Abstract-C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentamer (pentameric CRP) in plasma. The in vivo existence of monomeric (m)CRP has been postulated, but its function and source are not clear. We show that mCRP is deposited in human aortic and carotid atherosclerotic plaques but not in healthy vessels. pCRP is found neither in healthy nor in diseased vessels. As source of mCRP, we identify a mechanism of dissociation of pCRP to mCRP. We report that activated platelets, which play a central role in cardiovascular events, mediate this dissociation via lysophosphatidylcholine, which is present on activated but not resting platelets. Furthermore, the dissociation of pCRP to mCRP can also be mediated by apoptotic monocytic THP-1 and Jurkat T cells. The functional consequence is the unmasking of proinflammatory effects of CRP as demonstrated in experimental settings that are pathophysiologically relevant for atherogenesis: compared to pCRP, mCRP induces enhanced monocyte chemotaxis; monocyte activation, as determined by conformational change of integrin Mac-1; generation of reactive oxygen species; and monocyte adhesion under static and physiological flow conditions. In conclusion, we demonstrate mCRP generation via pCRP dissociation on activated platelets and H 2 O 2 -treated apoptotic THP-1 and Jurkat T cells, thereby identifying a mechanism of localized unmasking of the proinflammatory properties of CRP. This novel mechanism provides a potential link between the established cardiovascular risk marker, circulating pCRP, and localized platelet-mediated inflammatory and proatherogenic effects. Key Words: C-reactive protein Ⅲ atherosclerosis Ⅲ platelets C -reactive protein (CRP) is a highly conserved protein of the pentraxin family that consists of 5 noncovalently linked subunits of Ϸ23 kDa. It is mainly produced in the liver, but under certain conditions can also be secreted by smooth muscle cells 1 and endothelial cells. 2 It was first discovered as an acute phase reactant, with plasma levels increasing from a baseline level of 1 to 2 g/mL up to 100-to 1000-fold within 24 to 72 hours. Because of this rapid cytokinedriven response to tissue injury, infection, and inflammation, CRP is seen as the prototypic inflammatory marker.Small, 2-to 5-fold increases in the baseline level of plasma CRP in asymptomatic individuals have been associated with an increased risk for cardiovascular events such as stroke and myocardial infarction. 3,4 In the recently published Jupiter trial, mildly elevated CRP levels were used to guide primary prevention, resulting in a significant reduction of major cardiovascular events in apparently healthy individuals. 5 Although the exact role of CRP in atherosclerosis and its complications are unknown, evidence is now emerging to suggest that it may be a direct, causative factor. 6,7 In vitro, CRP has been reported to increase interleukin-8 production in monocytes, 8 inhibit endothelial nitric oxide synthase, 9 alter the antioxidant defenses, and promote...

Background-Strong evidence supports a role for CD40 ligand (CD40L) as marker and mediator of inflammatory diseases such as atherosclerosis. Despite extensive characterization of CD40, the classic receptor of CD40L, its role in immune defense against inflammatory diseases remains uncertain. The present study aimed to characterize the contribution of CD40 signaling to atherogenesis. Methods and Results-Surprisingly, mice deficient in both CD40 and the low-density lipoprotein receptor did not develop smaller lesions in the aortic arch, root, and thoracoabdominal aorta compared with mice deficient only in the low-density lipoprotein receptor that consumed an atherogenic diet for 8 and 16 weeks. By flow cytometry, radioactive binding assays, and immunoprecipitation, we demonstrate that CD40L interacts with the integrin Mac-1, which results in Mac-1-dependent adhesion and migration of inflammatory cells as well as myeloperoxidase release in vitro. Furthermore, mice deficient in CD40L show significantly reduced thioglycolate-elicited invasion of inflammatory cells into the peritoneal cavity compared with mice deficient in CD40 and wild-type controls. Inhibition of Mac-1 in low-density lipoprotein receptor-deficient mice attenuates lesion development and reduces lesional macrophage accumulation. Conclusions-These observations identify the interaction of CD40L and Mac-1 as an alternative pathway for CD40L-mediated inflammation. This novel mechanism expands understanding of inflammatory signaling during atherogenesis.

Objective-High-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. We investigated its role in the development of atherosclerosis in ApoEϪ/Ϫ mice. Methods and Results-Apolipoprotein E-deficient (ApoEϪ/Ϫ) mice fed a high-fat diet were administered a monoclonal anti-HMGB1 neutralizing antibody, and the effects on lesion size, immune cell accumulation, and proinflammatory mediators were assessed using Oil Red O, immunohistochemistry, and real-time polymerase chain reaction. As with human atherosclerotic lesions, lesions in ApoEϪ/Ϫ mice expressed HMGB1. Treatment with the neutralizing antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43%, and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression was attenuated by 48% and 72%, respectively. CD11cϩ dendritic cells were reduced by 65%, and the mature (CD83ϩ) population was reduced by 60%. Treatment also reduced CD4ϩ cells by nearly 50%. mRNAs in lesions encoding tumor necrosis factor-␣ and interleukin-1␤ tended to be reduced. Mechanistically, HMGB1 stimulated macrophage migration in vitro and in vivo; in vivo, it markedly augmented the accumulation of F4/80ϩGr-1(Ly-6C)ϩ macrophages and also increased F4/80ϩCD11bϩ macrophage numbers. Conclusion-HMGB1

Platelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

Background-Molecular imaging is a fast emerging technology allowing noninvasive detection of vascular pathologies.However, imaging modalities offering high resolution currently do not allow real-time imaging. We hypothesized that contrast-enhanced ultrasound with microbubbles (MBs) selectively targeted to activated platelets would offer high-resolution, real-time molecular imaging of evolving and dissolving arterial thrombi. Methods and Results-Lipid-shell based gas-filled MBs were conjugated to either a single-chain antibody specific for activated glycoprotein IIb/IIIa via binding to a Ligand-Induced Binding Site (LIBS-MBs) or a nonspecific single-chain antibody (control MBs). Successful conjugation was assessed in flow cytometry and immunofluorescence double staining. LIBS-MBs but not control MBs strongly adhered to both immobilized activated platelets and microthrombi under flow. Thrombi induced in carotid arteries of C57Bl6 mice in vivo by ferric chloride injury were then assessed with ultrasound before and 20 minutes after MB injection through the use of gray-scale area intensity measurement. Gray-scale units converted to decibels demonstrated a significant increase after LIBS-MB but not after control MB injection (9.55Ϯ1.7 versus 1.46Ϯ1.3 dB; PϽ0.01). Furthermore, after thrombolysis with urokinase, LIBS-MB ultrasound imaging allows monitoring of the reduction of thrombus size (PϽ0.001). Conclusion-We

This paper provides an update on the European Society of Cardiology task force report on the management of chest pain. Its main purpose is to provide an update on the decision algorithms and diagnostic pathways to be used in the emergency department for the assessment and triage of patients with chest pain symptoms suggestive of acute coronary syndromes.

BACKGROUND: Platelets store large amounts of serotonin that they release during thrombus formation or acute inflammation. This facilitates hemostasis and modulates the inflammatory response. METHODS:Infarct size, heart function, and inflammatory cell composition were analyzed in mouse models of myocardial reperfusion injury with genetic and pharmacological depletion of platelet serotonin. These studies were complemented by in vitro serotonin stimulation assays of platelets and leukocytes in mice and men, and by measuring plasma serotonin levels and leukocyte activation in patients with acute coronary syndrome. RESULTS:Platelet-derived serotonin induced neutrophil degranulation with release of myeloperoxidase and hydrogen peroxide (H 2 O 2 ) and increased expression of membrane-bound leukocyte adhesion molecule CD11b, leading to enhanced inflammation in the infarct area and reduced myocardial salvage. In patients hospitalized with acute coronary syndrome, plasmatic serotonin levels correlated with CD11b expression on neutrophils and myeloperoxidase plasma levels. Longterm serotonin reuptake inhibition-reported to protect patients with depression from cardiovascular events-resulted in the depletion of platelet serotonin stores in mice. These mice displayed a reduction in neutrophil degranulation and preserved cardiac function. In line, patients with depression using serotonin reuptake inhibition, presented with suppressed levels of CD11b surface expression on neutrophils and lower myeloperoxidase levels in blood. CONCLUSIONS:Taken together, we identify serotonin as a potent therapeutic target in neutrophil-dependent thromboinflammation during myocardial reperfusion injury.

Objective-Integrins are attractive therapeutic targets. Inhibition of integrin ␣ IIb ␤ 3 effectively blocks platelet aggregation.However, limitations with intravenous ␣ IIb ␤ 3 antagonists and failure of oral ␣ IIb ␤ 3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. Methods and Results-Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic ␣ IIb ␤ 3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of ␣ IIb ␤ 3 , (2) receptor clustering, (3)