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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.

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.

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti–Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK−

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti–Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1in megakaryocytes (MK) (St3gal1MK−/−). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK−/−mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H–positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK−/−BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell–like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

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.

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.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

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.

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.

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 Dnm2fl/flPf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2fl/flPf4-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.

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 Dnm2fl/flPf4-Cremice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2fl/flPf4-Cremice 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.

Despite its relatively estimated high occurrence, the characterization of pediatric upper extremity deep vein thrombosis (UE-DVT) and of UE postthrombotic syndrome (PTS) is still lacking. We investigated the occurrence, characteristics, and predictors of UE-PTS in a cohort of children with objectively confirmed UE-DVT. Patients were analyzed in 3 groups according to DVT pathogenesis and neonatal status: primary (G1), secondary neonates (G2neonates), and non-neonates (G2non-neonates). A total of 158 children (23 G1, 25 G2neonates, and 110 G2non-neonates) were included. The most common triggering factors were effort-related (87%) in G1 and central lines in G2neonates (100%) and in G2non-neonates (92%). PTS scores ≥1, as per the Modified Villalta Scale, were identified in 87% of primary patients, 16% of G2neonates, and 49% of G2non-neonates. Survival analysis showed that the time to PTS score ≥1 significantly differed among group (log-rank test P < .0001). A multivariable logistic regression showed that DVT pathogenesis and imaging-determined degree of thrombus resolution at the end of therapy were independent predictors of a PTS score ≥2. In conclusion, pediatric UE-PTS frequency and severity depend on UE-DVT pathogenesis (primary/secondary) and, within the secondary group, on patient’s age. Line-related UE-PTS has a more benign course, particularly in neonates.

Despite its relatively estimated high occurrence, the characterization of pediatric upper extremity deep vein thrombosis (UE-DVT) and of UE postthrombotic syndrome (PTS) is still lacking. We investigated the occurrence, characteristics, and predictors of UE-PTS in a cohort of children with objectively confirmed UE-DVT. Patients were analyzed in 3 groups according to DVT pathogenesis and neonatal status: primary (G1), secondary neonates (G2neonates), and non-neonates (G2non-neonates). A total of 158 children (23 G1, 25 G2neonates, and 110 G2non-neonates) were included. The most common triggering factors were effort-related (87%) in G1 and central lines in G2neonates(100%) and in G2non-neonates(92%). PTS scores ≥1, as per the Modified Villalta Scale, were identified in 87% of primary patients, 16% of G2neonates, and 49% of G2non-neonates. Survival analysis showed that the time to PTS score ≥1 significantly differed among group (log-rank test P< .0001). A multivariable logistic regression showed that DVT pathogenesis and imaging-determined degree of thrombus resolution at the end of therapy were independent predictors of a PTS score ≥2. In conclusion, pediatric UE-PTS frequency and severity depend on UE-DVT pathogenesis (primary/secondary) and, within the secondary group, on patient's age. Line-related UE-PTS has a more benign course, particularly in neonates.

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.

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.

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 Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-null mice that displayed thrombocytopenia similar 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-null, but not WAS−, megakaryocytes. Electron micrographs of CIP4-null 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.

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 Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-nullmice that displayed thrombocytopenia similar 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-null, but not WAS−, megakaryocytes. Electron micrographs of CIP4-nullmegakaryocytes 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.

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 C14orf133encoding 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.

Bacteria can enter the bloodstream in response to infectious insults. Bacteremia elicits several immune and clinical complications, including thrombocytopenia. A primary cause of thrombocytopenia is shortened survival of platelets. We demonstrate that pathogenic bacteria induce apoptotic events in platelets that include calpain-mediated degradation of Bcl-xL, 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-xLprotein in platelets. Bcl-xLprotein degradation is enhanced when platelets are exposed to pathogenic Escherichia colithat 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 nonpathogenic E coligain degrading activity when they are forced to express α-hemolysin. Like α-hemolysin, purified α-toxin readily degrades Bcl-xLprotein in platelets, as do clinical Staphylococcus aureusisolates that produce α-toxin. Inhibition of calpain activity, but not the proteasome, rescues Bcl-xLprotein degradation in platelets coincubated with pathogenic E coliincluding 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 bacterial pathogens might cause thrombocytopenia in patients with bloodstream infections.

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.

Bacteria can enter the bloodstream in response to infectious insults. Bacteremia elicits several immune and clinical complications, including thrombocytopenia. A primary cause of thrombocytopenia is shortened survival of platelets. We demonstrate that pathogenic bacteria induce apoptotic events in platelets that include calpain-mediated degradation of Bcl-xL, 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-xL protein in platelets. Bcl-xL protein degradation is enhanced when platelets are exposed to pathogenic Escherichia 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 nonpathogenic E coli gain degrading activity when they are forced to express α-hemolysin. Like α-hemolysin, purified α-toxin readily degrades Bcl-xL protein in platelets, as do clinical Staphylococcus aureus isolates that produce α-toxin. Inhibition of calpain activity, but not the proteasome, rescues Bcl-xL protein degradation in platelets coincubated 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 bacterial pathogens might cause thrombocytopenia in patients with bloodstream infections.

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.

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.

Platelets are classified as terminally differentiated cells that are incapable of cellular division. However, we observe that anucleate human platelets, either maintained in suspension culture or captured in microdrops, give rise to new cell bodies packed with respiring mitochondria and α-granules. Platelet progeny formation also occurs in whole blood cultures. Newly formed platelets are structurally indistinguishable from normal platelets, are able to adhere and spread on extracellular matrix, and display normal signal-dependent expression of surface P-selectin and annexin V. Platelet progeny formation is accompanied by increases in biomass, cellular protein levels, and protein synthesis in expanding populations. Platelet numbers also increase during ex vivo storage. These observations indicate that platelets have a previously unrecognized capacity for producing functional progeny, which involves a form of cell division that does not require a nucleus. Because this new function of platelets occurs outside of the bone marrow milieu, it raises the possibility that thrombopoiesis continues in the bloodstream.

Platelets are classified as terminally differentiated cells that are incapable of cellular division. However, we observe that anucleate human platelets, either maintained in suspension culture or captured in microdrops, give rise to new cell bodies packed with respiring mitochondria and α-granules. Platelet progeny formation also occurs in whole blood cultures. Newly formed platelets are structurally indistinguishable from normal platelets, are able to adhere and spread on extracellular matrix, and display normal signal-dependent expression of surface P-selectin and annexin V. Platelet progeny formation is accompanied by increases in biomass, cellular protein levels, and protein synthesis in expanding populations. Platelet numbers also increase during ex vivo storage. These observations indicate that platelets have a previously unrecognized capacity for producing functional progeny, which involves a form of cell division that does not require a nucleus. Because this new function of platelets occurs outside of the bone marrow milieu, it raises the possibility that thrombopoiesis continues in the bloodstream.

Atypical hemolytic uremic syndrome (aHUS) is associated with complement system dysregulation, and more than 25% of pediatric aHUS cases are linked to mutations in complement factor H (CFH) or CFH autoantibodies. The observation of thrombocytopenia and platelet-rich thrombi in the glomerular microvasculature indicates that platelets are intimately involved in aHUS pathogenesis. It has been reported that a releasable pool of platelet CFH originates from α-granules. We observed that platelet CFH can arise from endogenous synthesis in megakaryocytes and that platelets constitutively lacking α-granules contain CFH. Electron and high-resolution laser fluorescence confocal microscopy revealed that CFH was present throughout the cytoplasm and on the surface of normal resting platelets with no evident concentration in α-granules, lysosomes, or dense granules. Therapeutic plasma transfusion in a CFH-null aHUS patient revealed that circulating platelets take up CFH with similar persistence of CFH in platelets and plasma in vivo. Washed normal platelets were also observed to take up labeled CFH in vitro. Exposure of washed normal platelets to plasma of an aHUS patient with CFH autoantibodies produced partial platelet aggregation or agglutination, which was prevented by preincubation of platelets with purified CFH. This CFH-dependent response did not involve P-selectin mobilization, indicating a complement-induced platelet response distinct from α-granule secretion.

Atypical hemolytic uremic syndrome (aHUS) is associated with complement system dysregulation, and more than 25% of pediatric aHUS cases are linked to mutations in complement factor H (CFH) or CFH autoantibodies. The observation of thrombocytopenia and platelet-rich thrombi in the glomerular microvasculature indicates that platelets are intimately involved in aHUS pathogenesis. It has been reported that a releasable pool of platelet CFH originates from α-granules. We observed that platelet CFH can arise from endogenous synthesis in megakaryocytes and that platelets constitutively lacking α-granules contain CFH. Electron and high-resolution laser fluorescence confocal microscopy revealed that CFH was present throughout the cytoplasm and on the surface of normal resting platelets with no evident concentration in α-granules, lysosomes, or dense granules. Therapeutic plasma transfusion in a CFH-null aHUS patient revealed that circulating platelets take up CFH with similar persistence of CFH in platelets and plasma in vivo. Washed normal platelets were also observed to take up labeled CFH in vitro. Exposure of washed normal platelets to plasma of an aHUS patient with CFH autoantibodies produced partial platelet aggregation or agglutination, which was prevented by preincubation of platelets with purified CFH. This CFH-dependent response did not involve P-selectin mobilization, indicating a complement-induced platelet response distinct from α-granule secretion.

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg−/−) mice, and engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process. Furthermore, Fg−/− platelets significantly increased P-selectin expression following transfusion into β3 integrin–deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg−/−) mice, and engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process. Furthermore, Fg−/−platelets significantly increased P-selectin expression following transfusion into β3 integrin–deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

Turro: Tachyon Ventures: Other: Partner & Scientific Director.

No relevant conflicts of interest to declare.

GP1BAencodes the glycoprotein (GP) 1bα subunit of the GP1b/IX/V complex, the platelet receptor for von Willebrand factor (VWF) and a mediator of outside-in signalling that contributes to platelet activation. Biallelic variants in GPIBAthat prevent surface expression of GPIB/IX/V cause the recessive disorder Bernard Soulier Syndrome (BSS; OMIM #23100) in which there is often severe bleeding caused by thrombocytopenia and platelet dysfunction. Thrombocytopenia has also been associated with monoallelic missense variants in the GP1bα R loop (residues 227-242) which enhance VWF binding (platelet-type von Willebrand disease (pVWD); OMIM #177820). Other monoallelic GPIBAmissense variants such as the Bolzano variant (p.Ala172Val; OMIM #153670) have been associated with dominant thrombocytopenia without altered VWF levels.

The Thomsen-Friedenreich antigen (TF-antigen) occurs during exposure of the underlying Core-1 disaccharide (Gal-beta(1,3)GalNAc) through the loss of its capping sialic acid (Sia). Exposure of the cryptic TF-antigen occurs during inflammation, during acute infections with influenza viruses or bacteria, in malignancies, and is associated with thrombocytopenia. Exposure of the TF-antigen on circulating blood cells, including platelets and red blood cells (RBC), can lead to severe thrombocytopenia or hemolysis in hemolytic uremic syndrome and other immune diseases.

Bleeding problems are associated with defects in platelet α-granules, yet little is known about how these granules are formed and released. Mutations affecting VPS33B, a novel Sec1/Munc18 protein, have recently been linked to arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. We have characterized platelets from patients with ARC syndrome and observed reduced aggregation with arachidonate and adenosine diphosphate (ADP). Structural abnormalities seen in ARC platelets included increased platelet size, a pale appearance in blood films, elevated numbers of δ-granules, and completely absent α-granules. Soluble and membrane-bound α-granule proteins were significantly decreased or undetectable in ARC platelets, suggesting that both the releasable protein pools and membrane components of α-granules were absent. The role of VPS33B in platelet granule biogenesis was evaluated by immunofluorescence microscopy in normal human megakaryocytes. VPS33B colocalized appreciably with markers of α-granules, moderately with late endosomes/lysosomes, minimally with δ-granules/lysosomes, and not with cis-Golgi complexes. VPS33B protein expression determined by immunoblotting confirmed the presence of VPS33B in control fibroblasts but not in ARC fibroblasts, and in normal megakaryocytes but not in platelets. We conclude that like other Sec1/Munc18 proteins, VPS33B is involved in intracellular vesicle trafficking, being essential for the development of platelet α-granules but not for granule secretion.

Bleeding problems are associated with defects in platelet α-granules, yet little is known about how these granules are formed and released. Mutations affecting VPS33B, a novel Sec1/Munc18 protein, have recently been linked to arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. We have characterized platelets from patients with ARC syndrome and observed reduced aggregation with arachidonate and adenosine diphosphate (ADP). Structural abnormalities seen in ARC platelets included increased platelet size, a pale appearance in blood films, elevated numbers of δ-granules, and completely absent α-granules. Soluble and membrane-bound α-granule proteins were significantly decreased or undetectable in ARC platelets, suggesting that both the releasable protein pools and membrane components of α-granules were absent. The role of VPS33B in platelet granule biogenesis was evaluated by immunofluorescence microscopy in normal human megakaryocytes. VPS33B colocalized appreciably with markers of α-granules, moderately with late endosomes/lysosomes, minimally with δ-granules/lysosomes, and not with cis-Golgi complexes. VPS33B protein expression determined by immunoblotting confirmed the presence of VPS33B in control fibroblasts but not in ARC fibroblasts, and in normal megakaryocytes but not in platelets. We conclude that like other Sec1/Munc18 proteins, VPS33B is involved in intracellular vesicle trafficking, being essential for the development of platelet α-granules but not for granule secretion.

The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder associated with delayed bleeding and α-granule protein degradation. The degradation of α-granule, but not plasma, fibrinogen in patients with the QPD led to the investigation of their platelets for a protease defect. Unlike normal platelets, QPD platelets contained large amounts of fibrinolytic serine proteases that had properties of plasminogen activators. Western blot analysis, zymography, and immunodepletion experiments indicated this was because QPD platelets contained large amounts of urokinase-type plasminogen activator (u-PA) within a secretory compartment. u-PA antigen was not increased in all QPD plasmas, whereas it was increased more than 100-fold in QPD platelets (P< .00009), which contained increased u-PA messenger RNA. Although QPD platelets contained 2-fold more plasminogen activator inhibitor 1 (PAI-1) (P< .0008) and 100-fold greater u-PA–PAI-1 complexes (P< .0002) than normal platelets, they contained excess u-PA activity, predominantly in the form of two chain (tcu-PA), which required additional PAI-1 for full inhibition. There was associated proteolysis of plasminogen in QPD platelets, to forms that comigrated with plasmin. When similar amounts of tcu-PA were incubated with normal platelet secretory proteins, many α-granule proteins were proteolyzed to forms that resembled degraded QPD platelet proteins. These data implicate u-PA in the pathogenesis of α-granule protein degradation in the QPD. Although patients with the QPD have normal to increased u-PA levels in their plasma, without evidence of systemic fibrinogenolysis, their increased platelet u-PA could contribute to bleeding by accelerating fibrinolysis within the hemostatic plug. QPD is the only inherited bleeding disorder in humans known to be associated with increased u-PA.

The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder associated with delayed bleeding and α-granule protein degradation. The degradation of α-granule, but not plasma, fibrinogen in patients with the QPD led to the investigation of their platelets for a protease defect. Unlike normal platelets, QPD platelets contained large amounts of fibrinolytic serine proteases that had properties of plasminogen activators. Western blot analysis, zymography, and immunodepletion experiments indicated this was because QPD platelets contained large amounts of urokinase-type plasminogen activator (u-PA) within a secretory compartment. u-PA antigen was not increased in all QPD plasmas, whereas it was increased more than 100-fold in QPD platelets (P < .00009), which contained increased u-PA messenger RNA. Although QPD platelets contained 2-fold more plasminogen activator inhibitor 1 (PAI-1) (P < .0008) and 100-fold greater u-PA–PAI-1 complexes (P < .0002) than normal platelets, they contained excess u-PA activity, predominantly in the form of two chain (tcu-PA), which required additional PAI-1 for full inhibition. There was associated proteolysis of plasminogen in QPD platelets, to forms that comigrated with plasmin. When similar amounts of tcu-PA were incubated with normal platelet secretory proteins, many α-granule proteins were proteolyzed to forms that resembled degraded QPD platelet proteins. These data implicate u-PA in the pathogenesis of α-granule protein degradation in the QPD. Although patients with the QPD have normal to increased u-PA levels in their plasma, without evidence of systemic fibrinogenolysis, their increased platelet u-PA could contribute to bleeding by accelerating fibrinolysis within the hemostatic plug. QPD is the only inherited bleeding disorder in humans known to be associated with increased u-PA.

Callaghan: Bayer: Consultancy, Speakers Bureau; Alnylum: Equity Ownership; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.

Emerging data indicate that germline mutations in transcription factors involved in hematopoiesis can lead to a cascade of downstream molecular alterations that modify the function of megakaryocytes (MK) and platelets. Our group and others have found that mutations in ETV6lead to mild thrombocytopenia with a bleeding diathesis, red cell macrocytosis, and predisposition to lymphoblastic leukemia. The mechanisms responsible for thrombocytopenia and propensity for bleeding in patients with ETV6mutations are unknown. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.Pro214Leu mutation and non-affected family members, indicating that ETV6is critically involved in defining the molecular phenotype and function of platelets. We hypothesize that normal regulation and function of ETV6is essential for the transcriptional machinery that controls megakaryocyte differentiation and formation of platelets that function normally under homeostatic conditions.