Infusion Of Activated Protein C Research Articles

Activated Protein C Improves Macrovascular and Microvascular Reactivity in Human Severe Sepsis and Septic Shock

We tested the effects of activated protein C (APC) in macrovascular and microvascular beds within 60 min of treatment. Twelve patients treated with APC for severe sepsis were included. We assessed macrovascular reactivity by phenylephrine arterial dose response. Pharmacological modeling (EC50, Emax, and Hill coefficient) and individual dose-response curve were tested. Microvascular reactivity was tested with skin laser Doppler by using postocclusive reactive hyperemia with measurements of peak, time to peak (Tmax), time to half recovery (T1/2R), and myogenic and sympathetic tones. All measurements were done 30 min before, just before, and 30 and 60 min after APC infusion. Microvascular reactivity was also tested in eight healthy volunteers. In patients, arterial pressure did not increase significantly. However, 60 min after the beginning of APC infusion, reactivity to α-1 stimulation was improved: EC50 decreased from 15.3 (0.9-56) to 3.1 (1.0-6.2) (P = 0.04), and 5 of 12 patients improved their dose-response curve. As for microcirculatory parameters, as early as 30 min after the beginning of APC infusion, postocclusive reactive hyperemia peak increased from 102 (40-168) to 162 (35-196) (P = 0.04), Tmax was shorter: 30 s (14-52 s) versus 56 s (22-83 s) (P = 0.03), and the T1/2R also decreased, from 72.4 s (41.9-134.6 s) to 49.8 s (31.0-129.8 s) (P = 0.02). Myogenic tone increased (P = 0.03), whereas sympathetic tone decreased (P = 0.03), and myogenic tone was lower than controls before but not after APC treatment. In conclusion, APC improves vascular reactivity both at macrocirculatory and microcirculatory levels very quickly, suggesting that this is not due to protein synthesis or anticoagulant effect. The myogenic properties of vessels could partly drive this effect.

High dose infusion of activated protein C (rhAPC) fails to improve neuronal damage and cognitive deficit after global cerebral ischemia in rats

Purpose: Recent studies demonstrated anticoagulatory, antiinflammatory, antiapoptotic, and neuroprotective properties of activated protein C (APC) in rodent models of acute neurodegenerative diseases, suggesting APC as promising broad acting therapeutic agent. Unfortunately, continuous infusion of recombinant human APC (rhAPC) failed to improve brain damage following cardiac arrest in rats. The present study was designed to investigate the neuroprotective effect after global cerebral ischemia (GI) with an optimized infusion protocol. Methods: Rats were subjected to bilateral clip occlusion of the common carotid arteries (BCAO) and controlled hemorrhagic hypotension to 40mmHg for 14min and a subsequent 5h-infusion of rhAPC (2mg/kg bolus+6mg/kg/h continuous IV) or vehicle (0.9% NaCl). The dosage was calculated to maintain plasma hAPC activity at 150%. Cerebral inflammation, apoptosis and neuronal survival was determined at day 10. Results: rhAPC infusion did not influence cortical cerebral perfusion during reperfusion and failed to reduce neuronal cell loss, microglia activation, and caspase 3 activity. Conclusion: Even an optimized rhAPC infusion protocol designed to maintain a high level of APC plasma activity failed to improve the sequels following GI. Despite positive reports about protective effects of APC following, e.g., ischemic stroke, the present study supports the notion that infusion of APC during the early reperfusion phase does not result in sustained neuroprotection and fails to improve outcome after global cerebral ischemia.

Activated Protein C, Sepsis, and the Innate Immune Response to Infection

AbstractAbstract SCI-43The phase 3 PROWESS clinical trial in 2001 resulted in approval of recombinant human activated protein C (APC) using low-dose, 96 hour infusion therapy to reduce mortality for adult severe sepsis linked to bacterial infection (1). In that trial, APC reduced mortality from 30.8% to 24.7%, an absolute mortality reduction of 6.1% (19.4% relative risk reduction), but this therapy carried a risk of serious bleeding (4.0 vs. 1.5%, p=0.06). Trials of APC therapy for less than severe adult or pediatric sepsis failed to show benefit but confirmed increased risk for serious bleeding. Subsequently, 10 years after the PROWESS trial, the similarly designed large trial (PROWESS SHOCK) (2) failed to document therapeutic efficacy in adult severe sepsis, reporting mortalities of 26.4% vs. 24.2% and, curiously, no increased risk of serious bleeding (1.2 vs. 1.0%, p=0.81). Recombinant APC was thus withdrawn from the market in late 2011. A simple comparison of PROWESS to PROWESS-SHOCK is confounded by significant improvements in ICU standard of care for severe sepsis over a decade. These two trials were notable in being limited to therapy using low-dose APC infusion for four days. The success of PROWESS stimulated major research advances in 2002–2012 for understanding APC’s in vitro and in vivo mechanisms of action. In preclinical work, APC is highly effective for sepsis, non-infectious inflammatory disease, ischemic stroke and neurodegenerative disorders. This presentation will review current knowledge about mechanisms for APC’s antithrombotic and cellular cytoprotection, about new insights that might help explain the absence of efficacy in PROWESS-SHOCK, and about possible avenues towards translating the benefits of APC seen in preclinical studies to patients. This presentation will discuss the rationale for use of second generation APC variants that may reduce the risk of bleeding while retaining beneficial anti-inflammatory and cytoprotective functions. Recent preclinical animal studies indicate that such second-generation APC variants might widen the time-window of opportunity for successful therapies, including for lethal infections. Remarkably, APC reduces mortality caused by high-dose total body radiation in mice by preserving bone marrow cells (3). This presentation will review cellular and molecular mechanisms by which APC affects the response of bone marrow-derived innate immune cells to stress induced by infection or radiation. This presentation will provide evidence for the existence of a previously unrecognized role of coagulation Factor V as a modifier of the response to sepsis therapy with APC. Based on APC’s direct cellular effects, one would speculate that the use of second generation APC variants given in high-dose bolus regimens holds great promise for multiple maladies. Disclosures:Weiler:BloodCenter of Wisconsin: Patents & Royalties.

Protein S: An anticoagulant in its own right

Editorial on Heeb et al. Thromb Haemost 2012; 107: 690-698. Since the isolation of protein S in 1979, initial studies demonstrated that protein S acted as a natural anticoagulant by promoting the serine protease activity of activated protein C (APC) on its substrates, the coagulation factors Va and VIIIa. This interaction required binding to phospholipid membranes through the vitamin K-dependent Gla residues of protein S. Later work showed that protein S interacts with other blood components, including a regulator of complement (C4b-binding protein), other components of the coagulation cascade (tissue factor pathway inhibitor and factor Xa), plasma lipoproteins and cells. The later interaction is mediated through specific membrane receptor tyrosine kinases belonging to the TAM family. This choreography of interactions predicted new biological functions, and protein S is now recognised as a regulator of essential processes in the response to damage, including regulation of phagocytosis of apoptotic cells (efferocytosis), cell survival and activation of innate immunity. Still, the anticoagulant function of protein S is central to its biological role, as it was first proposed by the association of protein S deficiency and thrombosis in human cohorts. This has been experimentally corroborated by the recent characterisation of the protein S knockout mouse phenotype (1). In the present issue of Thrombosis and Haemostasis, Heeb et al. (2) provide further evidence of the use of protein S in therapeutic anticoagulation independently of the infusion of APC. One of the reasons behind the late recognition of the APC-independent anticoagulant function of protein S derives from basic biochemical reasons. This functional property depends on the presence of Zn2+ ions, which were often removed during protein S purification procedures (3). The present study shows promises of using protein S as an anticoagulant drug on its own, independently of APC. This would add a new therapeutic substance that could be especially interesting for situations where control of both anticoagulation and inflammation has to be provided. This includes treatments for sepsis and atherosclerotic disease, were the role of protein S has been recently evaluated (4). Protein S is a complex molecule with several posttranslational modifications, and needs suitable animal models where its functional requirements are validated. The present work is an important step forward in this direction.

Effects of a Novel Anticoagulant Compound (TV7130) in an Ovine Model of Septic Shock

We compared the effects of a new compound (TV7130) with those of activated protein C (APC) in a large animal model of septic shock. Thirty-two fasted, anesthetized, invasively monitored, mechanically ventilated female sheep received 1.5 g/kg body weight of feces into the abdomen to induce sepsis. Immediately after feces injection, all animals received a bolus followed by a continuous infusion of saline (n = 8, bolus 1.5 mL for 15 min, infusion 1.5 mL/[kg·h]), low-dose TV7130 (n = 8; 0.4 mg/kg bolus, 0.4 mg/[kg·h] infusion), high-dose TV7130 (n = 8; 0.8 mg/kg bolus, 0.8 mg/[kg·h] infusion), or APC (n = 8; saline bolus, APC infusion of 0.024 mg/[kg·h]). Experiments were pursued until each sheep's spontaneous death. There were no significant differences among groups in heart rate or cardiac index, but mean arterial pressure, systemic vascular resistance index, and left ventricular stroke work index decreased less in the high-dose TV7130 and APC groups than in the other groups. Gas exchange was preserved better in the high-dose TV7130 and APC groups. Interleukin 6 and lactate concentrations were lower in the high-dose TV7130 and APC groups than in the other groups. Functional capillary density and proportion of perfused vessels, evaluated in the sublingual region using sidestream dark-field videomicroscopy, were significantly higher in the TV7130 and APC groups than in the vehicle group at 16 h. Survival time was significantly longer in the high-dose TV7130 and APC groups than in the other groups (log-rank test, P = 0.0002). TV7130 has similar effects to APC and may be a promising agent for the management of severe sepsis.

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.

Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

Reprint of Crosstalk between inflammation and thrombosis

Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Multiple mechanisms are at play including up regulation of tissue factor leading to the initiation of clotting, amplification of the clotting process by augmenting exposure of cellular coagulant phospholipids, inhibition of fibrinolysis by elevating plasminogen activator inhibitor 1 (PAI-1) and decreases in natural anticoagulant pathways, particularly targeted toward down regulation of the protein C anticoagulant pathway through multiple mechanisms. The decreased function of the natural anticoagulant pathways may be particularly problematic because these appear to play a role in dampening inflammatory responses. The protein C anticoagulant pathway provides a useful model for the impact of inflammation on coagulation. This pathway plays a major role in preventing microvascular thrombosis. The pathway is initiated when thrombin binds to thrombomodulin (TM) on the surface of the endothelium. An endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin–TM complex more than 10-fold in vivo. EPCR is shed from the endothelium by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1 and appears to inhibit leukocyte extravisation. EPCR can undergo translocation from the plasma membrane to the nucleus where it redirects gene expression. During translocation it can carry activated protein C (APC) to the nucleus, possibly accounting for the ability of APC to modulate inflammatory mediator responses in the endothelium. TNF ∝ and other inflammatory mediators can down-regulate EPCR and TM and IL-6 can depress levels of protein S in experimental animals. Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury and leukocyte extravisation in response to endotoxin, processes that are decreased by infusion of APC. In vitro, APC inhibits TNF ∝ elaboration from monocytes and to block leukocyte adhesion to selectins. Since thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.

Use of Recombinant Activated Protein C in Therapy for Sepsis in Children after Cardiosurgical Interventions

Sepsis is the principal cause of hospital death among children and ranks fourth among all causes of death in infants under 1 year of age and second in 1-to-14-year-old children. Objective: to assess whether activated protein C (APC) infusion may be incorporated into the complex intensive therapy for sepsis in babies of the first year of life. Subjects and methods. In January 2005 to April 2007, APC was used in the complex intensive therapy for sepsis in 36 infants of the first year of life. APC therapy was initiated in the first 24 hours after the occurrence of organ dysfunction in 29 (80%) patients and in the first 48 hours in others cases. Results. Status stabilization and multiple organ dysfunction (MOD) regression were noted in most patients during APC infusion. Ten (28%) patients died; mortality rates in the groups of patients with early and late infusion were 17 and 71%, respectively. The baseline APC level failed to affect 28-day survival. By the end of infusion, the mean level of protein C was much higher in the group of survivors than that in the deceased. Conclusion. APC as a part of the complex intensive therapy for sepsis should be given to infants of the first year of life who had multiple organ dysfunctions within the first 24 hours after the occurrence of organ dysfunction. Key words: pediatric sepsis, activated a-drotrecogin, multiple organ dysfunction.

ACTIVATED PROTEIN C ATTENUATES MICROVASCULAR INJURY DURING SYSTEMIC HYPOXIA

In response to hypoxia, an inflammatory cascade is initiated and microvascular injury ensues. Specifically, within 10 min, leukocyte adherence to the endothelium begins, and leukocyte emigration and vascular leak soon follow. Activated protein C (APC) has been reported to have both anticoagulant and anti-inflammatory properties. Activated protein C is best described in its role as a treatment for sepsis. However, it has been used, with some success, in experimental models of hypoxic injury. We hypothesized that APC would be protective against microvascular injury during systemic hypoxia. Randomized prospective animal study. Adult male Sprague-Dawley rats. To characterize the microvascular response to APC exposure during hypoxia, four rat groups were used: saline control, APC infusion alone (100 mg/kg bolus), hypoxia alone (10% O2), and simultaneous hypoxia + APC infusion. Measurements of leukocyte adherence (no. per 100-microm venule), leukocyte emigration (no. per 4,000 microm(2)), and venular leak by fluorescein isothiacyanate-labeled albumin (Fo/Fi) were performed during intravital microscopy of the intact venular bed. Leukocyte adherence decreased from 14.5 (+/-1.2) cells/100-microm venule in hypoxic rats to 4.4 (+/-1.5) cells/100-microm venule in those treated with both hypoxic gas and APC infusion (P < 0.001). Similarly, leukocyte emigration in hypoxic rats reached 12.3 (+/- 2.2) cells/4,000-microm(2) venule, but was reduced to 3.5 (+/-0.3) cells/4,000-microm(2) venule (P <.001). Venular permeability to protein was also significantly decreased in the APC-treated group from 0.82 (+/-0.14) to 0.25 (+/-0.14) (P < 0.001). The infusion of APC attenuates the inflammatory response during systemic hypoxia at the microvascular level, as evidenced by measurements of leukocyte adherence, emigration, and venular permeability. Further investigation is needed to examine the potential role of APC in the treatment of hypoxic injury.

Beneficial effects of recombinant human activated protein C in a ewe model of septic shock*

To investigate the effects of activated protein C (APC) in a clinically relevant animal model of septic shock. Prospective, randomized, controlled study. University medical center research laboratory. Eighteen female sheep (body weight, 27-35 kg). Animals were fasted, anesthetized, invasively monitored, and mechanically ventilated before receiving 0.5 g/kg body weight of feces intraperitoneally to induce sepsis. Fluid resuscitation with Ringer lactate was titrated to maintain pulmonary artery occlusion pressure at baseline levels. No vasoactive agents or antibiotics were used. Two hours after the induction of sepsis, animals were randomized to receive an infusion of APC (24 microg x kg(-1) x hr(-1), n = 9) or an equivalent volume of vehicle (n = 9) throughout the experimental period. The APC-treated animals had significantly higher arterial pressure, urine output, PaO2/FIO2 ratios, and thoracopulmonary compliance than the control animals. They had lower pulmonary arterial pressure and arterial lactate concentrations than the control animals. Plasma colloid oncotic pressure was better maintained in the APC-treated group than in the control group (p < .05). Prothrombin time and activated partial thromboplastin time were altered less, and plasma D-dimer concentrations were significantly lower in the APC-treated group than in the control group (p < .05). The blood protein C concentration and platelet count were maintained better in the APC-treated group than in the control group (p < .05). APC administration was associated with significantly longer survival (median, 27 hrs vs. 20 hrs; p < .05). At postmortem examination, the lung wet/dry ratio was significantly lower in the APC group than in the control group (6.3 +/- 0.7 vs. 7.1 +/- 1.2, p < .05). In this clinically relevant model of septic shock due to fecal peritonitis, administration of APC had beneficial effects on hemodynamic variables, gas exchange, lactic acidosis, and coagulation abnormalities. Higher colloid oncotic pressures and lower lung wet/dry ratios at autopsy suggest preserved endothelial integrity. APC administration resulted in prolonged survival.

Activated protein C.

Protein C is a vitamin K-dependent plasma protein zymogen whose genetic mild or severe deficiencies are linked with risk for venous thrombosis or neonatal purpura fulminans, respectively. Studies over past decades showed that activated protein C (APC) inactivates factors (F) Va and VIIIa to down-regulate thrombin generation. More recent basic and preclinical research on APC has characterized the direct cytoprotective effects of APC that involve gene expression profile alterations, anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. These actions generally require endothelial cell protein C receptor (EPCR) and protease activated receptor-1. Because of these direct cytoprotective actions, APC reduces mortality in murine endotoxemia and severe sepsis models and provides neuroprotective benefits in murine ischemic stroke models. Furthermore, APC reduces mortality in patients with severe sepsis (PROWESS clinical trial). Although much remains to be clarified about mechanisms for APC's direct effects on various cell types, it is clear that APC's molecular features that determine its antithrombotic action are partially distinct from those providing cytoprotective actions because we have engineered recombinant APC variants with selective reduction or retention of either anticoagulant or cytoprotective activities. Such APC variants can provide relatively enhanced levels of either cytoprotective or anticoagulant activities for various therapeutic applications. We speculate that APC variants with reduced anticoagulant action but normal cytoprotective actions hold the promise of reducing bleeding risk because of attenuated anticoagulant activity while reducing mortality based on direct cytoprotective effects on cells.

Relative antithrombotic and antihemostatic effects of protein C activator versus low-molecular-weight heparin in primates

AbstractThe anticoagulant and anti-inflammatory enzyme, activated protein C (APC), naturally controls thrombosis without affecting hemostasis. We therefore evaluated whether the integrity of primary hemostasis was preserved during limited pharmacological antithrombotic protein C activator (PCA) treatment in baboons. The double-mutant thrombin (Trp215Ala/Glu217Ala) with less than 1% procoagulant activity was used as a relatively selective PCA and compared with systemic anticoagulation by APC and low-molecular-weight heparin (LMWH) at doses that inhibited fibrin deposition on thrombogenic segments of arteriovenous shunts. As expected, both systemic anticoagulants, APC (0.028 or 0.222 mg/kg for 70 minutes) and LMWH (0.325 to 2.6 mg/kg for 70 minutes), were antithrombotic and prolonged the template bleeding time. In contrast, PCA at doses (0.0021 to 0.0083 mg/kg for 70 minutes) that had antithrombotic effects comparable with LMWH did not demonstrably impair primary hemostasis. PCA bound to platelets and leukocytes, and accumulated in thrombi. APC infusion at higher circulating APC levels was less antithrombotic than PCA infusion at lower circulating APC levels. The observed dissociation of antithrombotic and antihemostatic effects during PCA infusion thus appeared to emulate the physiological regulation of intravascular blood coagulation (thrombosis) by the endogenous protein C system. Our data suggest that limited pharmacological protein C activation might exhibit considerable thrombosis specificity.

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.

Crosstalk between inflammation and thrombosis

Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Multiple mechanisms are at play including up regulation of tissue factor leading to the initiation of clotting, amplification of the clotting process by augmenting exposure of cellular coagulant phospholipids, inhibition of fibrinolysis by elevating plasminogen activator inhibitor 1 (PAI-1) and decreases in natural anticoagulant pathways, particularly targeted toward down regulation of the protein C anticoagulant pathway through multiple mechanisms. The decreased function of the natural anticoagulant pathways may be particularly problematic because these appear to play a role in dampening inflammatory responses. The protein C anticoagulant pathway provides a useful model for the impact of inflammation on coagulation. This pathway plays a major role in preventing microvascular thrombosis. The pathway is initiated when thrombin binds to thrombomodulin (TM) on the surface of the endothelium. An endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin–TM complex more than 10-fold in vivo. EPCR is shed from the endothelium by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1 and appears to inhibit leukocyte extravisation. EPCR can undergo translocation from the plasma membrane to the nucleus where it redirects gene expression. During translocation it can carry activated protein C (APC) to the nucleus, possibly accounting for the ability of APC to modulate inflammatory mediator responses in the endothelium. TNF α and other inflammatory mediators can down-regulate EPCR and TM and IL-6 can depress levels of protein S in experimental animals. Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury and leukocyte extravisation in response to endotoxin, processes that are decreased by infusion of APC. In vitro, APC inhibits TNF α elaboration from monocytes and to block leukocyte adhesion to selectins. Since thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.

Activated Protein C Prevents Neuronal Apoptosis via Protease Activated Receptors 1 and 3

Activated protein C (APC), a serine protease with anticoagulant and anti-inflammatory activities, exerts direct cytoprotective effects on endothelium via endothelial protein C receptor-dependent activation of protease activated receptor 1 (PAR1). Here, we report that APC protects mouse cortical neurons from two divergent inducers of apoptosis, N-methyl-D-aspartate (NMDA) and staurosporine. APC blocked several steps in NMDA-induced apoptosis downstream to nitric oxide, i.e., caspase-3 activation, nuclear translocation of apoptosis-inducing factor (AIF), and induction of p53, and prevented staurosporine-induced apoptosis by blocking caspase-8 activation upstream of caspase-3 activation and AIF nuclear translocation. Intracerebral APC infusion dose dependently reduced NMDA excitotoxicity in mice. By using different anti-PARs antibodies and mice with single PAR1, PAR3, or PAR4 deletion, we demonstrated that direct neuronal protective effects of APC in vitro and in vivo require PAR1 and PAR3. Thus, PAR1 and PAR3 mediate anti-apoptotic signaling by APC in neurons, which may suggest novel treatments for neurodegenerative disorders.

Coagulation and inflammation

The protein C anticoagulant pathway is critical for controlling microvascular thrombosis and is initiated when thrombin binds to thrombomodulin (TM) on the surface of the endothelium. Protein C activation is augmented by an endothelial cell protein C receptor (EPCR). EPCR is shed from the vasculature by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1 and appears to inhibit leukocyte extravasation. EPCR can undergo translocation from the plasma membrane to the nucleus where it re-directs gene expression. During translocation, EPCR can carry activated protein C (APC) to the nucleus, possibly accounting for the ability of APC to modulate inflammatory mediator responses in the endothelium. TNF-α and other inflammatory mediators can down-regulate EPCR and TM. Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury and leukocyte extravasation in response to endotoxin and infusion of APC reverses these processes. In vitro, APC has been reported to inhibit TNF-α elaboration from monocytes and to block leukocyte adhesion to selectins. Since thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.

Coagulation and inflammation.

The protein C anticoagulant pathway is critical for controlling microvascular thrombosis and is initiated when thrombin binds to thrombomodulin (TM) on the surface of the endothelium. Protein C activation is augmented by an endothelial cell protein C receptor (EPCR). EPCR is shed from the vasculature by inflammatory mediators and thrombin. EPCR binds to activated neutrophils in a process that involves proteinase 3 and Mac-1 and appears to inhibit leukocyte extravasation. EPCR can undergo translocation from the plasma membrane to the nucleus where it re-directs gene expression. During translocation, EPCR can carry activated protein C (APC) to the nucleus, possibly accounting for the ability of APC to modulate inflammatory mediator responses in the endothelium. TNF-alpha and other inflammatory mediators can down-regulate EPCR and TM. Inhibition of protein C pathway function increases cytokine elaboration, endothelial cell injury and leukocyte extravasation in response to endotoxin and infusion of APC reverses these processes. In vitro, APC has been reported to inhibit TNF-alpha elaboration from monocytes and to block leukocyte adhesion to selectins. Since thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.

Inhibition of Microarterial Thrombosis by Activated Protein C in a Rabbit Model

Protein C is the key component in a natural anticoagulant pathway. After its activation by the thrombin-thrombomodulin complex, it degrades the activated forms of coagulation cofactors VIII and V, which leads to downregulation of the coagulation process. Due to its specific anticoagulant activity, activated protein C (APC) is potentially interesting as an antithrombotic agent. The effect of bovine activated protein C on thrombus formation and haemostasis was investigated in a rabbit model of microarterial thrombosis. Segments of both central ear arteries were prepared and blood-flow interrupted with double vascular clamps. Longitudinal arteriotomies (7 mm) and deep vessel wall trauma (5 mm) were performed, whereafter the arteriotomies were closed with running sutures. Five minutes prior to opening of the clamps (reperfusion), boluses of APC (0.8 mg/kg bodyweight) or vehicle alone were administered to two groups, each of 10 rabbits, in a blind random fashion. Vessel patency-rates were drastically improved by the administration of APC compared to vehicle. Correspondingly, thrombus weights were significantly lower in the APC group than in the control group. The activated partial thromboplastin time was prolonged to approximately twice the baseline throughout the 2 h observation interval in the APC group. Levels of circulating platelets were unaffected by the APC infusion, but the arteriotomy bleeding times were significantly longer in the APC group. In summary, activated protein C exerted powerful and long-acting antithrombotic effects in a microarterial model of thrombosis in rabbits.

Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model

The in vivo antithrombotic properties of human plasma activated protein C (APC), a natural anticoagulant enzyme, were investigated in a baboon model of thrombus formation on prosthetic vascular grafts. Infusion of 0.25 to 1.1 mg/kg/h purified, human, APC inhibited blood clotting, as measured by the activated partial thromboplastin time (APTT), and reduced vascular graft platelet deposition by 40% to 70%, as determined by the real-time scintillation camera imaging of 111In-labeled platelet deposition. APC infusion also preserved graft patency. Hemostatic plug formation remained normal, as measured by the template bleeding times. These results suggest that APC administration may produce immediate antithrombotic effects under arterial flow conditions.