[show abstract][hide abstract] ABSTRACT: MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA). MYH9-RD is characterized by a considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end-stage renal disease (ESRD). We searched for genotype-phenotype correlations in the largest series of consecutive MYH9-RD patients collected so far (255 cases from 121 families). Association of genotypes with non-congenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9-RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early-onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the non-congenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC-IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N and C-terminal deletions had low risk of non-congenital defects. These findings are essential to patients' clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9-RD. This article is protected by copyright. All rights reserved.
[show abstract][hide abstract] ABSTRACT: Megakaryocytes generate platelets through extensive reorganization of the cytoskeleton and plasma membrane. Cdc42 interacting protein 4 (CIP4) is an F-BAR protein that localizes to membrane phospholipids through its BAR domain and interacts with WASP via its SH3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function we generated CIP4(-/-) mice, which displayed thrombocytopenia similarly to that of WAS(-/) mice. The number of megakaryocytes and their progenitors was not affected. However, the number of proplatelet protrusions was reduced in CIP4(-/-), but not WAS(-/), megakaryocytes. Electron micrographs of CIP4(-/-) megakaryocytes showed an altered demarcation membrane system. Silencing of CIP4, not WASP, expression resulted in fewer proplatelet-like extensions. Fluorescence anisotropy studies showed that loss of CIP4 resulted in a more rigid membrane. Micropipette aspiration demonstrated decreased cortical actin tension in megakaryocytic cells with reduced CIP4 or WASP protein. These studies support a new biophysical mechanism for platelet biogenesis whereby CIP4 enhances the complex, dynamic reorganization of the plasma membrane (WASP-independent) and actin cortex network (as known for WASP and cortical actin) to reduce the work required for generating proplatelets. CIP4 is a new component in the highly-coordinated system of megakaryocytic membrane and cytoskeletal remodeling affecting platelet production.
[show abstract][hide abstract] ABSTRACT: Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NBEAL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit splenomegaly, macrothrombocytopenia and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1 and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of Vps33B and Vps16B in Nbeal2(-/-) platelets suggests that Nbeal2 acts independently of Vps33B/Vps16B at a later stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function and they also indicate that abnormal α-granule formation in Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development and platelet production.
[show abstract][hide abstract] ABSTRACT: Once considered exceptionally rare, congenital thrombocytopenias are increasingly recognized as a heterogeneous group of disorders characterized by a reduction in platelet number and a bleeding tendency that may range from very mild to life threatening. Although some of these disorders affect only megakaryocytes and platelets, others involve different cell types and may result in characteristic phenotypic abnormalities. This review elaborates the clinical presentation and laboratory manifestations of common congenital thrombocytopenias in addition to exploring our understanding of the molecular basis of these disorders and therapeutic interventions available.
Hematology/oncology clinics of North America 06/2013; 27(3):465-494. · 2.05 Impact Factor
[show abstract][hide abstract] ABSTRACT: OBJECTIVES:: To assess potential hypercoagulability during diabetic ketoacidosis in children. DESIGN:: A prospective, controlled pilot study. SETTING:: University-affiliated pediatric critical care unit and emergency department in a tertiary care children's hospital. PATIENTS:: Children (1-18 yr) admitted with an episode of diabetic ketoacidosis and healthy children as controls. All patients with diabetic ketoacidosis managed according to a preestablished protocol. INTERVENTIONS:: Thromboelastography was performed using citrated whole-blood samples drawn at the time of admission and upon biochemical and clinical resolution of diabetic ketoacidosis. Citrated whole-blood samples drawn from healthy nondiabetic children acted as control samples. MEASUREMENTS AND MAIN RESULTS:: Fifteen patients (11.7 ± 4.1 yr) in the diabetic ketoacidosis group and 20 patients (8.9 ± 4.5 yr; p = 0.06) in the control group completed the study. Values for standard thromboelastography parameters (R and K time, α angle, maximum amplitude, coagulation index, and Ly30) in the diabetic ketoacidosis group, both on admission and resolution, were within the control range; thromboelastography profiles of diabetic ketoacidosis patients on admission were not significantly different from profiles obtained upon diabetic ketoacidosis resolution. The mean α-angle was significantly higher in known diabetic patients compared with newly diagnosed diabetics on admission; however, it still remained within the control normal range. CONCLUSIONS:: Thromboelastographic assay results do not reflect a hypercoagulable state in this group of children with diabetic ketoacidosis. Further investigation is required to examine the potential role of injured endothelium in the suspected hypercoagulability during diabetic ketoacidosis.
Pediatric Critical Care Medicine 02/2013; · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis.
[show abstract][hide abstract] ABSTRACT: Bacteria can enter the bloodstream in response to infectious insults, during surgery, or when catheters enter arteries or veins. Bacteremia elicits several immune and clinical complications, including thrombocytopenia. A primary cause of thrombocytopenia is shortened survival of activated platelets. Here, we demonstrate that pathogenic bacteria induce apoptotic events in platelets that include calpain-mediated degradation of Bcl x(L), an essential regulator of platelet survival. Specifically, bloodstream bacterial isolates from patients with sepsis induce lateral condensation of actin, impair mitochondrial membrane potential, and degrade Bcl x(L) protein in platelets. Bcl x(L) protein degradation is enhanced when platelets are exposed to pathogenic Escherichia coli (E. coli) that produce the pore-forming toxin α-hemolysin, a response that is markedly attenuated when the gene is deleted from E. coli. We also found that non-pathogenic E. coli gain degrading activity when they are forced to express α-hemolysin. Like α-hemolysin, purified α-toxin readily degrades Bcl x(L) protein in platelets as do clinical Staphylococcus aureus (S. aureus) isolates that produce α-toxin. Inhibition of calpain activity, but not the proteasome, rescues Bcl x(L) protein degradation in platelets co-incubated with pathogenic E. coli including α-hemolysin producing strains. This is the first evidence that pathogenic bacteria can trigger activation of the platelet intrinsic apoptosis program and our results suggest a new mechanism by which gram-negative and positive pathogens might cause thrombocytopenia in patients with bloodstream infections.
[show abstract][hide abstract] ABSTRACT: Vascular injury and atherothrombosis involve vessel infiltration by inflammatory leukocytes, migration of medial vascular smooth muscle cells to the intimal layer, and ultimately acute thrombosis. A strategy to simultaneously target these pathological processes has yet to be identified. The secreted protein, Slit2, and its transmembrane receptor, Robo-1, repel neuronal migration in the developing central nervous system. More recently, it has been appreciated that Slit2 impairs chemotaxis of leukocytes and vascular smooth muscle cells toward diverse inflammatory attractants. The effects of Slit2 on platelet function and thrombus formation have never been explored.
We detected Robo-1 expression in human and murine platelets and megakaryocytes and confirmed its presence via immunofluorescence microscopy and flow cytometry. In both static and shear microfluidic assays, Slit2 impaired platelet adhesion and spreading on diverse extracellular matrix substrates by suppressing activation of Akt. Slit2 also prevented platelet activation on exposure to ADP. In in vivo studies, Slit2 prolonged bleeding times in murine tail bleeding assays. Using intravital microscopy, we found that after mesenteric arteriolar and carotid artery injury, Slit2 delayed vessel occlusion time and prevented the stable formation of occlusive arteriolar thrombi.
These data demonstrate that Slit2 is a powerful negative regulator of platelet function and thrombus formation. The ability to simultaneously block multiple events in vascular injury may allow Slit2 to effectively prevent and treat thrombotic disorders such as myocardial infarction and stroke.
[show abstract][hide abstract] ABSTRACT: Antibody mediated rejection (AMR) activates the classical complement pathway and can be detrimental to graft survival. AMR can be accompanied by thrombotic microangiopathy (TMA). Eculizumab, a monoclonal C5 antibody prevents induction of the terminal complement cascade (TCC) and has recently emerged as a therapeutic option for AMR. We present a highly sensitized 13-year-old female with end-stage kidney disease secondary to spina bifida-associated reflux nephropathy, who developed severe steroid-, ATG- and plasmapheresis-resistant AMR with TMA 1 week post second kidney transplant despite previous desensitization therapy with immunoglobulin infusions. Eculizumab rescue therapy resulted in a dramatic improvement in biochemical (C3; creatinine) and hematological (platelets) parameters within 6 days. The patient was proven to be deficient in complement Factor H-related protein 3/1 (CFHR3/1), a plasma protein that regulates the complement cascade at the level of C5 conversion and has been involved in the pathogenesis of atypical hemolytic uremic syndrome caused by CFH autoantibodies (DEAP-HUS). CFHR1 deficiency may have worsened the severe clinical progression of AMR and possibly contributed to the development of donor-specific antibodies. Thus, screening for CFHR3/1 deficiency should be considered in patients with severe AMR associated with TMA.
American Journal of Transplantation 06/2012; 12(9):2546-53. · 6.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report on a consanguineous, Afghani family with two sisters affected with characteristic facial features, multiple contractures, progressive joint and skin laxity, hemorrhagic diathesis following minor trauma and multisystem fragility-related manifestations suggestive of a diagnosis of musculocontractural Ehlers-Danlos syndrome (EDS). This novel form of connective tissue disorder was recently reported in patients of Japanese, Turkish, and Indian descent who were formerly classified as having EDS type VIB and has now been recognized to be a part of spectrum including patients previously classified as having adducted thumb-clubfoot syndrome. We identified a previously unreported mutation in the CHST14 gene, which codes for the enzyme dermatan 4-O-sulfotransferase. We discuss the prenatal presentation, detailed clinical manifestations, and neurological findings in two sisters with this newly described musculocontractural EDS-CHST14 type. We demonstrate that fibroblasts from one of our patients produce more chondroitin sulfate than normal and show lower than normal deposition of collagens I and II and fibrillin 1-containing microfibrills. These findings suggest that the imbalance in the glycosaminoglycan content in developing tissues might interfere with normal deposition of other extracellular matrix components and ultimately contribute to the development of the phenotype observed in these patients. Furthermore, we ruled out the contribution of intrinsic platelet factors to the bleeding diathesis observed in some affected individuals.
American Journal of Medical Genetics Part A 05/2012; 158A(6):1344-54. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Retinal hemorrhage is a cardinal manifestation of abusive head injury. Thrombophilia is relatively common in the general population and in adults can be associated with retinal hemorrhage. The specificity of retinal hemorrhage for abusive head trauma in the presence of prothrombotic factors, in particular following non-abusive head trauma, has not been investigated. Our objective was to determine whether the hypothesis that prothrombotic factors affect specificity of retinal hemorrhage to AHT can be tested. This may have important ramifications both for diagnosis and expert witness testimony.
To investigate the feasibility of studying this issue, we conducted a prospective cohort study of children with abusive and non-abusive head trauma. Thrombophilia screening and ophthalmic examinations were performed.
Six of 30 admitted children were fully enrolled. Enrollment obstacles included caregiver stress, animosity towards allegations of abuse, child protection services involvement, and research phlebotomy coordination. Prevalence of thrombophilia was high in children with retinal hemorrhage and in 1 case the question of hemorrhage adjudicated as abuse was considered in light of a history of a fall.
We estimate that to answer the critical question of retinal hemorrhage specificity for abuse in the presence of thrombophilia will require 53 centers for a 1 year study or 18 centers for a 3-year study. We identify potential obstacles and interventions.
[show abstract][hide abstract] ABSTRACT: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half-life.
To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet-mediated events during thrombus formation.
The affinities of Diannexin and annexin A5 for PS-containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet-fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser-induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis.
Diannexin and annexin A5 bind PS with K(D) values of 0.6 and 5 nm, respectively, and both bind to the same subpopulation of PS-exposing platelets. Diannexin inhibited thrombin generation and platelet-fibrin clot formation in vitro at 10 nm (P<0.05-0.001 compared with control), and reduced platelet accumulation at 1 μg g(-1) (P<0.05) and activation at 0.25 μg g(-1) (P<0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 μg g(-1) (P<0.01).
Diannexin binds to PS with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation.
Journal of Thrombosis and Haemostasis 03/2012; 10(6):1109-19. · 6.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Unfractionated heparin (UFH) is a widely used anticoagulant. Current American College of Chest Physicians guidelines for infants extrapolated from adults recommend 28 U kg(-1) h(1) of UFH to achieve an anti-factor Xa level of 0.35-0.7 IU mL(-1).
To assess the profile of anti-FXa-based UFH dosing guidelines in infants.
We included all infants aged < 6 months treated with per-protocol intravenous UFH at the Hospital for Sick Children, Toronto, over a 3.5-year period.
Of 100 infants, 11% achieved sustained therapeutic anti-FXa levels with current dose recommendations. Only 15% achieved target anti-FXa levels within 24 h with per-protocol dose escalations. Seventeen per cent of patients never achieved therapeutic anti-FXa levels, despite up to 60 days of therapy and triple the recommended dose. The median dose needed to achieve therapeutic anti-FXa levels in the remaining 83 infants was 33 U kg(-1) h(-1) (interquartile range, 30-36). Two in three infants had decreased thrombus size at completion of therapy and no thrombus progression/recurrence, and 11/100 infants suffered major bleeding. Without exclusion of extracorporeal membrane oxygenation patients, an activated partial thromboplastin time (APTT) of > 180 s was detected as a risk factor for major bleeding.
UFH monitoring is challenging in infants. Despite their delay in reaching therapeutic anti-FXa levels, infants monitored with the adult-based anti-FXa range have a high thrombus resolution rate, no thrombus progression, but a relatively high bleeding rate. Extreme APTT elevation may contribute to this bleeding risk, particularly in critically ill patients. Current UFH guidelines for young infants may still be inadequate, and laboratory methods with age-appropriate ranges may be required to further improve clinical outcomes within this population.
Journal of Thrombosis and Haemostasis 03/2012; 10(3):368-74. · 6.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.
[show abstract][hide abstract] ABSTRACT: Inherited platelet disorders encompass a heterogeneous group of bleeding disorders where a variety of molecular defects can affect platelet number, function or both. The defects involve deficiencies or dysfunction of platelet receptors, signaling pathways, cytoskeletal proteins, granule contents and abnormalities in procoagulant activity. These disorders can be difficult to distinguish clinically as they present with the common symptom of mucocutaneous bleeding. Inherited thrombocytopenia needs to be considered in all patients suspected of having primary immune thrombocytopenia, where platelets may also have functional defects. After a careful history and physical examination, initial investigations include a complete blood count with a peripheral smear, followed by appropriate specific investigations that often require specialized referral centers. This article is a summary of the current data on clinical presentation, pathogenesis, diagnosis and management of inherited platelet disorders.
Expert Review of Hematology 08/2011; 4(4):455-72. · 2.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is a fatal recessive disorder caused by mutations in the VPS33B or VPS16B genes. Both encode homologues of the Vps33p and Vps16p subunits of the HOPS complex necessary for fusions of vacuoles in yeast. Here, we describe a mutation in the full-of-bacteria (fob) gene, which encodes Drosophila Vps16B. Flies null for fob are homozygous viable and fertile. They exhibit, however, a defect in their immune defense that renders them hypersensitive to infections with nonpathogenic bacteria. fob hemocytes (fly macrophages) engulf bacteria but fail to digest them. Phagosomes undergo early steps of maturation and transition to a Rab7-positive stage, but do not mature to fully acidified phagolysosomes. This reflects a specific requirement of fob in the fusion of phagosomes with late endosomes/lysosomes. In contrast, cargo of autophagosomes as well as endosomes exhibit normal lysosomal delivery in fob cells. These findings suggest that defects in phagosome maturation may contribute to symptoms of ARC patients including recurring infections.
The Journal of Cell Biology 02/2011; 192(3):383-90. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: The investigation of children with suspected inherited platelet disorders is challenging. The causes of mucocutaneous bleeding are many, and specialized testing for platelet disorders can be difficult to access or interpret. An algorithm developed for the investigation of suspected platelet disorders provides a sequential approach to evaluating both platelet function abnormalities and thrombocytopenia. Investigation begins with a clinical evaluation and laboratory testing that is generally available, including platelet counting, peripheral blood cell morphology, and aggregometry. Based on results of initial investigations, the algorithm recommends specialized testing for specific diagnoses, including flow cytometry, immunofluorescence microscopy, electron microscopy, and mutational analysis.
Pediatric Blood & Cancer 02/2011; 56(6):975-83. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by thrombocytopenia and the absence of α-granules in platelets. Patients with GPS present with mild to moderate bleeding and many develop myelofibrosis. The genetic cause of GPS is unknown. We present 2 Native American families with a total of 5 affected persons and a single affected patient of Pakistani origin in which GPS appears to be inherited in an autosomal recessive manner. Homozygosity mapping using the Affymetrix 6.0 chips demonstrates that all 6 GPS-affected persons studied are homozygous for a 1.7-Mb region in 3p21. Linkage analysis confirmed the region with a logarithm of the odds score of 2.7. Data from our families enabled us to significantly decrease the size of the critical region for GPS from the previously reported 9.4-Mb region at 3p21.