Platelet Surface Glycoprotein Research Articles

Dysregulation of platelet serotonin, 14–3–3, and GPIX in sudden infant death syndrome

Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal infant mortality, but the underlying cause(s) are unclear. A subset of SIDS infants has abnormalities in the neurotransmitter, serotonin (5-hydroxytryptamine [5-HT]) and the adaptor molecule, 14–3–3 pathways in regions of the brain involved in gasping, response to hypoxia, and arousal. To evaluate our hypothesis that SIDS is, at least in part, a multi-organ dysregulation of 5-HT, we examined whether blood platelets, which have 5-HT and 14–3–3 signaling pathways similar to brain neurons, are abnormal in SIDS. We also studied platelet surface glycoprotein IX (GPIX), a cell adhesion receptor which is physically linked to 14–3–3. In infants dying of SIDS compared to infants dying of known causes, we found significantly higher intra-platelet 5-HT and 14–3–3 and lower platelet surface GPIX. Serum and plasma 5-HT were also elevated in SIDS compared to controls. The presence in SIDS of both platelet and brainstem 5-HT and 14–3–3 abnormalities suggests a global dysregulation of these pathways and the potential for platelets to be used as a model system to study 5-HT and 14–3–3 interactions in SIDS. Platelet and serum biomarkers may aid in the forensic determination of SIDS and have the potential to be predictive of SIDS risk in living infants.

Treatment and Follow-up Data for Young Patients with a Gne Defect and Congenital Thrombocytopenia

Congenital thrombocytopenia can be associated not only with a reduced production, but also with an accelerated degradation of platelets. Sialic acid binds to platelet surface glycoproteins and protects platelets from degradation via the Ashwell-Morell receptor. The GNE gene encodes an enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid, a precursor of sialic acids. According to OMIM alterations in GNE are autosomal recessive associated with adult-onset progressive GNE myopathy (with or without thrombocytopenia) and autosomal dominant with sialuria. In recent years, biallelic GNE mutations located mainly in the N-acetylmannosamine (ManNAc) kinase domain of GNE have been described in children/young adults with otherwise unexplained thrombocytopenia. Recently, we identified compound heterozygous GNE variants in a young girl (Pt.1) with life-threatening intracranial bleeding and severe congenital thrombocytopenia (lowest platelet count 5 x 10 9/L), who needed weekly platelet transfusions. We showed decreased α2,3 sialic acid and increased terminal galactose and α2,6 sialic acid moieties in the girl´s platelets. She received hematopoietic stem cell transplantation (HSCT) at 2,3 years, which normalized platelet counts and function. Follow-up data showed normalized platelet function (20 months after HSCT) and counts (20, 21, and 23 months after HSCT). Following HSCT, the platelet lectin profile was markedly different from before transplantation with an increase in α2,3 sialic acid recognizing lectins WGA and MAL I compared with data before transplantation, indicating that sialic acid moieties have been normalized. Additionally, we identified a biallelic GNE variant in two siblings (Pt.2 and Pt.3) affected by congenital thrombocytopenia. Pt.2 is a 1,8-year-old boy with thrombocytopenia since birth (lowest platelet count 6 x10 9/L) who needed weekly platelet transfusions. His 17-year-old sister (Pt.3) presented with lifelong thrombocytopenia and suspected chronic immune thrombocytopenia; however, therapy with immunoglobulin and steroids did not elevate the platelets. The parents and two other siblings were clinically not affected. Flow cytometry performed for the affected siblings showed severely decreased thrombin-induced platelet CD62 and CD63 exposure hinting at an impaired α- and δ-granule secretion. NGS identified a homozygous GNE variant (NM_001128227.3:c.1727G>C, p.Gly576Ala) with damaging prediction to be present in both siblings. The parents and two healthy siblings are heterozygous carriers of this variant. The nucleotide change c.1726G>C leading to Gly576Arg has been described compound heterozygous in a patient with GNE myopathy. For the sister (Pt.3) lectin binding analysis showed decreased α-2,3 sialylation (MAA) and increased terminal galactose (RCA120) expression on platelets. This pattern is consistent with loss of sialic acid synthesis and indicative of rapid platelet clearance. She had received therapy with romiplostim since she was 12, resulting in stable platelet counts (> 50 x10 9/L). At the age of 1 year, Pt.2 also started with romiplostim therapy. During the 8-month treatment, platelet counts were mainly stable above 30 x 10 9/L. Only two transfusions were necessary when platelet counts dropped to 18 and 11 x 10 9/L. In conclusion, this research elucidates novel associations between GNE mutations, specifically within the ManNAc kinase domain, and congenital thrombocytopenia. These findings were derived from studying three patients carrying such mutations, underscoring this discovery's potential broad relevance. Importantly, this alteration in the GNE gene, traditionally associated with GNE myopathy, prompts us to anticipate and closely monitor the potential future development of this neuromuscular condition in these patients. Of particular interest is the effectiveness of romiplostim in ameliorating thrombocytopenia in these cases. Both siblings in our study exhibited stable, acceptable platelet counts in response to this therapy, suggesting that romiplostim might offer a promising therapeutic strategy for managing congenital thrombocytopenia linked to GNE mutations.

Hydroxysafflor yellow A inhibits the hyperactivation of rat platelets by regulating the miR-9a-5p/SRC axis

Pathological platelet activation plays a vital role in the prevalence of cardiovascular diseases. Hydroxysafflor yellow A (HSYA) has been shown to have significant anti-platelet aggregation and anti-activation effects, but its mechanism of action is unclear. Our study showed that HSYA inhibited the expression of platelet surface glycoproteins IIβ/III α (GPIIβ/III α) and thromboxane A2 (TXA2) during platelet activation and reduced platelet Ca2+ accumulation. HSYA significantly reduced the number of platelets and inhibited adrenaline-induced platelet hyperaggregation in rats. Transcriptomic analysis of platelets suggested that HSYA significantly suppressed SRC and MAPK3 (ERK1/2) gene expression. YEEI peptide, an SRC activator, could significantly reverse the inhibition of HSYA on the phosphorylation of SRC/PLCγ2/PKCδ/MEK/ERK1/2 pathway proteins and reverse the effect of HSYA on platelet activation-related markers GPIIβ/IIIα protein, TXA2 and cAMP. The SRC genes were further predicted by transcriptome analysis of HSYA-regulated miRNAs combined with bioinformatics techniques. The results suggested that HSYA could significantly upregulate the expression level of the miR-9a-5p gene and further confirmed that miR-9a-5p had a targeted regulatory relationship with SRC by dual-luciferase activity reporter and cell transfection experiments. The inhibitory effect of HSYA on the SRC/PLCγ2/PKCδ/MEK/ERK1/2 pathway was significantly reversed after platelets were transfected with the miR-9a inhibitor, while SRC siRNA attenuated the effect of the miR-9a inhibitor. SRC siRNA was able to attenuate the effect of the miR-9a inhibitor. In conclusion, this study suggests that HSYA can inhibit the activation of the SRC/PLCγ2/PKC δ/MEK/ERK1/2 axis by upregulating platelet miR-9a-5p, thereby reducing the activation of platelets and inhibiting platelet aggregation.

PLATELET SUPPRESSION BY TIROFIBAN AMELIORATES PULMONARY COAGULATION AND FIBRINOLYSIS ABNORMALITIES IN THE LUNGS OF MOUSE ANTIBODY-MEDIATED TRANSFUSION-RELATED ACUTE LUNG INJURY.

This study aimed to explore the ameliorating effects of the platelet surface glycoprotein IIb/IIIa receptor antagonist tirofiban on coagulation and fibrinolytic abnormalities in a mouse model of antibody-mediated transfusion-associated acute lung injury (ALI). This is important because ALI is a major cause of death attributable to the occurrence of adverse transfusion reactions. No information on a definite diagnosis or pathological mechanism exists, and targeted treatment options are not available. In this study, wild-type male Balb/c mice aged 8 to 10 weeks were randomly divided into the TRALI model, blank control, tirofiban intervention, and isotype control groups. After different treatment exposures, the mice were observed for 2 h before being killed, and lung tissue samples were collected. To explore the intervention effect of tirofiban, the degree of lung injury was quantified by estimating the lung wet/dry ratio, rectal temperature, survival rate, total protein, and myeloperoxidase and via hematoxylin-eosin staining. Furthermore, the coagulation, anticoagulation, and fibrinolysis assays were measured by automatic coagulation instrument and enzyme-linked immunosorbent assay kits, and the fluorescence densities of platelets and fibrin were quantified using immunofluorescence to analyze the effects of tirofiban on the platelet and fibrin interactions of TRALI. Compared with the TRALI model group, the lung injury indices in the tirofiban intervention group decreased significantly, and survival rates also improved. Furthermore, the level of coagulation and fibrinolytic abnormalities were obviously lower than those in the TRALI model group. In conclusion, our findings suggest that tirofiban might interfere with TRALI by inhibiting platelet activation and improving coagulation and fibrinolytic abnormalities.

Evaluation of platelet surface glycoproteins in inherited thrombocytopathy: relationship with bleeding severity

Evaluation of platelet surface glycoproteins in inherited thrombocytopathy: relationship with bleeding severity

Flow cytometric analysis of platelet surface glycoproteins in the diagnosis of thirty-two Turkish patients with Glanzmann thrombasthenia: a multicenter experience

Background/aimGlanzmann thrombasthenia (GT) is a rare autosomal recessively inherited bleeding disorder characterized by the quantitative (type 1 and type 2) or qualitative (type 3) deficiency in platelet membrane glycoprotein (GP) IIb/IIIa (CD41a/CD61) fibrinogen receptors. In type 1, 2, and 3, CD41a/CD61 expression is 5%, 5%–20% and above 20%, respectively. In this study, diagnosis of GT was confirmed and subgroups were identified in 32 Turkish patients by flow cytometry analysis.Materials and methodsCD41a/CD61 expression levels in platelet-rich plasma (PRP) obtained from peripheral venous EDTA blood samples were analyzed with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). GT subgroup analysis was performed by counting 50,000 events in the BD FACSDiva Software v6.1.3 program of the instrument.ResultsIn the present study, in blood samples of 32 patients from 23 families with GT and 22 healthy controls, co-expression levels of CD41a and CD61 in PRP was analyzed. 12 out of 23 families were consistent with type 1 GT (52.2%), 4 were consistent with type 2 GT (17.4%), and 7 were consistent with type 3 GT (30.4%).ConclusionEspecially due to consanguineous marriages, GT with various glycoprotein levels may be detected. As a result of the flow cytometry analysis of the present study with the highest GT patient population in Turkey, type 1 GT patients were the most common subgroup. In the determination of the GT subgroups; especially in the detection of type 3 GT, flow cytometry is the most sensitive glycoprotein analysis method. In addition to light transmission aggregometry, CD41a/CD61 study by flow cytometer confirms diagnosis when mutation analysis cannot be performed.

GPVI Intracellular Signaling Is Activated in Platelets from Patients with Essential Thrombocythemia

GPVI Intracellular Signaling Is Activated in Platelets from Patients with Essential Thrombocythemia

Decreased platelet surface phosphatidylserine predicts increased bleeding in patients with severe factor VIII deficiency

BackgroundBleeding phenotypes among individuals with severe factor VIII (FVIII) deficiency are variable, even with routine prophylactic FVIII administration. Activated platelets, in addition to their role in primary hemostasis, play a major role in coagulation by providing a phospholipid surface to which coagulation factors bind. ObjectivesThe aim of this study was to determine whether platelet function is associated with past and/or future bleeding in patients with severe FVIII deficiency on prophylaxis. Patients/MethodsPlatelet function quantified by platelet surface expression of phosphatidylserine, platelet surface glycoprotein (GP) VI, platelet surface activated GPIIb‐IIIa, platelet surface P‐selectin, the percentage of coated platelets, and the percentage of platelet‐derived microparticles in the presence or absence of in vitro activation by various agonists was assessed in 34 patients. ResultsDecreased platelet surface phosphatidylserine expression (identified by annexin V binding), both in the presence and absence of ADP/thrombin receptor activating peptide, demonstrated a significant association with both prior and subsequent bleeding in any location and specifically with hemarthrosis. No significant difference between patients with and without bleeding was observed in any of the other platelet activity markers. ConclusionsDecreased platelet surface phosphatidylserine expression measured by annexin V binding predicts increased bleeding in severe FVIII deficient patients on prophylaxis.

Acute Severe Thrombocytopenia Event Associated with Trimethoprim/Sulfamethoxazole Use.

Objective:To report a case of life-threatening thrombocytopenia associated with the use of trimethoprim/sulfamethoxazole (TMP/SMX) therapy.Report of the case:50-year-old woman with no significant past medical history who presented with one day of petechial rash on her arms, chest and legs. Patient reports that she had just completed a 7- day course of TMP/SMX (1-double strength tablet twice a day) for uncomplicated UTI by her PMD. On admission, the patient was hemodynamically stable, and complete blood cell count revealed a platelet count of 2000/uL. TMP/SMX was believed to be the most likely cause of thrombocytopenia. After discontinuation of TMP/SMX and treatment with 2 units of platelets, 1gm intravenous immunoglobulin (IVIG) and oral dexamethasone, repeat CBC showed a stable platelet count of 90,000/uL. Patient was successfully discharged on hospital day 3 with outpatient follow up with the hematology clinic for further monitoring.Conclusion and Discussion:Differential diagnosis of severe thrombocytopenia include drug induced thrombocytopenia (DITP), thrombotic thrombocytopenic purpura (TTP), post transfusion purpura (PTP), immune thrombocytopenic purpura (ITP), heparin induced thrombocytopenia (HIT), or catastrophic antiphospholipid antibody syndrome (APS). Drug-dependent antibodies are an unusual class of antibodies that bind firmly to specific epitopes on platelet surface glycoproteins only in the presence of the sensitizing drugs. DITP typically has an abrupt onset of severe thrombocytopenia, usually less than 20,000/uL. Thrombocytopenia usually begins to recover within 1-2 days after the offending drug is discontinued and platelet levels usually normalize within one week as demonstrated in our case report. Pharmacological treatment can include platelet transfusions in case of severe, overt bleeding, corticosteroids or IVIG administration. In most cases, however, discontinuation of the offending drug is sufficient.

Platelet surface GPIbα, activated GPIIb-IIIa, and P-selectin levels in adult veno-arterial extracorporeal membrane oxygenation patients

Our objective was to characterize platelet surface glycoprotein (GP)Ibα, activated GPIIb-IIIa, and P-selectin levels during and after extracorporeal membrane oxygenation (ECMO). We performed a single center cohort study of 10 adult patients on ECMO for cardiogenic shock. Patients had blood samples drawn on ECMO day 1 or 2, day 3, day 5, and 48–72 hours after ECMO decannulation. Platelets from untreated blood samples and samples treated with either adenosine diphosphate (ADP) or thrombin receptor agonist peptide (TRAP) had surface GPIbα, activated GPIIb-IIIa, and P-selectin levels measured using flow cytometry. Platelet surface GPIbα levels varied significantly by time on ECMO (p = .002) and were significantly higher on ECMO day 5 compared to ECMO day 1 (p = .01). GPIbα levels during ECMO did not differ significantly from levels after ECMO decannulation (p = .14). Activated GPIIb-IIIa levels did not change significantly during ECMO, but were significantly higher after ECMO decannulation (p = .04). There were no significant differences in P-selectin levels during ECMO (p = .87) or after ECMO decannulation (p = .41). Platelet surface GPIbα and P-selectin levels were similar during and after ECMO whereas activated GPIIb-IIIa levels were lower during ECMO, particularly in response to TRAP stimulation, potentially contributing to ECMO-induced coagulopathy.

Glycoside Residues on Platelet's Surface Regulate Platelet Function, Apoptosis and Binding of Coagulation Complexes in Patients with Immune Thrombocytopaenia

Introduction: Platelet surface glycoproteins (GPs) are highly glycosylated and are key elements for platelet function since most of them constitute receptors for adhesion ligands. However, exact role of their glycan composition is not clear. Under normal conditions, platelets contain sialic acid in the carbohydrate side chains of their GPs, and it has been described that alterations in the degree of their sialinization can affect the clearance of platelets. This mechanism has been proposed as involved in etiopathogenesis of immune thrombocytopaenia (ITP), mainly in those patients who do not respond to treatments. Thus, after the loss of sialic acid, there would be a greater exposure of galactose and of N-acetyl-glucosamine residues on the surface of circulating platelets to hepatic Ashwell-Morell receptors, which could induce their phagocytosis and platelet clearance. On the other hand, procoagulant platelets, defined as the platelet subpopulation that binds functional prothrombinase, exposed on their surface increased levels of P-selectin and GPIb, two glycan rich GPs. So, it is tempting to speculate that changes in glycan residues on platelet surface may induce changes in their function. Aim: We aimed to assess in ITP patients whether changes in platelet glycosylation, mainly the loss of sialic acid, may condition platelet function, apoptosis and binding of prothrombinase complex. Methods: This is an observational, prospective and transversal study approved by Ethics Committee from La Paz University Hospital. One hundred and eight patients with chronic primary ITP (68 with a platelet count ≥30x103 platelets/µL and 40 with a platelet count <30x103 platelets/µL) and 132 healthy controls were included after signing the informed consent. Platelet activation markers were determined in platelet rich plasma; whereas platelet glycosylation, binding of prothrombinase, annexin V and caspase's activities were assayed in washed platelets. Samples were analyzed by flow cytometry. Table 1 shows lectins tested and their sugar-binding specificity. Data were analyzed with GraphPad Prism 6.0 software. Results: Platelets from ITP patients with a platelet count <30x103/µL exposed less sialic acid in correspondence to an enhanced binding of lectins to non-sialylated residues. Moreover, levels of α1,6-Fucose, a glycan residue which could directly regulate antibody-dependent cellular cytotoxicity, and of α-Mannose, which could be recognized by the mannose binding lectin and activate complement pathway, were increased in platelets from these ITP patients. In accordance, sialic acid loss and consequent platelet surface exposure of other glycoside residues were inversely related to platelet count and ability to be activated (Table 1). These differences in glycosylation observed in ITP patients with a platelet count <30x103/µL were accompanied by a less ability of platelets to be activated (Figure 1), an increased exposure of phosphatidylserine and higher caspase activites (Figure 2). Moreover, increased exposure of phosphatidylserine and of N-acetyl-glucosamine residues (measured through the binding of WGA) enhanced binding of prothrombinase complex (Figure 3). Conclusion: Changes in glycoside composition of GPs on platelet's surface impaired their functional capacity, increases their apoptosis and modifies conditions for the binding of coagulation proteins. These modifications in platelet's glycoside residues seem to be related to severity of ITP. This work was supported by grants from FIS-FONDOS FEDER (PI19/00772) and and Platelet Disorder Support Association. EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Butta: Grifols: Research Funding; Novartis: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; SOBI: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Speakers Bureau. Alvarez Román:Grifols: Research Funding; Bayer: Consultancy; Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau. Martín:SOBI: Research Funding; Pfizer: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau. Rivas Pollmar:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Justo Sanz:Takeda: Current Employment. García Barcenilla:NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Pfizer,: Speakers Bureau; Roche: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Speakers Bureau. Canales:Celgene: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria; Roche: Honoraria; Gilead: Honoraria; Sandoz: Honoraria; iQone: Honoraria; Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Sandoz: Honoraria; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Janssen: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding.

Platelet count and function in umbilical cord blood versus peripheral blood in term neonates

Platelet function in neonates is sparsely investigated. The majority of previous studies investigated platelets in umbilical cord (UC) blood rather than in peripheral blood. We included 20 term neonates and sampled UC blood and peripheral blood within 20 min and 24 h after birth. Platelet count and mean platelet volume (MPV) were measured. Platelet surface glycoproteins (GP) and platelet activation (bound fibrinogen, CD63 and p-selectin) after agonist stimulation were examined by flow cytometry. Platelet aggregation was evaluated by impedance aggregometry. The significance level was set after Bonferroni correction. Platelet count and MPV did not differ between UC and peripheral blood (p-values >0.08). Expression of platelet surface GP was similar in UC and peripheral blood (all p-values >0.02). Platelet activation was lower in UC blood than in peripheral blood for bound fibrinogen (four out of eight p-values <0.001) but did not differ for CD63 (all p-values >0.01) or P-selectin (all p-values >0.01). Platelet aggregation was significantly higher in UC than in peripheral blood (p-values <0.001). In conclusion, platelet count, MPV and expression of platelet surface GP measured in term neonatal UC blood represented that of peripheral blood. Platelet activation and aggregation in UC blood did not reflect that of peripheral blood.

Common gene variants in ASGR1 gene locus associate with reduced cardiovascular risk in absence of pleiotropic effects

Background and aimsThe rare ASGR1 del12 variant is associated with a beneficial effect on coronary artery disease (CAD) that is disproportionate to the small reductions in plasma LDL cholesterol (LDLc). This unexplained benefit has sparked the debate on potential additional pleiotropic effects of ASGR1 variants. Since ASGR1 has also been implicated in platelet homeostasis, we evaluated platelet function in heterozygous ASGR1 del12 carriers and controls. In addition, we compared the magnitude of various LDLc lowering genetic scores in the UK-biobank using Mendelian randomization. MethodsDesialylation of platelet surface glycoproteins and platelet aggregation capacity were measured in 12 carriers and 10 controls. We selected 3 common genetic variants in the ASGR1 locus that were significantly associated with plasma LDLc and assessed the association with coronary artery disease (CAD) and compared it with the effects of HMCGR, LDLR, NCI1L1 and PCSK9 gene scores. ResultsPlatelet surface GlcNAC residues were significantly lower in carriers but platelet aggregation did not differ. The relative risk reduction of ASGR1 GRS on CAD and myocardial infarction per 10 mg/dl LDLc reduction was 23% (OR 0.77, 95% CI 0.62–0.96). This risk reduction was proportionally similar to the gene risk scores in HMCGR, NPC1L1, PCSK9, and LDLR. ConclusionsUnlike previous reports, we did not find any evidence for a pleiotropic effect of the rare del12 variant in the ASGR1 locus on CAD, as platelet function did not differ between carriers and controls. Moreover, the observed effect of ASGR1 variants on CAD risk was proportional to the reduction in plasma LDLc levels.

Acquired immune thrombocytopenia: An update on aspects of diagnosis and management relevant for intensive care medicine

Acquired thrombocytopenias represent a group of bleeding diseases, which can be mediated by immune or non-immune factors. Acquired immune thrombocytopenia (AITP) leads to an accelerated decrease in platelet count by platelet reactive antibodies arising from several mechanisms. In AITP, autoantibodies, alloantibodies or drug-dependent antibodies are usually targeting platelet surface glycoproteins. The consequence of this is a significant decrease in the number of circulating platelets, leading to clinic pathological disorders including immune thrombocytopenia, heparin-induced thrombocytopenia or drug-induced thrombocytopenia, respectively. The aforementioned disorders are characterized by a severe reduction in platelet count (< 20 × 109/l), which is, with the exception of HIT, associated with high bleeding risk. In this review we provide current insight into recent achievements regarding diagnosis and management of AITP.

Desialylation and Apoptosis Crosstalk to Modulate Platelet Clearance

Our understanding of cell biological processes involved in aging has advance greatly over the past decades. Platelets are small cells that circulate for 4-5 days in mice and 7-10 days in humans. And even though, platelets are anucleated cells, a growing body of evidence shows that platelet clearance is a well-regulated mechanism. We have recently demonstrated that platelets lose sialic acid as they circulate and age in blood and are rapidly cleared by the hepatic Ashwell-Morell receptor (AMR). And others have shown, in a series of studies using genetically modified mice or pharmacological inhibitors that platelets undergo apoptosis by triggering the intrinsic mitochondrial apoptotic machinery. Here, we investigate if desialylation and apoptosis are related events. First, using a newly developed state-of-the-art technique called dynamic BH3 profiling (DBP), we investigated the mitochondria readiness to undergo apoptosis on platelets derived from WT and AMR deficient (Asgr2-/-) mice. In our assay, digitonin-permeabilized platelets were exposed to activators signaling peptides (such as Bim, Bid and PUMA), and as cells undergo apoptosis due to peptide treatment, they released Cytochrome C. Our data showed that desialylated platelets derived from Asgr2-/-mice have high background levels of Cytochrome C release when compared to WT platelets in the presence of all activator peptides, indicating that desialylated platelets are highly primed to apoptosis. We also tested the level of dependence on pro-survival protein, by using sensitizer peptides (Bad, Hrk and MS1). We observed that desialylated platelets (Asgr2-/-platelets), and to a certain degree, WT platelets, are extremely sensitive to BCL-xL inhibition, as indicated by the extremely high response to Bad and Hrk peptides even at lower concentrations (0.1 and 1uM). Surprisingly, WT and Asgr2-/-platelets show very little response to the MS1 peptide, indicating that they are not dependent on MCL1 for survival, as otherwise suggested. Flow cytometry analysis revealed desialylated platelets from Asgr2-/-mice have a ~2-fold increase in Phosphatidylserine (PS) surface exposure when compared to WT platelets. In addition, western blot analysis showed increased expression of cleaved caspase 3 in Asgr2-/-platelets, but no changes in Bcl-xL protein expression between WT and Asgr2-/-platelets. Next, WT and Asgr2-/-mice received a single dose of the BH3 mimetic, ABT-737, which binds and inhibits pro-survivor proteins (Bcl-2, Bcl-xL and Bcl-w) inducing apoptosis in vivo. Approximately 2 hours after the injection of ABT-737, we observed a big drop on platelets counts in both WT (~42%) and Asgr2-/-(~59%) mice. Importantly, platelets from Asgr2-/-mouse were cleared more efficiently (~20%) from the circulation when compared to those in WT mice, consistent with the ~20% increment in platelet number observed in this mouse model and supporting the notion that the platelets that circulate longer in the Asgr2-/-mice are more sensitive to apoptotic events. To investigate if apoptosis could be triggering platelet desialylation, WT mice were treated with ABT-737 and 1hour later (time point before platelet count drop), platelets were collected and analyzed by flow cytometry. Interestingly, analysis of galactose exposure by RCA-I lectin showed no differences in desialylation between ABT-737 and PBS control groups. On the other hand, Phosphatidylserine (PS) exposure was significantly elevated on ABT-737 group, indicating that platelets were undergoing apoptosis without changing their sialylated status. To confirm our in vivodata, freshly isolated washed WT platelets were treated with ABT-737 to induce apoptosis or Neuraminidase (NA) to desialylated platelets. NA treatment induced platelet desialylation (increased RCA-I binding) in WT platelets, as expected, and interestingly triggered apoptosis, judge by increased PS exposure in both ABT-737 and NA treated groups. However, ABT-737 treatment wasn't able to induce desialylation as levels of RCA-I binding to platelets was the same when compared to PBS control platelets. Taken together, our data shows that desialylated platelets in circulation are prone to apoptosis. In addition, our findings strongly support the hypothesis that desialylation of platelet surface glycoproteins trigger the intrinsic apoptotic pathway in platelets in vivo. Disclosures No relevant conflicts of interest to declare.

Mechanisms of the antiplatelet and analgesic effects of dextromethorphan and its metabolites.

Objective:In the present study, we investigated the effects of dextromethorphan (DM) and its metabolites, including dextrorphan (LK2), 3-methoxymorphinan (LK3), and 3-hydroxymorphinan (LK4), on platelet aggregation in vitro and the inflammatory pain caused by carrageenan in rats, and their underlying mechanisms.Materials and Methods:Rabbit platelets were pretreated with DM or its metabolites to assess their effects on platelet aggregation and related target mediators. In addition, the analgesic activity and the underlying mechanisms of DM and LK3 were investigated in a carrageenan-evoked thermal hyperalgesia rat model.Results:The inhibitory potency of DM and its metabolites on platelet aggregation induced by arachidonic acid or collagen was LK3> DM > LK4>> LK2 as demonstrated by the half-maximal inhibitory concentration values. Moreover, the mechanisms of the antiplatelet effect of DM and LK3 may involve the inhibition of intracellular calcium mobilization, expression of platelet surface glycoprotein IIb/IIIa, the formation of thromboxane B2, and elevation of platelet membrane fluidity. DM and LK3 also exhibited analgesic effects on carrageenan-evoked thermal hyperalgesia by suppressing the production of pro-inflammatory cytokines, nitric oxide, prostaglandin E2, and neutrophil infiltration in inflammatory sites.Conclusion:DM and its metabolites, especially LK3, exhibit both antiplatelet and analgesic effects, and may, therefore, potentially ameliorate platelet hyperactivity and inflammatory-related diseases.

Helicobacter pylori: An Underrated Cause of Immune Thrombocytopenic Purpura. A Comprehensive Review.

Idiopathic thrombocytopenic purpura (ITP) is the autoimmune-mediated destruction of platelets. ITP is a diagnosis of exclusion after other identifiable etiologies have been ruled out. After the first report by Gasbarrini et al. (1998) showing rising platelet counts in ITP patients following Helicobacter pylori (HP) eradication therapy, there is growing evidence that highlights the role of HP in triggering ITP. However, the exact pathophysiology of HP-associated ITP is still unclear, but many theories have been implicated in this regard. According to various reports, the postulated mechanisms for the role of HP in cITP include molecular mimicry, increased plasmacytoid dendritic cell numbers, phagocytic perturbation, and variable host immune response to HP virulence factors. One famous theory suggested molecular mimicry between platelet surface antigen and bacterial virulence factor, i.e. cytotoxin-associated gene A (CagA). It is thought that a chronic inflammatory response following an HP infection induces the host autoantibodies' response against CagA, which cross-reacts with platelet surface glycoproteins; therefore, it may accelerate platelet destruction in the host reticuloendothelial system. However, further studies are mandated to better understand the causal link between ITP and HP and study the role of biogeography. Nowadays, it is recommended that every patient with ITP should undergo HP diagnostic testing and triple therapy should be administered in all those candidates who test positive for HP infection. In our review, there were a few pregnant female ITP patients who took HP eradication therapy mainly after 20 weeks of gestation without maternal or fetal worst outcomes. However, large-scale studies are advisable to study the adverse fetal outcomes following triple therapy use.

Elevated plasma levels of soluble C-type lectin-like receptor 2 (CLEC2) in patients with thrombotic microangiopathy

BackgroundThrombotic microangiopathy (TMA) is caused by activated platelets. The plasma C-type lectin-like receptor 2 (CLEC2) levels in 58 patients with TMA were examined and compared with those in healthy volunteers and other diseases. Materials and methodsThe plasma levels of soluble platelet surface glycoprotein VI (GPVI) and CLEC2 were measured in patients with TMA. ResultsPlasma CLEC2 levels in patients with DIC and TMA were significantly higher (p < 0.001) than those in thrombocytopenic patients with other hematological diseases, but no significant differences in the plasma CLEC2 levels were observed among patients with thrombotic thrombocytopenic purpura, hemolytic uremic syndrome (HUS), atypical HUS and other TMA. The plasma CLEC2 levels after the remission were significantly lower than those before treatment (p < 0.001). The plasma CLEC2 levels were poorly correlated with the levels of soluble GPVI in the plasma of patients with TMA. The plasma CLEC2 levels were not significantly differ between survivor and non-survivor in TMA patients, but were significantly higher in non-survivor in overall population (p < 0.001). ConclusionThe measurement of the plasma CLEC2 level is considered to be important for the diagnosis and evaluation of TMA.

Sialic Acid Content on Platelet Surface Glycoproteins Modulates Thrombin-Induced Activation

Platelets are fundamentally important in normal hemostasis and pathological thrombosis (i.e. cardiovascular diseases, stroke, etc.). Platelets mediate the initial first-step in hemostasis through surface glycoproteins like the GPIb-IX-V complex and integrin αIIbβ3 (GPIIbIIIa). Although the functions of platelet surface glycoproteins are well known, the roles of posttranslational modifications on those surface glycoproteins are poorly understood. We have recently shown that sialic acid is a key regulator of platelet survival. As platelets circulate and age in blood, they lose sialic acid and are rapidly cleared by the hepatocytes where they stimulate liver TPO production and consequently regulate thrombopoiesis. Here, we investigated the importance of glycosylation to platelet function by measuring the impact of sialic acid content on platelet responses to thrombin activation.Freshly isolated wild-type washed platelets were treated with a2-3, -6, -8 sialidase (neuraminidase, NA) to remove sialic acid from the platelet surface glycoproteins or with a competitive NA inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) to prevent sialic acid loss by the action of sialidases. After treatment with both NA and DANA, platelets were activated with Thrombin (THR, 0.1U/mL). As controls, aliquots of freshly isolated wild-type washed platelets were left untreated (Rest), only treated with neuraminidase (NA) or activated with Thrombin (THR). First, we measured b-galactose exposure using RCA-I lectin to test the efficacy of treatments. As expected, NA treated platelets showed significantly higher RCA-I binding when compared to Rest, THR and DANA treated platelets. Noteworthy, RCA-I binding to THR activated platelets was higher than Rest or DANA + THR platelets.We next investigated the effect of NA and DANA treatments of platelet degranulation. Thrombin activated platelets showed high level of P-selectin surface exposure when compared to Rest platelets. NA treatment alone caused low P-selectin exposure (~18% positive platelets) and NA + THR treated platelets showed high levels of P-selectin similar to THR only treatment. Interestingly, DANA +THR platelets showed a lower percentage of P-selectin positive platelets when compared to THR activation only (~50% compared to ~85%). In platelets, thrombin signaling is mediated by PARs, G-protein-coupled receptors that trigger several intracellular pathways, including phosphorylation of several proteins. We next investigated if glycan remodeling of surface glycoproteins could alter the intracellular signaling triggered by Thrombin. Our data shows that NA + THR platelets have increased phosphorylated Akt when compared to THR alone and pretreatment with DANA dampens the phosphorylation signal triggered by THR activation. These data suggest that the glycosylation status of surface glycoproteins on platelets regulates thrombin-induced activation.Neuraminidases are lysosome-resident enzymes, they act primarily intracellularly but can also be recruited to the cell surface. Studies have shown that Neu1, one of the neuraminidase isoforms, regulates lysosome exocytosis by desialylation of LAMP1. Flow cytometry analysis of LAMP1 surface expression showed that THR activation induced LAMP1 surface exposure when compared to Rest. NA treatment did not affect LAMP1 surface exposure caused by THR, but DANA treatment completely blocked LAMP1 translocation to the surface, suggesting that Neuraminidase is a regulator of lysosomal exocytosis in platelets.Taken together, our data shows that sialic acid is a potential regulator of platelet function. More studies are needed to identify platelet glycoproteins affected by sialic acid changes. Nonetheless, these data illustrate that glycan remodeling is ideally suited for therapeutic manipulation to prevent undesired platelet activation. DisclosuresNo relevant conflicts of interest to declare.

Identification of a Novel Heterozygous Mutation (c.2213T>G;p.Leu738Arg) in Platelet Glycoprotein ITGB3 gene in a Patient with Glanzmann's Thrombasthenia

BACKGROUNDGlanzmann's thrombasthenia (GT) is an inherited platelet disorder (IPD) that leads to clinically significant bleeding. Platelets from patients with GT can show qualitative or quantitative defects of platelet membrane glycoprotein (GP) IIb/IIIa complex. Most GT are caused by autosomal recessive genetic defects in ITGA2B and ITGB3 (genes for GPIIb and GPIIIa, respectively) with rare cases showing an autosomal dominant pattern. Accurate diagnosis of GT requires a constellation of both phenotypic and genetic studies. Here we report a unique case of autosomal dominant GT resulted from thorough phenotypic and genetic studies. PATIENTS AND METHODSA 19 year-old woman was recently evaluated for life-long history of easy bruising and severe menorrhagia that was only partially responsive to medroxyprogesterone. She has severe thrombocytopenia first recognized shortly after birth with a platelet count around 20x109/L. Platelet size was normal. She was presumed to have immune-mediated thrombocytopenia (ITP) since early infancy, but lacking responsiveness to the conventional treatments of ITP including immunomodulation, splenectomy and thrombopoietin receptor agonists. Family history was negative for a bleeding diathesis. Both von Willebrand factor antigen and activity were within normal range. Bone marrow aspirate and biopsy with associated chromosome studies were all normal. Platelet surface glycoprotein assessment by flow cytometry using antibodies to GP IIb, IIIa, Ia, Ib-a, VI, IX, and whole exome sequencing (WES) utilizing a predefined list of 53 clinically significant genes* related to genetically IPDs were performed. Due to thrombocytopenia platelet aggregation studies could not be performed.METHODS/RESULTS:Platelet surface expression of GPIIb (CD41) and GPIIIa (CD61) were markedly decreased suggestive of a variant of GT (Figure). WES performed with Illumina HiSeq 2500 sequencing system by using Agilent SureSelelct CRE kit V1 to capture and target the exonic regions showed presence of a heterozygous mutation in ITGB3 gene (c2213T>G; p.Leu738Arg). By in silico prediction modeling, this mutation is likely to be pathogenic and results in the substitution of hydrophobic leucine with hydrophilic arginine in the transmembrane domain of the β3 subunit of alpha IIb/beta 3 integrin (αIIbβ3), the platelet receptor that binds to fibrinogen. Interestingly, GPVI is also decreased which may be associated with GPIIb and GPIIIa deficiency or due to accelerated shedding since no GPVI mutation was identified. CONCLUSIONWe describe a patient with GT associated with a novel heterozygous autosomal dominant mutation in ITGB3 gene with substitution of leucine with arginine (c2213T>G; p.Leu738Arg). This deletion caused a low expression of αIIbβ3 integrin on her platelets surface and severe thrombocytopenia. This case underscores the importance of an integrated phenotyping and genotyping approach to render a definitive diagnosis of an IPD.*Platelet exome gene list: ABCG5, ABCG8, ACBD5, ACTN1, ANKRD26, ANO6, AP3B1, BLOC1S3, BLOC1S6, CYCS, DTNBP1, ETV6, FLI1, FLNA, GATA1, GFI1B, GP1BA, GP1BB, GP6, GP9, HOXA11, HPS1, HPS3, HPS4, HPS5, HPS6, ITGA2B, ITGB3, LYST, MASTL, MPL, MYH9, NBEAL2, ORAI1, PSRX1, P2RY12, PLA2G4A, PRKACG, RASGRP2, RBM8A, RUNX1, STIM1, STX11, STXBP2, TBXA2R, TBXAS1, THPO, TUBB1, UNC13D, VIPAS39, VPS33B, VPS45, WAS [Display omitted] DisclosuresNo relevant conflicts of interest to declare.