Activated Protein C Therapy Research Articles

Abstract 13370: Single-Cell RNA-seq Reveals Transcriptomic Modulation of Alzheimer’s Disease by Activated Protein C

Introduction: Alzheimer’s Disease (AD) causes neuroinflammation characterized by degradation of neurological functions resulting in memory loss and confusion. Activated Protein C (APC), a plasma zymogen, has shown promising cytoprotection, anti-inflammation, and anti-apoptotic properties. ScRNA-seq allows for examining the underlying mechanisms by which APC therapy on AD at the cellular level. Hypothesis: APC treatment can ameliorate AD symptoms of 5xFAD transgenic mice. The scRNA Seq analysis could provide detailed responses of various cell populations to Alzheimer's disease pathogenesis and molecular aspects of APC therapy. Methods: Whole brain tissue were collected at 7 months, APC-treated 5xFAD mice received daily recombinant APC or saline injections. The 10x Genomics Next Gem kit was used to dissociate the tissue for sequencing. Bioinformatic analysis was performed using Seurat in R and an integrated dataset was used to compare multiple single-cell samples. Results: Bioinformatic analysis of 5xFAD versus wild type (WT) C57BL/6J mouse brain revealed much of the sample consists of microglial cells. Interestingly, 5xFAD versus WT brains have notable decreases in astrocyte and neuron populations. Administration of recombinant APC rebounds these populations to comparable levels as WT while dramatically reduces the microglial population in 5xFAD versus WT brain. The largest proportion of oligodendrocytes are also recorded after APC treatment. Differential expression testing conveys transcriptomic regulation of known AD critical genes such as Apoe , Ctsb , Trem2 , and Tyrobp by APC. Consequently, GO term enrichment uncovers inflammatory biological processes such as microglial activation to be downregulated by APC-treated 5xFAD. Additionally, recuperation of nervous system developmental processes was found in APC-treated 5xFAD mice. Conclusions: APC treatment drastically impacts the cellular pathophysiology of AD. Differential expression testing of APC treated 5xFAD versus WT mice revealed transcriptomic downregulation of AD implicated genes throughout multiple cell-types. Administration of APC significantly impacts AD implicated genes associated with AD pathogenesis through enrichment analysis.

Activated Protein C Protects against Murine Contact Dermatitis by Suppressing Protease-Activated Receptor 2.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1β, IL-6 and TGF-β1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

Cytoprotective-selective activated protein C therapy for ischaemic stroke.

Despite years of research and efforts to translate stroke research to clinical therapy, ischaemic stroke remains a major cause of death, disability, and diminished quality of life. Primary and secondary preventive measures combined with improved quality of care have made significant progress. However, no novel drug for ischaemic stroke therapy has been approved in the past decade. Numerous studies have shown beneficial effects of activated protein C (APC) in rodent stroke models. In addition to its natural anticoagulant functions, APC conveys multiple direct cytoprotective effects on many different cell types that involve multiple receptors including protease activated receptor (PAR) 1, PAR3, and the endothelial protein C receptor (EPCR). Application of molecular engineered APC variants with altered selectivity profiles to rodent stroke models demonstrated that the beneficial effects of APC primarily require its cytoprotective activities but not its anticoagulant activities. Extensive basic, preclinical, and clinical research provided a compelling rationale based on strong evidence for translation of APC therapy that has led to the clinical development of the cytoprotective-selective APC variant, 3K3A-APC, for ischaemic stroke. Recent identification of non-canonical PAR1 and PAR3 activation by APC that give rise to novel tethered-ligands capable of inducing biased cytoprotective signalling as opposed to the canonical signalling provides a mechanistic explanation for how APC-mediated PAR activation can selectively induce cytoprotective signalling pathways. Collectively, these paradigm-shifting discoveries provide detailed insights into the receptor targets and the molecular mechanisms for neuroprotection by cytoprotective-selective 3K3A-APC, which is currently a biologic drug in clinical trials for ischaemic stroke.

Coagulation proteases and cardiovascular disease

There is increasing interest in the many possible mechanisms through which co-agulation proteases may promote arterial cardiovascular disease (CVD). Athero-sclerosis and thrombosis are the principal pathologies underlying coronary heart dis-ease (CHD), ischaemic stroke, and periph-eral arterial disease (PAD) (1). Circulating coagulation proteases (as well as their final substrate, fibrinogen) may be biomarkers for risk of arterial thrombosis and throm-bo-embolism; and may also influence the structure of fibrin clots and thrombi (2). Both coagulant and anticoagulant pro-teases can influence multiple cells in the vessel wall and in vital organs; and hence can influence processes including haemo-stasis, atherosclerosis, ischaemia and reper-fusion injury, inflammation, organ and vessel remodelling, and fibrosis (3) (Figure 1).Selective anticoagulation with new agents which target specific proteases has increased knowledge of this multitude of physiological and pathological actions; and clinical trials of NOACs (non-vitamin K-dependent oral anticoagulants; also re-ferred to as DOACs, for direct oral antico-agulants) have shown reduction in risk of major bleeding compared to traditional vit-amin K antagonists (VKAs), especially in intracranial bleeding; but in some trials in-creased risk of gastro-intestinal bleeding (4). These results require explanation of the possible mechanisms for organ-specific haemostasis.For this theme issue of the journal, we invited experts in several of these areas to provide brief reviews.Lowe and Rumley review evidence from prospective epidemiological studies for as-sociations of circulating levels of coagu-lation proteases, fibrinogen, and activation markers of coagulation and fibrinolysis with risk of CVD (5). To date, plasma fibri-nogen shows the strongest and most con-sistent associations with CVD; however, genetic studies do not support causality, and there is limited evidence that lowering fibrinogen levels reduces CVD risk. These associations may result from up-regulation of pro-inflammatory cytokines such as in-terleukin-6. In contrast, the common gen-etic polymorphisms for factors II, V and the von Willebrand factor:FVIII complex (non-O blood group) show associations with CHD risk, consistent with potential causal roles in arterial thrombosis, as with venous thrombosis. The associations of fi-brin D-dimer and tissue plasminogen acti-vator (t-PA) antigen levels with CHD risk are consistent with potential roles for acti-vation of coagulation, fibrinolysis and en-dothelial cells.Bjorkqvist et al. review in vivo activation and functions of the protease factor XII (FXII), the principal initiator of the contact system of coagulation, and an initiator of fibrinolysis and the classic complement system pathway of inflammation (6). They conclude that FXII influences thrombotic risk without altering haemostasis, and also plays a role in inflammation, sepsis and hereditary angioedema. These properties have renewed interest in FXII inhibition as a therapeutic strategy to treat aberrant vas-cular leakage and thrombotic disorders.Rezaie reviews protease-activated recep-tor (PAR) signalling by coagulation pro-teases in endothelial cells. PAR 1–4 are G-protein coupled receptors which can be specifically activated by thrombin and all vitamin K-dependent coagulation pro-teases (except FIXa) (7). While activation of PAR1 by thrombin induces pro-inflam-matory signaling responses, its activation by activated protein C (APC) elicits anti-inflammatory responses in cultured en-dothelial cells. Recombinant APC has been used as a therapeutic drug for treatment of adults with severe sepsis.Mosnier et al. review cytoprotective APC therapy for ischaemic stroke. APC is not only a natural anticoagulant, but also conveys multiple cytoprotective effects on many different cell types that involve multiple receptors including PAR1, PAR3, and the endothelial protein C receptor (EPRC) (7). Application of molecular en-gineered APC variants to rodent stroke models demonstrated that the beneficial ef-fects of APC require its cytoprotective ac-tivities but not its anticoagulant activities. The cytoprotective selective APC variant, 3K3A-APC is currently undergoing clinical trials in ischaemic stroke.Antoniak et al. review the role of tissue factor (TF)-FVII(a) activation of PARs in pathologic heart remodeling associated with myocardial infarction, viral myocardi-tis and hypertension (8). Cardiomyocyte TF is essential to maintain heart haemo-stasis. TF-dependent activation of coagu-lation and activation of PARs contribute to initial size of myocardial infarction, as well as pathologic heart remodelling including hypertrophic growth of cardiomyocytes and fibrosis.Bridge et al. review the associations of clot properties and CVD. Fibrin clot struc-ture is altered by levels of fibrinogen, thrombin, but also by metabolic disturb-ances including diabetes mellitus and hy-perhomocystinaemia; as well as treatment with aspirin, anticoagulants, statins and fi-brates (9). Change in fibrin clot structure in vitro, to a denser clot with smaller pores, which is more resistant to lysis, is strongly

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.

Activated protein C action in inflammation

Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti-inflammatory, anti-apoptotic, and endothelial barrier protection. Recently, a recombinant form of human APC (rhAPC or drotrecogin alfa activated; known commercially as 'Xigris') was approved by the US Federal Drug Administration for treatment of severe sepsis associated with a high risk of mortality. Sepsis, also known as systemic inflammatory response syndrome (SIRS) resulting from infection, is a serious medical condition in critical care patients. In sepsis, hyperactive and dysregulated inflammatory responses lead to secretion of pro- and anti-inflammatory cytokines, activation and migration of leucocytes, activation of coagulation, inhibition of fibrinolysis, and increased apoptosis. Although initial hypotheses focused on antithrombotic and profibrinolytic functions of APC in sepsis, other agents with more potent anticoagulation functions were not effective in treating severe sepsis. Furthermore, APC therapy is also associated with the risk of severe bleeding in treated patients. Therefore, the cytoprotective effects, rather than the anticoagulant effect of APC are postulated to be responsible for the therapeutic benefit of APC in the treatment of severe sepsis.

Activated Protein C is Neuroprotective and Mediates New Blood Vessel Formation and Neurogenesis After Controlled Cortical Impact

Activated protein C (APC) is neuroprotective in stroke models and promotes postischemic neovascularization and neurogenesis. We used a controlled cortical impact (CCI) in mice to determine the effects of APC on neuroprotection and angiogenesis and neurogenesis after traumatic brain injury (TBI). Mice were given (1) single-dose APC (0.8 mg/kg intraperitoneally) 15 minutes after injury, (2) multidose APC (0.8 mg/kg intraperitoneally) 15 minutes and 6 to 48 hours after injury, or (3) vehicle. We then assessed the effects of APC on posttraumatic motor function with the rotarod and wire grip and beam balance tasks, and we determined the lesion volumes and studied the formation of new blood vessels and markers of neurogenesis. Mice treated with single-dose or multidose APC, compared with vehicle, showed significantly improved motor function on all tests. In the single-dose and multidose APC treatment groups, at 7 days after treatment, lesion volume was significantly decreased by 30% and 50%, respectively. Multidose APC, but not single-dose APC, increased new blood vessel formation as shown by CD105(+)/Ki-67(+) double immunostaining by nearly 2-fold at 7 days. Multidose APC also promoted posttraumatic proliferation of neuroblasts in the subventricular zone (SVZ) and their migration from the SVZ to the perilesional area. Activated protein C improves functional outcome and is neuroprotective after TBI. It also promotes angiogenesis and survival and migration of neuroblasts from the SVZ to the perilesional area, but the exact role of these brain repair mechanisms remains to be determined. The present findings suggest that APC therapy may hold a significant therapeutic potential for TBI.

Activated Protein C Improves Heatstroke Outcomes Through Restoration of Normal Hypothalamic and Thermoregulatory Function

BackgroundHuman recombinant activated protein C (APC) has been found to be beneficial in treating heatstroke in both humans and rats. Here, we further investigated the possible mechanism underlying the therapeutic action exerted by APC in experimental heatstroke. MethodsUnanesthetized and unrestrained mice were exposed to an ambient temperature of 42.4°C for 1hour to induce heatstroke. They were given normal saline or APC (5mg/kg of body weight, intravenously) 1hour after the termination of heat stress. Their physiologic and biochemical parameters were continuously monitored for 4hours after cessation of heat stress. Mice that survived on day 4 of heat treatment were considered survivors. ResultsWhen the vehicle-treated mice underwent heat treatment, the fraction survival and core temperature at 4hours of body heating were found to be 0 of 12 and 33.8±0.6°C, respectively. Administration of APC 1hour after the cessation of heat stress rescued the mice from heat-induced death (fraction survival, 12 of 12) and reduced hypothermia (core temperature, 37.4±0.2°C). Heat-induced apoptosis in the hypothalamus was significantly reduced by APC. The increased levels of cellular ischemia (eg, glutamate, lactate-to-pyruvate ratio, nitrite, and dihydroxybenzoic acid) and damage (eg, glycerol) markers in the hypothalamus during heatstroke were also decreased significantly by APC therapy. The hypothalamic levels of tumor necrosis factor-α (a proinflammatory cytokine) that are upregulated in heat-stressed mice were significantly lower in APC-administered mice. ConclusionsOur results show that human recombinant APC improves heatstroke through restoration of normal hypothalamic and thermoregulatory function.

Protective mechanisms of activated protein C in severe inflammatory disorders

The protein C system is an important natural anticoagulant mechanism mediated by activated protein C (APC) that regulates the activity of factors VIIIa and Va. Besides well-defined anticoagulant properties, APC also demonstrates anti-inflammatory, anti-apoptotic and endothelial barrier-stabilizing effects that are collectively referred to as the cytoprotective effects of APC. Many of these beneficial effects are mediated through its co-receptor endothelial protein C receptor, and the protease-activated receptor 1, although exact mechanisms remain unclear and are likely pleiotropic in nature. Increased insight into the structure-function relationships of APC facilitated design of APC variants that conserve cytoprotective effects and reduce anticoagulant features, thereby attenuating the risk of severe bleeding with APC therapy. Impairment of the protein C system plays an important role in acute lung injury/acute respiratory distress syndrome and severe sepsis. The pathophysiology of both diseases states involves uncontrolled inflammation, enhanced coagulation and compromised fibrinolysis. This leads to microvascular thrombosis and organ injury. Administration of recombinant human APC to correct the dysregulated protein C system reduced mortality in severe sepsis patients (PROWESS trial), which stimulated further research into its mechanisms of action. Several other clinical trials evaluating recombinant human APC have been completed, including studies in children and less severely ill adults with sepsis as well as a study in acute lung injury. On the whole, these studies have not supported the use of APC in these populations and challenge the field of APC research to search for additional answers.

Mechanisms of human complement factor B induction in sepsis and inhibition by activated protein C

To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-alpha and IFN-gamma or stimulation with LPS demonstrated a time- and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and +128 bp was found to mediate IFN-gamma, TNF-alpha, and LPS responsiveness as well as the synergistic effect of IFN-gamma/TNF-alpha on hBf promoter activity. Site-directed mutagenesis of a IFN-gamma-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-gamma responsiveness. Mutagenesis of a nuclear factor (NF)-kappaB cis element at -466 to -456 bp abrogated TNF-alpha and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-gamma, TNF-alpha, and LPS is through GAS and NF-kappaB cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway.

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.

Preservation of Beneficial TAFI Functions by an Activated Protein C Variant with Normal Cytoprotective Functions but Minimal Anticoagulant Activity.

Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa), a carboxypeptidase B-like enzyme, both inhibits fibrinolysis and exerts anti-inflammatory effects via removal of C-terminal Arg residues of bradykinin (BK) and the complement anaphylatoxins, C3a and C5a. Proteolytic activation of TAFI is caused by thrombin or thrombin-thrombomodulin; thus, TAFI activation can be down-regulated by the anticoagulant, activated protein C (APC). Recombinant APC reduces mortality in severe sepsis patients (PROWESS trial), but it increases risk of serious bleeding. This risk is presumably due to APC's anticoagulant activity and possibly exacerbated by profibrinolytic effects derived from down-regulation of thrombin-mediated activation of TAFI. In addition, APC therapy might ironically aggravate sepsis by reducing TAFIa generation because TAFIa inactivates BK, C3a and C5a, well known pathological mediators in sepsis. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191–193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, J Exp Med, 2007). This current study compares the effects of this variant to wild type (wt) APC on profibrinolytic and TAFIa's anti-inflammatory activities in plasma-based assays. To determine the effects of APC on TAFIa-mediated inhibition of fibrinolysis, clot lysis was studied in a plasma system of thrombin-induced clot formation and tPA-mediated fibrinolysis. Compared to wt-APC (IC50=0.36 nM APC), almost 10-times more 5A-APC was required for 50% inhibition of clot lysis (IC50=3.1 nM 5A-APC) and 20-times more 5A-APC was needed to inhibit completely TAFI activation under the conditions employed. The effects of wt-APC and 5A-APC on TAFIa's anti-inflammatory activities were determined by following proteolytic inactivation of BK in plasma during tissue factor-induced clotting. In the absence of added thrombomodulin, angiotensin-converting enzyme, carboxypeptidase N, and TAFIa each inactivated approximately 10–15% of BK under the study conditions. But in the presence of thrombomodulin, BK was fully inactivated in plasma and converted to des-Arg9-BK (> 98%), an effect mainly attributable to TAFIa. Analysis of the time course of BK inactivation in the presence of thrombomodulin indicated that TAFIa rapidly converted BK to des-Arg9-BK with only a minor contribution from carboxypeptidase N. Under conditions where TAFIa fully converted BK to des-Arg9-BK, wt-APC, but not 5A-APC, dose-dependently diminished BK inactivation and des-Arg9-BK generation. At the highest tested concentrations of APC (40 nM), wt-APC completely inhibited TAFIa-dependent BK inactivation whereas non-anticoagulant 5A-APC permitted normal TAFI activation and showed no significant inhibition of TAFIa-dependent BK inactivation. Thus, 5A-APC left intact the TAFIa-mediated anti-inflammatory mechanism for inactivation of BK in plasma and, presumably, TAFIa's ability to inactivate the C3a and C5a anaphylatoxins. These studies show that genetic engineering can selectively alter APC's multiple activities to provide an APC variant that retains this enzyme's beneficial anti-inflammatory and cytoprotective effects but that diminishes bleeding risk due to reduction in APC's anticoagulant and profibrinolytic activities. Thus, these data support the hypothesis that preservation of TAFI activation in plasma by normally cytoprotective 5A-APC variant would preserve TAFIa-dependent anti-inflammatory effects that are lost with the use of wt-APC.

Mechanisms for Mortality Reduction by Activated Protein C in Severe Sepsis.

Recombinant wild type (wt) activated protein C (APC) reduces patient mortality in severe sepsis and multi-organ failure. APC can exert both anticoagulant activity and direct cytoprotective effects on cells (anti-inflammatory, anti-apoptotic, endothelial barrier stabilization, etc.). The contribution of distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. Lethal mouse endotoxemia (i.p. LPS administration) and bacterial sepsis (i.p. Staphylococcus aureus) models were used to clarify mechanisms for APC's beneficial mortality reduction effects and to distinguish the relative importance of APC anticoagulant effects vs. APC direct effects on cells. Murine rec wtAPC (APC) was administered as bolus plus i.v. infusion (over < 2 hr) in total doses ranging from 0.2 to 0.04 mg/kg and was given coincident with or at times up to 3 hr after challenge. Following induction of LPS-mediated septicemia in normal mice, APC markedly reduced mortality (eg., from 50% to 0–10% at LD-50 LPS doses). APC treatment did not alter the extent of circulating inflammatory cytokine levels at 3 or 24 hr after endotoxin exposure. The survival benefit conferred by wt APC infusions was abolished in mice with genetically reduced levels of endothelial protein C receptor (EPCR) (< 10% of normal) or in mice genetically lacking protease-activated receptor-1 (PAR-1). Murine APC variants with either 3 or 5 Ala substitutions, 3K3A-APC (KKK192-194AAA) or 5A-APC (RR230/231AA + KKK192-194AAA) that had reduced anticoagulant activity (25 % and < 10 % of wt APC, respectively), but normal cytoprotective activities, were as effective as wt APC in reducing mortality after LPS challenge. A murine APC variant lacking proteolytic activity (active site S360A) did not enhance survival after LPS, showing a requirement for APC's enzymatic activity. Thus, the survival-promoting efficacy of APC in this model requires the enzymatic active site of APC and the presence of two receptors, EPCR and PAR-1, that are known to mediate APC's in vitro beneficial cytoprotective effects on cells. In a whole bacteria sepsis model, when APC was given to mice at the time of initiation of peritoneal Staphylococcus aureus-induced sepsis and again at 24 hr, wt APC surprisingly increased mortality (100% mortality vs. 50% at LD-50 bacteria dose). In contrast, when 3K3A-APC or 5A-APC variants with attenuated anticoagulant activity was given at 0 and 24 hr, they prevented mortality due to bacterial sepsis (0–10% vs. 50% mortality for saline control at LD-50 dose). This implies that APC's anticoagulant action might impair beneficial coagulation-dependent host defense mechanisms in early stages of bacterial sepsis whereas the 5A-APC variant, with very low anticoagulant activity but normal cytoprotective activity, might provide beneficial cellular effects to help prevent death during bacterial sepsis. In summary, the full anticoagulant activity of APC is not required for protection against mortality in each of these models. These results highlight the importance of the cellular protein C pathway for APC therapy and suggest that APC variants with reduced anticoagulant action but normal potency for beneficial direct cellular effects merit further evaluation for sepsis therapy.

The promise of protein C

Protein C, a vitamin K-dependent serine protease zymogen that circulates in plasma, is converted by limited proteolysis to activated protein C (APC) by the thrombin–thrombomodulin complex. APC exerts anticoagulant, antiinflammatory, cytoprotective, and antiapoptotic activities. Recombinant APC therapy reduces mortality in severe sepsis patients. This review summarizes data from clinical observations, from in vitro studies, and from animal models of focal ischemic injury that provide a compelling rationale for clinical trials of APC for ischemic stroke.

A Novel Mechanism of Endothelial Protein C Receptor Release, in Microparticulate Form, by Activated Protein C.

Activated protein C (APC) administration is now used for treating patients with severe sepsis. We investigated its effect on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release from the cell surface. Exposure of human umbilical vein endothelial cells or monocytes to APC (from physiological levels of 0.5 up to 100nM) resulted in the increasing release of EPCR-containing microparticles (EPCR-MP), as demonstrated by confocal microscopy. Further characterisation through flow cytometry showed a concomitant fall in EPCR levels from the cell surface. This release of EPCR could not be inhibited by the metalloproteinase inhibitors 1, 10-phenanthroline or Ro31-9790, unlike soluble EPCR (sEPCR) that is metalloproteinase cleaved at the cell surface following thrombin or pro- inflammatory cytokine stimulation. Western blotting confirmed the molecular weight of EPCR-MP to be identical to the full-length membrane form (49 kD) and different from sEPCR (45 kDa). APC was also bound to EPCR-MP and could be quantified by ELISA using EPCR capture and APC detection by chromogenic substrate, S2366. Using an initial factor Va incubation step followed by a prothrombinase assay, the APC bound to EPCR-MP could significantly reduce thrombin generation. This was abrogated in the presence of excess α1-antitrypsin, an APC inhibitor. By contrast, APC bound to sEPCR could no longer inactivate factor Va. Further characterisation showed the APC induction of EPCR-MP to be time dependent with increasing release over 24 hours, as quantified by ELISA. The phenomenon also required the active site of APC. Neither protein C, heat-inactivated or D-Phe-Pro-Arg-chloromethylketone-blocked APC could induce EPCR-MP formation. Co-incubation with hirudin (6mM) did not alter the APC effect and excluded any role of contaminating thrombin. This novel observation provides new insights into the consequences of APC therapy in the septic patient as well as demonstrating for the first time that there can be 2 circulating forms of EPCR. Unlike sEPCR however, EPCR-MP can facilitate and potentially disseminate the anticoagulant activity of bound APC.

Activated Protein C Preserves Functional Islet Mass After Intraportal Transplantation

Clinical studies indicate that significant loss of functional islet mass occurs in the peritransplant period. Islets are injured as a result of detrimental effects of brain death, pancreas preservation, islet isolation, hypoxia, hyperglycemia, and immune-mediated events. In addition, recent studies demonstrated that islets are injured as a result of their exposure to blood and of activation of intrahepatic endothelial and Kupffer cells, resulting in inflammation and thrombosis. Activated protein C (APC) is an anticoagulant enzyme that also exerts anti-inflammatory and antiapoptotic activities by acting directly on cells. Here, we report that exogenous administration of recombinant murine APC (mAPC) significantly reduced loss of functional islet mass after intraportal transplantation in diabetic mice. Animals given mAPC exhibited better glucose control, higher glucose disposal rates, and higher arginine-stimulated acute insulin release. These effects were associated with reduced plasma proinsulin, intrahepatic fibrin deposition, and islet apoptosis early after the transplant. In vitro and in vivo data demonstrated that mAPC treatment was associated with a significant reduction of proinflammatory cytokine release after exposure of hepatic endothelial cells to islets. mAPC treatment also prevented endothelial cell activation and dysfunction elicited by intrahepatic embolization of isolated islets inherent to pancreatic islet transplantation (PIT). This study demonstrates multiple remarkable beneficial effects of mAPC for PIT and suggests that APC therapy may enhance the therapeutic efficacy of PIT in diabetic patients.

Activated protein C variants with normal cytoprotective but reduced anticoagulant activity

AbstractRecombinant activated protein C (APC), a well-defined anticoagulant enzyme, reduced mortality in severe sepsis patients in a phase 3 trial. However, 2 potent anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, failed to do so, implying the physiologic relevance of APC's less well-defined anti-inflammatory and antiapoptotic activities. Recombinant APC therapy conveys an increased risk of serious bleeding complications due to APC anticoagulant activity. To generate recombinant APC variants with reduced risk of bleeding due to reduced anticoagulant activity, we dissected APC's anticoagulant activity from its cytoprotective activity by site-directed mutagenesis. Using staurosporine-induced endothelial cell apoptosis assays, we show here that Ala mutations (RR229/230AA and KKK191_ 193AAA) in 2 APC surface loops that severely reduce anticoagulant activity result in 2 APC variants that retain normal antiapoptotic activity that requires protease activated receptor-1 and endothelial cell protein C receptor. Thus, it is possible to reduce anticoagulant activity while preserving antiapoptotic activity of recombinant APC variants. We suggest that therapeutic use of such APC variants may reduce serious bleeding risks while providing the beneficial effects of APC acting directly on cells. (Blood. 2004;104: 1740-1744)

Endogenous protein C activation in patients with severe sepsis.

To review how endogenous protein C activation might change in disease states and to discuss the implications of these findings in the context of severe sepsis. A review of the published literature in PubMed together with data from abstracts from 2001 to present. Activated protein C (APC) supplementation has been shown to significantly reduce mortality in patients with severe sepsis, presumably by virtue of its ability to down-regulate coagulation, inflammation, and apoptosis. In vivo, endogenous APC is generated in the circulation when protein C is activated by the thrombin-thrombomodulin complex. Protein C activation is augmented by the endothelial cell protein C receptor. Thus, thrombomodulin and the endothelial cell protein C receptor are components of the endothelium-based "machinery" required for efficient activation of protein C. In healthy individuals, the amount of APC formed is proportional to thrombin levels. In vitro studies have shown that thrombomodulin and the endothelial cell protein C receptor are down-regulated by inflammatory cytokines, and the levels of these receptors are reduced in the endothelium of skin-biopsy specimens in children with severe meningococcal sepsis. However, endothelial studies of excised blood vessels provide only a partial picture of the APC pathway in vivo. Knowledge of endogenous plasma levels of protein C, thrombin, and APC may be helpful in assessing the functional status of the protein C pathway in the systemic circulation. To date, there are few reports available on endogenous APC levels in patients with severe sepsis, perhaps due to the lack of available assays that permit both rapid and accurate measurements. A unique feature of our study is that we have developed an APC assay that, for the first time, permits rapid and accurate measurements of plasma APC levels. Preliminary studies using this assay suggest that adult patients with severe sepsis vary markedly in their ability to generate APC endogenously. These results are intriguing because they suggest that, depending on individual defects in the protein C pathway, some patients have impaired protein C activation and might require APC therapy, whereas others may benefit from administration of protein C. Although the clinical efficacy of recombinant human APC (drotrecogin alfa [activated]) in severe sepsis has been reported in a phase III clinical trial, the efficacy of protein C in severe sepsis remains to be determined. Preliminary results suggest that adult patients with severe sepsis vary markedly in their ability to convert endogenous protein C to APC. Additional research is required to establish whether endogenous APC activation profiles are useful in the clinical management of patients with severe sepsis.

Protein C anticoagulant pathway and its role in controlling microvascular thrombosis and inflammation.

To review the physiologic and biochemical mechanisms that suggest that protein C and activated protein C (APC) have unique properties that make them good candidates for the treatment of microvascular thrombosis, disseminated intravascular coagulation, and sepsis. A summary of published medical literature from MEDLINE search files and published reviews on protein C physiology, biochemical properties, and activity in experimental and human sepsis. Protein C is critical to the regulation of microvascular coagulation, as seen most clearly in humans born with congenital deficiency of protein C, who develop neonatal purpura fulminans. Protein C supplementation reverses the lesion formation. In primate models of sepsis, APC blocks disseminated intravascular coagulation initiated by Escherichia coli infusion, and inhibition of APC function exacerbates both the coagulant and inflammatory responses of the animals to sublethal levels of E. coli. In vitro experiments have shown that APC can inhibit neutrophil binding to selectins: Endothelial cell protein C receptor, a protein C/APC binding receptor, can bind to proteinase 3 bound to Mac-1 on leukocytes, potentially blocking tight leukocyte adhesion; and APC can inhibit tumor necrosis factor-alpha secretion by monocytes and other cell lines by interfering with nuclear factor-kappaB nuclear translocation. By blocking nuclear factor-kappaB nuclear translocation, cytokine- and endotoxin-mediated adhesion molecule up-regulation is decreased. These properties of APC are consistent with a large number of animal studies demonstrating that APC can diminish complications of crush injury and leukocyte damage to lung and other tissues in response to sepsis and decrease the inflammatory response. The animal studies are consistent with the phase 2 studies reported on APC use in the treatment of human sepsis. The protein C pathway is uniquely poised to interfere with the microvascular coagulation and inflammation that follows challenge with endotoxin. By limiting leukocyte activation, cytokine elaboration, and microvascular coagulation, APC has been shown to prevent organ damage in experimental models of sepsis. These results are consistent with the initial phase 2 reports of APC therapy in human sepsis suggesting a clinical benefit and demonstrating anti-inflammatory activity with several reports of apparent protein C effectiveness in severe sepsis, especially meningococcemia.