Tsai H M, Sussman I I, Nagel R L
Division of Hematology, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY 10467.
Blood. 1994 Apr 15;83(8):2171-9.
While von Willebrand factor (vWF) is secreted from endothelial cells as a very large polymer, it circulates as a series of multimers that are reducible to a 225-kD polypeptide and three proteolytic fragments of 189, 176, and 140 kD. Cleavage at the Tyr-842/Met-843 bond of the vWF polypeptide creates the 140- and 176-kD fragments. In the process of understanding vWF multimer formation, the role of shear stress in vWF proteolysis was investigated in this study. A shear-rate-dependent loss of the largest multimers was observed when normal plasma was perfused through long capillary tubings achieving shear rates normally encountered in the circulation. The shear-dependent vWF change was not observed when purified vWF or normal plasma containing calcium chelator EGTA or EDTA was perfused. As the large multimers decreased, an increase in the smaller multimers, including 200- and 350-kD bands, was detected. Elution and immunoblotting studies with peptide-specific antibodies LJ-7745 and VP-1 showed that the 200-kD band was a dimer of the 140-kD fragment, whereas the 350-kD band was a dimer of the 176-kD fragment. When analyzed after disulfide bonds were reduced, sheared plasma showed an increase in the 176- and 140-kD fragments, but not the 189-kD fragment. Finally, shearing of purified vWF enhanced its proteolytic cleavage when it was subsequently incubated with the cryosupernatant fraction of normal plasma or with cathepsin G, a leukocyte granule serine protease. These results show that shear stress is capable of enhancing the susceptibility of vWF to proteolytic cleavage. It promotes vWF proteolysis in normal plasma at a site that generates the 140-kD/176-kD fragments, leading to a decrease in multimer size. Shear stress might be involved in modulating the size of vWF in the circulation.
虽然血管性血友病因子（vWF）以内皮细胞分泌的一种非常大的聚合物形式存在，但它以一系列多聚体的形式循环，这些多聚体可还原为一条225-kD的多肽和189-kD、176-kD和140-kD的三个蛋白水解片段。vWF多肽在Tyr-842/Met-843键处的切割产生了140-kD和176-kD的片段。在了解vWF多聚体形成的过程中，本研究对剪切应力在vWF蛋白水解中的作用进行了研究。当正常血浆通过长毛细管灌注，达到循环中通常遇到的剪切速率时，观察到最大多聚体的剪切速率依赖性损失。当灌注纯化的vWF或含有钙螯合剂EGTA或EDTA的正常血浆时，未观察到剪切依赖性vWF变化。随着大多聚体减少，检测到较小多聚体增加，包括200-kD和350-kD条带。用肽特异性抗体LJ-7745和VP-1进行的洗脱和免疫印迹研究表明，200-kD条带是140-kD片段的二聚体，而350-kD条带是176-kD片段的二聚体。当二硫键还原后进行分析时，剪切后的血浆中176-kD和140-kD片段增加，但189-kD片段未增加。最后，纯化的vWF在随后与正常血浆的冷冻上清部分或与组织蛋白酶G（一种白细胞颗粒丝氨酸蛋白酶）孵育时，其剪切增强了蛋白水解切割。这些结果表明，剪切应力能够增强vWF对蛋白水解切割的敏感性。它在正常血浆中促进vWF在产生140-kD/176-kD片段的位点进行蛋白水解，导致多聚体大小减小。剪切应力可能参与调节循环中vWF的大小。
