Studies on coagulation-induced inflammation in mice - Chapter 2 Microvascular coagulopathy and disseminated intravascular coagulation

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Studies on coagulation-induced inflammation in mice

Schoenmakers, S.H.H.F.

Publication date

2004

Link to publication

Citation for published version (APA):

Schoenmakers, S. H. H. F. (2004). Studies on coagulation-induced inflammation in mice.

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Microvascularr coagulopathy and disseminated intravascular

coagulation n

Hugoo ten Cate,

'

Saskia H.H.F. Schoenmakers,

Rendrik Franco,

Janneke J.

Timmerman,

Angelique P. Groot,

C. Arnold Spek,

Pieter H. Reitsma

laboratoryy for Experimental Internal Medicine, Academic Medical Center, and

departmentt of Internal Medicine, Slotervaart Hospital, Amsterdam, The

Netherlands. .

Presented,, in part, at the Margaux Conference on Critical Illness, Margaux,

France,, November 8-12, 2000.

Chapterr 2

Abstract: :

Objective:Objective: To review the dual characteristics of disseminated intravascular

coagulationn (DIC), as both a contributor to multiple organ failure as well as a symptomm of severe underlying disease associated with systemic vascular changes.

DataData Sources: Published literature data and unpublished results from the authors. DataData Summary: Clinical and experimental studies strongly suggest that DIC

contributess to multiple organ failure and death in patients with severe systemic disorderss such as sepsis. DIC is evoked by systemic cytokine activity, and the inflammatoryy response aggravates vascular permeability, inflammation, and cell damagee in tissues. In addition to intravascular fibrin formation, thrombin and fibrinn generation in tissues is also an important aspect of DIC. An example of DIC att the organ level is adult respiratory distress syndrome, where fibrin in the lung is aa characteristic feature. Intravascular fibrin formation and occlusion may elicit a hypoxicc response with induction of hypoxia related transcription factors. The resultingg ischemic preconditioning may offer protective effects to the involved organ(s). .

Conclusions:Conclusions: Overall, the beneficial or harmful effects of activated coagulation

andd fibrin formation for organ pathology and recovery from DIC remain to be explored.. This may be a critical element in the assessment of ischemia-reperfusionn effects of specific anticoagulant therapy.

Introduction n

Disseminatedd intravascular coagulation (DIC) is a systemic syndrome characterizedd by enhanced activation of coagulation with some intravascular fibrinn formation and deposition, depending on the degree of activity.1 DIC is thoughtt to contribute to multi-organ failure and death in a variety of underlying conditionss for several reasons. First, pathologic studies have repeatedly demonstratedd the presence of intravascular fibrin in tissues of patients who had diedd from an illness associated with evidence of DIC, suggesting a causal relationship.. Second, cohort studies have indicated an increased mortality in patientss with DIC compared with those who have the same underlying disease but noo evidence of DIC. And third, experimental studies of DIC associated with sepsiss or low-grade activation of coagulation have repeatedly demonstrated that effectivee inhibition of DIC can indeed reduce mortality. In contrast, many investigatorss currently believe that it is not DIC, and particularly not fibrin formationn itself that is harmful, but rather it is the generation of serine proteases andd their potential interactions with pro-inflammatory mediators that contribute to organn failure and death.

Thee microvasculature is the critical interface for oxygen and energy delivery to thee tissues. Thus, any damage to or obstruction of the microvasculature may have potentiallyy harmful consequences. In diseases complicated by DIC, a systemic inflammatoryy response syndrome is a standard finding (Fig. 1). The generation of pro-inflammatoryy cytokines has several consequences for the microvasculature withh relation to blood coagulation and DIC. Vascular endothelial cells may be

perturbedd by the action of cytokines such as interleukins (IL)-l, -6, and -8, as well ass tumor necrosis factor-a (TNF-a).2 These cytokines change the general anticoagulantt phenotype of the endothelium into a procoagulant phenotype, at leastt under in vitro conditions, resulting in, among other features, reduced expressionn of thrombomodulin 3 and heparan sulfates 4 as well as potentially upregulatedd tissue factor (TF).5 Inducible TF is predominantly expressed by monocytess and macrophages. The expression of TF on monocytes is markedly stimulatedd by the presence of platelets and granulocytes in a P-selectin-dependent reaction.. This effect may be the result of nuclear factor-KB activation induced by bindingg of activated platelets to neutrophils and mononuclear cells. This cellular interactionn also markedly enhances the production of IL-ip, IL-8, monocyte chemoattractantt protein-1 (MCP-1), and TNF-a.7'8

Inflammation n

TF/cytokines s

Vascularr leakage Leukocytee activation

Figuree 1. Relation between disseminated intravascular coagulationn (DIC) and inflammation. TF, tissue factor.

55

DIC

C

Thrombosiss ^ ™ Extravascular fibrin n

Increasedd endothelial permeability facilitates the interaction of transmigrating leukocytess with the subendothelial space, such that extravascular inflammation andd coagulation may occur. The generation of procoagulant pathways, as well as theirr interactions with platelets and leukocytes, in the microvasculature may lead too intravascular fibrin formation, which, in turn, may cause occlusion of the smallerr vessels. We will now consider several aspects related to the generation of fibrin,, the consequences that vascular occlusion might have, and the site of fibrin depositionn in relation to inflammation.

GenerationGeneration of Fibrin and Its Deposition.

Fromm studies in human volunteers and in baboons challenged with lethal

EscherichiaEscherichia coli, it is known that DIC can be distinguished in various stages, in

relationn to the degree of procoagulant derangement.9'10 Initially, thrombin generationn and down-regulation of fibrinolysis occur," followed by intra-vascular fibrinn formation and endothelial cell activation, as indicated by increased levels of fairlyy specific endothelial cell molecules.9 Subsequently, increased vascular permeabilityy occurs, indicative of endothelial disruption and damage. In the latter stages,, fibrin deposition occurs intravascularly as well as in extravascular spaces, forr example, as seen in adult respiratory distress syndrome (ARDS). Because of depletionn of clotting factors and platelets, bleeding may be seen in this stage of DIC. .

Thee kinetics of fibrin deposition in organs was studied in a more systematic way byy analyzing the lungs of mice with a mutation in the thrombomodulin gene that

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weree challenged with endotoxin. In these studies, thrombomodulin mutated mice withh a mixed genetic background of Svl29 and C57B1/6 were utilized, which mightt have contributed to part of the observed effects on fibrin formation and inflammation122 (R Franco, et al., unpublished observations). Nevertheless, intravascularr fibrin formation occurred early after endotoxin challenge of mice (detectablee after 30 mins), was associated with signs of inflammation, and disappeared,, probably as a result of fibrinolysis, at 24 hrs. In plasma, evidence of DICC was provided by substantial elevations in the levels of thrombin-antithrombinn complexes, which followed a time course that was approximately parallell to the pattern of fibrin deposition. This study illustrated that intravascular fibrinn formation can be observed in a specific organ, and does not lead to overt organn damage except for transient evidence of inflammation. Furthermore, this processs is reversible under certain conditions as a result of fibrinolytic clearance off the microvasculature.

DICDIC and Vascular Occlusion

Theoretically,, when the trigger of DIC is stronger or more prolonged than anticoagulation,, or when the anticoagulant or fibrinolytic mechanisms fail to protect,, fibrin formation may persist and lead to prolonged vascular occlusion. Thee resulting hypoxia may contribute to organ ischemia and cell death (Fig. 2).

Vascular occlusion by fibrin inflammatory response

hypoxia ^ + tissue ischemias necrosis extravascular fibrin/inflammation

i i

multi organ damage

Systemicc hypoxia is known to cause fibrin formation as well. Several studies have indicatedd that, in the presence of either a defect in an anticoagulant pathway such ass thrombomodulin (S Schoenmakers et al., unpublished observations), or a defectt in the fibrinolytic system,14 hypoxia induced by keeping mice at 8% or less oxygenn causes fibrin formation in the lungs. Although, at lower oxygen pressure (6%),, fibrin may also accumulate in normal mice,15 these very low oxygen levels aree not well tolerated in normal or other mice (data not shown). Furthermore, it is unknownn to what extent fibrin formation occurs in tissues other than the lung. The proposedd procoagulant mechanism is enhanced expression of TF by monocytes as aa result of enhanced activity of transcription factors such as Egr-1 6 but it may be possiblee that endothelial cell-induced TF also plays a role in this process. Nevertheless,, it remains to be seen how the presence of fibrin influences the adjacentt tissue and whether inflammation and clotting may facilitate local apoptosiss and tissue damage. Local hypoxia also induces the expression of hypoxia-induciblee transcription factors that, via ischemic preconditioning, may

Figuree 2. Consequences of disseminated intravascularr coagulation.

defendd the organ against permanent damage.17 The relevance of these defense mechanismsmechanisms in DIC remains to be investigated.

Inn addition to intravascular fibrin formation, fibrin may be generated in or transferredd to extra vascular areas, where it may, in turn, be deposited.18 For example,, ARDS is frequently associated with intra-alveolar and intravascular fibrinn formation,19'20 most likely a result of both systemic and local mediators of procoagulantt reactions. Local production of TF, which is detectable in bronchoalveolarr lavage fluid, may trigger the procoagulant reactions.21

DICDIC and Inflammation

Thee inflammatory component involved in ARDS may be distinct from the pathwayy that leads to fibrin formation. In a rat model of E. coli induced pulmonaryy injury, a synthetic specific inhibitor of kallikrein prevented pulmonary vascularvascular injury, but did not inhibit DIC. In contrast, the active site-blocked factor Vilaa inhibited DIC but not pulmonary injury, suggesting that the inflammatory andd coagulation reaction in the lungs to endotoxin are not intimately associated.22 Severall studies suggest a direct effect of fibrin on inflammatory activity:

fibrinogenfibrinogen interacts with bacteria and modulates their activity, fibrin serves to encapsulatee bacteria, or fibrin cleavage peptides may trigger the release of

proinflammatoryy cytokines.23 Thus, in extravascular spaces such as the intraperitoneall cavity or pulmonary tissue, fibrin may be involved in the regulationn of inflammatory activity and tissue damage. It remains unknown whetherr fibrin plays an important role in this regard, and it is entirely unknown whetherr fibrin has "good" or "bad" properties in localized inflammatory processes. .

Inn an experimental ARDS model induced by installation of endotoxin endobronchial^,, a peribronchial inflammatory response occurs, with extravasationn of leukocytes (mostly granulocytes). The granulocytes stain positive withh specific antibodies against murine TF, possibly indicating that these cells mayy be involved in the local fibrin generating process. Indeed, local generation of thrombinn is indicated by elevated thrombin-antithrombin complexes in bronchoalveolarr lavage fluid (JJ Timmerman et al., unpublished observations) and fibrinfibrin appears to be localized at areas of inflammatory activity, suggesting a relationshipp between these elements.

Conclusion n

Fibrinn formation in the course of DIC may be an important determinant of organ morbidity.. However, direct evidence to support this is essentially absent. All favorablee studies suggesting a beneficial effect of anticoagulant treatment in humanss or animals may be explained by the inhibition of intermediate proteases off the coagulation cascade that have proinflammatory activities. Whether preventingg fibrin formation per se is helpful in limiting organ damage remains to bee established. With the emergence of powerful anticoagulant strategies such as

Chapterr 2

activatedd protein C,24 aspects involving ischemia-reperfusion damage and long-termm organ recovery become important to investigate.

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