Study on functinal domains of von Willebrand (vW) factor in patients with vW disease.

一、血管性血友病因子(VWF)功能结构域的分析及抗vWf.A.单抗的制备和鉴定VWF-纤溶酶或胰酶消化片段p-34(氨基酸残基1-298(273))经DEAE和肝素柱纯化。4种不同的抗p-34的单抗是由法国INSERM 143的D.Meyer与我合作生产的。现在，我将描述这些抗体的特征。血小板膜糖蛋白Ib(GPIB)结合域：两种不同的单抗(NMC/vW4来自我们实验室。322)对抑制VWF与GPIB结合的VWF的作用进行了鉴定，发现两种诱导剂，如抗生素司他西汀和蛇毒因子Botrocetin，与VWF结合的功能部位不同。已知金黄色三羧酸(ATA)能抑制瑞沙西汀诱导的血小板聚集，但不能抑制其他血小板聚集物。进一步研究了ATA阻断VWF-血小板相互作用的能力，发现ATA通过干扰VWF而不是与血小板来阻断VWF/GPIB通路，是预防血栓形成C早期的潜在工具。人胶原结合结构域：发现一株能抑制VWF-人III型胶原纤维结合的单抗(NMC/vW3)，其表位位于VWF亚基。即表位定位于911~1365位氨基酸残基之间。建立了多种VWF功能检测方法和VWF蛋白分析方法，对正常人和几种类型的von Willebrand病患者的血浆VWF进行了研究。发现II型vWD患者存在胶原结合异常。B.建立了微量样品分析血浆VWF亚基的方法。现在我们来检测几种类型vWD的血浆VWF。

I. The analysis of functional domains of von Willebrand factor(vWf) and the production and characterization of monoclonal antibodies (MAbs) against vWf.A. the factorVIII-binding domain: vWf-plasmin or trypsin digested fragment p-34(amino acid residue 1-298(273)) was purified using DEAE and Heparin column. 4 different MAbs against p-34 were produced by D.Meyer, INSERM 143 France, collaborated with me. Now, I'm going to characterize these antibodiesB. Platelet glycoprotein Ib(GPIb)-binding domain: Two different MAbs (NMC/vW4 from our lab. 322 from france) against vWf which inhibit the binding of vWf to GPIb were characterized, and I discovered the different functional site of GPIb-binding of vWf by two inducers, such as antibiotics ristocetin and venom factor botrocetin. and Aurin tricarboxylic acid (ATA) is known to inhibit ristocetin-induced platelet aggregation but not the other platelet aggregaters. Its capacity to abolish human vWf-platelet interaction was further in vestigated and was found that ATA which blocks the vWf/GPIb pathway by interfering with vWf and not with platelet, is a potential tool in prevention the early stages of thrombosisC. Human collagen-binding domain: One MAb(NMC/vW3) which inhibit the vWf-human typeIII fibrilar collagen binding was discovered and was shown the epitope in vWf subunit. That is, The epitope was localized between the amino acid residue 911 and 1365.II. The analysis of plasma von Willebrand factor in von Willebrand disease. I studied on the establishment of several vWf function assays and vWf protein analysis of plasma vWf in normal and several types of von Willebrand disease.A. The vWf-human collagen binding assay, using a minute plasma was established. The abnormality of the collagen- binding in type II vWd was discovered.B. The analytical method of vWf subunit in plasma vWf using a minute sample was established. Now the plasma vWf in several types of vWd is going to examine.