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Ann Thorac Surg 2003;75:S649-S655
© 2003 The Society of Thoracic Surgeons

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I: Pathophysiology of ischemic-reperfusion injury

Tissue factor and thrombin mediate myocardial ischemia-reperfusion injury

^a Division of Cardiothoracic Surgery, The University of Washington, Seattle, Washington, USA
^b Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California, La Jolla, California, USA

* Address reprint requests to Dr Pohlman, Division of Trauma and Critical Care, Department of Surgery, The University of Washington, Box 359796, 325 9th Avenue, Seattle, WA 98104, USA
e-mail: tpohlman{at}u.washington.edu

Presented at the 3^rd International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, June 2–6, 2002.

Abstract

Reperfusion of the ischemic heart is necessary to prevent irreversible injury of the myocardium, which leads to permanent organ dysfunction. However, reperfusion in itself leads to myocardial ischemia/reperfusion (I/R) injury, which is characterized by an acute inflammatory response mediated by activated inflammatory cells, chemokines, cytokines, and adhesion molecules. The molecular mechanisms of myocardial I/R injury are not completely known. Tissue factor (TF) and thrombin, two potent procoagulant and proinflammatory mediators, are recognized to play significant roles in myocardial I/R injury. To investigate the role of TF and thrombin in myocardial I/R injury, we used rabbit and murine in situ coronary artery ligation models. Increased TF mRNA, antigen, and activity were found in ischemic cardiomyocytes. Administration of an inhibitory antirabbit TF monoclonal antibody before or during the onset of ischemia resulted in a significant reduction in infarct size. Functional inhibition of thrombin with hirudin also reduced the infarct size. However, defibrinogenating rabbits with ancrod had no effect on infarct size, suggesting a requirement of thrombin generation but not fibrin deposition in myocardial I/R injury.

Reperfusion of the ischemic heart is essential to prevent irreversible injury that leads to myocardial necrosis. However, reperfusion of ischemic myocardium in and of itself can exacerbate tissue injury, with loss or dysfunction of potentially salvageable or functional tissue [1–4]. Further injury of ischemic tissue during reperfusion (ischemia-reperfusion [I/R] injury) is observed after percutaneous coronary angioplasty, pharmacologic thrombolysis, coronary artery bypass grafting, and termination of cardiac arrest at the completion of open heart procedures [5, 6]. Myocardial ischemia-reperfusion (I/R) injury in any circumstance may produce a spectrum of clinical manifestations, from myocardial stunning to permanent organ dysfunction and failure [1]. Myocardial stunning, first described by Heyndrickx and colleagues [7] in 1975, is defined as "prolonged [but reversible] postischemic dysfunction of viable tissue salvaged by reperfusion." Permanent organ dysfunction is characterized by tissue damage as a result of irreversible myocellular necrosis.

Reperfusion of the ischemic myocardium induces an inflammatory process caused by the local actions of chemokines and cytokines, and by the increased expression of adhesion molecules on endothelial cell surfaces and on cardiomyocytes [8–13]. This acute inflammatory response is characterized by the recruitment and activation of monocytes, platelets, and neutrophils that, in turn, accentuate the injury by producing inflammatory mediators [1, 4]. Neutrophils release cytodestructive enzymes such as myeloperoxidase and elastase and generate free radicals, contributing to tissue damage in I/R injury [14–17].

The mechanism of I/R injury remains largely unknown but seems to be multifactorial, involving mediators of both inflammation and coagulation elaborated by activated cells and humoral inflammatory cascades. Among these inflammatory and procoagulant mediators, tissue factor (TF) has been implicated in multiple pathophysiologic states involving ischemia and I/R injury. Tissue factor is a transmembrane, 47 kDa, high-affinity receptor and cofactor for plasma factors VII/VIIa [18]. Tissue factor initiates the extrinsic coagulation cascade, ultimately leading to thrombin generation and fibrin deposition (Fig 1) [19]. This procoagulant glycoprotein is expressed constitutively at numerous extravascular sites such as the vascular adventitia, where it serves to initiate coagulation when vascular integrity is breached (Fig 2) [20]. The endothelium, under normal conditions, prevents the exposure of TF in the subendothelial layers from contact with plasma coagulation factors. In the intravascular space, endothelial cells and monocytes express TF at low levels or not at all [21–23]. However, both cell types harbor the capability of upregulating TF expression in response to variety of stimuli including endotoxin (lipopolysaccharide), C5a, tumor necrosis factor–, interferon-, interleukin-1ß, and platelet-activating factor, leading to intravascular thrombus formation [10, 22]. Furthermore, we have demonstrated that thrombin also increases the expression of TF in monocytes and endothelial cells (Chong AJ, unpublished data). This process creates a possible positive feedback loop where thrombin-stimulated monocytes and endothelial cells produce TF, which in turn initiates the extrinsic coagulation cascade, thereby leading to generation of more thrombin and unregulated intravascular coagulation (Fig 1). Elevated TF antigen levels and activity are present in atherosclerotic lesions [24]. Disruption of the atherosclerotic plaque exposes TF to the intravascular serine protease coagulation proteins, initiating the process of coagulation, resulting in the generation of thrombus, occlusion of vessels, and ultimately producing cellular damage and tissue destruction.

View larger version (23K): [in this window] [in a new window]   Fig 1. Coagulation cascade. Tissue factor (TF) initiates the extrinsic coagulation cascade, leading to generation of thrombin and fibrin. Thrombin induces upregulation of TF expression in monocytes and endothelial cells, resulting in a positive feedback loop. X = coagulation factor X; Xa = activated coagulation factor X; Xase = coagulation factor X activation.

View larger version (65K): [in this window] [in a new window]   Fig 2. Expression of tissue factor (TF). Tissue factor is constitutively expressed at low-levels in monocytes and endothelial cells intravascularly, and in the vascular adventitial layer and cardiomyocytes extravascularly. The endothelium prevents the exposure of subendothelial TF from plasma coagulation factors.

Thrombin mediates its widespread actions on cells through the activation of G-protein–coupled protease-activated receptors (PARs) [36]. Four human PAR subtypes are known. PAR-1, PAR-3, and PAR-4 are activated by thrombin [37–41]. The PAR-2 subtype is not activated by thrombin, but it can be activated by trypsin, tryptase, and coagulation factors VIIa and Xa [36]. PAR-1, the prototype of PARs, has been studied extensively and is the receptor mediating thrombin signaling in endothelial cells. PAR-1 is activated when thrombin cleaves the PAR-1 amino-terminal extracellular domain [37, 38]. This proteolytic cleavage exposes a new amino sequence, which serves as a tethered ligand, binding intramolecularly to the body of the receptor, resulting in signaling. Because the thrombin-generated PAR ligand is tethered to PAR, signaling cannot be terminated by diffusion of the ligand away from the receptor [37]. Thrombin-generated PAR activation is terminated by internalization of the receptor into lysosomes within the cell. Thus cells activated by thrombin remain refractory to further thrombin signaling until new PAR molecules can be transcribed, translated, and processed on to the cell surface. Mutant PAR constructs that lack a cytoplasmic domain that directs internalization, signal indefinitely when activated with thrombin [36].

The procoagulant and proinflammatory effects of thrombin and TF have been shown to play a role in myocardial I/R injury [34, 42]. A recent study demonstrated that TF activity increases in hearts of rabbits subjected to myocardial I/R injury, and administration of an blocking antirabbit TF monoclonal antibody improved blood flow to the ischemic myocardium during reperfusion [42]. Although TF expression on endothelial cells (leading to intravascular thrombosis) may occur during myocardial ischemia and reperfusion injury, TF may contribute to myocardial I/R injury by potentially increasing extrasvascular thrombin generation and inflammation through thrombin activation of PAR in the myocardium. To examine this question, we used a rabbit model of myocardial I/R injury to identify the source of increased TF expression and to examine the mechanism by which TF and thrombin accentuate myocardial I/R injury [34].

Material and methods

The in situ coronary ligation model was described in detail previously [34]. In this rabbit model of regional myocardial I/R injury, adult New Zealand White rabbits weighing 3 to 4 kg were used. Rabbits were anesthetized with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). Rabbits were then endotracheally intubated and mechanically ventilated with 100% oxygen at a rate of 18 to 20 breaths/min with a tidal volume of 48 mL using an animal respirator. Anesthesia was maintained with inhaled 4% halothane for 2 minutes, which was decreased to 1% during the procedure. Lactated Ringer’s solution was administered intravenously at 5 mL · kg^-1 · h^-1 and the temperature of the rabbit was maintained with a warming pad. A 4.0-Vicryl (Ethicon, Somerville, NJ) suture was passed twice around a large anterolateral branch of the left main coronary artery, which perfused most of the left ventricle (LV). The ends of the suture were passed through a small length of polyethylene tubing to form a snare. After a 20- to 30-minute stabilization period, regional myocardial ischemia was produced by reversibly tightening the snare and occluding the artery for 45 minutes. The coronary was then subsequently released to allow 120 minutes of reperfusion. After 120 minutes of reperfusion, all animals were sacrificed with an intravenous bolus injection of pentobarbital. The myocardial tissue was isolated and processed for either calculation of infarct size or histologic analysis.

To assess the effect of functional inhibition of TF on myocardial I/R injury, 2 mg/kg of an inhibitory antirabbit TF monoclonal antibody was administered intravenously to rabbits either 15 minutes before (n = 6) or 30 minutes after (n = 5) the onset of ischemia. Control rabbits (n = 5) received normal saline. To determine the effects of inhibition of thrombin on myocardial I/R injury, rabbits (n = 5) were treated with recombinant hirudin. Hirudin is an irreversible inhibitor of thrombin activity through competitive inhibition of its catalytic site [43]. Hirudin treatment began with the intravenous administration of a 1-mg/kg bolus 30 minutes before ischemia, and a continuous infusion of 1 mg · kg^-1 · h^-1 for 1 hour and 0.5 mg · kg^-1 · h^-1 for 1 hour. This dosing protocol prolonged the activated partial thromboplastin time to greater than twice base line throughout the ischemia and reperfusion periods. Control rabbits (n = 4) received saline. To determine the contribution of fibrin deposition to myocardial I/R injury, rabbits (n = 5) were treated with ancrod, a defibrinogenating agent. Ancrod cleaves only the A-chains of fibrinogen producing soluble, uncrosslinked fibrin–fibrinogen degradation products that are cleared by the reticuloendothelial system [44]. Rabbits received ancrod intravenously beginning with a bolus dose of 1.0 IU/kg, followed by a second bolus dose (1.0 IU/kg) 1 hour later, and a third bolus dose (2.0 IU/kg) 3 hours later. The I/R protocol was initiated 6 hours after the first ancrod dose. This dosing schedule decreased circulating fibrinogen from 2.60 ± 0.09 mg/mL to undetectable levels ( < 0.20 mg/mL) after the first dose and throughout ischemia and reperfusion as determined by the von Clauss method [45]. No serious bleeding complications were noted during any of the treatments.

All quantitative data presented in this article are expressed as the mean ± SE. Statistically significant differences between groups were determined using the Mann-Whitney U test. Values of p less than 0.05 were considered to be statistically significant.

Results

Expression of TF in myocardial I/R injury
As shown in Figure 3, after myocardial I/R injury, TF antigen, TF mRNA, and TF activity (TF activity data not shown) in the LV at-risk (AR) areas were all significantly elevated compared with control animals. Therefore, these results confirmed the finding of Golino and colleagues [42] in that myocardial I/R significantly increased TF expression. Immunohistochemical analysis and in situ hybridization experiments of tissue sections of LV from I/R-injured rabbits demonstrated a regional increase in TF antigen and TF mRNA, identified as ischemic cardiomyocytes (Fig 4). These results indicated that TF antigen and mRNA expression were upregulated by ischemic cardiomyocytes, not endothelial cells.

View larger version (30K): [in this window] [in a new window]   Fig 3. Tissue factor mRNA (TF mRNA) expression after myocardial ischemia/reperfusion (I/R) injury. TF mRNA levels were determined by Northern blotting and normalized to levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH). (A) Determination of TF mRNA levels in two separate samples from non-AR areas (N) of LV of three independent sham-operated rabbits. (B) Comparison of TF mRNA levels in the non-AR areas (N) of LV with levels in the AR areas of LV of three independent I/R-injured rabbits. (Reprinted from [34], with permission.) AR = at-risk; LV = left ventricle.

View larger version (101K): [in this window] [in a new window]   Fig 4. Tissue factor (TF) antigen upregulation in ischemic cardiomyocytes. (A) Regional increase in TF staining of ischemic cardiomyocytes in fixed heart tissue from a rabbit after I/R injury. Tissue factor-positive areas stain dark red. (B) Serial section stained with acid-fuchsin to identify ischemic cardiomyocytes (reddish brown). (C) Tissue factor expression in the AR (at risk) area of LV (left ventricle) of I/R-injured rabbit compared with (D) normal (non-AR area) of same rabbit. (E) Minimal TF expression observed in vascular endothelium (arrowhead) and increased TF expression observed in cardiomyocytes (arrow). Tissue factor–positive cells stain brown. lu = lumen. (F) Serial section stained with control antibody. (G) The sheep antirabbit TF polyclonal antibody was detected with a fluorescein isothiocyanate–labeled antibody and stains green. The mouse anti-CD31 monoclonal antibody was detected with a Texas red–labeled antibody and stains red. (H) Analysis of TF and vWF expression. The anti-TF monoclonal antibody was stained green and the antirabbit vWF antibody was stained red. (Reprinted from [34], with permission.)

View larger version (95K): [in this window] [in a new window]   Fig 5. Ultrastructural analysis of myocardium from I/R-injured rabbits. (A) Normal capillary from the LV (left ventricle) myocardium of a control rabbit. (B) Injured capillary in an AR (at risk) area of myocardium of an I/R-injured rabbit (arrow indicates endothelial disruption). (Reprinted from [34], with permission.)

View larger version (44K): [in this window] [in a new window]   Fig 6. Effect of anti–tissue factor antibody (anti-TF Ab) treatment on infarct size. (A) Rabbits were treated with either saline (n = 5, hatched bars) or an antirabbit TF monoclonal antibody (n = 6, black bars) 15 minutes before ischemia. Similar AR areas of the left ventricle (LV) were observed in both groups (42 2% for antirabbit TF antibody group and 39% 5% for saline-treated rabbits). The infarct size was significantly reduced in the antibody treatment group (16% ± 1% for the antirabbit TF antibody group and 41% ± 1% for the saline-treated rabbits, p = 0.004). (B) Rabbits were treated with saline (n = 5) or antirabbit TF monoclonal antibody (n = 5) 30 minutes after onset of ischemia. Similar AR areas of LV were observed in both groups (47% ± 4% for antirabbit TF antibody group and 44% ± 1% for saline-treated rabbits). The infarct size was significantly reduced in the antibody treatment group (24% ± 4% for the antirabbit TF antibody rabbits and 43% ± 1% for saline-treated group, p = 0.014). Data are expressed as mean ± SE. (Reprinted from [34], with permission.)

Comment

The significant finding in this study is that administration of an inhibitory anti-TF antibody either before or after ischemia significantly reduces infarct size after I/R injury, indicating that TF contributes to myocardial I/R injury. Tissue factor activity may be increased after myocardial I/R injury by de novo protein synthesis as well as by deencryption of preexisting TF. There are a variety of cell types that may contribute to the pathologic expression of TF during myocardial I/R injury including cardiomyocytes, which constitutively express low levels of TF, and vascular cells, such as endothelial cells and circulating leukocytes, which can be induced to express TF [20, 22, 23, 46]. We found that TF expression was upregulated in cardiomyocytes but not in endothelial cells, in the AR areas of LV and anti-TF antibody administered in vivo bound to these cardiomyocytes. We observed structural and functional disruption of the endothelium, which is consistent with a previous report showing increased permeability of the coronary microvasculature after brief ischemia (15 minutes) and reperfusion [47]. Damage to the endothelial barrier would permit plasma-clotting factors to gain access to TF expressed by extravascular ischemic and nonischemic cardiomyocytes, suggesting that these cells may participate in local thrombin generation and fibrin deposition.

We further investigated the role of fibrin deposition in I/R injury by defibrinogenating the rabbits with ancrod, which reduced fibrinogen to undetectable levels. However, in the absence of fibrinogen and thrombus formation, infarct size remained unchanged compared to rabbits with normal fibrinogen levels and the ability to form clot. Consistent with these results, by immunohistochemistry and ultrastructural analysis we did not observe intravascular fibrin deposition or microvascular thrombosis in heart tissue of rabbits subjected to I/R injury, although we did observe a low level of extravascular fibrin deposition. Although these results do not exclude the possibility that a low level of intravascular fibrin deposition occurs in this model and impairs blood flow, our results suggest that TF contributed to myocardial I/R injury by mechanisms other than initiating intravascular fibrin deposition. Fibrin-independent mechanisms for the no-reflow phenomenon in I/R injury have been described, such as capillary plugging by leukocytes and erythrocytes [16, 48]. Other investigators found frequent leukocyte and erythrocyte capillary plugging and only occasional fibrin-containing microthrombi in the microvasculature by electron microscopy in a similar pig I/R model [49].

We found that inhibition of thrombin activity with hirudin treatment reduced the infarct size by 59%, which is similar to the effect observed using anti-TF antibody treatment. Thrombin stimulates endothelial cells to express chemoattractants, such as interleukin-8 and monocyte chemoattractant protein–1 (MCP-1), and adhesion molecules, such as intercellular adhesion molecule–1 (ICAM–1) and P-selectin [50–52]. These molecules are required for the recruitment and extravasation of polymorphonuclear (PMN) and monocytes, which constitute an inflammatory response. Previous studies have suggested that thrombin contributes to inflammation in septic shock and glomerulonephritis through PAR-1 signaling [53, 54]. Vascular smooth muscle cells and endothelial cells express PAR-1 and both cell types exhibit inducible expression of MCP-1 in response to thrombin [51]. Functional inhibition of thrombin decreased chemokine expression and inhibition of TF or thrombin reduced the infiltration of PMNs after myocardial I/R injury. PMN infiltration was assessed by counting the number of PMNs infiltrating into AR tissue. PMNs are an important component of myocardial cell death in I/R injury. Inhibition of PMN accumulation by blocking CD18 and ICAM-1 or by use of CD18- and ICAM-1–deficient mice has shown that PMNs contribute to infarct size in models of myocardial I/R injury [55–57]. We propose that extravascular TF, through the action of thrombin, has a proinflammatory role in myocardial I/R injury by increasing chemokine expression and enhancing the recruitment of leukocytes.

To further elucidate the roles of TF and thrombin in myocardial I/R injury, we have used a model of regional myocardial I/R injury in mice. In a murine model of myocardial I/R injury, we are able to examine the effect of exact genotypes on the pathogenesis of this injury. In preliminary studies we have examined the effect of a PAR-1 knockout in mice subjected to 30 minutes of regional ischemia of the left ventricle by temporary occlusion of the left anterior descending artery (LAD), followed by 2 hours of reperfusion after release of the LAD occlusion. Phenotypically, PAR-1 mice develop normally and form clot normally in response to injury. In contrast to humans, mice do not express PAR-1 on platelets and consequently platelets function normally in PAR-1 knockout mice. We have observed that in mice lacking PAR-1, infarct size was significantly reduced compared with littermate wild-type mice after both groups were subjected to myocardial I/R injury (p < 0.001; Hampton CR and Mackman N, unpublished data). These preliminary data indicate that thrombin generated during myocardial I/R injury interacts with PAR-1 on one or more cell types in the myocardium to sensitize the heart to ischemia or reperfusion after a brief period of ischemia. These results. together with those presented above, strongly suggest the significant role of TF-dependent generation of thrombin in myocardial I/R injury through a proinflammatory mechanism independent of coagulation and thrombus formation.

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