Shinichiro Kurosawa

The soluble endothelial protein C receptor binds to activated neutrophils: involvement of proteinase-3 and CD11b/CD18.

The protein C pathway is a primary regulator of blood coagulation and a critical component of the host response to inflammatory stimuli. The most recent member of this pathway is the endothelial protein C receptor (EPCR), a type I transmembrane protein with homology to CD1d/MHC class I proteins. EPCR accelerates formation of activated protein C, a potent anticoagulant and antiinflammatory agent. The current study demonstrates that soluble EPCR binds to PMA-activated neutrophils. Using affinity chromatography, binding studies with purified components, and/or blockade with specific Abs, it was found that soluble EPCR binds to proteinase-3 (PR3), a neutrophil granule proteinase. Furthermore, soluble EPCR binding to neutrophils was partially dependent on Mac-1 (CD11b/CD18), a β2 integrin involved in neutrophil signaling, and cell-cell adhesion events. PR3 is involved in multiple diverse processes, including hemopoietic proliferation, antibacterial activity, and autoimmune-mediated vasculitis. The observation that soluble EPCR binds to activated neutrophils via PR3 and a β2 integrin suggests that there may be a link between the protein C anticoagulant pathway and neutrophil functions.

The endothelial cell protein C receptor. Inhibition of activated protein C anticoagulant function without modulation of reaction with proteinase inhibitors.

A soluble form of the endothelial cell protein C receptor (EPCR) was analyzed for the ability to modulate the functional properties of protein C and activated protein C (APC). In a plasma clotting system initiated with factor Xa, EPCR blocked the anticoagulant activity of APC in a dose-dependent fashion. EPCR had no influence on clotting in the absence of APC. Consistent with the plasma results, EPCR slowed the proteolytic inactivation of factor Va by slowing both of the key proteolytic cleavages in the heavy chain of factor Va. EPCR did not prevent protein C activation by the soluble thrombin-thrombomodulin complex, did not alter the inactivation of APC by α1-antitrypsin or protein C inhibitor, and did not influence the kinetics of peptide paranitroanilide substrate cleavage significantly. We conclude that EPCR binds to an exosite on APC that selectively modulates the enzyme specificity in a manner reminiscent of the influence of thrombomodulin on thrombin.

Reduced neutrophil CD10 expression in nonhuman primates and humans after in vivo challenge with E. coli or lipopolysaccharide

CD10, also known as neutral endopeptidase or CALLA, is a major metalloproteinase that regulates levels of biologically active peptides that initiate inflammatory, cardiovascular, and neurogenic responses. Relative tissue expression levels of CD10, its peptide substrates, and their receptors constitute the basic regulatory mechanism. Neutrophils contain abundant CD10 and are rapid responders to an inflammatory septic challenge. Expression of neutrophil surface antigens in response to inflammation was studied in the primate model of Escherichia coli-mediated sepsis and in human volunteers injected with lipopolysaccharide (LPS). There was a rapid and profound (up to 95%) reduced baboon neutrophil CD10 expression in response to E. coli injections of 5.71 × 106 CFU/kg to 2.45 × 109 CFU/kg that gradually resolved to preinjection levels. The reduction was both dose and time dependent. Reduced CD10 antigen on mature baboon neutrophils and bands was observed by immunohistochemistry. Human volunteers challenged with 4ng/kg LPS experienced transient chills, nausea, fever, and myalgia. Up to ∼20% of their neutrophils had reduced CD10 expression, peaking at 2 to 8 h after injection. By 24 h, neutrophil CD10 expression resolved to preinjection levels. In contrast, in both the baboon and human studies, other neutrophil surface antigens were only slightly decreased (CD11a) or increased (CD11b, CD18, CD35, CD66b, and CD63). These data present the novel observation that neutrophil CD10 expression decreases significantly in response to in vivo inflammatory challenge. This decrease appears to be unique to CD10 and may contribute to a reduced regulation of bioactive peptides released in response to inflammatory challenge.

Interaction of Thrombin with Thrombomodulin

Product regulation of complex pathways is well established in metabolism. The role of product regulation, however, in protease cascades is less well established. Although thrombin has long been recognized as a positive feedback activator of coagulation, its potential for serving as a negative regulator is only recently becoming understood. Protein C activation and the subsequent expression of the activated protein C anticoagulant function provides one mechanism by which thrombin can serve as a negative regulator of coagulation. Thrombins critical role in this pathway is as the protein C activating enzyme.z4 Once thrombin participates in the protein C anticoagulant pathway, its function is transformed from a procoagulant to an anticoagulant. The mechanisms by which this transformation occurs constitute the central theme of this paper. One of the major problems of a product feedback system using proteolysis involves how to regulate rather than destroy the system. In the protein C pathway, this is accomplished at a minimum of two levels. First, activated protein C demonstrates remarkable specificity for activated factors V and VIII as compared to their precursor forms. s.6 A second feature of the pathway that imparts unique properties is that protein C activation occurs in response to formation of a complex between thrombin and thrombomodulin, an endothelial cell-specific This feature implies that protein C activation probably occurs primarily in the microcirculation where, because of the high surface to volume ratio, the concentration of thrombomodulin is relatively high? Activation probably occurs downstream from the site of injury and hence prevents propagation of coagulation reactions downstream. In addition to catalyzing protein C activation, the complex between thrombin and thrombomodulin has significantly reduced coagulant activity in terms of fibrinogen clotting activity, factor V activation, and platelet activation.- At least two models could account for this change in macromolecular specificity. First, as we have proposed, complex formation could alter either the substrate binding sites and/or the active site. Alternatively, complex formation could result in steric hindrance, preventing the interaction of thrombin with fibrinogen, platelets, or factor V. Such a model would predict that the substrates like fibrinogen might compete with thrombomodulin for interaction with thrombin. Such

The Membrane Proteinase 3 Expression on Neutrophils Was Downregulated After Treatment With Infliximab in Patients With Rheumatoid Arthritis

Proteinase 3 (PR3) expression on neutrophils was examined in rheumatoid arthritis (RA) patients before and after antitumor necrosis factor (TNF)-α therapy. Membrane PR3 expression from patients with either an infection or RA significantly increased. Membrane PR3 expression on neutrophils from RA patients treated with infliximab (anti-TNF-α antibody) therapy was less than in those without such treatment in a resting state, but the expression later increased after stimulation in vitro. Membrane PR3 expression increased because of the stimulation of TNFα, whereas it was significantly suppressed by plasma or α1-proteinase inhibitor. The condition of patients with RA improved after treatment with infliximab. Membrane PR3 expression on neutrophils in RA patients was downregulated by infliximab. As a result, PR3 might play an important role in the neutrophil-mediated inflammatory reaction in patients with either RA or an infection.

Inflammation: the protein C pathway*

Summary The protein C pathway serves as one of the most important negative regulatory mechanisms in coagulation. Unlike most pathways in coagulation, the protein C pathway can be downregulated by inflammatory mediators at the levels of protein C activation and function. Antiphospholipid antibodies can selectively block protein C function. In gram negative sepsis, protein C in humans and activated protein C (APC) in baboons and rodents have been shown to block the lethal response, at least in part by blocking the DIC response and probably by modulating the cytokine cascade. The mechanisms by which the pathway modulates inflammation remains unclear, but a novel endothelial cell specific receptor has recently been identified that binds APC and modulates enzyme specificity. Like thrombomodulin, the receptor is downregulated by the inflammatory cytokine, TNF. These observations provide a basis for a model in which the protein C pathway serves as a major regulatory mechanism in the control of coagulation and inflammation.

Normalization of Plasma Antithrombin Activity in Patients Requiring Hemodynamic and/or Respiratory Support has Anti-inflammatory Properties Related to Survival

The Italian Antithrombin Sepsis Study has shown that maintenance of antithrombin (AT) levels around 100% results in a 53% reduction in the 30-day mortality risk of intensive care unit patients with sepsis and/or post-surgical complications requiring hemodynamic and/or respiratory support [1, 2]. The changes in a series of coagulation and fibrinolysis parameters were evaluated with the aim of correlating such changes with the potential effect of AT treatment on survival and exploring the predictive value of laboratory tests on 30-day mortality [3]. Blood samples from 119 patients were taken at baseline and then daily until day 7 from the beginning of AT or placebo infusion. The parameters evaluated were: AT activity, protein C (PC) and S activity and antigen levels, α2-antiplasmin and plasminogen activity, fibrin and fibrinogen degradation products, plasmin-antiplasmin complex, prothrombin fragment 1.2, and thrombin-antithrombin (TAT) complex. Prealbumin was also measured to correct for impaired liver synthesis of coagulation and fibrinolysis factors and inhibitors. Improvement — but never normalization — in most of the laboratory parameters was observed over time. In addition to AT, treatment only affected TAT levels (p = 0.05). In a Cox survival regression model, including the presence of septic shock, the multiorgan failure (MOF) score and the type of treatment as covariates, baseline AT levels were an independent predictor of mortality in the entire series of patients (p = 0.003). After 24 h of treatment, TAT levels were negatively associated with survival (p = 0.05). On the last day of treatment, the levels of PC (p = 0.006) and of fibrinogen-degradation products (p = 0.005) were negatively and positively associated with mortality in the 91 survivors [3].