Protein C Pathway, Inflammation, and Pump Thrombosis in Patients With Left Ventricular Assist Devices.

Use of left ventricular assist devices (LVADs) for management of advanced heart failure is becoming increasingly common; however, device associated thrombosis remains an important cause of mortality in this patient population. We hypothesize that inflammation in LVAD implanted patients dysregulates the protein C pathway, creating a hypercoagulable state leading to thrombosis. Plasma samples from 22 patients implanted with the Thoratec HeartMate II LVAD were analyzed by commercial ELISAs. Retrospective sample selection included those collected 1-3 months prior to and within 1 month after a thrombotic or bleeding event. Unrelated to warfarin dosing, total protein S and free protein S (p = 0.033) levels were 20% lower in patients with LVAD-thrombosis than in patients with LVAD-bleeding. Levels of protein C, soluble endothelial cell protein C receptor, and soluble thrombomodulin were similar in both groups before and after the event. Compared to normal, C-reactive protein levels were 25-fold elevated in LVAD-thrombosis patients but only 9-fold elevated in LVAD-bleeding patients. This study suggests that protein S, influenced by the inflammatory state, is a gatekeeper for the function of protein C in patients with LVAD-associated thrombosis.

Podocyte Integrin-ß3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-ß3, a subunit of integrin-αvß3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvß3 induces transient binding of integrin-ß3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-ß3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-ß3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-ß3 interaction by specifically deleting podocyte integrin-ß3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvß3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvß3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

IgG plasma cells initiate changes in the protein C system in mouse ulcerative colitis through CD14+CD64+ macrophage activation.

BACKGROUND: Inhibition of the protein C system (PCS) might be one of the mechanisms of ulcerative colitis (UC). OBJECTIVES: The aim of the study was to explore the role of IgG plasma cells in changes in the PCS in UC. MATERIAL AND METHODS: Dextran sulfate sodium (DSS) was chosen to induce mouse UC. Inflammation was assessed using hematoxylin & eosin (H&E) staining and immunofluorescence. The profiling of colonic plasma cells and macrophages from colitis mice was analyzed with flow cytometry. After stimulation of macrophages with IgG type immune complex (IgG-IC), western blot was used to determine tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) protein levels. After co-incubation of colonic mucosa microvascular endothelial cells (MVECs) with TNF-α or IL-6, mitogen-activated protein kinase (MAPK) expression was detected. RESULTS: The DSS-colitis mice showed higher inflammatory indexes (p < 0.05 or p < 0.01), accompanied by greater infiltration of CD38+IgG+ plasma cells (p < 0.01), CD14+CD64+ macrophages (p < 0.01) and IgG-IC than healthy mice. Enhancement of TNF-α and IL-6 protein expression was demonstrated in this subset of macrophages when stimulated by IgG-IC (p < 0.01). After MVECs were incubated with TNF-α or IL-6, the expression of ß-arrestin1, pP38 MAPK and pJNK MAPK exhibited an increase (p < 0.05 or p < 0.01), but downregulation of endothelial protein C receptor (EPCR) expression was observed (p < 0.05 or p < 0.01); this inhibition of EPCR expression was reversed by SB203580, SP600125 or U0126 (p < 0.05 or p < 0.01). In addition, changes in activated protein C (APC) presented results similar to those for EPCR expression (p < 0.05 or p < 0.01). CONCLUSIONS: These results reveal that the PCS is inhibited during UC processing. There is a possibility that the interaction between IgG plasma cells and CD14+CD64+ macrophages, as well as further secretion of cytokines from CD14+CD64+ macrophages by the formation and stimulation of IgG-IC, subsequently influence MVECs through the ß-arrestin-MAPK pathway. Enhancement of PCS activity may represent a novel approach for treating UC.

The roles of activated protein C in experimental trauma models.

Trauma-induced coagulopathy is classified into primary and secondary coagulopathy, with the former elicited by trauma and traumatic shock itself and the latter being acquired coagulopathy induced by anemia, hypothermia, acidosis, and dilution. Primary coagulopathy consists of disseminated intravascular coagulation and acute coagulopathy of trauma shock (ACOTS). The pathophysiology of ACOTS is the suppression of thrombin generation and neutralization of plasminogen activator inhibitor-1 mediated by activated protein C that leads to hypocoagulation and hyperfibrinolysis in the circulation. This review tried to clarify the validity of activated protein C hypothesis that constitutes the main pathophysiology of the ACOTS in experimental trauma models.

Purpura Fulminans: Mechanism and Management of Dysregulated Hemostasis.

Purpura fulminans (PF) is a highly thrombotic subtype of disseminated intravascular coagulation that can accompany severe bacterial, and more rarely, viral infections. PF is associated with an extremely high mortality rate, and patients often die of overwhelming multisystemic thrombosis rather than septic shock. Survivors typically experience amputation of involved extremities and significant scarring in affected areas. Despite the devastating clinical course associated with this hemostatic complication of infection, the mechanism of PF remains poorly understood. Severe acquired deficiency of protein C and dysfunction of the protein C-thrombomodulin pathway as well as other systems that exert a negative regulatory effect on coagulation have been implicated. Management of PF involves treatment of the underlying infection, aggressive anticoagulation, and robust transfusion support aimed at correcting acquired deficiencies in natural anticoagulant proteins. In this review, we address the diagnosis and management of PF with a focus on a rational approach to this condition informed by the available data. Proposed mechanisms underlying the dysregulation of coagulation seen in PF are also covered, and implications for therapy are discussed.

Acute traumatic coagulopathy: The elephant in a room of blind scientists.

Acute traumatic coagulopathy (ATC) is the failure of coagulation homeostasis that can rapidly arise following traumatic injury, hemorrhage, and shock; it is associated with higher injury severity, coagulation abnormalities, and increased blood transfusions. Acute traumatic coagulopathy has historically been defined by a prolonged prothrombin time, although newer, more informative measurements of hemostatic function have been used to improve diagnosis and support clinical decision making. The underlying biochemical mechanisms of and best practice therapeutics for ATC remain under active investigation because of its significant correlation to poor outcomes. The wide range of hypothesized mechanisms for ATC results from the large number of symptoms, phenotypes, and altered states in these patients as observed by multiple research groups. Much like the ancient fable of blind men describing an elephant from their limited perspectives, the limited nature of clinical and laboratory tools used to diagnose coagulopathy or evaluate hemostatic function has made finding causation difficult. The prolonged prothrombin time, degree of fibrinolysis, depletion of coagulation factors and inhibitors, and general failure of the blood have all been identified as being primary indicators for ATC. Therapeutic interventions including recombinant coagulation factors, antifibrinolytics, and blood products have been used with varying degrees of success as they are used to address specific symptoms. To truly understand the causes of ATC, research efforts must recognize the complexity of the hemostatic system and get to the heart of the matter by answering the question: "Is ATC a pathological condition that develops from the observed deficiencies in coagulation, fibrinolysis, and autoregulation, or is ATC an adaptive response generated as the body attempts to restore perfusion and avoid massive organ failure?" Because patient management must proceed without definitive answers regarding the entire causative chain, the current therapeutic focus should be on using what knowledge has been gained to the patient's advantage: control hemorrhage, maintain appropriate homeostatic balances of coagulation proteins, and restore oxygen perfusion.

Disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by widespread intravascular activation of coagulation that can be caused by infectious insults (such as sepsis) and non-infectious insults (such as trauma). The main pathophysiological mechanisms of DIC are inflammatory cytokine-initiated activation of tissue factor-dependent coagulation, insufficient control of anticoagulant pathways and plasminogen activator inhibitor 1-mediated suppression of fibrinolysis. Together, these changes give rise to endothelial dysfunction and microvascular thrombosis, which can cause organ dysfunction and seriously affect patient prognosis. Recent observations have pointed to an important role for extracellular DNA and DNA-binding proteins, such as histones, in the pathogenesis of DIC. The International Society on Thrombosis and Haemostasis (ISTH) established a DIC diagnostic scoring system consisting of global haemostatic test parameters. This scoring system has now been well validated in diverse clinical settings. The theoretical cornerstone of DIC management is the specific and vigorous treatment of the underlying conditions, and DIC should be simultaneously managed to improve patient outcomes. The ISTH guidance for the treatment of DIC recommends treatment strategies that are based on current evidence. In this Primer, we provide an updated overview of the pathophysiology, diagnosis and management of DIC and discuss the future directions of basic and clinical research in this field.

Relationship between trauma-induced coagulopathy and progressive hemorrhagic injury in patients with traumatic brain injury.

Progressive hemorrhagic injury (PHI) can be divided into coagulopathy-related PHI and normal coagu- lation PHI. Coagulation disorders after traumatic brain injuries can be included in trauma-induced coagulopathy (TIC). Some studies showed that TIC is associated with PHI and increases the rates of disability and mortality. In this review, we discussed some mechanisms in TIC, which is of great importance in the development of PHI, including tissue factor (TF) hypothesis, protein C pathway and thrombocytopenia. The main mechanism in the relation of TIC to PHI is hypocoagulability. We also reviewed some coagulopathy parameters and proposed some possible risk factors, predictors and therapies.

Research progress of acute coagulopathy of trauma-shock.

Acute coagulopathy of trauma-shock (ACoTS) occurs in 25% of patients with severe trauma in the early phase, and the mortality of those patients is four-fold higher than patients without coagulopathy. The pathophysiology of this complicated phenomenon has been focused on in recent years. Tissue injury and hypoperfusion, activated protein C and Complements play important roles in the early phase after trauma. While the use of blood products, hypothermia, acidosis and inflammation are the main mechanism in late phase. Supplementing coagulation factors and platelets to improve ACoTS are inefficient. Only positive resuscitation from shock and improving tissue hypoperfusion have expected benefits.

Coagulopathy of Acute Sepsis.

Coagulopathy is common in acute sepsis and may range from subclinical activation of blood coagulation (hypercoagulability), which may contribute to venous thromboembolism, to acute disseminated intravascular coagulation, characterized by widespread microvascular thrombosis and consumption of platelets and coagulation proteins, eventually causing bleeding. The key event underlying this life-threatening complication is the overwhelming inflammatory host response to the pathogen leading to the overexpression of inflammatory mediators. The latter, along with the microorganism and its derivatives drive the major changes responsible for massive thrombin formation and fibrin deposition: (1) aberrant expression of tissue factor mainly by monocytes-macrophages, (2) impairment of anticoagulant pathways, orchestrated by dysfunctional endothelial cells (ECs), and (3) suppression of fibrinolysis because of the overproduction of plasminogen activator inhibitor-1 by ECs and thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor. Neutrophils and other cells, upon activation or death, release nuclear materials (neutrophil extracellular traps and/or their components such as histones, DNA, lysosomal enzymes, and High Mobility Group Box-1), which have toxic, proinflammatory and prothrombotic properties thus contributing to clotting dysregulation. The ensuing microvascular thrombosis-ischemia significantly contributes to tissue injury and multiple organ dysfunction syndromes. These insights into the pathogenesis of sepsis-associated coagulopathy may have implications for the development of new diagnostic and therapeutic tools.

Novel Functions of the Anticoagulant Activated Protein C in Maintaining Skin Barrier Integrity to Impact on Skin Disease.

The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. When the barrier is disrupted any of a number of diseases, such as chronic wounds, psoriasis, pemphigus, atopic dermatitis or toxic epidermal necrolysis, can take hold. Activated protein C (APC) or its precursor, protein C, is abundantly expressed by skin epidermal keratinocytes and stimulates their proliferation and migration, and inhibits apoptosis and inflammation, leading to a healing phenotype. Importantly, APC also increases the barrier function of keratinocytes by promoting expression and cell-cell contact redistribution of tight junction proteins. These cytoprotective properties of APC on epidermal keratinocytes place it as an exciting new therapy for skin disorders associated with the disruption of barrier function and inflammation.

Protease-activated receptor signalling by coagulation proteases in endothelial cells.

Endothelial cells express several types of integral membrane protein receptors, which upon interaction and activation by their specific ligands, initiate a signalling network that links extracellular cues in circulation to various biological processes within a plethora of cells in the vascular system. A small family of G-protein coupled receptors, termed protease-activated receptors (PAR1-4), can be specifically activated by coagulation proteases, thereby modulating a diverse array of cellular activities under various pathophysiological conditions. Thrombin and all vitamin K-dependent coagulation proteases, with the exception of factor IXa for which no PAR signalling has been attributed, can selectively activate cell surface PARs on the vasculature. Thrombin can activate PAR1, PAR3 and PAR4, but not PAR2 which can be specifically activated by factors VIIa and Xa. The mechanistic details of the specificity of PAR signalling by coagulation proteases are the subject of extensive investigation by many research groups worldwide. However, analysis of PAR signalling data in the literature has proved to be challenging since a single coagulation protease can elicit different signalling responses through activation of the same PAR receptor in endothelial cells. This article is focused on briefly reviewing the literature with respect to determinants of the specificity of PAR signalling by coagulation proteases with special emphasis on the mechanism of PAR1 signalling by thrombin and activated protein C in endothelial cells.

Mechanisms of phagocytosis and host clearance of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for a high incidence of acute and chronic pulmonary infection. These infections are particularly prevalent in patients with chronic obstructive pulmonary disease and cystic fibrosis: much of the morbidity and pathophysiology associated with these diseases is due to a hypersusceptibility to bacterial infection. Innate immunity, primarily through inflammatory cytokine production, cellular recruitment, and phagocytic clearance by neutrophils and macrophages, is the key to endogenous control of P. aeruginosa infection. In this review, we highlight recent advances toward understanding the innate immune response to P. aeruginosa, with a focus on the role of phagocytes in control of P. aeruginosa infection. Specifically, we summarize the cellular and molecular mechanisms of phagocytic recognition and uptake of P. aeruginosa, and how current animal models of P. aeruginosa infection reflect clinical observations in the context of phagocytic clearance of the bacteria. Several notable phenotypic changes to the bacteria are consistently observed during chronic pulmonary infections, including changes to mucoidy and flagellar motility, that likely enable or reflect their ability to persist. These traits are likewise examined in the context of how the bacteria avoid phagocytic clearance, inflammation, and sterilizing immunity.

An update on the coagulopathy of trauma.

Trauma remains the leading cause of death with bleeding as the primary cause of preventable mortality during the first 24 h following trauma. When death occurs, it happens quickly, typically within the first 6 h after injury. One of four patients to arrive in the emergency department after trauma is already in the state of acute traumatic coagulopathy and shock. The principal drivers of acute traumatic coagulopathy have been characterized by tissue hypoperfusion, inflammation, and the acute activation of the neurohumoral system. Hypoperfusion leads to an activation of protein C with cleavage of activated factors V and VIII and the inhibition of plasminogen activator inhibitor 1 with subsequent hyperfibrinolysis. Endothelial damage and activation result in Weibel-Palade body degradation and glycocalyx shedding associated with autoheparinization. In contrast, there is an iatrogenic coagulopathy that occurs secondary to uncritical volume therapy leading to acidosis, hypothermia, and hemodilution. This coagulopathy then may be an integral part of the "vicious cycle" when combined with acidosis and hypothermia. The present article summarizes an update on the principal mechanisms and triggers of the coagulopathy of trauma including traumatic brain injury.

Hemostasis, coagulation abnormalities, and liver disease.

Coagulopathy-related bleeding events are a major concern in the management of acute and chronic liver disease. The liver attempts to maintain a balance between procoagulant and anticoagulant factors, and providers struggle with poor prognostic indicators to manage bleeding and critical complications. Subtle changes in patient presentation that may require extensive provider-directed interventions, such as blood transfusions, intravenous fluid management, mitigating possible sepsis, and evaluating appropriate pharmacologic treatment, are discussed.

Endothelial protein C receptor polymorphisms and risk of severe sepsis in critically ill patients.

PURPOSE: Endothelial protein C receptor (EPCR) is expressed mainly in endothelial cells and is involved in regulation of the cytoprotective and anticoagulant pathways of protein C. We assessed whether haplotypes in the EPCR gene modify the risk of severe sepsis and/or septic shock (SS/SS) development in critically ill patients. METHODS: Three polymorphisms in the EPCR gene were genotyped in 389 Caucasian critically ill patients, hospitalized in the intensive care units of two major hospitals in Athens, Greece. Multivariate logistic regression analysis controlling for age, acute physiology and chronic health evaluation (APACHE) II and sequential organ failure assessment (SOFA) scores, sex, and diagnosis was performed to determine the effect of haplotypes H1 and H3 in the EPCR gene on the development of SS/SS. RESULTS: H2 carriers versus all other genotypes combined had a nonsignificant excess of SS/SS (p = 0.087). SS/SS occurred in 38.8% of critically ill patients carrying minor alleles belonging to both H1 and H3 haplotypes, in 58.0% of H1 carriers, 64.3% of H3 carriers, and 65.2% of patients carrying all common alleles (H2). Compared with H2 carriers, the odds ratios (OR) for developing SS/SS were 0.34 [95% confidence interval (CI) 0.16-0.76, p = 0.008] for simultaneous H1 and H3 carriers, 0.65 (95% CI 0.37-1.13, p = 0.123) for H1 carriers, and 0.82 (95 % CI 0.39-1.70, p = 0.590) for H3 carriers. CONCLUSIONS: Our results indicate that simultaneous carriers of minor alleles belonging to both the H1 and H3 haplotypes may be at reduced risk of developing SS/SS in this cohort of critically ill patients.

Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3.

The direct cytoprotective activities of activated protein C (APC) on cells convey therapeutic, relevant, beneficial effects in injury and disease models in vivo and require the endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1). Thrombin also activates PAR1, but its effects on cells contrast APC's cytoprotective effects. To gain insights into mechanisms for these contrasting cellular effects, protease activated receptor 3 (PAR3) activation by APC and thrombin was studied. APC cleaved PAR3 on transfected and endothelial cells in the presence of EPCR. Remarkably, APC cleaved a synthetic PAR3 N-terminal peptide at Arg41, whereas thrombin cleaved at Lys38. On cells, APC failed to cleave R41Q-PAR3, whereas K38Q-PAR3 was still cleaved by APC but not by thrombin. PAR3 tethered-ligand peptides beginning at amino acid 42, but not those beginning at amino acid 39, conveyed endothelial barrier-protective effects. In vivo, the APC-derived PAR3 tethered-ligand peptide, but not the thrombin-derived PAR3 peptide, blunted vascular endothelial growth factor (VEGF)-induced vascular permeability. These data indicate that PAR3 cleavage by APC at Arg41 can initiate distinctive APC-like cytoprotective effects. These novel insights help explain the differentiation of APC's cytoprotective versus thrombin's proinflammatory effects on cells and suggest a unique contributory role for PAR3 in the complex mechanisms underlying APC cytoprotective effects.