A novel germline mutation in GP1BA gene N-terminal domain in monoallelic Bernard-Soulier syndrome.

Mutations in the GP1BA gene have been associated with platelet-type von Willebrand disease and Bernard-Soulier syndrome. Here, we report a novel GP1BA mutation in a family with autosomal dominant macrothrombocytopenia and mild bleeding. We performed analyses of seven family members. Using whole-exome sequencing of germline DNA samples, we identified a heterozygous single-nucleotide change in GP1BA (exone2:c.176T>G), encoding a p.Leu59Arg substitution in the N-terminal domain, segregating with macrothrombocytopenia. This variant has not been previously reported. We also analysed the structure of the detected sequence variant in silico. In particular, we used the crystal structure of the human platelet receptor GP Ibα N-terminal domain. Replacement of aliphatic amino-acid Leu 59 with charged, polar and larger arginine probably disrupts the protein structure. An autosomal dominant mode of inheritance, a family history of mild bleeding episodes, aggregation pattern in affected individuals together with evidence of mutation occurring in part of the GP1BA gene encoding the leucine-rich repeat region suggest a novel variant causing monoallelic Bernard-Soulier syndrome.

The contribution of mouse models to the understanding of constitutional thrombocytopenia.

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.

Heat-shock protein gp96/grp94 is an essential chaperone for the platelet glycoprotein Ib-IX-V complex.

The platelet glycoprotein Ib-IX-V complex (GPIb-IX-IV) is the receptor for VWF and is responsible for VWF-mediated platelet activation and aggregation. Loss of the GPIb-IX-V complex is pathogenic for Bernard-soulier Syndrome (BSS), which is characterized by macrothrombocytopenia and impaired platelet function. It remains unclear how the GPIb-IX-V complex is assembled and whether there is a role for a specific molecular chaperone in the process. In the present study, we report that the assembly of the GPIb-IX-V complex depends critically on a molecular chaperone in the endoplasmic reticulum (ER): gp96 (also known as grp94 and HSP90b1). gp96/grp94 deletion in the murine hematopoietic system results in thrombocytopenia, prolonged bleeding time, and giant platelets that are clinically indistinguishable from human BSS. Loss of gp96/grp94 in vivo and in vitro leads to the concomitant reduction in GPIb-IX complex expression due to ER-associated degradation. We further demonstrate that gp96/grp94 binds selectively to the GPIX subunit, but not to gpIbα or gpIbß. Therefore, we identify the platelet GPIX subunit of the GPIb-IX-V complex as an obligate and novel client of gp96/grp94.

Bernard-Soulier syndrome caused by a hemizygous GPIbß mutation and 22q11.2 deletion.

BACKGROUND: Bernard-Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by giant platelets, thrombocytopenia, and a prolonged bleeding time, which is caused by homozygous mutations in the GPIbα, GPIbß, or GPIX genes. The 22q11.2 deletion syndrome (22q11.2DS) is caused by a microdeletion on chromosome 22, which includes the GPIbß gene, and is characterized by abnormal development of the pharyngeal apparatus and heart. Thus, patients with 22q11.2DS are obligate carriers for BSS. METHODS: We evaluated two infants with BSS and performed the genetic analysis of the GPIbα, GPIbß, or GPIX genes, and investigated the segregation of the mutation within the families. The status of the 22q11.2 deletion was examined by fluorescence in situ hybridization and single-nucleotide polymorphism array copy number analysis. RESULTS: DNA sequencing analysis revealed that the infants were compound heterozygous for a hemizygous mutation in the GPIbß gene (p.Trp148X and p.Leu97Phe, respectively) and 22q11.2 deletion in the other chromosome. Both infants had the common 3Mb 22q11.2 deletion but did not show major phenotypic features of 22q11.2DS, such as developmental delay, cardiac defects, dysmorphic facial features, palatal anomalies, hypocalcemia, and immune deficiency. The 22q11.2DS would not have become clear if detailed molecular genetic analyses of BSS had not been performed. CONCLUSIONS: Our cases illustrate that a suspicion of 22q11.2 deletion is warranted in pediatric BSS patients with a mutation in the GPIbß gene, even without remarkable symptoms.

Point-of-care platelet function testing results in a dog with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS), also known as hemorrhagiparous thrombocytic dystrophy (OMIA 002207-9615), is a rare defect in platelet function recognized in both dogs and humans. It is caused by a deficiency in glycoprotein 1b-IX-V, the platelet surface protein which acts as a receptor for the von Willebrand factor. The characteristic features of BSS in humans and dogs include macrothrombocytes and mild-to-moderate thrombocytopenia with a bleeding tendency. This condition has previously been reported in European Cocker Spaniel dogs; however, the results of platelet function tests in these animals have not been reported. This case report describes a European Cocker Spaniel dog with spontaneously occurring Bernard-Soulier syndrome and the results of point-of-care platelet function tests, including a prolonged buccal mucosal bleeding time (>8 min), prolongation (>300 s) of PFA-200 COL/ADP, COL/EPI, and P2Y closure times, and reduced aggregation (15%-48%) with Plateletworks ADP, but with normal aggregation (92%) with Plateletworks AA. This is the first description of the results of platelet function tests in canine Bernard-Soulier syndrome.

A A386G biallelic GPIbα gene mutation with anomalous behavior: a new mechanism suggested for Bernard-Soulier syndrome pathogenesis.

Platelet glycoprotein GPIbα mutations are the basic defect behind Bernard-Soulier syndrome, a rare inherited macrothrombocytopenia characterized by anomalies of the GPIbα, GPIbß and GPIX subunits of von Willebrand factor receptor. A 32-year old man was investigated for suspected Bernard-Soulier syndrome. Ristocetin induced agglutination was absent. Flow cytometry and Western blot analysis showed a severe reduction in GPIbα, but sequencing revealed only a biallelic c.386A>G substitution, theoretically leading to a p.Asn110Glu variation. To further clarify the data, megakaryocyte cultures were set. Though the maturation of megakaryocytes was normal, proplatelet formation was defective and GPIbα mRNA was not detectable. GPIX protein was slightly reduced and GPIbß polypeptide almost absent. Computational analysis showed that the c.386A>G mutation disrupted an exon splicing enhancer motif involved in the proper maturation of the GPIbα transcript. The c.386A>G mutation suggests a unique mutational mechanism causing the virtual absence of GPIbα without creating a stop codon.

Human platelets express and are activated by galectin-8.

Gals (galectins) are proteins with glycan affinity that are emerging as mediators of atherosclerosis. Despite the similarities in structure and sequence, different Gals exert distinct effects on their target cells. We have shown that Gal-1 triggers platelet activation, suggesting a role for Gals in thrombus formation. Since Gal-8 is expressed upon endothelial activation and also contributes to inflammation, to understand further the role of these lectins in haemostasis, we evaluated the effect of Gal-8 on human platelets. Gal-8 bound specific glycans in the platelet membrane and triggered spreading, calcium mobilization and fibrinogen binding. It also promoted aggregation, thromboxane generation, P-selectin expression and granule secretion. GP (glycoprotein) αIIb and Ib-V were identified as putative Gal-8 counter-receptors by MS. Studies performed using platelets from Glanzmann's thromboasthenia and Bernard-Soulier syndrome patients confirmed that GPIb is essential for transducing Gal-8 signalling. Accordingly, Src, PLC2γ (phospholipase C2γ), ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3-kinase)/Akt downstream molecules were involved in the Gal-8 signalling pathway. Gal-8 fragments containing either the N- or C-terminal carbohydrate-recognition domains showed that activation is exerted through the N-terminus. Western blotting and cytometry showed that platelets not only contain Gal-8, but also expose Gal-8 after thrombin activation. These findings reveal Gal-8 as a potent platelet activator, supporting a role for this lectin in thrombosis and inflammation.

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome.

BACKGROUND: Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia. DESIGN AND METHODS: To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbbeta. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells. RESULTS: The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbbeta(-/-) bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbbeta(-/-) megakaryocytes could be differentiated in culture from Lin(-) fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbbeta(-/-) cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbbeta(-/-) released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbbeta(-/-) proplatelet buds in cultured and circulating platelets. CONCLUSIONS: Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.

Thrombocytopenia in the neonate.

Thrombocytopenia is one of the commonest haematological problems in neonates, affecting at least 25% of all admissions to neonatal intensive care units (NICUs) [Murray NA, Howarth LJ, McCloy MP et al. Platelet transfusion in the management of severe thrombocytopenia in neonatal intensive care unit patients. Transfus Med 2002;12:35-41; Garcia MG, Duenas E, Sola MC et al. Epidemiologic and outcome studies of patients who received platelet transfusions in the neonatal intensive care unit. J Perinatol 2001;21:415-20; Del Vecchio A, Sola MC, Theriaque DW et al. Platelet transfusions in the neonatal intensive care unit: factors predicting which patients will require multiple transfusions. Transfusion 2001;41:803-8]. Although a long list of disorders associated with neonatal thrombocytopenia can be found in many textbooks, newer classifications based on the timing of onset of thrombocytopenia (early vs. late) are more useful for planning diagnostic investigations and day-to-day management. The mainstay of treatment of neonatal thrombocytopenia remains platelet transfusion although it is important to note that no studies have yet shown clinical benefit of platelet transfusion in this setting. Indeed some reports even suggest that there may be significant adverse effects of platelet transfusion in neonates, including increased mortality, and that the effects of transfusion may differ in different groups of neonates with similar degrees of thrombocytopenia [Bonifacio L, Petrova A, Nanjundaswamy S, Mehta R. Thrombocytopenia related neonatal outcome in preterms. Indian J Pediatr 2007;74:269-74; Kenton AB, Hegemier S, Smith EO et al. Platelet transfusions in infants with necrotizing enterocolitis do not lower mortality but may increase morbidity. J Perinatol 2005;25:173-7]. There is also considerable variation in transfusion practice between different countries and between different neonatal units. Here we review recent progress in understanding the prevalence, causes and pathogenesis of thrombocytopenia in the newborn, the clinical consequences of thrombocytopenia and developments in neonatal platelet transfusion.

Decreased thrombotic tendency in mouse models of the Bernard-Soulier syndrome.

OBJECTIVE: The platelet glycoprotein (GP)Ib-V-IX complex is a receptor required for normal hemostasis deficient in the Bernard-Soulier bleeding disorder. To evaluate the consequences of GPIb-V-IX deficiency in thrombosis we generated mouse models of the disease by targeting the GPIb beta subunit. METHODS AND RESULTS: Complete deletion (GPIb beta-/-) or an intracellular truncation (GPIb beta deltaIC-/-) reproduced typical and variant forms of Bernard-Soulier, with absent and partial (20%) expression of the complex on the platelet surface. Both strains exhibited thrombocytopenia and enlarged platelets with abnormal microtubular structures but normal granule composition. They exhibited prolonged tail bleeding times, which were less pronounced in GPIb beta deltaIC-/-. Decreased thrombus formation was observed after blood perfusion over a collagen coated surface at high shear. Resistance to vascular occlusion and an abnormal thrombus composition were observed in a model of FeCl3-induced lesion of carotid arteries. In a model of laser-induced lesion of mesenteric arterioles, thrombosis was strongly reduced in GPIb beta-/- mice, while a more modest effect was observed in GPIb beta deltaIC-/- animals. Finally, the two strains were protected against death in a model of systemic thromboembolism. CONCLUSIONS: This study provides in vivo evidence of a decreased thrombotic tendency linked to defective platelet GPIb-V-IX in mouse models of Bernard-Soulier syndrome.

Induced pluripotent stem cells derived from Bernard-Soulier Syndrome patient's peripheral blood cells with a p.Phe55Ser mutation in the GPIX gene.

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease.

Congenital and acquired platelet disorders: current dilemmas and treatment strategies.

Platelets are important for primary hemostasis. When a blood vessel is damaged, platelets adhere to exposed subendothelial connective tissue and form a hemostatic plug. Formation of the plug is contingent upon a series of processes, with adhesion, activation, and aggregation all being involved. Patients with quantitative platelet disorders have reduced numbers of platelets. Patients with qualitative disorders have platelets that exhibit abnormal functioning. Defects that impair function can affect platelet receptors, secretory responses, or intracellular signaling pathways. Examples of qualitative platelet disorders include Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS). The treatment of platelet disorders is primarily with platelet concentrates. However, in patients with abnormalities of their platelet surface receptors, platelet transfusion may provoke an immune response. Recombinant factor VIIa (rFVIIa) may provide hemostatically effective therapy in such patients.

Generation of induced pluripotent stem cells (iPSCs) from a Bernard-Soulier syndrome patient carrying a W71R mutation in the GPIX gene.

We generated an induced pluripotent stem cell (iPSC) line from a Bernard-Soulier Syndrome (BSS) patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR) profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

Impaired prothrombin consumption in Bernard-Soulier syndrome is corrected in vitro by human factor VIII.

The Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein Ib (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII:C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

[Platelet membrane glycoproteins and the molecular mechanisms of hereditary platelet dysfunction].

Glycoproteins, present on the outer surface of platelets, function in the aggregating and releasing reactions of these cells. Some of the hereditary diseases of platelet dysfunction, such as, thrombasthenia and Bernard-Soulier syndrome, have been shown to result from defects in some of these glycoproteins. Recent advances in molecular biology have enabled us to analyze the genes which encode the defective glycoproteins in these patients. The information from these studies also helps us to understand in detail the function of the glycoproteins. At present, the major glycoproteins, GP II b/IIIa and GP I b/IX, have been studied extensively and we know the details of the mechanisms through which they function. However, as to the minor glycoproteins, GP I a/II a, GP IV and GP VI, details remain to be elucidated.

Genetic deletion of platelet glycoprotein Ib alpha but not its extracellular domain protects from atherosclerosis.

The pathogenesis of atherosclerosis involves the interplay of haematopoietic, stromal and endothelial cells. Platelet interactions with endothelium and leukocytes are pivotal for atherosclerosis promotion. Glycoprotein (GP) Ibα is the ligand-binding subunit of the platelet GPIb-IX-V receptor complex; its deficiency causes the Bernard-Soulier syndrome (BSS), characterised by absent platelet GPIb-IX-V, macrothrombocytopenia and bleeding. We designed this study to determine the role of platelet GPIbα in the pathogenesis of atherosclerosis using two unique knockout models. Ldlr-/- mice were reconstituted with wild-type (wt), GPIbα-/- (lacks GPIbα) or chimeric IL-4R/GPIbα-Tg (lacks GPIbα extracellular domain) bone marrow and assayed for atherosclerosis development after feeding with pro-atherogenic "western diet". Here, we report that Ldlr-/-mice reconstituted with GPIbα-/- bone marrow developed less atherosclerosis compared to wt controls; accompanied by augmented accumulation of pro-inflammatory CD11b+ and CD11c+ myeloid cells, reduced oxLDL uptake and decreased TNF and IL 12p35 gene expression in the aortas. Flow cytometry and live cell imaging in whole blood-perfused microfluidic chambers revealed reduced platelet-monocyte aggregates in GPIbα-/- mice, which resulted in decreased monocyte activation. Interestingly, Ldlr-/-mice reconstituted with IL-4R/GPIbα-Tg bone marrow, producing less abnormal platelets, showed atherosclerotic lesions similar to wt mice. Platelet interaction with blood monocytes and accumulation of myeloid cells in the aortas were also essentially unaltered. Moreover, only complete GPIbα ablation altered platelet microparticles and CCL5 chemokine production. Thus, atherosclerosis reduction in mice lacking GPIbα may not result from the defective GPIbα-ligand binding, but more likely is a consequence of functional defects of GPIbα-/- platelets and reduced blood platelet counts.

Compound heterozygosity for a novel nine-nucleotide deletion and the Asn45Ser missense mutation in the glycoprotein IX gene in a patient with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder due to quantitative or qualitative abnormalities in the platelet glycoprotein (GP) Ib/IX/V complex, the major von Willebrand factor receptor. The complex comprises four subunits, each encoded by a separate gene. Several mutations have been described for each of the subunits, except for GPV, as a cause of BSS. We describe here the genetic basis of the disorder in a child with BSS. Flow-cytometric analysis of the patient's platelets showed a markedly reduced surface expression of all three glycoproteins of the GPIb/IX/V complex. DNA sequencing analysis showed the patient to be a compound heterozygote for two mutations in the GPIX gene, a novel nine-nucleotide deletion starting at position 1952 of the gene that changes asparagine 86 for alanine and eliminates amino acids 87, 88, and 89 (arginine, threonine, and proline) and a previously reported point mutation that changes the codon asparagine (AAC) for serine (AGC) at residue 45. Her mother was heterozygous for the Asn45Ser mutation, and her father, for the nine-nucleotide deletion. Our findings suggest that the additive effects of both mutations in the GPIX gene are responsible for the BSS phenotype of the patient.