Abnormal Large Platelets Are Associated Not Only with von Willebrand Disease (VWD) Type 2B (Including Type 2B/1 New York) but Also with Severe Type 3: A Potential Role for von Willebrand Factor (VWF) in Megakaryocytopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 101-101 ◽  
Author(s):  
Paquita Nurden ◽  
Alan T. Nurden ◽  
Jocelyne Enouf ◽  
Silvia La Marca ◽  
Luciano Baronciani ◽  
...  
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Gene Gain ◽  
Giant Platelets ◽  
Immunogold Staining ◽  
Platelet Production ◽  
Large Deletions ◽  
Von Willebrand

Abstract Background: VWD type 2B results from mutations in exon 28 of the VWF gene. Gain of function of this adhesive protein results in an increased affinity for the platelet glycoprotein (GP) Ib-IX-V complex. Recently we reported that impaired megakaryocytopoiesis results from an abnormal interaction between GPIb with newly synthesized VWF in megakaryocytes of a family with the R1308P mutation (Blood2006; 108:2587–95). Aim of the Study: to further examine the potential consequences of VWF abnormalities on platelet production we have studied a series of patients with different types of VWD. Patients, Methods: 13 VWD patients were enrolled in the study after informed consent. Diagnoses of VWD were performed according to the criteria of the ISTH-SSC-SC. Included were 8 VWD 2B patients from 6 families with the following mutations: R1306W (n=1), R1308C (n=1), I1309V (n=1), V1326M (n=2), R1341Q (n=2) and P1266L (n=1, 2B/1NY). Also studied were 5 additional VWD cases characterized by low/absent VWF in their platelets: VWD 2M (n=1, D1277-E78delInsL), VWD 3 without inhibitors against VWF (n=2, 276delT/257delA and 6182delT/6182delT) and VWD with isoantibodies against VWF (n=2, large deletions of the VWF gene). The platelet count was decreased at the time of examination for 6/8 VWD 2B patients and normal for 2/8 (n=1 V1316M and n=1 P1266L). Platelet counts were normal in the remaining 5 patients with VWD types 3, and 2M. Electron microscopy (EM) of platelets and immunolocalization of VWF were performed. Results: EM showed the presence of an increased population of giant platelets (15 to 40% versus <10% for controls) in all VWD 2B. Characteristics of these platelets were the presence of large vacuoles often filled with material and the presence of numerous membrane complexes. Additional abnormalities were observed in the patient with 2B/1NY; alpha-granule morphology was different with a population of enlarged granules, sometimes giant. There was also a heterogeneneous distribution with some platelets almost devoid of alpha-granules. Immunogold staining for all type 2B patients showed that VWF was present not only inside the granules but also in the surface-connected canalicular system. For 3/8 patients with VWD 2B, cleaved caspase was present in the platelets indicating abnormal caspase activity at least for R1341Q and V1316M. In VWD 2M (mutation D1277-E78delInsL) characterized by low platelet VWF content as well as in the VWD 3 (n=2) with a premature stop codon, no significant modification of platelet morphology was found. Some residual VWF was also seen in the alpha-granules of these 2 VWD 3 patients. In contrast, a significant number of enlarged platelets with numerous vacuoles were found in the 2 VWD 3 with large deletions and isoantibodies directed against VWF. Immunogold labelling for platelet VWF was completely negative for these two patients. Conclusions: Patients with VWD types 2B and 3 (undetectable VWF) show platelet production defects of varying severity, suggesting a major role of VWF in the fine regulation of megakaryocytopoiesis. Up-regulation or loss of the interaction between VWF and GPIb may lead to a variable proportion of giant platelets with or without thrombocytopenia.

Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor

2014 ◽  
Vol 112 (12) ◽  
pp. 1159-1166 ◽  
Author(s):  
Viviana Daidone ◽  
Giovanni Barbon ◽  
Elena Pontara ◽  
Grazia Cattini ◽  
Lisa Gallinaro ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Loss Of Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryCysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients’ plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted – apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients’ plasma VWF. The in vitro expression of the E244Lfs*211–VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.

Identification of a New Type 2M von Willebrand Disease Mutation also at Position 1324 of von Willebrand Factor

2002 ◽  
Vol 87 (04) ◽  
pp. 635-640 ◽  
Author(s):  
E. Fressinaud ◽  
A. S. Ribba ◽  
D. Meyer ◽  
C. Mazurier ◽  
L. Hilbert ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Von Willebrand Factor ◽  
Stop Codon ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Patient Reported ◽  
French Family ◽  
New Type ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2M von Willebrand disease (VWD) refers to variants with decreased platelet-dependent function that is not associated with the loss of high molecular weight (HMW) von Willebrand factor (VWF) multimers. This category includes the so-called “phenotype B” responsible for inexistent ristocetin-induced but normal botrocetin-induced binding of VWF to platelet glycoprotein Ib. The missense mutation G1324S was identified in the first patient reported to display “phenotype B”.We report here on the identification in four members of a French family of a missense mutation also affecting this glycine residue but changing it into an alanine residue. These individuals are heterozygous for this mutation and two of them display an additional quantitative VWF deficiency resulting from a stop codon at position 2470. After transient transfection in Cos-7 cells, the mutated recombinant protein harbouring the G1324A substitution was shown to exhibit normal multimers and inexistent ristocetin-induced but normal botrocetininduced binding to GPIb, confirming the classification of this new mutation as a type 2M VWD mutation.

Expression of Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program Study Reveals Defects in VWF Secretion and Multimerization

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1398-1398
Author(s):  
Azza Abdelaal ◽  
David Jakab ◽  
Pamela A Christopherson ◽  
Robert R Montgomery ◽  
Sandra L Haberichter
Keyword(s):  
Conflicts Of Interest ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Von Willebrand Disease ◽  
Sequence Variants ◽  
Cell Lysate ◽  
Expression Studies ◽  
Von Willebrand ◽  
The Impact

Abstract von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Type 1 is the most common form of VWD and results in a partial quantitative deficiency of von Willebrand Factor (VWF). The mechanisms underlying type 1 VWD are still not very well understood although reduced VWF secretion and increased VWF clearance have been implicated in causing VWD. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD in order to define the underlying mechanism and explore if SV in a particular domain are mechanistically similar. We utilized homozygous expression in human embryonic kidney cells (HEK-293T) to study the effect of VWF SV on VWF secretion, intracellular retention, multimerization, and function. Novel SV have been identified throughout the entire VWF protein. We introduced the following variants into a VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Sequence variants were confirmed by Sanger sequencing. Variant VWF cDNA is transfected homozygously into HEK-293T cells. The supernatants and cell lysates from 3 independent transfections are collected and analyzed by ELISA for VWF:Ag and VWF binding to collagen type III (VWF:CB). VWF multimer structure is analyzed by SDS-agarose gel electrophoresis and western blotting. The genotype-phenotype patient data is correlated with the data from expression studies to explore a model to predict the impact of the SV on the VWD phenotype. Variants V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F demonstrated secretion comparable to that of wild type (WT)-VWF. In contrast, VWF variants R960P and C2701Y showed reduced VWF secretion (<50% of WT) with increased VWF in the cell lysate. VWF variants W199X, C524Y, G994D, C996W, Q1353X, E1660X, C2199Y, and C2754Y demonstrated a complete absence of secreted VWF. Not unexpectedly, homozygous expression of stop codon variants W199X, Q1353X, and E1660X demonstrated no VWF in the cell lysate. However, non-secreted VWF variants C524Y, G994D, C996W, C2199Y, and C2754Y showed intracellular retention with detectable VWF in the cell lysate. SV occurring at cysteine residues (C524Y, C996W, C2199Y, C2701Y, and C2754Y) all had reduced secretion and increased intracellular retention, consistent with altered conformation leading to increased intracellular chaperone interaction and proteasomal degradation. VWF binding to collagen is dependent on the presence of high molecular weight multimers (HMWM). VWF:CB/VWF:Ag is used to predict multimer structure with VWF:CB/VWF:Ag < 0.7 indicative of loss of the HMWM. VWF variants V86M, M947V, R1763Q, Q2256H, P2524L, C2701Y had VWF:CB/VWF:Ag ≥ 0.7 consistent with normal multimer structure, while variants R960P, R1204W, T2282I, A2569E, and C2693F had VWF:CB/VWF:Ag < 0.7 indicating abnormal multimer structure. 47.3% of the 19 VWF variants studied had normal VWF secretion, 10.5% had reduced secretion with increased intracellular retention, and 26.3% revealed absent secretion with intracellular retention. Variants with a premature stop codon did not synthesize VWF at all. Some SV had normal secretion and multimerization (V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F) implying that the VWD phenotype in these patients results from yet unidentified mechanisms and may not be associated with these SV. Reduced plasma survival is unlikely as these patients had normal VWFpp/VWF:Ag level consistent with normal VWF clearance. Among the VWF variants with normal or decreased secretion, 45.4% had reduced VWF:CB/VWF:Ag consistent with abnormal multimer structure. Heterozygous expression, as observed in the patient, is expected to normalize these multimerization defects. The decreased or absent secretion observed for 52.7% of the variants studied correlates with the patient phenotype, indicating reduced secretion is the mechanism underlying these patients' type 1 VWD phenotype. No domain-specific correlation of VWF secretion or multimer abnormality was observed. In summary, VWF expression studies confirmed the causative nature of many, but not all of the novel sequence variants identified in type 1 VWD subjects in the Zimmerman Program. Disclosures No relevant conflicts of interest to declare.

Combined partial exon skipping and cryptic splice site activation as a new molecular mechanism for recessive type 1 von Willebrand disease

2006 ◽  
Vol 96 (12) ◽  
pp. 711-716 ◽  
Author(s):  
Lisa Gallinaro ◽  
Francesca Sartorello ◽  
Elena Pontara ◽  
Maria Cattini ◽  
Antonella Bertomoro ◽  
...  
Keyword(s):  
Splice Site ◽  
Stop Codon ◽  
Consensus Sequence ◽  
Premature Stop Codon ◽  
Exon Skipping ◽  
Von Willebrand Disease ◽  
Cryptic Splice Site ◽  
Recessive Type ◽  
Von Willebrand

SummaryWe describe the complex picture associated with a mutated splice junction in intron 13 of von Willebrand factor (VWF) gene. The proband, characterized by a marked decrease in plasma and platelet VWF and near normal multimer organization, was classified as recessive type 1 von Willebrand disease (VWD). Genetic analysis demonstrated that he was homozygous for the 1534–3C>A mutation in the consensus sequence of the acceptor splicing site of intron 13 of the VWF gene. Platelet mRNA analysis documented three VWF transcripts: a wild type generated by the correct recognition of the mutated splice site, a smaller transcript not containing exon 14, and a longer one that, in addition to exons 13 and 14, included a 62bp fragment corresponding to the end of intron 13. The small transcript derives from the skipping of exon 14, the long one from the activation of a cryptic splice site in intron 13; both show a premature stop codon inVWF propeptide, so the probandVWF derives entirely from the correct splice site recognition. Combined incomplete exon skipping and cryptic splice site activation are first recognized in VWD. Since the 1534–3C>A mutation does not abolish the normal processing of mRNA, it is unlikely to be found in type 3VWD. This mutation therefore appears to be peculiar to type 1 VWD.

Abnormal VWF modifies megakaryocytopoiesis: studies of platelets and megakaryocyte cultures from patients with von Willebrand disease type 2B

Blood ◽  
2010 ◽  
Vol 115 (13) ◽  
pp. 2649-2656 ◽  
Author(s):  
Paquita Nurden ◽  
Giuliana Gobbi ◽  
Alan Nurden ◽  
Jocelyne Enouf ◽  
Ibtissam Youlyouz-Marfak ◽  
...  
Keyword(s):  
Membrane System ◽  
Positive Influence ◽  
Disease Type ◽  
Von Willebrand Disease ◽  
Giant Platelets ◽  
Platelet Production ◽  
Platelet Receptor ◽  
Immunofluorescence Labeling ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) is an essential mediator of platelet adhesion to the vessel wall, but little is known about its role in megakaryocytopoiesis. VWF and its platelet receptor, glycoprotein Ibα (GPIbα), are both expressed during megakaryocyte (MK) maturation. This study was designed to evaluate whether the enhanced VWF-GPIbα interactions typical of patients with von Willebrand disease type 2B (VWD2B) modify platelet production. Platelets from 9 patients with VWD2B with 7 different gain-of-function mutations were examined by electron microscopy (EM) and immunofluorescence labeling. For the patients with VWD2B, EM characteristically showed variable numbers of structurally abnormal giant platelets, sometimes in agglutinates. Cultures of MKs from controls performed with or without purified VWF confirmed a positive influence of VWF on platelet production with specific inhibition by an antibody blocking VWF binding to GPIbα. VWD2B MK cultures examined by EM showed a disorganized demarcation membrane system and abnormal granule distribution. They produced platelets with structural abnormalities typical of VWD2B. Confocal examination of MK revealed limited extension of pseudopods with few large proplatelets. These results confirm that megakaryocytopoiesis is modified by the enhanced VWF-GPIbα interactions. These data obtained for controls and patients with VWD2B suggest a novel regulatory role of VWF-GPIbα interactions in platelet production.

Bernard-Soulier Syndrome with Severe Bleeding: Absent Platelet Glycoprotein lb alpha Due to a Homozygous One-base Deletion

1996 ◽  
Vol 76 (05) ◽  
pp. 670-674 ◽  
Author(s):  
Chaoyang Li ◽  
Dominick N Pasquale ◽  
Gerald J Roth
Keyword(s):  
Stop Codon ◽  
Surface Membrane ◽  
Genetic Defect ◽  
Premature Stop Codon ◽  
Platelet Glycoprotein ◽  
Severe Bleeding ◽  
Giant Platelets ◽  
Platelet Disorder ◽  
Oligonucleotide Hybridization ◽  
Bernard Soulier Syndrome

SummaryBernard-Soulier syndrome is a rare congenital platelet disorder that affects a surface membrane adhesion receptor, glycoprotein (GP) Ib-V-IX. Both the genetic defects and the bleeding diatheses associated with the syndrome are heterogeneous due, in part, to the complexity of the involved receptor which consists of four different members, GPs: Ibα-Mr 143 K (contains the von Willebrand factor-binding site), Ibβ-Mr 22 K, V-Mr 83 K and IX-Mr 20 K. We studied a kindred that includes a 40 year-old man with severe Bernard-Soulier syndrome: life-threatening gastrointestinal bleeding, thrombocytopenia, giant platelets and absent ristocetin-dependent platelet aggregation. By Southern blotting, PCR amplification/sequencing, hetero-duplex analysis, and allele-specific oligonucleotide hybridization, the Ib-V-IX genes were analyzed, and the molecular genetic defect was defined as a one-base deletion in the GPIbα gene, involving an adenine of codon 19. The mutation, K19R, homozygous in the propositus and heterozygous in the available unaffected relatives, leads to a frame shift in codons 19-21 and a premature stop codon after codon 21. No functional GPIbα can be produced from the mutant allele, implying that the platelets of the affected patient lack all GPIbα. Within the spectrum of Bernard-Soulier syndrome, this patient’s disorder exemplifies a severe or “classic” extreme; an “experiment of Nature” that illustrates the effect of a complete deficiency of the ligand-binding chain (GPIbα) of the GPIb-V-IX receptor.

Morphological Changes of Platelets from Patients with Von Willebrand Disease Types 2B and 3 Suggest That Von Willebrand Factor Intervenes in Platelet Formation from Megakaryocytes.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1238-1238
Author(s):  
Paquita Nurden ◽  
Alan T Nurden ◽  
Jean-Max Pasquet ◽  
Jocelyne Enouf ◽  
Cécile Faurie-Bonnafous ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Morphological Changes ◽  
Stress Protein ◽  
Morphological Characteristics ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Gain Of Function ◽  
Giant Platelets ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The interaction between von Willebrand factor (VWF) and platelets plays a key role in hemostasis. VWF gene abnormalities result in the most common inherited bleeding disorder, von Willebrand disease (VWD). VWD2B, characterized by gain-of-function binding of VWF to platelet glycoprotein (GP)Ib, is due to mutations located within a portion of exon 28 of the VWF gene, coding for the VWFA1 domain. We have previously shown in a family with a R1308P mutation that the enhanced interaction between VWF and GPIb can also occur in the bone marrow leading to a premature release of platelets from megakaryocytes (MKs) with circulating large platelets and agglutinates (Nurden et al, Blood 2006). We have now widened our study to see if these changes are common in VWD2B. As a control, we also included platelets from patients lacking VWF. In total, we have analyzed the platelets from 9 VWD2B patients with the following mutations: P1266L, R1306W, R1308C, I1309V, V1316M, R1341Q, R1341W; platelets from 2 VWD3 patients with a large deletion of the VWF gene were also included. We have used electron microscopy (EM) and immunofluorescence (IF) to evaluate platelet morphological characteristics; included in the morphometric studies was the measure of platelet size. We have also analyzed platelet VWF content and performed plasma/platelet VWF multimeric analyses. PMCA4B belonging to the SERCA proteins (a substrate of caspase) and GRP78 a stress protein were evaluated as markers of abnormal megakaryocytopoiesis. At the time of blood withdrawal, 4 patients were thrombocytopenic; nevertheless, for all patients with VWD2B a pool of giant platelets was found. Agglutinates were often but not always present. Globally, the percentage of platelets with the longest diameter (LD) &gt; than 3μm was 58 ±16% (controls 24 ± 13%) and the shortest diameter (SD) &gt; 2μm was 33% ± 13 (controls 4 ± 0.6%). Thus platelets were enlarged without being round. The largest platelets were found for the patient with the V1316M mutation. The content of alpha granules appeared normal, as was the quantity of internal membrane pools. To our surprise, giant platelets were also present for the two VWD3 patients; 66 and 57% of the platelets have a LD &gt; 3μm and 21 and 19% a SD &gt; than 2μm. The EM evaluation for both type 3 patients suggested that some giant platelets resembled large fragments prematurely detached from MKs. Examining further by IF and EM coupled with immunogold labelling with a mixture of anti-GPIIb-IIIa and GPIb-IX MoAbs revealed occasional circulating MKs. Nuclei surrounded by cytoplasmic remnants were also seen. Analysis of PMCA4B and GRP 78, showed that their levels were increased for VWD2B patients with the R1341Q mutation but borderline for the patient with the R1308C mutation (contrasting with increased levels for R1308P) showing that premature apoptosis was not a constant feature. These results were normal for the VWD3 patients. Our results confirm that megakaryocytopoiesis can be affected by both an abnormal gain-of-function (VWD2B) and completely deficient (VWD3) VWF. However, the consequences of up-regulated VWF and VWF deficiency may be different.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

The Interaction of Botrocetin with Normal or Variant von Willebrand Factor (Types IIA and IIB) and Its Inhibition by Monoclonal Antibodies that Block Receptor Binding

1992 ◽  
Vol 68 (04) ◽  
pp. 464-469 ◽  
Author(s):  
Y Fujimura ◽  
S Miyata ◽  
S Nishida ◽  
S Miura ◽  
M Kaneda ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Normal Individuals ◽  
Rag 1 ◽  
The One ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca2+-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957–64).Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an “activated complex” formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GPIb, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

Characterization of Partial Gene Deletions in Type III von Willebrand Disease with Alloantibody Inhibitors

1994 ◽  
Vol 72 (02) ◽  
pp. 180-185 ◽  
Author(s):  
David J Mancuso ◽  
Elodee A Tuley ◽  
Ricardo Castillo ◽  
Norma de Bosch ◽  
Pler M Mannucci ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Pcr Analysis ◽  
Gene Deletions ◽  
Factor Gene ◽  
Type Iii ◽  
Large Deletions ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor gene deletions were characterized in four patients with severe type III von Willebrand disease and alloantibodies to von Willebrand factor. A PCR-based strategy was used to characterize the boundaries of the deletions. Identical 30 kb von Willebrand factor gene deletions which include exons 33 through 38 were identified in two siblings of one family by this method. A small 5 base pair insertion (CCTGG) was sequenced at the deletion breakpoint. PCR analysis was used to detect the deletion in three generations of the family, including two family members who are heterozygous for the deletion. In a second family, two type III vWD patients, who are distant cousins, share an -56 kb deletion of exons 22 through 43. The identification and characterization of large vWF gene deletions in these type III vWD patients provides further support for the association between large deletions in both von Willebrand factor alleles and the development of inhibitory alloantibodies.

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