The Sec1-Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B.

Loss-of-function variants of vacuolar protein sorting proteins VPS33B and VPS16B (VIPAS39) are causative for arthrogryposis, renal dysfunction, and cholestasis syndrome, where early lethality of patients indicates that VPS33B and VPS16B play essential cellular roles. VPS33B is a member of the Sec1-Munc18 protein family and thought to facilitate vesicular fusion via interaction with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, like its paralog VPS33A in the homotypic fusion and vacuole sorting complex. VPS33B and VPS16B are known to associate, but little is known about the composition, structure, or function of the VPS33B-VPS16B complex. We show here that human VPS33B-VPS16B is a high molecular weight complex, which we expressed in yeast to perform structural, composition, and stability analysis. Circular dichroism data indicate VPS33B-VPS16B has a well-folded α-helical secondary structure, and size-exclusion chromatography-multiangle light scattering revealed a molecular weight of ∼315 kDa. Quantitative immunoblotting indicated a VPS33B:VPS16B ratio of 2:3. Expression of arthrogryposis, renal dysfunction, and cholestasis syndrome-causing VPS33B missense variants showed L30P disrupts complex formation but not S243F or H344D. Truncated VPS16B (amino acids 143 to 316) was sufficient to form a complex with VPS33B. Small-angle X-ray scattering and negative-staining EM revealed a two-lobed shape for VPS33B-VPS16B. Avidin tagging indicated that each lobe contains a VPS33B molecule, and they are oriented in opposite directions. We propose a structure for VPS33B-VPS16B that allows the VPS33B at each end to interact with separate SNARE bundles and/or SNAREpins, plus associated membrane components. These observations reveal the only known potentially bidirectional Sec1-Munc18 protein complex.

Moderate-intensity aerobic exercise vs desmopressin in adolescent males with mild hemophilia A: a randomized trial.

Persons with mild hemophilia A (HA) may use intranasal desmopressin prior to sports participation. Desmopressin is expensive and can cause vomiting, headache, palpitation, and occasionally seizures. Our group has previously documented a 2.3-fold increase in factor VIII activity (FVIII:C) in adolescents with mild HA after moderate-intensity aerobic exercise. Herein, we report principal findings of a randomized trial of intranasal desmopressin vs a standardized, moderate-intensity aerobic exercise regimen in adolescents with mild HA. Our primary objective was to compare the change in FVIII:C associated with these 2 interventions. We also examined changes in hemostatic parameters arising from their sequential administration. The study was conducted simultaneously at the Hospital for Sick Children, Canada, and Nationwide Children's Hospital, USA. Thirty-two eligible male adolescents (mean age ± standard deviation: 16.1 ± 2.6 years) with mild HA (mean baseline FVIII:C: 27.9% ± 18.4%) were randomized to 1 of 4 study arms (desmopressin followed by exercise, desmopressin alone, exercise followed by desmopressin, and exercise alone). Blood work was obtained at baseline and at 3 subsequent time-points. Participants randomized to exercise cycled on an ergometer for approximately 12 minutes, with the final 3 minutes at 85% of their predicted maximum heart rate. Standard weight-based dosing of desmopressin was used. Mean immediate increase in FVIII:C was 1.7-fold with exercise compared with 1.9-fold with desmopressin (noninferiority, P = .04). Exercise-induced improvement in hemostatic parameters including FVIII:C was brief compared with more sustained improvements seen with desmopressin. More than 60% of participants randomized to receive both exercise and desmopressin achieved normal (>50%) FVIII:C, 75 and 135 minutes into the study protocol.

Evaluation of human platelet granules by structured illumination laser fluorescence microscopy.

Many roles of human platelets in health and disease are linked to their ability to transport and secrete a variety of small molecules and proteins carried in dense (δ-) and α-granules. Determination of granule number and content is important for diagnosis of platelet disorders and for studies of platelet structure, function, and development. We have optimized methods for detection and localization of platelet proteins via antibody and lectin staining, imaging via structured illumination laser fluorescence microscopy (SIM), and three-dimension (3D) image analysis. The methods were validated via comparison with published studies based on electron microscopy and high-resolution fluorescence microscopy. The α-granule cargo proteins thrombospondin-1 (TSP1), osteonectin (SPARC), fibrinogen (FGN), and Von Willebrand factor (VWF) were localized within the granule lumen, as was the proteoglycan serglycin (SRGN). Colocalization analysis indicates that staining with fluorescently labeled wheat germ agglutinin (WGA) allows detection of α-granules as effectively as immunostaining for cargo proteins, with the advantage of not requiring antibodies. RAB27B was observed to be concentrated at dense granules, allowing them to be counted via visual scoring and object analysis. We present a workflow for counting dense and α-granules via object analysis of 3D SIM images of platelets stained for RAB27B and with WGA.Abbreviation: SIM: structured illumination microscopy; WGA: wheat germ agglutinin; FGN: fibrinogen; TSP1: thrombospondin 1; ER: endoplasmic reticulum.

Platelets support blood clotting, wound healing, and other essential processes. These functions rely on the ability of platelets to transport and release small molecules like serotonin carried in dense granules and a wide range of proteins carried in alpha granules. Several conditions have been linked to abnormalities in one or more of platelet granule number, content, structure, and function. These conditions can be difficult to diagnose because platelet granules are so small they cannot be consistently resolved by conventional light microscopy, while higher power electron microscopy is not widely accessible.The goal of this study was to develop a method for counting and examining platelet dense and alpha granules without the need of electron microscopy. Key to this was the discovery that alpha granules can be reliably stained with the plant lectin wheat germ agglutinin, which has the advantages of being a smaller and less expensive molecule than the antibodies commonly used to detect alpha granule cargo proteins. We also establish that dense granules can be detected with high specificity via antibody staining of the membrane-associated protein RAB27B. We used structured illumination laser fluorescence microscopy to obtain high-resolution images of stained platelets. These were assembled into 3D renders using image analysis software, which was used to validate a protocol for rapidly counting granules within individual platelets.Our method supports the relatively rapid, accurate, and cost-effective assessment of platelet granules. We have already shown that it can confirm dense granule deficiency, and we anticipate that this approach will also prove useful in diagnosing and studying alpha granule abnormalities.

The endoplasmic reticulum protein SEC22B interacts with NBEAL2 and is required for megakaryocyte α-granule biogenesis.

Studies of inherited platelet disorders have provided many insights into platelet development and function. Loss of function of neurobeachin-like 2 (NBEAL2) causes gray platelet syndrome (GPS), where the absence of platelet α-granules indicates NBEAL2 is required for their production by precursor megakaryocytes. The endoplasmic reticulum is a dynamic network that interacts with numerous intracellular vesicles and organelles and plays key roles in their development. The megakaryocyte endoplasmic reticulum is extensive, and in this study we investigated its role in the biogenesis of α-granules by focusing on the membrane-resident trafficking protein SEC22B. Coimmunoprecipitation (co-IP) experiments using tagged proteins expressed in human HEK293 and megakaryocytic immortalized megakaryocyte progenitor (imMKCL) cells established binding of NBEAL2 with SEC22B, and demonstrated that NBEAL2 can simultaneously bind SEC22B and P-selectin. NBEAL2-SEC22B binding was also observed for endogenous proteins in human megakaryocytes using co-IP, and immunofluorescence microscopy detected substantial overlap. SEC22B binding was localized to a region of NBEAL2 spanning amino acids 1798 to 1903, where 2 GPS-associated missense variants have been reported: E1833K and R1839C. NBEAL2 containing either variant did not bind SEC22B coexpressed in HEK293 cells. CRISPR/Cas9-mediated knockout of SEC22B in imMKCL cells resulted in decreased NBEAL2, but not vice versa. Loss of either SEC22B or NBEAL2 expression resulted in failure of α-granule production and reduced granule proteins in imMKCL cells. We conclude that SEC22B is required for α-granule biogenesis in megakaryocytes, and that interactions with SEC22B and P-selectin facilitate the essential role of NBEAL2 in granule development and cargo stability.

Recent advances in inherited platelet disorders.

PURPOSE OF REVIEW: The increasing use of high throughput sequencing and genomic analysis has facilitated the discovery of new causes of inherited platelet disorders. Studies of these disorders and their respective mouse models have been central to understanding their biology, and also in revealing new aspects of platelet function and production. This review covers recent contributions to the identification of genes, proteins and variants associated with inherited platelet defects, and highlights how these studies have provided insights into platelet development and function. RECENT FINDINGS: Novel genes recently implicated in human platelet dysfunction include the galactose metabolism enzyme UDP-galactose-4-epimerase in macrothrombocytopenia, and erythropoietin-producing hepatoma-amplified sequence receptor transmembrane tyrosine kinase EPHB2 in a severe bleeding disorder with deficiencies in platelet agonist response and granule secretion. Recent studies of disease-associated variants established or clarified roles in platelet function and/or production for the membrane receptor G6b-B, the FYN-binding protein FYB1/ADAP, the RAS guanyl-releasing protein RASGRP2/CalDAG-GEFI and the receptor-like protein tyrosine phosphatase PTPRJ/CD148. Studies of genes associated with platelet disorders advanced understanding of the cellular roles of neurobeachin-like 2, as well as several genes influenced by the transcription regulator RUNT-related transcription factor 1 (RUNX1), including NOTCH4. SUMMARY: The molecular bases of many hereditary platelet disorders have been elucidated by the application of recent advances in cell imaging and manipulation, genomics and protein function analysis. These techniques have also aided the detection of new disorders, and enabled studies of disease-associated genes and variants to enhance understanding of platelet development and function.

The Birth and Death of Platelets in Health and Disease.

Blood platelets are involved in a wide range of physiological responses and pathological processes. Recent studies have considerably advanced our understanding of the mechanisms of platelet production and clearance, revealing new connections between the birth and death of these tiny, abundant cells. Key insights have also been gained into how physiological challenges such as inflammation, infection, and chemotherapy can affect megakaryocytes, the cells that produce platelets.

NBEAL2 (Neurobeachin-Like 2) Is Required for Retention of Cargo Proteins by α-Granules During Their Production by Megakaryocytes.

Objective- Human and mouse megakaryocytes lacking NBEAL2 (neurobeachin-like 2) produce platelets where α-granules lack protein cargo. This cargo is mostly megakaryocyte-synthesized, but some proteins, including FGN (fibrinogen), are endocytosed. In this study, we examined the trafficking of both types of cargo within primary megakaryocytes cultured from normal and NBEAL2-null mice, to determine the role of NBEAL2 in α-granule maturation. We also examined the interaction of NBEAL2 with the granule-associated protein P-selectin in human megakaryocytes and platelets. Approach and Results- Fluorescence microscopy was used to compare uptake of labeled FGN by normal and NBEAL2-null mouse megakaryocytes, which was similar in both. NBEAL2-null cells, however, showed decreased FGN retention, and studies with biotinylated protein showed rapid loss rather than increased degradation. Intracellular tracking via fluorescence microscopy revealed that in normal megakaryocytes, endocytosed FGN sequentially associated with compartments expressing RAB5 (Ras-related protein in brain 5), RAB7 (Ras-related protein in brain 7), and P-selectin, where it was retained. A similar initial pattern was observed in NBEAL2-null megakaryocytes, but then FGN passed from the P-selectin compartment to RAB11 (Ras-related protein in brain 11)-associated endosomes before release. Megakaryocyte-synthesized VWF (Von Willebrand factor) was observed to follow the same route out of NBEAL2-null cells. Immunofluorescence microscopy revealed intracellular colocalization of NBEAL2 with P-selectin in human megakaryocytes, proplatelets, and platelets. Native NBEAL2 and P-selectin were coimmunoprecipitated from platelets and megakaryocytes. Conclusions- NBEAL2 is not required for FGN uptake by megakaryocytes. NBEAL2 is required for the retention of both endocytosed and megakaryocyte-synthesized proteins by maturing α-granules, and possibly by platelet-borne granules. This function may involve interaction of NBEAL2 with P-selectin.

NBEAL2 mutations and bleeding in patients with gray platelet syndrome.

Homozygosity/compound heterozygosity for loss of function mutations in neurobeachin-like 2 (NBEAL2) is causative for Gray platelet syndrome (GPS; MIM #139090), characterized by thrombocytopenia and large platelets lacking α-granules and cargo. Most GPS-associated NBEAL2 mutations generate nonsense codons; frameshifts causing premature translation termination and/or changes in mRNA splicing have also been observed. Data regarding NBEAL2 protein expression in GPS patients is limited. We observed absence of NBEAL2 in platelets from GPS patients with 3 different genotypes, and reduced/truncated platelet NBEAL2 has been reported for others. GPS is commonly associated with mild bleeding, but lifethreatening bleeding has been reported in some cases. A common long-term complication in GPS patients is myelofibrosis; splenomegaly is less common but sometimes of sufficient severity to merit splenectomy. Like GPS patients, mice lacking NBEAL2 expression exhibit macrothrombocytopenia, deficiency of platelet α-granules, splenomegaly, myelofibrosis, impaired platelet function and abnormalities in megakaryocyte development. Animal studies have also reported impaired platelet function in vivo using laser injury and thrombo-inflammation models. NBEAL2 is a large gene with 54 exons, and several putative functional domains have been identified in NBEAL2, including PH (pleckstrin homology) and BEACH (beige and Chediak-Higashi) domains shared with other members of a protein family that includes LYST and LRBA, also expressed by hematopoietic cells. Potential NBEAL2-interacting proteins have recently been identified, and it is expected that current and future efforts will reveal the cellular mechanisms by which NBEAL2 facilitates platelet development and supports hemostatic function.

Dynamin 2-dependent endocytosis is required for normal megakaryocyte development in mice.

Dynamins are highly conserved large GTPases (enzymes that hydrolyze guanosine triphosphate) involved in endocytosis and vesicle transport, and mutations in the ubiquitous and housekeeping dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in thrombopoiesis, we generated Dnm2(fl/fl) Pf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2(fl/fl) Pf4-Cre mice had severe macrothrombocytopenia with moderately accelerated platelet clearance. Dnm2-null bone marrow MKs had altered demarcation membrane system formation in vivo due to defective endocytic pathway, and fetal liver-derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2-dependent endocytosis plays a major role in MK membrane formation and thrombopoiesis. Endocytosis of the thrombopoietin receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating thrombopoietin levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by marked expansion of hematopoietic stem and progenitor cells, MK hyperplasia, myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. Taken together, our data demonstrate that unrestrained MK growth and proliferation results in rapid myelofibrosis and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis, and bone marrow homeostasis.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.

Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases.

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.

α-granule biogenesis: from disease to discovery.

Platelets are critical to hemostasis and thrombosis. Upon detecting injury, platelets show a range of responses including the release of protein cargo from α-granules. This cargo is synthesized by platelet precursor megakaryocytes or endocytosed by megakaryocytes and/or platelets. Insights into α-granule biogenesis have come from studies of hereditary conditions where these granules are immature, deficient or absent. Studies of Arthrogryposis, Renal dysfunction, and Cholestasis (ARC) syndrome identified the first proteins essential to α-granule biogenesis: VPS33B and VPS16B. VPS33B and VPS16B form a complex, and in the absence of either, platelets lack α-granules and the granule-specific membrane protein P-selectin. Gray Platelet Syndrome (GPS) platelets also lack conventionally recognizable α-granules, although P-selectin containing structures are present. GPS arises from mutations affecting NBEAL2. The GPS phenotype is more benign than ARC syndrome, but it can cause life-threatening bleeding, progressive thrombocytopenia, and myelofibrosis. We review the essential roles of VPS33B, VPS16B, and NBEAL2 in α-granule development. We also examine the existing data on their mechanisms of action, where many details remain poorly understood. VPS33B and VPS16B are ubiquitously expressed and ARC syndrome is a multisystem disorder that causes lethality early in life. Thus, VPS33B and VPS16B are clearly involved in other processes besides α-granule biogenesis. Studies of their involvement in vesicular trafficking and protein interactions are reviewed to gain insights into their roles in α-granule formation. NBEAL2 mutations primarily affect megakaryocytes and platelets, and while little is known about NBEAL2 function some insights can be gained from studies of related proteins, such as LYST.

Von Willebrand factor regulates complement on endothelial cells.

Atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury.

Abnormalities in the alternative pathway of complement in children with hematopoietic stem cell transplant-associated thrombotic microangiopathy.

Hematopoietic stem cell transplant (HSCT)-associated thrombotic microangiopathy (TMA) is a complication that occurs in 25% to 35% of HSCT recipients and shares histomorphologic similarities with hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). The hallmark of all thrombotic microangiopathies is vascular endothelial cell injury of various origins, resulting in microangiopathic hemolytic anemia, platelet consumption, fibrin deposition in the microcirculation, and tissue damage. Although significant advances have been made in understanding the pathogenesis of other thrombotic microangiopathies, post-HSCT TMA remains poorly understood. We report an analysis of the complement alternative pathway, which has recently been linked to the pathogenesis of both the Shiga toxin mediated and the atypical forms of HUS, with a focus on genetic variations in the complement Factor H (CFH) gene cluster and CFH autoantibodies in six children with post-HSCT TMA. We identified a high prevalence of deletions in CFH-related genes 3 and 1 (delCFHR3-CFHR1) and CFH autoantibodies in these patients with HSCT-TMA. Conversely, CFH autoantibodies were not detected in 18 children undergoing HSCT who did not develop TMA. Our observations suggest that complement alternative pathway dysregulation may be involved in the pathogenesis of post-HSCT TMA. These findings shed light on a novel mechanism of endothelial injury in transplant-TMA and may therefore guide the development of targeted treatment interventions.

Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice.

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 NEABL2 (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.

Eculizumab therapy in children with severe hematopoietic stem cell transplantation-associated thrombotic microangiopathy.

We recently observed that dysregulation of the complement system may be involved in the pathogenesis of hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA). These findings suggest that the complement inhibitor eculizumab could be a therapeutic option for this severe HSCT complication with high mortality. However, the efficacy of eculizumab in children with HSCT-TMA and its dosing requirements are not known. We treated 6 children with severe HSCT-TMA using eculizumab and adjusted the dose to achieve a therapeutic level >99 µg/mL. HSCT-TMA resolved over time in 4 of 6 children after achieving therapeutic eculizumab levels and complete complement blockade, as measured by low total hemolytic complement activity (CH50). To achieve therapeutic drug levels and a clinical response, children with HSCT-TMA required higher doses or more frequent eculizumab infusions than currently recommended for children with atypical hemolytic uremic syndrome. Two critically ill patients failed to reach therapeutic eculizumab levels, even after dose escalation, and subsequently died. Our data indicate that eculizumab may be a therapeutic option for HSCT-TMA, but HSCT patients appear to require higher medication dosing than recommended for other conditions. We also observed that a CH50 level ≤ 4 complement activity enzyme units correlated with therapeutic eculizumab levels and clinical response, and therefore CH50 may be useful to guide eculizumab dosing in HSCT patients as drug level monitoring is not readily available.

The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis.

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.