Binding of the von Willebrand factor A1 domain to histone

Human cytochrome P450 2D6 metabolizes more than 50 common drugs and is polymorphically expressed, with 5-10% of the population lacking expression caused by mutant genes. This may result in a defective and toxic response in deficient individuals treated with 2D6 drug substrates. Baculovirus-expressed 2D6 was used to immunize mice for hybridoma production and two clones yielded monoclonal antibodies, that were positive against 2D6 by ELISA and inhibited 2D6 catalysed metabolism of bufuralol, dextromethorphan and phenanthrene by more than 90%. The inhibitory activity was highly specific to 2D6 and the monoclonal antibodies did not bind to 11 other P450s, nor inhibit seven human P450s tested. Analysis of eight human liver microsome samples showed that their basal bufuralol 1'-hydroxylase activity varied from 6.7-83.5 pmol min-1 nmol-1 P450. The monoclonal antibody 512-1-8 inhibited 2D6-dependent bufuralol 1'-hydroxylase in these samples by 10-70% indicating a widely variable role for 2D6 in human liver bufuralol 1'-hydroxylase activity and a role for other P450s in bufuralol metabolism. Independent analysis of several recombinant human P450s showed that 2D6, 2C8, 2C9, 2C19 and 1A2 exhibited bufuralol 1'-hydroxylase activity with 2D6 and 2C19 being the most active. Further analysis of three liver samples was made with individual inhibitory monoclonal antibodies. Inhibitory antibodies to 2D6, 2B6, 2E1, 2C8/9/19, 3A4 and 1A2 were added to the microsomes either singly or additively. Inhibitory activity of bufuralol 1'-hydroxylase was observed with antibodies to 2D6 (14-76%), 2C8/9/19 (24-69%) and 1A2 (2-25%) indicating a variable and different role for each of these P450s in the bufuralol 1'-hydroxylase of human liver. The monoclonal antibodies to 2B6, 2E1 and 3A4 were not inhibitory, indicating that these enzymes play no role in bufuralol 1'-hydroxylase metabolism. When the three antibodies to 2D6, 2C8/9/19 and 1A2, respectively, were all added, the total bufuralol 1'-hydroxylase of the liver samples was inhibited by more than 90%, indicating that the latter P450s catalyse all of liver bufuralol 1'-hydroxylase metabolism. These studies demonstrate that inhibitory monoclonal antibodies offer a simple and precise method for assessing the quantitative role of each P450 in the metabolism of a P450 substrate in a tissue, which include drugs, carcinogens, mutagens, toxic chemicals and endobiotics.     

Activation of human platelets by the membrane-expressed A1 domain of von Willebrand factor

Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF-A1-domains to interact with human platelets in vitro. Both human and porcine vWF-A1-domains expressed as glycosyl phosphatidylinositol-linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.     

Dominant type 1 von Willebrand disease caused by mutated cysteine residues in the D3 domain of von Willebrand factor

No defects have been reported in moderately severe type 1 von Willebrand disease (vWD) with a clear autosomal dominant inheritance pattern, and the mechanism underlying this form of vWD remains obscure. We have studied a type 1 vWD family with such a dominant phenotype. The entire coding sequence of the von Willebrand factor (vWF) gene was analyzed by direct sequencing of DNA fragments amplified by polymerase chain reaction. Only one candidate mutation T(3445)-->C in exon 26 was detected that predicts a replacement of cysteine (C) at position 386 of the mature vWF subunit by arginine (R). Both mutant and normal vWF alleles were expressed as shown by analysis of platelet mRNA. This substitution segregates with vWD in the family and was not found in 100 unrelated individuals. The recombinant mutant vWF(C386R) was characterized by expression in 293T cells. The secretion of vWF(C386R) was greatly impaired due to retention in the endoplasmic reticulum. In cotransfections of normal and mutant vWF constructs, the vWF(C386R) subunits caused a dose-dependent decrease in the secretion of vWF. The multimer pattern remained nearly normal and consistent with a dominant vWD type 1 phenotype. The importance of the cysteine residues in the D3 domain of vWF in the pathogenesis of dominant type 1 vWD was further shown by the detection of another cysteine mutation, Cys367-->Phe, in two additional unrelated patients with a similar dominant type 1 vWD phenotype. We conclude that the loss of cysteine pairing in the D3 domain, leaving one free cysteine, can induce a purely quantitative deficiency of vWF by dominantly suppressing the secretion of normal vWF.     

Mechanism of platelet aggregation induced by a monoclonal antibody requiring Fc portion

A monoclonal antibody designated Apt4, which is IgG1, was produced by fusion of mouse myeloma cells to spleen cells from a BALB/c mouse immunized with normal human platelets. Apt4 whole IgG caused the aggregation of both platelet rich plasma (PRP) and washed platelets from normal subjects and a patient with Bernard Soulier syndrome but not those from two patients with the Type 1 Glanzmann's thrombasthenia. No aggregation was observed when Apt4 F(ab')2 fragments were used. Immunofluorescence study showed that both whole IgG and F(ab')2 fragments of Apt4 bound to fresh or formalin fixed platelets from normal subjects and a patient with Bernard Soulier syndrome but not to those from two patients with Glanzmann's thrombasthenia. Aggregation induced by Apt4 IgG was inhibited by EDTA (10 mM), PGE1 (1 mM), 2-deoxy-D-glucose/antimycin (1.4 uM), and apyrase (20 units/ml). Preincubation of normal PRP with monoclonal anti-GPIIb/IIIa or anti-GPIb antibodies completely or partially inhibited the Apt4-induced aggregation, whereas anti-GPIIIa antibodies have no effects on this activation. Monoclonal ant-Fc gamma RII antibody (IV.3) inhibited Apt4 induced aggregation. Immunoprecipitation of 125I-labeled platelet membrane lysate by Apt4 IgG showed two protein bands with a molecular weight of 145,000 and 95,000 daltons respectively under non-reducing condition, which are corresponding to GPIIb and GPIIIa. In conclusion, Apt4 antibody binds to GPIIb/IIIa complex and induces aggregation, requiring energy metabolism, calcium, ADP release and Fc portion of IgG to interact with Fc receptor, but independent of thromboxane A2 formation.     

Conformational changes in the A1 domain of von Willebrand factor modulating the interaction with platelet glycoprotein Ibalpha

The interaction between von Willebrand factor (vWF) A1 domain and platelet glycoprotein Ib alpha occurs in the presence of high shear stress or when vWF becomes immobilized onto a surface but not appreciably in the normal circulation. To investigate the structural properties regulating A1 domain function, we have used recombinant fragments prepared either in cyclic form with oxidized Cys509-Cys695 disulfide bond or reduced and alkylated. Interaction with glycoprotein Ibalpha was assessed by testing inhibition of monoclonal antibody LJ-Ib1 binding to platelets and inhibition of shear-induced platelet aggregation mediated by native vWF. Fragments exposed to pH between 2.5 and 3.5 adopted the molten globule conformation with loosened tertiary structure intermediate between native and completely unordered state. Maximal receptor binding activity was observed when fragments kept at acidic pH, particularly after reduction of the Cys509-Cys695 disulfide bond, were subjected to quick refolding by rapid pH increase. In contrast, slow refolding by incremental pH change over several hours resulted in at least 20-fold lower activity. A specific single point mutation (I546V) resulted in enhanced receptor binding, whereas another mutation (S561G) caused markedly reduced binding. These results provide experimental evidence that conformational transitions can modulate function of the vWF A1 domain in solution.     

Crystal structure of NMC-4 fab anti-von Willebrand factor A1 domain

We have solved the crystal structure of the Fab fragment of NMC-4, a mouse monoclonal antibody that binds to the A1 domain of von Willebrand factor (vWF). Two Asp and three Tyr residues in the complementarity determining regions 1 and 3 of the heavy chain exhibited a spatial orientation suggestive of a dominant role in establishing contact with the antigen. A cluster of Asp and Tyr residues occurs also in a region of the platelet glycoprotein (GP) Ib alpha amino terminal domain known to be critically involved in vWF binding. Thus, the structural information obtained with NMC-4 may prove relevant to understand the stereochemical bases of the GP Ib alpha-vWF interaction essential for thrombus formation at sites of vascular lesion.     

von Willebrand disease in the RIIIS/J mouse is caused by a defect outside of the von Willebrand factor gene

An animal model for human type I von Willebrand disease (vWD) has been previously described in the inbred mouse strain RIIIS/J. Murine vWD is characterized by a prolonged bleeding time, normal von Willebrand factor (vWF) multimer distribution, autosomal dominant inheritance, and proportionately decreased plasma vWF antigen, ristocetin cofactor, and factor VIII (FVIII) activities. To study the molecular genetics of murine vWD, a portion of the vWF gene surrounding exon 28 was cloned, sequenced, and used to develop two informative DNA sequence polymorphisms for rapid genotyping by DNA polymerase chain reaction. RIIIS/J mice were crossed with PWK/Ph mice, an inbred line of Mus musculus musculus, and the F1 progeny backcrossed to the parental PWK/Ph strain. vWF antigen levels in F1 mice were not significantly different from the parental RIIIS/J strain but were markedly decreased compared with the parental PWK/Ph mice. Genetic linkage analysis of 104 backcross progeny showed no correlation between vWF antigen level and vWF genotype. These data indicate that murine vWD is caused by a defect at a novel genetic locus, distinct from the murine vWF gene. The distribution of vWF antigen levels among backcross progeny suggests the presence of one major dominant vWD gene in the RIIIS/J mouse with possible modifying contributions from one or more additional minor loci. These observations may provide new insights into the molecular basis and variable expressivity of human vWD.     

A novel mutation in the D3 domain of von Willebrand factor markedly decreases its ability to bind factor VIII and affects its multimerization

In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient's vWF gene showed, at heterozygous state, a G-->A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C-->T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient's vWF. The monoclonal antibody 31H3 against D' domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D' domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.     

von Willebrand factor as a regulator of intrinsic factor X activation

Factor VIII is an important cofactor in the intrinsic activation of factor X. To function effectively as a cofactor, factor VIII must be activated. In plasma, factor VIII circulates in a complex with von Willebrand factor, and although thrombin can activate complexed factor VIII, the activation by activated factor X is inhibited by von Willebrand factor. In this study, the effect of von Willebrand factor on the generation of factor Xa by the factor IXa-VIII complex was investigated. Purified human factors VIII, IXa, and X were incubated on human umbilical vein endothelial cells or phospholipid vesicles in the presence of calcium ions, and the generation of factor Xa was followed. In the presence of von Willebrand factor, a prolonged lag-phase and a dose-dependent inhibition of factor X activation was observed. These effects were not observed when von Willebrand factor was preincubated with a monoclonal antibody directed against von Willebrand factor that blocks factor VIII binding. When factor VIII was activated with thrombin before the incubation, neither the monoclonal antibody nor von Willebrand factor had an effect on the rate of factor X activation. Preincubation of endothelial cells with the monoclonal antibody resulted in a somewhat higher rate of factor X activation. When endothelial cells from a patient with von Willebrand's disease type I were used, preincubation of the monoclonal antibody had no effect on the rate of factor X activation. We conclude that von Willebrand factor on the surface of endothelial cells can modulate the intrinsic factor X activation. This effect is greatly enhanced, however, by the addition of exogenous von Willebrand factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary binding domain of bovine von Willebrand factor fragment expressed in E. coli

Bovine vWF cDNA has been cloned from a bovine endothelial cell library. A fragment of this cDNA, corresponding to amino acid sequence Leu 469-Ser 723, called primary adhesion domain (PAD-1), and containing the binding sites for platelet glycoprotein Ib (GPIb), heparin and collagen, has been expressed in E. coli. The reduced and alkylated form of fragment PAD-1 inhibited native vWF binding to GPIb. Fragment PAD-1 bound to heparin and botrocetin in a specific and dose dependent manner as did the native vWF. In a solid-phase assay, fragment PAD-1 bound to calf skin collagen in contrast to a human vWF recombinant fragment (Ser 445-Val 733) which was inactive in the same assay. The studies presented in this paper demonstrated that the A1 domain of bovine vWF contained the GPIb, heparin, botrocetin as well as collagen binding sites and that integrity of the disulfide bond (Cys 509-Cys 695), did not seem to be essential for binding of bovine vWF fragment to GPIb.     

Binding of factor VIII to von willebrand factor is enabled by cleavage of the von Willebrand factor propeptide and enhanced by formation of disulfide-linked multimers

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.     

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.     

Inhibition of platelet-derived growth factor autocrine growth stimulation by a monoclonal antibody to the human alpha platelet-derived growth factor receptor

A potent neutralizing monoclonal antibody to the human alpha platelet-derived growth factor (PDGF) receptor (alpha PDGFR) was raised by immunizing BALB/c mice with 32D cells expressing the human alpha PDGFR. This monoclonal antibody, designated alpha R1, immunoprecipitated human, monkey, rabbit, pig, dog, and cat, but not hamster, rat, or mouse alpha PDGFRs. Comparison with PR292, a monoclonal antibody previously generated against the alpha PDGFR, showed that both recognized alpha PDGFR extracellular domains, but neither demonstrated reactivity against the beta PDGFR. In vitro binding studies revealed that alpha R1, but not PR292, detection of the alpha PDGFR was blocked by either PDGF AA or PDGF BB. These results strongly suggest that the receptor ligand-binding domain spatially overlapped with the alpha R1 epitope. Monoclonal antibody alpha R1 also inhibited PDGF stimulation of [3H]thymidine uptake by 32D cells expressing the alpha PDGFR (32D alpha R) as well as autocrine growth stimulation of 32D alpha R cells transfected with and expressing PDGF AA or PDGF BB. Therefore, monoclonal antibody alpha R1 may be useful in the detection and growth inhibition of malignancies in which PDGF autocrine stimulation and/or alpha PDGFR overexpression plays an important role(s).     

Organization of von Willebrand factor on surface-activated platelets

The distribution and organization of von Willebrand factor (vWF) multimers on platelets after surface activation have not been fully characterized. In the present study, washed human platelets were allowed to interact with Formvar-coated, electron microscope grids for 20 minutes at 37 degrees C and then fixed. After fixation, cells were washed and then incubated with buffer alone, human plasma, human plasma preincubated with ristocetin (1.2 mg/mL), purified human vWF plus ristocetin, or bovine plasma. Macromolecular complexes were revealed by ultrastructural immunocytochemistry employing a polyclonal antibody against vWF and protein A-gold (PAG) as the electron-dense probe. vWF multimers were not present in discoid platelets but appeared on the central zone of dendritic cells and over larger central areas of fully spread platelets. Exposure to human plasma alone did not affect the distribution of electron-dense probes for vWF in central regions of surface-activated cells. Incubation of spread platelets with ristocetin-activated human plasma or bovine plasma resulted in the appearance of randomly dispersed, mottled areas of increased density covering the surface from edge to edge. Exposure to vWF antibody and PAG resulted in specific labeling of the dense areas in a serpentine, linear array. The gold-probe distribution suggested that the vWF multimers were not superimposed and were distributed in a random, irregular manner from edge to edge with label-free, clear areas between them. The results extend previous observations demonstrating that glycoprotein Ib-IX receptors are not spontaneously cleared from the plasma membranes of surface-activated platelets by showing that the receptor function of glycoprotein Ib-IX complex remains unchanged.     

Autocrine regulation of endothelial exocytosis: von Willebrand factor release is induced by prostacyclin in cultured endothelial cells

OBJECTIVE: To find out whether recombinant human erythropoietin (r-HuEPO) given perioperatively has any effect on haemostatic activity in patients undergoing elective colorectal resection. DESIGN: A placebo-controlled double-blind study. SETTING: Odense university hospital, Denmark. SUBJECTS: 24 patients undergoing elective colorectal resection, 13 of whom were given r-HuEPO (Eprex) and 11 placebo. MAIN OUTCOME MEASURES: Concentrations of haemoglobin, tissue-type plasminogen activator and plasminogen activator inhibitor-1; activated partial thromboplastin time; prothrombin time; platelet and reticulocyte counts; blood loss; and transfusions. RESULTS: There was no significant change in fibrinolytic activity, prothrombin time, or activated prothrombin time in the treatment group. Platelet counts differed slightly but not significantly, being higher in the r-HuEPO group. There was a significant increase in reticulocyte counts in the r-HuEPO group. CONCLUSION: R-HuEPO given perioperatively significantly increased erythropoiesis in patients undergoing elective colorectal operations but had no influence on haemostatic activity.     

Quantitative analysis of von Willebrand factor and its propeptide in plasma in acquired von Willebrand syndrome

Measurement of the von Willebrand factor (vWF) propeptide, also known as von Willebrand antigen II, has been suggested to be helpful in the discrimination of congenital von Willebrand disease type I from type 2 and in assessing the extent of activation of the endothelium. We performed a quantitative analysis of mature vWF and its propeptide in plasma in 8 patients with acquired von Willebrand syndrome (AvWS) and in 20 normal individuals. Mature vWF levels were significantly lower in AvWS as compared with normal individuals (13.4+/-3.5 vs 35.6+/-3.3 nM, p <0.001). In contrast, propeptide levels were significantly higher in AvWS (11.4+/-1.1 vs 4.7+/-0.2 nM, p <0.001), probably reflecting a compensatory increase in vWF synthesis or increased perturbation of the endothelium in AvWS. After treatment with DDAVP, propeptide and mature vWF levels rose 5-fold in AvWS, whereas propeptide levels were not altered by the infusion of a vWF concentrate or treatment with high dose intravenous immunoglobulins, indicating that plasma propeptide levels are a reliable reflection of vWF synthesis. Measurement of propeptide levels may provide additional information in AvWS as to whether decreased levels of mature vWF in the circulation are due to a decrease in synthesis or due to an accelerated removal of vWF from the circulation.