逆锁键调控野生型和功能增强型ADAMTS13与VWF A2结构域的相互作用

Catch⁃Bond Regulates the Interactions of WT and GOF ADAMTS13 with VWF A2 Domain

目的探究瞬时接触时间下野生型(wild⁃type,WT)和功能增强型(gain⁃of⁃function,GOF)金属蛋白酶ADAMTS13与VWF A2结构域的相互作用,阐明其动力学调控的分子机制。方法利用原子力显微镜测量在不同外力作用下WT和GOF ADAMTS13分子与VWF A2的黏附频率、断裂力和分子键寿命,并利用Bell⁃Evans模型提取相关动力学参数。结果WT ADAMTS13与VWF A2结合的能障宽度为0.41 nm,0 N力下的解离速率为1.50 s^(-1);GOF ADAMTS13与VWF A2结合的能障宽度为0.29 nm,0 N力下的解离速率为3.28 s^(-1)。进一步利用AFM clamp模式测量在不同作用力下WT/GOF ADAMTS13⁃VWF A2复合物的分子键生存寿命和解离速率,发现两组复合物的结合均呈现“逆锁键”⁃“滑移键”转换的双相力学依赖特性。结论WT ADAMTS13⁃VWF A2相互作用的机械强度与力学稳定性高于GOF ADAMTS13⁃VWF A2,WT/GOF ADAMTS13与VWF A2结构域的相互作用具有“逆锁键”⁃“滑移键”转换的双相力学响应特性。研究结果有助于深入理解ADAMTS13与VWF的相互作用,为抗血栓药物的开发提供新思路。

Objective To investigate the regulatory mechanism of wild⁃type(WT)and gain⁃of⁃function(GOF)ADAMTS13 interaction with VWF A2.Methods The adhesion frequency,rupture force,and bond lifetime between WT or GOF ADAMTS13 and VWF A2 under different external forces were measured by atomic force microscope(AFM).The kinetic parameters were derived by fitting with the Bell⁃Evans model.Results The widths of the potential barrier along the direction of force were 0.41 nm and 0.29 nm,and the dissociation rates under 0 N force were 1.50 s^(-1) and 3.28 s^(-1) for the WT ADAMTS13⁃VWF A2 complex and the GOF ADAMTS13⁃VWF A2 complex,respectively.Furthermore,the lifetime of bond and dissociation rate of the complexes under different applied forces were measured by AFM clamp mode.The result revealed that the interaction between WT or GOF ADAMTS13 and VWF A2 exhibited the characteristics of biphasic force⁃dependent‘catch⁃slip’transition bond.Conclusions The mechanical strength and stability of the WT ADAMTS13⁃VWF A2 complex are higher than those of the GOF ADAMTS13⁃VWF A2 complex.Both the binding of these two complexes exhibits the mechanical response characteristics of the‘catch⁃slip’transition bond.The research findings contribute to further understanding the interaction between ADAMTS13 and VWF,so as to provide new ideas for the development of antithrombotic drugs.