Acidification of α-granules in megakaryocytes by vacuolar-type ATPase is essential for organelle biogenesis.

BACKGROUND: Platelets coordinate blood coagulation at sites of vascular injury and play fundamental roles in a wide variety of (patho)physiological processes. Key to many platelet functions is the transport and secretion of proteins packaged within α-granules, organelles produced by platelet precursor megakaryocytes. Prominent among α-granule cargo are fibrinogen (FGN) endocytosed from plasma, and endogenously-synthesized Von Willebrand factor (VWF). These and other proteins are known to require acidic pH for stable packaging. Luminal acidity has been confirmed for mature α-granules isolated from platelets, but direct measurement of megakaryocyte granule acidity has not been reported. OBJECTIVE: To determine the luminal pH of α-granules and their precursors in megakaryocytes, and assess the requirement of vacuolar-type adenosine triphosphatase (V-ATPase) activity establish and maintain the luminal acidity and integrity of these organelles. RESULTS: Using cresyl violet staining, we show that most of the acidic granules detected in megakaryocytes appear to be α-granules/precursors. Endocytosis of FGN tagged with the pH-sensitive fluorescent dye FITC was used to load a subset of these organelles, and ratiometric fluorescence analysis established a median luminal pH of 5.2 (interquartile range 5.0 - 5.5). Inhibition of megakaryocyte V-ATPase activity led to enlargement of cargo-containing compartments detected by fluorescence microscopy and electron microscopy. CONCLUSION: These observations reveal that V-ATPase activity is required to establish and maintain a luminal acidic pH in megakaryocyte α-granules/precursors, confirming its importance for stable packaging of cargo proteins such as VWF.

Constitutive hypercoagulability in pediatric sickle cell disease patients with hemoglobin SS genotype.

Background: Constitutive inflammation and hemostatic activation have been identified as key contributors to the pathophysiology of sickle cell disease (SCD), leading to clinical consequences such as vaso-occlusive crises and stroke. Patients with hemoglobin SS (HbSS) and hemoglobin SC (HbSC) genotypes are reported to have different symptoms, as do patients in steady-state and crisis situations. Differences among these groups remain unclear in pediatric patients. Objectives: To compare hemostatic activity in HbSS and HbSC pediatric patients during steady state, in crisis, and in clinical follow-up and compare HbSS and HbSC patients with normal healthy children. Methods: Whole-blood coagulation assay thromboelastography (TEG) was used to assess hemostatic activity. In parallel, flow cytometry was used to assess procoagulant surface expression of platelets and red blood cells. Results: TEG results indicated no significant differences in clotting onset (R time), clot maximum amplitude, or maximum rate of thrombus generation among steady-state, crisis, and follow-up subgroups of HbSS and HbSC patients. TEG parameters did not differ significantly between HbSC patients and healthy children, while HbSS patients showed significantly shorter R time and greater maximum amplitude and maximum rate of thrombus generation, all indicative of a constitutive hypercoagulable state. Flow cytometry results did not detect increased platelet integrin αIIbß3 activation or red blood cell procoagulant surface expression in SCD patients compared with unaffected children. Conclusion: Our results indicate that pediatric SCD patients with the HbSS genotype have constitutively activated hemostasis relative to HbSC patients and healthy children. It remains to be determined how treatments that improve clinical outcomes in SCD patients affect this constitutively hypercoagulable state.

Human platelets contain a pool of free zinc in dense granules.

Background: Activated platelets release procoagulant factors that include Ca2+ and Zn2+. Releasable Ca2+ stores have been identified in platelet dense granules and the dense tubular system, but similar stores of free Zn2+ have not been identified. Objectives: Guided by studies of platelet Ca2+, we employed minimally disruptive methods to identify and localize concentrated free Zn2+ in human platelets. Methods: Resting platelets from normal donors (NDs), patients with gray platelet syndrome (GPS) lacking α-granules, and patients with Hermansky-Pudlak syndrome (HPS) deficient in dense granules were loaded with cell-permeant fluorescent probes specific to free Ca2+ or Zn2+. Ion concentrations were detected in fixed cells as bright puncta via high-resolution confocal microscopy. Ions were also directly detected via transmission electron microscopy and energy dispersive X-ray analysis. Levels of total platelet Ca, Zn, and Mg were measured via inductively coupled plasma optical emission spectroscopy. Results: Fluorescent Zn2+ puncta counts were similar in ND and GPS platelets and markedly lower in HPS platelets, pointing to dense granules as likely reservoirs of free Zn2+. This localization was supported by direct detection of Ca2+, Zn2+, and Na+ in platelet dense granules via transmission electron microscopy and energy dispersive X-ray analysis. Measurements of total platelet Ca, Zn, and Mg via inductively coupled plasma optical emission spectroscopy indicated that free Zn2+ represents a small proportion of total platelet zinc, consistent with the strong affinity of Zn2+ for binding proteins, including several abundant in platelet α-granules. Conclusion: We conclude that normal human platelets contain a pool of free Zn2+ concentrated in dense granules that is available for secretion upon platelet activation and potentially contributes to hemostasis.

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.

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.

Thromboelastography and thrombin generation assessments for pediatric severe hemophilia A patients are highly variable and not predictive of clinical phenotypes.

Background: Severe hemophilia A (SHA) patients vary in severity of bleeding, arthropathy, and requirements for replacement factor VIII (FVIII). Baseline hemostatic activity assays using calibrated automated thrombography (CAT) and thromboelastography (TEG) may offer insights into the physiological basis of clinical heterogeneity. Objectives: Use CAT and TEG to measure baseline hemostatic activity in a cohort of 30 pediatric SHA patients with available clinical data. Determine effect of contact activation inhibition with corn trypsin inhibitor (CTI). Assess heterogeneity among patients for baseline hemostatic activity and examine correlations between assay results and clinical parameters including FVIII dosing regimen, von Willebrand factor level, and Pettersson arthropathy score. Methods: SHA blood after FVIII washout was subjected to TEG, and platelet-rich (PRP) and platelet-poor plasma was used for CAT assays. Varying concentrations of tissue factor (TF) were used. Statistical analysis examined relationships between assay results, and clinical parameters. Results: CTI treatment was required to obtain TEG and CAT results representative of baseline hemostatic activity. Weak activity was observed in assays with low TF concentrations (0.5-2 pM), and most but not all samples approached normal activity levels at high TF concentrations (10-20 pM). A significant positive correlation was observed between results of TEG and CAT-PRP assays. Correlations were not detected between hemostatic assay results and clinical parameters. Conclusions: In vitro hemostatic assay results of samples containing platelets showed concordance. Assay results were not predictive of FVIII requirements or correlated with other clinical parameters. SHA patient heterogeneity is influenced by factors other than baseline hemostatic activity.

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.

Platelet VPS16B is dependent on VPS33B expression, as determined in two siblings with arthrogryposis, renal dysfunction, and cholestasis syndrome.

BACKGROUND: Platelet α-granule biogenesis in precursor megakaryocytes is critically dependent on VPS33B and VPS16B, as demonstrated by the platelet α-granule deficiency seen in the rare multisystem disorder arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome associated with biallelic pathogenic variants in VPS33B and VIPAS39 (encoding VPS16B). VPS33B and VPS16B are ubiquitously expressed proteins that are known to interact and play key roles in protein sorting and trafficking between subcellular locations. However, there remain significant gaps in our knowledge of the nature of these interactions in primary cells from patients with ARC syndrome. OBJECTIVES: To use primary cells from patients with ARC syndrome to better understand the interactions and roles of VPS33B and VPS16B in platelets and precursor megakaryocytes. PATIENTS/METHODS: The proband and his male sibling were clinically suspected to have ARC syndrome. Confirmatory genetic testing and platelet phenotyping, including electron microscopy and protein expression analysis, was performed with consent in a research setting. RESULTS: We describe the first case of ARC syndrome identified in Costa Rica, associated with a novel homozygous nonsense VPS33B variant that is linked with loss of expression of both VPS33B and VPS16B in platelets. CONCLUSION: These results indicate that stable expression of VPS16B in platelets, their precursor megakaryocytes, and other cells is dependent on VPS33B. We suggest that systematic evaluation of primary cells from patients with a range of VPS33B and VIPAS39 variants would help to elucidate the interactions and functions of these proteins.

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.

Vascular endothelial cells evade complement-mediated membrane injury via Weibel-Palade body mobilization.

BACKGROUND: Defective complement inhibition can lead to the formation of membrane attack complexes (MAC; C5b-9) on the plasma membranes of vascular endothelial cells, resulting in injury that drives the progression of thrombotic microangiopathy (TMA), a key pathology in kidney disease. OBJECTIVE/METHODS: We examined the response of human endothelial cells to complement-mediated damage using blood outgrowth endothelial cells (BOECs) derived from healthy donors. BOECs were sensitized to complement factors present in normal human serum to induce the formation of C5b-9 on their plasma membranes. RESULTS: This triggered an expected abrupt rise in intracellular Ca2+ reflecting membrane leakage. Remarkably, while intracellular Ca2+ remained elevated, membrane leakage ceased within 30 minutes, and cells did not show significant death. Extensive mobilization of Weibel-Palade bodies (WPBs) was observed along with secretion of von Willebrand factor (VWF). The potential role of WPBs and VWF in mitigating complement-mediated damage was examined by comparing the effects of C5b-9 on BOECs derived from von Willebrand disease (VWD) patients expressing reduced amounts of VWF, lacking expression of functional VWF, or lacking both VWF and WPBs. BOECs lacking WPBs were not resistant to complement-mediated damage, but became resistant when transfected to express VWF (and thus WPBs). CONCLUSION: We conclude that BOECs exposed to C5b-9 attack respond by mobilizing WPBs, which mitigate and repair damage by fusing with the plasma membrane. We propose that a similar cell-specific response may protect the vascular endothelium from complement-mediated damage in vivo.

A Hypoxia-Inducible HIF1-GAL3ST1-Sulfatide Axis Enhances ccRCC Immune Evasion via Increased Tumor Cell-Platelet Binding.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and the major cause of mortality for individuals with von Hippel-Lindau (VHL) disease. ccRCC is characterized most frequently by inactivation of VHL tumor suppressor protein that mediates degradation of the alpha subunit of the hypoxia-inducible factor (HIF) transcription factor family. HIF has been implicated in disease progression and the aim of this study was to identify novel HIF target genes that may contribute to ccRCC. We show that GAL3ST1, an enzyme that catalyzes the sulfonation of the plasma membrane sulfolipid sulfatide, is among the top 50 upregulated genes in ccRCC tissue relative to matched normal tissue. Increased expression of GAL3ST1 in primary ccRCC correlates with decreased survival. We show that GAL3ST1 is a HIF target gene whose expression is induced upon VHL loss leading to the accumulation of its enzymatic product sulfatide. Notably, platelets bind more efficiently to renal cancer cells with high GAL3ST1-sulfatide expression than to GAL3ST1-sulfatide-negative counterparts, which protects ccRCC cells against natural killer cell-mediated cytotoxicity. These results suggest that GAL3ST1 is a HIF-responsive gene that may contribute to ccRCC development via promoting cancer cell evasion of immune surveillance. IMPLICATIONS: Cancer development is in part dependent on evasion of immune response. We identify a HIF target gene product GAL3ST1 that may play a role in this critical process.

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.

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.

Imaging Platelets and Megakaryocytes by High-Resolution Laser Fluorescence Microscopy.

Microscopy is central to studies of platelets and their precursor megakaryocytes. Here we describe methods to rapidly obtain high resolution images of fixed platelets, megakaryocytes and megakaryocytic cells via immunofluorescence microscopy. Protocols covered include: (1) isolation and preparation of cells suitable for fluorescence staining; (2) staining with antibodies and other molecules; (3) imaging via spinning-disc confocal and structured illumination laser fluorescence microscopy; (4) processing and presentation of images. Also included is a list of primary antibodies we have validated for use in staining specific proteins and subcellular structures in platelets and megakaryocytes.

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.

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.

Relative antibacterial functions of complement and NETs: NETs trap and complement effectively kills bacteria.

Neutrophil extracellular traps (NETs) are web-like DNA structures released by activated neutrophils. These structures are decorated with antimicrobial proteins, and considered to trap and kill bacteria extracellularly. However, the exact functions of NETs remain elusive, and contradictory observations have been made with NETs functioning as an antimicrobial or a pathogentrapping mechanism. There is a disconnect in the interpretation of the involvement of other major immune mechanisms, such as the complement system, as effectors of the function of NETs. We have recently shown that NETs activate complement. In this study, we aimed to elucidate the relative antimicrobial roles of NETs in the absence and presence of complement. Using primary human neutrophils, human serum (normal, heat inactivated, and C5-depleted), P. aeruginosa (at multiplicity of infection, MOI, of 1 or 10), S. aureus (MOI of 1), colony-counting assays and confocal microscopy, we demonstrate that most bacteria trapped by NETs remain viable, indicating that NETs have limited bactericidal properties. By contrast, complement effectively killed bacteria, but NETs decreased the bactericidal ability of complement and degrading NETs by DNases restored complement-mediated killing. Experiments with conditions allowing for specific pathway activation showed that the complement classical and lectin, but not the alternative, pathway lead to bacterial killing. NETs under static conditions showed limited killing of bacteria while NETs under dynamic conditions showed enhanced bacteria trapping and reduced killing. Furthermore, NETs incubated with normal human serum depleted complement and reduced the hemolytic capacity of the serum. This report, for the first time, clarifies the relative bactericidal contributions of NETs and complement. We propose that - while NETs can ensnare bacteria such as P. aeruginosa - complement is necessary for efficient bacterial killing.