Severe Macrothrombocytopenia Research Articles

Novel GNE missense variants impair de novo sialylation and cause defective angiogenesis in the developing brain in mice

AbstractGlucosamine (UDP-N-acetyl)-2-epimerase and N-acetylmannosamine (ManNAc) kinase (GNE) is a cytosolic enzyme in de novo sialic acid biosynthesis. Congenital deficiency of GNE causes an autosomal recessive genetic disorder associated with hereditary inclusion body myopathy and macrothrombocytopenia. Here, we report a pediatric patient with severe macrothrombocytopenia carrying 2 novel GNE missense variants, c.1781G>A (p.Cys594Tyr, hereafter, C594Y) and c.2204C>G (p.Pro735Arg, hereafter, P735R). To investigate the biological significance of these variants in vivo, we generated a mouse model carrying the P735R mutation. Mice with homozygous P735R mutations exhibited cerebral hemorrhages as early as embryonic day 11 (E11), which subsequently progressed to large hemorrhages in the brain and spinal cord, and died between E11.5 and E12.5. Defective angiogenesis such as distended vascular sprouts were found in neural tissues and embryonic megakaryocytes were abnormally accumulated in the perineural vascular plexus in mutant mouse embryos. Furthermore, our in vitro experiments indicated that both C594Y and P735R are loss-of-function mutations with respect to de novo sialic acid biosynthesis. Overall, this study reveals a novel role for GNE-mediated de novo sialic acid biosynthesis in mouse embryonic angiogenesis.

Thrombocytopenia Associated with Myelofibrosis Is Characterized By a Reduced Proportion of Megakaryocyte (MK) Biased Hematopoietic Stem Cells, Defects in MK Maturation and Increased Levels of Pro-Inflammatory Cytokines

The clinical course of patients with advanced forms of myelofibrosis (MF) can frequently be punctuated by thrombocytopenia. Marrow biopsies from these MF patients uniformly reveal megakaryocytic (MK) hyperplasia and dysplasia irrespective of the patient's platelet count. We have performed studies to better understand the origins of MF associated thrombocytopenia. We first examined the expression of G6b-B (MK-lineage specific immunoreceptor tyrosine-based inhibition motif) which is essential in promoting MK maturation, normal platelet release, platelet size and function (Becker. Blood Adv 2022). Recent reports have indicated that genetic deletion in G6b-B and loss-of-function mutations in both murine models and man lead to severe macro-thrombocytopenia and increased bone marrow fibrosis (Becker. Blood Adv 2022; Hofmann. Blood 2018; Melhem. Eur J Hematol 2017), indicating a possible role for this receptor in the development of thrombocytopenia in MF patients. We generated populations of MKs from mononuclear cells or CD34 + from thrombocytopenic or non-thrombocytopenic MFs and healthy donors (HDs) using a two-step culture system as previously described (Mosoyan. Leukemia 2017). A progressive increase of MK maturation (CD41 +CD42 +) was observed in HDs (28.5%±7) and non-thrombocytopenic MFs (32%±13.6) with a concomitant increase of total G6B expression 27%±9 and 38%±14 respectively. By contrast, MKs generated from thrombocytopenic MFs patient cells had limited CD41 +CD42 + (7%±5) and G6B (8.7%±4.7) expression (p<0.05). In addition, morphological analysis revealed that MKs from thrombocytopenic MF patients were small with hypolobulated nuclei while the MKs generated from HDs and non-thrombocytopenic MFs were larger and contained greater numbers of lobes ( Figure 1A). Psaila and coworkers have previously reported that MF is characterized by MK biased hematopoietic stem cells (HSCs) identified by increased expression of G6B (Psaila. Mol Cell 2020). We, however, determined that a reduced proportion of CD34 +CD38 - HSCs from thrombocytopenic patients expressed G6B as compared to non-thrombocytopenic MF or HD CD34 +CD38 - cells (p<0.05) ( Figure 1B). These studies indicate a reduced proportion of MK biased HSCs in thrombocytopenic MFs which likely leads to impaired MKs maturation. We next explored if the MF associated pro-inflammatory milieu also contributes to the MF MK maturation block. Thrombocytopenic MF plasma contained higher levels of YKL-40 (chitinase-3-like-1 protein), TNFα and TPO than HD plasma. For instance, MF plasma was characterized by higher levels of YKL-40 (median141,321 pg/mL) in comparison to HDs (median 19,380 pg/mL). Furthermore, YKL-40 levels were significantly higher in thrombocytopenic (median 187,696 pg/mL; p<0.05) than non-thrombocytopenic (median 78,082 pg/mL) MFs. In addition, TNFα and TPO plasma levels were also significantly higher in plasma from thrombocytopenic than non-thrombocytopenic MFs (p<0.05). We also observed that addition of either rYKL40 or rTNFα to HD-MK cultures resulted in delayed MK maturation as assessed by a reduced proportion of cells expressing CD41 +CD42 + (from 30% to 16.5% and 6.5% respectively) and G6B (from 35.5% to 22.5% and 9.5% respectively). We have previously reported that the treatment of MFs with a protein trap for TGFβ1 led to increases in platelet numbers (Mascarenhas. Clin Cancer Res 2023). The plasma from these patients prior to treatment not only had elevated levels of TGFβ1 (median 6433,917 pg/mL) but also YKL40 (median 275,640 pg/mL). Treatment of the patients with the TGFβ protein trap reduced both TGFβ1 (median 608,087 pg/mL) and YKL-40 (median 160,733 pg/mL) plasma levels, suggesting a regulatory loop between YKL-40 and TGFβ. We further explored this loop by treating the JAK2V617F+ HEL cell line with rYKL-40 in combination with rTGFβ1, which resulted in greater expression of YKL-40 (1.6 FI) than treated cells with rYKL-40 alone. In addition, treatment of HEL cells with a pan TGFβ inhibitor reduced YKL-40 protein expression (1.55 to 0.38). These data suggest the existence of an interplay between YKL40 and TGFβ on MF-MK maturation. Overall, these experiments indicate that thrombocytopenia in MF patients is due to intrinsic abnormalities in G6B originating at the level of the HSC compartment resulting in impaired MK maturation which can be further enhanced by the actions of a number of pro-inflammatory cytokines.

Successful Kidney Transplantation in MYH-9-Related Disease Presenting with Severe Macrothrombocytopenia

Successful Kidney Transplantation in MYH-9-Related Disease Presenting with Severe Macrothrombocytopenia

Treatment of congenital thrombocytopenia and decreased collagen reactivity in G6b-B–deficient mice

AbstractMice lacking the immunoreceptor tyrosine-based inhibition motif-containing co-inhibitory receptor G6b-B (Mpig6b, G6b knockout, KO) are born with a complex megakaryocyte (MK) per platelet phenotype, characterized by severe macrothrombocytopenia, expansion of the MK population, and focal myelofibrosis in the bone marrow and spleen. Platelets are almost completely devoid of the glycoprotein VI (GPVI)-FcRγ-chain collagen receptor complex, have reduced collagen integrin α2β1, elevated Syk tyrosine kinase activity, and a subset has increased surface immunoglobulins. A similar phenotype was recently reported in patients with null and loss-of-function mutations in MPIG6B. To better understand the cause and treatment of this pathology, we used pharmacological- and genetic-based approaches to rescue platelet counts and function in G6b KO mice. Intravenous immunoglobulin resulted in a transient partial recovery of platelet counts, whereas immune deficiency did not affect platelet counts or receptor expression in G6b KO mice. Syk loss-of-function (R41A) rescued macrothrombocytopenia, GPVI and α2β1 expression in G6b KO mice, whereas treatment with the Syk kinase inhibitor BI1002494 partially rescued platelet count but had no effect on GPVI and α2β1 expression or bleeding. The Src family kinase inhibitor dasatinib was not beneficial in G6b KO mice. In contrast, treatment with the thrombopoietin mimetic romiplostim rescued thrombocytopenia, GPVI expression, and platelet reactivity to collagen, suggesting that it may be a promising therapeutic option for patients lacking functional G6b-B. Intriguingly, GPVI and α2β1 expression were significantly downregulated in romiplostim-treated wild-type mice, whereas GPVI was upregulated in romiplostim-treated G6b KO mice, suggesting a cell intrinsic feedback mechanism that autoregulates platelet reactivity depending on physiological needs.

GNE – Related Severe Congenital Macrothrombocytopenia

Congenital thrombocytopenia results from genetic mutations in genes implicated in megakaryocyte differentiation and/or platelet formation and clearance. We report the case of an 11-month-old girl who presented with severe macrothrombocytopenia since birth and subsequently developed an intracranial bleed. She was diagnosed to have GNE gene mutation. GNE gene encodes the key enzyme in sialic acid biosynthesis, glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE/MNK). Its mutation is responsible for the reduction in sialic acid biosynthesis and consequently leads to severe congenital thrombocytopenia and/or myopathy. Although no sign of myopathy is observed in our patient; it is possible myopathy can be developed later, thus long-term follow-up with neurology is highly advisable. We recommend the genetic counseling and a segregation analysis of this variant in other affected individuals in the family.

The impact of aberrant von Willebrand factor-GPIbα interaction on megakaryopoiesis and platelets in humanized type 2B von Willebrand disease model mouse.

Type 2B von Willebrand disease (VWD) is caused by gain-of-function mutations in von Willebrand factor (VWF). Increased VWF affinity for GPIbα results in loss of high molecular weight multimers and enhanced platelet clearance, both contributing to the bleeding phenotype. Severity of the symptoms vary among type 2B VWD patients, with some developing thrombocytopenia only under stress conditions. Efforts have been made to study underlying pathophysiology for platelet abnormalities, but animal studies have been limited because of species specificity in the VWF-GPIbα interaction. Here, we generated a severe form of type 2B VWD (p.V1316M) knockin mice in the context of human VWF exon 28 (encoding A1 and A2 domains) and crossed them with human GPIbα transgenic strain. Heterozygous mutant mice recapitulated the phenotype of type 2B VWD in autosomal dominant manner and presented severe macrothrombocytopenia. Of note, platelets remaining in the circulation had extracytoplasmic GPIbα shed-off from the cell surface. Reciprocal bone marrow transplantation determined mutant VWF produced from endothelial cells as the major cause of the platelet phenotype in type 2B VWD mice. Moreover, altered megakaryocyte maturation in the bone marrow and enhanced extramedullary megakaryopoiesis in the spleen were observed. Interestingly, injection of anti-VWF A1 blocking antibody (NMC-4) not only ameliorated platelet count and GPIbα expression, but also reversed MK ploidy shift. In conclusion, we present a type 2B VWD mouse model with humanized VWF-GPIbα interaction which demonstrated direct influence of aberrant VWF-GPIbα binding on megakaryocytes.

G6b-B regulates an essential step in megakaryocyte maturation

AbstractG6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif–containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, whereas proplatelet release is hampered, but the underlying molecular mechanism remains unclear. We report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis, and osteosclerosis. As the mutation is based on a single-nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller, displayed a less-developed demarcation membrane system, and had a reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1 and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b-null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.

Severe Congenital Thrombocytopenia Characterized by Decreased Platelet Sialylation and Moderate Complement Activation Caused by Novel Compound Heterozygous Variants in GNE.

BackgroundHereditary thrombocytopenias constitute a genetically heterogeneous cause of increased bleeding. We report a case of a 17-year-old boy suffering from severe macrothrombocytopenia throughout his life. Whole genome sequencing revealed the presence of two compound heterozygous variants in GNE encoding the enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, crucial for sialic acid biosynthesis. Sialic acid is required for normal platelet life span, and biallelic variants in GNE have previously been associated with isolated macrothrombocytopenia. Furthermore, sialic acid constitutes a key ligand for complement factor H (FH), an important inhibitor of the complement system, protecting host cells from indiscriminate attack.MethodsSialic acid expression and FH binding to platelets and leukocytes was evaluated by flow cytometry. The binding of FH to erythrocytes was assessed indirectly by measuring the rate of complement mediated hemolysis. Complement activation was determined by measuring levels of C3bBbP (alternative pathway), C4d (classical/lectin pathway) and soluble terminal complement complex assays.ResultsThe proband exhibited markedly decreased expression of sialic acid on platelets and leukocytes. Consequently, the binding of FH was strongly reduced and moderate activation of the alternative and classical/lectin complement pathways was observed, together with an increased rate of erythrocyte lysis.ConclusionWe report two previously undescribed variants in GNE causing severe congenital macrothrombocytopenia in a compound heterozygous state, as a consequence of decreased platelet sialylation. The decreased sialylation of platelets, leukocytes and erythrocytes affects the binding of FH, leading to moderate complement activation and increased hemolysis.

Identification By Whole Exome Sequencing of the Molecular Defect in a Novel Gene Related to Glycosylation in Two Unrelated Families with Syndromic Macrothrombocytopenia

IntroductionInherited thrombocytopenias (ITs) are a heterogeneous group of rare platelet disorders. which lead not only to increased bleeding, but also to syndromic forms. ITs are caused by genetic alterations in megakaryopoiesis-related genes. In the last years, whole-exome sequencing (WES) has allowed the identification of novel genes involved in IT.AimTo perform the molecular, clinical and platelet characterization of two unrelated families with syndromic IT, to unveil the underlaying alteration leading to the disease. To explore the functional role of the identified alterations during megakaryocytic (Mk) differentiation.MethodsWES was performed in two unrelated non-consanguineous families with lifelong severe macrothrombocytopenia (MCT), bleeding, and extra-hematological manifestations. Bleeding score (BS) was recorded by ISTH-BAT. Platelet phenotyping included platelet count (P), blood film, aggregometry (LTA) and flow cytometry (FC). UDP-galactose-4-epimerase enzymatic activity was measure by HPLC/MS/MS. In vitro functional studies were performed through overexpression of GALE genetic variants in human K562 cell line to elucidate its role in Mk differentiation, by measuring cell ploidy and expression of CD41, CD61 and CD42b surface markers after 7-days of PMA treatment.ResultsFamily pedigrees are shown in Figure 1. Three patients (A.II.1, A.II.2 and B.II.1) were referred due to lifelong severe MCT and moderate-severe bleeding tendency (Figure 1). Moreover, they presented mental retardation, mitral insufficiency, and increased bilirubin levels. Blood film revealed enlarged, giant, and grey platelets (A.II.1: 36%, 6% and 54%, respectively; A.II.2: 56%, 4%, 34%, respectively; B.II.1: 32%, 46%, 12%).LTA showed moderate/severe impaired aggregation with ADP, TRAP-6, CRP, epinephrine, arachidonic acid, and ristocetin. FC confirmed null secretion of alpha and dense granules in A.II.1, A.II.2 and reduced levels in B.II.1 (7.8%, 8.1%, 28.3% respectively, vs. 51.7% control platelets with ADP 10µM; 10.8%, 7.8%, 36.7% respectively, vs. 95.6% control platelets with TRAP6 25µM).WES revealed that both pedigrees carried compound heterozygous variants in GALE (NM_001127621.2): c.230_231insTGTT; p.Lys78Valfs*32 (exon 3), and c.449C>T; p.Thr150Met (exon 5) in A.II.1 and A.II.2 patients; and, c.668T>C, p.Leu223Pro (exon 7), and c.382G>A, p.Val128Met (exon 5) in B.II.1 (Figure 1). Enzymatic activity of the GALE-encoded protein UDP-galactose-4-epimerase was severely reduced in the affected patients: both A.II.1, A.II.2 patients had 1.3 μmol/h/g hemoglobin (control: 8.8 μmol/h/g hemoglobin), and B.II.1 patient had 0.6 μmol/h/g hemoglobin (control: 8 μmol/h/g hemoglobin).Furthermore, in vitro overexpression assays between wild-type GALE and p.Thr150Met, p.Leu223Pro and p.Val128Met variants, confirmed a delayed maturation of Mks upon PMA treatment (at 3, 5 and 7 days). characterized by a significant reduction in the expression of the megakaryocytic surface markers CD41, CD61 and CD42b.ConclusionWES has allowed us to identify pathogenic variants in GALE, which were associated with syndromic IT characterized by severe macrothrombocytopenia. Patients harboring these pathogenic variants presented moderate to severe bleeding tendency associated with cardiovascular and neurological abnormalities. Regarding the platelet phenotype, the presence of giant and grey platelets and the absence of both platelet granules were the most remarkable features reported. Moreover, these GALE variants led to an alteration in in vitro Mk maturation, supporting the thrombocytopenic phenotype observed in patients.FundingISCIII (PI17/01966, PI 17/01311, PI20/00926), GRS (GRS2061A/19, GRS2135/A/2020), Fundación Séneca (19873/GERM/15), Fundación Mutua Madrileña (AP172142019), Premio López Borrasca (2019), Grupo Trabajo Patología Hemorrágica-SETH (2020). [Display omitted] DisclosuresHernández-Rivas: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes.

Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids invivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and β1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.

Severity of Megakaryocyte-Driven Osteosclerosis in Mpig6b-Deficient Mice Is Sex-Linked.

Patients with chronic myelofibrosis often suffer from osteosclerosis, which is associated with bone pain and may lead to bone marrow failure. The pathogenesis of myelofibrosis is linked to aberrant megakaryocyte development and function. Null and loss-of-function mutations in MPIG6B, which codes for the inhibitory heparan sulfate receptor G6b-B, result in severe macrothrombocytopenia, large megakaryocyte clusters, and focal primary myelofibrosis in mice and humans. We investigated the development of osteosclerosis in Mpig6b null (Mpig6b-/- ) mice. Although male and female Mpig6b-/- mice presented with elevated bone marrow megakaryocyte number and macrothrombocytopenia, female Mpig6b-/- mice developed progressive splenomegaly starting at 8 weeks of age. Micro-computed tomography (μCT) of femurs showed that female Mpig6b-/- mice had increased cortical thickness and reduced bone marrow area starting at 8 weeks of age and developed occlusion of the medullary cavity by trabeculae by 16 weeks of age. In contrast, male Mpig6b-/- mice developed only a small number of trabeculae in the medullary cavity at the proximal diaphysis and demonstrated a temporary decrease in bone volume fraction and trabecular thickness at 16 weeks. Ovariectomy of 10-week-old female Mpig6b-/- mice prevented the development of medullary cavity osteosclerosis, whereas orchiectomy of male Mpig6b-/- mice did not exacerbate their disease. Importantly, ovariectomized female Mpig6b-/- mice also demonstrated improvement in spleen weight compared to sham-operated Mpig6b-/- mice, establishing estrogen as a contributing factor to the severity of the megakaryocyte-driven osteosclerosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1

AbstractRearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.

Shared roles for Scl and Lyl1 in murine platelet production and function

AbstractThe stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

Whole Genome Sequencing of 10,000 Rare Disease Patients Reveals Digenic Inheritance of Blood Disorders

Inherited bleeding, thrombotic and platelet disorders (BPDs) affect approximately 3M people worldwide and an appreciable portion have a disorder of megakaryopoiesis and the production and function of platelets, including the formation of granules. While genetic variants in 76 genes have been implicated in BPDs, many patients remain without a molecular diagnosis (Lentaigne et al, Simeoni et al). We hypothesised that some of these disorders may be caused by compound inheritance of variants in two different genes, a mode of inheritance thus far never implicated in BPDs.For the pilot phase of the 100 000 Genomes Project we have sequenced the whole genomes of 10,000 individuals consisting of probands with molecularly unexplained rare disorders and their close relatives, with 3,000 having inherited disorders of the blood and immune system. We searched for causal variants in known BPD-related genes and employed a new statistical method for Bayesian evaluation of rare variants in Mendelian disease (BeviMed) (Greene et al) to identify novel marginal associations between rare variants and disease status. Where the identified variants could not individually explain the phenotype in full within the pedigrees, we searched for additional variants affecting other BPD-related genes or novel genes identified using BeviMed.First, we have identified a large pedigree in which certain members with mild thrombocytopenia (lower 10th percentile of the population distribution) are affected by a single variant encoding a premature stop at residue 69 in the major isoform of the tropomyosin gene TPM4 expressed in megakaryocytes (Pleines et al). Other members of the family have distinctly more severe macrothrombocytopenia (with platelet counts as low as 24x109/l) and this is due to inheritance of a second variant in the actinin gene ACTN1 leading to a Thr340Met substitution, demonstrating a compound additive effect of variants in two genes encoding cytoskeletal proteins important for actin polymerization and thereby causing inadequate platelet formation. It is noteworthy that both of these genes have been identified in a genome wide association study of the count and mean volume of platelets (Gieger et al). Second, we used BeviMed to identify genes containing variants that are marginally associated with a syndrome defined by a platelet granule defect combined with familial autism (Bijl et al). The strongest association is due to splice variants in a granule-related gene on Chr17p13 but in all four unrelated cases at least one additional variant is required to explain the observed segregation patterns. Two of these unrelated cases harbor variants in two other marginally associated genes at Chr9q32 and Chr4q31 and a third harbors a de novo copy number variant. These additional variants likely explain why the children in three of the families are affected by this syndrome while their parents are not.In conclusion, we have used whole genome sequencing, pedigree building, detailed platelet phenotyping and new association approaches to identify the first cases of digenic inheritance of BPDs. Our results illustrate the cooperative role of different cytoskeletal proteins in platelet formation and cement the role of granule biology in the function of both platelets and neurons.

LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B.

von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein's multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics.

Lentiviral gene rescue of a Bernard–Soulier mouse model to study platelet glycoprotein Ibβ function

Essentials A signaling role of glycoprotein (GP)Ibβ is postulated but not formally demonstrated in platelets. Lentiviral-mediated rescue in knock-out mice can be used to evaluate GPIbβ function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb-IX deficiency Deletion of intracellular 159-170 segment increased thrombosis, 150-160 removal increased bleeding. Background The platelet glycoprotein (GP)Ib-V-IX complex is required for normal hemostasis and megakaryopoiesis. A role in GPIb-dependent responses has been ascribed to the less well characterized GPIbβ subunit using a specific antibody and GPIb-IX transfected cells. Objectives Our aim was to evaluate, in vivo, the role of the GPIbβ in hemostasis and thrombosis. Methods GPIbβ(null) Sca-1(+) progenitors transduced with viral particles harboring hGPIbβ were transplanted into lethally irradiated GPIbβ(-/-) recipient mice. Results hGPIbβ transplanted into the bone marrow of GPIbβ(null) mice rescued GPIb-IX expression in 97% of circulating platelets. These platelets efficiently bound von Willebrand factor (VWF) and extended filopodia on a VWF matrix, demonstrating the restoration of GPIb-dependent adhesive and signaling properties. These mice exhibited less severe macrothrombocytopenia and had normal tail bleeding times as compared with GPIbβ(null) mice. This strategy was employed to manipulate and evaluate the role of the GPIbβ intracellular domain. Removal of the membrane proximal segment (Δ(150-160) ) decreased GPIb-IX expression by 43%, confirming its involvement in receptor assembly and biosynthesis, and resulted in increased bleeding times and decreased thrombosis in a mechanical injury model in the aorta. On the other hand, deletion of the C-flanking 159-170 segment allowed normal GPIb-IX expression, VWF-dependent responses and bleeding times, but resulted in enhanced arterial thrombosis. Conclusion This pointed to a repressor role of GPIbβ in thrombus formation in vivo that was not predicted in studies of heterologous cells. These results highlight the utility of this lentiviral strategy for the structure-function evaluation of GPIb-IX in platelets.

Thrombocytopenia and Postpartum Hemorrhage in a Woman with Chromosome 22q11.2 Deletion Syndrome.

Chromosome 22q11.2 deletion syndrome, also known as DiGeorge or velocardiofacial syndrome, is associated with a wide spectrum of phenotypic features. It is known to be associated with severe macrothrombocytopenia. Postpartum hemorrhage is a leading cause of maternal morbidity and mortality globally. Chromosome 22q11.2 deletion syndrome is rare cause of thrombocytopenia that can be a significant risk factor for life-threatening postpartum hemorrhage. We report a case of postpartum hemorrhage in a woman with 22q11.2 deletion syndrome causing severe macrothrombocytopenia.

Dynamin 2 (DNM2) and PACSIN2 Regulate Megakaryocyte Demarcation Membrane System Formation and Platelet Production in Concert

Blood platelets are produced in the bone marrow by megakaryocytes (MKs) in a process that requires extensive intracellular membrane rearrangements. These include the formation of the demarcation membrane system (DMS), the surface-connected membrane extension that invaginates into the cell body and further develops to provide membranes for future platelets. The precise molecular mechanisms responsible for these unique membrane rearrangements remain poorly understood. We have recently shown that Dnm2fl/fl Pf4-Cre mice specifically lacking the large GTPase dynamin 2 (DNM2) in MKs develop severe macrothrombocytopenia due to impaired receptor-mediated endocytosis (RME) (Bender, Giannini et al. Blood. 2015;125(6):1014-1024). Specifically, Dnm2fl/fl Pf4-Cre MKs accumulate arrested endocytic clathrin-coated vesicles that obstruct DMS formation. The actin nucleating factor Arp2/3 complex and polymerized actin clustered with clathrin at sites of impaired RME in Dnm2fl/fl Pf4-Cre MKs. We hypothesized that a DNM2 partner recruits actin-regulatory proteins at sites of RME and investigated the contribution of the F-BAR protein PACSIN2, an internal component of the initiating DMS (Jurak Begonja, Pluthero et al. Blood. 2015;126(1):80-88), in DMS formation and platelet production, as PACSIN2 interacts with DNM2 and actin-regulatory proteins such as N-WASP and filamin A (FlnA). Pacsin2-/- mice developed mild thrombocytopenia with slightly enlarged and shallow platelets. The DMS appeared less well defined and platelet territories were not readily visualized in Pacsin2-/- MKs. Pacsin2-/- Dnm2fl/fl Pf4-Cre mice lacking both PACSIN2 and DNM2 in MKs were further generated to determine the contribution of PACSIN2 in clathrin and actin clustering in Dnm2fl/fl Pf4-Cre MKs. Strikingly, PACSIN2 genetic deletion significantly improved the severe thrombocytopenia of Dnm2fl/fl Pf4-Cre mice. Specifically, PACSIN2 deletion abrogated the accumulation of clathrin and actin clusters, thereby unclogging DMS formation, which appeared as elongated maze-like membrane tubules in Pacsin2-/- Dnm2fl/fl Pf4-Cre MKs. Our results show that DNM2 terminates PACSIN2-dependent actin polymerization that accompanies RME, thereby allowing membrane rearrangements required for DMS formation. DisclosuresNo relevant conflicts of interest to declare.

CXCL12/SDF‐1 Stimulates Megakaryocyte β‐1,4‐Galactosyltransferase 1 Activity to Enhance Platelet Production in Vivo

Platelet recovery following bone marrow (BM) transplant and chemotherapy is crucial to prevent bleeding complications. The chemokine SDF‐1 promotes megakaryocyte (MK) migration towards BM sinusoids increasing platelet production. Here we show that SDF‐1 upregulates expression of the glycan structure Type‐2‐Lactosaminoglycan (LacNAc), synthesized by β4Galactosyltransferase1 (β4galt1). We hypothesized that SDF‐1 increases LacNAc synthesis promoting platelet production. Supporting this hypothesis, β4galt1–/– mice had severe macrothrombocytopenia (80% reduction) with increased BMMK numbers (35%), which localized poorly with BM sinusoids (50%), compared to WT MKs (72%). β4galt1–/– hematopoietic stem cells (HSCs) transplanted into lethally irradiated WT mice restored BMMKs, which failed to migrate to sinusoids and produce circulating platelets. We next treated WT and β4galt1–/– mice with the myeloablative chemotherapeutic agent 5‐Fluorouracil (5‐FU), which decreased platelet counts by 50% in both phenotypes. SDF‐1 injections increased platelet counts by 70% in 5‐FU treated WT mice but not in β4galt1–/– mice. Importantly, WT but not β4galt1–/– platelets had increased LacNAc expression after SDF‐1 injections.β4galt1–/– BMMKs had increased β1 integrin expression. Expression of laminin, β1 integrin ligand, was upregulated in β4galt1–/– MKs and sinusoids. Our results suggest that SDF‐1 upregulates β4GalT1‐dependentLacNAc expression promoting MK interaction with BM sinusoids, likely regulating β1 integrin expression and interaction with components of the extracellular matrix, specifically laminin.