From the Experimental Dermatology Division, Department of Dermatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany (S.G., V.H., K.I.P., C.G., S.W.S.); Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany (C.A.-S., F.G.); Theory Department, Fritz Haber Institute of the Max Planck Society, Berlin, Germany (C.B.); and Department of Pediatric Hematology and Oncology, University Medical Center, Hamburg-Eppendorf, Hamburg, Germany (R.S., T.O.).
Arterioscler Thromb Vasc Biol. 2014 Jul;34(7):1382-9. doi: 10.1161/ATVBAHA.113.303016. Epub 2014 May 1.
Inflammatory conditions provoke essential processes in the human vascular system. It leads to the formation of ultralarge von Willebrand factor (VWF) fibers, which are immobilized on the endothelial cell surface and transform to highly adhesive strings under shear conditions. Furthermore, leukocytes release a meshwork of DNA (neutrophil extracellular traps) during the process of the recently discovered cell death program NETosis. In the present study, we characterized the interaction between VWF and DNA and possible binding sites to underline the role of VWF in thrombosis and inflammation besides its function in platelet adhesion.
Both functionalized surfaces and intact cell layers of human umbilical vein endothelial cells were perfused with isolated, protein-free DNA or leukocytes from whole blood at distinct shear rates. DNA-VWF interaction was monitored using fluorescence microscopy, ELISA-based assays, molecular dynamics simulations, and electrostatic potential calculations. Isolated DNA, as well as DNA released by stimulated leukocytes, was able to bind to shear-activated, but not inactivated, VWF. However, DNA-VWF binding does not alter VWF degradation by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13. Moreover, DNA-VWF interaction can be blocked using unfractionated and low-molecular-weight heparin, and DNA-VWF complexes attenuate platelet binding to VWF. These findings were supported using molecular dynamics simulations and electrostatic calculations of the A1- and A2-domains.
Our findings suggest that VWF directly binds and immobilizes extracellular DNA released from leukocytes. Therefore, we hypothesize that VWF might act as a linker for leukocyte adhesion to endothelial cells, supporting leukocyte extravasation and inflammation.
炎症会引发人体血管系统的基本过程。这会导致超大 von Willebrand 因子 (VWF) 纤维的形成，这些纤维固定在血管内皮细胞表面，并在切变条件下转化为高粘性的纤维。此外，白细胞在最近发现的细胞死亡程序 NETosis 过程中释放 DNA 网（中性粒细胞胞外诱捕网）。在本研究中，我们描述了 VWF 与 DNA 的相互作用以及可能的结合位点，以强调 VWF 在血栓形成和炎症中的作用，以及其在血小板黏附中的功能。
用分离的、无蛋白的 DNA 或全血中的白细胞以不同的切变率灌注人脐静脉内皮细胞的功能化表面和完整细胞层。使用荧光显微镜、基于 ELISA 的测定、分子动力学模拟和静电势计算监测 DNA-VWF 相互作用。分离的 DNA 以及受刺激的白细胞释放的 DNA 能够与剪切激活但不失活的 VWF 结合。然而，DNA-VWF 结合不会改变 a 型血小板反应蛋白 13 金属蛋白酶与血小板反应蛋白 1 型基序（a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13）对 VWF 的降解。此外，使用未分级和低分子量肝素可以阻断 DNA-VWF 相互作用，并且 DNA-VWF 复合物可减弱血小板与 VWF 的结合。这些发现得到了分子动力学模拟和 A1 域和 A2 域的静电计算的支持。
我们的研究结果表明，VWF 直接结合并固定从白细胞释放的细胞外 DNA。因此，我们假设 VWF 可能作为白细胞与内皮细胞黏附的连接物，支持白细胞渗出和炎症。
