Hypercoagulability Inhibits Monocyte Transendothelial Migration Through Protease-Activated Receptor-1-, Phospholipase-Cβ-, Phosphoinositide 3-Kinase-, and Nitric Oxide-Dependent Signaling in Monocytes and Promotes Plaque Stability

Abstract

Background-Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis. Methods and Results-De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TM Pro/Pro ApoE Ϫ/Ϫ and FVL Q/Q ApoE Ϫ/Ϫ mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-C␤, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells. Conclusions-Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-C␤, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis. (Circulation. 2009;120:774-784.)Key Words: atherosclerosis Ⅲ blood coagulation Ⅲ endothelium Ⅲ plaque Ⅲ monocytes A therosclerosis is a slowly progressive disease characterized by vascular remodeling and intraplaque accumulation of monocyte-derived macrophages. The role of thrombin and the protease-activated receptor-1 (PAR-1) in atherosclerosis is generally perceived as being detrimental on the basis of their pathogenic role in acute atherosclerotic complications or after vascular injury. [1][2][3] This perception is fostered by a study evaluating de novo atherogenesis in experimental murine atherosclerosis, which showed larger plaques in hypercoagulable factor V Leiden mice (FVL Q/Q ). 4 However, several large studies evaluating the role of genetic risk factors of hypercoagulability (eg, factor V Leiden [FV G1691A] or prothrombin variant [FII G20210A]) failed to show an association between hypercoagulability and the prevalence of Received January 8, 2009; accepted June 29, 2009. From the Department of Medicine I and Clinical Chemistr...

Cytoprotective signaling by activated protein C requires protease-activated receptor-3 in podocytes

“…Proteinuria levels remained significantly elevated in aPC-treated PAR-3 Ϫ/Ϫ mice (0.50 vs 0.29 mg/mL in aPC-treated wild-type mice, P Ͻ .005, Figure 7A) or in mice pretreated with the cell penetrating lipid-conjugated PAR-1 antagonist Ppal12S (0.68 mg/mL vs 0.29 mg/mL in aPC-treated mice, P Ͻ .005, Figure 7A). [32][33][34] Expression of nephrin was markedly reduced in LPS-treated wild-type mice, reflecting podocyte injury (1.4 AU vs 2.4 AU in PBS-treated controls, P ϭ .02, Figure 7B). 22 Nephrin expression was maintained in aPC-treated wild-type mice (2.3 AU in LPS ϩ aPC-treated mice vs 1.4 AU in LPS only mice, P ϭ .03, Figure 7B), but not in aPC-treated PAR-3 Ϫ/Ϫ mice or in mice concomitantly treated with aPC and the PAR-1 antagonist Ppal12S (1.2 AU in PAR-3 Ϫ/Ϫ and 0.9 AU in Ppal12S-treated vs 2.3 AU in aPC-treated mice, P ϭ .03 and P ϭ .01, respectively, Figure 7B).…”

Cytoprotective activated protein C averts Nlrp3 inflammasome–induced ischemia-reperfusion injury via mTORC1 inhibition

“…For the myocardial IRI model (left anterior descending artery ligation for 90 minutes followed by 24 hours of reperfusion), mice were injected with phosphate-buffered saline (PBS; control; equal volume intraperitoneally [IP]), aPC (1 mg/kg IP), 24 aPC-HAPC1573 complex (aPC was preincubated before injection with HAPC1573 antibody at a 1:1 molar ratio for 10 minutes under gentle agitation to block its anticoagulant activity), [24][25][26][27] an aPC variant lacking specifically anticoagulant function (3K3A-aPC; 1 mg/kg IP), 28,29 the inhibitory PAR-1 pepducin (P1pal-12S; 2.5 mg/kg subcutaneously) followed by aPC (1 mg/kg IP), 26,30,31 or parmodulin-2 (5 mg/kg IV) 32 reperfusion), 25 mice were injected with PBS (control; 1 mg/kg IP) or aPC (1 mg/kg IP) 30 minutes before IRI. P1pal-12S and parmodulin-2 have been previously described.…”

“…P1pal-12S and parmodulin-2 have been previously described. [30][31][32][33] Infarct size was determined as described in the supplemental Methods.…”

“…Before myocardial IRI, mice were exposed to PBS (control), aPC alone, the aPC-HAPC1573 complex (the antibody HAPC1573 specifically blocks the anticoagulant function of aPC), [24][25][26][27] or an aPC variant specifically lacking anticoagulant function (3K3A-aPC), 28,29 or they were first treated with an inhibitory PAR-1 pepducin (P1pal-12S) 26,30,31 followed by aPC treatment. Treatment with 3K3A-aPC or preincubation of aPC with the HAPC1573 antibody did not abolish the protective effect of aPC, as reflected by reduced infarct size ( Figure 5D-F), reduced myocardial Nlrp3 expression and reduced cl-Casp1 and cl-IL-1b ( Figure 5G; supplemental Figure 4A), reduced myocardial caspase-1 activity (FLICA-Casp1 assay; Figure 5H; supplemental Figure 4B), and reduced plasma IL-1b and IL-18 levels ( Figure 5I-J).…”

Novel agonists & antagonists for protease-activated receptor 2 and C3a receptor

“…PAR4 KO mice with an atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) background, fed a high cholesterol diet, display unaffected plaque formation and indifferent plaque surface area coverage . Interestingly, consistent with the observation that PAR1 does not mediate murine platelet response to thrombin, a recent report has indicated that it is the action of thrombin on monocytes via PAR1 that promotes plaque stability during atherogenesis in mice by inhibiting transendothelial migration to reduce macrophage accumulation (Seehaus et al, 2009). Further, use of PAR1 KO mice demonstrated a potential role for PAR1 in regulation of murine vasculature independent of platelet activation in response to injury, likely mediated by tissue remodeling and extracellular matrix synthesis .…”