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Abstract: Children and adults with sickle cell disease (SCD) have increases in morbidity and mortality with COVID-19 infections. The American Society of Hematology Research Collaborative Sickle Cell Disease Research Network performed a prospective COVID-19 vaccine study to assess antibody responses and analyze whether messenger RNA (mRNA) vaccination precipitated any adverse effects unique to individuals with SCD. Forty-one participants received 2 doses of the Pfizer-BioNTech vaccine and provided baseline blood samples before vaccination and 2 months after the initial vaccination for analysis of immunoglobulin G (IgG) reactivity against the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike protein. Six-month IgG reactivity against the viral RBD was also available in 37 patients. Postvaccination reactogenicity was common and similar to the general population. There were no fevers that required inpatient admission. Vaso-occlusive pain within 2 to 3 days of first or second vaccination was reported by 5 participants (12%) including 4 (10%) who sought medical care. Twenty-seven participants (66%) were seropositive at baseline, and all 14 initially seronegative participants (34%) converted to seropositive after vaccination. Overall, mRNA vaccination had a good risk-benefit profile in individuals with SCD. This mRNA vaccine study also marks the first evaluation of vaccine safety and antibody response in very young children with SCD. This trial was registered at www.ClinicalTrials.gov as #NCT05139992., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Platelets use signal transduction pathways facilitated by class I phosphatidylinositol transfer proteins (PITPs). The 2 mammalian class I PITPs, PITPα and PITPβ, are single PITP domain soluble proteins that are encoded by different genes and share 77% sequence identity, although their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominantly expressed murine PITP isoform, had no effect on hemostasis but impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we found that mice lacking the less expressed PITPβ in their platelets exhibited a similar phenotype. However, in contrast to PITPα-null platelet lysates, which have impaired lipid transfer activity, PITPβ-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs led to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our study also demonstrated that PITP isoforms are required to maintain endogenous phosphoinositide PtdInsP2 levels and agonist-stimulated second messenger formation. The data shown here demonstrate that the 2 isoforms are functionally overlapping and that a single isoform is able to maintain the homeostasis of platelets. However, both class I PITP isoforms contribute to phosphoinositide signaling in platelets through distinct biochemical mechanisms or different subcellular domains., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Because of the unique biology of sickle cell disease (SCD) as well as the societal disadvantages and racial inequities suffered by these patients, individuals with SCD have not benefited from the same remarkable advances in care and therapeutics as those with other hematologic disorders. Life expectancy of individuals with SCD is shortened by ∼20 years even with optimal clinical care, and infant mortality continues to be a major concern in low-income countries. As hematologists, we must do more. The American Society of Hematology (ASH) and the ASH Research Collaborative have instituted a multipronged initiative to improve the lives of individuals living with this disease. Here, we describe 2 components of this ASH initiative, the Consortium on Newborn Screening in Africa (CONSA) to improve the early diagnosis of infants in low-resource countries and the SCD Clinical Trial Network to accelerate the development of more effective therapeutics and care for those with this disorder. The combination of SCD-focused initiatives, ASH Research Collaborative, CONSA, and Sickle Cell Clinical Trials Network has enormous potential to dramatically alter the course of SCD worldwide. We believe that the timing is ripe to embark on these critical and worthwhile initiatives and improve the lives of individuals with this disease., (© 2023 by The American Society of Hematology.)

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

Billions of individuals worldwide have benefited from the unprecedented large-scale rollout of COVID-19 vaccines. Given the sheer number of people that have received these vaccines, it is not surprising that rare side effects are reported that were not previously detected in the phase III vaccine trials. This review addresses one rare complication called SARS-CoV-2 vaccination-induced thrombotic thrombocytopenia (VITT). It occurs in approximately 1/50,000 to 1/100,000 recipients of the adenovirus vector-based COVID-19 vaccines made by AstraZeneca-Oxford or Johnson & Johnson. Information on VITT syndrome was disseminated quickly via social media and publications after it was first discovered. Initial observations associating VITT with specific patient populations, thrombus locations, and outcomes associated with heparin therapy have since been refined with additional clinical experience. In this review, we discuss what is currently known about the incidence, pathophysiology, diagnosis, and treatment of VITT.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect., Competing Interests: SH has received consultancy fees from Sanofi Pasteur, Lumen, Novavax, and Merck for work unrelated to this report. AH is a consultant for Immunai. EW is consulting or is an advisor for Merck, Elstar, Janssen, Related Sciences, Synthekine and Surface Oncology. EW is a founder of Surface Oncology and Arsenal Biosciences. EW is an inventor on a patent (US Patent number 10,370,446) submitted by Emory University that covers the use of PD-1 blockade to treat infections and cancer. NM reports funding to her institution from Quantum Leap Healthcare Collaborative, Athersys, Inc., Biomarck, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Apostolidis, Sarkar, Giannini, Goel, Mathew, Suzuki, Baxter, Greenplate, Alanio, Abdel-Hakeem, Oldridge, Giles, Wu, Chen, Huang, Belman, Pattekar, Manne, Kuthuru, Dougherty, Weiderhold, Weisman, Ittner, Gouma, Dunbar, Frank, Huang, Vella, The UPenn COVID Processing Unit, Reilly, Hensley, Rauova, Zhao, Meyer, Poncz, Abrams and Wherry.)

The ASH Research Collaborative is a nonprofit organization established through the American Society of Hematology's commitment to patients with hematologic conditions and the science that informs clinical care and future therapies. The ASH Research Collaborative houses 2 major initiatives: (1) the Data Hub and (2) the Clinical Trials Network (CTN). The Data Hub is a program for hematologic diseases in which networks of clinical care delivery sites are developed in specific disease areas, with individual patient data contributed through electronic health record (EHR) integration, direct data entry through electronic data capture, and external data sources. Disease-specific data models are constructed so that data can be assembled into analytic datasets and used to enhance clinical care through dashboards and other mechanisms. Initial models have been built in multiple myeloma (MM) and sickle cell disease (SCD) using the Observational Medical Outcomes Partnership (OMOP) Common Data Model (CDM) and Fast Healthcare Interoperability Resources (FHIR) standards. The Data Hub also provides a framework for development of disease-specific learning communities (LC) and testing of health care delivery strategies. The ASH Research Collaborative SCD CTN is a clinical trials accelerator that creates efficiencies in the execution of multicenter clinical trials and has been initially developed for SCD. Both components are operational, with the Data Hub actively aggregating source data and the SCD CTN reviewing study candidates. This manuscript describes processes involved in developing core features of the ASH Research Collaborative to inform the stakeholder community in preparation for expansion to additional disease areas., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2-a STAT5-dependent lipid binding protein downstream of erythropoietin-as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis.

Macrophages are professional phagocytes that are essential for host defense and tissue homeostasis. Proper membrane trafficking and degradative functions of the endolysosomal system are known to be critical for the function of these cells. We have found that PIKfyve, the kinase that synthesizes the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate, is an essential regulator of lysosomal biogenesis and degradative functions in macrophages. Genetically engineered mice lacking PIKfyve in their myeloid cells ( PIKfyve fl/fl LysM-Cre ) develop diffuse tissue infiltration of foamy macrophages, hepatosplenomegaly, and systemic inflammation. PIKfyve loss in macrophages causes enlarged endolysosomal compartments and impairs the lysosomal degradative function. Moreover, PIKfyve deficiency increases the cellular levels of lysosomal proteins. Although PIKfyve deficiency reduced the activation of mTORC1 pathway and was associated with increased cleavage of TFEB proteins, this does not translate into transcriptional activation of lysosomal genes, suggesting that PIKfyve modulates the abundance of lysosomal proteins by affecting the degradation of these proteins. Our study shows that PIKfyve modulation of lysosomal degradative activity and protein expression is essential to maintain lysosomal homeostasis in macrophages., (Copyright © 2019 American Society for Microbiology.)

We hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα -/- and pitpα -/- /β -/- ) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα -/- /β -/- BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα -/- and pitpα -/- /β -/- BM MKs contained higher levels of transforming growth factor β1 (TGF-β1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα -/- and pitpα -/- /β -/- mice had higher concentrations of TGF-β1. CM from pitpα -/- and pitpα -/- /β -/- MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti-TGF-β antibody, and treatment of pitpα -/- /β -/- mice in vivo with anti-TGF-β antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-β and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα -/- /β -/- MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-β, are stored in MK α-granules and were also elevated in CM of cultured pitpα -/- /β -/- MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-β1., (© 2018 by The American Society of Hematology.)

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)-dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins., (© 2018 by The American Society of Hematology.)

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) stimulate PtdIns-4-P synthesis and signaling in eukaryotic cells, but to what biological outcomes such signaling circuits are coupled remains unclear. Herein, we show that two highly related StART-like PITPs, PITPNA and PITPNB, act in a redundant fashion to support development of the embryonic mammalian neocortex. PITPNA/PITPNB do so by driving PtdIns-4-P-dependent recruitment of GOLPH3, and likely ceramide transfer protein (CERT), to Golgi membranes with GOLPH3 recruitment serving to promote MYO18A- and F-actin-directed loading of the Golgi network to apical processes of neural stem cells (NSCs). We propose the primary role for PITP/PtdIns-4-P/GOLPH3/CERT signaling in NSC Golgi is not in regulating bulk membrane trafficking but in optimizing apically directed membrane trafficking and/or apical membrane signaling during neurogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.

Platelets are increasingly recognized for their contributions to tumor metastasis. Here, we show that the phosphoinositide signaling modulated by phosphatidylinositol transfer protein type α (PITPα), a protein which shuttles phosphatidylinositol between organelles, is essential for platelet-mediated tumor metastasis. PITPα-deficient platelets have reduced intracellular pools of phosphoinositides and an 80% reduction in IP 3 generation upon platelet activation. Unexpectedly, mice lacking platelet PITPα form thrombi normally at sites of intravascular injuries. However, following intravenous injection of tumor cells, mice lacking PITPα develop fewer lung metastases due to a reduction of fibrin formation surrounding the tumor cells, rendering the metastases susceptible to mucosal immunity. These findings demonstrate that platelet PITPα-mediated phosphoinositide signaling is inconsequential for in vivo hemostasis, yet is critical for in vivo dissemination. Moreover, this demonstrates that signaling pathways within platelets may be segregated into pathways that are essential for thrombosis formation and pathways that are important for non-hemostatic functions.

Objectives: The clinical diagnosis of qualitative platelet disorders (QPDs) based on light transmission aggregometry (LTA) requires significant blood volume, time, and expertise, all of which can be barriers to utilization in some populations and settings. Our objective was to develop a more rapid assay of platelet function by measuring platelet-mediated clot contraction in small volumes (35 µL) of whole blood using T2 magnetic resonance (T2MR)., Methods: We established normal ranges for platelet-mediated clot contraction using T2MR, used these ranges to study patients with known platelet dysfunction, and then evaluated agreement between T2MR and LTA with arachidonic acid, adenosine diphosphate, epinephrine, and thrombin receptor activator peptide., Results: Blood from 21 healthy donors was studied. T2MR showed 100% agreement with LTA with each of the four agonists and their cognate inhibitors tested. T2MR successfully detected abnormalities in each of seven patients with known QPDs, with the exception of one patient with a novel mutation leading to Hermansky-Pudlak syndrome. T2MR appeared to detect platelet function at similar or lower platelet counts than LTA., Conclusions: T2MR may provide a clinically useful approach to diagnose QPDs using small volumes of whole blood, while also providing new insight into platelet biology not evident using plasma-based platelet aggregation tests., (© American Society for Clinical Pathology, 2016. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.)

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1(+) and Rab7(+) vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1(+)perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis.

In this issue of Blood, Valet et al1 report a novel regulatory role of class II phosphoinositide 3-kinase (PI3K)-C2α in the morphology and remodeling of platelet membranes and its implications in platelet maturation and arterial thrombosis.

Acquired thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and microangiopathic hemolytic anemia (MAHA) without an obvious cause, and may include fever, mild renal failure, and neurologic deficits. It is characterized by a deficiency of the von Willebrand factor (VWF) cleaving enzyme, ADAMTS13 (a disintegrin and metalloproteinase, with a thrombospondin type 1 motif, member 13), resulting in formation of microthrombi in the high sheer environment of the microvasculature. This causes microvascular occlusion, MAHA, and organ ischemia. Diagnosis is based on the presence of clinical symptoms, laboratory aberrations consistent with MAHA, decreased ADAMTS13 activity, and possibly presence of anti-ADAMTS13 autoantibodies. Upfront treatment of acute TTP includes plasma exchange and corticosteroids. A significant number of patients are refractory to this treatment and will require further interventions. There are limited data and consensus on the management of the refractory TTP patient. Management involves simultaneously ruling out other causes of thrombocytopenia and MAHA, while also considering other treatments. In this article, we describe our management of the patient with refractory TTP, and discuss use of rituximab, increased plasma exchange, splenectomy, and immunosuppressive options, including cyclophosphamide, vincristine, and cyclosporine. We also review recent evidence for the potential roles of bortezomib and N-acetylcysteine, and explore new therapeutic approaches, including recombinant ADAMTS13 and anti-VWF therapy., (© 2015 by The American Society of Hematology.)

Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding diathesis, and other variable symptoms. The bleeding diathesis has been attributed to δ storage pool deficiency, reflecting the malformation of platelet dense granules. Here, we analyzed agonist-stimulated secretion from other storage granules in platelets from mouse HPS models that lack adaptor protein (AP)-3 or biogenesis of lysosome-related organelles complex (BLOC)-3 or BLOC-1. We show that α granule secretion elicited by low agonist doses is impaired in all 3 HPS models. High agonist doses or supplemental adenosine 5'-diphosphate (ADP) restored normal α granule secretion, suggesting that the impairment is secondary to absent dense granule content release. Intravital microscopy following laser-induced vascular injury showed that defective hemostatic thrombus formation in HPS mice largely reflected reduced total platelet accumulation and affirmed a reduced area of α granule secretion. Agonist-induced lysosome secretion ex vivo was also impaired in all 3 HPS models but was incompletely rescued by high agonist doses or excess ADP. Our results imply that (1) AP-3, BLOC-1, and BLOC-3 facilitate protein sorting to lysosomes to support ultimate secretion; (2) impaired secretion of α granules in HPS, and to some degree of lysosomes, is secondary to impaired dense granule secretion; and (3) diminished α granule and lysosome secretion might contribute to pathology in HPS., (© 2015 by The American Society of Hematology.)

PIKfyve is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and for the regulation of endolysosomal membrane dynamics in mammals. PtdIns(3,5)P2 deficiency causes neurodegeneration in mice and humans, but the role of PtdIns(3,5)P2 in non-neural tissues is poorly understood. Here we show that platelet-specific ablation of PIKfyve in mice leads to accelerated arterial thrombosis, and, unexpectedly, also to inappropriate inflammatory responses characterized by macrophage accumulation in multiple tissues. These multiorgan defects are attenuated by platelet depletion in vivo, confirming that they reflect a platelet-specific process. PIKfyve ablation in platelets induces defective maturation and excessive storage of lysosomal enzymes that are released upon platelet activation. Impairing lysosome secretion from PIKfyve-null platelets in vivo markedly attenuates the multiorgan defects, suggesting that platelet lysosome secretion contributes to pathogenesis. Our findings identify PIKfyve as an essential regulator for platelet lysosome homeostasis, and demonstrate the contributions of platelet lysosomes to inflammation, arterial thrombosis and macrophage biology.

Protein arginylation by arginyl-transfer RNA protein transferase (ATE1) is emerging as a regulator protein function that is reminiscent of phosphorylation. For example, arginylation of β-actin has been found to regulate lamellipodial formation at the leading edge in fibroblasts. This finding suggests that similar functions of β-actin in other cell types may also require arginylation. Here, we have tested the hypothesis that ATE1 regulates the cytoskeletal dynamics essential for in vivo platelet adhesion and thrombus formation. To test this hypothesis, we generated conditional knockout mice specifically lacking ATE1 in their platelets and in their megakaryocytes and analyzed the role of arginylation during platelet activation. Surprisingly, rather than finding an impairment of the actin cytoskeleton structure and its rearrangement during platelet activation, we observed that the platelet-specific ATE1 knockout led to enhanced clot retraction and in vivo thrombus formation. This effect might be regulated by myosin II contractility since it was accompanied by enhanced phosphorylation of the myosin regulatory light chain on Ser19, which is an event that activates myosin in vivo. Furthermore, ATE1 and myosin co-immunoprecipitate from platelet lysates. This finding suggests that these proteins directly interact within platelets. These results provide the first evidence that arginylation is involved in phosphorylation-dependent protein regulation, and that arginylation affects myosin function in platelets during clot retraction.

Phosphatidylinositol and its phosphorylated derivatives, phosphoinositides, are minor constituents of phospholipids at the cellular membrane level. Nevertheless, phosphatidylinositol and phosphoinositides represent essential components of intracellular signaling that regulate diverse cellular processes, including platelet plug formation. Accumulating evidence indicates that the metabolism of phosphoinositides is temporally and spatially modulated by the opposing effects of specific phosphoinositide-metabolizing enzymes, including lipid kinases, lipid phosphatases, and phospholipases. Each of these enzymes generates a selective phosphoinositide or second messenger within precise cellular compartments. Intriguingly, phosphoinositide-metabolizing enzymes exist in different isoforms, which all produce the same phosphoinositide products. Recent studies using isoform-specific mouse models and chemical inhibitors have elucidated that the different isoforms of phosphoinositide-metabolizing enzymes have nonredundant functions and provide an additional layer of complexity to the temporo-spatial organization of intracellular signaling events. In this review, we will discuss recent advances in our understanding of phosphoinositide organization during platelet activation.

RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion.

Disseminated intravascular coagulation (DIC) profoundly increases the morbidity and mortality of patients who have sepsis. Both laboratory and clinical research advanced the understanding of the biology and pathophysiology of DIC. This, in turn, gave rise to improved therapies and patient outcomes. Beginning with a stimulus causing disruption of vascular integrity, cytokines and chemokines cause activation of systemic coagulation and inflammation. Seemingly paradoxically, the interplay between coagulation and inflammation also inhibits endogenous anticoagulants, fibrinolytics, and antiinflammatory pathways. The earliest documented and best-studied microbial cause of DIC is the lipopolysaccharide endotoxin of Gram-negative bacteria. Extensive microvascular thrombi emerge in the systemic vasculature due to dysregulation of coagulation. The result of this unrestrained, widespread small vessel thromboses multiorgan system failure. Consumption of platelets and coagulation factors during this process can lead to an elevated risk of hemorrhage. The management of these patients with simultaneous hemorrhage and thrombosis is complex and challenging. Definitive treatment of DIC, and attenuation of end-organ damage, requires control of the inciting cause. Currently, activated protein C is the only approved therapy in the United States for sepsis complicated by DIC. Further research is needed in this area to improve clinical outcomes for patients with sepsis., (© 2012 Wiley Periodicals, Inc.)

Understanding platelet biology has been aided by studies of mice with mutations in key megakaryocytic transcription factors. We have shown that point mutations in the GATA1 cofactor FOG1 that disrupt binding to the nucleosome remodeling and deacetylase (NuRD) complex have erythroid and megakaryocyte lineages defects. Mice that are homozygous for a FOG1 point mutation (ki/ki), which ablates FOG1-NuRD interactions, have platelets that display a gray platelet syndrome (GPS)-like macrothrombocytopenia. These platelets have few α-granules and an increased number of lysosomal-like vacuoles on electron microscopy, reminiscent of the platelet in patients with GATA1-related X-linked GPS. Here we further characterized the platelet defect in ki/ki mice. We found markedly deficient levels of P-selectin protein limited to megakaryocytes and platelets. Other α-granule proteins were expressed at normal levels and were appropriately localized to α-granule-like structures. Treatment of ki/ki platelets with thrombin failed to stimulate Akt phosphorylation, resulting in poor granule secretion and platelet aggregation. These studies show that disruption of the GATA1/FOG1/NuRD transcriptional system results in a complex, pleiotropic platelet defect beyond GPS-like macrothrombocytopenia and suggest that this transcriptional complex regulates not only megakaryopoiesis but also α-granule generation and signaling pathways required for granule secretion.

Unlabelled: Summary. , Background: Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin's function in platelets is unknown., Objective: To determine whether vinculin is required for the functions of platelets and their major integrin, α(IIb) β(3) ., Methods: The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4-Cre mice. Platelet and integrin functions were studied in vivo and ex vivo., Results: Vinculin was undetectable in platelets from Vcl fl/fl Cre(+) mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on α(IIb) β(3) with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to α(IIb) β(3) , aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild-type littermates in response to carotid artery injury with FeCl(3) ., Conclusion: Despite promoting membrane cytoskeleton integrity when mechanical force is applied to α(IIb) β(3) , vinculin is not required for the traditional functions of α(IIb) β(3) or the platelet actin cytoskeleton., (© 2010 International Society on Thrombosis and Haemostasis.)

The predominant pathway for phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) synthesis is thought to be phosphorylation of phosphatidylinositol 4-phosphate at the 5 position of the inositol ring by type I phosphatidylinositol phosphate kinases (PIPK): PIPKIalpha, PIPKIbeta, and PIPKIgamma. PIPKIgamma has been shown to play a role in PI(4,5)P(2) synthesis in brain, and the absence of PIPKIgamma is incompatible with postnatal life. Conversely, mice lacking PIPKIalpha or PIPKIbeta (isoforms are referred to according to the nomenclature of human PIPKIs) live to adulthood, although functional effects in specific cell types are observed. To determine the contribution of PIPKIalpha and PIPKIbeta to PI(4,5)P(2) synthesis in brain, we investigated the impact of disrupting multiple PIPKI genes. Our results show that a single allele of PIPKIgamma, in the absence of both PIPKIalpha and PIPKIbeta, can support life to adulthood. In addition, PIPKIalpha alone, but not PIPKIbeta alone, can support prenatal development, indicating an essential and partially overlapping function of PIPKIalpha and PIPKIgamma during embryogenesis. This is consistent with early embryonic expression of PIPKIalpha and PIPKIgamma but not of PIPKIbeta. PIPKIbeta expression in brain correlates with neuronal differentiation. The absence of PIPKIbeta does not impact embryonic development in the PIPKIgamma knock-out (KO) background but worsens the early postnatal phenotype of the PIPKIgamma KO (death occurs within minutes rather than hours). Analysis of PIP(2) in brain reveals that only the absence of PIPKIgamma significantly impacts its levels. Collectively, our results provide new evidence for the dominant importance of PIPKIgamma in mammals and imply that PIPKIalpha and PIPKIbeta function in the generation of specific PI(4,5)P(2) pools that, at least in brain, do not have a major impact on overall PI(4,5)P(2) levels.

Purpose of Review: Heparin-induced thrombocytopenia (HIT) is a significant cause of morbidity and mortality in hospitalized patients, due to life and limb-threatening thrombosis. Prompt recognition, laboratory testing, and alternate anticoagulation are essential. At present, HIT remains an underdiagnosed and undertreated condition. This review will discuss the relative merits of the approved treatment options, as well as address additional anticoagulants that show promise for the future., Recent Findings: Argatroban and lepirudin are well studied and approved drugs for treatment of HIT. Both of these drugs are equal in efficacy, and differences in pharmacokinetic profiles allow the choice of drug to be tailored to the clinical scenario. Bivalirudin and fondaparinux have been used to treat HIT in small case series. New oral anticoagulants, such as factor IIa and factor Xa inhibitors, may provide a novel treatment approach in HIT., Summary: First-line therapies for HIT are argatroban or lepirudin. Patient-specific factors determine which drug should be used, and taking advantage of their differences allows effective anticoagulation with minimal risk of bleeding. Bivalirudin and fondaparinux require further study before they can be recommended. Once proven well tolerated and effective for treating thrombosis, these new oral anticoagulants should next be studied for treating HIT.

Thrombocytopenia is a predictor of adverse outcomes in patients with acute coronary syndromes and in critically ill patients. The Complications After Thrombocytopenia Caused by Heparin (CATCH) registry was designed to explore the incidence, management, and clinical consequences of in-hospital thrombocytopenia occurring during heparin-based anticoagulation in diverse clinical settings. We conducted a prospective observational study of 37 United States hospitals participating in the CATCH registry to assess the relation of in-hospital thrombocytopenia to long-term outcomes. A total of 2,104 patients at increased risk of developing in-hospital thrombocytopenia or thrombosis were identified, and the 6-month mortality and rehospitalization rates were determined. Thrombocytopenia was not a significant predictor of 6-month mortality. In an adjusted model for in-hospital death in this cohort, thrombocytopenia had an odds ratio of 3.59 (95% confidence interval 2.24 to 5.77). The postdischarge mortality rate at 6 months was 9.7%. No significant difference was observed in the long-term mortality between patients who developed thrombocytopenia and those who did not. Thrombocytopenia was a weak, but statistically significant, predictor of a composite of mortality and rehospitalization at 6 months (hazards ratio 0.80, 95% confidence interval 0.65 to 0.98, p = 0.03). In conclusion, the 6-month mortality rate among heparin-treated patients with thrombocytopenia is high, although the risk independently related to thrombocytopenia appears to be restricted to the acute hospital phase.

Macromolecules can be transported into the cells by endocytosis, either by phagocytosis or by pinocytosis. Typically, phagocytosis involves the uptake of solid large particles mediated by cell-surface receptors, whereas pinocytosis takes up fluid and solutes. The synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 plays fundamental roles in all forms of endocytosis. Curiously, almost all eukaryotic cells have multiple isoforms of the kinases that synthesize these critical phosphatidylinositols. In this issue of the Biochemical Journal, Namiko Tamura, Osamu Hazeki and co-workers report that the subunit p110alpha of the type I PI3K (phosphoinositide 3-kinase) is implicated in the phagocytosis and the pinocytosis of large molecules, whereas the receptor-mediated pinocytosis and micropinocytosis of small molecules do not seem to be controlled by this mechanism. The present commentary discusses recent literature that has begun to unravel why cells need so many phosphatidylinositol kinase isoforms, which were previously believed to be redundant.

Pleckstrin, the platelet and leukocyte C kinase substrate, is a prominent substrate of PKC in platelets, monocytes, macrophages, lymphocytes, and granulocytes. Pleckstrin accounts for 1% of the total protein in these cells, but it is best known for containing the 2 prototypic Pleckstrin homology, or PH, domains. Overexpressed pleckstrin can affect polyphosphoinositide second messenger-based signaling events; however, its true in vivo role has been unknown. Here, we describe mice containing a null mutation within the pleckstrin gene. Platelets lacking pleckstrin exhibit a marked defect in exocytosis of delta and alpha granules, alphaIIbbeta3 activation, actin assembly, and aggregation after exposure to the PKC stimulant, PMA. Pleckstrin-null platelets aggregate normally in response to thrombin, but they fail to aggregate in response to thrombin in the presence of PI3K inhibitors, suggesting that a PI3K-dependent signaling pathway compensates for the loss of pleckstrin. Although pleckstrin-null platelets merged their granules in response to stimulation of PKC, they failed to empty their contents into the open canalicular system. This might be attributable to impaired actin assembly present in cells lacking pleckstrin. These data show that pleckstrin regulates the fusion of granules to the cell membrane and is an essential component of PKC-mediated exocytosis.

Background: Thrombocytopenia and heparin-induced thrombocytopenia (HIT) are potentially devastating paradoxical side effects of heparin therapy. We explored the evaluation, management, and clinical consequences of thrombocytopenia occurring during heparin therapy in diverse clinical settings., Methods: CATCH was a prospective observational study that enrolled 3,536 patients in 48 US hospitals. Data were collected on 3 strata: patients receiving any form of heparin for > or =96 hours (n = 2,420); cardiac care unit (CCU) patients treated with heparin who developed thrombocytopenia (n = 1,090); patients who had an HIT assay performed (n = 449)., Results: Thrombocytopenia occurred in 36.4% of patients in the prolonged heparin stratum and was associated with an increased risk of death or thromboembolic complication (OR 1.5, 95% CI 1.2-1.9). Among a subset of patients whose clinical presentation suggested they were at high risk for HIT, suspicion for HIT was uncommon (prolonged heparin stratum 19.8%, CCU stratum 37.6%) and often did not arise until > or =1 day after patients developed thrombocytopenia. Often patients were not evaluated for HIT until after they had had a thromboembolic complication (prolonged heparin stratum 43.8%, CCU stratum 61%). Even after HIT was suspected, patients often continued to receive heparin. Direct thrombin inhibitor use was infrequent (prolonged heparin stratum 29.4%, CCU stratum 35.6%). Among the few patients who underwent evaluation, HIT was confirmed in 46.7% of the prolonged heparin stratum and 31.4% of the CCU stratum., Conclusions: Thrombocytopenia is common among patients receiving heparin, and it is associated with substantial risk for catastrophic complications. Despite the high risk for HIT in this population, recognition, evaluation, and appropriate treatment are infrequent and delayed.

The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gamma and alpha isoforms, which synthesize PIP(2), during phagocytosis. PIP5K-gamma-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgammaR clustering. Delivery of exogenous PIP(2) rescued these defects. PIP5K-gamma knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-gamma by RNA interference inhibits particle attachment. In contrast, PIP5K-alpha knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation. We propose that PIP5K-gamma acts upstream of Rac/Rho and that the differential regulation of PIP5K-gamma and -alpha allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.

Decades of basic science and clinical research have led to an increased understanding of the pathophysiology of immune thrombocytopenic purpura (ITP), the processes underlying thrombopoiesis, and the treatment of chronic ITP. Now, new agents are available to treat ITP in a nonimmunosuppressive fashion. Lessons learned from the clinical trials of recombinant human thrombopoietin (TPO) have led to the development of a novel class of compounds: nonimmunogenic agonists of the thrombopoietin receptor. Representing the first nonimmunosuppressive agents to treat chronic refractory ITP in decades, medications such as romiplostim and eltrombopag were recently approved by the US Food and Drug Administration. These new agents offer physicians a new tool for treating difficult cases of ITP in their medical armamentarium. Additional TPO mimetics are also being developed that show promise in vitro, and await future development.

The three isoforms of PIP5KI (alpha, beta, and gamma) synthesize PI4,5P(2) (PIP(2)) by phosphorylating PI4P. Therefore, it is not clear why platelets, like all eukaryotic cells, have more than one isoform. To test the hypothesis that PIP5KI isoforms have nonoverlapping functions, we generated a murine line containing a null mutation of PIP5KIbeta and analyzed the effect on platelet signaling. PIP5KIbeta-null mice had normal platelet counts. In contrast to platelets lacking PIP5KIalpha, platelets lacking PIP5KIbeta exhibited impaired aggregation accompanied by disaggregation. Although platelets lacking PIP5KIbeta had only a moderate deficiency of PIP(2) under basal conditions, they had a striking deficiency in PIP(2) synthesis and IP(3) formation after thrombin stimulation. We have also observed that platelets lacking both PIP5KIalpha and PIP5KIbeta have a complete loss of thrombin-induced IP(3) synthesis even though they still contain PIP5KIgamma, the predominant PIP5KI isoform in platelets. These results demonstrate that PIP5KIbeta, like PIP5KIalpha, contributes to the rapid synthesis of a pool of PIP(2) that is required for second-messenger formation, whereas the pool of PIP(2) synthesized by PIP5KIgamma does not contribute to this process. Additionally, we found that PIP5KIbeta-null platelets failed to form arterial thrombi properly in vivo. Together, these data demonstrate that PIP5KIbeta is required for rapid PIP(2) synthesis, second-messenger production, and stable platelet adhesion under shear in vivo. These results also demonstrate that after stimulation of a G protein-coupled receptor, IP(3) is completely derived from a rapidly synthesized discrete pool of PIP(2) synthesized by PIP5KIalpha and PIP5KIbeta.

The canonical Wnt-beta-catenin signaling pathway is initiated by inducing phosphorylation of one of the Wnt receptors, low-density lipoprotein receptor-related protein 6 (LRP6), at threonine residue 1479 (Thr1479) and serine residue 1490 (Ser1490). By screening a human kinase small interfering RNA library, we identified phosphatidylinositol 4-kinase type II alpha and phosphatidylinositol-4-phosphate 5-kinase type I (PIP5KI) as required for Wnt3a-induced LRP6 phosphorylation at Ser1490 in mammalian cells and confirmed that these kinases are important for Wnt signaling in Xenopus embryos. Wnt3a stimulates the formation of phosphatidylinositol 4,5-bisphosphates [PtdIns (4,5)P2] through frizzled and dishevelled, the latter of which directly interacted with and activated PIP5KI. In turn, PtdIns (4,5)P2 regulated phosphorylation of LRP6 at Thr1479 and Ser1490. Therefore, our study reveals a signaling mechanism for Wnt to regulate LRP6 phosphorylation.

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP76), an adaptor that plays a critical role in platelet activation in vitro, contains three N-terminal tyrosine residues that are essential for its function. We demonstrate that mice containing complementary tyrosine to phenylalanine mutations in Y145 (Y145F) and Y112 and Y128 (Y112/128F) differentially regulate integrin and collagen receptor signaling. We show that mutation of Y145 leads to severe impairment of glycoprotein VI (GPVI)-mediated responses while preserving outside-in integrin signaling. Platelets from Y112/128F mice, although having mild defects in GPVI signaling, exhibit defective actin reorganization after GPVI or alpha IIb beta 3 engagement. The in vivo consequences of these signaling defects correlate with the mild protection from thrombosis seen in Y112/128F mice and the near complete protection observed in Y145F mice. Using genetic complementation, we further demonstrate that all three phosphorylatable tyrosines are required within the same SLP76 molecule to support platelet activation by GPVI.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the alpha, beta, and gamma isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of PIP(2) in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KIgamma(-/-) megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wild-type PIP5KIgamma, but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KIbeta- and PIP5KIgamma(-/-) cells had impaired PIP(2) synthesis. However, PIP5KIbeta-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KIbeta in PIP5KIgamma(-/-) cells failed to revert this defect. Megakaryocytes lacking the PIP5KIgamma-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KIgamma(-/-) cells. These findings demonstrate a unique role for PIP5KIgamma in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells.

Background: Despite widespread heparin use in clinical practice, the associated development of thrombocytopenia is an underrecognized and undertreated complication., Methods: We analyzed data from consecutive hospitalized patients treated with heparin (unfractionated or low molecular weight) for 4 days or longer to determine the incidence, predictors, prognostic significance, and management of "thrombocytopenia," defined as a platelet count less than 150 x 10(9)/L, reduction in platelet count of 50% or more from the admission level, or both., Results: We enrolled 2420 patients (median age, 65.2 years; 43.8% women) in 48 US hospitals. Thrombocytopenia occurred in 881 patients (36.4%; 95% confidence interval [CI], 34.5%-38.3%). Of those who developed thrombocytopenia, 5.1% died, compared with 1.6% of those without thrombocytopenia (odds ratio [OR], 3.4; 95% CI, 2.1-5.6; P< .001). Thrombocytopenia was also associated with greater risk of myocardial infarction (OR, 2.1; 95% CI, 1.5-2.8; P< .001) and congestive heart failure (OR, 1.3; 95% CI, 1.1-1.6; P= .01). After adjustment for important covariates, thrombocytopenia remained an independent predictor of thrombotic and hemorrhagic events. A relative reduction in platelet count of more than 70% was the strongest independent predictor of death (OR, 13.4; 95% CI, 6.5-27.6; P< .001), followed by a relative reduction in platelet count of 50% to 70%, worse Killip class, occurrence of thromboembolic complications, older age, and longer duration of heparin therapy., Conclusions: Thrombocytopenia occurs frequently after prolonged heparin therapy and is strongly associated with worse short-term clinical outcome. The relative reduction in platelet count is a powerful independent predictor of all-cause mortality in hospitalized patients.

In recent years, great advances have been made in elucidating the pathogenesis of thrombocytopenia and the mechanisms of thrombopoiesis. Drawing upon decades of basic science and clinical research, the pathways behind the immune-mediated destruction of platelets have opened new avenues. This has led to the application of safer and more efficacious immunosuppressive agents, such as the anti-CD20 monoclonal antibody rituximab, or potentially altering the co-stimulatory pathways of the immune system with an anti-CD40L (CD154) monoclonal antibody. This has been coupled with the discoveries of the genetic and molecular pathways in thrombopoiesis, including the identification and cloning of thrombopoietin (TPO) and its receptor. The use of recombinant TPO, such as PEG-rHuMGDF and rhTPO, to treat thrombocytopenia have paved the way for alternative peptidyl and nonpeptidyl thrombopoietic agents that are considered to be nonimmunogenic. Those that have undergone evaluation in humans include the Amgen megakaryopoiesis protein (AMG) 531 compound, eltrombopag, and AKR-501. Additional TPO mimetics show promise in vitro and await future development.

The malignant cells in Sezary syndrome express the skin trafficking molecules' cutaneous lymphocyte associated antigen (CLA) and chemokine receptor 4 (CCR4). High levels of the CCR4 ligand, thymus, and activation-regulated chemokine (TARC), have been reported in the blood and skin of patients. The rexinoid X-receptor specific retinoid, bexarotene, has contributed to the resolution of cutaneous disease among patients. To evaluate the effects of bexarotene on skin trafficking molecule expression and chemotaxis, peripheral blood mononuclear cells from Sezary syndrome patients and healthy controls were treated with bexarotene in vitro. CCR4 and CLA expression levels and chemotaxis in response to TARC (6.25 ng/ml) were evaluated among lymphocytes before and after treatment with bexarotene (10 microM). Flow cytometric analysis was performed to evaluate CD4, CD26, CLA, and CCR4 cell surface expression. Transwell migration assays were performed to evaluate chemotaxis to TARC. Prior to treatment, malignant cells exhibited higher CCR4 expression (45-90%) and greater than four times more chemotaxis to TARC compared with healthy controls. After treatment with bexarotene for 36-96 hr, a 28% reduction in CCR4 expression was noted (P < 0.05) among the malignant population with an associated 9% decrease in chemotaxis to TARC (P < 0.05). Our results show that bexarotene may inhibit malignant cell trafficking to the skin through an ability to suppress CCR4 expression among Sezary syndrome lymphocytes., (2007 Wiley-Liss, Inc)

Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in G(alphai) protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCbeta is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCbeta isoforms (PLCbeta2 and PLCbeta3), and demonstrate that loss of PLCbeta2 and PLCbeta3 significantly impaired T cell migration. Because second messengers generated by PLCbeta lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCbeta-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1alpha was undetectable in PLCbeta2beta3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCbeta play a critical role in T lymphocyte chemotaxis.

Most new genes arise by duplication of existing gene structures, after which relaxed selection on the new copy frequently leads to mutational inactivation of the duplicate; only rarely will a new gene with modified function emerge. Here we describe a unique mechanism of gene creation, whereby new combinations of functional domains are assembled at the RNA level from distinct genes, and the resulting chimera is then reverse transcribed and integrated into the genome by the L1 retrotransposon. We characterized a novel gene, which we termed PIP5K1A and PSMD4-like (PIPSL), created by this mechanism from an intergenic transcript between the phosphatidylinositol-4-phosphate 5-kinase (PIP5K1A) and the 26S proteasome subunit (PSMD4) genes in a hominoid ancestor. PIPSL is transcribed specifically in the testis both in humans and chimpanzees, and is post-transcriptionally repressed by independent mechanisms in these primate lineages. The PIPSL gene encodes a chimeric protein combining the lipid kinase domain of PIP5K1A and the ubiquitin-binding motifs of PSMD4. Strong positive selection on PIPSL led to its rapid divergence from the parental genes PIP5K1A and PSMD4, forming a chimeric protein with a distinct cellular localization and minimal lipid kinase activity, but significant affinity for cellular ubiquitinated proteins. PIPSL is a tightly regulated, testis-specific novel ubiquitin-binding protein formed by an unusual exon-shuffling mechanism in hominoid primates and represents a key example of rapid evolution of a testis-specific gene.

All eukaryotic cells contain the phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP2) that serves multiple roles in signal transduction cascades. Type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) catalyzes the synthesis of PIP2 by phosphorylating phosphatidylinositol 4 phosphate. Although the classical isoforms of PIP5KI (designated as alpha, beta, and gamma) all generate the same phospholipid product, they have significantly dissimilar primary structures and expression levels in different tissues, and they appear to localize within different compartments within the cell. Therefore, it appears likely that PIP5KI isoforms have overlapping, but not identical, functions. Here we show that targeted disruption of PIP5KIgamma causes widespread developmental and cellular defects. PIP5KIgamma-null embryos have myocardial developmental defects associated with impaired intracellular junctions that lead to heart failure and extensive prenatal lethality at embryonic day 11.5 of development. Loss of PIP5KIgamma also results in neural tube closure defects that were associated with impaired PIP2 production, adhesion junction formation, and neuronal cell migration. These data, along with those of other PIP5KI isoforms, indicate that individual PIP5KI isoenzymes fulfill specific roles in embryonic development.