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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Taylor Jr, F.B. Articles by Hedner, U. Search for Related Content PubMed PubMed Citation Articles by Taylor Jr, F.B. Articles by Hedner, U. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 91 No. 5 (March 1), 1998: pp. 1609-1615 Active Site Inhibited Factor VIIa (DEGR VIIa) Attenuates the Coagulant and Interleukin-6 and -8, but not Tumor Necrosis Factor, Responses of the Baboon to LD100 Escherichia coli By F.B. Taylor Jr, A.C.K. Chang, G. Peer, A. Li, M. Ezban, and U. Hedner From the Oklahoma Medical Research Foundation, Oklahoma City; the University of Oklahoma Health Sciences Center, Oklahoma City; and Novo Nordisk, Gentofte, Denmark.     ABSTRACT Abstract Introduction Methods Results Discussion References Antitissue factor antibody attenuated the coagulopathic and lethal responses to LD100 Escherichia coli, whereas active site inhibited factor Xa inhibited only the coagulopathic response. In this study, we wished to determine: (1) whether active site inhibited factor VIIa blocks the coagulopathic and/or attenuates the lethal effects of LD100 E coli and (2) whether these effects are accompanied by attenuation of the inflammatory cytokine response to LD100 E coli. Eight baboons infused for 2 hours with LD100 E coli also were given five bolus infusions of DEGR VIIa of 280 µg/kg at T = 10 minutes, +2, 4, 6, and 8 hours and observed for changes in vital signs, and the concentrations of hemostatic components (fibrinogen, platelets, fibrin degradation products) and inflammatory mediators (tumor necrosis factor [TNF], interleukin-6 [IL-6], IL-8) at T = 0, 1, 2, 4, 6, and 8 hours. Eight control baboons were also infused with LD100 E coli alone and followed as described above. Four of the eight baboons treated with DEGR VIIa were permanent 7-day survivors versus none in the control group. The mean survival times for the treated and control groups were 116 ± 22 and 26 ± 8 hours, respectively. These values differed significantly from each other, (P = .0008). The decrease in platelet and fibrinogen concentrations and the increase in fibrin degradation products observed in the control group were significantly attenuated in the treated group, as was thrombosis of renal glomerular capillaries. Treatment with DEGR VIIa showed no effect on the peak TNF response to LD100 E coli at T = 2 hours (170 ± 32 v 120 ± 35 ng/mL). DEGR VIIa, however, did attenuate the IL-6 and IL-8 responses at T = 8 hours (ie, the IL-6 concentrations were 81 ± 10 for treated and 1,256 ± 236 for the control groups and the IL-8 concentrations were 28 ± 3.9 for the treated and 60 ± 8.2 for the control group). These values for IL-6 and IL-8 differed significantly from each other between the treated and control groups (P = .0001 and .0074, respectively). It should be noted that the initial responses of IL-6 and IL-8 up to T = 4 hours were not attenuated. We concluded that DEGR VIIa treatment attenuates inflammatory, as well as hemostatic system responses to LD100 E coli. We hypothesize that this occurs through interference with the assembly and/or interactions of tissue factor/VIIa complexes.     INTRODUCTION Abstract Introduction Methods Results Discussion References IN PREVIOUS STUDIES, we have shown that tissue factor plays an important role in mediation of the disseminated intravascular coagulopathic (DIC) response to LD100 Escherichia coli in the baboon.1 We found that of several monoclonal antibodies against tissue factor, only one antibody inhibited both tissue factor coagulant activity in vitro and attenuated the coagulopathic response to and protected the baboon from the lethal effects of LD100 E coli. While this observation was important in describing the role of tissue factor in producing DIC in this model, we wished to intervene in this model using factor VIIa, the active site of which was inhibited with Dansyl-Glu-Gly-Arg chloromethylketone (DEGR VIIa) to further understand the role and importance of additional factors mediating the DIC and the lethal responses to E coli. In these studies, we addressed the following three questions: First, does factor VIIa also participate in mediating the DIC response and does active site inhibited fVIIa also protect against the lethal effects of LD100 E coli? If DEGR VIIa attenuated these responses, this would suggest that the tissue factor/fVIIa complex contributes to them and strengthens evidence supporting the role already described for tissue factor alone. Second, how does intervention with DEGR VIIa compare with intervention with DEGR Xa? Previously, we showed that DEGR Xa inhibited only the coagulopathic response to LD100 E coli.2 If DEGR VIIa, in contrast to DEGR Xa, inhibited both the coagulopathic and both the inflammatory and lethal responses to LD100 E coli, this would suggest that those factors tightly associated with tissue factor (ie, fVIIa) might play a role different from those less tightly associated with tissue factor (ie, fXa). Third, to what extent and at what point during these responses to LD100 E coli does DEGR VIIa attenuate elements of the inflammatory response (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8)? If DEGR VIIa exhibits distinct antiinflammatory, as well as anticoagulant effects, this would support current evidence linking the TF/fVII complex to the inflammatory, as well as to coagulopathic responses to LD100 E coli1,3-5 and support the concept that control of coagulant events involving proximal (upstream) coagulant factors might also influence cellular signaling and inflammatory events.     MATERIALS AND METHODS Abstract Introduction Methods Results Discussion References Materials E coli: Escherichia coli 086:K61H of 33985 American Type Culture Collection, Rockville, MD. These bacteria were previously isolated from a stool specimen at Children's Memorial Hospital, Oklahoma City, OK. They were stored in the lyophilized state at 4°C after growth in tryptic soybean agar and were reconstituted and characterized as described by Hinshaw et al.6 Recombinant human factor VIIa was purified from baby hamster kidney cell culture medium as described previously.7 Recombinant factor VIIa was inactivated by incubation with Dansyl-Glu-Gly-Arg chloromethyl ketone (DEGRck) as described by Wildgoose et al.8 Preexperimentation and Experimentation Procedures Papio c cynocephalus or Papio c anubis baboons were purchased from a breeding colony maintained at the University of Oklahoma Health Sciences Center or from Biomedical Research Foundation, Inc, Houston, TX. Animals weighed 4 to 14 kg, had leukocyte concentrations not exceeding 10,000/µL, and hematocrits above 36%. They were screened for tuberculosis. These animals were held for 30 days at the OUHSC animal facility on campus where the infusion studies were performed. The animals were observed continuously during the first 8 hours postinfusion. All animals were followed for up to 7 days, at which time they were killed and tissues examined at postmortem. Infusion Procedures Experiments were performed on 16 baboons. Animals were fasted overnight before the study, administered water ad libitum, and immobilized on the morning of the experiment with Ketamine (14 ± 0.5 mg/kg intramuscularly). Animals then were administered sodium pentobarbital (2 mg/kg) through a percutaneous catheter in the cephalic vein of the forearm and maintained at a light level of surgical anesthesia with supplemental bolus infusions, approximately every 20 to 40 minutes for 8 hours. The animals were intubated orally and allowed to breath spontaneously. A superficial femoral vein was exposed aseptically and cannulated for sampling blood. This cannulization involved insertion of the catheter into the superficial femoral vein and advanced into the inferior vena cava. Each baboon was placed on its side in contact with a controlled temperature heating pad. The blood pressure and rectal temperature were monitored using a Dinamap Research monitor, model no. 1255 (Critikon, Inc, Tampa, FL) and a telethermometer (Yellow Springs Instrument Co, Yellow Springs, OH), respectively. The percutaneous catheter in the cephalic vein was used to infuse DEGR VIIa and E coli organisms. At the conclusion of the initial 8-hour observation period, both the cephalic and superficial femoral vein catheters were removed, the superficial femoral vein was ligated, and the animals were returned to their cages. Sampling The vital signs and cardiovascular responses were monitored, and blood samples were collected at 0, 1, 2, 4, 6, and 8 hours. T = 0 designated the point at which the infusion of E coli was started. The whole blood samples at each drawing included: 1.0 mL anticoagulated with EDTA for complete blood count (CBC), hematocrit, platelet count, and differential; 1.8 mL anticoagulated with 3.8% sodium citrate for fibrinogen,9 TNF, IL-6, IL-8, and DEGR VIIa; 1.0 mL in trasylol/thrombin for fibrin degradation products10; 1.0 mL of clotted blood for blood urea nitrogen,11 creatinine,12 and serum glutamic-pyruvic transaminase13; and 0.5 mL whole blood collected at T-0 and T = 2 hours for E coli colony counts.6 This totaled approximately 5.3 mL of blood at each sampling period. Not more than 10% of the baboon's total blood volume was withdrawn over the 8-hour monitoring period. This study protocol received prior approval by the Institutional Animal Care and Use Committees of the Oklahoma Medical Research Foundation and the University of Oklahoma Health Sciences Center. Experimental Groups and Plasma Concentration of DEGR VIIa Table 1 lists the animals, their sex and weight, the concentrations of E coli organisms, and DEGR VIIa used in the experiments, and the duration of their survival. E coli (approximately 2 to 3 × 1010 colony-forming unit [CFU]/kg) infusion was begun at T-0 and given as a continuous infusion until T = 2 hours. DEGR VIIa (280 µg/kg) had a T1/2 of approximately 20 minutes. Therefore, it was given as a bolus at T-10 minutes and again at T = 2, 4, 6, and 8 hours. Figure 1 shows an in vitro assay demonstrating a dose-dependent inhibition of thromboplastin coagulant activity by DEGR VIIa. Figure 2 shows the concentrations of DEGR VIIa antigen in the treated animals versus untreated animals.   View this table: [in this window] [in a new window]   Table 1. Summary of Sex, Weight, E coli Dose, DEGR VIIa Dose, and Survival of the DEGR VIIa-Treated and Control Groups View larger version (16K): [in this window] [in a new window]   Fig 1. Dose-response curve of inhibition of thromboplastin-induced coagulant activity of baboon plasma by increasing concentrations of DEGR VIIa. A baboon thromboplastin standard curve was constructed using dilutions of baboon thromboplastin in normal baboon plasma. Active site inhibited fVIIa was added to 1:5 dilution of thromboplastin (100%) and the clotting times obtained were converted to thromboplastin activity using the standard curve. The plot was then obtained by plotting DEGR VII concentration versus thromboplastin activity. View larger version (22K): [in this window] [in a new window]   Fig 2. Average concentration of DEGR VIIa observed in the DEGR VIIa treated (X-X, N = 8) and control group (-, N = 8). Samples were drawn just before each bolus infusion. Assays TNF enzyme-linked immunosorbent assay (ELISA).   Microtiter plates (Dynatech, Chantilly, VA) were coated overnight at 4°C with a polyclonal antibody against human (r)TNF (R & D Systems no. AB-210-NA, Minneapolis, MN), 5 µg/mL in 50 mmol/L carbonate buffer, pH 9.6, 50 µL/well. The wells were washed three times with 20 mmol/L Tris-HCl, 0.15 mol/L NaCl, 0.1% wt/vol Tween 20, pH 7.5 (wash buffer), and then incubated at room temperature for 1 hour with 20 mmol/L Tris-HCl, 0.15 mol/L NaCl, 1% wt/vol bovine serum albumin (BSA), pH 7.5, 50 µL/well. After washing three times again, citrated plasma samples, which had been diluted in 20 mmol/L Tris-HCl, 0.15 mol/L NaCl, 0.1% wt/vol BSA, pH 7.5 (diluting buffer), were added in duplicate to the wells, 50 µL/well. A standard curve of normal baboon plasma, diluted 1:10, to which human (r)TNF had been added was run from 0.5 to 7.0 ng/mL. The plate was incubated for 1 hour at 37°C and washed. To detect bound TNF, antibody against human (r)TNF (R & D Systems no. AB-210-NA) was biotinylated by the method of Gretch et al14 and 50 µL of 1 µg/mL biotinylated antibody was added to the plate and incubated for 1 hour at 37°C. After washing, 50 µL of 0.25 µg/mL streptavidin-alkaline phosphatase conjugate (BRL no. 9589SA; GIBCO-BRL, Gaithersburg, MD) was added, and the plate was incubated for 1 hour at 37°C. After thorough washing, nicotinamide adenine dinucleotide phosphate (NADPH) substrate stock solution from the ELISA amplification kit (BRL no. 9589SA) was added, 50 µL/well, and the plate incubated 10 minutes at room temperature. Amplifier solution was then added, 50 µL/well, and incubation continued at room temperature for 20 minutes, until color development. The A490 was measured with the microplate reader. The data was analyzed using a 4-parameter curve fitting Softmax program (Molecular Devices, Menlo Park, CA). IL-6 ELISA.   The IL-6 ELISA assay was performed in a manner similar to that described for TNF. Microtiter plates (Nunc, Roskilde, Denmark) were coated overnight with 100 µL of 0.5 µg/mL MoAb  IL-6 under the same conditions and with the same buffers as described for TNF. The MoAb  IL-6 clone 8 at 5.0 mg/mL stock was a gift of Dr L. Arden (BCL, Amsterdam, Holland). The succeeding wash steps, blocking with BSA, and incubation of plasma samples were as described above. Biotinylated goat anti-IL-6 (1 µg/mL), (R&D Systems, no. AB-206-NA) plus streptavidin-horseradish peroxidase congugate and trimethyl benzamodine (TMB) substrate also were used in quantitating the antibody reaction as described above. IL-8 ELISA.   The IL-8 ELISA assay was also performed in a manner similar to that described for TNF. Microtiter plates (Costar no. 2596; Costar, Cambridge, MA) were coated overnight with 50 µL of 0.5 µg/mL MoAb  IL-8 (R&D Systems, no. MAB208) under the same conditions and with the same buffers as described above for TNF. The succeeding wash steps, blocking with BSA, and incubation of plasma samples were as described above. Biotinylated goat anti-IL-8 (1 µg/mL) (R&D Systems, no. AB-208-NA) plus strepovidin-horseradish peroxidase congugate and TMB substrate in quantitating the antibody reactions also were used as described above. DEGR VIIa ELISA.   Total VIIa antigen levels were determined using a solid-phase double antibody enzyme-linked immunoassay.15 Statistical Analysis and Scoring of Tissue Histopathology Statistical analysis.   All data are presented as mean ± standard error of mean (SEM). The survival data in Table 1 was analyzed using the Kaplan-Meier Product-Limit Estimate of Survival Function to compare the survival times of treated versus control groups, as the data included censored observations (ie, times of survival were measured only up to 168 hours, "permanent survivors"). The clinical laboratory data in Table 2 were analyzed using an analysis of variance with Duncan's multicomparison test to determine significant differences (P < .05) between groups at given times and within certain variables. An analysis of variance with Dunnett's multicomparison test was also used to determine significant differences (P < .05) between time 0 (T-0) or baseline and subsequent times for a given variable and a given group. The cytokine data in Table 4 were analyzed using the Wilcoxon-Rank Sum test to compare the IL-6 and IL-8 assay results at T = 8 hours of the treated versus the control groups. The pathologic lesions of adrenal glands, kidneys, and lungs were analyzed by dividing their description into five categories: thrombosis, hemorrhage, congestion, white cell influx, and necrosis. The tissues were rated according to the severity of the histopathologic lesions. The scale ranged from 1 to +4, with 4 being the most severe. All microscopic sections were read by Dr Kosanke (Oklahoma Health Sciences Center, Oklahoma City), a veterinary pathologist, who was blinded as to which study was being analyzed. The Kruskal-Wallis test, a nonparametric test, was used to determine significant differences (P < .05) between groups for a given pathologic lesion.   View this table: [in this window] [in a new window]   Table 2. Summary of Clinical Laboratory Parameters of DEGR VIIa-Treated and Control Groups   View this table: [in this window] [in a new window]   Table 4. IL-6 and IL-8 Concentrations at T = 8 Hours of the DEGR VIIa-Treated and Control Groups     RESULTS Abstract Introduction Methods Results Discussion References Table 1 shows that coinfusion of DEGR VIIa with LD100 E coli was associated with an average survival time of 116 hours versus 26 hours for the control group. There were four permanent (168 hours) survivors in the treated versus none in the control group. Table 2 summarizes the clinical laboratory studies. The decrease in platelet, fibrinogen concentrations and increase in fibrin degradation product (FDP) concentration observed in the control group at T = 6 to 8 hours were attenuated significantly in the treated group. The mean systemic arterial blood pressure (MSAP) of the treated group was significantly lower than that of the control group at T = 6 to 8 hours. Coinfusion of DEGR VIIa with LD100 E coli had no effect, however, on the responses of other clinical laboratory parameters including the white cells and markers of renal (blood urea nitrogen [BUN], creatinine [CR]) and liver (serum glutamic-pyruvic transaminase [SGPT]) function. Table 3 shows that renal glomerular capillary thrombosis was almost completely inhibited in the treated versus that observed in the control group. The presence or absence of thrombotic lesions appear to be independent of the length of survival.   View this table: [in this window] [in a new window]   Table 3. Renal Glomerular Capillary Thrombosis Scores and Survival Times of the DEGR VIIa-Treated and Control Groups Table 4 shows that the IL-6 and IL-8 concentrations at T = 8 hours were significantly lower in the treated than in the control group. This table illustrates that while the IL-6 and IL-8 concentrations of the treated and control groups were comparable at T = 4 hours, that of the treated group decreased after T = 4 hours, while that of the control group remained elevated out to T + 8 hours. In contrast to IL-6 and IL-8, there was no difference in the TNF response between the treated and control groups. Peak TNF concentrations at T = 2 hours were 170 ± 32 ng/mL and 120 ± 35 ng/mL in the treated and control groups, respectively. Given the reports associating IL-6 cytokine with procoagulant activity,16-18 the association between IL-6 concentration and extent of renal glomerular capillary thrombosis was examined. Figure 3 shows that there is a significant correlation between the extent of renal glomerular capillary thrombosis and the plasma concentration of IL-6. View larger version (9K): [in this window] [in a new window]   Fig 3. Correlation between IL-6 concentration at T + 8 hours (ng/mL) and degree of renal glomerular capillary thrombosis (0 to 4+) observed at postmortem. DEGR VIIa treated group (-, N = 8), control group (-, N = 6). R = .88, P = .0001.     DISCUSSION Abstract Introduction Methods Results Discussion References The first question raised in the introduction was does DEGR VIIa attenuate DIC and prolong survival? DEGR VIIa clearly inhibited fibrinogen consumption and the generation of fibrin degradation products. It also attenuated to a lesser degree the consumption of platelets, while dramatically inhibiting renal glomerular capillary thrombosis. The renal pathology observed did not appear to be affected by differences in times of survival. The extent of thrombosis in tissues recovered from animals in the control group that died in 14 to 26 hours, did not differ greatly from those that died in 86 hours. Likewise, little or no thrombosis was observed in tissues recovered from animals in the treated group regardless of whether they died in 31 to 119 hours or were permanent survivors (168 hours). Thus, we concluded that the extent of lesions appeared to be more dependent on whether the animals were infused with DEGR VIIa and not on duration of survival. Although the difference in the survival times of the treated and the untreated groups did reach significance (P = .0008), the attenuation of the response to LD100 E coli by DEGR VIIa did not appear to be uniform, as the treated group was split between four permanent survivors and four nonsurvivors. This raised the question of a subgroup within the treated group, which is more responsive to treatment. We know from experience over the last 10 years that the response to infusion of LD100 E coli alone can vary from a capillary leak, shock, and death within 12 hours at one extreme to microvascular thrombosis, renal failure, and death in 3 to 4 days.19 The incidence of these two extremes was 10% and 8%, respectively. The majority of animals (approximately 75%) exhibited a classical consumptive coagulopathy and succumbed between 12 and 30 hours.19 Although the small number of animals studied preclude drawing a conclusion, these results raise the possibility that the differences in the effectiveness of DEGR VIIa in mitigating the response to LD100 E coli may be related to these variations of the host response to E coli. The second question raised in the introduction was how does intervention with DEGR VIIa compare with intervention with DEGR Xa, as both are efficient inhibitors of the DIC response to E coli? We concluded that DEGR VIIa could mitigate the lethal effects of E coli, whereas DEGR Xa,2 heparin, and hirudin20 do not. This again suggests that like tissue factor pathway inhibitor (TFPI),3 DEGR VIIa interacts with tissue factor or tissue factor/fVIIa complex in such a manner as to attenuate lethal inflammatory, as well as procoagulant responses. This observation raised the third question with respect to the effect of DEGR VIIa intervention on cytokine release and what, if any, correlation might exist between the alteration of cytokine release and development of renal glomerular capillary thrombosis. We concluded that in addition to inhibiting the DIC response and mitigating the lethal effects of LD100 E coli, DEGR VIIa also inhibited the elevation of IL-6 and to a lesser degree that of IL-8 observed beyond T = 4 hours after the infusion of E coli. This broadens the effects of DEGR VIIa to include antiinflammatory effects, as postulated by Taylor.21 Evidence supporting this concept is offered by Rottingen et al.4 They observed that addition of factor VIIa caused Ca2+ transients in 100% of cells from the kidney cell line MDCK, a constitutive tissue factor producer. They concluded that tissue factor mediates a cytosolic Ca2+ signal on interaction with its ligand, factor VIIa. This is consistent with the characterization of tissue factor as being related to the class 2 cytokine receptor superfamily. These investigators later showed that DEGR VIIa did not illicit this calcium flux.5 Both of these observations lend support to the hypothesis that DEGR VIIa by blocking tissue factor interaction with native factor VII not only blocks the coagulant response, but also may attenuate the inflammatory response by inhibiting this calcium flux. This also may explain why DEGR Xa fails to protect while inhibiting the coagulant response, as it does not interfere with the tissue factor-factor VIIa interaction. These data also raised the question of where this action of DEGR VIIa is taking place in the vasculature. The fact that the TNF response is not inhibited at all and that the IL-6 and IL-8 responses are attenuated only after T = 3 to 4 hours raises the question of the target of DEGR VIIa action within the vasculature. Monocytes, fixed macrophages, and endothelium are all in contact with the blood and contribute to varying degrees to the cytokine response to LD100 E coli. Because DEGR VIIa and tissue factor pathway inhibitor (TFPI) both exhibit this selective attenuation, we concluded that the phenomenon is real. This raises the question of whether these selective effects reflect action on either different tissues (endothelium v monoctye/macrophage) or at different stages (early v late) of the response of the same tissue. Finally, we concluded that there was a correlation between the plasma concentration of IL-6 and the degree of renal glomerular capillary thrombosis. This supports previous studies associating IL-6 with increased procoagulant activity.16-18 IL-6 levels also returned to normal in animals infused with sublethal concentrations of E coli (data not reported). Thus, the treated animals receiving LD100 E coli and control animals receiving sublethal E coli showed similar patterns of IL-6 release, peaking at T = 3 to 4 hours followed by a return toward normal by T = 6 to 8 hours. In contrast, those control animals receiving only LD100 E coli showed a similar release of IL-6 for the first 3 to 4 hours which, however, remained elevated out to T + 8 hours. It is interesting to note, however, that while a return of IL-6 toward normal was associated with survival, this pattern did not differentiate between the four of eight treated animals that survived from the four of eight treated animals that died. IL-6 returned toward normal in all eight cases of the treated animals regardless of whether they survived. This raises the question of whether in those four animals other overriding factors, which determine survival, came into play (eg, target cell sensitivity to TNF, etc).     FOOTNOTES    Submitted March 4, 1997; accepted October 27, 1997.    Supported in part by National Institutes of Health (Bethesda, MD) Grant No. 2R01 GM37704 (to F.B.T.).    Address reprint requests to F.B. Taylor, Jr, MD, Cardiovascular Biology Research, Oklahoma Medical Research Foundation, 825 NE 13th St, Oklahoma City, OK 73l04.    The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.     ACKNOWLEDGMENT We acknowledge the excellent assistance in preparing the manuscript performed by Joy Albert-Gorr and Penny Antkowiak and figures by Richard Irish.     REFERENCES Abstract Introduction Methods Results Discussion References 1. Taylor FB Jr, Chang AC, Ruf W, Morrissey JH, Hinshaw LB, Blick KE, Edgington TS: Lethal septic shock is prevented by blocking tissue factor. Circ Shock 33:127, 1991[Medline] [Order article via Infotrieve] 2. Taylor FB Jr, Chang AK, Mather T, Blick K, Catlett R, Esmon CT: DEGR-factor Xa blocks disseminated intravascular coagulation initiated by E. coli without preventing shock or organ damage. Blood 78:364, 1991[Abstract/Free Full Text] 3. Creasey AA, Chang AC, Fiegen L, Wun T-C, Taylor FB Jr, Hinshaw LB: Tissue factor pathway inhibitor (TFPI) reduces mortality from Escherichia coli septic shock. J Clin Invest 91:2850, 1993 4. Rottingen JA, Enden T, Camerer E, Iversen JG, Prydz H: Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor. J Biol Chem 270:4650, 1995[Abstract/Free Full Text] 5. Camerer E, Rottingen JA, Iversen JG, Prydz H: Coagulation factors VII and X induce Ca2+ oscillations in Madin-Darby canine kidney cells only when proteolytically active. J Biol Chem 271:29034, 1996[Abstract/Free Full Text] 6. Hinshaw LB, Bracket DJ, Archer LT, Beller L, Wilson MF: Detection of "hyperdynamic state" of sepsis in the baboon during lethal E. coli infusion. J Trauma 23:361, 1983[Medline] [Order article via Infotrieve] 7. Thim L, Bjoern S, Christensen M, Nicolaisen EM, Lund HT, Pedersen AH, Hedner U: Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry 27:7785, 1988[Medline] [Order article via Infotrieve] 8. Wildgoose P, Berkner KL, Kisiel W: Synthesis, purification and characterization of an Arg 152->Glu site-directed mutant of recombinant human blood clotting factor VII. Biochemistry 29:3413, 1990[Medline] [Order article via Infotrieve] 9. Hougie C: Methods for estimating fibrinogen concentrations. Thrombin time test, in Williams WJ, Beutler E, Erslev AJ, Lichtman MA (eds): Hematology (ed 3). New York, NY, McGraw Hill, 1983, p 1667 10. Wellcome Research Laboratories. Fibrinogen degradation production. Thromb-Welcotest (Rapid Latex Kit), Southern, Purley, Surrey, UK, 1984 11. Spayd RN, Bruschi B, Burdick BA, Dappen GM, Eikenberry JN, Esders TW, : Multilayer film elements for clinical analysis: Applications to representative determinations. Clin Chem 24:1343, 1978[Abstract/Free Full Text] 12. Technical Information in Test Methodologies. Rochester, NY, Eastman Kodak Co, 1985 13. Wroblewski F, LaDue JS: Serum glutamic pyruvate transaminase in cardiac and hepatic disease. Proc Soc Exp Biol Med 91:569, 1956 14. Gretch DR, Suter M, Stinski MF: The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus. Anal Biochem 163:270, 1987[Medline] [Order article via Infotrieve] 15. (abstr) Lund-Hansen T, Olsen AA, Selmer J: A double monoclonal ELISA for human coagulation factor VII. Thromb Haemost 62:55, 1989 16. Mestries JC, Kruithof EKO, Gascon MP, Herodin F, Agay D, Ythier A: In vivo modulation of coagulation and fibrinolysis by recombinant glycosylated human interleukin-6 in baboons. Eur Cytokine Netw 5:275, 1994[Medline] [Order article via Infotrieve] 17. Van der Poll T, Levi M, Hack CE, ten Cate H, Van Deventer SJH, Eerenberg AJM, De Groot ER, Jansen J, Gallati H, Büller HR, ten Cate JW, Aarden LA: Elimination of interleukin 6 attenuates coagulation activation in experimental endotoxemia in chimpanzees. J Exp Med 179:1253, 1994[Abstract/Free Full Text] 18. Robson SC, Shepard EG, Kirsch RE: Fibrin degradation product D-dimer induces the synthesis and release of biologically active Il-1, IL-6 and plasminogen activator inhibitors from monocytes in vivo. Br J Haematol 86:322, 1994[Medline] [Order article via Infotrieve] 19. Taylor FB Jr, Kosanke SD, Randolph MM, Emerson TE Jr, Hinshaw LB, White GL, Chang AC, Peer G, Blick K: Retrospective description and experimental reconstitution of three different responses of the baboon to lethal E. coli. Circ Shock 42:92, 1994[Medline] [Order article via Infotrieve] 20. (abstr) Taylor FB Jr, Chang AC, Hoogendorne H, Maraganore JM: Evidence that thrombin mediates secondary fibrinolysis in disseminated intravascular coagulation. Thromb Haemost 65:1959, 1991 21. Taylor FB Jr: Role of tissue factor in the coagulant and inflammatory response to LD100 E. coli sepsis and in the early diagnosis of DIC in the baboon, in Muller-Berghaus G (ed): DIC: Pathogenesis, Diagnosis and Therapy of Disseminated Intravascular Fibrin Formation. New York, NY, Elsevier Science, 1993, p 19  © 1998 by The American Society of Hematology.   0006-4971/98/91-0034$3.00/0 CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: A. Kinev and R. Roubey Tissue factor in the antiphospholipid syndrome Lupus, October 1, 2008; 17(10): 953 - 959. [Abstract] [PDF] L. I. Pahlman, E. Malmstrom, M. Morgelin, and H. 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