shown that blocking endothelial protein C receptor (EPCR)-protein C interaction results in about an 88% decrease in circulating activated protein C (APC) levels generated in response to thrombin infusion and exacerbates the response to Escherichia coli. To determine whether higher levels of EPCR expression on endothelial cells might further enhance the activation of protein C and protect the host during septicemia, we generated a transgenic mouse (Tie2-EPCR) line which placed the expression of EPCR under the control of the Tie2 promoter. The mice express abundant EPCR on endothelial cells not only on large vessels, but also on capillaries where EPCR is generally low. Tie2-EPCR mice show higher levels of circulating APC after thrombin infusion. Upon infusion with factor Xa and phospholipids, Tie2-EPCR mice generate more APC, less thrombin and are protected from fibrin/ogen deposition compared with wild type controls. The Tie2-EPCR animals also generate more APC upon lipopolysaccharide (LPS) challenge and have a survival advantage. These results reveal that overexpression of EPCR can protect animals against thrombotic or septic challenge.