Identification of an IgD/IgT chimera in the European sea bass (Dicentrarchus labrax L.).

Three classes of immunoglobulins have been identified in Teleosts: IgM, IgT/Z and IgD. They are fundamental for fish immune responses and, therefore, their functional activities are heavily investigated. In this paper, we describe the identification of a new IgD/IgT chimera in sea bass (Dicentrarchus labrax) from a gills transcriptome. This transcript joined the first six constant domains of the IgD chain with the two terminal constant domains of IgT, generating a long in-frame coding sequence with a junction between the canonical δ6 exon splicing donor site and the τ3 exon splicing acceptor site. Studies performed on genomic DNA confirmed the presence of the sequence and identifies and intronic region of 656 bp within this joining region. The basal expression of the IgD/IgT chimera was investigated both in silico and in vivo: high level of expression was found in gills, gut and head kidney. Moreover, IgD/IgT transcripts were up-regulated after in vitro stimulation of sea bass HK leukocytes with LPS. The IgD/IgT chimera was found also in two congener species, Morone saxatilis and Morone chrysops. It is not possible to have a precise idea on the evolutionary scenario that lead to the appearance of this sequence due to the lack of genomic information, but we could speculate that an ancestral duplication of the entire IgH locus was followed by the chimerization of Cδ/Cτ in one of the two loci. Finally, the IgD/IgT high basal expression in tissues and organs fundamental for sea bass immune response and its modulation after LPS stimulation provide a very preliminary indication that this unusual Ig variant could have a functional activity.

IgD-Fc-Ig fusion protein, a new biological agent, inhibits T cell function in CIA rats by inhibiting IgD-IgDR-Lck-NF-κB signaling pathways.

IgD-Fc-Ig fusion protein, a new biological agent, is constructed by linking a segment of human IgD-Fc with a segment of human IgG1-Fc, which specifically blocks the IgD-IgDR pathway and selectively inhibits the abnormal proliferation, activation, and differentiation of T cells. In this study we investigated whether IgD-Fc-Ig exerted therapeutic effects in collagen-induced arthritis (CIA) rats. CIA rats were treated with IgD-Fc-Ig (1, 3, and 9 mg/kg) or injected with biological agents etanercept (3 mg/kg) once every 3 days for 40 days. In the PBMCs and spleen lymphocytes of CIA rats, both T and B cells exhibited abnormal proliferation; the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1(CD4+IFN-γ+), and Th17(CD4+IL-17+) were significantly increased, whereas the Treg (CD4+CD25+Foxp3+) cell percentage was decreased. IgD-Fc-Ig administration dose-dependently decreased the indicators of arthritis; alleviated the histopathology of spleen and joint; reduced serum inflammatory cytokines levels; decreased the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1 (CD4+IFN-γ+), and Th17(CD4+IL-17+); increased Treg (CD4+CD25+Foxp3+) cell percentage; and down-regulated the expression of key molecules in IgD-IgDR-Lck-NF-κB signaling (p-Lck, p-ZAP70, p-P38, p-NF-κB65). Treatment of normal T cells with IgD (9 µg/mL) in vitro promoted their proliferation. Co-treatment with IgD-Fc-Ig (0.1-10 µg/mL) dose-dependently decreased IgD-stimulated T cell subsets percentages and down-regulated the IgD-IgDR-Lck-NF-κB signaling. In summary, this study demonstrates that IgD-Fc-Ig alleviates CIA and regulates the functions of T cells through inhibiting IgD-IgDR-Lck-NF-κB signaling.

Molecular cloning, characterization and expression of immunoglobulin D on pathogen challenge and pathogen associated molecular patterns stimulation in freshwater carp, Catla catla.

The primordial immunoglobulin class, IgD, was the first non-IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (CcIgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of CcIgD exhibited greatest similarity with Ctenopharyngodon idella. qRT-PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post-Aeromonas hydrophila challenge. CcIgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in CcIgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.

Molecular Cloning and Expression Analysis of IgD in Nile Tilapia (Oreochromis niloticus) in Response to Streptococcus agalactiae Stimulus.

IgD is considered to be a recently-evolved Ig and a puzzling molecule, being previously found in all vertebrate taxa, except for birds. Although IgD likely plays an important role in vertebrate immune responses, the function of IgD in Nile tilapia (Oreochromis niloticus) is virtually unknown. In the present study, a membrane form of IgD (mIgD) heavy chains were cloned from the GIFT strain of Nile tilapia (designated On-mIgD). The On-mIgD heavy chain's cDNA is composed of 3347 bp with a 31 bp of 5'-UTR, 3015 bp open reading frame (ORF) and 301 bp 3'-UTR, encoding a polypeptide of 1004 amino acids (GenBank accession no: KF530821). Phylogenetic analysis revealed that On-mIgD heavy chains showed the highest similarity to Siniperca chuatsi. Quantitative real-time PCR (qRT-PCR) analysis showed that On-mIgD expression occurred predominately in head kidney, thymus, spleen, and kidney. After Streptococcus agalactiae infection, transcripts of On-mIgD increased and reached its peak at 48 h in the head kidney and thymus, and 72 h in the spleen, respectively. Taken together, these results collectively indicated that IgD could possibly have a key role to play in the immune response when bacterial infections in Nile tilapia.

10-valent pneumococcal non-typeable Haemophilus influenzae protein-D conjugate vaccine (PHiD-CV) induces memory B cell responses in healthy Kenyan toddlers.

Memory B cells are long-lived and could contribute to persistence of humoral immunity by maintaining the plasma-cell pool or making recall responses upon re-exposure to an antigen. We determined the ability of a pneumococcal conjugate vaccine to induce anti-pneumococcal memory B cells. Frequencies of memory B cells against pneumococcal capsular polysaccharides from serotypes 1, 6B, 14, 19F and 23F were determined by cultured B cell enzyme-linked immunospot (ELISPOT) in 35 children aged 12-23 months who received pneumococcal non-typeable Haemophilus influenzae protein-D conjugate vaccine (PHiD-CV). The relationships between plasma antibodies and memory B cell frequencies were also assessed. After two doses of PHiD-CV, the proportion of subjects with detectable memory B cells against pneumococcal capsular polysaccharides increased significantly for serotypes 1 (3-45%; P < 0·01), 19F (21-66%; P < 0·01) and 23F (13-36%; P = 0·02), but not serotypes 6B (24-42%; P = 0·24) and 14 (21-40%; P = 0·06). Correlations between antibodies and memory B cells were weak. Carriage of serotype 19F at enrolment was associated with poor memory B cell responses against this serotype at subsequent time-points (day 30: non-carriers, 82% versus carriers, 0%, P < 0·01; day 210: non-carriers, 72% versus carriers, 33%, P = 0·07). PHiD-CV is capable of inducing memory B cells against some of the component pneumococcal capsular polysaccharides.

Conformational dynamics of individual antibodies using computational docking and AFM.

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. In this study, we aimed at analyzing the conformational variability of receptors portrayed by monoclonal antibodies that have been individually imaged using atomic force microscopy (AFM). Individual antibodies were chemically coupled to activated mica surface, and they have been imaged using AFM in ambient conditions. The resulting topographical surface of antibodies was used to assemble the three subunits constituting antibodies: two antigen-binding fragments and one crystallizable fragment using a surface-constrained computational docking approach. Reconstructed structures based on 10 individual topographical surfaces of antibodies are presented for which separation and relative orientation of the subunits were measured. When compared with three X-ray structures of antibodies present in the protein data bank database, results indicate that several arrangements of the reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X-ray structure consequence of the antibody flexibility. We conclude that high-resolution AFM imaging with appropriate computational reconstruction tools is adapted to study the conformational dynamics of large individual macromolecules deposited on mica.

Human rotavirus-specific IgM Memory B cells have differential cloning efficiencies and switch capacities and play a role in antiviral immunity in vivo.

Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM(+) mBc, we found that 62% and 21% of total (non-antigen-specific) IgM(+) and RV-IgM(+) mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM(+) (17%) and RV-IgM(+) (7%) mBc were lower than those of the corresponding switched (IgG(+) IgA(+)) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM(+) RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2Rγ(null)) immunodeficient mice were adoptively transferred highly purified human IgM(+) mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM(+) mBc are phenotypically diverse and significantly enriched in the IgM(hi) IgD(low) subset. Thus, RV-IgM(+) mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.

Antibodies in a heavy chain knock-in mouse exhibit characteristics of early heavy chain rearrangement.

Studies in autoantibody transgenic mice have demonstrated receptor editing rearrangements at Ab H and L chain loci. However, the physiologic role of H chain editing (V(H) replacement and rearrangement on the second allele) has been called into question. It is unclear if additional rounds of H chain rearrangement are driven by BCR specificity. In this study, we analyze the manner in which B cells undergo additional H chain rearrangements in an anti-DNA H chain knock-in mouse, B6.56R. We find that rearrangements in 56R(+) B cells tend to involve the D gene locus on both alleles and the most J(H)-proximal V(H) gene segments on the endogenous allele. As a result, some B cells exhibit V(D)J rearrangements on both H chain alleles, yet allelic exclusion is tightly maintained in mature 56R B cells. As B cells mature, a higher proportion expresses the nontransgenic H chain allele. Rearrangements on both H chain alleles exhibit junctional diversity consistent with TdT-mediated N-addition, and TdT RNA is expressed exclusively at the pro-B cell stage in B6.56R. Collectively, these findings favor a single, early window of H chain rearrangement in B6.56R that precedes the expression of a functional BCR. B cells that happen to successfully rearrange another H chain may be favored in the periphery.

Functional characteristics of small proteins (70 amino acid residues) forming protein-nucleic acid complexes.

The proteins composed of short polypeptides (about 70 amino acid residues) participating in large complexes (ribosome) and proteins interacting with DNA/RNA were taken for analysis and classified according to the hydrophobicity excess/deficiency distribution as a measure of structural and functional specificity and similarity. The characterization of this group of proteins is the introductory part to the analysis of the so called "Never Born Proteins" (NBP) in search for protein compounds exhibiting biological activity that may be valuable in pharmacological research. The entropy scale (classification between random and deterministic limits) organized in ranking list allows the comparative analysis of the proteins under consideration. The comparison of the hydrophobicity deficiency appeared to be useful for similarity recognition, the examples of which are shown in the paper. The specificity of proteins participating in large protein-nucleic acid complexes generation is presented.

IgD in the reptile leopard gecko.

Immunoglobulin D (IgD) has been a mysterious antibody ever since it was discovered in mammals 40 years ago. It shares with IgM the role of antigen-receptor in the membrane of mature B cells. The absence of IgD in birds and its description in bony fishes contributed to the confusion about its evolutionary origins. Recent studies have established the presence of IgD in the amphibian Xenopus tropicalis. It is essential to study IgD genes in reptiles in order to better understand the evolution of this immunoglobulin in vertebrates. We describe in this report the IgM and IgD genes of the reptile Eublepharis macularius. The IgM gene has characteristics that are similar to those described in other species whereas IgD gene departs from the normal structure described for this antibody class in other species. It is made up of 11 immunoglobulins domains without evidence of recent intragenic duplications of exons as described in IgD genes of fish and X.tropicalis. It is possible that the immunoglobulin is comprised of domains inherited from earlier species and that this form of IgD is close to that present in animals that left the sea to live on land. Furthermore, domains CH7 and CH8 of E. macularius IgD are orthologues to domains CH2 and CH3 of mammalian IgD. The present study also describes a second IgD (IgD2) which must have appeared recently by duplication of an older immunoglobulin gene and recombination with the IgA-like gene described in this specie. Tissue expression of IgD and IgD2 mRNA is similar to that of IgM mRNA, suggesting a functional role of reptilian IgD.

Teleost IgD+IgM- B Cells Mount Clonally Expanded and Mildly Mutated Intestinal IgD Responses in the Absence of Lymphoid Follicles.

Immunoglobulin D (IgD) is an ancient antibody with dual membrane-bound and fluid-phase antigen receptor functions. The biology of secreted IgD remains elusive. Here, we demonstrate that teleost IgD+IgM- plasmablasts constitute a major lymphocyte population in some mucosal surfaces, including the gut mucosa. Remarkably, secreted IgD binds to gut commensal bacteria, which in turn stimulate IgD gene transcription in gut B cells. Accordingly, secreted IgD from gut as well as gill mucosae, but not the spleen, show a V(D)J gene configuration consistent with microbiota-driven clonal expansion and diversification, including mild somatic hypermutation. By showing that secreted IgD establishes a mutualistic relationship with commensals, our findings suggest that secreted IgD may play an evolutionary conserved role in mucosal homeostasis.

Deletion of a single mevalonate kinase (Mvk) allele yields a murine model of hyper-IgD syndrome.

In the current study our objective was to develop a murine model of human hyper-IgD syndrome (HIDS) and severe mevalonic aciduria (MA), autoinflammatory disorders associated with mevalonate kinase deficiency (MKD). Deletion of one Mvk allele (Mvk (+/-)) yielded viable mice with significantly reduced liver Mvk enzyme activity; multiple matings failed to produce Mvk (-/-) mice. Cholesterol levels in tissues and blood, and isoprene end-products (ubiquinone, dolichol) in tissues were normal in Mvk (+/-) mice; conversely, mevalonate concentrations were increased in spleen, heart, and kidney yet normal in brain and liver. While the trend was for higher IgA levels in Mvk (+/-) sera, IgD levels were significantly increased (9-12-fold) in comparison to Mvk (+/+) littermates, in both young (<15 weeks) and older (>15 weeks) mice. Mvk (+/-) animals manifested increased serum TNF-alpha as compared to wild-type littermates, but due to wide variation in levels between individual Mvk (+/-) mice the difference in means was not statistically significant. Mvk (+/-) mice represent the first animal model of HIDS, and should prove useful for examining pathophysiology associated with this disorder.

A patient with hyper-IgD syndrome responding to anti-TNF treatment.

The hyperimmunoglobulinemia D periodic fever syndrome (HIDS) is caused by recessive mutations in the mevalonate kinase gene, which encodes an enzyme involved in cholesterol and nonsterol isoprenoid biosynthesis. The pathogenesis and treatment remains unclear. We describe a 6-year-old Turkish girl with severe disease. Her clinical features were accompanied with very high acute-phase reactants including a very high serum amyloid A level. The patient responded well to anti-tumor necrosis factor treatment. Our findings support the use of this anti-cytokine treatment in HIDS.

Immunogenicity and Safety of 10-valent Pneumococcal Nontypeable Haemophilus influenzae Protein D Conjugate Vaccine (PHiD-CV) Administered to Children With Sickle Cell Disease Between 8 Weeks and 2 Years of Age: A Phase III, Open, Controlled Study.

BACKGROUND: Immunogenicity, safety and reactogenicity of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) were evaluated in children with sickle cell disease (SCD), who are at increased risk for infections. METHODS: In this phase III, open-label, single-center, controlled study in Burkina Faso (NCT01175083), children with SCD (S) or without SCD (NS) were assigned to 6 groups (N = 300): children 8-11 weeks of age (<6 months; <6S and <6NS groups) received 3 primary doses and a booster dose of PHiD-CV coadministered with routine childhood vaccines; children 7-11 months of age (7-11S and 7-11NS groups) received 2 primary doses and a booster dose of PHiD-CV; children 12-23 months of age (12-23S and 12-23NS groups) received 2 catch-up doses of PHiD-CV. Pneumococcal antibody responses were measured using 22F-inhibition enzyme-linked immunosorbent assay and functional opsonophagocytic activity. Responses to other antigens were measured by enzyme-linked immunosorbent assay. Adverse events were recorded. RESULTS: One month postprimary vaccination, for each vaccine serotype ≥98% of infants in the <6S and <6NS groups had antibody concentrations ≥0.2 µg/mL, except for 6B (≥85%) and 23F (≥89%). Immune responses to PHiD-CV after age-appropriate vaccination in children <2 years did not appear influenced by SCD. All infants were seroprotected/seropositive for diphtheria, tetanus and Bordetella pertussis antigens postprimary and booster vaccination. Safety and reactogenicity profiles were similar in children with or without SCD. CONCLUSIONS: PHiD-CV was immunogenic with an acceptable safety profile in children with and without SCD starting vaccination at 8 weeks to 23 months of age.

Semi-extended solution structure of human myeloma immunoglobulin D determined by constrained X-ray scattering.

Human immunoglobulin D (IgD) occurs most abundantly as a membrane-bound antibody on the surface of mature B cells (mIgD). IgD possesses the longest hinge sequence of all the human antibody isotypes, with 64 residues connecting the Fab and Fc fragments. A novel rapid purification scheme of secreted IgD from the serum of an IgD myeloma patient using thiophilic (T-gel) and lectin affinity chromatography gave a stable, homogeneous IgD preparation. Synchrotron X-ray scattering and analytical ultracentrifugation of IgD identified the solution arrangement of its Fab and Fc fragments, and thereby its hinge structure. The Guinier X-ray radius of gyration R(G) of 6.9(+/-0.1)nm showed that IgD is more extended in solution than the immunoglobulin subclass IgA1 (R(G) of 6.1-6.2nm). Its distance distribution function P(r) showed a single peak at 4.7nm and a maximum dimension of 23nm. Velocity experiments gave a sedimentation coefficient of 6.3S, which is similar to that for IgA1 at 6.2S. The complete IgD structure was modelled using molecular dynamics to generate IgD hinge structures, to which homology models for the Fab and Fc fragments were connected. Good scattering curve fits were obtained with 18 semi-extended best fit IgD models that were filtered from 8500 trial models. These best-fit models showed that the IgD hinge does not correspond to an extended polypeptide structure. The averaged solution structure arrangement of the Fab and Fc fragments in IgD is principally T-shaped and flexible, with contribution from Y-shaped and inverted Y-shaped structures. Although the linear sequence of the IgD hinge is much longer, comparison with previous scattering modelling of IgA1 and IgA2(m)1 suggests that the hinge of IgA1 and IgD are more similar than might have been expected, Both possess flexible T-shaped solution structures, probably reflecting the presence of restraining O-linked sugars.

Anakinra is safe and effective in controlling hyperimmunoglobulinaemia D syndrome-associated febrile crisis.

Hyper-IgD and periodic fever syndrome (HIDS) is a hereditary autoinflammatory syndrome, characterized by recurrent inflammatory attacks. Treatment of HIDS is difficult. Recently, the IL-1ra analogue anakinra was reported to be successful in aborting the IgD inflammatory attacks in a vaccination model. We report a clinical case of spectacular reduction of febrile attacks in a severe HIDS patient.

More frequent IgD and reduced CD200 expression in Chinese patients younger than 50 years old with multiple myeloma: a multicenter analysis.

We retrospectively analyzed the presenting features and survival of 194 newly diagnosed patients with multiple myeloma in the People's Republic of China. Compared with older patients, younger patients had a higher percentage of IgD isotype, lower percentage of International Staging System Stage 3 disease, higher albumin level, and lower frequency of high ß2-microglobulin and CD200 expression. There was no difference in sex, Durie-Salmon stage, bone lesion degree, creatinine, lactate dehydrogenase, fluorescence in situ hybridization, and expression of other antigens. Among all 940 newly diagnosed patients with multiple myeloma, those younger than 50 years had better overall survival and progression-free survival than older patients. Of these patients, 457 were treated with a bortezomib-containing regimen, and 450 received conventional therapy. Younger patients treated with bortezomib had better overall survival and progression-free survival than older patients. However, younger patients treated with conventional therapy had the same survival as older patients.

Evaluation of the immunogenic property of NT H. influenzae protein D with Neisseria meningitidis OMV in BALB/c.

INTRODUCTION: Identifying ideal non typeable Haemophilus influenzae (NTHi) vaccine candidates has not been easy due to extensive sequence and antigenic variation among gene products interacting with the immune system. Protein D (PD) is a highly conserved 42 kDa surface lipoprotein available in all H. influenzae, including NTHi. METHODOLOGY: In this study, the gene encoding PD was cloned from H. influenzae and expressed in Escheriachia coli TOPO10 cell in pBAD vector. Arabinose was used to express recombinant protein. In order to purify the protein, Ni-NTA agarose was used to perform affinity chromatography. Purified PD and PD mixed with outer membrane vesicle (OMV) and alum adjuvant were used for subcutaneous immunization in BALB/c mice. After vaccination, IgG responses to PD-OMV, PD-alum, and PD alone were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The recombinant PD containing His6 residues showed a molecular weight of 42 kDa. Anti-PD IgG was detected after first immunization in all groups of mice compared to the negative control group, and it increased after first vaccination, but results showed that the addition of OMV to PD led to a remarkable increase in IgG responses. CONCLUSIONS: Our results suggest an important role for OMV as an adjuvant and show how it could potentially be used when conjugated to H. influenzae PD or other safe subunit vaccine candidates.

Characterization of a myeloma patient with a life-threatening hemorrhagic diathesis: presence of a lambda dimer protein inhibiting shear-induced platelet aggregation by binding to the A1 domain of von Willebrand factor.

We have identified a patient with IgD lambda-type multiple myeloma who was characterized by a severe bleeding tendency, especially after puncture of arterial vessels. Both the bleeding time (>25 min) and activated partial thromboplastin time (APTT) were prolonged. To clarify the underlying pathogenesis, we purified the APTT-prolonging activity from the patient's serum. The purified protein was a highly negatively-charged homodimer of the lambda light chain. The lambda dimer protein (M-protein) inhibited ristocetinand high shear-induced platelet aggregation, dependent on platelet glycoprotein Ibalpha (GPIbalpha), but not epinephrine-, collagen-, ADP-, thrombin-, or botrocetin-induced platelet aggregation. The lambda dimer protein inhibited the binding of platelets to immobilized or ristocetin-treated von Willebrand factor (VWF). Furthermore, a 39/34 kD fragment of VWF encompassing the A1 domain specifically bound to the immobilized lambda dimer protein in the presence of ristocetin, suggesting that the lambda dimer protein directly binds to the A1 domain of VWF. To help elucidate the binding site within the A1 domain, binding of ristocetin-treated VWF to the immobilized lambda dimer protein was assayed in the presence of various anti-A1 domain monoclonal antibodies. Based on these data, we conclude that the lambda dimer protein binds to the region of the A1 domain composed of helices alpha3 and alpha4 and thus interferes with VWF-GPIbalpha interaction. The existence of a protein that inhibits high shear-induced platelet aggregation in acquired von Willebrand disease (VWD) has only rarely been reported. The results suggest that the hemostatic function in arteries with high shear force is profoundly disrupted if the binding of GPIbalpha to VWF is abrogated, supporting the relevance of shear-induced VWF interaction with GPIbalpha in the initiation of the hemostatic process.