Perspectives in Pharmacology Role of Inflammatory Mediators in Thrombogenesis

The role of inflammation in cardiovascular disease and especially in thrombogenesis has become increasingly recognized as an important component of the overall disease process. Plaque rupture promotes activation of the inflammatory response and increased expression of tissue factor (TF), which in turn acts as one of the major initiators of extrinsic coagulation. It is becoming apparent that the expression of TF on endothelial cells, underlying smooth muscle cells and monocytes is regulated, in part, by proinflammatory cytokines including tumor necrosis factor and IL-1. In addition to initiating coagulation, interaction of TF with the adhesion molecule, P-selectin, has been demonstrated to accelerate the rate and extent of fibrin formation and deposition. P-selectin is expressed on activated platelets and endothelium and serves as the receptor for the endogenous ligand, P-selectin glycoprotein-1 (PSGL-1), expressed on various leukocytic cell types. In addition to mediating transient interactions between endothelial cells and leukocytes, P-selectin has been reported to mediate adherence of platelets to monocytes and neutrophils via specific interaction with PSGL-1. P-selectin is rapidly cleaved off the surface of the platelet membrane and appears in the circulation as a soluble form, which has been reported to be elevated in patients with acute coronary syndromes including unstable angina and nonQ-wave myocardial infarction. This review will focus on the role of cytokines in mediating TF expression and also explore the significance of the relationship between P-selectin and tissue factor in thrombus generation. In addition, possible pharmacological mechanisms to interrupt this disease process will be discussed. Role of Cytokines in Promotion of Thrombosis The expression of proinflammatory cytokines has been implicated in mediating the pathogenesis of a number of cardiovascular diseases including ischemia/reperfusion injury, heart failure, and atherosclerosis (Zhou et al., 1999). However, the role of these inflammatory mediators in unrestrained coagulation remains to be fully understood. Several lines of evidence derived from both preclinical and clinical studies demonstrate a link between inflammation and coagulation. Foremost is the increased coagulation in Gram-negative sepsis/endotoxemia and increased circulating levels of thrombin following infusion of IL-6 in cancer patients (Stouthard et al., 1996; Grignani and Maiolo, 2000). While the involvement of cytokines in mediating thrombosis has focused primarily on sepsis, it is becoming increasingly apparent that the expression of these mediators can shift the intravascular environment from hemodynamically stable to a procoagulative state, even in nonseptic conditions. Proinflammatory cytokines, IL-1 , IL-6, MCP-1, and TNF , have been shown to be up-regulated in the setting of thrombosis and may be involved in maintaining the balance between coagulation and fibrinolysis. However, during progression of the inflammatory response the balance between anticoagulant and prothrombotic activity is shifted toward the procoagulant state by the ability of these molecules to down-regulate antithrombotic proteins (i.e., thrombomodulin/protein C pathway) while up-regulating prothrombotic proteins (ten Cate et al., 1997). One of the primary consequences of increased proinflammatory cytokines in the vasculature is the increased expression of a number of proteins that serve to regulate coagulation. Foremost among these proteins is tissue factor (TF, coagulation factor III, CD142), which acts to regulate the activation state of the extrinsic pathway of coagulation. Tissue factor is a 46-kDa transmembrane glycoprotein that serves as one of the primary initiators of blood coagulation (Giesen and Nemerson, 2000). Cellanchored TF interacts with soluble factor VIIa (FVIIa) to ABBREVIATIONS: IL, interleukin; TNF, tumor necrosis factor; TF, tissue factor; PSGL-1, P-selectin glycoprotein-1; rPSGL-1, recombinant PSGL-1. 0022-3565/02/3003-729–735$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 300, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 900056/964814 JPET 300:729–735, 2002 Printed in U.S.A. 729 at A PE T Jornals on A ril 0, 2017 jpet.asjournals.org D ow nladed from induce factor Xa (FXa) activation, leading to cleavage of prothrombin to form proteolytically active thrombin. Thrombin in turn is responsible for conversion of plasma fibrinogen to fibrin, which envelopes and stabilizes developing thrombi/ blood clots. Thrombin also has other diverse biological actions such as inducing platelet aggregation and influencing cell growth and migration signaling pathways. With respect to procoagulant activity of TF, the majority of cell surface TF activity is normally “encrypted” in which state it is capable of binding FVIIa but does not express full activity as it does not bind FXa. To become fully activated, TF must be “de-encrypted” by some form of cellular perturbation, which may involve plasma membrane phosphatidylserine-dependent and independent mechanisms (Bach and Moldow, 1997). Agents such as Annexin V, which binds to and inhibits phosphatidylserine, may block the de-encryption process or selectively inhibit the de-encrypted form of TF. Identification of the primary cell types expressing TF is critical to understanding the role of the inflammatory response in mediating thrombosis. TF is constitutively expressed by a variety of cell types including fibroblasts, glomerular epithelial cells, and tumor cells (Hair et al., 1996; Mackman, 1997). TF is also constantly present in the adventitia of blood vessels and is thought to provide a protective barrier that serves an important role in maintaining hemostasis of the vascular system. TF also has recently been described to circulate as TF-rich microparticles (Giesen et al., 1999) that may interact with blood-borne FVIIa to initiate coagulation. Although expressed constitutively by a number of nonvasculature-associated cell types (i.e., fibroblasts), TF activity is rapidly up-regulated by monocytes and endothelial cells in response to various chemical and mechanical stimuli (Grabowski and Lam, 1995; Lorenzet et al., 1998). It is becoming evident that the cytokine-mediated expression of TF by endothelial cells and inflammatory cells acts as one of the primary initiators of thrombosis (Dosquet et al., 1995). The initiation of the inflammatory response and the subsequent stimulation of the vascular endothelium by TNF and/or IL-1 result in increased TF expression, thereby shifting the vascular environment to the prothrombotic state. Monocytes and smooth muscle cells in atherosclerotic plaques strongly express TF. Presumably, these cells are responding to inflammatory signals within the plaque. Rupture of plaques exposes active TF directly to blood in the lumen of the vessel and is thought to be the triggering event that causes myocardial infarction and ischemic stroke. Recent studies suggest the accumulation of TF in atherosclerotic plaques plays a major role in determining plaque thrombogenicity (Taubman et al., 1997). However, the classical view that active, constitutively expressed TF present on the atherosclerotic plaque itself was the primary initiator of thrombosis has been contested (Libby, 2000). It is becoming increasingly apparent that the inappropriate expression of TF by circulating monocytes plays an important role in pathological conditions characterized by hypercoagulation such as that noted in acute thrombotic episodes (Esmon, 2001). In cell culture, monocytes and endothelial cells can be induced by TNF, IL-1, MCP-1, or IL-6 to strongly express tissue factor on their cell surfaces (Grabowski and Lam, 1995; Ernofsson and Siegbahn, 1996). In addition to the other thrombotic events associated with the molecule, TF expression on the monocyte surface facilitates the interaction of the monocyte with activated platelets and endothelial cells via binding of P-selectin (Fig. 1). The end result of the ability of inflammatory mediators to increase the expression of TF on monocytes and endothelial cells is the acceleration of the rate and extent of fibrin formation and deposition in thrombus. It is apparent that circulating monocytes may represent one of the principle players in the cross-talk between the inflammatory and coagulative pathways (Napoleone et al., 1997). In addition to the evidence derived from in vitro studies, there is substantial in vivo data supporting the important role of cytokines in mediating thrombogenesis. In sepsis, TF is strongly expressed by circulating monocytes. In contrast, expression of TF on endothelium in vivo is very rare, even in fulminant sepsis. So, it is suspected that the coagulopathy seen in sepsis is driven largely by the de novo expression of tissue factor on circulating monocytes. In animal models of sepsis, specific antagonists of tissue factor or factor VII block the coagulopathy and lead to survival of animals that would have died without intervention (Taylor et al., 1991; Uchiba et al., 1997). These antagonists include inhibitory antibodies to tissue factor and factor VII, and active site-blocked factor VIIa (VIIai). Fig. 1.Role of proinflammatory cytokines and adhesion molecules in thrombus initiation and stabilization. Disruption of the endothelial layer and generation of proinflammatory cytokines promotes the expression of TF on circulating monocytes and vascular endothelium. Activated platelets express P-selectin, which is subsequently shed and circulates as a soluble form as does TF. Increased TF expression on these cell surfaces, and as a soluble microparticle form, facilitates the interactions between inflammatory cells, platelets, and disrupted endothelial cells via interactions of Pselectin and PSGL-1, resulting in fibrin deposition and thrombus formation and stabilization at sites of vascular injury or plaque rupture.