Protein C Replacement in Severe Meningococcemia: Rationale and Clinical Experience

Severe meningococcemia, which is associated with hemodynamic instability, purpura fulminans and disseminated intravascular coagulation, still has a high mortality rate, and patients who survive are often left invalids because of amputations and organ failure. Clinical studies have shown that levels of protein C are markedly decreased in patients with severe meningococcemia and that the extent of the decrease correlates with a negative clinical outcome. There is a growing body of data demonstrating that activated protein C, in addition to being an anticoagulant, is also a physiologically relevant modulator of the inflammatory response. The dual function of protein C may be relevant to the treatment of individuals with severe meningococcal sepsis. In the present review we give a basic overview of the protein C pathway and its anticoagulant activity, and we summarize experimental data showing that activated protein C replacement therapy clearly reduces the mortality rate for fulminant meningococcemi

Shedding of endothelial protein C receptor contributes to vasculopathy and renal injury in lupus: In vivo and in vitro evidence1

Shedding of endothelial protein C receptor contributes to vasculopathy and renal injury in lupus: In vivo and in vitro evidence.BackgroundCandidate biomarkers for vasculopathy in systemic lupus erythematosus (SLE) include circulating endothelial cells and the recently identified endothelial protein C receptor (EPCR) which, when shed, promotes a thrombotic diathesis. This study sought correlation between plasma levels of soluble EPCR and disease manifestation/severity, with a focus on lupus nephritis.MethodsIn 81 SLE patients (evaluated cross-sectionally and longitudinally) and 59 healthy controls, levels of soluble EPCR and soluble E-selectin were assessed by sandwich enzyme-linked immunosorbent assay (ELISA), circulating endothelial cells isolated by immunomagnetic separation, and EPCR gene polymorphisms determined. Mechanisms of vascular injury were addressed in vitro in human aortic endothelial cells (HAEC) cultured in the presence and absence of interferon-γ (IFN-γ).ResultsThe mean level of soluble EPCR was significantly higher in SLE patients (263 ± 13ng/mL) than controls (174 ± 11ng/mL) (P < 0.0001). Patients with active or past renal involvement had significantly higher mean soluble EPCR levels (306 ± 21ng/mL) (N = 40) than patients without nephritis (228 ± 14ng/mL) (N = 41) (P = 0.0033). Mean soluble EPCR correlated positively with serum creatinine (R = 0.3429, P < 0.0001). The prevalence of the enhanced-shedding EPCR polymorphism A6936G was higher in SLE (41%) (N = 27) than controls (7%) (N = 29) (P = 0.0039). Patient and control plasma were also interrogated for soluble E-selectin, a comparator plasma marker. The results suggest that soluble E-selectin and soluble EPCR are not equivalent end points of vasculopathy and endothelial perturbation in SLE. Although in SLE patients the absence or diminished expression of membrane EPCR on circulating endothelial cells varied, the rare circulating endothelial cells detected in controls invariably expressed membrane-bound EPCR. IFN-γ-treated HAEC expressed less membrane-bound EPCR [133 relative fluorescence units (rfu)] than untreated HAEC (275 rfu); more soluble EPCR was detected in IFN-γ-treated (1.1ng/106 cells) than untreated HAEC (0.65ng/106 cells) (P = 0.027).ConclusionThe results obtained from this cross-sectional/longitudinal study support the hypothesis that the vascular dysfunction characteristic of SLE may be related to a dramatically altered distribution of EPCR, both soluble and membrane-bound forms

Limited Ability to Activate Protein C Confers Left Atrial Endocardium A Thrombogenic Phenotype: A Role in Cardioembolic Stroke?

Background and Purpose—Atrial fibrillation is the most important risk factor for cardioembolic stroke. Thrombi form in the left atrial appendage rather than in the right. The causes of this different thrombogenicity are not well-understood. The goal herein was to compare the activation of the anticoagulant protein C and the thrombomodulin and endothelial protein C receptor/activated protein C receptor expression on the endocardium between right and left atria. Methods—We harvested the atria of 6 monkeys (Macaca fascicularis) and quantified their ability to activate protein C ex vivo and we measured the thrombomodulin and endothelial protein C receptor expression by immunofluorescence. Results—We found the ability to activate protein C decreased by half (P 0.028) and there was lower expression of thrombomodulin in the left atrial endocardium than the right (52.5 19.9 and 72.1 18.8 arbitrary intensity units, mean standard deviation; P 0.028). No differences were detected in endothelial protein C receptor expression. Conclusions—Impaired protein C activation on the left atrial endocardium attributable to low thrombomodulin expression may explain its higher thrombogenicity and play a role in cardioembolic stroke

Cytoprotective Activated Protein C Averts Nlrp3 Inflammasome–Induced Ischemia-Reperfusion Injury Via Mtorc1 Inhibition

Cytoprotection by activated protein C (aPC) after ischemia-reperfusion injury (IRI) is associated with apoptosis inhibition. However, IRI is hallmarked by inflammation, and hence, cell-death forms disjunct from immunologically silent apoptosis are, in theory, more likely to be relevant. Because pyroptosis (ie, cell death resulting from inflammasome activation) is typically observed in IRI, we speculated that aPC ameliorates IRI by inhibiting inflammasome activation. Here we analyzed the impact of aPC on inflammasome activity in myocardial and renal IRIs. aPC treatment before or after myocardial IRI reduced infarct size and Nlrp3 inflammasome activation in mice. Kinetic in vivo analyses revealed that Nlrp3 inflammasome activation preceded myocardial injury and apoptosis, corroborating a pathogenic role of the Nlrp3 inflammasome. The constitutively active Nlrp3A350V mutation abolished the protective effect of aPC, demonstrating that Nlrp3 suppression is required for aPC-mediated protection from IRI. In vitro aPC inhibited inflammasome activation in macrophages, cardiomyocytes, and cardiac fibroblasts via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Accordingly, inhibiting PAR-1 signaling, but not the anticoagulant properties of aPC, abolished the ability of aPC to restrict Nlrp3 inflammasome activity and tissue damage in myocardial IRI. Targeting biased PAR-1 signaling via parmodulin-2 restricted mTORC1 and Nlrp3 inflammasome activation and limited myocardial IRI as efficiently as aPC. The relevance of aPC-mediated Nlrp3 inflammasome suppression after IRI was corroborated in renal IRI, where the tissue protective effect of aPC was likewise dependent on Nlrp3 inflammasome suppression. These studies reveal that aPC protects from IRI by restricting mTORC1-dependent inflammasome activation and that mimicking biased aPC PAR-1 signaling using parmodulins may be a feasible therapeutic approach to combat IRI

Role of Coagulation Factors in Cerebral Venous Sinus and Cerebral Microvascular Thrombosis

Thrombus formation can occur in both macroscopic and microscopic blood vessels. In the brain, cerebral venous sinus thrombosis (CVST) and focal cortical infarctions can result from the formation of thrombi in these different sized vessels. In this study we define the relative contributions of three major pro- and anti-coagulation pathways (heparin-antithrombin, protein C, and tissue factor (TF)) in the thrombogenic responses that occur in large and small vessels of the brain

sPLA2-V inhibits EPCR anticoagulant and antiapoptotic properties by accommodating lysophosphatidylcholine or PAF in the hydrophobic groove

The endothelial protein C receptor (EPCR) plays an important role in cardiovascular disease by binding protein C/activated protein C (APC). EPCR structure contains a hydrophobic groove filled with an unknown phospholipid needed to perform its function. It has not been established whether lipid exchange takes place in EPCR as a regulatory mechanism of its activity. Our objective was to identify this phospholipid and to explore the possibility of lipid exchange as a regulatory mechanism of EPCR activity driven by the endothelially expressed secretory group V phospholipase A2 (sPLA2-V). We identified phosphatidylcholine (PCh) as the major phospholipid bound to human soluble EPCR (sEPCR). PCh in EPCR could be exchanged for lysophosphatidylcholine (lysoPCh) and platelet activating factor (PAF). Remarkably, lysoPCh and PAF impaired the protein C binding ability of sEPCR. Inhibition of sPLA2-V, responsible for lysoPCh and PAF generation, improved APC binding to endothelial cells. EPCR-dependent protein C activation and APC antiapoptotic effect were thus significantly enhanced. In contrast, endothelial cell supplementation with sPLA2-V inhibited both APC generation and its antiapoptotic effects. We conclude that APC generation and function can be modulated by changes in phospholipid occupancy of its endothelial cell receptor

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

Protein C anticoagulant system—anti-inflammatory effects

Activated protein C (APC) plays active roles in preventing progression of a number of disease processes. These include thrombosis due to its direct anticoagulant activity which is likely augmented by its cytoprotective activity, thereby limiting exposure of procoagulant cellular membrane surfaces on cells. Beyond that, the pathway signals the cells to prevent apoptosis, to dampen inflammation, to increase endothelial barrier function, and to selectively downregulate some genes implicated in disease progression. Most of these functions are manifested to APC binding to endothelial protein C receptor (EPCR) allowing PAR1 activation, but activation of other PARS is also implicated in some cases. In addition to EPCR orchestrating these changes, CD11b is also capable of supporting APC signaling. Selective control of these pathways offers potential in new therapeutic approaches to disease