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Thrombin, Inflammation, and Cardiovascular Disease
Thrombin, Inflammation, and Cardiovascular Disease* An Epidemiologic Perspective Russell P. Tracy, PhD
The exploration of coagulation led to identifying inflammation as a major factor in arterial disease throughout life. “Integrative molecular physiology” reflects our emerging understanding of how coagulation and inflammation integrate with one another, in both normal physiology and in pathophysiology. Our own responses to environmental challenge provide much of the damage that cumulatively results in chronic cardiovascular disease. Only by intervening in exquisitely precise ways can we hope to effectively and safely modify the course of lifelong chronic diseases, such as atherosclerosis. (CHEST 2003; 124:49S–57S) Key words: cardiovascular disease risk; coagulation; epidemiology; fibrinolysis; inflammation; thrombosis Abbreviations: APC ⫽ activated protein C; CRP ⫽ C-reactive protein; CVD ⫽ cardiovascular disease; FDP ⫽ fibrin degradation product; IL ⫽ interleukin; MI ⫽ myocardial infarction; PAI-1 ⫽ plasminogen activator inhibitor-1; PAR ⫽ protease-activated receptor; TNF ⫽ tissue necrosis factor; TNFR ⫽ tissue necrosis factor-␣ receptor
and resulting clinical cardiovascular disA therosclerosis ease (CVD) are complex pathophysiologic processes
involving a large number of genes and gene products in complicated interactions with a variety of environmental influences. An enormous body of research over the last 20 years has helped us understand that many of the biochemical processes involved in CVD are the same as those involved in ongoing defense against a hostile world, including blood coagulation, inflammation, and immune response. From this same body of research, we are beginning to understand that these processes are intricately linked and, in fact, are interdependent.1 While in its infancy, this understanding (which can be termed integrative molecular physiology) has already yielded major health advances, such as the use of activated protein C (APC), an anticoagulant, in the clinical setting of sepsis, an hyperinflammatory infectious condition.2 Table 1 lists some of the ways in which population studies, clinical
*From the Laboratory for Clinical Biochemistry Research, College of Medicine, University of Vermont, Colchester, VT. Funding was provided in part by grants from the National Institutes of Health (HL46696, HL58329) and from AstraZeneca LP. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Russell P. Tracy, PhD, Director, Laboratory for Clinical Biochemistry Research, University of Vermont, 208 South Park Dr, Suite 2, Colchester, VT 05446; e-mail: [email protected] www.chestjournal.org
trials, and more basic investigations have supported the interplay between coagulation, fibrinolysis, and inflammation. Within this framework, the generation of thrombin is a key event, with many ramifications. This article will attempt to review the possible roles for thrombin in CVD; discuss whether a preexisting hypercoagulable state predisposes a patient to CVD; highlight some of the known and hypothesized relationships of coagulation and fibrinolysis, and coagulation and inflammation; and review the known associations of coagulation and inflammation factors with CVD.
Connections of Thrombin to Clinical CVD CVD is the major cause of death for Americans,14 and incidence rates around the world are reaching epidemic proportions.15 A major form of CVD is myocardial infarction (MI); in the early 1970s, it became evident, through the work of DeWood and colleagues16,17 and others, that the proximal event in MI is commonly an occlusive blood clot in a coronary artery. The term vulnerable plaque has been used to describe a form of atherosclerosis characterized by rapid, focal lipid accumulation, with the development of a large pool of subendothelial fat covered by a thin, mechanically fragile cap.18 –20 There is often little intrusion into the lumen of the vessel, with considerable remodeling into the vessel wall to accommodate the lipid build-up. As the cap ruptures, the cells and soluble factors of the coagulant system are exposed to this large pool of presumably procoagulant lipid, along with many subendothelial components deep into the arterial wall, which results in platelet activation and aggregation, thrombin generation, and the development of a large often occlusive thrombus. If not cleared quickly, significant myocardial damage ensues. Arterial clots are often so-called “white clots” (ie, platelet-rich), suggesting less of a role for fibrin formation; however, the direct role of thrombin as the ultimate clotting enzyme in this setting is obvious. As others in this supplement will illustrate in detail, thrombin is not only responsible for the cleavage of fibrinogen resulting in fibrin formation, but is also the most powerful platelet agonist, and is believed to play a critical role in the growth of platelet aggregates. CVD encompasses more than MI. Other coronary syndromes, such as sudden coronary death, may not have a thrombotic component to the same degree or extent. For example, autopsy studies21 have revealed that sudden coronary death is associated with coronary thrombosis in ⬎ 70% of the cases in younger adults (when the precipitating cause is most frequently incident MI), but that this prevalence decreases to less than a third in older adults. The precipitating cause is less apparent in these cases, and one may speculate that transient platelet aggregation at the site of lesion fracture or erosion, which may be tolerated if occurring in younger hearts, may prove fatal in older, weaker hearts. The atherosclerotic burden associated with events later in life is much greater than that associated with events in younger people, where the actual plaque burden may be limited.22,23 CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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Table 1—Findings Supporting the Interplay Between Coagulation, Fibrinolysis, and Inflammation Area of Science Population studies
Clinical trials
Biochemistry and cell biology
Findings CRP levels correlate strongly with d-dimer, plasmin-antiplasmin complex levels3 CRP levels correlate weakly, but significantly, with many coagulation and fibrinolysis factors, such as factor VIII, factor X, PAI-13,4 APC is used successfully in sepsis5 Aspirin prevents thrombosis-dependent incident MI in a CRP-dependent manner6 Statins prevent incident and secondary MI in a CRP-dependent manner7 IL-6 stimulates monocyte tissue factor expression8 CRP stimulates monocyte tissue factor expression9 FDPs stimulates monocyte IL-6 expression10 Thrombin stimulates monocyte IL-6 expression11 Thrombin inhibits IL-6–induced Stat3 signaling12 Statins cause decrease in tissue factor expression13
This raises the question of whether thrombin might participate in atherosclerotic heart disease in ways that do not directly involve thrombus formation. The answer is that thrombin generation has numerous possible nonthrombotic associations with atherosclerosis and heart disease, and more pathways are certain to be identified.24 Many of these pathways are discussed in more detail in other sections of this article. Some center on the role of thrombin as a signaling molecule, through thrombin receptors (protease-activated receptors [PARs]). As discussed by Dr. Brass elsewhere in this supplement and by Patterson et al25 in a review article, these signaling events concern virtually all aspects of vascular biology, including vessel tone, cellular differential, migration and proliferation (especially smooth-muscle cells), angiogenesis and vascular development, and vascular pathology such as atherosclerosis. At least three PARs are expressed by human cells, with attendant G protein-coupled signaling cascades and physiologic effects. Thrombin plays a central role in the activation of all three PARs. Moreover, these signaling events can coordinate with other receptor-based intracellular signaling to modify the resulting effect. In total, then, the possible effects of thrombin in vascular wall biology are large in number and key to both normal and pathologic vascular physiology. Thrombin is also responsible for the activation of a newly discovered major thrombosis regulator, thrombinactivated fibrinolysis inhibitor,26 also discussed in more detail by Dr. Nesheim elsewhere in this supplement. Thrombin-activated fibrinolysis inhibitor appears to play a major role in down-regulating fibrinolysis, which may have important implications in heart disease, as has been proposed.27 Interestingly, along with being the key final enzyme in fibrin formation, and the most powerful known platelet activator, thrombin is also a major anticoagulant in its role as the thrombomodulin-associated activator of protein C28 (see the accompanying article by Dr. Esmon in this supplement). APC is a powerful anticoagulant that has recently been established as a critical new therapeutic weapon in the setting of sepsis.2 It is unlikely that in this role thrombin has a major effect on atherosclerosis and clinical heart disease, however, since low protein C levels are not CVD risk factors.29 50S
This theory is supported by the fact that genotypes associated with low protein C levels (or with cofactors that are refractive to APC activity, eg, Factor V Leiden) have not been linked to CVD risk.30 We hypothesize that this lack of association may be due to two factors: the heavy dependence of arterial thrombosis on atherosclerotic plaque rupture and/or erosion, and the high flow rates of blood around areas of stenosis, making it likely that available thrombomodulin may reside downstream of the forming thrombus. However, the anticoagulant role of thrombin may be important in arterial disease once an occlusive thrombus has formed with its attendant stasis, just as it is important in venous thrombosis, which is also predominantly stasis dependent. This hypothesis remains to be tested in a clinical setting.
Is There a Hypercoagulable State That Predisposes to CVD? Meade and colleagues31 at the Northwick Park Heart Study produced pioneering research into the molecular epidemiology of coagulation and fibrinolysis, which suggested that a preexisting hypercoagulable state might be present in those most at risk for MI and CVD death. While the research that followed their work has provided little support for this as a general condition—for example, it does not appear that elevations in factor VIIc levels are a major risk factor,32–34 as they originally proposed—their results for fibrinogen and factor VIIIc have held up in many other studies. We now interpret these results for fibrinogen and factor VIII as probably best supportive of inflammation rather than hypercoagulability.32–34 The plasma level of the fibrin degradation product d-dimer is an integrated marker of coagulant and subsequent fibrinolytic activity. As the population under study becomes older and has greater atherosclerotic burden, d-dimer does predict events. In studies of older middleaged men with mixed health status and in a study of men with peripheral vascular disease, d-dimer levels were predictive of CVD events.35,36 In addition, in the elderly cohort of the Cardiovascular Health Study, where most individuals had moderate-to-extensive vascular disease, d-dimer and the marker of plasmin activation, plasminantiplasmin complex, were strongly predictive of events.37 Thrombin: Physiology and Pathophysiology
In contrast, studies of d-dimer as a CVD risk factor in younger, healthier populations have been negative. In preliminary findings, d-dimer levels did not predict early calcification in younger people.38 Also, levels were not associated with incident events in a healthy middle-aged population, such as the Physician’s Health Study.39 Overall, a meta-analysis35 demonstrated moderate risk prediction for d-dimer. The most likely interpretation of these data are that the degree of atherosclerosis and vascular damage causes changes in coagulation status, not vice versa; ie, it does not appear that a preexisting hypercoagulable state is in the causal pathway of atherosclerotic disease and CVD events. This position is supported by the findings such as those of Lowe et al,40 in which future ischemic events were predicted by d-dimer levels but not by other markers of procoagulant activity, such as prothrombin fragment F1 ⫹ 2 and thrombin-antithrombin complex (measures of thrombin generation), or by factor VII coagulant activity (factor VIIc, which at least partly reflects factor VII activation). Genetic studies also provide some information regarding the possible role of hypercoagulability in precipitating CVD events. For example, there are unambiguous and compelling associations between genetic protein C deficiency (lack of an anticoagulant), factor V Leiden genotype (a procoagulant resistant to inactivation), and prothrombin 20210A genotype (increased procoagulant zymogen levels), and the prevalence and incidence of venous thrombotic disease.41,42 The general statement is that the presence of genetic factors that result in either reduced anticoagulation or increased procoagulation are directly in the causal pathway for venous thrombosis. However, key to our interpretation of hypercoagulability, in general none of these factors are risk factors for arterial disease.30 There are rare exceptions, such as atypical arterial thrombosis occurring in otherwise healthy younger women.43 Overall, the preponderance of data indicates a lack of a compelling argument supporting the importance of a preexisting hypercoagulable state as a major risk factor for atherothrombotic disease.
There Is a Complex Interplay Among Atherosclerosis, Thrombosis, and Inflammation Atherosclerotic coronary heart disease commonly manifests itself clinically via a thrombotic event: so-called “atherothrombosis,” especially in younger men. As mentioned above, this has been clearly understood only since the early 1970s.16 As an overview, the process of blood clotting is comprised of coagulation, limited and controlled by anticoagulation; and the counterbalancing process of fibrinolysis, limited and controlled by antifibrinolysis. It has been known for a long time that increases or decreases in specific factor levels are associated with risk of venous clotting or bleeding. For example, the lack of factor VIII or factor IX leads to hemophilia A or B, and deficiency of the anticoagulant factor protein C leads to a propensity to form clots, such as occurs in deep vein thrombosis. Because of this knowledge, and the knowlwww.chestjournal.org
edge that blood clots are important in the risk of MI, researchers in the early 1980s pursued the importance of specific factor levels in atherothrombotic risk. Meade and colleagues44 pioneered this work in the Northwick Park Heart Study, and demonstrated the clear importance of fibrinogen as a CVD risk factor. This research was quickly confirmed and extended by others,45– 47 and it became clear that fibrinogen, factor VIII, and several other proteins found in abundance in plasma were risk factors, at least in part, because they reflected chronic, low-level inflammation. Said another way, the specific factors that were elevated in the presence of clinical CVD, and whose levels in otherwise healthy people predicted the occurrence of future CVD, were in general known as acutephase reactants, ie, proteins known to respond to inflammatory stimulation via the effects of proinflammatory cytokines, such as interleukin (IL)-6. To support this position, studies of the plasma levels of prothrombin (a key procoagulant protein, but not an inflammation-sensitive protein) and/or the prothrombin G20210A genotype associated with plasma levels, generally have been null for CVD risk in most cases48 while consistently positive for venous thrombosis.49 Also, several studies have examined the anticoagulant proteins and their relationship to CVD. As an ancillary study29 to the Thrombolysis in Myocardial Infarction Phase II trial of thrombolytic therapy, we demonstrated that in those entering the health system with MIs, anticoagulant proteins, such as protein C and antithrombin, were elevated, not decreased. We have also demonstrated that in otherwise healthy adults tissue factor pathway inhibitor levels were higher, not lower, in those with increased measures of subclinical disease based on ankle-brachial BP index and carotid ultrasonography.50 Also, Folsom and colleagues34 showed in the Atherosclerosis Risk in Communities study that protein C was weakly, but positively, associated with CVD, even though protein C is not a strong acute phase reactant. This is counter to expectations based on the hypercoagulable hypothesis (ie, lower levels, not higher levels, of anticoagulants would be found in those either with, or at risk for, CVD), but rather supports the inflammatory hypothesis: inflammation, rather than the process of clotting, is more related to CVD risk. Having suggested that the principal mechanism for association of some coagulation factors with CVD is through their nature as inflammation-related factors, it remains possible that these factors may also reflect risk because, once they are elevated, higher levels increase the likelihood of blood clot formation. Using fibrinogen as an example, possible mechanisms by which higher levels of fibrinogen might be related to increased likelihood of blood clotting include increased platelet crosslinking, increased fibrin clot formation, and increased blood viscosity, among others.51 While it remains unproven whether any of these mechanisms are actually at play in situ, given all the available data it seems likely that higher fibrinogen may well not only reflect the low-grade inflammation caused by the atherothrombosis, but also participate in that process, allowing it to proceed at a faster rate. This “positive feedback” is illustrated in Figure 1. Along with their work in clotting, Meade and colCHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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Figure 1. The complex interplay of atherothrombotic disease with inflammation and clotting. Inflammation and the proinflammatory cytokines, eg, IL-6, are increased by a variety of mechanisms in atherothrombosis. In addition, there is activation of the coagulation system, with subsequent activation of fibrinolysis, leading to a complex interplay. An example concerns the feedback control of fibrinogen levels. The use of fibrinogen in clotting, including the increased clotting of atherosclerotic progression, results in the production of FDPs. FDPs are bound by monocytes, which in turn increase production of IL-6. This IL-6 acts via the endocrine system to increase the hepatic synthesis of fibrinogen.10 As discussed in the text, increased plasma fibrinogen may feedback in a positive manner, increasing the likelihood of atheroprogression. In addition, other causes of inflammation feed into this system, such as chronic infections and diabetes. Also, the elaboration of proinflammatory cytokines is affected by an individual’s capacity for inflammatory response, such as the extent of visceral adiposity.52 A wide variety of genetic influences are easily conceived, and as we have recently observed, increased IL-6 itself may accelerate the atherothrombotic process,53 possibly acting as a potent cell growth regulator, an activator of monocytes and/or other cells, an amplifier of the innate immune response exacerbating uptake of lipid particles by macrophage, or by other potential mechanisms. The plasma proteins affected by IL-6, such as CRP, fibrinogen, and others, may also directly participate in the atherothrombotic process to make it worse. Adapted with permission.54 ICAM ⫽ intracellular adhesion molecule; HDL ⫽ high-density lipoprotein.
leagues31 were also the first to propose that deficiencies in fibrinolysis might also be associated with CVD and that levels of these factors might predict future CVD events. In this same vein, others have demonstrated that levels of the major plasma antifibrinolysis protein, plasminogen activator inhibitor-1 (PAI-1), are elevated in those with existing clinical CVD and did offer some risk prediction for future secondary CVD events.55,56 However, this finding, like those in the coagulation system, was not always confirmed in future studies.57 It has been suggested that the degree of predictive power may be a function of the adjustment done for other variables,58 since PAI-1 levels are associated with inflammation,59 plasma lipid levels, and, most strongly, with adiposity and insulin levels.4 These factors are all CVD risk factors in their own right, and it remains 52S
uncertain whether the concept of fibrinolytic capacity is valid and in fact a predisposing risk factor for arterial disease. Sobel60 invoked a possible role for PAI-1 in the arterial wall, where it may play more of a role than in blood. Plaques particularly prone to rupture and that precipitate relatively large thrombus formation are characterized by minimal cellularity, among other factors. Migration of cells into the plaque region is likely to require collagenase activity, which is in turn provided by plasminmediated activation of collagenase zymogens. PAI-1 in the wall may inhibit plasmin formation and contribute to a lack of cellularity and resultant instability.61 PAI-1 levels are associated with insulin, suggesting that the role of PAI-1 may be particularly important in people with the metabolic syndrome or type 2 diabetes. The Thrombin: Physiology and Pathophysiology
regulation of PAI-1 levels—at least in blood—is in part mediated by regulators of glycemic control and inflammation: insulin and proinsulin can stimulate endothelial cells and hepatocytes to produce PAI-1,62,63 and PAI-1 is believed to be a weak acute-phase reactant.59 In addition, adipocytes can directly synthesize and secrete PAI-1, helping to explain the known association of PAI-1 levels with body mass index.64 All of these factors associated with PAI-1 levels are also correlated with each other; this high degree of covariance makes it difficult to establish independent associations. To help make these connections, we performed a factor analysis as part of the data analysis of the Cardiovascular Health Study. Factor analysis is a statistical approach that computes a set of hypothetical uncorrelated “factors” from a set of covariate variables. This method has been used to analyze the components of the metabolic syndrome; in several studies,65– 67 it has consistently yielded four factors: BP, body mass, insulin/glucose, and lipids. We entered these variables, as well as variables related to inflammation and hemostasis; this analysis yielded the established four factors, plus three new factors, which we termed inflammation, vitamin K-dependent coagulation factors, and procoagulant activity.4 PAI-1 was associated with the insulin/glucose factor and the body mass factor, and not with the others, supporting the notion that PAI-1 reflects both insulin level and adiposity, but only weakly, if at all, inflammation or ongoing coagulant activity.
Inflammation Markers and CVD Risk Research done over the last 10 years has played an important role in identifying inflammation as a key process in atherosclerosis and CVD. We should note that “inflammation” as the term is used here does not mean the full form of the condition (warmth, redness, swelling, and pain), but rather implies a “micro-inflammation.” A person with this condition is characterized by being in the upper part of the “normal” distribution for inflammation status, without the signs and symptoms of overt, clinical inflammation. Molecular epidemiology has played a key role in identifying the role of inflammation in CVD, and recently
it has become clear that inflammation is connected to the metabolic syndrome in a complex and important manner. Acute-phase proteins are a class of secreted proteins, primarily from the liver, that either rise or fall in concentration in response to inflammatory stimuli, such as tissue damage and infection. This change in most cases represents no more than a doubling or tripling in concentration (eg, fibrinogen) or a 30 to 50% decrease (eg, albumin). A few of the known proteins, such as C-reactive protein (CRP), may increase in concentration ⬎ 1,000-fold.68 Virtually all of the proteins that have been studied have been shown to be associated with CVD (Table 2). Most of the known acute-phase proteins are produced in the liver in response to IL-6. Although these markers have many different functions, they all are moderately to strongly associated with the presence of CVD (in all cases clinical CVD; in some cases subclinical CVD as defined by such techniques as carotid artery ultrasonography); in almost every case, an argument can be made—at least hypothetically—that the protein in question is not only a marker of the process but might also participate in the process. In population studies or clinical research, inflammation is usually estimated by the measurement of a plasma acute-phase protein, such as CRP or fibrinogen. However, a wide variety of activities, such as innate immunity, coagulation, and others, fall under the term inflammation. Each of these activities plays an important role in our response to trauma and/or environmental challenge: coagulation and fibrinolysis in restricting blood loss and in wound repair69; complement activation and T-cell differentiation as part of innate and adaptive immunity70,71; endothelial cell, neutrophil, and monocyte activation in immunity and wound repair72; antioxidation in response to oxidative challenge73; and others. In addition, variances in the genes responsible for the overall regulation of this system may play important roles in disease susceptibility; eg, IL-6 and tumor necrosis factor (TNF)-␣ genes.74,75 Finally, other environmental factors (eg, diet, smoking, etc)3,76 and the volume of inflammation-mediating tissue (eg, visceral fat)52 also appear to be important. It is clear, therefore, that the relationship of inflammation to athero-
Table 2—Acute-Phase Reactants Associated With CVD Acute-Phase Reactants Positive Fibrinogen Factor VIII CRP Ceruloplasmin PAI-1 Serum amyloid A Ferritin ␣1-acid glycoprotein Negative Albumin High-density lipoprotein
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Proposed Mechanisms Increased coagulant activity; decreased fibrinolysis due to increased clot density; increased platelet aggregation; increased plasma viscosity Increased coagulant activity Increased tissue factor expression on monocytes; increased cell adhesion molecule-mediated cell attachment to endothelial cells; increased complement activation Increased oxidative damage due to higher plasma copper levels Decreased fibrinolytic potential; decreased plaque cellularity None proposed to date Increased oxidative damage due to higher plasma iron levels None proposed to date Decreased antioxidation due to decreased plasma bilirubin level Increased low-density lipoprotein levels due to decreased removal of tissue cholesterol
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sclerosis is complex and much additional research will be needed before we can truly understand these processes and their relationships to health and disease. A number of inflammatory markers have been shown to predict future CVD events, all with a certain degree of similarity. For example, ceruloplasmin—the major copper-transporting protein in plasma— had similar risk prediction to CRP in the Monitoring Trends and Determinants in Cardiovascular Disease-Augsburg cohort.77 In the Cardiovascular Health Study, fibrinogen, factor VIII, and CRP each independently predicted future CVD events32 with similar relative risks. Also, in the Iowa 65⫹ Rural Health Study, both CRP and IL-6 predicted future fatal CVD events, again with similar risk prediction.78 These similarities, however, do not necessarily mean that one marker can be directly substituted for another. In some cases, there are significant differences in the strengths of association. Also, each marker may participate in the CVD process in a different way. In a side-by-side comparison,79 CRP had the strongest risk prediction, but a meta-analysis80 observed no real difference between CRP and fibrinogen in risk prediction.
Inflammation and the Metabolic Syndrome: an Important Relationship It has been observed that markers of inflammation are associated with the components of the metabolic syndrome4; this finding is in addition to the known association of inflammation markers, as well as markers of coagulant activity, with diabetes status.81,82 For example, CRP values increase with the number of components of the metabolic syndrome present,83 and fibrinogen levels are correlated with albumin excretion, even in those patients without demonstrable impaired glucose tolerance (N. Jenny, PhD; unpublished data). Despite these strong relationships, as mentioned before, factor analysis indicates that inflammation represents a distinct underlying pathophysiologic pathway.4 To help better understand the metabolic pathways associated with inflammation, we and others3,4,76,83,84 have studied the correlates of CRP. The major correlates are adiposity and insulin sensitivity status. CRP is also associated with coagulation activity status, as estimated by markers such as d-dimer (a fibrin degradation product [FDP] that reflects first the formation of fibrin and then fibrinolysis) and plasmin-antiplasmin complex (a marker reflecting plasmin formation rate, itself depending on the stimulation of fibrin formation, as well as the levels of tissue plasminogen activator and PAI-1).3,4,85 Increases in coagulation status in the metabolic syndrome and diabetes have been well documented.86,87 The exact mechanism for the association of CRP with coagulation status is not clear; however the work of Ritchie and colleagues10 suggests a possible pathway. Monocytes appear capable of binding FDPs and subsequently producing IL-6, which goes to the liver and affects the wide range of proteins known to be in the acute-phase response. This has been proposed to be the mechanism by which fibrinogen consumption is replaced, and may be an important mechanism connecting coagulation and inflammation. This theory may have far54S
reaching implications both in the general biology we have been discussing and in such areas as drug development, where the coagulation-inflammation connection in sepsis, as described by Esmon et al,69 has been recently exploited.5 Adipose tissue is a key producer of inflammatory cytokines (so-called “adipokines”), among which IL-6 is a major pathophysiologic mediator of diabetes and the metabolic syndrome, as well as acute-phase proteins, such as CRP.88 In this way, the metabolic syndrome may be a contributor to inflammatory response through the visceral obesity component: increased visceral fat may lead to increased proinflammatory response to a variety of stimulants, resulting in a chronic up-regulation of IL-6 production. Chronic up-regulation of IL-6 may predispose a patient to atherosclerosis in a number of ways. We have demonstrated in murine atherosclerosis that chronic injections of small amounts of IL-6 (yielding a minor chronic acute-phase response) resulted in a twofold to fivefold increase in lesion size.53 In humans, IL-6 activities range from acute-phase response to tissue factor expression by monocytes, and many others.8 The association of CRP with insulin level is at least partially independent of adiposity, and is strongly related to insulin sensitivity.4 This statistical association may be mediated by the key proinflammatory cytokine (also an adipokine) TNF-␣, which is a so-called first-wave cytokine influencing IL-6 production,89 as well as many other cellular functions. TNF-␣ receptor (TNFR) I signals programmed cell death, while TNFR II may signal survival or proliferation through cytoplasmic TNFRassociated proteins, which can both negatively regulate apoptosis and positively promote survival.90 Although the role (if any) of circulating TNF-␣ in humans remains uncertain, TNF-␣ induces insulin resistance in tissue culture and in animal models.91,92 Plasma levels are also elevated in the metabolic syndrome, and are associated with insulin resistance in humans.93
Conclusions We now know that thrombin generation is critical in atherosclerotic heart disease in at least two ways: as the ultimate clotting and platelet-activating enzyme, and as an important cell-signaling effector molecule. While thrombosis remains a major contributor to the morbidity and mortality of atherosclerotic disease, the other roles of thrombin in vascular biology may prove to be the more critical in the overall scheme. We also now know that coagulation, fibrinolysis, and inflammation are critically interconnected, and markers related to all of these activities are associated cross-sectionally with both subclinical and clinical heart disease, and epidemiologically are predictors of future clinical events. These new insights highlight the need to engage in integrative molecular physiology, with the goal of understanding in detail not just the individual pathways, but the ways they intersect and interact. In particular, we will need to understand how to interrupt “pathologic” activities (eg, thrombosis or foam cell proliferation) without interrupting homologous “physiologic” activities (eg, hemostasis or wound repair), which often involve the same mediators and effectors. It is likely Thrombin: Physiology and Pathophysiology
that in the future important therapeutic and preventive advances will have to be made with such integrated knowledge in mind.
References 1 Tracy R. Atherosclerosis, thrombosis and inflammation: a question of linkage. Fibrinolysis Proteolysis 1997; 11(suppl 1):137–142 2 Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344:699 –709 3 Tracy RP, Psaty BM, Macy E, et al. Lifetime smoking exposure affects the association of C-reactive protein with cardiovascular disease risk factors and subclinical disease in healthy elderly subjects. Arterioscler Thromb Vasc Biol 1997; 17:2167–2176 4 Sakkinen PA, Wahl P, Cushman M, et al. Clustering of procoagulation, inflammation, and fibrinolysis variables with metabolic factors in insulin resistance syndrome. Am J Epidemiol 2000; 152:897–907 5 Ely EW, Bernard GR, Vincent JL. Activated protein C for severe sepsis. N Engl J Med 2002; 347:1035–1036 6 Ridker PM, Cushman M, Stampfer MJ, et al. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med 1997; 336:973–979 7 Ridker PM, Rifai N, Pfeffer MA, et al. Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events (CARE) Investigators. Circulation 1998; 98:839 – 844 8 Neumann FJ, Ott I, Marx N, et al. Effect of human recombinant interleukin-6 and interleukin-8 on monocyte procoagulant activity. Arterioscler Thromb Vasc Biol 1997; 17: 3399 –3405 9 Cermak J, Key NS, Bach RR, et al. C-reactive protein induces human peripheral blood monocytes to synthesize tissue factor. Blood 1993; 82:513–520 10 Ritchie DG, Levy BA, Adams MA, et al. Regulation of fibrinogen synthesis by plasmin-derived fragments of fibrinogen and fibrin: an indirect feedback pathway. Proc Natl Acad Sci U S A 1982; 79:1530 –1534 11 Tokunou T, Ichiki T, Takeda K, et al. Thrombin induces interleukin-6 expression through the cAMP response element in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2001; 21:1759 –1763 12 Bhat GJ, Raghu G, Gunaje JJ, et al. Alpha-thrombin inhibits interleukin-6-induced Stat3 signaling and gp130 gene expression in primary cultures of human lung fibroblasts. Biochem Biophys Res Commun 1999; 256:626 – 630 13 Eto M, Kozai T, Cosentino F, et al. Statin prevents tissue factor expression in human endothelial cells: role of Rho/Rhokinase and Akt pathways. Circulation 2002; 105:1756 –1759 14 American Heart Association. Heart disease and stroke statistics–2003 update. Dallas, TX: American Heart Association, 2002 15 Sans S, Kesteloot H, Kromhout D. The burden of cardiovascular diseases mortality in Europe: Task Force of the European Society of Cardiology on Cardiovascular Mortality and Morbidity Statistics in Europe. Eur Heart J 1997; 18:1231– 1248 16 DeWood MA, Spores J, Notske R, et al. Prevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med 1980; 303:897–902 17 DeWood M, Spores J, Notske R, et al. Prevalence of total coronary occlusion during the early hours of transmural www.chestjournal.org
myocardial infarction. N Engl J Med 1980; 303:897–902 18 Kullo IJ, Edwards WD, Schwartz RS. Vulnerable plaque: pathobiology and clinical implications. Ann Intern Med 1998; 129:1050 –1060 19 Fuster V, Badimon JJ, Chesebro JH. Atherothrombosis: mechanisms and clinical therapeutic approaches. Vasc Med 1998; 3:231–239 20 Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation 2001; 104:365–372 21 Burke AP, Farb A, Pestaner J, et al. Traditional risk factors and the incidence of sudden coronary death with and without coronary thrombosis in blacks. Circulation 2002; 105:419 – 424 22 Fuster V, Badimon L, Badimon JJ, et al. The pathogenesis of coronary artery disease and the acute coronary syndromes (1). N Engl J Med 1992; 326:242–250 23 Fuster V, Badimon L, Badimon JJ, et al. The pathogenesis of coronary artery disease and the acute coronary syndromes (2). N Engl J Med 1992; 326:310 –318 24 Harker LA, Hanson SR, Runge MS. Thrombin hypothesis of thrombus generation and vascular lesion formation. Am J Cardiol 1995; 75:12B–17B 25 Patterson C, Stouffer GA, Madamanchi N, et al. New tricks for old dogs: nonthrombotic effects of thrombin in vessel wall biology. Circ Res 2001; 88:987–997 26 Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 1995; 270:14477–14484 27 Juhan-Vague I, Morange PE, Aubert H, et al. Plasma thrombin-activatable fibrinolysis inhibitor antigen concentration and genotype in relation to myocardial infarction in the north and south of Europe. Arterioscler Thromb Vasc Biol 2002; 22:867– 873 28 Esmon CT. The protein C anticoagulant pathway. Arterioscler Thromb Vasc Biol 1992; 12:135–145 29 Callas PW, Tracy RP, Bovill EG, et al. The association of anticoagulant protein concentrations with acute myocardial infarction in the Thrombolysis in Myocardial Infarction Phase II (TIMI II) Trial. J Thromb Thrombolysis 1998; 5:53– 60 30 Juul K, Tybjaerg-Hansen A, Steffensen R, et al. Factor V Leiden: the Copenhagen City Heart Study and 2 metaanalyses. Blood 2002; 100:3–10 31 Meade TW, Mellows S, Brozovic M, et al. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet 1986; 2:533–537 32 Tracy RP, Arnold AM, Ettinger W, et al. The relationship of fibrinogen and factors VII and VIII to incident cardiovascular disease and death in the elderly: results from the cardiovascular health study. Arterioscler Thromb Vasc Biol 1999; 19:1776 –1783 33 Tracy R, Psaty B, Bovill E, et al. Fibrinogen and factor VIII, but not factor VII, are positively associated with prevalent cardiovascular disease in older adults: analyses from the Cardiovascular Heart Study [abstract P31]. Paper presented at the 32nd Annual Conference on Cardiovascular Disease Epidemiology, March 19 –21, 1992; Memphis, TN 34 Folsom AR, Wu KK, Rosamond WD, et al. Prospective study of hemostatic factors and incidence of coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) Study. Circulation 1997; 96:1102–1108 35 Danesh J, Whincup P, Walker M, et al. Fibrin D-dimer and coronary heart disease: prospective study and meta-analysis. Circulation 2001; 103:2323–2327 36 Folsom AR, Aleksic N, Park E, et al. Prospective study of fibrinolytic factors and incident coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) Study. Arterioscler Thromb Vasc Biol 2001; 21:611– 617 CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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37 Cushman M, Lemaitre RN, Kuller LH, et al. Fibrinolytic activation markers predict myocardial infarction in the elderly: The Cardiovascular Health Study. Arterioscler Thromb Vasc Biol 1999; 19:493– 498 38 Jacobs J, Tracy R, Gross M, et al. Reactive protein and vitamin C are associated with coronary calcification in young adults: The Cardia Study [abstract]. Circulation 2000; 101:720 39 Ridker PM, Hennekens CH, Cerskus A, et al. Plasma concentration of cross-linked fibrin degradation product (Ddimer) and the risk of future myocardial infarction among apparently healthy men. Circulation 1994; 90:2236 –2240 40 Lowe GD, Rumley A, Sweetnam PM, et al. Fibrin D-dimer, markers of coagulation activation and the risk of major ischaemic heart disease in the caerphilly study. Thromb Haemost 2001; 86:822– 827 41 Bertina RM, Rosendaal FR. Venous thrombosis: the interaction of genes and environment. N Engl J Med 1998; 338: 1840 –1841 42 Folsom AR, Cushman M, Tsai MY, et al. A prospective study of venous thromboembolism in relation to factor V Leiden and related factors. Blood 2002; 99:2720 –2725 43 Rosendaal FR, Siscovick DS, Schwartz SM, et al. Factor V Leiden (resistance to activated protein C) increases the risk of myocardial infarction in young women. Blood 1997; 89:2817– 2821 44 Meade TW, North WR, Chakrabarti R, et al. Haemostatic function and cardiovascular death: early results of a prospective study. Lancet 1980; 1:1050 –1054 45 Wilhelmsen L, Svardsudd K, Korsan-Bengtsen K, et al. Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med 1984; 311:501–505 46 Stone MC, Thorp JM. Plasma fibrinogen: a major coronary risk factor. J R Coll Gen Pract 1985; 35:565–569 47 Kannel WB, Wolf PA, Castelli WP, et al. Fibrinogen and risk of cardiovascular disease: The Framingham Study. JAMA 1987; 258:1183–1186 48 Smiles AM, Jenny NS, Tang Z, et al. No association of plasma prothrombin concentration or the G20210A mutation with incident cardiovascular disease: results from the Cardiovascular Health Study. Thromb Haemost 2002; 87:614 – 621 49 Bertina RM. The prothrombin 20210 G to A variation and thrombosis. Curr Opin Hematol 1998; 5:339 –342 50 Sakkinen PA, Cushman M, Psaty BM, et al. Correlates of antithrombin, protein C, protein S, and TFPI in a healthy elderly cohort. Thromb Haemost 1998; 80:134 –139 51 Tracy R, Bovill E. Hemostasis and risk of ischemic disease: epidemiologic evidence with emphasis on the elderly. In: Califf R, Mark D, Wagner G, eds. Acute coronary care in the thrombolytic era. 2nd ed. St. Louis, MO: Mosby Year Book; 1995; 27– 43 52 Tracy RP. Is visceral adiposity the “enemy within”? Arterioscler Thromb Vasc Biol 2001; 21:881– 883 53 Huber SA, Sakkinen P, Conze D, et al. Interleukin-6 exacerbates early atherosclerosis in mice. Arterioscler Thromb Vasc Biol 1999; 19:2364 –2367 54 Tracy RP. Epidemiological evidence for inflammation in cardiovascular disease. Thromb Haemost 1999; 82:826 – 831 55 Hamsten A, Wiman B, de Faire U, et al. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med 1985; 313:1557–1563 56 Hamsten A, de Faire U, Walldius G, et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet 1987; 2:3–9 57 Iacoviello L, Burzotta F, Di Castelnuovo A, et al. The 4G/5G polymorphism of PAI-1 promoter gene and the risk of 56S
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myocardial infarction: a meta-analysis. Thromb Haemost 1998; 80:1029 –1030 Juhan-Vague I, Alessi M, Morange P. PAI-1, obesity, and insulin resistance. In: Reaven G, Laws A, eds. Insulin resistance: the metabolic syndrome X. Totowa, NJ: Humana Press; 1999; 317–332 Kluft C, Verheijen JH, Jie AF, et al. The postoperative fibrinolytic shutdown: a rapidly reverting acute phase pattern for the fast-acting inhibitor of tissue-type plasminogen activator after trauma. Scand J Clin Lab Invest 1985; 45:605– 610 Sobel BE. Coronary artery disease and fibrinolysis: from the blood to the vessel wall. Thromb Haemost 1999; 82(suppl 1):8 –13 Sobel BE. Increased plasminogen activator inhibitor-1 and vasculopathy: a reconcilable paradox. Circulation 1999; 99: 2496 –2498 Alessi MC, Juhan-Vague I, Kooistra T, et al. Insulin stimulates the synthesis of plasminogen activator inhibitor 1 by the human hepatocellular cell line Hep G2. Thromb Haemost 1988; 60:491– 494 Schneider DJ, Absher PM, Ricci MA. Dependence of augmentation of arterial endothelial cell expression of plasminogen activator inhibitor type 1 by insulin on soluble factors released from vascular smooth muscle cells. Circulation 1997; 96:2868 –2876 Loskutoff DJ, Samad F. The adipocyte and hemostatic balance in obesity: studies of PAI-1. Arterioscler Thromb Vasc Biol 1998; 18:1– 6 Hanley AJ, Karter AJ, Festa A, et al. Factor analysis of metabolic syndrome using directly measured insulin sensitivity: The Insulin Resistance Atherosclerosis Study. Diabetes 2002; 51:2642–2647 Meigs JB. Invited commentary: insulin resistance syndrome? Syndrome X? Multiple metabolic syndrome? A syndrome at all? Factor analysis reveals patterns in the fabric of correlated metabolic risk factors. Am J Epidemiol 2000; 152:908 –911 Edwards KL, Austin MA, Newman B, et al. Multivariate analysis of the insulin resistance syndrome in women. Arterioscler Thromb Vasc Biol 1994; 14:1940 –1945 Kushner I. C-reactive protein and the acute-phase response. Hosp Pract (Off Ed) 1990; 25:3,16,21–28 Esmon CT, Taylor FB Jr, Snow TR. Inflammation and coagulation: linked processes potentially regulated through a common pathway mediated by protein C. Thromb Haemost 1991; 66:160 –165 Lagrand WK, Visser CA, Hermens WT, et al. C-reactive protein as a cardiovascular risk factor: more than an epiphenomenon? Circulation 1999; 100:96 –102 Hansson GK. Immune mechanisms in atherosclerosis. Arterioscler Thromb Vasc Biol 2001; 21:1876 –1890 Ross R. Inflammation, growth regulatory molecules and atherosclerosis. J Cell Biochem 1992; 48(suppl 16A):1–30 Berliner JA, Navab M, Fogelman AM, et al. Atherosclerosis: basic mechanisms; oxidation, inflammation, and genetics. Circulation 1995; 91:2488 –2496 Fishman D, Faulds G, Jeffery R, et al. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998; 102:1369 –1376 Jenny NS, Tracy RP, Ogg MS, et al. In the elderly, interleukin-6 plasma levels and the ⫺ 174G⬎C polymorphism are associated with the development of cardiovascular disease. Arterioscler Thromb Vasc Biol 2002; 22:2066 –2071 Mendall MA, Patel P, Ballam L, et al. C reactive protein and its relation to cardiovascular risk factors: a population based Thrombin: Physiology and Pathophysiology
cross sectional study. BMJ 1996; 312:1061–1065 77 Koenig W, Sund M, Frohlich M, et al. C-reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men: results from the MONICA (Monitoring Trends and Determinants in Cardiovascular Disease) Augsburg Cohort Study, 1984 to 1992. Circulation 1999; 99:237–242 78 Harris TB, Ferrucci L, Tracy RP, et al. Associations of elevated interleukin-6 and C-reactive protein levels with mortality in the elderly. Am J Med 1999; 106:506 –512 79 Ridker PM, Stampfer MJ, Rifai N. Novel risk factors for systemic atherosclerosis: a comparison of C-reactive protein, fibrinogen, homocysteine, lipoprotein(a), and standard cholesterol screening as predictors of peripheral arterial disease. JAMA 2001; 285:2481–2485 80 Danesh J, Collins R, Appleby P, et al. Association of fibrinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analyses of prospective studies. JAMA 1998; 279:1477–1482 81 Schmidt MI, Duncan BB, Sharrett AR, et al. Markers of inflammation and prediction of diabetes mellitus in adults (Atherosclerosis Risk in Communities study): a cohort study. Lancet 1999; 353:1649 –1652 82 Juhan-Vague I, Alessi MC, Vague P. Thrombogenic and fibrinolytic factors and cardiovascular risk in non-insulindependent diabetes mellitus. Ann Med 1996; 28:371–380 83 Festa A, D’Agostino R Jr, Howard G, et al. Chronic subclinical inflammation as part of the insulin resistance syndrome: the Insulin Resistance Atherosclerosis Study (IRAS). Circulation 2000; 102:42– 47 84 Yudkin JS, Stehouwer CD, Emeis JJ, et al. C-reactive protein in healthy subjects: associations with obesity, insulin resis-
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tance, and endothelial dysfunction: a potential role for cytokines originating from adipose tissue? Arterioscler Thromb Vasc Biol 1999; 19:972–978 Sakkinen PA, Cushman M, Psaty BM, et al. Relationship of plasmin generation to cardiovascular disease risk factors in elderly men and women. Arterioscler Thromb Vasc Biol 1999; 19:499 –504 Fuller JH, Keen H, Jarrett RJ, et al. Haemostatic variables associated with diabetes and its complications. BMJ 1979; 2:964 –966 Yudkin JS. Abnormalities of coagulation and fibrinolysis in insulin resistance: evidence for a common antecedent? Diabetes Care 1999; 22(suppl 3):C25–C30 Yudkin JS, Kumari M, Humphries SE, et al. Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link? Atherosclerosis 2000; 148:209 –214 Aggarwal B, Puri R. Human cytokines: their role in disease and therapy. Cambridge, MA: Blackwell Science, 1995 Arch RH, Gedrich RW, Thompson CB. Tumor necrosis factor receptor-associated factors (TRAFs): a family of adapter proteins that regulates life and death. Genes Dev 1998; 12:2821–2830 Qi C, Pekala PH. Tumor necrosis factor-␣-induced insulin resistance in adipocytes. Proc Soc Exp Biol Med 2000; 223:128 –135 Hotamisligil GS. The role of TNF-␣ and TNF receptors in obesity and insulin resistance. J Intern Med 1999; 245:621– 625 Nilsson J, Jovinge S, Niemann A, et al. Relation between plasma tumor necrosis factor-␣ and insulin sensitivity in elderly men with non-insulin-dependent diabetes mellitus. Arterioscler Thromb Vasc Biol 1998; 18:1199 –1202
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The exploration of coagulation led to identifying inflammation as a major factor in arterial disease throughout life. “Integrative molecular physiology” reflects our emerging understanding of how coagulation and inflammation integrate with one another, in both normal physiology and in pathophysiology. Our own responses to environmental challenge provide much of the damage that cumulatively results in chronic cardiovascular disease. Only by intervening in exquisitely precise ways can we hope to effectively and safely modify the course of lifelong chronic diseases, such as atherosclerosis. (CHEST 2003; 124:49S–57S) Key words: cardiovascular disease risk; coagulation; epidemiology; fibrinolysis; inflammation; thrombosis Abbreviations: APC ⫽ activated protein C; CRP ⫽ C-reactive protein; CVD ⫽ cardiovascular disease; FDP ⫽ fibrin degradation product; IL ⫽ interleukin; MI ⫽ myocardial infarction; PAI-1 ⫽ plasminogen activator inhibitor-1; PAR ⫽ protease-activated receptor; TNF ⫽ tissue necrosis factor; TNFR ⫽ tissue necrosis factor-␣ receptor
and resulting clinical cardiovascular disA therosclerosis ease (CVD) are complex pathophysiologic processes
involving a large number of genes and gene products in complicated interactions with a variety of environmental influences. An enormous body of research over the last 20 years has helped us understand that many of the biochemical processes involved in CVD are the same as those involved in ongoing defense against a hostile world, including blood coagulation, inflammation, and immune response. From this same body of research, we are beginning to understand that these processes are intricately linked and, in fact, are interdependent.1 While in its infancy, this understanding (which can be termed integrative molecular physiology) has already yielded major health advances, such as the use of activated protein C (APC), an anticoagulant, in the clinical setting of sepsis, an hyperinflammatory infectious condition.2 Table 1 lists some of the ways in which population studies, clinical
*From the Laboratory for Clinical Biochemistry Research, College of Medicine, University of Vermont, Colchester, VT. Funding was provided in part by grants from the National Institutes of Health (HL46696, HL58329) and from AstraZeneca LP. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Russell P. Tracy, PhD, Director, Laboratory for Clinical Biochemistry Research, University of Vermont, 208 South Park Dr, Suite 2, Colchester, VT 05446; e-mail: [email protected] www.chestjournal.org
trials, and more basic investigations have supported the interplay between coagulation, fibrinolysis, and inflammation. Within this framework, the generation of thrombin is a key event, with many ramifications. This article will attempt to review the possible roles for thrombin in CVD; discuss whether a preexisting hypercoagulable state predisposes a patient to CVD; highlight some of the known and hypothesized relationships of coagulation and fibrinolysis, and coagulation and inflammation; and review the known associations of coagulation and inflammation factors with CVD.
Connections of Thrombin to Clinical CVD CVD is the major cause of death for Americans,14 and incidence rates around the world are reaching epidemic proportions.15 A major form of CVD is myocardial infarction (MI); in the early 1970s, it became evident, through the work of DeWood and colleagues16,17 and others, that the proximal event in MI is commonly an occlusive blood clot in a coronary artery. The term vulnerable plaque has been used to describe a form of atherosclerosis characterized by rapid, focal lipid accumulation, with the development of a large pool of subendothelial fat covered by a thin, mechanically fragile cap.18 –20 There is often little intrusion into the lumen of the vessel, with considerable remodeling into the vessel wall to accommodate the lipid build-up. As the cap ruptures, the cells and soluble factors of the coagulant system are exposed to this large pool of presumably procoagulant lipid, along with many subendothelial components deep into the arterial wall, which results in platelet activation and aggregation, thrombin generation, and the development of a large often occlusive thrombus. If not cleared quickly, significant myocardial damage ensues. Arterial clots are often so-called “white clots” (ie, platelet-rich), suggesting less of a role for fibrin formation; however, the direct role of thrombin as the ultimate clotting enzyme in this setting is obvious. As others in this supplement will illustrate in detail, thrombin is not only responsible for the cleavage of fibrinogen resulting in fibrin formation, but is also the most powerful platelet agonist, and is believed to play a critical role in the growth of platelet aggregates. CVD encompasses more than MI. Other coronary syndromes, such as sudden coronary death, may not have a thrombotic component to the same degree or extent. For example, autopsy studies21 have revealed that sudden coronary death is associated with coronary thrombosis in ⬎ 70% of the cases in younger adults (when the precipitating cause is most frequently incident MI), but that this prevalence decreases to less than a third in older adults. The precipitating cause is less apparent in these cases, and one may speculate that transient platelet aggregation at the site of lesion fracture or erosion, which may be tolerated if occurring in younger hearts, may prove fatal in older, weaker hearts. The atherosclerotic burden associated with events later in life is much greater than that associated with events in younger people, where the actual plaque burden may be limited.22,23 CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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Table 1—Findings Supporting the Interplay Between Coagulation, Fibrinolysis, and Inflammation Area of Science Population studies
Clinical trials
Biochemistry and cell biology
Findings CRP levels correlate strongly with d-dimer, plasmin-antiplasmin complex levels3 CRP levels correlate weakly, but significantly, with many coagulation and fibrinolysis factors, such as factor VIII, factor X, PAI-13,4 APC is used successfully in sepsis5 Aspirin prevents thrombosis-dependent incident MI in a CRP-dependent manner6 Statins prevent incident and secondary MI in a CRP-dependent manner7 IL-6 stimulates monocyte tissue factor expression8 CRP stimulates monocyte tissue factor expression9 FDPs stimulates monocyte IL-6 expression10 Thrombin stimulates monocyte IL-6 expression11 Thrombin inhibits IL-6–induced Stat3 signaling12 Statins cause decrease in tissue factor expression13
This raises the question of whether thrombin might participate in atherosclerotic heart disease in ways that do not directly involve thrombus formation. The answer is that thrombin generation has numerous possible nonthrombotic associations with atherosclerosis and heart disease, and more pathways are certain to be identified.24 Many of these pathways are discussed in more detail in other sections of this article. Some center on the role of thrombin as a signaling molecule, through thrombin receptors (protease-activated receptors [PARs]). As discussed by Dr. Brass elsewhere in this supplement and by Patterson et al25 in a review article, these signaling events concern virtually all aspects of vascular biology, including vessel tone, cellular differential, migration and proliferation (especially smooth-muscle cells), angiogenesis and vascular development, and vascular pathology such as atherosclerosis. At least three PARs are expressed by human cells, with attendant G protein-coupled signaling cascades and physiologic effects. Thrombin plays a central role in the activation of all three PARs. Moreover, these signaling events can coordinate with other receptor-based intracellular signaling to modify the resulting effect. In total, then, the possible effects of thrombin in vascular wall biology are large in number and key to both normal and pathologic vascular physiology. Thrombin is also responsible for the activation of a newly discovered major thrombosis regulator, thrombinactivated fibrinolysis inhibitor,26 also discussed in more detail by Dr. Nesheim elsewhere in this supplement. Thrombin-activated fibrinolysis inhibitor appears to play a major role in down-regulating fibrinolysis, which may have important implications in heart disease, as has been proposed.27 Interestingly, along with being the key final enzyme in fibrin formation, and the most powerful known platelet activator, thrombin is also a major anticoagulant in its role as the thrombomodulin-associated activator of protein C28 (see the accompanying article by Dr. Esmon in this supplement). APC is a powerful anticoagulant that has recently been established as a critical new therapeutic weapon in the setting of sepsis.2 It is unlikely that in this role thrombin has a major effect on atherosclerosis and clinical heart disease, however, since low protein C levels are not CVD risk factors.29 50S
This theory is supported by the fact that genotypes associated with low protein C levels (or with cofactors that are refractive to APC activity, eg, Factor V Leiden) have not been linked to CVD risk.30 We hypothesize that this lack of association may be due to two factors: the heavy dependence of arterial thrombosis on atherosclerotic plaque rupture and/or erosion, and the high flow rates of blood around areas of stenosis, making it likely that available thrombomodulin may reside downstream of the forming thrombus. However, the anticoagulant role of thrombin may be important in arterial disease once an occlusive thrombus has formed with its attendant stasis, just as it is important in venous thrombosis, which is also predominantly stasis dependent. This hypothesis remains to be tested in a clinical setting.
Is There a Hypercoagulable State That Predisposes to CVD? Meade and colleagues31 at the Northwick Park Heart Study produced pioneering research into the molecular epidemiology of coagulation and fibrinolysis, which suggested that a preexisting hypercoagulable state might be present in those most at risk for MI and CVD death. While the research that followed their work has provided little support for this as a general condition—for example, it does not appear that elevations in factor VIIc levels are a major risk factor,32–34 as they originally proposed—their results for fibrinogen and factor VIIIc have held up in many other studies. We now interpret these results for fibrinogen and factor VIII as probably best supportive of inflammation rather than hypercoagulability.32–34 The plasma level of the fibrin degradation product d-dimer is an integrated marker of coagulant and subsequent fibrinolytic activity. As the population under study becomes older and has greater atherosclerotic burden, d-dimer does predict events. In studies of older middleaged men with mixed health status and in a study of men with peripheral vascular disease, d-dimer levels were predictive of CVD events.35,36 In addition, in the elderly cohort of the Cardiovascular Health Study, where most individuals had moderate-to-extensive vascular disease, d-dimer and the marker of plasmin activation, plasminantiplasmin complex, were strongly predictive of events.37 Thrombin: Physiology and Pathophysiology
In contrast, studies of d-dimer as a CVD risk factor in younger, healthier populations have been negative. In preliminary findings, d-dimer levels did not predict early calcification in younger people.38 Also, levels were not associated with incident events in a healthy middle-aged population, such as the Physician’s Health Study.39 Overall, a meta-analysis35 demonstrated moderate risk prediction for d-dimer. The most likely interpretation of these data are that the degree of atherosclerosis and vascular damage causes changes in coagulation status, not vice versa; ie, it does not appear that a preexisting hypercoagulable state is in the causal pathway of atherosclerotic disease and CVD events. This position is supported by the findings such as those of Lowe et al,40 in which future ischemic events were predicted by d-dimer levels but not by other markers of procoagulant activity, such as prothrombin fragment F1 ⫹ 2 and thrombin-antithrombin complex (measures of thrombin generation), or by factor VII coagulant activity (factor VIIc, which at least partly reflects factor VII activation). Genetic studies also provide some information regarding the possible role of hypercoagulability in precipitating CVD events. For example, there are unambiguous and compelling associations between genetic protein C deficiency (lack of an anticoagulant), factor V Leiden genotype (a procoagulant resistant to inactivation), and prothrombin 20210A genotype (increased procoagulant zymogen levels), and the prevalence and incidence of venous thrombotic disease.41,42 The general statement is that the presence of genetic factors that result in either reduced anticoagulation or increased procoagulation are directly in the causal pathway for venous thrombosis. However, key to our interpretation of hypercoagulability, in general none of these factors are risk factors for arterial disease.30 There are rare exceptions, such as atypical arterial thrombosis occurring in otherwise healthy younger women.43 Overall, the preponderance of data indicates a lack of a compelling argument supporting the importance of a preexisting hypercoagulable state as a major risk factor for atherothrombotic disease.
There Is a Complex Interplay Among Atherosclerosis, Thrombosis, and Inflammation Atherosclerotic coronary heart disease commonly manifests itself clinically via a thrombotic event: so-called “atherothrombosis,” especially in younger men. As mentioned above, this has been clearly understood only since the early 1970s.16 As an overview, the process of blood clotting is comprised of coagulation, limited and controlled by anticoagulation; and the counterbalancing process of fibrinolysis, limited and controlled by antifibrinolysis. It has been known for a long time that increases or decreases in specific factor levels are associated with risk of venous clotting or bleeding. For example, the lack of factor VIII or factor IX leads to hemophilia A or B, and deficiency of the anticoagulant factor protein C leads to a propensity to form clots, such as occurs in deep vein thrombosis. Because of this knowledge, and the knowlwww.chestjournal.org
edge that blood clots are important in the risk of MI, researchers in the early 1980s pursued the importance of specific factor levels in atherothrombotic risk. Meade and colleagues44 pioneered this work in the Northwick Park Heart Study, and demonstrated the clear importance of fibrinogen as a CVD risk factor. This research was quickly confirmed and extended by others,45– 47 and it became clear that fibrinogen, factor VIII, and several other proteins found in abundance in plasma were risk factors, at least in part, because they reflected chronic, low-level inflammation. Said another way, the specific factors that were elevated in the presence of clinical CVD, and whose levels in otherwise healthy people predicted the occurrence of future CVD, were in general known as acutephase reactants, ie, proteins known to respond to inflammatory stimulation via the effects of proinflammatory cytokines, such as interleukin (IL)-6. To support this position, studies of the plasma levels of prothrombin (a key procoagulant protein, but not an inflammation-sensitive protein) and/or the prothrombin G20210A genotype associated with plasma levels, generally have been null for CVD risk in most cases48 while consistently positive for venous thrombosis.49 Also, several studies have examined the anticoagulant proteins and their relationship to CVD. As an ancillary study29 to the Thrombolysis in Myocardial Infarction Phase II trial of thrombolytic therapy, we demonstrated that in those entering the health system with MIs, anticoagulant proteins, such as protein C and antithrombin, were elevated, not decreased. We have also demonstrated that in otherwise healthy adults tissue factor pathway inhibitor levels were higher, not lower, in those with increased measures of subclinical disease based on ankle-brachial BP index and carotid ultrasonography.50 Also, Folsom and colleagues34 showed in the Atherosclerosis Risk in Communities study that protein C was weakly, but positively, associated with CVD, even though protein C is not a strong acute phase reactant. This is counter to expectations based on the hypercoagulable hypothesis (ie, lower levels, not higher levels, of anticoagulants would be found in those either with, or at risk for, CVD), but rather supports the inflammatory hypothesis: inflammation, rather than the process of clotting, is more related to CVD risk. Having suggested that the principal mechanism for association of some coagulation factors with CVD is through their nature as inflammation-related factors, it remains possible that these factors may also reflect risk because, once they are elevated, higher levels increase the likelihood of blood clot formation. Using fibrinogen as an example, possible mechanisms by which higher levels of fibrinogen might be related to increased likelihood of blood clotting include increased platelet crosslinking, increased fibrin clot formation, and increased blood viscosity, among others.51 While it remains unproven whether any of these mechanisms are actually at play in situ, given all the available data it seems likely that higher fibrinogen may well not only reflect the low-grade inflammation caused by the atherothrombosis, but also participate in that process, allowing it to proceed at a faster rate. This “positive feedback” is illustrated in Figure 1. Along with their work in clotting, Meade and colCHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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Figure 1. The complex interplay of atherothrombotic disease with inflammation and clotting. Inflammation and the proinflammatory cytokines, eg, IL-6, are increased by a variety of mechanisms in atherothrombosis. In addition, there is activation of the coagulation system, with subsequent activation of fibrinolysis, leading to a complex interplay. An example concerns the feedback control of fibrinogen levels. The use of fibrinogen in clotting, including the increased clotting of atherosclerotic progression, results in the production of FDPs. FDPs are bound by monocytes, which in turn increase production of IL-6. This IL-6 acts via the endocrine system to increase the hepatic synthesis of fibrinogen.10 As discussed in the text, increased plasma fibrinogen may feedback in a positive manner, increasing the likelihood of atheroprogression. In addition, other causes of inflammation feed into this system, such as chronic infections and diabetes. Also, the elaboration of proinflammatory cytokines is affected by an individual’s capacity for inflammatory response, such as the extent of visceral adiposity.52 A wide variety of genetic influences are easily conceived, and as we have recently observed, increased IL-6 itself may accelerate the atherothrombotic process,53 possibly acting as a potent cell growth regulator, an activator of monocytes and/or other cells, an amplifier of the innate immune response exacerbating uptake of lipid particles by macrophage, or by other potential mechanisms. The plasma proteins affected by IL-6, such as CRP, fibrinogen, and others, may also directly participate in the atherothrombotic process to make it worse. Adapted with permission.54 ICAM ⫽ intracellular adhesion molecule; HDL ⫽ high-density lipoprotein.
leagues31 were also the first to propose that deficiencies in fibrinolysis might also be associated with CVD and that levels of these factors might predict future CVD events. In this same vein, others have demonstrated that levels of the major plasma antifibrinolysis protein, plasminogen activator inhibitor-1 (PAI-1), are elevated in those with existing clinical CVD and did offer some risk prediction for future secondary CVD events.55,56 However, this finding, like those in the coagulation system, was not always confirmed in future studies.57 It has been suggested that the degree of predictive power may be a function of the adjustment done for other variables,58 since PAI-1 levels are associated with inflammation,59 plasma lipid levels, and, most strongly, with adiposity and insulin levels.4 These factors are all CVD risk factors in their own right, and it remains 52S
uncertain whether the concept of fibrinolytic capacity is valid and in fact a predisposing risk factor for arterial disease. Sobel60 invoked a possible role for PAI-1 in the arterial wall, where it may play more of a role than in blood. Plaques particularly prone to rupture and that precipitate relatively large thrombus formation are characterized by minimal cellularity, among other factors. Migration of cells into the plaque region is likely to require collagenase activity, which is in turn provided by plasminmediated activation of collagenase zymogens. PAI-1 in the wall may inhibit plasmin formation and contribute to a lack of cellularity and resultant instability.61 PAI-1 levels are associated with insulin, suggesting that the role of PAI-1 may be particularly important in people with the metabolic syndrome or type 2 diabetes. The Thrombin: Physiology and Pathophysiology
regulation of PAI-1 levels—at least in blood—is in part mediated by regulators of glycemic control and inflammation: insulin and proinsulin can stimulate endothelial cells and hepatocytes to produce PAI-1,62,63 and PAI-1 is believed to be a weak acute-phase reactant.59 In addition, adipocytes can directly synthesize and secrete PAI-1, helping to explain the known association of PAI-1 levels with body mass index.64 All of these factors associated with PAI-1 levels are also correlated with each other; this high degree of covariance makes it difficult to establish independent associations. To help make these connections, we performed a factor analysis as part of the data analysis of the Cardiovascular Health Study. Factor analysis is a statistical approach that computes a set of hypothetical uncorrelated “factors” from a set of covariate variables. This method has been used to analyze the components of the metabolic syndrome; in several studies,65– 67 it has consistently yielded four factors: BP, body mass, insulin/glucose, and lipids. We entered these variables, as well as variables related to inflammation and hemostasis; this analysis yielded the established four factors, plus three new factors, which we termed inflammation, vitamin K-dependent coagulation factors, and procoagulant activity.4 PAI-1 was associated with the insulin/glucose factor and the body mass factor, and not with the others, supporting the notion that PAI-1 reflects both insulin level and adiposity, but only weakly, if at all, inflammation or ongoing coagulant activity.
Inflammation Markers and CVD Risk Research done over the last 10 years has played an important role in identifying inflammation as a key process in atherosclerosis and CVD. We should note that “inflammation” as the term is used here does not mean the full form of the condition (warmth, redness, swelling, and pain), but rather implies a “micro-inflammation.” A person with this condition is characterized by being in the upper part of the “normal” distribution for inflammation status, without the signs and symptoms of overt, clinical inflammation. Molecular epidemiology has played a key role in identifying the role of inflammation in CVD, and recently
it has become clear that inflammation is connected to the metabolic syndrome in a complex and important manner. Acute-phase proteins are a class of secreted proteins, primarily from the liver, that either rise or fall in concentration in response to inflammatory stimuli, such as tissue damage and infection. This change in most cases represents no more than a doubling or tripling in concentration (eg, fibrinogen) or a 30 to 50% decrease (eg, albumin). A few of the known proteins, such as C-reactive protein (CRP), may increase in concentration ⬎ 1,000-fold.68 Virtually all of the proteins that have been studied have been shown to be associated with CVD (Table 2). Most of the known acute-phase proteins are produced in the liver in response to IL-6. Although these markers have many different functions, they all are moderately to strongly associated with the presence of CVD (in all cases clinical CVD; in some cases subclinical CVD as defined by such techniques as carotid artery ultrasonography); in almost every case, an argument can be made—at least hypothetically—that the protein in question is not only a marker of the process but might also participate in the process. In population studies or clinical research, inflammation is usually estimated by the measurement of a plasma acute-phase protein, such as CRP or fibrinogen. However, a wide variety of activities, such as innate immunity, coagulation, and others, fall under the term inflammation. Each of these activities plays an important role in our response to trauma and/or environmental challenge: coagulation and fibrinolysis in restricting blood loss and in wound repair69; complement activation and T-cell differentiation as part of innate and adaptive immunity70,71; endothelial cell, neutrophil, and monocyte activation in immunity and wound repair72; antioxidation in response to oxidative challenge73; and others. In addition, variances in the genes responsible for the overall regulation of this system may play important roles in disease susceptibility; eg, IL-6 and tumor necrosis factor (TNF)-␣ genes.74,75 Finally, other environmental factors (eg, diet, smoking, etc)3,76 and the volume of inflammation-mediating tissue (eg, visceral fat)52 also appear to be important. It is clear, therefore, that the relationship of inflammation to athero-
Table 2—Acute-Phase Reactants Associated With CVD Acute-Phase Reactants Positive Fibrinogen Factor VIII CRP Ceruloplasmin PAI-1 Serum amyloid A Ferritin ␣1-acid glycoprotein Negative Albumin High-density lipoprotein
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Proposed Mechanisms Increased coagulant activity; decreased fibrinolysis due to increased clot density; increased platelet aggregation; increased plasma viscosity Increased coagulant activity Increased tissue factor expression on monocytes; increased cell adhesion molecule-mediated cell attachment to endothelial cells; increased complement activation Increased oxidative damage due to higher plasma copper levels Decreased fibrinolytic potential; decreased plaque cellularity None proposed to date Increased oxidative damage due to higher plasma iron levels None proposed to date Decreased antioxidation due to decreased plasma bilirubin level Increased low-density lipoprotein levels due to decreased removal of tissue cholesterol
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sclerosis is complex and much additional research will be needed before we can truly understand these processes and their relationships to health and disease. A number of inflammatory markers have been shown to predict future CVD events, all with a certain degree of similarity. For example, ceruloplasmin—the major copper-transporting protein in plasma— had similar risk prediction to CRP in the Monitoring Trends and Determinants in Cardiovascular Disease-Augsburg cohort.77 In the Cardiovascular Health Study, fibrinogen, factor VIII, and CRP each independently predicted future CVD events32 with similar relative risks. Also, in the Iowa 65⫹ Rural Health Study, both CRP and IL-6 predicted future fatal CVD events, again with similar risk prediction.78 These similarities, however, do not necessarily mean that one marker can be directly substituted for another. In some cases, there are significant differences in the strengths of association. Also, each marker may participate in the CVD process in a different way. In a side-by-side comparison,79 CRP had the strongest risk prediction, but a meta-analysis80 observed no real difference between CRP and fibrinogen in risk prediction.
Inflammation and the Metabolic Syndrome: an Important Relationship It has been observed that markers of inflammation are associated with the components of the metabolic syndrome4; this finding is in addition to the known association of inflammation markers, as well as markers of coagulant activity, with diabetes status.81,82 For example, CRP values increase with the number of components of the metabolic syndrome present,83 and fibrinogen levels are correlated with albumin excretion, even in those patients without demonstrable impaired glucose tolerance (N. Jenny, PhD; unpublished data). Despite these strong relationships, as mentioned before, factor analysis indicates that inflammation represents a distinct underlying pathophysiologic pathway.4 To help better understand the metabolic pathways associated with inflammation, we and others3,4,76,83,84 have studied the correlates of CRP. The major correlates are adiposity and insulin sensitivity status. CRP is also associated with coagulation activity status, as estimated by markers such as d-dimer (a fibrin degradation product [FDP] that reflects first the formation of fibrin and then fibrinolysis) and plasmin-antiplasmin complex (a marker reflecting plasmin formation rate, itself depending on the stimulation of fibrin formation, as well as the levels of tissue plasminogen activator and PAI-1).3,4,85 Increases in coagulation status in the metabolic syndrome and diabetes have been well documented.86,87 The exact mechanism for the association of CRP with coagulation status is not clear; however the work of Ritchie and colleagues10 suggests a possible pathway. Monocytes appear capable of binding FDPs and subsequently producing IL-6, which goes to the liver and affects the wide range of proteins known to be in the acute-phase response. This has been proposed to be the mechanism by which fibrinogen consumption is replaced, and may be an important mechanism connecting coagulation and inflammation. This theory may have far54S
reaching implications both in the general biology we have been discussing and in such areas as drug development, where the coagulation-inflammation connection in sepsis, as described by Esmon et al,69 has been recently exploited.5 Adipose tissue is a key producer of inflammatory cytokines (so-called “adipokines”), among which IL-6 is a major pathophysiologic mediator of diabetes and the metabolic syndrome, as well as acute-phase proteins, such as CRP.88 In this way, the metabolic syndrome may be a contributor to inflammatory response through the visceral obesity component: increased visceral fat may lead to increased proinflammatory response to a variety of stimulants, resulting in a chronic up-regulation of IL-6 production. Chronic up-regulation of IL-6 may predispose a patient to atherosclerosis in a number of ways. We have demonstrated in murine atherosclerosis that chronic injections of small amounts of IL-6 (yielding a minor chronic acute-phase response) resulted in a twofold to fivefold increase in lesion size.53 In humans, IL-6 activities range from acute-phase response to tissue factor expression by monocytes, and many others.8 The association of CRP with insulin level is at least partially independent of adiposity, and is strongly related to insulin sensitivity.4 This statistical association may be mediated by the key proinflammatory cytokine (also an adipokine) TNF-␣, which is a so-called first-wave cytokine influencing IL-6 production,89 as well as many other cellular functions. TNF-␣ receptor (TNFR) I signals programmed cell death, while TNFR II may signal survival or proliferation through cytoplasmic TNFRassociated proteins, which can both negatively regulate apoptosis and positively promote survival.90 Although the role (if any) of circulating TNF-␣ in humans remains uncertain, TNF-␣ induces insulin resistance in tissue culture and in animal models.91,92 Plasma levels are also elevated in the metabolic syndrome, and are associated with insulin resistance in humans.93
Conclusions We now know that thrombin generation is critical in atherosclerotic heart disease in at least two ways: as the ultimate clotting and platelet-activating enzyme, and as an important cell-signaling effector molecule. While thrombosis remains a major contributor to the morbidity and mortality of atherosclerotic disease, the other roles of thrombin in vascular biology may prove to be the more critical in the overall scheme. We also now know that coagulation, fibrinolysis, and inflammation are critically interconnected, and markers related to all of these activities are associated cross-sectionally with both subclinical and clinical heart disease, and epidemiologically are predictors of future clinical events. These new insights highlight the need to engage in integrative molecular physiology, with the goal of understanding in detail not just the individual pathways, but the ways they intersect and interact. In particular, we will need to understand how to interrupt “pathologic” activities (eg, thrombosis or foam cell proliferation) without interrupting homologous “physiologic” activities (eg, hemostasis or wound repair), which often involve the same mediators and effectors. It is likely Thrombin: Physiology and Pathophysiology
that in the future important therapeutic and preventive advances will have to be made with such integrated knowledge in mind.
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