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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 5
Tissuee factor pathway inhibitor (TFPI) does not
influencee inflammatory pathways during human
endotoxemia a
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) Vascularr Medicine, Academic Medical Center, University of Amsterdam; (4) Centrall Laboratory of the Red Cross Blood Transfusion Service, Amsterdam, Thee Netherlands; (5) Searle Research & Development, Skokie, Illinois and
(3)) Chiron Corporation, Emeryville, California.
Abstract t
Activationn of coagulation induces a pro-inflammatory response in in vitro andd animal experiments. Inhibition of the tissue factor dependent pathway of coagulationn inhibits cytokine release and prevents death in gram-negative sepsis modelss in primates. In this study we investigated the influence of blocking the activationn of the coagulation system by tissue factor pathway inhibitor (TFPI) on endotoxin-inducedd inflammatory responses in healthy humans. Eight healthy humann subjects were studied in a double-blind, randomized, placebo-controlled cross-overr study. Each subject was studied on two separate occasions. They receivedd a bolus intravenous injection of 4 ng/kg endotoxin, followed by a 6-hr continuouss infusion of TFPI (0.2 mg/kg/hr following a bolus of 0.05 mg/kg ). Endotoxinn induced-activation of coagulation, as measured by prothrombin fragmentt F1+2 and TAT, was completely prevented by TFPI. In contrast, TFPI didd not influence leukocyte activation (neutropenia followed by neutrophilia, increasedd plasma concentrations of elastase and lactoferrin), chemokine release (IL-8,, MCP-1 and MIP-lp), endothelial cell activation (release of von Willebrandd factor and sE-selectin) and the acute phase response (CRP and LBP). Wee conclude that complete prevention of coagulation activation by TFPI does nott influence activation of other inflammatory pathways during human endotoxemia. .
TFPIdoesTFPIdoes not influence inflammatory pathways during human endotoxemia
Introduction n
Severee sepsis is associated with activation of multiple inflammatory pathways.. Activation of the coagulation system, which may ultimately lead to thee clinical syndrome of disseminated intravascular coagulation, is an important manifestationn of the systemic inflammatory response of the host to severe infection.. Other features of this response include activation of neutrophilic leukocytes,, endothelial cell activation, and release of cytokines and chemokines.11 Recent studies have suggested that during sepsis activation of the coagulationn system and induction of inflammatory responses may be linked in a bimodall manner. Indeed, while cytokines are involved in the changes in the coagulationn system following infection or endotoxemia2, a significant body of evidencee supports the concept that in turn, activated coagulation factors can provokee a proinflammatory response. Ex vivo studies with human blood have revealedd that coagulation activation can stimulate interleukin-8 (IL-8) and IL-6 productionn by monocytes and endothelial cells.3 Several coagulation factors, likee thrombin,3"9 factor Xa,10;11 and fibrin12 may contribute to this proinflammatoryy response.
Tissuee factor pathway inhibitor (TFPI) is a natural anticoagulant acting by directt factor Xa inhibition and, in a factor Xa dependent manner, by feedback inhibitionn of the TF/VTIa complex.13 In animal sepsis models, TFPI was able to blockk the coagulant response and to prevent death with concurrent reduction of cytokinee release.14"17 The inflammatory and coagulant responses after administrationn of endotoxin to healthy humans are well-defined.18 We recently showedd that intravenous infusion of TFPI dose-dependently attenuates the activationn of coagulation following endotoxin administration in healthy humans.199 In the present study, we sought to determine whether TFPI, given at a dosee that completely prevented the activation of coagulation, influences the endotoxin-inducedd inflammatory response.
Methods s
StudyStudy design
Thee present investigation was performed simultaneously with a study on thee effect of TFPI on endotoxin-induced activation of coagulation and fibrinolysis,, of which the results have been published previously.19 The study wass performed as a randomized, double-blind, placebo-controlled cross-over experiment.. Written informed consent was obtained from each subject before
thee start of the study, and the study was approved by the institutional scientific andd ethics committees. Eight healthy, male volunteers (age 1 9 - 2 9 years) participatedd in the study. None had abnormalities on physical examination or routinee laboratory investigation. Tests for Hepatitis B and C and HIV were negative.. They did not not take any medication and did not smoke or use illicit drugs.. Each subject was studied on two occasions 6 weeks apart. The subjects fastedd overnight before endotoxin administration. At 7.00 a.m. 2 intravenous canulass were inserted, one for endotoxin administration and blood collection, thee other for infusion of TFPI or placebo. Endotoxin {Escherichia coli lipopolysaccharide,, lot G , United States Pharmacopeia Convention Inc, 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 0.05 mg/kgg body weight followed by a continuous 6-hour infusion off 0.2 mg/kg/hr. In the control experiments the same solution used for diluting TFPII was given as placebo.
BloodBlood collection
Bloodd was obtained from an intravenous canula at 20 minutes before endotoxinn administration and at l/2, 1, 114, 2, 3, 4, 5, 6, 8, 12 and 24 hours
thereafter.. Blood for coagulation assays was collected in siliconized vacutainer tubess (Becton Dickinson, Plymouth, England) containing 0.105M sodium citrate;; the ratio of anticoagulant to blood was 1:9 (v/v). Blood for all other assayss was collected in K3-EDTA containing tubes. Leukocyte counts and differentialss were assessed by a Stekker analyzer (counter STKS, Coulter counter,, Bedfordshire, U.K.). All blood samples, except those for determination off leukocyte count and differentials, were centrifuged at 3000 rpm for 15 minutess at 4° C and plasma was stored at -20° C until assays were performed.
Assays Assays
Thee plasma concentrations of prothrombin fragment F1+2 and thrombin-antithrombinn complexes (TAT) were measured by ELISA's (Beringwerkee AG, Marburg, Germany). Chemokine concentrations were measuredd by ELISA. MCP-1 was measured using purified monoclonal mouse anti-humann MCP-1 (PharMingen, San Diego, CA) as coating antibody, biotinylatedd rabbit anti-human MCP-1 (PharMingen) as detecting antibody and humann recombinant MCP-1 (PharMingen) as standard. For determination of
TFPIdoesTFPIdoes not influence inflammatory pathways during human endotoxemia
Macrophagee Inflammatory Protein (MIP)-ip levels, purified monoclonal mouse anti-humann MIP-lp was used as coating antibody (R&D Systems, Abingdom, Unitedd Kingdom), biotinylated affinity purified goat IgG anti-human MIP-1$ as detectingg antibody (R&D Systems) and recombinant human MIP-1$ as standard (R&DD Systems). Detection limits of the assays were 8.2 pg/ml (MCP-1) and
15.66 pg/ml (MIP-lp). IL-8 was measured by ELISA according to the instructionss of the manufacturer (Central Laboratory of the Netherlands Red Crosss Blood Transfusion Service [CLB], Amsterdam, the Netherlands). The detectionn limit was 1 pg/ml. Antigenic levels of von Willebrand factor (vWF)20 andd sE-selectin21 were measured by ELISA as described previously. LPS-bindingg protein (LBP) was measured by ELISA,22 using polyclonal rabbit anti-humann LBP as capturing antibody, biotinylated polyclonal rabbit anti-humann LBP as labeling antibody and recombinant LBP as standard. Plasma CRPP levels were determined by ELISA, using polyclonal rabbit anti-human CRP ass catching antibody, biotinylated anti-CRP mAb as detecting antibody and CRP (Beringwerkee AG, Marburg, Germany) as standard.23 Concentrations of elastase-al-antitrypsinn complex (elastase) and lactoferrin were determined using radioimmunoo assays as describedd previously.24
StatisticalStatistical analysis
Valuess are given as means SEM. Differences in results between TFPI andd control experiments were tested by repeated measurements analysis of variance.. A p-value < 0.05 was considered to represent a significant difference.
Results s
ActivationActivation of coagulation
Ass published previously, administration of endotoxin resulted in activationn of coagulation as reflected by increased levels of prothrombin activationn fragment F1+2 and TAT. TFPI given at the dose studied in the present investigationn completely prevented the rise in both markers of thrombin generation.19 9
LeukocyteLeukocyte activation (fig 1)
Endotoxinn injection elicited activation of neutrophilic granulocytes, as reflectedd by a biphasic change in neutrophil counts, involving an initial neutropeniaa (lh: 1 x 109/L) followed by neutrophilia (12 h: 3 x
109/L)) and systemic release of elastase-ccl -antitrypsin complexes and lactoferrin (alll p< 0.05). The plasma concentrations of elastase and lactoferrin peaked after 44 hr, rising from 44 to 244 ng/ml and from 97 to 695 ng/ml respectively. None off these endotoxin-induced changes were influenced by TFPI.
o o X X 18 8 15 5 12 2 9 9 6 6 3 3 0 0 500 0 400 0 E E |>> 300 200 0 100 0 0 0 8000 -gg 600 C 4 0 0 0 200 0 0 0 Neutrophils s p=NS S Elastase e p=NS S -#--Lactoferrin n p=NS S "II 1 I 1 >V I 00 4 8 12 24 timee (hr)
Figuree 1. Mean SEM numberr of neutrophilic granulocytes,, and plasma concentrationss of elastase-ccl-antitrypsinn complexes andd lactoferrin after endotoxinn administration andd infusion of TFPI or placebo.. Endotoxin (4 ng/kg)) was given as a bolus injectionn at t=0. Infusion of TFPII started at t=0 and was continuedd until t=6 hr. P valuess indicate difference betweenn TFPI and placebo e x p e r i m e n t s .. NS = nonsignificant. .
EndothelialEndothelial cell response (fig 2)
Endotoxinn elicited endothelial eel activation, as indicated by increases in thee plasma concentrations of vWF (from 77 9 % to 364 9 % after 5 hr, p <
TFPITFPI does not influence inflammatory pathways during human endotoxemia
0.05)) and sE-selectin (from 73 18 to 457 49 ng/ml after 6 hr, p<0.05). TFPI infusionn did not influence this endothelial cell response.
5000 4000 -%% 300 2000 1000 00 8000 EE 600 - #4000 2000 00 -sE-selectin n p=NS S
ii 1
r~"—i
44 8 12 24 timee (hr)Figuree 2. Mean SEM plasmaa concentrations of vonn Willebrand factor and sE-selectinn after endotoxin administrationn and infusion off TFPI or placebo. Endotoxinn (4 ng/kg) was givenn as a bolus injection at t=0.. Infusion of TFPI startedd at t=0 and was continuedd until t=6 hr. P valuess indicate difference betweenn TFPI and placebo e x p e r i m e n t s .. NS = nonsignificant. .
ChemokinesChemokines (fig 3)
MIP-ipp levels rapidly increased after endotoxin injection (from 31 5 to 94799 537 pg/ml after 2 hr, p<0.05). Also, IL-8 and MCP-1 levels increased, reachingg their maximum values at 3 hr after endotoxin administration (from 5
11 to 1539 235 pg/ml and from 205 49 to 75923 6370 pg/ml resp, both p < 0.05).. Chemokine release induced by endotoxin was not influenced by TFPI.
22 - i
Figuree 3. Mean SEM plasmaa concentrations of IL-8,, MIP-ip and MCP-1 a f t e rr e n d o t o x i n administrationn and infusion off TFPI or placebo. Endotoxinn (4 ng/kg) was givenn as a bolus injection at t=0.. Infusion of TFPI startedd at t=0 and was continuedd until t=6 hr. P valuess indicate difference betweenn TFPI and placebo experiments.. NS = nonsignificant. .
AcuteAcute phase response (table 1)
CRPP and LBP levels increased after endotoxin, peaking at 24 and 12 hr respectively.. TFPI infusion did not alter these responses.
TFPIdoesTFPIdoes not influence inflammatory pathways during human endotoxemia
Tablee 1. TFPI does not influence the acute phase protein response to endotoxin
timee (hr) -- 20 min 12 2 24 4 CRPP (ug/ml) endotoxinn endotoxin p ++ TFPI 0.77 2 0.4 1 NS 29.55 5.7 34.8 3.0 NS 60.22 0 65.5 8 NS LBPP (ug/ml) endotoxinn endotoxin p ++ TFPI 16.11 0 14.2 5 NS 56.55 7 42.7 4 NS 50.44 4 42.1 0 NS
Meann SEM plasma concentrations of C-reactive protein (CRP) and Lipoprotein binding protein (LBP)) after intravenous injection of endotoxin (4 ng/kg at t=0) followed by infusion of TFPI (0.05 mg/kgg bolus followed by a 6-hr infusion of 0.2 mg/kg/hr) or placebo. P values indicate difference betweenn TFPI and placebo experiments.
Discussion n
Activationn of the TF/Vila pathway is considered crucial for the initiation off the coagulation system during bacteremia and endotoxemia. Under physiologicall conditions, tissue factor (TF) can not be detected on the luminal surfacee of the vascular endothelium25 and only in very low quantities on circulatingg blood cells.26"28 However, during infection and after stimulation with endotoxinn or tumor necrosis factor (TNF) TF is rapidly induced on blood mononuclearr cells26;29;30 and on vascular endothelium.133132 Elimination of TF activityy in endotoxemic or bacteremic primates results in a complete inhibition off coagulation activation.14;17;33;34 In accordance, we recently found that infusion off TFPI caused a dose-dependent reduction of the procoagulant response to endotoxinn in healthy humans in vivo.19 The main conclusion of the present study iss that TFPI infused at a dose that prevented endotoxin-induced thrombin generationn did not influence other proinflammatory effects of endotoxin, includingg leukocyte activation, endothelial cell activation, chemokine release andd the acute phase protein response. These data suggest that low dose endotoxinn elicits systemic inflammation by a coagulation independent mechanism. .
Ourr findings are in contrast with in-vitro and animal experiments suggestingg that the activation of coagulation contributes to various proinflammatoryy pathways. IL-6 and IL-8 production by monocytes and
endotheliall cells is stimulated during ex vivo clotting of human blood.3 Several coagulationn factors may contribute to this inflammatory response. Thrombin stimulatess the release of cytokines like IL-1, IL-6, IL-8 and TNF,3'7 and MCP-1.8;99 The thrombin-induced cytokine response by monocytes and endothelial cellss likely is mediated by thrombins catalytic activity and can be prevented by hirudin.3;355 Factor Xa, by binding to effector cell protease receptor-1 (EPR-1), hass been found to trigger acute inflammatory responses in vivo, characterized by prominentt perivascular accumulation of activated mast cells, and unaffected by specificc inhibition of thrombin.10 Exposure of cultured human endothelial cells too factor Xa stimulated the production of IL-6, IL-8, MCP-1 and the expression off the adhesion molecules sE-selectin, intercellular adhesion molecule
(ICAM)-11 and vascular cell adhesion molecule (VCAM)-l, by a mechanism independent off thrombin and EPR-1.11 Fibrin was shown to induce IL-8 production by endotheliall cells12 and fibrin degradation products are potent chemoattractants.36 Activatedd protein C, an endogenous anticoagulant, has several effects on the inflammatoryy response like down-regulation of TNF production by macrophages,377 blocking neutrophil activation38 inhibiting sE-selectin mediated celll adhesion39 and up-regulating IL-6 and IL-8 production.40
Thee role of the TF/VIIa dependent pathway of coagulation on inflammatoryy responses in vivo has been studied in lethal sepsis models in non-humann primates. In these models, blocking TF/VIIa by TFPI or active site degradedd factor Vila both inhibited thrombin generation and IL-6 and IL-8 release,, and prevented organ failure and death. There are several possible explanationss for the different findings in our study compared to the experiments inn primate models of sepsis. If one assumes that thrombin does contribute to inflammatoryy responses, it can be hypothesized that the amount of thrombin generatedd following low dose endotoxin administration is not sufficient to stimulatee inflammatory responses. The much higher amounts of thrombin formedd during lethal sepsis on the other hand could contribute to the IL-6 and IL-88 response. Alternatively, it is possible that cytokines are produced by differentt cell types during low-grade endotoxemia and severe sepsis. The prolongedd IL-6 and IL-8 response that is found during sepsis in primates14 could bee produced by endothelial cells, that are known to predominantly produce these twoo cytokines upon stimulation,11;40whereas the more transient rise in cytokines observedd after low dose endotoxin injection could be attributed to monocytes. Hence,, it can be speculated that TFPI attenuates the cytokine response by endotheliall cells, with a much smaller effect on endotoxin-induced cytokine productionn by monocytes. Yet another possible explanation is that not thrombin
TFPIdoesTFPIdoes not influence inflammatory pathways during human endotoxemia
and/orr other coagulation factors, but organ failure and ischemia contribute to the inflammatoryy response during sepsis. If so, anticoagulation would be able to attenuatee this inflammatory response during severe bacteremia by preventing disseminatedd intravascular coagulation leading to organ failure. Endotoxin administrationn in healthy humans, on the other hand, does not induce organ failure,, explaining why anticoagulation does not influence signs of inflammation inn this model. Finally, it has been hypothesized that TF has direct pro-inflammatoryy effects independent of activation of coagulation.41 In that case, it iss possible that our endotoxin model does not induce enough TF expression to contributee to inflammatory changes.
Thee present study, in contrast with previous animal experiments, shows noo influence of TFPI on endotoxin-induced inflammatory pathways in human subjects.. More studies are required to determine the exact role of activation of coagulationn on inflammatory pathways during sepsis and endotoxemia.
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