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Pathophysiology and management of coagulation disorders in critical care
medicine
de Jonge, E.
Publication date 2000
Link to publication
Citation for published version (APA):
de Jonge, E. (2000). Pathophysiology and management of coagulation disorders in critical care medicine.
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ChapterChapter 4
Tissuee factor pathway inhibitor (TFPI)
dose-dependentlyy inhibits coagulation activation
withoutt influencing the fibrinolytic and cytokine
responsee during human endotoxemia
Evertt de Jonge1, Pascale E.P. Dekkers2, Abla A. Creasey3, C. Erik Hack4, Susan K.. Paulson5, Aziz Karim5, Jozef Kesecioglu1, Marcel Levi6,
Sanderr J.H. van Deventer2, Tom van der Poll2
Departmentss of (1) Intensive Care, (2) Experimental Internal Medicine and (6)) Vascular Medicine, Academic Medical Center, University of Amsterdam,
(4)) Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam,, The Netherlands, (5) Searle Research & Development, Skokie,
Illinoiss and (3) Chiron Corporation, Emeryville, California
Abstract t
Inhibitionn of the tissue factor pathway has been shown to attenuate the activationn of coagulation and to prevent death in a gram negative bacteremia primatee model of sepsis. It has been suggested that tissue factor influences inflammatoryy cascades other than the coagulation system. In this study, we soughtt to determine the effects of two different doses of recombinant tissue factorr pathway inhibitor (TFPI) on endotoxin-induced coagulant, fibrinolytic andd cytokine responses in healthy humans. Two groups of eight healthy men weree studied in a double-blind, randomized, placebo-controlled cross-over study.. Each subject was studied on two different occasions. They received a boluss intravenous injection of 4 ng/kg endotoxin followed by a 6-hour continuouss infusion of TFPI or placebo. Eight subjects received 0.05 mg/kg/hr TFPII following a bolus of 0.0125 mg/kg (low-dose group), and eight subjects 0.22 mg/kg/hr following a bolus of 0.05 mg/kg (high-dose group). Endotoxin injectionn induced activation of coagulation, activation and subsequent inhibitionn of fibrinolysis, and release of proinflammatory and antiinflammatory cytokines.. TFPI infusion induced a dose-dependent attenuation of thrombin generation,, as measured by plasma F1+2 and thrombin-antithrombin complexes, withh a complete blockade of coagulation activation following high-dose TFPI. Endotoxin-inducedd changes in the fibrinolytic system and cytokine levels were nott altered by either low-dose or high-dose TFPI. We conclude that TFPI effectivelyy and dose-dependently attenuates the endotoxin-induced coagulation activationn in humans without influencing the fibrinolytic and cytokine response.
Introduction n
Disseminatedd intravascular coagulation (DIC) is a frequent complication of severee infection and in patients with septic shock, a strong predictor of death'. A pivotall mechanism in the pathogenesis of DIC is the activation of the (extrinsic) tissuee factor/factor Vila dependent pathway of coagulation.2 Under physiologicall conditions, tissue factor (TF) can not be detected on the luminal surfacee of the vascular endothelium3 and only in very low quantities on circulatingg blood cells.4"6 However, during infection and after stimulation with endotoxinn or tumour necrosis factor TF can be rapidly induced on blood mononuclearr cells4;7;8 and on vascular endothelium.9"11
Evidencee for the role of TF/factor Vila in activation of the coagulation system is derivedd from studies in primates, showing that the coagulant response during bacteremiaa or endotoxemia could be completely blocked by monoclonal antibodiess to TF12;13 or factor Vila,14 by active site-inhibited factor Vila15, and byy infusion of tissue factor pathway inhibitor (TFPI).16;17 Blockade of the TF drivenn pathway of coagulation by TFPI16:17 or antibodies to TF13 not only resultedd in decreased activation of the coagulation system, but also in prevention off death. It is unlikely that inhibition of the TF pathway reduced mortality duringg severe bacteremia merely by preventing DIC.18 Indeed, an alternative methodd of blocking the generation of thrombin by administration of active-site blockedd factor Xa, did not protect against organ failure and death after
EscherichiaEscherichia coli sepsis in baboons.19 It has been suggested that TF may modulatee the inflammatory response by a mechanism other than by initiating
bloodd coagulation.20 In accordance with this hypothesis are findings that inhibitingg the activity of the TF/VIIa pathway reduced the release of interleukin 66 (IL-6) and IL-8 during severe bacteremia.1*16
TFPII is a natural anticoagulant acting by direct factor Xa inhibition and, in aa factor Xa dependent manner, by feedback inhibition of the TF/VIIa complex.21 Inn animal sepsis models, TFPI was able to completely block the coagulant responsee and to prevent death with concurrent reduction of the cytokine response.16;17;22;233 Knowledge of the effect of TFPI in humans is highly limited. Therefore,, in the present study, we used the well-characterized human model of endotoxemiaa to determine the effect of TFPI, given as a 6-hour infusion in one off two doses, on coagulant, fibrinolytic and cytokine responses.
Methods s
StudyStudy design
Thee study was performed as a randomized, double-blind, placebo-controlled cross-overr experiment. Written informed consent was obtained from each subjectt before the start of the study, and the study was approved by the institutionall scientific and ethics committees. Sixteen healthy, male volunteers (agee 19-29 years) participated in the study. None had abnormalities on physical examinationn or routine laboratory investigation. Tests for hepatitis B and C and HIVV were negative.They did not not take any medication and did not smoke or usee illicit drugs. Each subject was studied on two occasions 6 weeks apart. Two dosess of TFPI were evaluated. Eight volunteers were studied after endotoxin and low-dosee TFPI/placebo, and eight subjects were studied after endotoxin and high-dosee TFPI/placebo. The subjects fasted overnight before endotoxin administration.. At 7.00 a.m. 2 intravenous canulas were inserted, one for endotoxinn administration and blood collection, the other for infusion of TFPI or placebo.. Endotoxin (Escherichia coli lipopolysaccharide, lot G , UPS, Rockville,, MD) was administered at 9.00 a.m. as a bolus intravenous injection at aa dose of 4 ng/kg body weight. TFPI (recombinant human TFPI/SC-59735, Chironn Corp., Emeryville, CA) was given immediately after endotoxin injection ass a bolus of 0.0125 mg/kg body weight followed by a continuous 6-hour infusionn of 0.05 mg/kg/hr (low-dose group) or as a bolus of 0.05 mg/kg body weightt followed by a continuous 6-hour infusion of 0.2 mg/kg/hr (high-dose group).. In the control experiments the same solution used for diluting TFPI was givenn as placebo. Oral temperature, blood pressure, heart rate and oxygen saturationn were measured at half-hourly intervals (Dinamapl846 SX, Critikon, Tampa,, FL.). Clinical symptoms such as headache, shivers, nausea, vomiting, tirednesss and malaise were recorded throughout the study periods using a graded scalee (0 as absent, 1 as weakly, 2 as moderately and 3 as severely present).
BloodBlood collection
Bloodd was obtained from an intravenous canula at 20 minutes before endotoxinn administration and at Vi, 1, VAt 2, 3, 4, 5, 6, 8, 12 and 24 hours thereafter.. Blood for coagulation and fibrinolysis assays was collected in siliconizedd vacutainer tubes (Becton Dickinson, Plymouth, England) containing 0.105MM sodium citrate; the ratio of anticoagulant to blood was 1:9 (v/v). Blood forr cytokine assays and leukocytes was collected in K3-EDTA containing tubes.
Leukocytee counts and differentials were assessed by a Stekker analyzer (counter STKS,, Coulter counter, Bedfordshire, U.K.). All blood samples, except those forr determination of leukocyte count and differentials, were centrifuged at 3000 rpmm for 15 minutes at 4° C and plasma was stored at -20° C until assays were performed. .
Assays Assays
Plasmaa levels of TFPI were measured in a validated sandwich immunoassay.. The assay uses a monoclonal antibody directed against the first Kunitzz domain of TFPI for capture and a fluorescein-labeled polyclonal antibodyy to TFPI for detection. These antibodies also recognize endogenous nativee human TFPI. All samples were assayed in triplicate. The lower limit of quantitationn was 40 ng/mL. Prothrombin time (PT) and activated partial thromboplastinn time (aPTT) were measured by one-stage clotting assays with thromboplastinn PT-Fibrinogen and thromboplastin APTT-SP respectively on an ACLL 7000 analyser (Instrumentation Laboratory, Lexington, MA). The plasma concentrationss of prothrombin fragment F1+2 and thrombin-antithrombin complexess (TATc) were measured by ELISA's (Beringwerke AG, Marburg, Germany).. Tissue-type plasminogen activator (tPA) antigen and plasminogen activatorr inhibitor type 1 (PAI-1) antigen were assayed by ELISA's (Asserachromm tPA, Diagnostica Stago, Asnieres-sur-Seine, France and PAI-ELISAA kit, Monozyme, Charlottenlund, Denmark). Plasmin-a2-antiplasmin complexess (PAPc) complexes were measured by ELISA (Enzygnost PAP micro, Behringg Diagnostics GmbH, Marburg, Germany). Tumor necrosis factor-(TNF),, interleukin-6 (IL-6) and IL-10 were measured by ELISA's according to thee instructions of the manufacturer (Central Laboratory of the Netherlands Red Crosss Blood Transfusion Service, CLB, Amsterdam, The Netherlands). Soluble TNFF receptor type I was measured by an enzyme-linked immunobound assay producedd by Hoffmann La Roche Ltd (Basel, Switzerland) as described previously.24 4
StatisticalStatistical analysis
Valuess are given as means SEM. Differences in results between the TFPI andd control experiments were tested by repeated measurements analysis of variance.. Changes in time within one group were analysed by one-way analysis off variance. A p-value < 0.05 was considered to represent a significant difference. .
Results s
TFPIplasmaTFPIplasma concentrations
Endogenouss TFPI plasma concentrations did not increase after endotoxin administrationn (fig 1). After TFPI infusion peak plasma concentrations increased fromm 54 4 to 175 8 ng/ml (p < 0.01, TFPI vs placebo) in the low-dose group andd from 65 6 to 456 34 ng/ml in the high-dose group (p<0.01, TFPI vs placebo). . 6000 -500 0 11 400 "3) ) cc 300 2000 1000 -00 -1 TFPI I low-dosee group p<0.01 1 =fi i I I 4 4 -#--6000 -5000 -ËË 400 -CC 300 -2000 -1000 00 -TFPI I high-dosee group p<0.01 1 -//--//-ii |—^~i ~ i 1 1 r 88 12 24 0 4 8 12 timee (hr) time (hr)
Figg 1. Mean SEM plasma TFPI concentrations after endotoxin administration and infusion of
TFPII (circles) or placebo (triangles). Endotoxin (4 ng/kg) was given as a bolus injection at t=0. Infusionn of TFPI started at t=0 and was continued until t=6 hr. P- values indicate difference betweenn TFPI and placebo experiments.
1 1
24 4
ClinicalClinical features and hematologic responses
Injectionn of endotoxin induced a febrile response, peaking after 3.5 hours, togetherr with tachycardia and transient flu-like symptoms, including headache, nausea,, malaise and chills. In addition, endotoxin administration resulted in a biphasicc change in white blood cell counts, characterized by an initial leukopeniaa followed by leukocytosis. None of these changes were influenced by TFPII (table 1 and data not shown). No adverse events attributable to TFPI infusionn were observed. There were no episodes of increased bleeding tendency.
Tablee 1. Clinical features and hematological responses
Peakk temperature (°C) Timee to temp, peak (hr) Heartt rate (peak, bpm) Systolicc blood pressure (nadir,, mmHg)
Whitee blood count (xl07L) ) -- nadir -peak k Low-dosee group TFPI I 2 2 2 2 9 9 1 1 1 1 7 7 Placebo o 2 2 3.6+0.2 2 110+3.0 0 104+2.0 0 2.1+0.2 2 14.6+1.4 4 P--value e NS S NS S NS S NS S NS S NS S High-dosee group TFPI I 2 2 3.9+0.2 2 4 4 105+2.7 7 2.0+0.2 2 8 8 Placebo o 38.2+0.2 2 3.9+0.4 4 99+3.0 0 9 9 1.9+0.1 1 13.1+1.3 3 P--value e NS S NS S NS S NS S NS S NS S
ActivationActivation of the coagulation system
Administrationn of endotoxin resulted in activation of thrombin generation ass reflected by increases in the plasma levels of the prothrombin fragment F1+2 andd TATc (p = 0.001, fig 2). The endotoxin-induced increase in F1+2 was diminishedd by low-dose TFPI (peak values 2.69 0.73 and 8.31 2.54 nmol/1 forr TFPI and placebo respectively, pO.01), and completely abolished by high-dosee TFPI (peak value 1.29 0.30 and 9.95 2.83 nmol/1 for TFPI and placebo respectively,, p<0.01). The endotoxin-induced increase in TATc was almost completelyy prevented by high-dose TFPI (peak values 17.9 3.9 vs 95.6 30.2 ug/L,, pO.01). Low-dose TFPI also decreased TATc formation, but this decrease didd not reach statistical significance (peak values 52.6 17.2 and 92.6 35.3 Ug/LL for TFPI and placebo respectively, p=0.19).
TATc c low-dosee group p=NS S 150 0 120 0 «« 90 60 0 30 0 0 0 TATc c high-dosee group p<0.01 1 155 122 -II 9-1 66 33 -00 -1 F1+2 2 low-dosee group p<0.01 1 I I 0 0 I I 4 4 11 1 88 12 timee (hr) I I 24 4 15 5 12 2 II 9 66 33 -00 -1 F1+2 2 high-dosee group p<0.01 1
Figg 2. TFPI dose-dependently inhibits coagulation activation. Mean SEM plasma concentrationss of thrombin-antithrombin (TAT) complexes and prothrombin fragment F1+2 afterr endotoxin administration and infusion of TFPI (circles) or placebo (triangles). Endotoxin (44 ng/kg) was given as a bolus injection at t=0. Infusion of TFPI started at t=0 and was continuedd until t=6 hr. P values indicate difference between TFPI and placebo experiments. .
APTTT values decreased after endotoxin injection, reaching a nadir after 3 hourss (p<0.01, fig 3). TFPI increased the aPTT initially and in the high-dose experiments,, it prevented the endotoxin-induced decrease in aPTT (p < 0.01, fig 3).. PT values slightly increased after endotoxin, reaching its maximum value afterr 5 hours (p<0.01 fig 3). Treatment with TFPI resulted in additional prolongationn of PT. In the low-dose group, PT increased from 12.7 0.1 to 14.5 0.2 sec during TFPI treatment versus 12.8 0.1 to 13.8 0.2 sec in the placeboo study period (p=0.04). In the high-dose group PT values increased from
12.22 0.2 to 15.6 0.4 sec in the TFPI study period and from 12.4 0.2 to 13.2 0.2 sec in the placebo study period (pO.01, fig 3).
455 400 355 300 255 -aPTT T low-dosee group p=NS S -\-\ 1 r 44 8 12 timee (hr) 24 4 45 5 40 0 35 5 30 0 255 -1 aPTT T high-dosee group p<0.01 1 177 -i 155 133 -111 -1 PT T low-dosee group p=0.04 4 nn i r 44 8 12 timee (hr) 24 4 177 - i 155 133 -111 -1 PT T high-dosee group p<0.01 1 TT 1 r 44 8 12 timee (hr) 24 4
Figg 3. Mean SEM values of PT and aPTT after endotoxin administration and
infusionn of TFPI (circles) or placebo (triangles). Endotoxin (4 ng/kg) was given ass a bolus injection at t=0. Infusion of TFPI started at t=0 and was continued untill r=6 hr. P values indicate difference between TFPI and placebo experiments.
ActivationActivation of the fibrinolytic system
Injectionn of endotoxin was associated with an early release of tPA (peaking afterr 3 hours ) followed by the appearance of PAI-1 (peaking after 4 hours) (bothh p < 0.01). Activation of the fibrinolytic system was confirmed by a transientt increase in the plasma concentrations of PAPc, peaking after 2 hours (p << 0.01). Neither low-dose TFPI nor high-dose TFPI influenced the endotoxin-inducedd release of tPA, PAI-1 or PAPc (fig 4).
100 0 __ 80 E E o)) 60 40 0 20 0 0 0 300 0 __ 250 II 200 O) ) CC 150 100 0 500 ' 00 ' tPA A low-dosee group p=NS S PAI-1 1 low-dosee group p=NS S PAPc c low-dosee group p=NS S 100 0 __ 80 E E oii 60 40 0 20 0 0 0 3000 __ 250 ff 200 -en n CC 150 100 0 50 0 0 0 tPA A high-dosee group p=NS S PAI-1 1 high-dosee group p=NS S PAPc c high-dosee group p=NS S
Figg 4. TFPI does not influence the fibrinolytic response. Mean SEM plasma
concentrationss of tPA, PAI-1 and PAPc after endotoxin administration and TFPII infusion (circles) or placebo (triangles). Endotoxin (4 ng/kg) was given ass a bolus injection at t=0. Infusion of TFPI started at t=0 and was continued untill t=6 hr. P values indicate difference between TFPI and placebo experiments. .
Cytokines Cytokines
TNFF plasma levels increased after 1 hour following endotoxin administration,, reaching peak values after 2 hours (p < 0.01). The TNF response uponn endotoxin injection was not influenced by either low or high-dose TFPI (figg 5). IL-6 levels increased from 90 min after endotoxin and peaked after 3 hourss (p < 0.01). IL-6 response after endotoxin was diminished after low-dose TFPII as compared to placebo but this difference was not statistically significant. Noo difference was observed in IL-6 response between the high-dose TFPI group andd the placebo treated subjects (fig 5). Endotoxin also elicited an antiinflammatoryy cytokine response, as reflected by transient increases in the plasmaa levels of the type I soluble TNF receptor (sTNF-Rl) and IL-10. Neither
off these endotoxin-induced increases was influenced by low or high-dose TFPI (dataa not shown).
2000 0 16000 H §12000 H 800 0 4000 oo 60000 -TNF F low-dosee group _1600 p=NS S 20000 -21200 0 800 0 400 0 0 0 TNF F high-dosee grou; p=NS S ooo o 12 2 ~1 1 24 4 IL-66 6000 low-dosee group P=NSS 4500 15000 -00 -1 IL-6 6 high-dosee group p=NS S
Figg 5. Mean SEM plasma concentrations of TNF and IL-6 after endotoxin
administrationn and infusion of TFPI (circles) or placebo (triangles). Endotoxin (4 ng/kg) wass given as a bolus injection at t=0. Infusion of TFPI started at t=0 and was continued untill t=6 hr. P values indicate difference between TFPI and placebo experiments
Discussion n
Activationn of the TF/VIIa pathway is considered crucial for the initiation of thee coagulation system during bacteremia and endotoxemia. It has been suggestedd that the TF/VIIa pathway, besides its effect on coagulation, can influencee other inflammatory mediator systems. Therefore, we considered it of interestt to determine the effect of TFPI on the coagulant, fibrinolytic and
cytokinee responses during human endotoxemia. The present study is the first to showw the anticoagulant effect of recombinant TFPI in man. In the high-dose TFPII experiments, endotoxin-induced thrombin generation, as determined by increasess in plasma F1+2 and TATc levels, was almost completely prevented, andd even in the low-dose TFPI studies, a reduction in thrombin production was observed.. Hence, our data confirm the importance of TF in the endotoxin-inducedd procoagulant response in man, and further demonstrate that the effect of TFPII on thrombin generation is dose-dependent. However, TFPI was without anyy effect on fibrinolysis or cytokine release. The results suggest that, at least duringg low grade endotoxemia, TFPI selectively attenuates coagulation activation. .
Thee role of endogenous TFPI in sepsis and endotoxemia is not completely clear.. Exposure of TF to circulating blood initiates the coagulation cascade by bindingg to factor Vila, after which the TF/VIIa complex activates factor X and
factorr IX. Recent evidence suggests that TF may be present in an inactive, encryptedd form and that the mere presence of TF on the cell surface is not sufficientt for initiating blood coagulation. Some additional stimulus may be requiredd to express this latent procoagulant activity.25126 TFPI is an approximatelyy 43-KD, trivalent, Kunitz-type inhibitor that directly inhibits factorr Xa with its second Kunitz domain. After factor Xa is bound, it rapidly inhibitss the TF/VIIa complex with the first Kunitz domain.27 The third Kunitz domainn has no known inhibitory role, but may be involved in the lipoprotein bindingg of TFPI.28 Most of total body TFPI is located in association with endotheliall cells and only 10-25% is found in circulating blood. Circulating TFPII is predominantly bound to lipoproteins.29 Blood platelets also carry native TFPII (about 10% of the plasma pool), which is released following stimulation byy thrombin.30 In vitro studies suggest that there might be a slight increase in
TFPII produced by endothelial cells and monocytes by stimulation with endotoxin.311 Furthermore, (slightly) increased levels of TFPI have been observedd in a number of illnesses, including malignancy and septicemia. " In previouss studies, plasma concentrations of TFPI only increased after severe injury.. Thus, a sublethal dose of E. coli only induced a minimal, approximately
1.2-fold,, increase in plasma TFPI concentrations, while infusion of a LD100 dose
E.coliE.coli resulted in a 2-fold rise in plasma TFPI levels.36 In our study, endogenous TFPII did not increase in plasma after endotoxin administration, which might be consideredd as a relatively mild stimulus. Together, these data suggest that the
dosee of endotoxin used in our human volunteer studies was not sufficient to elicitt an endogenous TFPI response in plasma.
Inn our experiments TFPI infusion was started immediately after endotoxin administrationn and was continued for 6 hours following a bolus injection. Two differentt dosages were compared. The two dosages were chosen based on pharmacokineticc studies in healthy humans, to achieve plasma TFPI levels of 86 andd 346 ng/ml respectively (Searle Research & Development, Investigational brochure).. In earlier studies these plasma levels were efficacious in reducing
mortalityy in a sepsis model in rabbits.23 Low-dose TFPI infusion resulted in
meann peak plasma TFPI concentrations of 175 ng/ml (3Vi-fold higher than
baseline)) and steady-state concentrations of 2!/2-fold baseline. In the high-dose
experimentss mean peak concentrations of 456 ng/ml (8-fold baseline) and
steady-statee concentrations of 41A-fold base-line were achieved. These TFPI
concentrationss resulted in minor prolongations of PT values of 1.8 and 3.4 sec respectively,, that were slightly larger than the prolongation (1 sec) attributable too endotoxin observed in the control experiments. Endotoxin induced a decrease inn aPTT values. Previous observations and the time course of his effect suggest thatt the most likely explanation might be the endotoxin-induced release of von Willebrandd factor (vWF) from the endothelium and the associated rise in factor
VIIII levels.37*8 Indeed, we could confirm a rapid release of vWF upon endotoxin
infusionn in our study (data not shown). Interestingly, the endotoxin-induced decreasee of aPTT values was attenuated by high-dose TFPI. Since vWF levels weree not affected by TFPI, this effect was probably due to a slight direct effect off TFPI on the aPTT as well.
Inn accordance with earlier studies,39140 the activation of coagulation after
endotoxinn administration was preceded by a rapid activation and subsequent inhibitionn of the fibrinolytic system, as reflected by increased levels of tPA and PAPcc followed by an increase in PAI-1 levels. Infusion of TFPI did not have anyy effect on the fibrinolytic response. These findings show that during low-gradee endotoxemia in humans, the fibrinolytic response occurs independent of thee generation of thrombin. Previous studies of low-grade endotoxemia in chimpanzeess have revealed that blockade of endotoxin-induced coagulation activationn by the administration of various anticoagulant agents, including anti-TFF and anti-factor Vila monoclonal antibodies, and the specific thrombin inhibitorr hirudin, likewise did not result in inhibition of plasmin
generation.12;14;411 TNF is considered the major denominator of activation of
endotoxin-inducedd activation of fibrinolysis in humans was not influenced by TFPI, is the observationn that TNF plasma concentrations were also unaffected by TFPI.
Itt should be noted that blocking the TF pathway in lethal Escherichia coli sepsiss in baboons not only prevented DIC, but also resulted in protection against lethality.13;16;177 Also the administration of other physiological coagulation inhibitorss has been shown not only to ameliorate the coagulation defect but also too prevent mortality.45 More downstream interventions in the coagulation cascade,, by administration of active-site degraded factor Xa (DEGR-Xa), failed too influence lethality of bacteremic baboons, while completely inhibiting the activationn of coagulation.19 Since it has been suggested that the TF pathway exertss effects on other inflammatory responses besides its effects on coagulation20,, inhibition of these effects by TFPI may have contributed to the TFPII mediated protection against lethality. Indeed, in lethal sepsis models inhibitionn of TF attenuated the IL-6 and IL-8 responses following E.coli infusion inn baboons.15"17 At present, it is uncertain how TFPI may influence cytokine production.. Interestingly, clotting blood has been found to produce IL-8, but not IL-66 in vitro. Addition of endotoxin to coagulating blood resulted in a synergisticc enhancement of IL-8 production which could be attenuated by the thrombinn inhibitor hirudin or TFPI.46 In addition, end products of the coagulationn cascade, i.e. factor Xa, thrombin and fibrin, can induce the synthesis off IL-6 and/or IL-8 by various cell types in vitro.47'50 Hence, inhibition of coagulationn by TFPI per se may reduce IL-6 and IL-8 release during sepsis. Furthermore,, in vitro TFPI has been found to bind endotoxin and to block endotoxinn effects on cells by interference with endotoxin transfer to CD14.51 In thee current study, we did not find any influence of TFPI on endotoxin-induced cytokinee responses, as reflected by unaltered plasma concentrations of TNF, IL-6,, IL-10 and sTNF-Rl. Also, IL-8 release was not changed (data not shown). Ourr model differs from the lethal primate models in many important aspects. Whilee endotoxin infusion in healthy humans leads to moderate activation of coagulationn without organ dysfunction, lethal sepsis models induce massive thrombinn generation with marked thrombus deposition at autopsy, leading to organn failure and death. The fact that inhibition of coagulation by TFPI attenuatess cytokine production in lethal sepsis models but not in the human endotoxinn model could be a reflection of the amounts of thrombin formed in the differentt models. An alternative explanation could be that monocytes are the predominantt source of cytokines during low-grade endotoxemia (which is associatedd with transient release of cytokines), and that the endothelium
contributess to the more prolonged release of especially IL-6 and IL-8 found in modelss of lethal sepsis. Indeed, endothelial cells predominantly produce IL-6 andd IL-8 following stimulation.50152 In septic baboons, TFPI attenuated the IL-6 andd IL-8 response without affecting the early and transient TNF peak.16 Hence, itt can be speculated that TFPI in part attenuates the cytokine response by endotheliall cells, with a much smaller effect on endotoxin-induced cytokine productionn by monocytes. Finally, it also could be that cytokine production in thee lethal sepsis model is not caused by thrombin, but due to ischaemia and organn failure resulting from occlusive thrombi in the microcirculation. If so, effectivee inhibition of coagulation during sepsis could prevent the development off organ failure and thereby the secondary increase in cytokine levels.
Knowledgee of the mechanisms involved in activation of the hemostatic mechanismm during severe infection has increased considerably over the past years.. The present study confirms in humans the pivotal role of the TF/VIIa pathwayy in endotoxin-induced coagulation activation and the anticoagulant potentiall of TFPI. Recombinant TFPI dose-dependently inhibited the activation off coagulation after endotoxin administration to healthy humans, without influencingg the fibrinolytic and cytokine responses. TFPI is a selective anticoagulantt drug during low grade human endotoxemia.
Acknowledgments s
Wee thank Dr. Abraham van den Ende and the staff of the Hemostasis Laboratoryy for excellent technical assistance.
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