Bridging Thrombosis and Inflammation
Thrombus formation within a coronary vessel is the precipitating event in unstable coronary syndromes. The angiographic severity of coronary stenoses may not predict sites of subsequent acute coronary events, inasmuch as rupture of atheromatous plaque with thrombosis in relatively mildly stenosed vessels may underlie many acute coronary syndromes. The intact endothelium normally prevents platelet activation, but the intimal injury associated with endothelial denudation and plaque rupture exposes subendothelial collagen and von Willebrand factor, which support prompt platelet adhesion and activation. Circulating platelets can adhere either directly to collagen or indirectly, via the binding of von Willebrand factor, to the glycoprotein Ib/IX complex. Local platelet activation promotes thrombus formation and additional platelet recruitment by supporting cell surface thrombin formation and releasing potent platelet agonists, such as adenosine 5′-diphosphate, serotonin, and thromboxane A2. The central role of platelet activation in acute coronary syndromes is supported by increased platelet-derived thromboxane and prostaglandin metabolites detected in patients with acute coronary syndromes1 and the clear clinical benefit of treatment with aspirin for prevention of acute coronary events.2
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The importance of platelet-dependent thrombosis has made the platelet aggregate a common therapeutic target in acute coronary syndromes. Recently, such therapies have included the use of aspirin, thienopyridines, and direct glycoprotein IIb/IIIa inhibitors. Although the mechanism of action of these various agents differs, they all inhibit fibrinogen-dependent platelet–platelet associations (ie, platelet aggregation). However, as highlighted by the recent disappointing clinical results of trials using oral glycoprotein IIb/IIIa inhibitors,3 merely preventing the process of platelet aggregation may not always translate into clinical efficacy.
Recent data suggest that patients with acute coronary syndromes have not only increased interactions between platelets (homotypic aggregates) but also increased interactions between platelets and leukocytes (heterotypic aggregates) detectable in circulating blood. These aggregates form when platelets are activated and undergo degranulation, after which they adhere to circulating leukocytes. Early work suggested that these heterotypic aggregates form in inflammatory states.4 Platelets bind via P-selectin (CD62P) expressed on the surface of activated platelets to the leukocyte receptor, P-selectin glycoprotein ligand-1 (PSGL-1).5 This initial association leads to increased expression of CD11b/CD18 (Mac-1) on leukocytes,6 which itself supports interactions with platelets, perhaps because bivalent fibrinogen links this integrin with its platelet surface counterpart glycoprotein IIb/IIIa.7,8⇓ The importance of platelet–leukocyte aggregates in vascular disease is supported by a recent study demonstrating that the infusion of recombinant human PSGL-1 in an animal model of vascular injury reduced myocardial reperfusion injury and preserved vascular endothelial function.9
In acute coronary syndromes, markers of cardiac necrosis by themselves do not provide information about plaque disruption; measures of platelet–leukocyte aggregates may be a better reflection of plaque instability and ongoing vascular thrombosis and inflammation. Platelet–leukocyte aggregates also may be a more sensitive marker of platelet activation than is surface P-selectin expression because degranulated platelets rapidly lose surface P-selectin in vivo but continue to be detected in circulation.10 This is supported by a recent study by Michelson et al,11 which demonstrates that after acute myocardial infarction, circulating monocyte–platelet aggregates are a more sensitive marker of in vivo platelet activation than is platelet surface P-selectin. In addition, a study by Ott et al12 of neutrophil activation and neutrophil–platelet adhesion demonstrated that patients with unstable angina had a significant increase in neutrophil–platelet binding compared with patients with stable angina.
In this issue of Circulation, Sarma et al13 add to the growing body of information about platelet–leukocyte aggregates in atherothrombosis and acute coronary syndromes. They studied 25 patients admitted with an acute coronary syndrome and compared them with 27 subjects with noncardiac chest pain, using flow cytometry to measure platelet–leukocyte binding. Compared with the subjects with noncardiac chest pain, the patients with acute coronary syndromes had increased levels of circulating platelet–monocyte aggregates. Platelet–monocyte binding was significantly decreased by saturating concentrations of a monoclonal antibody directed against PSGL-1 and the blockade of P-selectin, suggesting that monocyte binding to platelets occurs primarily through PSGL-1 and P-selectin.
The study by Sarma et al13 has several significant limitations, including the small sample size and the demographic differences between the groups. The patients who had myocardial infarctions were older and had a higher prevalence of diabetes mellitus, both of which are states in which enhanced platelet reactivity has been previously demonstrated. In addition, a higher incidence of previous myocardial infarction in patients with noncardiac chest pain makes it difficult to differentiate these subjects from those in the unstable angina group in the absence of testing for cardiac ischemia.
Despite these limitations, the study by Sarma et al13 supports a recent report by Furman and colleagues,14 who examined 61 patients with acute myocardial infarction and compared them with 150 control subjects. Furman et al controlled for age and found that the number of circulating monocyte–platelet aggregates was greatest in patients with acute myocardial infarction presenting within 4 hours of symptom onset. They also made the important finding that the patients presenting with acute myocardial infarction who had a normal creatine kinase isoenzyme ratio at the time of hospital presentation (just over 50%) also had elevated levels of circulating platelet–monocyte aggregates.
The binding of platelets and monocytes in acute coronary syndromes highlights the interaction between inflammation and thrombosis in cardiovascular disease. Plaque rupture promotes activation of the inflammatory response, and increased expression of tissue factor initiates extrinsic coagulation. The expression of tissue factor on both endothelial cells and monocytes is partially regulated by proinflammatory cytokines, including tumor necrosis factor and interleukin-1.15 In addition to initiating coagulation, tissue factor interacts with P-selectin, accelerating fibrin formation and deposition.15 Platelet surface P-selectin also induces the expression of tissue factor on monocytes,16 enhances monocyte cytokine expression,17 and promotes CD11b/CD18 expression.6
CD40–CD40 ligand (CD40L or CD154) interactions also are known to play a role in atherosclerotic, thrombotic, and inflammatory processes.18 On endothelial cells or monocytes, the engagement of CD40 with CD40L leads to the synthesis of adhesion molecules, chemokines, and tissue factor and causes the activation of matrix metalloproteinases. CD40L also is upregulated on platelets in a fresh thrombus.18 Thus, a potential additional mechanism for binding between the platelet and monocyte in the setting of acute myocardial infarction is the CD40–CD40L, representing a unique link between thrombosis and inflammation.
The study by Sarma et al13 also suggests that, in addition to the interaction via P-selectin and PSGL-1, cation-independent interactions may play a role in platelet–monocyte interactions. Incubation with a calcium chelator decreased the binding of platelets to monocytes among patients with acute myocardial infarction to a greater extent than among patients with unstable angina or noncardiac chest pain. Although intriguing, it is difficult to draw any definitive conclusions from these results because most of the platelet–monocyte binding was inhibited by the chelation, and the differences between the groups were small. External chelation does not eliminate potential contribution from pathways mediated by internal Ca2+ stores because platelets contain ≈20 nmol of Ca2+/108 cells, mostly stored in dense granules. This may be relevant because surface expression of the active conformation of glycoprotein IIb/IIIa and of CD40L has been shown to be dependent on internal Ca2+ stores.19
The studies by Sarma et al13 and others11,12,14⇓⇓ of platelet–monocyte aggregates suggest the potential utility of this measurement in diagnosing plaque activation and acute myocardial infarction. Although the study by Furman and colleagues14 first showed that circulating platelet–monocyte aggregates are an early marker of acute myocardial infarction, as compared with creatine kinase-MB, a direct comparison with troponin levels would be necessary to establish the true diagnostic utility of this measurement. It is important to note that the ease of troponin measurements greatly exceeds the significant limitations involved in the use of flow cytometry, making routine measurement of platelet–monocyte aggregates comparatively impractical at the present time. A simpler method of measuring platelet–monocyte binding would first need to be established. Possibly more important than its use as a marker of acute myocardial infarction is the fact that platelet–monocyte aggregates represent a novel index of platelet activation. Perhaps equally important, the platelet–leukocyte heterotypic aggregate represents a potential new therapeutic target for limiting thrombotic and inflammatory responses in acute coronary syndromes.
All of these observations lead to the central, yet unanswered, question: Why does platelet–leukocyte binding occur in acute coronary syndromes, and what are its pathobiological consequences (if any)? Furthermore, are these aggregates merely an epiphenomenal reflection of the inflammatory and thrombotic processes occurring in the setting of acute coronary syndromes, or do they directly contribute to the ongoing atherothrombogenesis? Do they represent an initial step in the phagocytotic clearance of “spent” platelets (thrombophagocytosis) by monocytic cells? If so, what is the consequence of this phagocytosis? Early work from one of us (J.L.) showed that thrombophagocytosis by monocytic cells leads to cholesteryl ester accumulation in monocytic cells and foam cell formation in the absence of hyperlipidemia, suggesting a unique link between thrombosis and atherogenesis.20 Future studies should be directed at understanding the role of platelet–leukocyte aggregates in vascular disease, their use as a diagnostic tool, and the possible therapeutic consequences of inhibiting their formation.
The authors thank Stephanie Tribuna and Jalna Ross for expert secretarial assistance.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Antiplatelet Trialists Collaboration. Collaborative overview of randomised trials of antiplatelet therapy: I: Prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. BMJ. 1994; 308: 81–106.
- ↵Chew DP, Bhatt DL, Sapp S, et al. Increased mortality with oral platelet glycoprotein IIb/IIIa antagonists: a meta-analysis of phase III multicenter randomized trials. Circulation. 2001; 103: 201–206.
- ↵Arber N, Berliner S, Pras E, et al. Heterotypic leukocyte aggregation in the peripheral blood of patients with leukemia, inflammation and stress. Nouv Rev Fr Hematol. 1991; 33: 251–255.
- ↵Rinder HM, Bonan JL, Rinder CS, et al. Dynamics of leukocyte-platelet adhesion in whole blood. Blood. 1991; 78: 1730–1737.
- ↵Gawaz MP, Loftus JC, Bajt ML, et al. Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIB- IIIA) dependent homotypic and heterotypic cell-cell interactions. J Clin Invest. 1991; 88: 1128–1134.
- ↵Simon DI, Ezratty AM, Francis SA, et al. Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway. Blood. 1993; 82: 2414–2422.
- ↵Hayward R, Campbell B, Shin YK, et al. Recombinant soluble P-selectin glycoprotein ligand-1 protects against myocardial ischemic reperfusion injury in cats. Cardiovasc Res. 1999; 41: 65–76.
- ↵Michelson AD, Barnard MR, Hechtman HB, et al. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci U S A. 1996; 93: 11877–11882.
- ↵Michelson AD, Barnard MR, Krueger LA, et al. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation. 2001; 104: 1533–1537.
- ↵Ott I, Neumann FJ, Gawaz M, et al. Increased neutrophil-platelet adhesion in patients with unstable angina. Circulation. 1996; 94: 1239–1246.
- ↵Sarma J, Laan KA, Alam S, et al. Increased platelet binding to circulating monocytes in acute coronary syndromes. Circulation. 2002; 105: 2166–2171.
- ↵Shebuski RJ, Kilgore KS. Role of inflammatory mediators in thrombogenesis. J Pharmacol Exp Ther. 2002; 300: 729–735.
- ↵Celi A, Pellegrini G, Lorenzet R, et al. P-selectin induces the expression of tissue factor on monocytes. Proc Natl Acad Sci U S A. 1994; 91: 8767–8771.
- ↵Neumann FJ, Marx N, Gawaz M, et al. Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets. Circulation. 1997; 95: 2387–2394.
- ↵Mendelsohn ME, Loscalzo J. Role of platelets in cholesteryl ester formation by U-937 cells. J Clin Invest. 1988; 81: 62–68.