Crosstalk of Inflammation and Coagulation in Infectious Disease and Their Roles in Disseminated Intravascular Coagulation

Introduction

Viral, bacterial, fungal, and parasitic infections may all cause disturbances in hemostasis, which can eventually lead to thrombohemorrhagic complications such as disseminated intravascular coagulation (DIC), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), or even vasculitis. Symptoms and signs may be absent in cases of subclinical activation of the coagulation cascade, or they may be overt in cases of bleeding, thrombosis, or both. None of the clinical signs and symptoms is pathogen specific, and in general, signs and symptoms depend on the severity of the infection and the response of the host. This chapter discusses general aspects and pathogen-specific aspects of the crosstalk between coagulation and inflammation. The focus is on the pathophysiology of gram-negative sepsis as a prototypic model of an inflammation-coagulation–regulated syndrome. In addition, recently developed and clinically tested anticoagulants designed to intervene in DIC and sepsis are discussed.

DIC is associated with a high rate of mortality in both bacterial and nonbacterial disease; for this reason the acronym has been said to mean “Death Is Coming.” According to criteria developed by the International Society on Thrombosis and Haemostasis (ISTH), DIC (see Chapter 10 ) is “an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes. It can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction.” One consequence of DIC may indeed be occlusion of small and midsize vessels by fibrin contributing to multiorgan failure. Because of consumption of coagulation factors and activation of the fibrinolytic system, life-threatening hemorrhage may occur. The severity of bleeding ranges from localized oozing from arterial or venous puncture sites to more systemic complications, such as petechiae, purpura, ecchymosis, and internal bleeding, as evidenced by hematemesis, hemoptysis, or frank hematuria. Purpura fulminans is observed in the course of bacterial infection with meningococci and pneumococci, as well as in other bacterial and viral infections. The typical thrombohemorrhagic syndrome described by Waterhouse and Friderichsen in 1911 includes fever, cyanosis, a purpuric rash, and circulatory collapse. Internal bleeding within organs may be confined and asymptomatic but can also cause systemic circulatory failure when localized in critical organs such as the adrenals.

Often, a patient with DIC has microvascular fibrin formation and bleeding at the same time, which hampers the clinician's choice of appropriate treatment. Local thromboembolic disease (i.e., deep vein thrombosis [DVT] and pulmonary embolism [PE]) may theoretically occur in patients with viral and bacterial infections, particularly in bedridden patients with comorbidity. In a thromboembolism prevention study of low-dose subcutaneous standard heparin therapy for hospitalized patients with infectious diseases, morbidity due to thromboembolic disease was significantly reduced in the heparin group compared with the group that was given no prophylaxis. However, no beneficial effect of prophylaxis was noted on mortality caused by thromboembolic complications.

HUS and TTP are frequently regarded as subtypes of a single syndrome (thrombotic microangiopathy) characterized by thrombocytopenia, hemolytic anemia, fever, renal abnormalities, and neurologic disturbances. For a discussion of TTP and HUS, the reader is referred to Chapters 24 and 25 . Vasculitis is characterized by local or more generalized vascular changes, resulting from ischemia caused by occlusion of the lumina of small blood vessels in the upper part of the dermis by thrombi or bleeding caused by local tissue damage (see Chapter 10 ).

General Aspects of Primary Hemostasis, Coagulation, and Fibrinolysis

The hemostatic mechanism consists of primary hemostasis and coagulation, on the one hand, and natural anticoagulant mechanisms and fibrinolysis, on the other hand. Under physiologic conditions, these mechanisms are balanced. The tissue factor (TF) pathway is considered to be the main route for activation of the coagulation cascade in sepsis. Initiating factors are the membrane-bound glycoprotein TF and plasma protein factor VIIa. TF is induced by proinflammatory mediators, including cytokines, C-reactive protein, and advanced glycated end products in circulating blood cells and on microparticle fragments. On expression at the cell surface, TF interacts with factor VII in its zymogen or activated form, and the catalytic complex activates factors IX and X, which results in the generation of thrombin and subsequently fibrin ( Fig. 13.1 ). The contact activation system consists of four plasma proteins, namely factor XII, factor XI, plasma prekallikrein, and high-molecular-weight kininogen. The contact activation system starts procoagulant and proinflammatory reactions via the intrinsic pathway and the kallikrein-kinin system, respectively. This system is initiated by factor XIIa. The physiologic role of factor XII in coagulation has long been questioned, because hereditary factor XII deficiency in humans is not associated with an increased bleeding risk. Nonetheless, studies performed in mice showed that factor XII is not irrelevant for procoagulant activity, because platelet-dependent thrombosis is reduced in factor XII–deficient mice. Furthermore, factor XII is important for the innate immune response. Factor XIIa can initiate the complement cascade by activation of factor C1. The kinin-kallikrein system, which is part of the contact activation system, plays a role in the regulation of vascular tone and permeability, also through interactions with the complement system.

Coagulation is balanced by different inhibitory mechanisms. The first mechanism is composed of circulating inhibitors of blood coagulation (e.g., antithrombin III [ATIII], protein C [PC], protein S [PS], and the TF pathway inhibitor [TFPI]). A second inhibitory mechanism consists of the glycocalyx-associated glycosaminoglycans such as heparan sulfate, endothelial protein C receptor (EPCR), and thrombomodulin (TM), all of which facilitate the inhibitory activity of ATIII and activated protein C (APC). The third mechanism, fibrinolysis, may be activated primarily and thus independently of activation of the coagulation cascade, or secondarily in response to fibrin formation. The fibrinolysis process is activated by tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) after their synthesis and release from endothelial cells. These activators initiate the conversion of plasminogen to plasmin, which hydrolyzes polymerized fibrin strands into soluble fibrin degradation products. An important inhibitor of this system is plasminogen activator inhibitor-1 (PAI-1). Severe infections result in an imbalance between primary hemostasis and coagulation, and anticoagulant mechanisms and fibrinolysis, as is explained later. During these events, endothelial cells at the tissue-blood interface become of crucial importance.

Endothelial Activation and Its Effects on Coagulation During Inflammation

Vascular endothelial cells play a central role in all mechanisms that contribute to inflammation-induced activation of coagulation. During acute infection, the endothelium is directly activated by pathogens or indirectly via inflammatory mediators, and the major regulatory antithrombotic properties become inactivated ( Fig. 13.2 ). Proinflammatory cytokines, including interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and IL-6, induce TF within endothelial cells, which may be shed in part as soluble TF. Shedding of soluble TF may perhaps explain why it has been difficult to detect endothelial TF by immunohistochemistry in animal studies. It remains uncertain whether endothelial cells contribute to TF production in sepsis. Knocking out the TF gene selectively in endothelial cells did not attenuate the level of activated coagulation measured as thrombin-ATIII complexes when mice were challenged with lipopolysaccharide (LPS). The same proinflammatory cytokines appear to downregulate the anticoagulant receptors TM and EPCR, as well as cellular glycosaminoglycans. Proteolytic cleavage of the receptors TM and EPCR may also occur owing to enzymes such as elastase, which are secreted by activated neutrophils. Soluble (unbound) TF probably does not activate clotting, yet soluble TM may continue to function as a cofactor for activation of thrombin-activatable fibrinolysis inhibitor (TAFI), which would impede fibrinolysis and worsen DIC. Given interactions with inflammation (see later discussion), it is also most likely that loss of TM and EPCR from the cell surface, such as has been demonstrated in the microvasculature of patients with sepsis, leads to enhanced inflammatory responses in vivo. The net effect of these changes in endothelial cell function likely is procoagulant and proinflammatory.

TFPI is synthesized and stored in endothelial cells. However, no evidence suggests that depletion or dysfunction of TFPI occurs as part of an infectious disease such as sepsis, and its clinical role in inflammation and infection must be further substantiated.

Endothelial cells are also able to express adhesion molecules and growth factors that may not only promote the inflammatory response but also increase the coagulation response. Interactions between platelets and endothelial cells, as well as between platelets and neutrophils, are important links in the onset of inflammation. In endothelial cells, Weibel-Palade bodies secrete von Willebrand factor (vWF) and P-selectin, which support platelet rolling. Inflamed endothelium supports leukocyte rolling, and activated platelets in contact with leukocytes and endothelial cells release a number of mediators of the inflammatory response. Such mediators include CD40 ligand, lipoxygenases, and prostaglandins. Of potential procoagulant importance are microparticles that are released on activation and apoptosis of cells and that originate from virtually any blood cell. Microparticles provide significant procoagulant activity by carrying TF or other enzymes in a number of disease states, including meningococcal sepsis. Microparticles appear to have several other biologic properties that modulate the cardiovascular system. In sepsis, microparticles regulate different inflammatory reactions in an organ-specific manner and may play a role in the distribution of proteins like APC.

Modulation of Inflammation by Coagulation in vivo

Communication between inflammation and coagulation is bidirectional, such that coagulation can also modulate inflammatory activity. Coagulation proteases and protease inhibitors interact not only with coagulation protein zymogens but also with specific cell receptors to induce signaling pathways.

A pivotal mechanism by which coagulation proteases modulate inflammation is by binding to protease-activated receptors (PARs). Four types (PAR 1–4) have been identified, all belonging to the family of transmembrane domain, G protein–coupled receptors. A typical feature of PARs is that they serve as their own ligand. Proteolytic cleavage by an activated coagulation factor leads to exposure of a neo-amino terminus, which activates the same receptor (and possibly adjacent receptors), initiating transmembrane signaling. PARs are localized in the vasculature on endothelial cells, mononuclear cells, platelets, fibroblasts, and smooth muscle cells. PARs 1, 3, and 4 are thrombin receptors and PAR 1 can also serve as receptor for the TF–factor VIIa complex and factor Xa. PAR 2 cannot bind thrombin but can be activated by the TF–factor VIIa complex or factor Xa. Binding of thrombin to its cellular receptor may induce the production of several cytokines and growth factors. Binding of TF–factor VIIa to PAR 2 also results in upregulation of inflammatory responses (production of reactive oxygen species and expression of major histocompatibility complex class II and cell adhesion molecules) in macrophages and was shown to affect neutrophil infiltration and proinflammatory cytokine (TNF-α, IL-1β) expression. The in vivo relevance of PARs has been confirmed in various experimental studies using PAR inhibitors or PAR-deficient mice. Studies have shown that the PC system is also critically important in inflammation and infection. On binding thrombin, TM catalyzes PC activation, and (recombinant) APC appears to be a modifier of inflammation, apoptosis, wound healing, and ischemia-reperfusion injury. The TM molecule has two properties that differentiate it from PC. First, the lectin-like domain of TM has several direct antiinflammatory properties in mice, including inhibition of leukocyte adhesion to endothelial cells, inhibition of complement pathways, neutralization of LPS, and sequestration and degradation of proinflammatory high-mobility group box 1 (HMGB1) protein. EPCR, by it sequence homology with major histocompatibility complex class I CD1, may also play a role in inflammation. Overexpression of EPCR enhances the level of APC in mice; such mice are also protected against LPS.

Although thrombin plays the preferred role in PAR 1 activation, data show that the cellular microenvironment containing lipid rafts is an important determinant in changing the substrate specificity of EPCR from thrombin to APC. Binding of APC to EPCR results in migration of EPCR out of the lipid rafts so that it may then interact with other G-coupled proteins. The zymogen PC also confers cytoprotective effects by binding to EPCR, inducing cell signaling. Importantly, although occupancy of EPCR (with PC/APC) is an important requirement for PAR 1 signaling, it is not sufficient to protect against sepsis because administration of active site–inactivated APC did not confer protection against mortality in mouse endotoxemia models. EPCR is a critical receptor in the protection against sepsis, as shown in several studies in septic baboons, in which mortality was provoked in animals challenged with sublethal LPS in the presence of an EPCR-blocking antibody. Of note, although a central role of endothelial cell EPCR may be postulated, both EPCR ⁺ dendritic cells and hematopoietic cellular precursors are probably more important for APC-mediated protection against mortality in sepsis.

ATIII directly mediates cellular responses through antiinflammatory effects that are partly antagonized by heparin (which may explain in part why patients with sepsis administered ATIII did worse when comedicated with heparin). In septic baboons, high doses of ATIII markedly attenuated proinflammatory activity.

Observations have pointed to an important role of extracellular DNA and DNA-binding proteins (such as histones and HMGB1 protein) in the pathogenesis of DIC. These cell-free DNA and DNA-binding components are released from nucleosomes of degraded cells and may form a surface on which assembly of activated coagulation factor complexes may be greatly facilitated. In addition, histones activate platelets and thereby stimulate thrombin generation. Activation and binding of neutrophils by DNA components results in the formation of neutrophil extracellular traps (NETs) that have been identified as important contributors to vascular thrombosis and inflammation. NETs may provide the availability of inflammatory cells expressing TF. Activation of coagulation is further enhanced by the proteolytic cleavage of physiologic anticoagulants by abundant neutrophilic elastase in NETs. NETs may also induce endothelial cell death and detrimental inflammatory activity, an effect likely mediated by NET-associated proteases or cationic proteins, including histones.

Coagulation and Inflammatory Disorders Associated With Various Pathogens