Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pa.
Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pa.
J Thorac Cardiovasc Surg. 2015 Jan;149(1):281-9. doi: 10.1016/j.jtcvs.2014.09.031. Epub 2014 Sep 18.
Bleeding is an important source of morbidity in patients with a left ventricular assist device. Evidence suggests a major role for von Willebrand factor. However, limited data exist to explain the mechanism(s) of von Willebrand factor degradation during left ventricular assist device support. We investigated whether left ventricular assist device-related shear stress and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13, the von Willebrand factor protease) altered von Willebrand factor metabolism.
Whole blood was collected from patients (n = 8) with a left ventricular assist device. von Willebrand factor multimers and degradation fragments were characterized with electrophoresis and immunoblotting. To investigate mechanisms, an in vitro model was developed to generate the supraphysiologic shear stress of a continuous-flow left ventricular assist device. Normal human blood (n = 8) was cycled in a laboratory vortexer (∼2400 rpm, shear stress ∼175 dyne/cm(2), 4 hours) to reproduce the pathologic degradation of von Willebrand factor that occurs during left ventricular assist device support. To investigate the specific mechanistic roles of shear stress and ADAMTS-13 in von Willebrand factor degradation, purified von Willebrand factor protein ± ADAMTS-13 protease were exposed to supraphysiologic shear stress in the vortexer. von Willebrand factor multimers and 11 von Willebrand factor degradation fragments were characterized with electrophoresis and immunoblotting.
Left ventricular assist device support reduced large von Willebrand factor multimers and significantly increased 10/11 von Willebrand factor degradation fragments (P < .05). Normal human blood exposed to supraphysiologic shear stress in the vortexer demonstrated the same profile of von Willebrand factor degradation fragments as in a patient with a left ventricular assist device. Supraphysiologic shear stress alone caused modest mechanical demolition of large von Willebrand factor multimers into smaller multimers but did not greatly generate von Willebrand factor fragments. In the presence of supraphysiologic shear stress, ADAMTS-13 completely eliminated large von Willebrand factor multimers and generated statistically significant amounts of 11/11 von Willebrand factor degradation fragments (P < .05). The profile of von Willebrand factor fragments generated was identical to the profile that was observed in vivo in patients with a left ventricular assist device.
Supraphysiologic shear stress alone causes physical demolition of large von Willebrand factor multimers into smaller von Willebrand factor multimers. In the setting of supraphysiologic shear stress, ADAMTS-13 cleaves large von Willebrand factor multimers into von Willebrand factor degradation fragments. ADAMTS-13 may be a therapeutic target to reduce von Willebrand factor degradation and bleeding complications in patients with a left ventricular assist device.
出血是左心室辅助装置患者发病和致残的重要原因。有证据表明，血管性血友病因子（von Willebrand factor，VWF）在此过程中起着重要作用。然而，目前关于左心室辅助装置支持期间 VWF 降解机制的研究数据十分有限。本研究旨在探讨左心室辅助装置相关切变力和 a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13（ADAMTS-13，VWF 蛋白酶）是否会改变 VWF 的代谢。
采集 8 例左心室辅助装置患者的全血。采用电泳和免疫印迹法对 VWF 多聚体和降解片段进行特征分析。为了研究其机制，我们建立了体外模型以产生左心室辅助装置的超生理切变力。将正常人体血液（n=8）在实验室漩涡器（转速约 2400rpm，切变力约 175dyne/cm2，持续 4 小时）中循环，以模拟左心室辅助装置支持过程中 VWF 发生的病理降解。为了研究切变力和 ADAMTS-13 在 VWF 降解中的具体作用机制，将纯化的 VWF 蛋白±ADAMTS-13 蛋白酶暴露于漩涡器中的超生理切变力下。采用电泳和免疫印迹法对 VWF 多聚体和 11 种 VWF 降解片段进行特征分析。
左心室辅助装置支持降低了大 VWF 多聚体的含量，显著增加了 10/11 种 VWF 降解片段（P<0.05）。将正常人体血液暴露于漩涡器中超生理切变力下，也会出现与左心室辅助装置患者相同的 VWF 降解片段谱。单纯超生理切变力会导致大 VWF 多聚体发生适度的机械破坏，转化为较小的多聚体，但不会大量产生 VWF 片段。在超生理切变力存在的情况下，ADAMTS-13 可完全消除大 VWF 多聚体，并产生统计学意义上大量的 11/11 种 VWF 降解片段（P<0.05）。生成的 VWF 片段谱与在左心室辅助装置患者体内观察到的谱完全一致。
单纯超生理切变力会导致大 VWF 多聚体发生物理性破坏，转化为较小的 VWF 多聚体。在超生理切变力的作用下，ADAMTS-13 可将大 VWF 多聚体切割成 VWF 降解片段。ADAMTS-13 可能成为治疗靶点，以减少左心室辅助装置患者的 VWF 降解和出血并发症。
