Portal de Periódicos da CAPES

Procoagulant protein levels are differentially increased during human endotoxemia

2003; Elsevier BV; Volume: 1; Issue: 5 Linguagem: Inglês

10.1046/j.1538-7836.2003.00237.x

1538-7933

Pieter H. Reitsma, Judith Branger, Bernt van den Blink, Sebastiaan Weijer, Tom van der Poll, Joost C.M. Meijers,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

SummaryOn the basis of plasma interleukin levels it was suggested that there is an inflammatory component to the risk of venous thrombotic disease. Other evidence shows that elevated levels of coagulation factor (F)VIII, FIX, FX and FXI are risk indicators for venous thrombosis, but the reasons for elevation remain unclear. We tested the hypothesis that the elevated levels could reflect an inflammatory reaction by measuring coagulation factor levels during experimental human endotoxemia. Male volunteers received endotoxin (4 ng kg−1), and blood samples were obtained before and at multiple time points after the challenge. Plasma was used for a panel of coagulation tests. Antigen levels of FVIII, von Willebrand factor (VWF), FIX, and FX were increased after endotoxin administration, reaching peak levels between 2 and 5 h. Within 24 h levels normalized, except for FVIII and VWF levels that remained at > 200%. Fibrinogen levels, and to a lesser extent FXI levels, also responded with an increase, but slower. These levels did not return to normal during the observation period. FVII levels were strongly depressed. FVIII, FIX and FX reacted immediately and strongly to endotoxin administration. The time pattern of this response is different from the slower so-called acute phase response, which appeared to be followed by FXI and fibrinogen. These increased levels of coagulation factors during an inflammatory state provide new ways of explaining why elevated levels of FVIII, FIX and FXI behave as risk indicators disease. On the basis of plasma interleukin levels it was suggested that there is an inflammatory component to the risk of venous thrombotic disease. Other evidence shows that elevated levels of coagulation factor (F)VIII, FIX, FX and FXI are risk indicators for venous thrombosis, but the reasons for elevation remain unclear. We tested the hypothesis that the elevated levels could reflect an inflammatory reaction by measuring coagulation factor levels during experimental human endotoxemia. Male volunteers received endotoxin (4 ng kg−1), and blood samples were obtained before and at multiple time points after the challenge. Plasma was used for a panel of coagulation tests. Antigen levels of FVIII, von Willebrand factor (VWF), FIX, and FX were increased after endotoxin administration, reaching peak levels between 2 and 5 h. Within 24 h levels normalized, except for FVIII and VWF levels that remained at > 200%. Fibrinogen levels, and to a lesser extent FXI levels, also responded with an increase, but slower. These levels did not return to normal during the observation period. FVII levels were strongly depressed. FVIII, FIX and FX reacted immediately and strongly to endotoxin administration. The time pattern of this response is different from the slower so-called acute phase response, which appeared to be followed by FXI and fibrinogen. These increased levels of coagulation factors during an inflammatory state provide new ways of explaining why elevated levels of FVIII, FIX and FXI behave as risk indicators disease. Venous thrombosis is an episodic disease in which a complex interplay between genetic and acquired risk factors plays a determining role. During the past two decades several of the genetic risk factors have been discovered and extensively characterized. These include inherited deficiencies in natural anticoagulants like protein C, protein S and antithrombin, or gain of function mutations in procoagulant genes like factor (F)V Leiden and prothrombin 20210 A [1Franco R.F. Reitsma P.H. Genetic risk factors of venous thrombosis.Hum Genet. 2001; 109: 369-84Crossref PubMed Scopus (226) Google Scholar]. The latter mutation appears to be associated with increased levels of otherwise normal prothrombin [2Poort S.R. Rosendaal F.R. Reitsma P.H. Bertina R.M. A common genetic variation in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis.Blood. 1996; 88: 3698-703Crossref PubMed Google Scholar]. At about the same time the prothrombin mutation was discovered, it was found that an elevated level of coagulation FVIII was also a risk factor for venous thrombotic disease [3Koster T. Blann A.D. Briët E. Vandenbroucke J.P. Rosendaal F.R. Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis.Lancet. 1995; 345: 152-5Abstract PubMed Scopus (1024) Google Scholar, 4O'Donnell J. Tuddenham E.G. Manning R. Kemball-Cook G. Johnson D. Laffan M. High prevalence of elevated factor VIII levels in patients referred for thrombophilia screening: role of increased synthesis and relationship to the acute phase reaction.Thromb Haemost. 1997; 77: 825-8Crossref PubMed Google Scholar]. These findings prompted studies aimed at determining whether elevated levels of other procoagulant proteins also are risk factors or indicators of venous thrombotic disease. This indeed appears to be the case for FIX and FXI [5Van Hylckama Vlieg A. Van Der Linden I.K. Bertina R.M. Rosendaal F.R. High levels of factor IX increase the risk of venous thrombosis.Blood. 2000; 95: 3678-82Crossref PubMed Google Scholar, 6Meijers J.C. Tekelenburg W.L. Bouma B.N. Bertina R.M. Rosendaal F.R. High levels of coagulation factor XI as a risk factor for venous thrombosis.N Engl J Med. 2000; 342: 696-701Crossref PubMed Scopus (558) Google Scholar]. Genetic determinants for plasma levels of FVIII, FIX and FXI have not been found [7Kamphuisen P.W. Eikenboom J.C. Rosendaal F.R. Koster T. Blann A.D. Vos H.L. Bertina R.M. High factor VIII antigen levels increase the risk of venous thrombosis but are not associated with polymorphisms in the von Willebrand factor and factor VIII gene.Br J Haematol. 2001; 115: 156-8Crossref PubMed Scopus (0) Google Scholar, 8Mansvelt E.P. Laffan M. McVey J.H. Tuddenham E.G. Analysis of the F8 gene in individuals with high plasma factor VIII. C levels and associated venous thrombosis.Thromb Haemost. 1998; 80: 561-5PubMed Google Scholar]. Such genetic determinants are an important tool in establishing whether a risk factor is in causative relationship with disease, or whether it represents an innocent bystander that reflects an unknown underlying disease process that increases plasma levels. Therefore the direct importance of elevated levels of FVIII, FIX and FXI for venous thrombotic disease remains uncertain. On the basis of plasma interleukin 8 levels, we have recently suggested that there may be an inflammatory component to the risk of venous thrombotic disease [9Van Aken B.E. Reitsma P.H. Rosendaal F.R. Interleukin 8 and venous thrombosis: evidence for a role of inflammation in thrombosis.Br J Haematol. 2002; 116: 173-7Crossref PubMed Scopus (108) Google Scholar, 10Van Aken B.E. Den Heijer M. Bos G.M. Van Deventer S.J. Reitsma P.H. Recurrent venous thrombosis and markers of inflammation.Thromb Haemost. 2000; 83: 536-9Crossref PubMed Scopus (124) Google Scholar]. This inflammatory component appeared to be independent of the acute phase reaction since adjustment for C-reactive protein levels failed to nullify the risk imparted by increased interleukin 8 levels. Inflammation is associated with elevated levels of fibrinogen and FVIII [11Noe D.A. Murphy P.A. Bell W.R. Siegel J.N. Acute-phase behavior of factor VIII procoagulant and other acute-phase reactants in rabbits.Am J Physiol. 1989; 257: R49-56PubMed Google Scholar, 12Gabay C. Kushner I. Acute-phase proteins and other systemic responses to inflammation.N Engl J Med. 1999; 340: 448-54Crossref PubMed Scopus (5038) Google Scholar, 13Korkmaz C. Ozdogan H. Kasapcopur O. Yazici H. Acute phase response in familial Mediterranean fever.Ann Rheum Dis. 2002; 61: 79-81Crossref PubMed Google Scholar]. We therefore wondered whether there might be a more general relationship between inflammation and coagulation factor levels. This encouraged us to study coagulation factor levels in a controlled model of human inflammation. In this model, male volunteers are challenged with a low dose of endotoxin, which results in a strong but temporary inflammatory response. We observed that plasma levels of FVIII, FIX, and FX respond quickly and strongly to endotoxin. The time course of this immediate inflammatory response is quite different from a classic acute phase reaction, which appears to be followed by FXI and fibrinogen. The human endotoxemia model was used to induce an acute sterile inflammatory response [14Van der Poll T. Van Deventer S.J.H. Endotoxemia in healthy subjects as a human model of inflammation.in: Cohen J Marshall J The Immune Response in the Critically Ill. Springer Verlag, 1999Google Scholar]. Eight male volunteers, aged 19–29 years, participated in the study. Each had a clean medical history, a normal physical examination and normal results in routine laboratory tests. Participation in the study was excluded if prior to the study any infectious disease had occurred within 1 month, if any acute illness was reported within 1 week, or if any medication was taken within 2 weeks. None of the volunteers smoked or used illicit drugs. Written informed consent was obtained from all participants, and the institutional scientific and ethics committees approved the study. Volunteers were admitted to a special clinical research unit in pairs. During the whole procedure emergency equipment was immediately available, and two supervising physicians were continuously present. Endotoxin (Escherichia coli lipopolysaccharide, lot G; UPS, Rockville, MD, USA) was injected intravenously in 1 min in an antecubital vein at a dose of 4 ng kg−1 body weight. Blood was obtained through an indwelling intravenous catheter at the following time points relative to the endotoxin administration: – 0.1, 0.5, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 10, and 24 h. For coagulation assays, blood were collected in siliconized vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA) containing 0.105 mol L−1 sodium citrate. All blood samples were centrifuged at 1000 × g for 15 min at 4 °C, and plasma was stored at −20 °C until assays were performed. Coagulation factor assays [prothrombin time (PT), activated partial thromboplastin time (PTT), and fibrinogen] were performed on an automated coagulation analyzer (Behring Coagulation System) with reagents and protocols from the manufacturer (Dade Behring, Marburg, Germany). FVII, FVIII and FIX antigen levels were determined with ELISA's from Diagnostica Stago (Asnières-sur-Seine, France). FII, FX and von Willebrand factor (VWF) antigen levels were determined with ELISAs developed in our laboratory using antibodies from Dako (Glostrup, Denmark). FV and FXI antigen levels were assayed using ELISAs with antibodies from Affinity Biologicals (Kordia, Leiden, the Netherlands). For each parameter determined, all samples of an individual were assayed in duplicate (except PT, PTT and fibrinogen) in a single experiment. As a reference for the antigen determinations, the reagent from the manufacturer (in case of FVII, FVIII and FIX) was taken, and for the other assays, a plasma pool of 150 healthy hospital personnel was used. All data are presented as mean ± standard error of the mean. Changes in time were analyzed by one-way analysis of variance followed by Dunnett's t-test. P < 0.05 was considered to represent a statistically significant effect. All volunteers responded strongly to the LPS challenge and developed all the signs of endotoxemia like fever, nausea and other flu-like symptoms [14Van der Poll T. Van Deventer S.J.H. Endotoxemia in healthy subjects as a human model of inflammation.in: Cohen J Marshall J The Immune Response in the Critically Ill. Springer Verlag, 1999Google Scholar]. As expected, plasma levels of a variety of cytokines and chemokines were also increased after endotoxin administration reaching peak levels, depending on the kind of the mediator, between 1 and 3 h (data not shown but similar to what is described in [14Van der Poll T. Van Deventer S.J.H. Endotoxemia in healthy subjects as a human model of inflammation.in: Cohen J Marshall J The Immune Response in the Critically Ill. Springer Verlag, 1999Google Scholar, 15Pajkrt D. Van der Poll T. Levi M. Cutler D.L. Affrime M.B. Van den Ende A. Ten Cate J.W. Van Deventer S.J. Interleukin-10 inhibits activation of coagulation and fibrinolysis during endotoxemia.Blood. 1997; 89: 2701-5Crossref PubMed Google Scholar, 16De Jonge E. Dekkers P.E. Creasey A.A. Hack C.E. Paulson S.K. Karim A. Kesecioglu A. Levi M. Van Deventer S.J. Van der Poll T. Tissue factor pathway inhibitor dose-dependently inhibits coagulation activation without influencing the fibrinolytic and cytokine response during human endotoxemia.Blood. 2000; 95: 1124-9Crossref PubMed Google Scholar, 17Verbon A. Dekkers P.E. Ten Hove T. Hack C.E. Pribble J.P. Turner T. Souza S. Axtelle T. Hoek F.J. Van Deventer S.J. Van der Poll T. IC14, an anti-CD14 antibody, inhibits endotoxin-mediated symptoms and inflammatory responses in humans.J Immunol. 2001; 166: 3599-605Crossref PubMed Google Scholar]). In addition, the coagulation system was activated as reflected by increases in thrombin–antithrombin complexes and prothrombin fragment 1 + 2 (data not shown, see also [15Pajkrt D. Van der Poll T. Levi M. Cutler D.L. Affrime M.B. Van den Ende A. Ten Cate J.W. Van Deventer S.J. Interleukin-10 inhibits activation of coagulation and fibrinolysis during endotoxemia.Blood. 1997; 89: 2701-5Crossref PubMed Google Scholar, 16De Jonge E. Dekkers P.E. Creasey A.A. Hack C.E. Paulson S.K. Karim A. Kesecioglu A. Levi M. Van Deventer S.J. Van der Poll T. Tissue factor pathway inhibitor dose-dependently inhibits coagulation activation without influencing the fibrinolytic and cytokine response during human endotoxemia.Blood. 2000; 95: 1124-9Crossref PubMed Google Scholar, 17Verbon A. Dekkers P.E. Ten Hove T. Hack C.E. Pribble J.P. Turner T. Souza S. Axtelle T. Hoek F.J. Van Deventer S.J. Van der Poll T. IC14, an anti-CD14 antibody, inhibits endotoxin-mediated symptoms and inflammatory responses in humans.J Immunol. 2001; 166: 3599-605Crossref PubMed Google Scholar]). Concentrations of coagulation factors were also altered which is clearly illustrated by the response of the activated partial thromboplastin time (APTT) and PT (Fig. 1). The APTT was shortened (probably because of a large increase in FVIII, see below) and returned to near normal levels within the observation period. The PT became longer over time (because of a solid drop in FVII levels, see below), and remained slightly elevated at 24 h, the last time point that blood was sampled. All these data are in complete agreement with previously published data from human endotoxin challenges [15Pajkrt D. Van der Poll T. Levi M. Cutler D.L. Affrime M.B. Van den Ende A. Ten Cate J.W. Van Deventer S.J. Interleukin-10 inhibits activation of coagulation and fibrinolysis during endotoxemia.Blood. 1997; 89: 2701-5Crossref PubMed Google Scholar, 16De Jonge E. Dekkers P.E. Creasey A.A. Hack C.E. Paulson S.K. Karim A. Kesecioglu A. Levi M. Van Deventer S.J. Van der Poll T. Tissue factor pathway inhibitor dose-dependently inhibits coagulation activation without influencing the fibrinolytic and cytokine response during human endotoxemia.Blood. 2000; 95: 1124-9Crossref PubMed Google Scholar]. Figure 2, Figure 3 show the antigen data for FVIII, FIX, FX, and VWF. It is clear from these figures that these procoagulant factors responded sharply to the endotoxin challenge. In terms of magnitude of increase the almost parallel responses of FVIII and VWF were the most impressive with mean peak values reaching almost 400% of pretest levels. It seems that FVIII levels responded slightly faster than VWF levels.Figure 3Response pattern 2: time course of FIX and FX antigen levels. Levels rise sharply and return to normal between 10 and 24 h. See also legend to Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) FIX increases were more modest with a mean peak value of about 115%. The response of FX levels reached a peak of almost 120%. The time course of these responses was quite comparable. As early as 1 h after the endotoxin challenge an increase in level was noticeable. The peak was reached after 3 h, which interestingly coincides with peak levels of interleukin 6 and 8 [14Van der Poll T. Van Deventer S.J.H. Endotoxemia in healthy subjects as a human model of inflammation.in: Cohen J Marshall J The Immune Response in the Critically Ill. Springer Verlag, 1999Google Scholar]. Thereafter FIX and FX levels dropped rapidly and returned to baseline within 10 h. Plasma levels of FVIII and VWF, however, did not return to normal within the observation period. Figure 4 shows the time series for two other liver derived procoagulant proteins: FXI and fibrinogen. There was a small decrease in fibrinogen and FXI within 1 h of the challenge. As anticipated, an increased level of fibrinogen was only seen after 24 h, which is in keeping with its behavior as an acute phase protein [12Gabay C. Kushner I. Acute-phase proteins and other systemic responses to inflammation.N Engl J Med. 1999; 340: 448-54Crossref PubMed Scopus (5038) Google Scholar]. FXI levels responded similarly with a tendency for high levels at the end of the observation period, but the increase was not significant. Not all coagulation proteins increased in concentration. Notably, FVII concentrations became much lower during the course of the study, reaching minimum levels at 24 h after the start of the challenge (Fig. 5). The aim of the present study was to verify whether increased plasma levels of a set of coagulation proteins could be related to an inflammatory reaction. The results presented here give credence to this hypothesis. Indeed many coagulation factors responded with an increase in plasma concentration, but the responses were much more complex than we had anticipated. The change in fibrinogen level follows the pattern of a classic ‘acute phase’ response. This response, other than the name suggests, was quite slow and only evident at the 24-h time point, which parallels the slow rise in C-reactive protein (CRP) in this model [17Verbon A. Dekkers P.E. Ten Hove T. Hack C.E. Pribble J.P. Turner T. Souza S. Axtelle T. Hoek F.J. Van Deventer S.J. Van der Poll T. IC14, an anti-CD14 antibody, inhibits endotoxin-mediated symptoms and inflammatory responses in humans.J Immunol. 2001; 166: 3599-605Crossref PubMed Google Scholar]. It seems that FXI followed the same pattern with a modest increase at 24 h, which indicates that this coagulation protein may also behave as an acute phase protein like fibrinogen and CRP. Antigen levels of FVIII and VWF followed a second type of pattern. We observed an immediate inflammatory response reflected by a steep increase maximizing between 3 and 5 h after the administration of endotoxin; at 24 h the levels were still up at around 300% of normal. The observation that FVIII levels remained high in our study contrasts with earlier findings. In a similar endotoxin challenge model, Gralnick et al. [18Gralnick H.R. McKeown L.P. Wilson O.M. Williams S.B. Elin R.J. von Willebrand factor release induced by endotoxin.J Lab Clin Med. 1989; 113: 118-22PubMed Google Scholar] observed that FVIII levels returned to normal within 24 h. It should be noted that our study examines FVIII antigen levels, whereas the older study relied on activity measurements. In fact, when we examine FVIII activity levels in our volunteers we also see a return to almost pretest values (data not shown) at 24 h. Apparently, measuring FVIII levels in an activity assay does not faithfully reflect the underlying antigen levels in the human endotoxin model. The responses of FVIII and VWF were almost parallel, both in time-course and in magnitude. It seems that the initial response is faster for FVIII than for VWF. This is somewhat surprising in view of the consideration that VWF is the prime carrier of plasma FVIII, even though the two proteins are synthesized in different cellular compartments (endothelial cells of large vessels vs. hepatocytes/non-parenchymal (sinusoidal endothelial) cells, respectively) [19Hollestelle M.J. Thinnes T. Crain K. Stiko A. Kruijt J.K. Van Berkel T.J. Loskutoff D.J. Van Mourik J.A. Tissue distribution of factor viii gene expression in vivo - a closer look.Thromb Haemost. 2001; 86: 855-61Crossref PubMed Google Scholar]. This suggests that the initial elevation of FVIII is not solely dependent on complex formation with VWF that is released from endothelial Weibel–Palade storage granules. More likely, endotoxin has a direct effect on liver-derived FVIII release. The third type of response is followed by FIX and FX. These plasma proteins responded immediately to the endotoxin challenge, and returned to normal shortly thereafter. Such a pattern was not observed for FVII or prothrombin (data not shown), which indicates that this response is not typical for all vitamin K-dependent coagulation proteins. We presume that the response is due to either a direct and immediate effect of endotoxin on the hepatocyte, the natural cellular source for these proteins, or indirectly due to a mediator that is induced by endotoxin-like tumor necrosis factor (TNF) or other cytokines. On the basis of the rapidity of the increases we assume that in the case of a direct effect of endotoxin on the hepatocyte, the surge in antigen may have resulted from newly synthesized protein. In the case of a mechanism where intermediate cytokines or chemokines need to be induced first, the time course of the changes suggests release from a storage pool of these proteins. The fourth type of response was observed for FVII. We were quite surprised that the levels of FVII were depressed so strongly by the endotoxin challenge. It seems hard to explain this decrease from consumption of FVII in activated coagulation. More likely, synthesis of FVII is slowed down. Apparently FVII falls in the category of negative acute phase proteins like albumin [12Gabay C. Kushner I. Acute-phase proteins and other systemic responses to inflammation.N Engl J Med. 1999; 340: 448-54Crossref PubMed Scopus (5038) Google Scholar]. On the basis of these results, we conclude that an acute inflammatory challenge results in diverse effects on the levels of coagulation proteins. Although the effects appear to be transient in this model, it is not difficult to imagine that a chronic and persistent inflammatory state could result in lasting elevated levels of at least some coagulation proteins. Therefore these results open new ways of explaining why elevated levels of coagulation proteins behave as risk indicators of disease. We would like to thank nurses and technicians of the AMC for their help in performing this study. Joost Meijers is an Established Investigator of the Netherlands Heart Foundation (grant D96.021), and was also supported by a grant from the Dutch Thrombosis Foundation (99 003). Bernt van den Blink was supported by a grant from the Meelmeijer fund. Sebastiaan Weijer was supported by a grant from the Netherlands Organization of Scientific Research.