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.