Proteolytic studies on the structure of bovine von Willebrand factor.
Proteolytic studies on the structure of bovine von Willebrand factor.
复制标题
牛血管性血友病因子结构的蛋白水解研究。
DOI：
10.1021/bi00404a030
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Kirby,EP
中科院分区：
文献类型：
--
作者：
Mascelli,MA;Kirby,EP
Revised Manuscript Received October 16, 1987 abstract: Bovine von Willebrand factor (vWF) was digested with protease I (PI), a metalloprotease isolated from rattlesnake venom. Digestion of vWF for 24 h with PI yielded a terminal digest consisting of an equimolar mixture of two major fragments (apparent Mr 250K and 200K). The 250-kilodalton (kDa) fragment consists of a 125-kDa chain from one subunit and a 45-and 78-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment consists of a 97-kDa chain from one subunit and a 35-and61-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment binds to heparin, and the heparin binding domain is located on the 97-kDa polypeptide chain. This fragment also competes with labeled, native vWF for binding to formalin-fixed humanplatelets, with an IC50 of 12.5 pg/mL (65 nM). However, native vWF has an IC50 of 2.5 pg/mL, indicating that the affinity of the 200-kDa fragment for platelets is approximately one-fifth that of vWF. The 200-kDa fragment agglutinates platelets, but its agglutinating ability is only 5% that of the native molecule. Only the 200-kDa fragment is recognized by monoclonal antibodies 2 and H-9, which are directed against vWF and inhibit vWF binding to platelet glycoprotein lb (GPIb). Immunological studies, using nine monoclonal antibodies directed against vWF, and the demonstration that the heparin and GPIb binding domains are located on only one fragment suggest that the two fragments are composed of different regions of the vWF subunit. Analysis of the PI cleavage pattern suggests that all vWF subunits are not cleaved in the same fashion. The first cleavage on half of the subunits generates the 45-kDaterminal and 175-kDa intermediate digest products. The 175-kDa chain is again cleaved, producing the 97-and 78-kDa terminal polypeptide chains. However, the first cleavage of the other subunits generates the 35-kDa terminal and the 186-kDa intermediate digest product, which upon cleavage produces the 125-and 61-kDa terminal polypeptide chains. Immunological data support the asymmetric cleavage pattern. An epitope for a monoclonal antibody is present on both the 186-and 175-kDa intermediate digest products but is only found on one terminal digest fragment, the 78-kDa polypeptide chain, suggesting that the 186-and 175-kDa polypeptides are cleaved at different sites. Epitope mapping of the fragments also indicates that they share a common region of the subunit. The presence of an overlapping region is possible if adjacent fragments are not cleaved in the same fashion. The basic protomer of vWF is a dimer of 230-kDa polypeptide chains. Analysis of proteolytic patterns of iodinated vWF suggests that physical differences exist between the two chains of a dimer. One of the chains contains a region which is highly susceptible to iodination; this region is located on the 175-kDa intermediate and 78-kDa terminal digest products. A structural model of bovine vWF has been developedin which the subunits are linked by disulfide bonds that alternatebetween two carboxyl-terminal and two amino-terminal regions in a head-to-head/tail-to-tail fashion. The proteolytic cleavage patterns and preferential iodination of certain subunits indicate that structural differencesexist between adjacent subunits. von Willebrand factor (vWF) is a high molecular weight, oligomeric, plasma glycoprotein which is required for platelets to recognize damaged endothelial surfaces (Baumgartner et al., 1980). It can act as a bridge between platelet membranes and subendothelial tissue at sites of endothelial damage (Sakariassen et al., 1979). In the presence of ristocetin, vWF …
