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Intraplaque release of inflammatory cytokines from macrophages is implicated in atherogenesis by inducing the proliferation and migration of media smooth muscle cells (SMCs). PCSK9 is present and released by SMCs within the atherosclerotic plaque but its function is still unknown. In the present study, we tested the hypothesis that PCSK9 could elicit a pro-inflammatory effect on macrophages. THP-1-derived macrophages and human primary macrophages were exposed to different concentrations (0.250 ÷ 2.5 µg/ml) of human recombinant PCSK9 (hPCSK9). After 24 h incubation with 2.5 µg/ml PCSK9, a significant induction of IL-1β, IL-6, TNF-α, CXCL2, and MCP1 mRNA, were observed in both cell types. Co-culture of THP-1 macrophages with HepG2 overexpressing hPCSK9 also showed the induction of TNF-α (2.4 ± 0.5 fold) and IL-1β (8.6 ± 1.8 fold) mRNA in macrophages. The effect of hPCSK9 on TNF-α mRNA in murine LDLR−/− bone marrow macrophages (BMM) was significantly impaired as compared to wild-type BMM (4.3 ± 1.6 fold vs 31.1 ± 6.1 fold for LDLR−/− and LDLR+/+, respectively). Finally, a positive correlation between PCSK9 and TNF-α plasma levels of healthy adult subjects (males 533, females 537) was observed (B = 8.73, 95%CI 7.54 ÷ 9.93, p < 0.001). Taken together, the present study provides evidence of a pro-inflammatory action of PCSK9 on macrophages, mainly dependent by the LDLR.

Summary. Background: Blood-borne tissue factor (TF) plays a crucial role in thrombogenesis. Aim: To study whether polymorphonuclear leukocytes (PMN) are a source of TF. Methods and Results: Human PMN were carefully separated from other blood cells and stimulated for 3 min with purified P-selectin or the chemotactic peptide formyl-MetLeuPhe (fMLP): they expressed both TF procoagulant activity, as identified by specific TF MoAb and inactivated factor VIIa blockade; and TF:Ag (four to six times), as shown by flowcytometry and immunocytochemistry. About 40% of permeabilized PMN, both resting and stimulated, contained TF:Ag, indicating that stimulation only modifies the location of TF:Ag within PMN. By real time-polymerase chain reaction (RT-PCR), a very low amount of TF mRNA was detectable in resting PMN, but a 3-to 5-fold increase was observed after 1-h stimulation with P-selectin or fMLP, respectively. Conclusions: These findings suggest that TF is not constitutively expressed in peripheral PMN, but can be up-regulated and produced upon stimulation and specific gene transcription, as for instance during contact with activated platelets or endothelium. The stored TF is rapidly expressed in vitro as a functional molecule on the surface of activated PMN. The availability of PMN TF supports the relevance of inflammatory cells and their interaction with platelets for fibrin deposition and thrombus formation.

Objective-Tissue factor (TF) is the main activator of the coagulation cascade occurring in physiologic and pathologic conditions. Recent data suggest that human platelets might contain TF that is possibly derived from leukocytes. In this study, we investigated whether intraplatelet TF can be exposed on the membrane by platelet agonists. The modulation of this process by antiplatelet drugs has been evaluated as well. Methods and Results-Flow cytometric analysis of unstimulated platelets showed a small amount of membrane-associated immunoreactive TF (irTF) in whole blood, platelet-rich plasma, and washed platelets isolated from healthy subjects. ADP, thrombin receptor-activating peptide, and epinephrine significantly increased functionally active, membraneassociated irTF. ADP induced irTF exposure in a concentration-and time-dependent fashion. Agonist-induced irTF expression was completely inhibited by iloprost but not by aspirin. Interestingly, glycoprotein IIb/IIIa antagonists did not inhibit but rather potentiated the stimulatory effect of ADP on platelet irTF expression. Real-time polymerase chain reaction experiments showed detectable amounts of TF mRNA in unstimulated platelets. Conclusions-These findings indicate that platelet agonists and antiplatelet drugs might modulate platelet-associated irTF expression. Regulated TF expression establishes the potential for a previously unrecognized role for platelets in sustaining thrombus formation and growth via coagulation-mediated mechanisms. (Arterioscler Thromb Vasc Biol. 2003;23:1690-1696.)Key Words: platelets Ⅲ tissue factor Ⅲ coagulation Ⅲ thrombosis Ⅲ antiplatelet agents C linical and experimental evidence indicates that tissue factor (TF), a 47-kDa glycoprotein, triggers activation of the coagulation cascade that occurs in several human diseases, such as sepsis and other systemic conditions. 1 In addition, TF has been shown to be present in atherosclerotic plaques. 2,3 In particular, TF protein and activity have been found to be increased in coronary plaques, thus playing a crucial role in human coronary syndromes. 4 Vessel wallassociated TF, however, does not entirely explain the thrombogenic potential of vascular lesions when they are exposed to flowing blood. It has been proposed that thrombus growth might be promoted by circulating (ie, microparticle or platelet-associated) TF. 5 Indeed, to interact with plasma coagulation proteins and thus sustain thrombus growth, TF might diffuse from its source to the initial platelet layer. Because the time to capture any diffusing species increases as the square of distance, the shortened distance resulting from TF associated with circulating platelets might have very significant effects on subsequent reaction velocities.Considerable data now support the hypothesis that platelets actively modulate the propagation of coagulation by expressing specific, high-affinity receptors for coagulation proteases, zymogens, and cofactors that contribute to localize thrombin generation at the site of vascular injury. 6 It has be...

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Extracellular vesicles (EVs) are well-established mediators of cell-to-cell communication. EVs can be released by every cell type and they can be classified into three major groups according to their biogenesis, dimension, density, and predominant protein markers: exosomes, microvesicles, and apoptotic bodies. During their formation, EVs associate with specific cargo from their parental cell that can include RNAs, free fatty acids, surface receptors, and proteins. The biological function of EVs is to maintain cellular and tissue homeostasis by transferring critical biological cargos to distal or neighboring recipient cells. On the other hand, their role in intercellular communication may also contribute to the pathogenesis of several diseases, including thrombosis. More recently, their physiological and biochemical properties have suggested their use as a therapeutic tool in tissue regeneration as well as a novel option for drug delivery. In this review, we will summarize the impact of EVs released from blood and vascular cells in arterial and venous thrombosis, describing the mechanisms by which EVs affect thrombosis and their potential clinical applications.

Angiotensin-converting enzyme (ACE) inhibitors reduce the risk of recurrent myocardial infarction in patients with left ventricular dysfunction. Tissue factor (TF), the initiator of blood coagulation, plays a pivotal role in arterial thrombosis that occurs after atherosclerotic plaque fissuring. Because monocytes synthesize TF and contain several components of the renin-angiotensin system, we investigated the possibility that ACE inhibitors could modulate monocyte TF expression. Mononuclear leukocytes from healthy volunteers were incubated with endotoxin in the presence or absence of different ACE inhibitors. Captopril reduced TF expression in endotoxin-stimulated mononuclear leukocytes, as measured by a 1-stage clotting assay and ELISA analysis, by approximately 60%. The effect was dose-dependent and was attributable to ACE inhibition, given that other ACE inhibitors, such as idrapril or fosinopril, and losartan, an antagonist of the angiotensin II AT(1) receptor, caused a comparable reduction in TF activity. Reverse transcriptase-polymerase chain reaction indicated that endotoxin-mediated increased levels of TF mRNA were inhibited by ACE inhibitors. Moreover, endotoxin-induced nuclear factor-kappaB translocation to the promoter region of the gene encoding for TF was markedly inhibited by captopril. The finding that ACE inhibitors and angiotensin II AT(1) antagonists can potentially modulate TF expression by mononuclear cells has important biological and therapeutic implications for the evolution of thrombi. Our results suggest that the anti-ischemic effect of these drugs might be explained, at least in part, by their ability to reduce TF expression in monocytes.