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GW24-e2203 Effect of cigarette smoking extract on the interaction between thrombomodulin and thrombin by live-cell single-molecule force spectroscopy
  1. Wei Yujie,
  2. Liu Huiliang
  1. Department of Cardiology, General Hospital of Chinese People’s Armed Police Forces

Abstract

Objectives To study the effect of cigarette smoking extract (CSE) on the single-molecule interactional force between TM and thrombin by live-cell single-molecule force spectroscopy.

Methods CSE was prepared by a modification of Nakamura’s method. Total RNA was extracted from HUVECs and construct the plasmid of TM-GFP. COS-7 cells were transfected with the recombinant plasmid TM-GFP and the expression of TM-GFP was detected by fluorescence microscopy and laser scanning confocal microscopy. The transfected COS-7 cells were grouped (1) GFP-thrombin group (2) TM-thrombin group (3) CSE- GFP-thrombin group (4) CSE-TM-thrombin-1h group (5) CSE-TM-thrombin-6h group. Force measurements with the thrombin modified AFM tips on the living cell surface were carried out on PicoSPM II with a Pico-Scan 3000 controller and a larger scanner. The force curves measured in living cells were recorded by PicoScan 5 software and analysed by MATLAB R2009a Metlab. Drawn affinity between tip and cell of the Gaussian probability distribution and bonding probility were analysed by Origin7.0 software.

Results (1) The single-molecule binding force of Thrombomodulin and thrombin (TM-Thr) was determined 60.90 ± 0.82 pN. The binding probability for TM-Thr was about 22.58 ± 3.95 %. Antibody blocking binding probability for TM-Thr was 2.58 ± 2.0 %. (2) Compared to TM-THr group, the binding probability for GFP-Thr group, CSE-TM-Thr 1h group, CSE-TM-Thr 6h group, CSE-GFP-Thr group significantly decreased (P < 0.0001).

Conclusions CSE significantly decreased the binding probability for TM-Thr. Our findings indicate that cigarette smoking reduced the binding probability for TM and thrombin to lead to intravascular thrombosis.

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