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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 6
Activationn of coagulation by administration of
recombinantt factor Vila elicits interleukin (IL)-6
andd IL-8 release in healthy human subjects
Evertt de Jonge1, Philip W. Friederich2, Marcel Levi2, Tom van der Poll3
Departmentss of (1) Intensive Care, (2) Vascular Medicine and (3) Laboratory of Experimentall Internal Medicine, Academic Medical Center, University of
Abstract t
Activationn of coagulation has been shown to contribute to pro-inflammatoryy responses in animal and in vitro experiments. In primate modelss of sepsis, specific inhibition of coagulation attenuated the cytokine responsee and prevented death. We sought to determine whether coagulation per see can also lead to a proinflammatory response in humans in vivo. Six healthy humann subjects were studied on two occasions in a randomized, controlled crossoverr study. They received a bolus injection of 90 (ig/kg recombinant factor Vilaa (rVIIa) to induce thrombin generation, 4 hr after pretreatment with either placeboo or a subcutaneous injection of 3.5 ug/kg of recombinant rNAPc2, a potentt and specific inhibitor of tissue factor. Administration of rVIIa resulted in aa marked generation of thrombin that was partly prevented by pretreatment with rNAPc2.. Peak plasma levels of thrombin-antithrombin complexes (TATc) were
10.77 1.5 and 14.0 1.3 pg/ml after rVIIa/rNAPc2 and rVIIa/placebo
respectively,, p<0.05); peak plasma levels of prothrombin fragment F1+2 were 3.11 1 vs 4.0 0.3 nmol/L, p=NS). Activation of coagulation resulted in increasedd plasma concentrations of interleukin (IL)-6 and IL-8 (IL-6: from <1.2 too 5.5 2.5 pg/ml vs. from <1.2 to 2.9 1.6 pg/ml, p=0.05; IL-8: from <2 to 8.3
33 vs. from <2 to 3.7 1.1 pg/ml, p<0.05 after rVIIa/placebo and rVIIa/rNAPc22 respectively). Plasma levels of tumor necrosis factor (TNF), solublee TNF receptor type 1, interleukin-1 receptor antagonist and soluble E-selectinn were not affected by rVIIa or rVIIa/rNAPc2. We conclude that a modestt activation of coagulation by administration of factor Vila can elicit IL-6 andd IL-8 release in healthy humans.
ActivationActivation of coagulation elicits IL-6 andIL-8 release in human subjects
Introduction n
Activationn of coagulation and inflammation during sepsis appear to be linkedd in a bimodal way. While cytokines are involved in the procoagulant state thatt follows endotoxemia or severe infection, recent studies have shown that activatedd coagulation factors in turn are capable of eliciting a proinflammatory response.11 Ex vivo clotting of human blood stimulates interleukin-8 (IL-8) and IL-66 production by monocytes and endothelial cells2;3. Recent in vitro studies havee shown that several coagulation factors, like factor Vila, factor Xa and thrombinn can activate cells directly and provoke a variety of proinflammatory responses1.. The in vivo relevance of this observation in humans has, however, nott been demonstrated yet. In this study we sought to determine whether activationn of coagulation in vivo might elicit a proinflammatory response. We thereforee measured the cytokine response following the generation of thrombin byy intravenous administration of factor Vila to healthy human subjects. Previous studiess have shown that infusion of factor Vila leads to factor Xa-dependent thrombinn formation in chimpanzees4 as well as in humans [Friederich PW et al, manuscriptt submitted].
Methods s
Thee study was approved by the research and medical ethical committees off the Academic Medical Center, University of Amsterdam. Written informed consentt was obtained from all subjects. The primary aim of this study was to investigatee whether recombinant factor Vila might be able to overcome the anticoagulantt response caused by the anti-tissue factor agent rNAPc2. The coagulantt responses to these treatments are described in detail in another report [Friederichh PW et al. manuscript submitted].
StudyStudy design
Thee study was performed as a randomised, placebo-controlled crossover study.. Six healthy male volunteers (21-26 years of age) were enrolled in the study.. Medical history, physical examination, and screening hematology and biochemistryy laboratory tests were completely normal in all subjects. The subjectss did not use any medication and had no signs or symptoms of illness in thee month preceding the start of the study. The volunteers were screened within 144 days prior to the start of the first study period. The study was performed underr Good Clinical Practice guidelines in a specially equipped and staffed
researchh unit in the Academic Medical Center, Amsterdam. Blood pressure, heartt rate, EKG and oxygen saturation were measured at hourly intervals (Dinamapp 1846 SX; Critikon, Tampa, FL). Each subject was studied on two occasionss at least six weeks apart. Treatment consisted of an intravenous bolus injectionn recombinant factor Vila, preceded by either rNAPc2 or placebo.
StudyStudy agents
Recombinantt factor Vila (rVIIa, NovoSeven, Novo Nordisk, Denmark) is aa pro-hemostatic agent that is mainly used in the treatment of bleeding patients withh severe coagulation abnormalities such as patients with antibodies to factor VIIII or IX. rVIIa was administered as an intravenous bolus injection at a dose of 900 ng/kg in 10 ml saline, four hours after the administration of rNAPc2 or placebo.. The dose rationale for rVIIa was based on the clinically effective dose inn patients with bleeding disorders and previous studies with rVIIa in human individualss using acenocoumarol.5 In the study period in which rVIIa was given inn combination with placebo, an intravenous injection with 0.15 ml/kg saline wass administered. Recombinant Nematode Anticoagulant Protein c2 (rNAPc2, Corvass Inc, San Diego, CA) forms a ternary inhibitory complex with factor Vila/tissuee factor following the binding to factor Xa at a site that is distinct from thee catalytic center. This small protein anticoagulant is derived from the family off Nematode Anticoagulant Proteins (NAPs), which were originally isolated fromfrom hematophagous hookworm nematodes and is currently produced as a recombinantt protein under Good Manufacturing Practices established by the Unitedd States Food and Drug Administration.^7 Previous studies have shown a bioavailabilityy of 90-100% after subcutaneous administration in animals and an apparentt terminal half-life of greater than 50 hours in humans following a single subcutaneouss administration.8 After dilution in sterile phosphate-buffered saline (655 mM sodium phosphate, 80 mM NaCl, pH 7.0), rNAPc2 was administered in aa volume of 0.3 mL at a dose of 3.5 u.g/kg as a single subcutaneous injection in thee abdomen at the start of the study. The rationale for the dose of rNAPc2 was basedd on previous phase-I studies in human volunteers, in which rNAPc2 was consideredd to be safe and well tolerated in doses up to 5 ug/kg.8
BloodBlood collection
Bloodd was drawn from the forearm by separate venipunctures before and att 0.5, 1, 1.5, 2, 3, 4, 6 and 9 hours after the administration of rVIIa. Blood for measurementt of clotting times, prothrombin fragment F1+2 and thrombin-antithrombinn (TAT) complexes was collected in tubes containing 3.2% (wt/vol)
ActivationActivation of coagulation elicits IL-6 and IL-8 release in human subjects
sodiumm citrate; the ratio of anticoagulant to blood was 1:9 (vol/vol). Blood for thee measurement of factor IX and X activation peptide was taken in a tube containingg 38 mM citric acid, 75 mM sodium citrate, 136 mM dextrose, 6 mM EDTA,, 6 mM adenosine and 25 U/ml heparin; the ratio of anticoagulant to bloodd was 1:5 (vol/vol). These samples were placed in melting ice immediately andd centrifuged at 2000 x g for 20 minutes at 4°C. For determination of plasma levelss of factor Vila, blood was collected in tubes, containing a mixture of 1 M benzamidine,, 100 mM EDTA and 1 mg/ml soybean trypsin inhibitor; the ratio off anticoagulant to blood was 1:9 (vol/vol). Blood for cytokine assays was collectedd in K3-EDTA-containing tubes. Plasma was obtained by centrifugation att room temperature for 20 minutes at 1600 x g. All plasma samples were stored att -70°C until assayed.
Assays Assays
Thrombinn generation was assessed by measuring the prothrombin activationn fragment F1+2 and thrombin-antithrombin (TAT) complexes. Both parameterss were assayed by ELISA's (Behring, Marburg, Germany). For measurementt of factor X activation, factor X activation peptide was assayed by aa radioimmunoassay, as previously described.*10 The plasma levels of factor Vilaa were determined by using a newly developed enzyme capture assay for determiningg factor Vila activity in human plasma, according to a previously describedd principle.11 Briefly, solid-phase bound monoclonal antibodies raised againstt recombinant factor Vila (rVIIa) enable capturing of rVIIa. In the next step,, bound rVIIa is allowed to convert a chromogenic substrate during incubationn in TRIS-BSA buffer at pH 7.9. Light absorption at 405 run minus 540 nmm was shown to be linearly correlated with rVIIa concentrations.12 Tumor necrosiss factor (TNF), IL-6 and IL-8 were measured by ELISA according to the manufacturer'ss instructions (Central Laboratory of the Netherlands Red Cross Bloodd Transfusion Service, Amsterdam, The Netherlands). Soluble TNF receptorr type 1 was measured by an enzyme-linked immunobound assay producedd by Hoffmann La Roche (Basel, Switzerland) as described previously.13 IL-11 receptor antagonist (IL-lra) was measured by ELISA using mouse anti-humann IL-lra mAb (Antibody Solutions SARL, Illkirch, France) as coating antibody,, biotinylated goat anti-human IL-lra (R&D Systems, Abingdon, Unitedd Kingdom) as detecting antibody, and recombinant human IL-lra (R&D Systems)) as standard (detection limit 41 pg/ml). Antigenic levels of sE-selectin weree measured by ELISA as described previously.14
StatisticalStatistical analysis
Valuess are given as means SEM. Differences in results between the two treatmentt groups were tested by repeated measurements analysis of variance. A p-valuee < 0.05 was considered to represent a significant difference.
Results s
PlasmaPlasma concentrations of factor Vila
Thee plasma levels of factor Vila were below the limit of detection prior to administrationn of rVIIa. Peak plasma levels of 23.7 2.4 nmol/L were reached att 30 min after administration. Pretreatment with rNAPc2 had no effect on the plasmaa levels of factor Vila (data not shown).
66 "I 22 -16 6 122 -E -E ~5) ) 00 -1 F1+2 2 p=NS S ii i i > i — i 1 r 00 1 2 3 4 6 TATc c p<0.05 5 ii i i i i i i i r 00 1 2 3 4 6 timee (hr)
Figuree 1. Mean SEM
plasmaa concentrations of prothrombinn fragment F1+22 and thrombin-antithrombinn complexes (TATc)) after administrationn of recombinantt factor Vila (rVIIa)) and placebo (circles)) or rVIIa and recombinantt NAPc2 (triangles).. P values indicatee the differences in resultss of both treatment groups. .
ActivationActivation of coagulation elicits IL-6 and IL-8 release in human subjects
ActivationActivation of coagulation (fig 1 and 2)
Administrationn of rVIIa resulted in substantial activation of thrombin generation,, as reflected by increased levels of F1+2 (from 0.7 0.1 nmol/L at baselinee to 4.0 0.3 nmol/L at 60 min) and TATc (from 3.3 0.8 pg/ml at baselinee to 14.0 1.3 pg/ml at 90 min). Pretreatment with rNAPc2 attenuated thee increase in Fl+2 (from 0.8 0.2 to 3.1 2.1 nmol/L at 60 min, p=NS for differencee between two treatment groups) and TATc (from 2.4 0.2 to 10.7 1.55 pg/ml at 90 min, p<0.05 for difference between treatment groups). Maximal plasmaa levels of factor X activation peptide were reached between at 60 minutes followingg the administration of rVIIa. Pre-treatment with rNAPc2 resulted in lowerr rVIIa-induced peak levels of factor X activation peptide as compared with injectionn of rVIIa alone (from 87 9 pmol/L to 302 25 pmol/L after rVIIa/placeboo and from 116 6 to 387 6 pmol/L after rVIIa preceded by rNAPc2,, p<0.05 for difference between treatment groups)
Figuree 2. Mean SEM plasmaa concentrations of factorr X activation peptide afterr administration of recombinantt factor Vila (rVIIa)) and placebo (circles)) or rVIIa and r e c o m b i n a n tt NAPc2 (triangles).. P values indicatee the differences in resultss of both treatment groups. .
InflammatoryInflammatory response (fig 3)
TNFF levels were below the limit of detection (8.2 pg/ml) in both treatment groupss throughout the experiment. IL-6 plasma levels increased from below the limitt of detection (1.2 pg/ml) at baseline to 5.5 2.5 pg/ml at 4 hr after administrationn of rVIIa and to 2.9 1.6 pg/ml at 6 hr after rVIIa preceded by rNAPc22 (p=0.05 for difference between treatment groups). IL-8 levels increased fromfrom <2 pg/ml to 8.3 3.3 pg/ml at 6 hr after administration of rVIIa and to 3.7
ouu u FF X act. peptide p<0.05 5 ,4000 " o o E E o. . 200200 -00 -1 11 1 0 0 1 1 1 1 11 1 2 2 11 1 33 4 timee (hr) I I 6 6 1 1 9 9
11 pg/ml at 4 hr after rVIIa/rNAPc2 (p<0.05 for difference between treatment groups).. In contrast, the plasma levels of the cytokine inhibitors sTNF-Rl and IL-lraa and of sE-selectin (marker for endothelial cell activation) were not influencedd by administration of rVIIa (data not shown).
'' i i i i 166 122 -~Ë -~Ë ^5> > °° 8 44 -oo -J '' I i I i I 00 1 2
x~i i
II I 33 4 time e (hr) ) IL-8 8 p<0.05 5 T T I I 66 <Figuree 3. Mean SEM plasmaa concentrations of plasmaa levels of Interleukin (IL)-66 and IL-8 after a d m i n i s t r a t i o nn o f recombinantt factor Vila >> ( r V I I a ) a n d p l a c e b o
(circles)) or rVIIa and r e c o m b i n a n tt N A P c 2 ( t r i a n g l e s ) .. P v a l u e s indicatee the differences in resultss of both treatment groups. .
) )
Inn vitro and animal experiments have shown that the activation of coagulationn may contribute to the induction of various inflammatory pathways. Thiss is the first study to show that the activation of coagulation can also induce releasee of cytokines in humans. We observed an increase in plasma levels of IL-66 and IL-8 after administration of rVIIa-initiated thrombin generation. TNF, sTNF-Rll and sE-selectin levels were not influenced by rVIIa administration. Pre-treatmentt with the tissue factor inhibitor rNAPc2 decreased the rVIIa
ActivationActivation of coagulation elicits IL-6 and IL-8 release in human subjects
inducedd thrombin generation and prevented the increase in circulating IL-6 and IL-8.. Our observation that IL-6 and IL-8 release is stimulated by activation of coagulationn is consistent with in vitro studies with human whole blood also revealingg that coagulation activation stimulates IL-8 and to a lesser extent IL-6, butt not TNF production.2 Furthermore, in primate models of sepsis IL-6 and IL-8,, but not TNF levels were significantly attenuated by anticoagulant interventionss with tissue factor pathway inhibitor (TFPI)15 or antithrombin.16
Severall coagulation factors could potentially contribute to the release of IL-66 and IL-8. rVIIa administration resulted in a marked thrombin generation as reflectedd by increased levels of F1+2 and TATc. Thrombin has been shown to stimulatee IL-1, IL-6, IL-8, TNF and monocyte chemotactic protein-1 (MCP-1) releasee by monocytes and endothelial cells,3;1722 probably mediated by thrombinss catalytic activity,*23 and by the cleavage of cell surface protease activatedd receptors (PARs).24 Another candidate for eliciting an inflammatory responsee is factor Xa. Factor Xa, by binding to effector cell protease receptor-1 (EPR-1)) and independent of thrombin, has been found to trigger acute inflammatoryy responses in animal experiments.25 Factor Xa was shown to stimulatee cultured human endothelial cells to produce IL-6, IL-8, MCP-1 and the adhesionn molecules sE-selectin, intercellular adhesion molecule (ICAM)-l and vascularr cell adhesion molecule (VCAM)-l, by a mechanism independent of thrombinn and EPR-1.26 Indeed, a prolonged increase of factor X activation peptidee was found following the administration of rVIIa. Finally, factor Vila itselff could contribute to the observed increase in cytokine levels. It has been shownn that factor Vila, independent of other coagulation proteins, can induce proinflammatoryy changes in mononuclear cells,27 possibly by activation of protease-activatedd receptor 2 (PAR 2) and to a lesser degree PAR l.28 In the presencee of factor Xa low picomolar concentrations of factor Vila caused robust signalingg in cells expressing TF and PAR 2.28 It is worth noting that these concentrationss are much lower than the nanomolar concentrations of factor Vila thatt were measured in our experiments after administration of rVIIa.
Ourr observations indicate that activation of coagulation in humans can elicitt cytokine release. Clearly, this inflammatory response is only small with IL-66 and IL-8 levels below 10 pg/ml. This limited response could be caused by thee short-lasting and relatively modest effect on thrombin generation by the intravenouss bolus injection of rVIIa. A more prolonged activation of coagulation couldd potentially have more influence on inflammatory responses. Furthermore, coagulationn could have a more profound effect on other inflammatory cascades whenn these are already activated to some extent by a common stimulus, such as
bacteriall infection or endotoxin. Alternatively, the limited effect of rVIIa on the cytokinee response could be explained by the absence of the expression of its cofactorr TF on circulating cells of healthy subjects. In situations with increased TFF expression on circulating blood cells, like during severe sepsis, activation of coagulationn could potentially induce a larger proinflammatory response. However,, in the mild inflammation model of endotoxemia in healthy humans, in whichh we prevented the endotoxin-induced activation of coagulation in humans byy tissue factor pathway inhibitor (TFPI) this inhibition of thrombin generation didd not affect the cytokine response.29
Inn conclusion, we demonstrate that the activation of coagulation by administrationn of factor Vila can elicit a IL-6 and IL-8 response in healthy humann subjects. This cytokine response can be prevented by attenuating the activationn of coagulation by the inhibitor of tissue1 factor rNAPc2. More research iss needed to determine whether this coagulation initiated inflammatory response mayy be quantitatively relevant in clinical situations like sepsis with disseminated intravascularr coagulation.
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