Abstract 3740: Cigarette Smoke Exposure Of Human Monocytic Cells Increases The Generation Of Pro-thrombotic Microvesicles Through Activation Of Erk: A Novel Mechanism For Pathologic Hypercoagulability in Smoking
Cigarette smoking substantially increases the risk of atherothrombotic vascular disease. Smokers exhibit higher circulating levels of tissue factor (TF) than do non-smokers, and smoking just 2 cigarettes in a row significantly increases plasma TF levels in humans (Circulation 107:973, 2003). Nevertheless, the mechanisms responsible for increased circulating TF in smokers have not been reported. Because TF released from cells into plasma is always carried by cell membrane microvesicles (MVs), we now examined the effect of cigarette smoke extract (CSE) on MV generation by human THP-1 monocytic cells. Our stock CSE was made by bubbling smoke from 5 research cigarettes through 10ml of RPMI with 0.2% BSA. Exposure of THP-1 cells to 5–15% CSE significantly increased total MV generation in a time-and dose-dependent manner, as assessed by flow cytometry. CSE treatment also up-regulated TF expression on the surface of THP-1 cells and increased release of TF-positive MVs. Most importantly, CSE treatment of THP-1 cells for 20hrs more than tripled the procoagulant activity of MVs isolated from the conditioned medium (3266±709 with CSE vs 944±137 pM/L/hr from controls without CSE, p=0.016; 2 million cells used in each condition). To examine mechanisms of MV generation, we found that CSE treatment of THP-1 cells induced dose-dependent apoptosis, as shown by surface exposure of phosphatidylserine and TUNEL staining (15% CSE: 25.1±1.8%; vs control: 2.9±0.7% TUNEL positive, p<0.001). In addition, CSE treatment activated ERK, p38, and JNK MAP kinases in THP-1 cells. Pre-treatment with an ERK inhibitor (10 μM U0126), but not with p38 and JNK inhibitors, significantly blunted CSE-induced apoptosis measured by TUNEL (17.1±1.8% for the CSE+U0126 group, p=0.02). Moreover, pre-treatment with U0126 decreased the procoagulant activity of isolated MVs released from CSE-treated THP-1 cells by 60% (1265±198 pM/L/hr in CSE+U0126 group, p<0.05). Overall, our results indicate that exposure of THP-1 monocytes to CSE induces the generation of pro-coagulant MVs, in part through ERK activation and apoptosis. We conclude that the generation of biologically active MVs could be a novel mechanism for pathologic hypercoagulability in cigarette smokers.
This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).