Platelet changes and bleeding symptoms in children, adolescents, and adults with 22q11.2 deletion syndrome.

BACKGROUND: The deletion region of 22q11.2 deletion syndrome (22q11.2DS) contains a gene encoding glycoprotein Ibß (GPIbß), which is required to express the GPIb/IX/V complex on the platelet surface. Therefore, patients with 22q11.2DS may have congenital platelet disorders. However, information is limited on platelets and bleeding symptoms. In this study, we investigated clinical information, including bleeding symptoms, platelet counts, and GPIb expression levels in children and adolescents/adults with 22q11.2DS. PROCEDURE: Thirty-two patients with 22q11.2DS were enrolled in a prospective cohort study between 2022 and 2023 at outpatient clinics within our institute. RESULTS: The median platelet counts in adolescents/adults with 22q11.2DS were significantly lower than those in children (p < .0001). A gradual decrease was found along with increasing age (p = .0006). Values of median GPIb expression on platelet surfaces (66% in children and 70% in adolescents/adults) were significantly lower than those in healthy controls (p < .0001 and p = .0002). Bleeding symptoms included surgery-related bleeding (52%), purpura (31%), and epistaxis (22%); most of them were minor. The median International Society on Thrombosis and Hemostasis bleeding assessment tool score was not significantly different between children and adolescents/adults (p = .2311). CONCLUSION: Although there was an age-related decrease in platelet count and a disease-related decrease in GPIb expression, no difference in bleeding symptoms was found between children and adolescents/adults. 22q11.2DS overall had minor bleeding symptoms in daily life, and the disease had little effect on spontaneous bleeding. However, some patients had major bleeding events; further accumulation of data on hemostasis during surgery and trauma is required.

[Rare VPS33B gene mutation combined with GP1BA mutation causes severe decrease in plasma VWF levels: a case report and literature review].

A 28-year-old woman was found to have coagulation factor Ⅷ activity (FⅧ∶C) <1% and von Willebrand factor antigen (VWF∶Ag) <1% during routine prenatal examinations. No pathogenic variation was found in the exon region of the VWF gene using next-generation sequencing. The clinical presentation of this patient does not match the clinical characteristics of type Ⅲ hemophilia [von Willebrand disease (VWD) ]; therefore, third-generation sequencing technology was used to perform whole-genome sequencing on the patient and her family members. Multiple members of the patient's paternal family carried a heterozygous variant of VPS33B, c.869G>C. The family members carrying this variant all had varying degrees of reduced VWF levels (39% -56% ). Moreover, the proband was detected with the heterozygous variant c.1474dupA in GP1BA. The ACMG and Clinvar databases determined that this variation was associated with platelet-type pseudo VWD. The decrease in VWF levels caused by heterozygous variations in VPS33B in families is the first international report, and no previous studies have reported cases of severe decrease in plasma VWF levels caused by double heterozygous variations in VPS33B and GP1BA.

Novel GPIb-independent platelet aggregation induced by botrocetin: implications for diagnosis and antithrombotic therapy.

BACKGROUND: Snake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand disease and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis. OBJECTIVES: To explore the alternative receptor(s)/mechanisms that participate in botrocetin-induced platelet aggregation. METHODS: The effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic, ITGA2B and ITGB3-deficient mice, and Bernard-Soulier syndrome and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing enzyme-linked immunosorbent assay. Botrocetin-αIIbß3 and botrocetin-GPIbα interactions were measured using enzyme-linked immunosorbent assay and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber. RESULTS: Botrocetin could induce aggregation of platelets from a Bernard-Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with αIIbß3 and facilitated αIIbß3-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting αIIbß3. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to αIIbß3 and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited αIIbß3- and GPIb-mediated platelet aggregation, spreading, and thrombus formation. CONCLUSION: Our study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both αIIbß3 and GPIbα.

Bernard-Soulier syndrome caused by a novel GP1BB variant and 22q11.2 deletion.

Bernard-Soulier syndrome (BSS) is caused by defects in GP1BA, GP1BB, or GP9 genes. Patients with 22q11.2 deletion syndrome (22q11.2DS) are obligate carriers of BSS because GP1BB resides on chromosome 22q11.2. A 15-month-old girl without bleeding symptoms had giant platelets and thrombocytopenia. Physical findings and macrothrombocytopenia suggested 22q11.2DS, which was confirmed by fluorescence in situ hybridization. Flow cytometry showed decreased GPIbα on the platelets. Gene panel testing revealed a novel variant in GP1BB, p.(Val169_Leu172del). These findings confirmed that the patient had BSS. This case suggests that any patient with 22q11.2DS and macrothrombocytopenia should be further tested for BSS.

Genetic Polymorphisms of GP1BA, PEAR1, and PAI-1 may be Associated with Serum sIgE and Blood Eosinophil Levels in Chinese Patients with Allergic Diseases.

BACKGROUND: It has been suggested that genetic factors may be substantially linked to allergy disorders. OBJECTIVE: This study aims to investigate the relationship between the serum specific Immunoglobulin E (sIgE), blood eosinophil, and the polymorphisms of glycoprotein Ib alpha gene (GP1BA) rs6065, platelet endothelial aggregation receptor 1 gene (PEAR1) rs12041331, and plasminogen activator inhibitor 1 gene (PAI-1) rs1799762. METHODS: From the Peking Union Medical College Hospital, this study enrolled 60 healthy participants and 283 participants with allergic diseases. TaqMan-minor groove binder (MGB) quantitative polymerase chain reaction (qPCR) was used to examine the gene polymorphisms in each group. RESULTS: The TaqMan-MGB qPCR results were completely consistent with the DNA sequencing results, according to other studies in this medical center (Kappa =1, p <0.001). The GP1BA rs6065, PEAR1 rs12041331, and PAI-1 rs1799762 polymorphisms did not show different distribution between allergy patients and healthy individuals. Concerning allergy patients, the CT (n=33) genotype of GP1BA rs6065 had higher blood eosinophil level than the CC (n=250) genotype (0.59, IQR 0.32-0.72 vs 0.31, IQR 0.15-0.61, *109/L, p =0.005). The serum sIgE of AA (n=46) genotype of PEAR1 rs12041331 was lower (median 3.7, interquartile quartiles (IQR) 0.2-16.8, kU/L) than the GA (n=136) and GG (n=101) genotypes (GA median 16.3, IQR 3.1-46.3, kU/L, p = 0.002; GG median 12.9, IQR 3.0-46.9, kU/L, p =0.003). The GA genotypes of PEAR1 rs12041331were with higher blood eosinophil levels (median 0.42, IQR 0.17-0.74 *109/L) than the AA genotype (median 0.25, IQR 0.15-0.41*109/L, p =0.012). The sIgE of the 5G5G (n=44) genotype of PAI-1 rs1799762 was lower (median 5.0, IQR 0.1-22.8, kU/L) than the 4G5G (n=144) (median 17.3, IQR 3.7-46.0, kU/L, p = 0.012). CONCLUSION: The GP1BA rs6065, PEAR1 rs12041331, and PAI-1 rs1799762 polymorphisms may be associated with the genetic susceptibility of serum sIgE or blood eosinophil in Chinese allergic disease patients.

GPIbα-filamin A interaction regulates megakaryocyte localization and budding during platelet biogenesis.

ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.

A new case of platelet-type von Willebrand disease supports the recent findings of gain-of-function GP1BA variants outside the C-terminal disulphide loop enhances affinity for von Willebrand factor.

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder characterized by an increased ristocetin-induced platelet aggregation (RIPA) and enhanced affinity of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF). To date, only seven variants have been described with this gain-of-function effect, most of them located in the C-terminal disulphide loop of the VWF-binding domain of GPIbα. We herein describe a patient with moderate bleeding symptoms, mild thrombocytopenia and increased RIPA. By direct sequencing of GP1BA, a novel leucine-rich repeat heterozygous variant was identified (c.580C>T; predictably p.Leu194Phe), strongly suggestive as being the underlying cause for the PT-VWD phenotype of our patient.

[The Effect of Ena/VASP Family on the Expression of GPIb-IX Complex in Human Megakaryoblastic Leukemia Dami Cells].

OBJECTIVE: To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells. METHODS: SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using LipofectamineTM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry. RESULTS: We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down. CONCLUSION: Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.

The Glycoprotein (GP)Ib-IX-V Complex on Platelets: GPIbα Protein Expression Is Reduced in HeartMate 3 Patients with Bleeding Complications within the First 3 Months.

Non-surgical bleeding (NSB) remains the most critical complication in patients under left ventricular assist device (LVAD) support. It is well known that blood exposed to high shear stress results in platelet dysfunction. Compared to patients without NSB, decreased surface expression of platelet receptor GPIbα was observed in LVAD patients with NSB. In this study, we aimed to compare the expression level of glycoprotein (GP)Ib-IX-V platelet receptor complex in HeartMate 3 (HM 3) patients with and without bleeding complications to investigate the alterations of the platelet transcriptomic profile on platelet damage and increased bleeding risk. Blood samples were obtained from HM 3 patients with NSB (bleeder group, n = 27) and without NSB (non-bleeder group, n = 55). The bleeder group was further divided into patients with early NSB (bleeder ≤ 3 mo, n = 19) and patients with late NSB (bleeder > 3 mo, n = 8). The mRNA and protein expression of GPIbα, GPIX and GPV were quantified for each patient. Non-bleeder, bleeder ≤ 3 mo and bleeder > 3 mo were comparable regarding the mRNA expression of GPIbα, GPIX and GPV (p > 0.05). The protein analysis revealed a significantly reduced expression level of the main receptor subunit GPIbα in bleeders ≤ 3 mo (p = 0.04). We suggest that the observed reduction of platelet receptor GPIbα protein expression in patients who experienced their first bleeding event within 3 months after LVAD implantation may influence platelet physiology. The alterations of functional GPIbα potentially reduce the platelet adhesion capacities, which may lead to an impaired hemostatic process and the elevated propensity of bleeding in HM 3 patients.

Biological, clinical features and modelling of heterozygous variants of glycoprotein Ib platelet subunit alpha (GP1BA) and glycoprotein Ib platelet subunit beta (GP1BB) genes responsible for constitutional thrombocytopenia.

Constitutional thrombocytopenias are rare disorders, often difficult to discriminate from acquired thrombocytopenias. More than 80 genes have been described as being at the origin of these diseases. Among them, several variants of the glycoprotein Ib platelet subunit alpha (GP1BA) and glycoprotein Ib platelet subunit beta (GP1BB) genes, coding for the GpIb-IX-V glycoprotein complex, have been reported in the literature. The study reported here aimed at describing newly identified monoallelic anomalies affecting the GP1BA and GP1BB genes on a clinical, biological and molecular level. In a cohort of nine patients with macrothrombocytopenia, eight heterozygous variants of the GP1BA or GP1BB genes were identified. Five of them had never been described in the heterozygous state. Computer modelling disclosed structure/function relationships of these five variants.

Characterization of zebrafish gp1ba mutant and modelling Bernard Soulier syndrome.

The aim of this study is to model classical Bernard Soulier Syndrome in the zebrafish by targeting Gp1ba. We obtained gp1ba mutant embryos from Zebrafish International Resource Center and grew them to adulthood. The tail clips from these fish were used to prepare DNA and sequenced to identify heterozygotes. They were then bred to obtain homozygotes. The mutation was confirmed by DNA sequencing as a termination codon UAA in place of AAA codon at position 886 in the gp1ba transcript. Thus, at the Pro-295, the Gp1ba protein could be terminated. The blood from gp1ba homozygous and heterozygous mutants showed decreased ristocetin-mediated agglutination in the whole blood agglutination assay. The gp1ba heterozygous and homozygous larvae were subjected to a laser-assisted arterial thrombosis assay, and the results showed the prolonged occlusion in the caudal artery. These results suggested that the gp1ba mutant had a bleeding phenotype. The blood smears from the adult gp1ba, heterozygous and homozygous mutants, showed macrothrombocytes, similar to the human GP1BA deficiency that showed giant platelets. The bleeding assay on these heterozygous and homozygous mutants showed greater bleeding than wildtype, confirming the above findings. Taken together, the characterization of gp1ba zebrafish mutant suggested an autosomal dominant mode of inheritance. The zebrafish gp1ba mutant models classical Bernard Soulier Syndrome and could be used for reversing this phenotype to identify novel factors by the genome-wide piggyback knockdown method.

ASSOCIATIONS OF POLYMORPHISMS NOS3-T-786C, MTHFR-C667T, P2RY12-T-744C, (GPIBα) -C482T AND GENE INTERACTIONS IN MACROANGIOPATHIES IN PATIENTS WITH COMBINED HYPERTENSION AND TYPE DIABETES MELLITUS 2.

OBJECTIVE: The aim: To establish the role of allelic polymorphisms NOS3-T-786C, MTHFR-C667T, P2RY12--744C, (GPIbα)-C482T in the development of vascular lesions in patients with hypertension and diabetes mellitus type 2. PATIENTS AND METHODS: Materials and methods: The study included 100 patients with hypertension and diabetes mellitus type 2 (main group) and 50 patients without type 2 diabetes (control group). Patients underwent echocardiography, color duplex scanning of extracranial, brachiocephalic and femoral vessels. The distribution of allelic polymorphisms was investigated by isolation DNA from leukocytes and polymerase chain reaction (PCR). RESULTS: Results: The risk of vascular damages increases 2-fold when carrying all 4 risk alleles in monozygotic genotypes of polymorphic loci in patients with hypertension with concomitant type 2 diabetes (p<0,05). In gene-gene interaction, the values of contributions and directions of interaction between alleles of polymorphic loci are established (p<0,05). Genes create a paired hierarchy of interaction according to their functional activity; the largest contribution to the probable vascular damage depends on the allelic polymorphism NOS3-786CT (p<0,05), the lowest - on the allelic polymorphism P2RY12-744CC (H2H2). The genetic polymorphism of the MTHFR gene is independent of the influence of other studied polymorphisms (p<0,05); the genes P2RY12-744CT and GPIbα 482CT act synergistically with the gene NOS3-786CT, being in a weak negative interaction with each other. CONCLUSION: Conclusions: Phenotypic manifestations of endothelial dysfunction may be modified by allelic polymorphism of genes associated with endothelial and platelet functions with the risk of vascular complications.

The p.P1127S pathogenic variant lowers von Willebrand factor levels through higher affinity for the macrophagic scavenger receptor LRP1: Clinical phenotype and pathogenic mechanisms.

BACKGROUND: The index case is a 21-year-old Italian woman with a mild hemorrhagic syndrome and von Willebrand factor antigen (VWF:Ag) = 34.3 U/dl, VWF recombinant glycoprotein Ib (VWF:GpIbR) = 32.8 U/dl, and factor VIII (FVIII) = 55.3 IU/dl. AIMS: The aim of this study is to characterize from a genetic and biochemical standpoint this low VWF phenotype. METHODS: Coagulation and biochemical methods were used to study the structural and functional pattern of VWF multimers in the index case's plasma. Recombinant wild-type and p.P1127S VWF variants were produced using human embryonic kidney (HEK)-293 cells. In addition, genetic screening was carried out to detect single nucleotide variants of some scavenger VWF/FVIII receptor genes such as CLEC4M, STAB2, and ASGR2. RESULTS: Genetic investigation revealed that the index case inherited from her mother the heterozygous missense mutation c.3379C > T (VWF exon 25), causing the p.P1127S substitution in the VWF D'D3 domain. The index case was also homozygous for the scavenger receptor ASGR2 c.-95 CC-genotype. Desmopressin normalized the VWF level of the patient, although its clearance was faster (t1/2  = 6.7 h) than in normal subjects (t1/2  = 12 ± 0.7 h). FVIII-VWF interaction, A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif-13 levels, ristocetin-induced-platelet-aggregation, and VWF multimeric pattern were normal. The p.P1127S variant was normally synthesized and secreted by HEK-293 cells, and molecular modeling predicts a conformational change showing higher affinity for the macrophagic scavenger receptor lipoprotein receptor-related protein 1 (LRP1), as also experimentally verified. CONCLUSIONS: The p.P1127S variant may cause a low VWF phenotype, stemming from an increased VWF affinity for the scavenger receptor LRP1 and, consequently, an accelerated clearance of VWF.

Murine models of glycoprotein Ib-IX.

The utility of mouse models to dissect the molecular basis of hemostasis and thrombosis is now well established. The anucleate properties of circulating blood platelet and their specialized release from mature megakaryocytes makes the use of in vivo models all the more informative and powerful. Indeed, they are powerful but there do exist limitations. Here, we review the contributions of mouse models to the pathogenesis of the Bernard-Soulier syndrome, their use in platelet-specific gene expression, the recent development of mice expressing both human GPIb-IX and human von Willebrand factor (VWF), and finally the use of GPIb-IX mouse models to examine the impact of platelet biology beyond clotting. The humanization of the receptor and ligand axis is likely to be a major advancement in the characterization of therapeutics in the complex pathogenesis that drives thrombosis. When appropriate, we highlight some limitations of each mouse model, but this is not to minimize the contributions these models to the field. Rather, the limitations are meant to provide context for any direct application to the important mechanisms supporting human primary hemostasis and thrombosis.

A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

A novel frameshift GP1BB mutation causes autosomal dominant macrothrombocytopenia with decreased vWF receptor expression but normal platelet aggregation.

GP1bß is a component of the von Willebrand factor (vWF) receptor complex that is necessary for platelet formation and activation. A novel frameshift variant in GP1BB has been identified in a family with macrothrombocytopenia. The variant leads to a protein that is 101 amino acids longer than wild type with loss of the transmembrane domain. As there is no defect in platelet aggregation, the family are classified as heterozygous carriers of a Bernard-Soulier syndrome-related mutation. The levels of the vWF receptor on platelets are reduced to 50% of the controls, with the presence of large platelets but normal platelet aggregation demonstrating that decreased vWF receptor expression impacts proplatelet formation but not platelet function.

Platelet dysfunction in platelet-type von Willebrand disease due to the constitutive triggering of the Lyn-PECAM1 inhibitory pathway.

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.