Sequencing of the primary adhesion domain of bovine von Willebrand factor.

A cDNA library, constructed from bovine heart endothelial cell poly(A)+ RNA, was screened using a BstXI fragment of human von Willebrand and factor (vWF) cDNA as a probe. This probe codes for the major adhesion domain of vWF that includes the GPIb, collagen and heparin binding domains. Of the ten positive clones obtained, a clone that spanned the region of interest was sequenced by the dideoxynucleotide method yielding a sequence of 1550 bp. This region of the bovine cDNA codes for amino acids corresponding to #262 to #777 in human vWF and encompasses the entire pro adhesion domain. Both the nucleotide sequence and the deduced amino acid sequence are 82% homologous to those of human vWF. Cysteine residues #471, 474, 509 and 695, which form intrachain bonds in human vWF, are also present in the bovine vWF sequence.     

Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin, and collagen and characterization of its three distinct functional sites

We have used purified proteolytic fragments of von Willebrand factor (vWF) to characterize three related functional sites of the molecule that support interaction with platelet glycoprotein Ib, collagen, and heparin. A fragment of 116 kDa was found to be dimeric and consisted of disulfide-linked subunits which, after reduction and alkylation, corresponded to the previously described 52/48-kDa fragment extending from residue 449 to 728. Fragment III-T2, also a dimer, was composed of two pairs of disulfide-linked subunits, two 35-kDa heavy chains (residues 273-511) and two 10-kDa light chains (residues 674-728). The 116-kDa fragment, but not the constituent 52/48-kDa subunit, supported ristocetin-induced platelet aggregation and retained 20% (on a molar basis) of the ristocetin cofactor activity of native vWF; fragment III-T2 retained less than 5% activity. All three fragments, however, inhibited vWF interaction with glycoprotein Ib. Both 116-kDa and 52/48-kDa fragments inhibited vWF binding to heparin with similar potency, while fragment III-T2 had no effect in this regard. Only the 116-kDa fragment inhibited vWF binding to collagen. These results indicate that dimeric fragments containing two glycoprotein Ib-binding sites possess the minimal valency sufficient to support ristocetin-induced aggregation. The sequence comprising residues 512-673, missing in fragment III-T2, is necessary for binding to heparin and collagen and may be crucial for anchoring vWF to the subendothelium. Immunochemical and functional data suggest that the same sequence, although not essential for interaction with glycoprotein Ib, may influence the activity of the glycoprotein Ib-binding site. Only binding to collagen has absolute requirement for intact disulfide bonds. Thus, the three functional sites contained in the 116-kDa domain of vWF are structurally distinct.     

Recombinant von Willebrand factor Arg578-->Gln. A type IIB von Willebrand disease mutation affects binding to glycoprotein Ib but not to collagen or heparin.

von Willebrand factor (vWF) is a multimeric plasma glycoprotein that mediates platelet adhesion to the subendothelium via binding to platelet glycoprotein Ib (GPIb) and to components of the vessel wall. Recently, missense mutations that cause type IIB von Willebrand disease (vWD) were described, clustered within a disulfide loop in the A1 domain of vWF that has binding sites for GPIb, collagen, and heparin. In type IIB vWD, plasma vWF exhibits increased affinity for platelet GPIb, but decreased binding to collagen and heparin. The effect was studied of a type IIB vWD mutation, Arg578-->Gln, on the interaction of vWF with GPIb, collagen, and heparin. Recombinant wild type rvWF and mutant rvWF(R578Q) were expressed in COS-7 cells. Ristocetin-induced binding of rvWF(R578Q) to GPIb was markedly increased compared with rvWF, confirming that the Arg578-->Gln mutation causes the characteristic gain-of-function abnormality of type IIB vWD; botrocetin-induced binding was only slightly increased. Binding to collagen type III and heparin-agarose was compared for rvWF(R578Q) and plasma vWF from patients with four different type IIB mutations: Arg543-->Trp, Arg545-->Cys, Val553-->Met, Arg578-->Gln. For all of the plasma samples, binding to collagen and to heparin was reduced compared with normal plasma. In contrast, binding of rvWF(R578Q) to collagen and heparin was normal compared with wild type rvWF. Therefore, the Arg578-->Gln mutation increases the affinity of vWF for GPIb but does not directly impair vWF interaction with collagen or heparin. Arg578 may therefore be necessary to prevent normal vWF from interacting with GPIb. In type IIB vWD, the defective binding of plasma vWF to collagen and heparin may be secondary to post-synthetic modifications that occur in vivo, such as the loss of high molecular weight vWF multimers.     

Functional analysis of a recombinant glycoprotein Ib alpha polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen.

By deletion mutagenesis and transient expression in COS cells, a 96-amino acid hydrophilic sequence in the glycoprotein Ib alpha polypeptide located between L220 and L318 was identified which appeared to contain its von Willebrand factor- (vWF) binding site. The cDNA encoding this fragment was then expressed in Escherichia coli and purified from the bacterial cell lysate. The recombinant polypeptide, rGpIb alpha Q221-L318, was monomeric and had an apparent molecular weight of 14,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It inhibited both ristocetin-induced binding of 125I-vWF to fixed washed platelets and ristocetin-induced platelet agglutination. The recombinant polypeptide also inhibited the binding of 125I-vWF to immobilized type I and III collagen. Inhibition of 125I-vWF binding to platelets and collagen was dose-dependent, with IC50 values of 500 and 200 nM rGpIb alpha Q221-L318, respectively. Fifty % inhibition of ristocetin-induced platelet agglutination required 500 nM rGpIb alpha Q221-L318. Although rGpIb alpha Q221-L318 inhibited vWF binding to collagen it did not, itself, bind to collagen-coated surfaces. Reduction of the disulfide bond between C248 and C264 abolished activity. 125I-rGpIb alpha Q221-L318 bound directly to GpIb/IX sites on multimeric vWF. These studies document that a portion of the sequence between Q221 and L318 is needed for recognition and binding to vWF and that binding requires an intact disulfide bond between C248 and C264. The binding of this recombinant polypeptide to vWF multimers inhibits vWF interaction with two important substrates, platelet GpIb/IX and collagen.     

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.     

Proteolytic studies on the structure of bovine von Willebrand factor

Bovine von Willebrand factor (vWF) was digested with protease I (P-I), a metalloprotease isolated from rattlesnake venom. Digestion of vWF for 24 h with P-I 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- and 61-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 human platelets, with an IC50 of 12.5 micrograms/mL (65 nM). However, native vWF has an IC50 of 2.5 micrograms/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 Ib (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 P-I 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-kDa terminal 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.(ABSTRACT TRUNCATED AT 400 WORDS)     

人内皮抑素在毕赤酵母中的表达、纯化与生物功能研究

内皮抑素（Endostatin）是近年来新发现的一种内源性新生血管生成（Angiogenesis）抑制因子,通过抑制新血管生成而抑制肿瘤的形成和转移且不会引起耐药性,具有极高的临床应用前景。巴斯德毕赤酵母（Pichia pastoris)具有表达率高、产物可分泌、可对高等真核生物蛋白正确进行翻译后加工、遗传稳定、发酵工艺成熟等优点被用来进行重组人Endostatin的表达。本研究用PCR的方法从人胎肝cDNA文库中扩增出人Endostatin的cDNA,测序正确后转入毕赤巴斯德甲醇酵母,并获得了高效可溶型表达,用肝素亲和层析的方法进行纯化,纯化后产物经SDSPAGE薄层扫描分析纯度达987％以上,质谱测定分子量为2043kD与理论值一致,蛋白质N端序列测定结果为SPPAHTHRDFQPVLH与天然序列一致。生物活性检测证明可抑制鸡胚尿囊绒毛膜（CAM）的新生血管生成（Angiogenesis),并可抑制血管内皮细胞的增殖。因此用酵母表达系统可以得到具有生物活性的内皮抑素,经纯化后可用于进一步的生物功能和作用机理试验。     

人von Willebrand因子A1和A3功能结构域嵌合体的表达及生物学功能

vWF介导血小板粘附到细胞外基质 ,在血栓形成过程中发挥重要作用 ,可通过阻断vWF与细胞外基质的结合阻止血小板的粘附 .应用RT PCR方法从人脐静脉内皮细胞中克隆vWF分子A1、A3区基因并在大肠杆菌中表达 ,表达的重组蛋白量占菌体总蛋白 12 6 % ,包涵体经过变性剂溶解、纯化和复性 ,获得重组蛋白 (rvWF A1 A3) .应用流式细胞术检测rvWF A1 A3与血小板膜糖蛋白 (GPⅠb)的结合功能 ;血小板聚集实验观察rvWF A1 A3对瑞斯托霉素 (ristocetin)诱导的血小板聚集 (RIPA)的影响 ;ELISA胶原结合实验及竞争抑制实验分析rvWF A1 A3与胶原的结合活性 .结果显示 :rvWF A1 A3嵌合体与血小板的结合阳性率为 70 4 % .rvWF A1 A3嵌合体不能引起血小板的聚集 ,但rvWF A1 A3嵌合体与血小板温育后可以阻断ristocetin诱导人血浆vWF对血小板的聚集作用 ,而且呈剂量依赖性 ,IC50 的rvWF A1 A3浓度为 0 76 μmol L ,当浓度为 1 17μmol L时抑制率最高达 76 8% .rvWF A1 A3具有良好的胶原结合活性 ,同时它可以竞争性抑制vWF与Ⅲ型胶原的结合 ,抑制率为 76 % .表明rvWF A1 A3可作为阻断剂用于干预vWF介导的血小板粘附过程 ,同时又可以阻断血浆vWF与血小板GPIb结合抑制血小板聚集 ,具有良好的抗栓应用前景 .     

A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor

We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal 272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two-dimensional crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease (SP), FVIII binding was seen only with the amino-terminal SP fragment III and not with the carboxyl-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed to polystyrene beads. This binding was inhibited in a dose-dependent manner by whole vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced and alkylated whole vWF. SP fragment II caused only minimal inhibition. Trypsin cleavage of SP fragment III produced a monomeric 35-kDa fragment containing the amino-terminal 272 amino acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited the binding of FVIII to vWF in a dose-dependent manner. These studies demonstrate that a major FVIII binding site resides within the amino-terminal 272 amino acid residues of vWF.     

Primary structure of the heparin-binding site of type V collagen

The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.     

人vWF-A1多肽的融合表达及生物学活性鉴定

血管性血友病因子 (vWF)通过与血小板膜糖蛋白结合介导血小板的粘附和聚集 ,在血栓形成过程中发挥重要作用 .通过阻断血小板与vWF的结合可抑制血栓形成 .应用RT PCR方法从人脐带内皮细胞中克隆vWF A1区基因并在原核细胞内进行表达 ,经过纯化、复性 ,获得重组蛋白(rvWF A1) .用流式细胞术检测rvWF A1与转染了糖蛋白Ib(GPⅠb)的CHO K1细胞和血小板GPⅠb的结合能力 ,血小板聚集仪测定rvWF A1对瑞斯托霉素 (ristocetin)诱导的血小板聚集作用的影响 .重组表达载体pET 2 0b(+ ) vWF A1在大肠杆菌BL2 1(DE3)plus中得到有效表达 ,表达的重组蛋白量占菌体总蛋白 30 % .次氮基三乙酸镍琼脂糖 (Ni NTAagarose)柱纯化后 ,其纯度为 95 % .经复性的rvWF A1蛋白具有良好的生物学活性 ,它可与转染了GPⅠb的CHO K1细胞和血小板结合 ,阳性率分别为 96 90 %与 78 6 0 % ,且可以抑制ristocetin诱导的血小板聚集 ,其抑制效应呈剂量依赖性 .IC50 的rvWF A1浓度为 0 5 6 μmol L ,当浓度为 1 4 μmol L时抑制率最高达 84 70 % .结果表明 ,在原核细胞中表达人rvWF A1区蛋白可抑制血浆中野生型vWF与血小板的结合 ,具有抗血栓形成的潜在应用前景     

Identification of the collagen-binding site of the von Willebrand factor A3-domain

Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962-966, 981-997, and 1022-1026. Alanine mutants were constructed of residues Arg(963), Glu(987), His(990), Arg(1016), and His(1023), located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His(1023) abolished collagen binding, whereas mutation of Arg(963) and Arg(1016) reduced collagen binding by 25-35%. These residues are part of loops alpha3beta4 and alpha1beta2 and alpha-helix 3, respectively, and lie near the bottom face of the domain. His(1023) and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains.     

Dual polarization interferometry characterization of carbohydrate-protein interactions

Dual polarization interferometry (DPI) is an analytical technique that allows the simultaneous determination of thickness, density, and mass of a biological layer on a sensing waveguide surface in real time. The technique was applied to the analysis of carbohydrate-protein interactions. The selected system involved a 12-kDa recombinant fragment of collagen V (HepV) and heparin, a complex polysaccharide. Here we report on the analysis of thickness, density, and mass of surface structures obtained during the binding of HepV to heparin, which is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. This system, which was initially studied for its biological relevance, displayed anomalous behavior in kinetic studies using surface plasmon resonance (SPR) assays that has been attributed to putative conformational changes. It was this putative conformational change that prompted us to investigate the binding using an alternative analytical approach. While using DPI to monitor binding events, a streptavidin layer (surface coverage 2.105 ng mm(-2)) was bound to the sensor surface (92% coverage), which captured 0.105 ng mm(-2) of biotinylated heparin (a stoichiometric ratio of 1:6 heparin-streptavidin). The heparin inserted into the streptavidin layer but was still found to be capable of binding 0.154 ng mm(-2) of HepV, which was also observed to insert into the streptavidin layer. This allowed the reliable calculation of the stoichiometric ratio for the HepV-heparin complex ( approximately 1.7:1.0), which has proved to be difficult to evaluate by SPR assays. Furthermore, real-time analysis of the heparin-HepV interaction by DPI suggested that there was some surface loss (probably of streptavidin) while the binding was occurring rather than the putative conformational change that has been suggested on the basis of kinetic data alone. This gives further insight into the binding mechanism of HepV to heparin.     

Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ib alpha demonstrating negatively charged residues involved in von Willebrand factor binding.

We have expressed in mammalian cells a fragment (residues 1-302) of the alpha chain of platelet glycoprotein (GP) Ib containing the von Willebrand factor- (vWF) binding site. The secreted soluble protein had an apparent molecular mass of 45 kDa and reacted with conformation-dependent monoclonal antibodies that bind only to native GP Ib, thus demonstrating its proper folding. After insolubilization on nitrocellulose membrane, the recombinant GP Ib alpha fragment bound soluble vWF in the presence of ristocetin or botrocetin with a dissociation constant similar to that exhibited by GP Ib.IX complex on platelets. Moreover, the interaction was blocked by anti-GP Ib monoclonal antibodies known to inhibit vWF binding to platelets. The sequence of GP Ib alpha between residues 269-287 has a strong net negative charge due to the presence of 10 glutamic or aspartic acid residues; 5 of these are contained in the sequence of a synthetic peptide (residues 251-279) previously shown to inhibit vWF-platelet interaction. In order to evaluate the possible functional role of these acidic residues, we employed site-directed mutagenesis to express two mutant GP Ib alpha fragments containing asparagine or glutamine instead of aspartic or glutamic acid, respectively. Mutant 1, with substitutions between residues 251-279, failed to bind vWF whether in the presence of ristocetin or botrocetin; in contrast, vWF binding to Mutant 2, with substitutions between residues 280-302, was nearly normal in the presence of ristocetin, but markedly decreased in the presence of botrocetin. Thus, mammalian cells transfected with a truncated cDNA sequence encoding the amino-terminal domain of GP Ib alpha synthesize a fully functional vWF-binding site; acidic residues in the sequence 252-287 are essential for normal function.     

Effect of glycosaminoglycans on fibronectin-mediated cell attachment

In this study, the role of glycosaminoglycans in fibronectin-mediated cell attachment to collagen has been investigated. While it has been established that fibronectin possesses binding sites for several glycosaminoglycans, it was found that only dextran sulfate and macromolecular heparin could decrease the initial rate of cell attachment to collagen. Low molecular weight heparin was inactive. This study suggests that the glycosaminoglycan binding site of fibronectin plays a role in the mechanism of cell attachment.     

Cloning,characterization, and functional studies of a 47-kDa platelet receptor for type III collagen

A 1.2-kb cDNA fragment encoding a platelet 47-kDa protein has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide of the sequenced amino terminus of the purified platelet protein with a poly(dT)(12).(dG) by polymerase chain reaction. A computer search revealed that the cDNA represents the coding sequence of a protein with a fragmentary homology to several proteins. Using a prokaryotic expression system, pBad TOPO-47 cDNA, a 47-kDa recombinant protein was obtained and purified to apparent homogeneity by nickel-nitrilotriacetic acid resin and collagen affinity column. The recombinant protein binds to type III but not type I collagen-Sepharose 2B affinity columns. Anti-47-kDa but not anti-65-kDa antibody inhibits the binding of the recombinant protein to the type III collagen-coated micro titer wells in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type III collagen-induced platelet aggregation also in a dose-dependent manner. We have defined two active peptides from the cloned deduced amino acid sequence. Both peptides inhibit type III but not type I collagen-induced platelet aggregation in a dose-dependent fashion. These results suggest that the active binding site of the platelet receptor to type III collagen resides in these portions of the protein.     

Full-length von Willebrand factor (vWF) cDNA encodes a highly repetitive protein considerably larger than the mature vWF subunit.

Full-length human von Willebrand factor (vWF) cDNA was assembled from partial, overlapping vWF cDNAs. This cDNA construct includes a coding sequence of 8439 nucleotides which encode a single-chain precursor of 2813 amino-acid residues, representing a putative signal peptide, a prosequence and mature vWF of 22, 741 and 2050 amino acids, respectively. This represents the longest coding sequence determined to date. In-vitro expression of full-length vWF cDNA revealed the synthesis of a polypeptide with a mol. wt corresponding with that of the unglycosylated precursor. The precursor is a highly repetitive protein which consists of two duplicated (B, C), a triplicated (A), a quadruplicated (D) and a partly duplicated domain (D'), in the following order: H-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-C1-C2-OH. Both the prosequence, composed of two D domains (D1, D2), and mature vWF harbor an arg-gly-asp ('R-G-D') sequence which has been implicated in cell-attachment functions. It is argued that the pro-sequence is equivalent to von Willebrand Antigen II (vW AgII).     

Heparin type IV collagen interactions: equilibrium binding and inhibition of type IV collagen self-assembly

Interactions between type IV collagen and heparin were examined under equilibrium conditions with rotary shadowing, solid-phase binding assays, and affinity chromatography. With the technique of rotary shadowing and electron microscopy, heparin appeared as thin, short strands and bound to the following three sites: the NC1 domain, and in the helix, at 100 and 300 nm from the NC1 domain. By solid-phase binding assays the binding of [3H]heparin in solution to type IV collagen immobilized on a solid surface was found to be specific, since it was saturable and could be displaced by an excess of unlabeled heparin. Scatchard analysis indicated three classes of binding sites for heparin-type IV collagen interactions with dissociation constants of 3, 30, and 100 nM, respectively. Furthermore, by the solid-phase binding assays, the binding of tritiated heparin could be competed almost to the same extent by unlabeled heparin and chondroitin sulfate side chains. This finding indicates that chondroitin sulfate should also bind to type IV collagen. By affinity chromatography, [3H]heparin bound to a type IV collagen affinity column and was eluted with a linear salt gradient, with a profile exhibiting three distinct peaks at 0.18, 0.22, and 0.24 M KCl, respectively. This suggested that heparin-type IV collagen binding was of an electrostatic nature. Finally, the effect of the binding of heparin to type IV collagen on the process of self-assembly of this basement membrane glycoprotein was studied by turbidimetry and rotary shadowing. In turbidity experiments, the presence of heparin, even in small concentrations, drastically reduced maximal aggregation of type IV collagen which was prewarmed to 37 degrees C. By using the morphological approach of rotary shadowing, lateral associations and network formation by prewarmed type IV collagen were inhibited in the presence of heparin. Thus, the binding of heparin resulted in hindrance of assembly of type IV collagen, a process previously described for interactions between various glycosaminoglycans and interstitial collagens. Such regulation may influence the assembly of basement membranes and possibly modify functions. Furthermore, qualitative and quantitative changes of proteoglycans which occur in certain pathological conditions, such as diabetes mellitus, may alter molecular assembly and possibly permeability functions of several basement membranes.     

Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains

The effects of Staphylococcus aureus V8 protease (V8) on the multimeric structure of human von Willebrand factor (vWF) were studied to test and expand our model for the substructure of vWF. Electron microscopy of V8 digests of vWF revealed that the multimers were cleaved where the flexible rod (R) domains join the large elongated globular (G) domains. The resulting two major fragments, which were purified by affinity and hydrophobic interaction chromatography and by glycerol-gradient ultracentrifugation, are disulfide-linked homodimers of these domains (i.e. RR and GG) and are morphologically identical to the alternating RR and GG domains of intact vWF. The glycoprotein fragment GG (6.5 X 35 nm) has mass 343 kDa by sedimentation equilibrium and the amino-terminal sequence of intact plasma vWF. It contains the binding site for heparin within 300 residues of its amino terminus and a separate site for the platelet GPIb receptor responsible for platelet agglutination in the presence of ristocetin. With approximately 18% alpha-helix and approximately 15% beta-pleated sheet, fragment GG accounts for most of the ordered secondary structure present in whole vWF. The two thin flexible rod domains (1.8-2.0 X 30-34 nm) of fragment RR are joined at a small central nodule (approximately 5 nm diameter) and also have a small nodule at each free end. Fragment RR contains an extraordinarily high cystine content, lower than average amounts of other hydrophobic residues, and essentially no alpha-helix, as judged by circular dichroism. The amino-terminal sequence and amino acid composition of fragment RR corresponded to that of the COOH-terminal 685 residues of the intact vWF subunit (Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., and Fujikawa, K. (1986) Biochemistry 25, 3171-3184). This sequence analysis gives a mass of 180 kDa for glycosylated fragment RR, somewhat higher than the 130 kDa we obtained by sedimentation equilibrium. Our sequence analysis of a 110-kDa plasmic vWF peptide also permitted identification of a major plasmin cleavage site 705 residues from the COOH terminus and a half-cystine residue (1360) involved in maintaining the multimeric structure of plasmin-degraded vWF.(ABSTRACT TRUNCATED AT 400 WORDS)