香烟提取物对血栓调节蛋白与凝血酶结合力的影响

Effect of cigarette smoke extract on the interaction between thrombomodulin and thrombin by live-cell single-molecule force spectroscopy

目的利用单分子力谱法检测香烟提取物(cigarette smoke extract,CSE)对血栓调节蛋白(thrombomodulin,TM)与凝血酶间单分子水平相互作用,探讨吸烟致血管内血栓形成机制。方法构建TM-GFP质粒并转染至COS-7细胞,应用原子力显微镜(atomic force microscope,AFM)分组测力:(1)空白质粒对照组(GFP-Thr);(2)TM-Thr对照组;(3)5%CSE孵育TM-Thr细胞组(CSE-TM-Thr);(4)5%CSE孵育空白质粒细胞组(CSE-GFP-Thr)。结果 (1)TM与凝血酶的相互作用力为(60.90±0.82)pN,成键几率为(22.58±3.95)%,抗体阻断后成键几率为(2.58±2.0)%。(2)与TM-Thr组比较,GFP-Thr组、CSE-TM-Thr组、CSE-GFP-Thr组,3组AFM成键几率明显减低,P<0.05;但3组间两两比较差异无统计学意义。在定量TM修饰的硅片表面检测证实CSE同样可减少TM与凝血酶的成键几率。结论吸烟可能通过减少TM与凝血酶结合几率,抑制TM的抗凝作用,从而导致血管内血栓形成。

Aim To study the effect of CSE （ cigarette smoke extract ） on the single-molecule interactional force between thrombomodulin and thrombin by live- cell single-molecule force spectroscopy. Methods CSE was prepared by a previously reported method.The plasmid of TM-GFP was constructed and transfect- ed in COS-7 cells. The expression of TM-GFP was de- tected by fluorescence microscopy and laser scanning confocal microscopy. The transfected COS-7 cells were grouped （ 1 ） GFP -thrombin group （ 2 ） TM-thrombingroup （ 3 ） CSE-TM-thrombin group （ 4 ） CSE- GFP- thrombin group. Force measurements with the thrombin modified AFM tips on the living cell surface were car- fled out on PicoSPM II with a Pico-Scan 3000 control- ler and a larger scanner. The force curves measured in living cells were recorded by PicoScan 5 software and analyzed by MATLAB R2009aMetlab. Results 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 proba- bility for TM-Thr was （ 2. 58 ± 2.0 ） %. The binding probabilities for GFP-Thr group, CSE-TM-Thr group and CSE-GFP-Thr group were significantly decreased compared with TM-Thr group （ P 〈 0.05 ）. The meanvalue of the most probable single molecular interaction force of thrombin/TM-ECD was determined as （45.30 ± 1.37） pN, the binding probability of thrombin and TM-ECD was （23.25 ± 7.02） %. When the binding was blocked with the TM-MAb solution, the binding probability decreased to （4. 64 ± 2. 31 ） %. The bind- ing probability was （8.31 ± 1.06）% in the CSE-TM- thr-S group. When further blocked with TM-MAb, the binding probability was （5. 17 ± 2.96） %. Conclusion CSE significantly decreases the binding probability for TM-Thr to induce intravascular thrombosis.