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D J Stearns-Kurosawa

Publications and source records attributed to D J Stearns-Kurosawa.

17 recordsLinked to original sources
PubMed2005

EPCR Ser219Gly: elevated sEPCR, prothrombin F1+2, risk for coronary heart disease, and increased sEPCR shedding in vitro.

We have progressively analysed three studies of coronary heart disease (CHD) for a variant in EPCR (Ser219Gly). Initially, in a prospective study, NPHSII, while no overall CHD-risk was identified in heterozygotes, homozygotes for 219Gly exhibited a three-fold elevated risk (HR 3.3, CI 1.22-8.96). In diabetics within NPHSII, there was a suggestion that 219Gly+ was associated with elevated CHD-risk (HR 1.89, CI 0.39-9.06) although numbers were small. To further assess the effect of the variant in diabetes, a case-control study of MI, HIFMECH, was used, in which previous analysis had defined a group with metabolic syndrome, by factor analysis. A significant CHD-risk interaction was identified between genotype and the 'metabolic syndrome' factor (interaction p=0.009). To further assess CHD-risk for this variant in type-2 diabetes and to assess the effect of the variant upon thrombin generation and plasma levels of soluble EPCR, a cross-sectional study of type-2 diabetes was used. A significant CHD-risk was identified for European Whites (OR 2.84, CI 1.38-5.85) and Indian Asians in this study (OR 1.6, CI 1.00-2.57) and the frequency of 219Gly was two-fold higher in Indian Asians. Soluble EPCR levels were strongly associated with genotype, with homozygotes for 219Gly having four-fold higher levels (p<0.0001). In vitro studies of EPCR-transfected cells suggested increased basal release of sEPCR from cells expressing the 219Gly EPCR phenotype. Furthermore, in base-line samples from NPHSII and in the diabetic study, a significant increase in prothrombin F1+2 level was observed for 219Gly. The increased CHD-risk and thrombin generation appears to be acting through increased shedding of the Gly allele from the cell surface.

Animals↗
PubMed2003

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 x 106 CFU/kg to 2.45 x 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 approximately 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.

Animals↗
PubMed2002

Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener's granulomatosis.

Elevated soluble thrombomodulin (sTM) levels are an accepted marker of endothelial damage. The physiological significance of plasma endothelial protein C receptor (sEPCR) levels is not known. To assess the relevance of this plasma protein in Wegener's granulomatosis (WG), sEPCR levels were measured in sera obtained from WG patients and related to disease activity, sTM levels, and other known markers of disease activity. In total, 129 sera (37 at active disease, 92 during follow-up) from 31 WG patients were tested. During active disease, eight (22%) and 17 (46%) out of 37 active sera had elevated levels of sEPCR and sTM, respectively (NS); sEPCR (r = 0.39; P = 0.02) and sTM (r = 0.53; P <0.01) levels correlated with disease activity (Birmingham Vasculitis Activity Score). Analysis of longitudinal sera revealed a significant increase in sEPCR (P = 0.01) and sTM (P = 0.04) levels prior to the moment of a relapse. Corrected for renal function, the increase in sEPCR remained significant (P =0.04) whereas sTM did not (NS). Levels of sEPCR correlated with sTM levels (r = 0.32; P < 0.001). Plasma levels of sEPCR respond to changes in the disease in patients with WG.

Adult↗
PubMed2000

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 beta(2) 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 beta(2) integrin suggests that there may be a link between the protein C anticoagulant pathway and neutrophil functions.

Blood Coagulation Factors↗
PubMed2000

The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis.

The influence of the endothelial protein C receptor (EPCR) on the host response to Escherichia coli was studied. Animals were treated with 4 separate protocols for survival studies and analysis of physiologic and biochemical parameters: (1) monoclonal antibody (mAb) that blocks protein C/activated protein C binding to EPCR plus sublethal numbers of E coli (SLEC) (n = 4); (2) mAb to EPCR that does not block binding plus SLEC (n = 3); (3) SLEC alone (n = 4); and (4) blocking mAB alone (n = 1). Those animals receiving blocking mAb to EPCR plus sublethal E coli died 7 to 54 hours after challenge, whereas all animals treated with the other protocols were permanent survivors. Histopathologic studies of tissues from animals receiving blocking mAb plus SLEC removed at postmortem were compared with those animals receiving SLEC alone killed at T+24 hours. The animals receiving the blocking mAb exhibited consumption of fibrinogen, microvascular thrombosis with hemorrhage of both the adrenal and renal cortex, and an intense influx of neutrophils into the adrenal, renal, and hepatic microvasculature, whereas the tissues from animals receiving only sublethal E coli exhibited none of these abnormal histopathologic changes. Compared with the control animals, the animals receiving the blocking mAb exhibited significantly elevated serum glutamic pyruvic transaminase, anion gap, thrombin-antithrombin complex, IL-6, IL-8, and soluble thrombomodulin. The levels of circulating activated protein C varied too widely to allow a clear determination of whether the extent of protein C activation was altered in vivo by blocking protein C binding to EPCR. We conclude that protein C/activated protein C binding to EPCR contributes to the negative regulation of the coagulopathic and inflammatory response to E coli and that EPCR provides an additional critical step in the host defense against E coli. (Blood. 2000;95:1680-1686)

Animals↗
PubMed1999

Inflammation, sepsis, and coagulation.

The molecular links between inflammation and coagulation are unquestioned. Inflammation promotes coagulation by leading to intravascular tissue factor expression, eliciting the expression of leukocyte adhesion molecules on the intravascular cell surfaces, and down regulating the fibrinolytic and protein C anticoagulant pathways. Thrombin, in turn, can promote inflammatory responses. This creates a cycle that logically progresses to vascular injury as occurs in septic shock. Most complex systems are regulated by product inhibition. This inflammation-coagulation cycle seems to follow this same principle with the protein C pathway serving as the regulatory mechanism. The molecular basis by which the protein C pathway functions as an anticoagulant is relatively well established compared to the mechanisms involved in regulating inflammation. As one approach to identifying the mechanisms involved in regulating inflammation, we set out to identify novel receptors that could modulate the specificity of APC in a manner analogous to the mechanisms by which thrombomodulin modulates thrombin specificity. This approach led to the identification of an endothelial cell protein C receptor (EPCR). To understand the mechanism, we obtained a crystal structure of APC (lacking the Gla domain). The crystal structure reveals a deep groove in a location analogous to anion binding exosite 1 of thrombin, the location of interaction for thrombomodulin, platelet thrombin receptor and fibrinogen. Thrombomodulin blocks the activation of platelets and fibrinogen without blocking reactivity with chromogenic substrates or inhibitors. Similarly, in solution, EPCR blocks factor Va inactivation without modulating reactivity with protease inhibitors. Thus, these endothelial cell receptors for the protein C system share many properties in common including the ability to be modulated by inflammatory cytokines. Current studies seek to identify the substrate for the APC-EPCR complex as the next step in elucidating the mechanisms by which the protein C pathway modulates the response to injury and inflammation.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Regulation and functions of the protein C anticoagulant pathway.

The protein C pathway plays a critical role in the negative regulation of the blood clotting process. We recently identified an endothelial cell receptor for protein C/activated protein C (APC). The receptor is localized almost exclusively on endothelial cells of large vessels and is present at only trace levels or indeed absent from capillaries in most tissues. Patients with sepsis or lupus erythematosus exhibit elevated levels of plasma EPCR which migrates on gels as a single band and is fully capable of binding protein C/APC. There is no correlation with thrombomodulin levels, probably due to different vascular localizations and/or cellular release mechanisms. To understand the mechanisms by which EPCR plasma levels are elevated, we examined EPCR mRNA expression in a rat endotoxin shock model. The EPCR mRNA gene exhibited an early immediate gene response to endotoxin with the mRNA levels increasing nearly 4 fold in the first 3-6 hrs, before returning toward baseline. Plasma levels of EPCR also rose about 4 fold with little change in tissue EPCR levels. Both processes were markedly attenuated by hirudin suggesting that thrombin was responsible for increases in mRNA and plasma EPCR levels. At the level of mRNA, the induction is mediated by a thrombin response element in the 5' flanking region of the gene. Direct thrombin infusion and cell culture experiments support this contention. On endothelium, thrombin is capable of releasing cell surface EPCR and this process is blocked by the metalloproteinase inhibitor orthophenanthroline. Taken together these studies indicate that elevation in soluble plasma EPCR reflects endothelial cell activation in the larger vessels and is likely to be an indication of local thrombin generation near these vessel surfaces.

Animals↗
PubMed1998

A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg506 and Arg306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (Kd approximately 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had approximately 5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

Amino Acid Sequence↗
PubMed1997

Identification of functional endothelial protein C receptor in human plasma.

The endothelial cell protein C receptor (EPCR) binds protein C and facilitates activation by the thrombin-thrombomodulin complex. EPCR also binds activated protein C (APC) and inhibits APC anticoagulant activity. In this study, we detected a soluble form of EPCR in normal human plasma. Plasma EPCR appears to be approximately 43, 000 D, and circulates at approximately 100 ng/ml (98.4+/-27.8 ng/ml, n = 22). Plasma EPCR was purified from human citrated plasma using ion exchange, immunoaffinity, and protein C affinity chromatography. Flow cytometry experiments demonstrated that plasma EPCR bound APC with an affinity similar to that previously determined for recombinant soluble EPCR (Kdapp = 30 nM). Furthermore, plasma EPCR inhibited both protein C activation on an endothelial cell line and APC anticoagulant activity in a one-stage Factor Xa clotting assay. The physiological function of plasma EPCR is uncertain, but if the local concentrations are sufficiently high, particularly in disease states, the present data suggest that the soluble plasma EPCR could attenuate the membrane-bound EPCR augmentation of protein C activation and the anticoagulant function of APC.

Anticoagulants↗
PubMed1996

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.

Antibodies, Monoclonal↗
PubMed1996

The endothelial cell protein C receptor. Cell surface expression and direct ligand binding by the soluble receptor.

Expression of the endothelial cell protein C receptor (EPCR) gene in mammalian cells imparts the capacity to bind activated protein C (APC) or protein C. Immunochemical analysis of CCD41, apparently the murine homologue of EPCR, suggested centrosomal localization, raising questions about the location of the EPCR gene product and its role in protein C binding. In this study, we express a soluble form of EPCR, demonstrate EPCR expression on the cell surface, and direct binding between soluble EPCR and protein C/APC. Affinity purified polyclonal and a monoclonal antibody against EPCR bound to the cell surface of EPCR-transfected cells but not to control cells. A 49-kDa protein, a mass similar to soluble EPCR, was immunoprecipitated from the cell surface of endothelium and cells transfected with human EPCR but not from control cells. The FLAGtrade mark antibody and APC bound to cells expressing an EPCR construct containing the FLAGtrade mark epitope located in a putative extracellular domain, whereas an EPCR construct truncated just before the putative transmembrane domain produced only soluble EPCR antigen. Soluble EPCR inhibited APC binding to EPCR expressing cells in a concentration-dependent fashion, Kd (app) = 29 nM and bound to immobilized protein C in a Ca2+-dependent fashion. Thus, EPCR is a type 1 transmembrane protein that binds directly to APC.

Animals↗
PubMed1996

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 alpha1-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.

Amidohydrolases↗
PubMed1994

The effect of calcium ionophore A23187 on tissue factor activity and mRNA in endothelial cells.

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cofactor for blood coagulation factor VIIa. The induction of TF on the surface of endothelial cells is initiated by various kinds of stimuli including lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha). The mechanisms leading to induction of TF are largely unknown and the present study explores the influence of calcium influx on TF induction in LPS-stimulated human umbilical vein endothelial cells. TF cofactor activity was measured on cell surfaces and in lysates by a two-stage chromogenic assay after the cells were incubated under a variety of conditions. TF activity of cell surfaces increased 3.3-fold above control values after LPS stimulation (100 ng/ml, 4 h). Addition of 20 microM A23187, a calcium ionophore, to the LPS-stimulated cells just before the TF assay, resulted in an additional 8.8-fold enhancement. TF activity of lysed cells increased 10.5-fold above control values after LPS stimulation (100 ng/ml). Incubation with lower concentrations of A23187 and 100 ng/ml-1 micrograms/ml LPS for 4 h resulted in activity twice that of LPS stimulation alone. The TF mRNA signal of LPS plus 1 microM A23187-treated cells was also increased in addition to LPS treated cells.

Base Sequence↗