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Either the glycoprotein (GP) Ib deficiency or hyper-function in humans can cause macrothrombocytopenia, the molecular mechanisms of which remain unclear. Herein, the investigations for disease pathogenesis were performed in the human induced pluripotent stem cell (hiPSC) model. The hiPSCs carrying a gain-of-function GP1BA p.M255V mutation which was described in platelet-type von Willebrand disease (PT-VWD) were generated using CRISPR/Cas9. The GP1BA-null hiPSCs were previously derived from a Bernard-Soulier syndrome (BSS) patient. After full megakaryocyte differentiation in culture, both hiPSC mutations showed large proplatelet tips under fluorescence microscopy and yielded fewer but larger platelets compared with those of wild-type cells. The Capillary Western analyses revealed the lower ERK1/2 activation and higher MLC2 (Myosin light chain 2) phosphorylation in megakaryocytes with mutated GPIb. Adding a mitogen-activated protein kinase (MAPK) pathway inhibitor to wild-type hiPSCs recapitulated the phenotypes of GPIb mutations and increased MLC2 phosphorylation. Notably, a ROCK inhibitor which could inhibit MLC2 phosphorylation rescued the macrothrombocytopenia phenotypes of both GPIb alterations and wild-type hiPSCs with a MAPK inhibitor. In conclusion, the genetically modified hiPSCs can be used to model disorders of proplatelet formation. Both loss- and gain-of-function GPIb reduced MAPK/ERK activation but enhanced ROCK/MLC2 phosphorylation resulting in dysregulated platelet generation.

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant platelet bleeding disorder, with 55 patients reported worldwide so far, probably frequently misdiagnosed. Currently, there are no clear guidelines for the diagnosis and management of PT-VWD and this may contribute to misdiagnosis and thus to inappropriate treatment of these patients. This report provides expert opinion-based consensus recommendations for the standardized diagnostic and management approach to PT-VWD. Tests essential in the diagnostic workup are platelet count and size, ristocetin-induced platelet agglutination with mixing studies, and sequencing of platelet GP1BA gene. Platelet transfusions and von Willebrand factor-rich concentrates (if VWF is low) are the most effective treatments. This consensus may help to avoid misdiagnosis and guide appropriate management of patients with this disease.

Platelet-type von Willebrand disease (PT-VWD) is an inherited platelet disorder. It is characterized by macrothrombocytopenia and mucocutaneous bleeding, of variable severity, due to gain-of-function variants of GP1BA conferring to glycoprotein Ibα (GPIbα) enhanced affinity for von Willebrand factor (VWF). The bleeding tendency is conventionally attributed to thrombocytopenia and large VWF-multimer depletion. However, while some indications suggest that platelet dysfunction may contribute to the bleeding phenotype, no information on its characteristics and causes are available. The aim of the present study was to characterize platelet dysfunction in PT-VWD and shed light on its mechanism. Platelets from a PT-VWD patient carrying the p.M239V variant, and from PT-VWD mice carrying the p.G233V variant, showed a remarkable platelet function defect, with impaired aggregation, defective granule secretion and reduced adhesion under static and flow conditions. VWFbinding to GPIbα is known to trigger intracellular signaling involving Src-family kinases (SFK). We found that constitutive phosphorylation of the platelet SFK Lyn induces a negative-feedback loop downregulating platelet activation through phosphorylation of PECAM1 on Tyr686 and that this is triggered by the constitutive binding of VWF to GPIbα. These data show, for the first time, that the abnormal triggering of inhibitory signals mediated by Lyn and PECAM1 may lead to platelet dysfunction. In conclusion, our study unravels the mechanism of platelet dysfunction in PT-VWD caused by deranged inhibitory signaling. This is triggered by the constitutive binding of VWF to GPIbα which may significantly contribute to the bleeding phenotype of these patients.

Hemophilia A, Hemophilia B and von Willebrand disease both can present as bleeding diathesis with isolated prolonged PTT. The two diseases can sometimes be difficult to differentiate and furthermore can concurrently occur, which can further complicate diagnosis and management. This chapter will discuss hemophilia A and B in terms of diagnostic criteria and factor VIII and IX physiology. In addition, this chapter will also look at both quantitative and qualitative von Willebrand disease. Quantitative von Willebrand disease includes type 1 and 3 disorders. On the other hand, qualitative disease implies function defects either in coagulation function which includes type 2A, 2B, and 2M disease or in factor VIII carrier function as seen in 2N disease. Pseudo-von Willebrand disease, as its name suggested, is not a true von Willebrand disease, but a platelet glycoprotein Ib receptor gain-of-function disorder which results in laboratory findings that mimic type 2B disease. Acquired von Willebrand syndrome represents a collection of acquired von Willebrand factor abnormalities that have been associated with a number of known diseases such as aortic stenosis and hypothyroidism. The laboratory findings of acquired von Willebrand syndrome often resemble congenital von Willebrand disease subtypes.

SummaryPlatelet defects due to hyper-responsive GPIbα causing enhanced VWF interaction, counter-intuitively result in bleeding rather than thrombosis. The historical explanation of platelet/VWF clearance fails to explain mechanisms of impaired haemostasis particularly in light of reported poor platelet binding to fibrinogen. This study aimed to evaluate the defects of platelets with hyper-responsive GPIbα and their contribution to impaired in vivo thrombosis. Using the PT-VWD mouse model, platelets from the hTgG233V were compared to control hTgWT mice. Platelets’ pro-coagulant capacity was evaluated using flowcytometry assessment of P-selectin and annexin V. Whole blood platelet aggregation in response to ADP, collagen and thrombin was tested. Clot kinetics using laser injury thrombosis model and the effect of GPIbα inhibition in vivo using 6B4; a monoclonal antibody, were evaluated. Thrombin-induced platelet P-selectin and PS exposure were significantly reduced in hTgG233V compared to hTgWT and not signifi-cantly different when compared to unstimulated platelets. The hTgG233V platelets aggregated normally in response to collagen, and had a delayed response to ADP and thrombin, when compared to hTgWT platelets. Laser injury showed significant impairment of in vivo thrombus formation in hTgG233V compared to hTgWT mice. There was a significant lag in in vitro clot formation in turbidity assay but no impairment in thrombin generation was observed using thromboelastography. The in vivo inhibition of GPIbα facilitated new – unstable – clot formation but did not improve the lag. We conclude platelets with hyper-responsive GPIbα have complex intrinsic defects beyond the previously described mechanisms. Abnormal signalling through GPIbα and potential therapy using inhibitors require further investigations.Supplementary Material to this article is available online at www.thrombosis-online.com.

Background Mutations in the β-switch of GPIbα cause gain-of-function in the platelet-type von Willebrand disease. Structures of free and A1-bound GPIbα suggest that the β-switch undergoes a conformational change from a coil to a β-hairpin. Objectives Platelet-type von Willebrand disease (VWD) mutations have been proposed to stabilize the β-switch by shifting the equilibrium in favor of the β-hairpin, a hypothesis predicated on the assumption that the complex crystal structure between A1 and GPIbα is the high-affinity state. Methods Hydrogen-deuterium exchange mass spectrometry is employed to test this hypothesis using G233V, M239V, G233V/M239V, W230L, and D235Y disease variants of GPIbα. If true, the expectation is a decrease in hydrogen-deuterium exchange within the β-switch as a result of newly formed hydrogen bonds between the β-strands of the β-hairpin. Results Hydrogen-exchange is enhanced, indicating that the β-switch favors the disordered loop conformation. Hydrogen-exchange is corroborated by differential scanning calorimetry, which confirms that these mutations destabilize GPIbα by allowing the β-switch to dissociate from the leucine-rich-repeat (LRR) domain. The stability of GPIbα and its A1 binding affinity, determined by surface plasmon resonance, are correlated to the extent of hydrogen exchange in the β-switch. Conclusion These studies demonstrate that GPIbα with a disordered loop is binding-competent and support a mechanism in which local disorder in the β-switch exposes the LRR-domain of GPIbα enabling high-affinity interactions with the A1 domain.

Platelet-type von Willebrand disease is an inherited platelet disorder characterized by thrombocytopenia with large platelets caused by gain-of-function variants in GP1BA leading to enhanced GPIbα-von Willebrand factor (vWF) interaction. GPIbα and vWF play a role in megakaryocytopoiesis, thus we aimed to investigate megakaryocyte differentiation and proplatelet-formation in platelet-type von Willebrand disease using megakaryocytes from a patient carrying the Met239Val variant and from mice carrying the Gly233Val variant. Platelet-type von Willebrand disease megakaryocytes bound vWF at an early differentiation stage and generated proplatelets with a decreased number of enlarged tips compared to control megakaryocytes. Moreover, they formed proplatelets upon contact with collagen, differently from normal megakaryocytes. Similarly, collagen triggered megakaryocytes showed defective activation of the RhoA-MLC2 axis, which prevents proplatelet formation, and increased phosphorylation of Lyn, which acts as a negative regulator of GPVI signaling, thus preventing ectopic proplatelet-formation on collagen. Consistently, human and murine bone marrow contained an increased number of extravascular platelets compared to controls. In addition, platelet survival of mutant mice was shortened compared to control mice, and the administration of desmopressin, raising circulating vWF, caused a marked drop in platelet count. Taken together, these results show for the first time that thrombocytopenia in platelet-type von Willebrand disease is due to the combination of different pathogenic mechanisms, i.e. the formation of a reduced number of platelets by megakaryocytes, the ectopic release of platelets in the bone marrow, and the increased clearance of platelet/vWF complexes.

Background: Primary haemostasis defects comprise von Willebrand disease (VWD) and platelet disorders (PD). Although presenting with mild to moderate bleeding tendency in most cases, severe bleeding and blood loss may occur unexpectedly in trauma and surgery. Diagnosis of VWD and PD often remains difficult owing to the wide spectrum of clinical and laboratory manifestations. Platelet-type von Willebrand disease (PT-VWD) is frequently misdiagnosed as type 2B VWD. Discrimination between type 2B VWD and PT-VWD is crucial as treatment differs. Methods and results: A literature review revealed difficulties in diagnostic work-up and choice of optimal treatment of PT-VWD. Guidelines favour the therapeutic use of platelet concentrates. A telephone survey of diagnostic practice with regard to type 2B VWD/PT-VWD was conducted. The prevalence and incidence of type 2B and PT-VWD remained unclear, but PT-VWD may be underestimated. Discussion: An international study estimated that PT-VWD constitutes up to 15% of the total number of patients diagnosed with type 2B VWD. Our survey confirmed difficulties with diagnosis and showed that some centres did not exclude PT-VWD in type 2B patients. Some authors emphasize that genetic testing is the gold standard for diagnosis, but functional testing allows immediate diagnosis. Due to the important therapeutic implications we suggest that type 2B VWD be confirmed by genetic testing and that in case of a negative result PT-VWD should be excluded. Conclusion: PT-VWD should be excluded in all suspected cases of type 2B. PT-VWD should be treated with platelet concentrates.

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations of ristocetin. Diagnosis of either condition is not easy and the differential diagnosis between the two entities is especially challenging as evidenced by high levels of misdiagnosis of both conditions, but particularly PT-VWD. Five mutations in the GP1BA gene related to PT-VWD and less than 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macrothrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, von Willebrand factor ristocetin cofactor (VWF:RCo) to antigen (VWF:Ag) T located at nucleotide 3805 in the g.DNA of the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L). This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue W246 is located within the VWF-binding region and exists in a strongly conserved position in the phylogenetic tree, which is expected to be unable to tolerate substitutions without changing its functional characteristics. These findings argue strongly in favor of the view that this substitution does not represent a polymorphism and is therefore responsible for the PT-VWD phenotype of the patient Fil: Woods, Adriana Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Sánchez Luceros, Analía Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Bermejo, Emilse. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Paiva, Juvenal. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Alberto, Maria Fabiana. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Grosso, Silvia H.. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Kempfer, Ana Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Lazzari, María Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

SummaryInteraction between von Willebrand factor (VWF) and platelet GPIbα is required for primary haemostasis. Lack or loss-of-function in the ligand-receptor pair results in bleeding complications. Paradoxically, gain-of-function mutations in VWF or GPIbα also result in bleeding complications as observed in type 2B von Willebrand disease (VWD) and platelet-type- (PT-) VWD, respectively. A similar phenotype is observed with increased ristocetin-induced platelet agglutination and disappearance of the highest molecular weight multimers of VWF. We evaluated a patient with a bleeding disorder and a biological presentation compatible with type 2B VWD. VWF and platelet functional assays, sequencing of the VWF and GP1BA genes, and expression studies in HEK cells were performed. Sequencing of the VWF gene in the propositus revealed a heterozygous p.Pro1266Leu mutation previously found in type 2B VWD Malmö/New York. These variants are characterised by a mild phenotype and a normal VWF multimer composition suggesting the presence of a second mutation in our propositus. Sequencing of the GP1BA gene revealed a heterozygous c.765G>A substitution changing Met at position 255 of GPIbα to Ile. This new mutation is located in the β-switch domain where five other gain-of-function mutations have been reported in PT-VWD. Expression of GPIbα Ile255 in HEK GPIb-IX cells resulted in enhanced VWF binding compared to wild-type, similar to known PT-VWD mutations (p.Val249, p.Ser249 and p.Val255) indicating that it contributes to the propositus defects. This first report associating PT-with type 2B VWD illustrates the importance of combining biological assays with genetic testing to better understand the clinical phenotype.

Platelet-type von Willebrand disease (PT-VWD) is an autosomal dominant rare bleeding disorder characterized by hyperresponsive platelets. This inherent platelet function defect is due to a gain-of-function mutation within the GP1BA gene coding for the platelet surface glycoprotein Ib alpha protein, the receptor for the adhesive protein von Willebrand factor (VWF). The defect results in excessive and unnecessary platelet-VWF interaction with subsequent removal of the hemostatically efficient high molecular weight VWF as well as platelets from the circulation, leading to thrombocytopenia and bleeding diathesis. Patients with PT-VWD present with mild to moderate mucocutaneous bleeding, which becomes more pronounced during pregnancy and following aspirin ingestion or drugs that have antiplatelet activity. Laboratory testing shows low VWF:ristocetin cofactor and low or normal VWF:antigen and characteristically an enhanced ristocetin-induced platelet agglutination (RIPA). These laboratory features are also indicators of the closely similar and more common bleeding disorder type 2B VWD. Simplified RIPA mixing assays, cryoprecipitate challenge, and flow cytometry can differentiate between the two disorders. However, the gold standard is to identify mutations within the VWF gene (indicating type 2B VWD) or the platelet GP1BA gene (confirming PT-VWD). Treatment is based on making a correct diagnosis of PT-VWD where platelet concentrates instead of VWF/factor VIII preparations should be administered. A recent fairly large retrospective/prospective registry-based international study showed that PT-VWD is very rare, likely to be misdiagnosed as type 2B VWD or idiopathic thrombocytopenic purpura, and represents 15% of type 2B VWD diagnoses.

Glycoprotein Ibα (GPIbα) interacts with von Willebrand factor (VWF) inducing the tethering of platelets to injured vessel walls and subsequent hemostasis process. We have previously shown that the conformation of the β-switch region of GPIbαN can be regulated by flow. Flow induces a loop-to-β-hairpin conformational change in this region, which is a suggested mechanism for the flow-enhanced binding of GPIbα to VWF-A1. To further evaluate the mechanism and obtain more complete evidences, here we performed flow molecular dynamics simulations of wild type and a number of mutants of the β-switch. The results demonstrate that the gain-of-function mutations G233V, D235V, and K237V promote the conformational transition toward β-hairpin, while the loss-of-function mutation Q232V impedes the transition. The promotion is caused mainly by the improved polarity similarity of the paired residues on the β-hairpin, and also by the decreased flexibility of one strand of the β-switch. The gain-of-function mutations exert the influence locally, affecting only hydrogen bonds near the mutated residues. The impediment of the loss-of-function mutant may be non-essential hydrophobic interactions blocking the conformational change.

Platelet-type von Willebrand disease (PT-VWD) is a bleeding disorder of the platelet glycoprotein Ib-IX/von Willebrand factor (VWF) axis caused by mutations in the glycoprotein Ib-IX receptor that lead to an increased affinity with VWF. In this report, platelets from a mouse expressing a mutation associated with PT-VWD have been visualized using state-of-the art image collection and processing. Confocal analysis revealed that VWF bound to the surface of single platelets and bridging micro-aggregates of platelets. Surface-bound VWF appears as a large, linear structure on the surface of 50% of the PT-VWD platelets. In vivo thrombus formation after chemical injury to the carotid artery revealed a severe impairment to occlusion as a consequence of the PT-VWD mutation. In vitro stimulation of PT-VWD platelets with adenosine diphosphate or thrombin demonstrates a significant block in their ability to bind fibrinogen. The impairment of in vivo thrombus formation and in vitro fibrinogen binding are more significant than might be expected from the observed platelet binding to VWF polymers over a small portion of the plasma membrane. Visualization of the receptor/ligand interaction and characterization of a severe antithrombotic phenotype provide a new understanding on the molecular basis of bleeding associated with the PT-VWD phenotype.

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder which is due to a mutation in the gene encoding for platelet glycoprotein Ibalpha (GPIbalpha) resulting in enhanced affinity for von Willebrand factor (VWF). PT-VWD is often mistakenly diagnosed as type 2B VWD for the similarities between these two conditions. We characterized a new case of PT-VWD and evaluated the usefulness of a flow cytometric assay in the differential diagnosis between PT-VWD (n=1) and type 2B VWD (n=4). The flow cytometric assay was able to highlight the increased affinity of VWF for GPIbalpha as much as did RIPA and to differentiate the two diseases through mixing tests. Genetic analysis revealed a heterozygous point mutation in codon 239 of the GPIbalpha gene leading to a methionine to valine substitution (M239V). Flow cytometry represents a useful tool for the diagnosis of PT-VWD.

Interaction of glycoprotein Ibα (GPIbα) with von Willebrand factor (VWF) initiates platelet adhesion to injured vascular wall to stop bleeding. A major contact between GPIbα and VWF involves the β-switch region, which is a loop in the unliganded GPIbα but switches to a β-hairpin in the complex structure. Paradoxically, flow enhances rather than impedes GPIbα-VWF binding. Gain-of-function mutations (e.g., M239V) in the β-switch reduce the flow requirement for VWF binding, whereas loss-of-function mutations (e.g., A238V) increase the flow requirement. These phenomena cannot be explained by crystal structures or energy calculations. Herein we demonstrate that the β-hairpin is unstable without contacting VWF, in that it switches to a loop in free molecular dynamics simulations. Simulations with a novel flow molecular dynamics algorithm show that the loop conformation is unstable in the presence of flow, as it switches to β-hairpin even without contacting VWF. Compared with the wild-type, it is easier for the M239V mutant but harder for the A238V mutant to switch to β-hairpin in the presence of flow. These results elucidate the structural basis for the two mutants and suggest a regulatory mechanism by which flow activates GPIbα via inducing a loop-to-β-hairpin conformational transition on the β-switch, thereby promoting VWF binding.

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) have different etiologies although both present with a similar clinical bleeding and basic laboratory phenotype. Both PT-VWD and 2B-VWD represent gain-of-function mutations that lead to enhanced binding between plasma von Willebrand factor (VWF) and its platelet ligand, glycoprotein Ib alpha (GP1BA). However, 2B-VWD results from a functionally abnormal VWF molecule arising from mutations in the VWF gene, whereas PT-VWD is caused by hyperresponsive platelets resulting from mutations in the platelet GP1BA gene. A definitive diagnosis of PT-VWD versus 2B-VWD is critical for treatment decisions (as differential therapies might be respectively required) and also for family counseling. However, laboratory discrimination of PT-VWD versus 2B-VWD is problematic because simple phenotypic testing will not permit their differentiation, and the more complex testing approaches that might permit their differentiation are rarely applied, or are perhaps poorly applied. Although differential identification of PT-VWD versus 2B-VWD can most definitively be achieved by identifying the gene defect at either the VWF or GP1BA loci, such tests are not commonly available, not always successful, and even if available and successful might not be readily available to serve time-critical treatment decisions. Accordingly, simple laboratory tools to enable discrimination of the two disorders would be a valuable addition to the test repertoire of the hemostasis laboratory. This article provides a review of PT-VWD-related literature and an overview of a phenotypic laboratory test process that should enable the effective identification of PT-VWD and its discrimination from 2B-VWD.

The platelet glycoprotein Ib-IX receptor binds surface-bound von Willebrand factor and supports platelet adhesion to damaged vascular surfaces. A limited number of mutations within the glycoprotein Ib-IX complex have been described that permit a structurally altered receptor to interact with soluble von Willebrand factor, and this is the molecular basis of platelet-type von Willebrand disease. We have developed and characterized a mouse model of platelet-type von Willebrand disease (G233V) and have confirmed a platelet phenotype mimicking the human disorder. The mice have a dramatic increase in splenic megakaryocytes and splenomegaly. Recent studies have demonstrated that hematopoetic cells can influence the differentiation of osteogenic cells. Thus, we examined the skeletal phenotype of mice expressing the G233V variant complex. At 6 months of age, G233V mice exhibit a high bone mass phenotype with an approximate doubling of trabecular bone volume in both the tibia and femur. Serum measures of bone resorption were significantly decreased in G233V animals. With decreased bone resorption, cortical thickness was increased, medullary area decreased, and consequently, the mechanical strength of the femur was significantly increased. Using ex vivo bone marrow cultures, osteoclast-specific staining in the G233V mutant marrow was diminished, whereas osteoblastogenesis was unaffected. These studies provide new insights into the relationship between the regulation of megakaryocytopoiesis and bone mass.

Summary. Background: Interaction between platelet glycoprotein (GP)Ibα and von Willebrand factor (VWF) has critical roles in both physiological hemostasis and thrombosis. Platelet-type von Willebrand disease (plt-VWD) is a congenital bleeding disorder characterized by gain-of-function mutations of GPIbα. To date, two mutations in GPIbα, G233V and M239V, have been reported in four unrelated families with plt-VWD. Objective: The present study aimed to determine whether G233S of GPIbα, a new mutation observed in plt-VWD patients, causes the plt-VWD phenotype and to examine whether conversions to other residues at this position affect VWF binding. Patients and methods: The propositus was a 3-year-old Japanese male. He displayed bleeding symptoms and moderate thrombocytopenia. His brother was similarly affected. Platelets from both patients were analyzed by ristocetin- or shear-induced platelet aggregation. DNA sequencing was performed to analyze the GPIbα sequence. We examined the 125I-labeled VWF binding using a series of recombinant GPIbα fragments with different residues at position 233 (G233S, G233A, G233K, and G233D) together with naturally occurring mutations previously reported in patients (G233V and M239V). Results: Platelet function analysis indicated that platelets from both patients had a typical plt-VWD phenotype. DNA sequencing analysis showed a heterozygous mutation of Gly to Ser at residue 233 of GPIbα in both patients. The 125I-labeled VWF binding to mutant compared with the wild type displayed three patterns, gain-of-function (G233S, G233V, and M239V), equivalent function (G233A), and loss-of-function (G233K and G233D). Conclusions: The G233S is a molecular basis of plt-VWD, and residue 233 plays critical roles in regulating VWF binding.

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.

We are pleased to highlight in this issue of Seminars in Thrombosis & Hemostasis, the study by Wood et al,1 wherein the authors describe a novel mutation in the platelet GP1BA gene creating a hyperresponsive GPIbα protein—a receptor for von Willebrand factor (VWF)—and causing platelet-type von Willebrand disease (PT-VWD). This is a new naturally occurring mutation in a 23-year-old male patient and is considered the sixth reported mutation thus far in patients described with this disease worldwide. Despite being a rare bleeding disorder, PT-VWD represents a significant challenging clinical problem as it may cause life-threatening bleeding, if not appropriately treated, particularly in situations related to surgeries and childbirth. The clinical diagnosis challenge stems from the close similarity of PT-VWD to the more common bleeding disorder, type 2BVWD.2,3 The discrimination and correct diagnosis can only be made after carefully assessing less commonly performed laboratory tests,3–8 and confirmed only after DNA analysis of the binding regions in the two genes VWF and GP1BA9 has been performed. The report by Woods et al1 highlights some important issues that add to our understanding of this rare but potentially life-threatening bleeding disorder. First, the patient presented with severe bleeding symptoms (rather than mild/moderate bleeding symptoms, and unlike previously reported cases) while also showing other typical laboratory phenotypic data known in this disease such as macrothrombocytopenia, mild spontaneous platelet aggregation, absence from plasma of high-molecular-weight VWF multimers, positive ristocetin-induced platelet aggregation (RIPA) at 0.3 and 0.4 mg/mL, VWF ristocetin cofactor (VWF:RCo) T GP1BA gene mutation that predicted the protein change Try246Leu. This mutation was absent in the unaffected mother and also in 100 healthy control subjects. Second, in that report,1 an attempt was made to quantify the bleeding symptoms in a patient with PT-VWD (bleeding score of 13). The knowledge about variation in bleeding symptoms and the ability to objectively assess these symptoms have proven to be important inmanagement of bleeding disorders and can help predict disease outcome and also aid in treatment.10 The only other such attempt was in a small case series reported recently. This series showed considerable variability in bleeding severity among PT-VWD that is independent of age or gender. The phenotypic variability in type 2B VWD has been reviewed in relation to various mutations and different patients’ cohorts.12 However, a systematic analysis of PT-VWD phenotype and the genotype–phenotype relationship remains to be investigated. Third, for the first time, the level of VWF propeptide (VWFpp) and VWFpp/VWF:Ag ratio was reported in a patient with PT-VWD.1 VWFpp is a 741 amino acid portion that gets cleaved from mature VWF by proteolysis. After cleavage, the VWFpp remains in noncovalent association with the VWF multimers, and both are stored together in the α-granules (megakaryocytes/platelets) or Weibel–Palade bodies (endothelial cells). Upon release and under physiologic pH, the VWF multimers and VWFpp dissociate and are secreted in 1:1 stoichiometric amounts.13 Studies have shown a regulatory role for VWFpp as an intramolecular chaperone for the mature VWF protein and an aid to its storage and multimerization.14,15 The VWFpp circulates in the plasma for a short time, with a half-life of approximately 2 to 3 hours and plasma levels of approximately 1 μg/mL, whereas multimeric VWF circulates with a half-life of approximately 8 to 12 hours and plasma levels of approximately 10 μg/mL.16,17 Recent

To maintain hemostasis under shear conditions, there must be an interaction between the platelet glycoprotein (GP) Ib-IX receptor and the plasma ligand von Willebrand factor (vWf). In platelet-type von Willebrand disease (Pt-vWD), hemostasis is compromised. Two mutations in the GPIbα polypeptide chain have been identified in these patients—a glycine-233 to valine change and a methionine-239 to valine change. For this investigation, these mutant proteins have been expressed in a Chinese hamster ovary cell model system. Ligand-binding studies were performed at various concentrations of ristocetin, and adhesion assays were performed under flow conditions. The Pt-vWD mutations resulted in a gain-of-function receptor. vWf binding was increased at all concentrations of ristocetin examined, and adhesion on a vWf matrix was enhanced in terms of cell tethering, slower rolling velocity, and decreased detachment with increasing shear rate. Two other mutations were also introduced into the GPIbα chain. One mutation, encompassing both the Pt-vWD mutations, created an increase in the hydrophobicity of this region. The second mutation, involving a valine-234 to glycine change, decreased the hydrophobicity of this region. Both mutations also resulted in a gain-of-function receptor, with the double mutation producing a hyperreactive receptor for vWf. These data further support the hypothesis that ligand binding is regulated by conformational changes in the amino-terminal region of GPIbα, thereby influencing the stability of the GPIbα–vWf interaction.

Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibalpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys(209)-Cys(248) disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on (125)I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibalpha. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibalpha, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibalpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.

SummaryPseudo-von Willebrand disease (p-VWD) and type 2B von Willebranddisease (VWD) have similar phenotypic parameters and clinical symptoms,but different aetiologies. Fourteen individuals from five families with ahistorical diagnosis of type 2B VWD but with no mutation in the vonWillebrand factor gene were re-investigated for the possibility of p-VWD,using platelet aggregation in the presence of cryoprecipitate. p-VWD wasconfirmed by targeted DNA sequencing of the glycoprotein Iba gene,identifying a heterozygous Glycine 233 Valine substitution. This studysuggests that p-VWD may be under diagnosed, and that platelet aggregationin the presence of cryoprecipitate is useful in differentiating this disorderfrom type 2B VWD.Keywords: pseudo-von Willebrand disease, type 2B von Willebrand disease,platelet aggregation with cryoprecipitate, mutation, GPIba gene. short report a 2006 The Authorsdoi:10.1111/j.1365-2141.2006.06078.x Journal Compilation a 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 664–666

Platelet-type von Willebrand disease (vWD) is a congenital bleeding disorder characterized by heightened ristocetin-induced platelet aggregation caused by abnormally high affinity between the platelet membrane glycoprotein (GP) Ib/IX complex and von Willebrand factor (vWF ). Two distinct point mutations, Gly233 to Val and Met239 to Val, have been reported in GPIbα. We have constructed a recombinant GPIbα fragment containing the latter mutation, Met239 to Val (M239V) and characterized the mutant molecule using two methods, ie, interaction between soluble vWF and immobilized M239V and inhibition of platelet aggregation by purified soluble M239V. Spontaneous binding (ie, binding without any inducers) was observed between 125I-vWF and immobilized M239V but not between 125I-vWF and immobilized wild-type (WT) GPIbα. The addition of low concentrations of ristocetin (0.2 mg/mL) induced specific 125I-vWF binding to immobilized M239V, but not to WT GPIbα. At high concentrations of ristocetin (1.2 mg/mL), both WT GPIbα and M239V specifically bound to 125I-vWF. Thus, M239V reproduced the unique functional abnormality of the GPIb/IX complex in platelet-type vWD. Moreover, the purified soluble M239V inhibited platelet aggregation induced by low concentration of ristocetin (0.3 mg/mL) in platelet-rich plasma from a patient having Met239 to Val mutation, whereas purified WT did not. These results provide direct evidences that the reported point mutation is the responsible molecular basis of this disorder.

Compared with coagulation factor defects, little attention is given to defects of platelet function as causes of rare bleeding disorders. Platelet-type von Willebrand disease (PT-VWD) is an autosomal dominant bleeding disorder and is unique among platelet disorders because it is characterized by platelet hyperresponsiveness rather than decreased function. The disease is caused by gain-of-function mutations in the platelet GP1BA gene, which codes for the platelet von Willebrand factor (VWF) receptor, GPIbα. Only five mutations (four missense and one deletion) have so far been reported. Affected patients suffer from mild to moderate mucocutaneous bleeding, low VWF activity compared with antigen, decreased high-molecular-weight VWF multimers, variable degree of thrombocytopenia and typically platelet aggregation in response to low concentrations of ristocetin. All reported PT-VWD missense mutations occur within the R-loop of GPIbα and it was speculated that the introduction of short branched chain mutations such as Val in PT-VWD stabilized the extended β-hairpin. Examination of this theory by surveying all the available GPIbα structures showed that a distinct conformation predominates for the R-loop when GPIbα is not bound to VWF-A1 and this provides the framework of a new hypothesis for the molecular basis of PT-VWD. Worldwide efforts to improve diagnosis of PT-VWD continue, and international systematic studies are required to further our understanding of the phenotype and the influence of the hyperresponsive GPIbα beyond hemostasis.

Genomic DNA was studied from four patients with platelet-type von Willebrand disease (vWD) from two Japanese families previously reported. The entire coding region of platelet glycoprotein (GP) Ib alpha, a component of the platelet receptor for von Willebrand factor (vWF), was examined by polymerase chain reaction (PCR) followed by direct DNA sequence analysis. A single point mutation was found in all patients resulting in substitution of Val (GTG) for Met (ATG) at residue 239 of GPIb alpha. All patients were heterozygous for the mutation, whereas none of the unaffected family members had an amino acid substitution at residue 239. Because the nucleotide substitution destroys an NIa III restriction site on GPIb alpha, PCR products were subjected to digestion with this enzyme; DNA fragments from both normal and mutant alleles were detected in all affected individuals. In allele- specific PCR, DNA was amplified from patients' genomic DNA using either adenine- or guanine-containing primers, whereas only adenine-containing primer successfully amplified DNA from normal individuals. Cloning of amplified DNA into bacteriophage M13mp19 and subsequent DNA sequence analysis confirmed the mutation in these families. The absence of the amino acid substitution at residue 239 of GPIb alpha in the normal individuals tested, together with the linkage of this substitution to the phenotypic expression of disease in these two families and in a family recently described suggest that this amino acid change is a molecular basis for platelet-type vWD, and the substitution may produce a quite similar phenotype to the one reported previously (Gly to Val at residue 233 of GPIb alpha).

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder with a defect in platelet glycoprotein Ib α (GP1bα), the receptor for the adhesive glycoprotein von Will...

Almost thirty years has passed since the description of platelet-type von Willebrand disease. Named initially pseudo-von Willebrand disease, this mild mucocutaneous bleeding disorder has an intrinsic defect in platelets rather than VWF. Due to its unique features and its similarity to the more common type 2B VWD, many investigators were intrigued by its nature, pathophysiology and molecular genetics aspects. The challenges to diagnose this disorder have created debate in literature about the best method of discrimination from type 2B VWD. The recent development of the PT-VWD mouse model carries huge potential for further understanding of the disease as well as the platelet defect and its influence on other biological processes. This review provides a basic as well as updated knowledge about the PT-VWD including history, clinical and laboratory features and treatment in addition to the diagnostic challenges with a focus on molecular genetics aspects and PT-VWD research.

The platelet GP Ib-IX receptor supports platelet adhesion and activation by binding to vWf in the exposed subendothelial matrix. An abnormal GP Ib-IX complex exists in platelet-type or pseudo-von Willebrand disease and has a characteristic increased affinity for soluble vWf resulting in impaired hemostatic function due to the removal of larger vWf multimers from the circulation. Genetic studies within an afflicted family have demonstrated that the disease is linked to a Gly233-->Val amino acid substitution within the alpha-subunit of the oligomeric GP Ib-IX complex (Miller, J.L., D. Cunningham, V.A. Lyle, and C. L. Finch. 1991. Proc. Natl. Acad. Sci. USA. 88:4761-4765). To evaluate the functional consequences of this mutation, we constructed a recombinant analogue of the alpha-subunit of GP Ib containing Val233. Experiments comparing molecules with either Gly233 or Val233 revealed that the Val substitution generates a molecule with increased affinity for vWf. The recombinant fragment reproduces the functional abnormality of the GP Ib-IX complex in platelet-type von Willebrand disease, thus establishing the molecular basis of the bleeding disorder within this family. Moreover, it becomes apparent that structural elements responsible for the regulation of hemostasis through modulation of vWf affinity for platelets reside within the alpha-subunit of the GP Ib-IX complex.

Pseudo (platelet-type)-von Willebrand disease is an autosomal dominant bleeding disorder caused by the hyperfunction of a receptor on the platelet surface. The abnormal receptor, glycoprotein Ib, displays increased affinity for its ligand, von Willebrand factor. Four members (normal mother/affected father/two affected daughters) of a family with pseudo-von Willebrand disease were studied to determine the molecular genetic basis for their congenital platelet defect. Segments of the platelet glycoprotein Ib alpha gene were amplified by means of the polymerase chain reaction, cloned, and sequenced. A point mutation (A to G, codon 239) was found in segments from the affected individuals but not from the normal. The mutation results in a single amino acid substitution (valine-mutant for methionine-normal) at residue 239 within the Ib alpha binding site for von Willebrand factor. Both the mutant and the normal sequence were found in affected individuals, suggesting a heterozygous state. Amplified DNA from family members and from 58 normal individuals was analyzed by allele-specific oligonucleotide hybridization. Only the normal sequence was found in the mother and the normal individuals, whereas both the normal and the mutant alleles were found in the affected family members. The described mutation is associated with the pseudo-von Willebrand disease phenotype seen in this kindred. The resultant single amino acid substitution in glycoprotein Ib alpha relates to increased receptor function and to excessive binding of von Willebrand factor to the platelet surface.

SummaryLess than 50 patients are reported with platelet type von Willebrand disease (PT-VWD) worldwide. Several reports have discussed the diagnostic challenge of this disease versus the closely similar disorder type 2B VWD. However, no systematic study has evaluated this dilemma globally. Over three years, a total of 110 samples/data from eight countries were analysed. A molecular approach was utilised, analysing exon 28 of the von Willebrand factor (VWF) gene, and in mutation negative cases the platelet GP1BA gene. Our results show that 48 cases initially diagnosed as putative type 2B/PT-VWD carried exon 28 mutations consistent with type 2B VWD, 17 carried GP1BA mutations consistent with a PT-VWD diagnosis, three had other VWD types (2A and 2M) and five expressed three non-previously published exon 28 mutations. Excluding 10 unaffected family members and one acquired VWD, 26 cases did not have mutations in either genes. Based on our study, the percentage of type 2B VWD diagnosis is 44% while the percentage of misdiagnosis of PT-VWD is 15%. This is the first large international study to investigate the occurrence of PT-VWD and type 2B VWD worldwide and to evaluate DNA analysis as a diagnostic tool for a large cohort of patients. The study highlights the diagnostic limitations due to unavailability/poor application of RIPA and related tests in some centres and proposes genetic analysis as a suitable tool for the discrimination of the two disorders worldwide. Cases that are negative for both VWF and GP1BA gene mutations require further evaluation for alternative diagnoses.

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patient platelets exhibit an abnormally increased binding of circulating von Willebrand factor (vWF). We have recently shown that this abnormality is associated with a point mutation resulting in substitution of Val for Gly 233 in platelet membrane glycoprotein Ib alpha (GPIb alpha), a major component of the platelet GPIb/IX receptor for vWF. To investigate the effect of this substitution on the three-dimensional structure of this region of the protein, we have generated the allowed (low energy) conformations of the region of the GPI alpha protein containing residues 228-238 (with 5 residues on either side of the critical residue 233) with Gly 233 (wild type) and Val 233 (PT-vWD) using the computer program ECEPP (Empirical Conformational Energies of Peptides Program). The wild-type sequence is Tyr-Val-Trp-Lys-Gln-Gly-Val-Asp-Val-Lys-Ala. We find that the Gly 233-containing peptide can exist in two low energy conformers. The lowest energy conformer is a structure containing a beta-turn at Gln 232-Gly 233 while the alternative conformation is an amphipathic helical structure. Only the amphipathic helical structure is allowed for the Val 233-containing peptide which contains a hydrophobic 'face' consisting of Val 229, Val 233 and Val 236 and another hydrophilic surface composed of such residues as Lys 231 and Asp 235. No such surfaces exist for the lowest energy bend conformer for the Gly 233-containing peptide, but do exist in the higher energy helical structure. The amphipathic surfaces in the 228-238 region of the Val 233-containing GPIb alpha protein may associate strongly with complementary surfaces during vWF binding to the GPIb/IX receptor complex and may help explain heightened association of vWF with this receptor in PT-vWD.

Platelet-type von Willebrand disease (PT-VWD), or pseudo-VWD, is a rare inherited platelet disorder characterized by an increased affinity of the platelet membrane glycoprotein Ibalpha receptor for normal von Willebrand factor leading to characteristic platelet hyperaggregability. As PT-VWD shares most of the clinical and laboratory features of subtype 2B VWD, the differential diagnosis between these two inherited bleeding disorders requires either platelet-mixing or molecular genetic studies. In this review, the main clinical, laboratory and therapeutic characteristics of PT-VWD are concisely reported.

Platelet-type von Willebrand disease (PT-VWD, or pseudo-VWD) and type 2B VWD share a common bleeding phenotype with different etiologies. Both PT-VWD and type 2B VWD represent an enhanced binding between the plasma von Willebrand factor (VWF) to its platelet ligand, glycoprotein Ib alpha ( GP1BA). However, type 2B VWD results from a functionally abnormal VWF molecule, whereas PT-VWD is caused by hyperresponsive platelets due to defects in the platelet GP1BA gene. The laboratory discrimination between the two disorders can be a challenge because simple phenotypic testing will not differentially identify the disorders, and the more complex testing approaches are often poorly applied. Definitive diagnosis is critical for treatment decisions and can be most definitively achieved by identifying the gene defect at either the VWF or GP1BA loci. A systematic international molecular genetic study would be helpful to address the question of whether PT-VWD is being misdiagnosed as type 2B VWD. Such a study can be facilitated by an international online database/disease registry to enhance international awareness about this otherwise long-recognized diagnostic dilemma.