GPIb Expression Research Articles

Impact of Pathogen Reduction Technology Treatment and Storage in New Generation Platelet Additive Solutions (PAS) on Platelet Function.

Abstract 2115Poster Board II-92New strategies for preparing platelet concentrates (PCs) are investigating the combination of pathogen reduction technologies (PRT) with storage of platelets in additive solutions (PAS) containing phosphate buffer. While these approaches have several advantages by reducing adverse effects, improving biological safety and increasing plasma availability, there is a reasonable concern on how these strategies could affect platelet function. In the present study we have evaluated the effect of Marisol® PRT treatment (CaridianBCT Biotechnologies, LLC) in combination with storage in PAS-III (Intersol; Baxter Healthcare Corp) or PAS-IIIM (SSP+; MacoPharma) on analytical and functional characteristics of apheresis PCS.PCs (2.5-3.4×109plts/ml) were split into three aliquots. One was kept untreated and stored in plasma as a control (CON-PPP). One was PRT-treated and stored in the corresponding PAS (PRT-PASIII or PRT-IIIM) with a 35% plasma carryover to achieve a final concentration of 0.7-1.5 × 109 plt/ml. The last aliquot was stored in the corresponding PAS (CON-PASIII or CON-PASIIIM), not subjected to PRT treatment. PCs were stored under standardized conditions and evaluations were performed on days 0, 5 and 7. Cell quality parameters (pH, swirl, lactate and glucose), flow cytometry analysis of major platelet glycoproteins and activation dependent antigens were assessed. Adhesive and aggregating functions of platelets were evaluated using an ex vivo perfusion model with flowing reconstituted blood recirculating through damaged vascular segments.A slight reduction in platelet counts was noticed on day 7 compared to day 0 in PRT-treated PCs (p<0.05), however no statistical differences were observed between the different study groups. All measured cell quality parameters were within ranges previously published. Flow cytometry analysis revealed comparable levels of the analyzed glycoproteins for all conditions evaluated. Only moderate reductions in the expression of GPIb were observed on day 7 of storage in all groups except for CON-PASIIIM. A progressive increase in the expression of P-selectin was observed in both controls and PRT-treated PCs during storage. Lysosomal integral membrane protein (LIMP) expression was increased in PRT-PASIII and PRT-PASIIIM and CON-PASIII, but not CON-PASIIIM PCs. PRT-PASIII PCs showed the highest levels of annexin V binding after 7 days of storage. Studies under flow conditions showed comparable levels of adhesion and aggregation to subendothelium for PRT-treated platelets stored in PAS solutions at 5 days of storage. A detailed morphometric analysis revealed slightly enhanced adhesive functions in CON-PASIII on day 0 and moderate reductions in adhesive properties in PRT-PASIII, but not PRT-PASIIIM PCs after 7 days of storage.Our investigations show that cell quality parameters, glycoproteins levels and platelet functions were preserved in PRT-treated concentrates and stored in new generation PAS for up to 5 days. The relevance of our experimental data on the in vivo performance of PCs exposed to PRT and stored in new generation PAS deserve further investigation in the clinical setting. Disclosures:Escolar:Caridian BCT Technologies: Consultancy, Research Funding. Marschner:Caridian BCT Technologies: Employment. Goodrich:Caridian BCT Technologies: Employment. Galan:Caridian BCT Technologies: Research Funding.

Platelet Mitochondrial Potential and Function Is Altered During Severe Septic Shock.

Abstract 5074 BackgroundSeptic shock is a condition characterized by a systemic inflammatory response associated to organ dysfunction. Primary hemostasis is impaired in sepsis, and multiple causative mechanisms have been proposed. MethodsIn this study we evaluated platelet function in patients with septic shock. In particular platelet mitochondrial potential was studied as a possible cause of platelet function impairment in septic shock. To test the hypothesis we enrolled 21 consecutive patients admitted to our intensive care unit (ICU) with diagnosis of septic shock. All patients were analysed within 24 hours from admission. 30 ml of citrated blood were withdrawn from each patient and the platelet function tests were performed. Platelet mitochondrial potential (Dy) was assessed by flow cytometry through JC1 staining, espressed as the ratio of FL-1 and FL-2 fluorescence. Platelet aggregation was performed on platelet rich plasma in 5 patients not assuming any drug known to influence platelet function with the following aggregating agents: ADP (final concentration 4 μM), collagen (2 μg/ml), U46619 (10 μM) and TRAP (10 μM). Platelet ATP secretion was quantified and intraplatelet δ-granules content dosage was performed. Expression of GpIb, of GpIIb/IIIa, and of annexin V binding were evaluated by flow cytometry. The study was approved by the Hospital Ethical Committee. Resultsat admission platelet mitochondrial potential was reduced in septic patients in comparison to normal controls (2.6±1.4 vs 3.4±0.3 p=0.06). At day one there was a good correlation between platelet mitochondrial impairment and the Sepsis-related Organ Failure Assessment (SOFA) score, an indicator of sepsis severity (R2=0,39 p<0.001 n=18) with higher values indicating higher degree of severity. Platelets taken from patients with a SOFA score above the group median value (>9, n=8) had Dy values significantly lower than those taken from less severely ill patients (SOFA score ' 9, n=10) and healthy volunteers (1.6±0.6 vs. 3.3±1.4 vs. 3.4±0.3; p<0.01 one way analysis of variance). At day one platelet aggregation was severely impaired, especially with strong aggregating agents as collagen (3/5 of patients with maximum platelet aggregation below 25%). The maximum aggregation caused by collagen seemed to correlate to platelet mitochondrial potential (R2=0,72 n=5 p=0.07). Platelet secretion was equally defective at admission, with pathological secretion to collagen stimulus in 40% of tested patients. Intraplatelet ADP content resulted low in 66% tested (n= 12), while serotonin content was pathological in 58% of patients (n= 12). Platelet GpIIb/IIIa and GpIb expression and annexin V binding were within normal limits. Conclusionwe demonstrated in a very well selected group of patients that during the early phase of a septic shock a mitochondrial dysfunction occurs, and this may have an impact on platelets dysfunction observed in this condition. DisclosuresNo relevant conflicts of interest to declare.

P2Y12 Inhibitors Restore the Ability of GPIb-IX to Inhibit Outside-in Signaling Across alphaIIbbeta3.

Abstract 3006Poster Board II-982P2Y12 inhibitors decrease the reactivity of platelets. Since the P2Y12 receptor acts through Gαai-induced signals, which inhibit adenyl cyclase, P2Y12 inhibitors are thought to act through phosphorylation of protein kinase A (PKA) substrates. One PKA substrate is serine166 (S166) of GPIbβ. Using a phospho-specific antibody, Western blots, and FACS of whole blood, we found that ADP and epinephrine, which activate P2Y12 receptors caused a dose-dependent dephosphorylation of S166 while apyrase and a P2Y12 inhibitor prevented this dephosphorylation. Since S166 functions with S609 of GPIbαa to bind 14-3-3, we determined whether i) dephosphorylation was associated with release of 14-3-3 from GPIb, and ii) 14-3-3 regulated αaIIbβ3 function. GPIb bound ∼91 ± 5% of the 14-3-3, as determined by its insolubility in streptolysin O-permeabilized platelets and its solubilization by phospho-S609 and -S166 peptides. Quantitation of 14-3-3 in GPIb immunoprecipitates showed that graded Gαai-induced dephosphorylation of S166 was accompanied by graded release of 14-3-3 from GPIb. Evidence that 14-3-3 participates in αaIIbβ3 signaling came from its co-immunoprecipitation with αaIIbβ3 from sheared platelets. Moreover, when CHO/αaIIbβ3 cell 14-3-3 was sequestered by expression of GPIb, αaIIbβ3-induced activation of RhoGTPases and cell spreading were inhibited; overexpression of 14-3-3 restored these functions. These studies: i) show that 14-3-3 is critical for αaIIbβ3-induced spreading; ii) describe a novel function of GPIb in dampening the transition to firm adhesion through the regulated sequestration of 14-3-3; and iii) suggest that the availability of 14-3-3 for αaIIbβ3-mediated adhesion is determined by the Gai-modulated level of GPIb phosphorylation. Disclosures:No relevant conflicts of interest to declare.

Filamin A Deficiency in Platelets Reveals Functional Impairment in ITAM-Based Signaling.

Abstract 769Filamin A (FlnA), encoded by the FLNA gene on the X-chromosome, crosslinks F-actin and controls the topology of the Von Willebrand factor (VWF) receptor and the integrin αIIbβ3 on platelets by connecting them to the underlying cytoskeleton. In mice, FlnA deficiency is embryonic lethal to hemizygous males. Heterozygous (HT) females are born, but experience premature mortality. FlnA confers survival value to platelets since HT female mice have a mild thrombocytopenia and 95% of their circulating platelets are FlnA positive. Mice specifically lacking FlnA expression in platelets were generated by breeding FlnAloxP/loxP females with GATA1-Cre males. FlnAloxP/y GATA1-Cre males have a severe macrothrombocytopenia and increased tail bleeding time. Their platelets lack a normal cytoskeleton and have decreased GPIbα expression, which lacks cytoskeletal linkage and has an altered surface distribution by electron microscopy. Binding of VWF to FlnA-null platelets was decreased in response to botrocetin. Under arterial shear conditions (shear rate 1,500/s), fluorescent FlnA-null platelets covered ∼70% less of the collagen-coated area via collagen-bound VWF than WT platelets. Unexpectedly, immunoreceptor tyrosine-based activation motif (ITAM)-mediated signals were severely compromised in the absence of FlnA. FlnA-null platelets had decreased α-granule secretion and integrin αIIbβ3 activation in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2 (CLEC-2). Protein tyrosine phosphorylation, including that of the protein tyrosine kinase Syk and phospholipase C-γ2, was severely impaired in FlnA-null platelets stimulated through GPVI and CLEC-2. Interaction between FlnA and Syk was detected using immunoprecipitation and purified proteins. The data reveal a novel interaction between FlnA and Syk regulating ITAM-containing receptor signaling and platelet function. Disclosures:No relevant conflicts of interest to declare.

Critical role for ERK1/2 in bone marrow and fetal liver–derived primary megakaryocyte differentiation, motility, and proplatelet formation

ObjectiveMegakaryopoiesis and platelet formation is a multistep process through which hematopoietic progenitor cells develop into mature megakaryocytes (MKs) and form proplatelets. The present study investigates the regulation of different steps of megakaryopoiesis (i.e., differentiation, migration, and proplatelet formation) by extracellar signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) in two models of primary murine MKs derived from bone marrow (BM) cells and fetal liver (FL) cells.Materials and MethodsA preparation of MKs was generated from BM obtained from femora and tibiae of C57BL6 mice. FL-derived MKs were obtained from the liver of mouse fetuses aged 13 to 15 days.ResultsFor both cell populations, activation of MEK-ERK1/2 pathway by thrombopoietin was found to have a critical role in MK differentiation, regulating polyploidy and surface expression of CD34, GPIIb, and GPIb. The MEK-ERK1/2 pathway plays a major role in migration of BM-derived MKs toward a stromal-cell−derived factor 1α (SDF1α) gradient, whereas unexpectedly, FL-derived cells fail to migrate in response to the chemokine due to negligible expression of its receptor, CXCR4. The MEK-ERK1/2 pathway also plays a critical role in the generation of proplatelets. In contrast, p38MAPK pathway was not involved in any of these processes.ConclusionThis report demonstrates a critical role of MEK-ERK1/2 pathway in MK differentiation, motility, and proplatelet formation. This study highlights several differences between BM- and FL-derived MKs, which are discussed.

Differential expression of zebrafish gpia and gpib during development

Glucose 6-phosphate isomerase (GPI), alternatively named phosphoglucose isomerase (PGI), autocrine motility factor (AMF) or neuroleukin (NLK), is a sugar metabolic enzyme catalyzing the interconversion between glucose-6-phosphate and fructose-6-phosphate. When secreted out of the cell, it can induce cellular activities of neighboring cells. As a prerequisite to study the function of gpi during development, we analyzed the sequences and expression patterns of zebrafish gpia and gpib. Phylogenetic analyses indicate that gpia and gpib are two paralogs resulting from the teleost fish-specific whole genome duplication. In adult zebrafish, gpia is widely expressed in many tissues, whereas gpib is only expressed in heart, muscle, and at lower abundance in eye. During embryonic development, gpia mRNA is maternally deposited in cleavage and blastula embryos, but gpib mRNA is not. Zygotic expression of gpia mRNA initiates in the primitive gut of segmentation embryos, and its expression increases in eye, cerebellum, hindbrain, heart, pharyngeal arches, pectoral fin buds and gut of hatching embryos at 60 hpf (hours post fertilization). At 72 hpf, gpia mRNA is strongly expressed in the retina of eye, tectum, hindbrain, and gut derivatives such as liver, intestine and circular smooth muscle layer of the swim bladder. By contrast, gpib is expressed in the yolk syncytial nuclei of late blastula, gastrula and segmentation embryos. Somitic gpib expression initiates in late pharyngula embryos, and it is most prominent in the muscle of hatching embryos around 48–60 hpf. Transcript of gpib is also expressed in the pectoral fins and portions of the pharyngeal arches in the hatching period. The differential expression of gpia and gpib in zebrafish suggests partition or divergence of gpi functions between the two duplicates.

Markers of platelet activation and apoptosis during storage of apheresis‐ and buffy coat–derived platelet concentrates for 7 days

The different production methods for platelet concentrates (PCs) result in products with variable in vitro quality and in vivo viability. The aim of this study was to compare in vitro variables of PCs produced by apheresis (AP-PC) or the buffy coat (BC-PC) method by applying a number of new and established assays. Standard TRIMA Accel (Gambro BCT) AP-PCs (n = 20) and BC-PCs (n = 20) were stored in 100 percent plasma and changes in mitochondrial membrane potential (DeltaPsi(m)) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethlybenzimidazolcarbocyanine iodide (JC-1) dye on Days 1, 3, 5, and 7. The capacity of platelets (PLTs) for oxidative phosphorylation was also monitored by measuring oxygen consumption using a Clark-type electrode. PLT viability was measured using a new assay that utilizes the vital stains calcein-AM and FM4-64. Expression of phosphatidylserine (PS), CD42b, CD47, CD61, and CD62P was also assessed. Although the JC-1 ratio (FL2/FL1) decreased significantly in both preparations, the percentage of PLTs with depolarized DeltaPsi(m) increased significantly in BC-PCs but not in AP-PCs. However, no significant change was detected in the PLTs' ability to consume oxygen in both preparations. PLTs in BC-PCs also showed significantly lower GPIb, CD47, and CD61 expression than AP-PCs on Day 1. PLTs in both preparations, however, showed a similar increase in CD62P and PS expression during storage, without significant loss of viability. PLTs in AP-PCs and BC-PCs undergo different degrees of deterioration in mitochondrial integrity and thus may undergo different degrees of apoptosis. Interventions that maintain mitochondrial integrity may improve PLT viability in vitro.

Role of PI3-K and Mlck during Activation of Thrombin Receptors

Object In order to compare the influences of wortmannin on platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptors activation, then to investigate the role of phosphatidylinositol 3-kinase (PI3-K) and myosin light chain kinase (MLCK) in the course of thrombin receptors activation.Methods Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP)were used for stimulating platelet, then the alterations of platelet aggregation and GPIb were analyzed in the involvement of 100nM wortmannin (inhibitor of PI3-K) and 10μM wortmannin (inhibitor of MLCK).Results Platelet activation was influenced by either concentration of wortmannin in response to PARs stimulation. Platelet aggregation was apparently inhibited by 10μM wortmannin following both peptides, and slightly inhibited by 100nM wortmannin only upon PAR1-AP activation. In addition, GPIbα interalisation is partly inhibited by 100nM wortmannin in response to PAR1 (P <0.05 at 1, 2, 5min) and PAR4 (P <0.05 at 2, 5, 10min) activation. Meanwhile, 10μM wortmannin induces little alteration for GPIbα centralisation in the course of PAR activation, with a delayed restoration of surface GPIbα observed upon PAR1-AP activation, and no change of GPIbα redistribution exists upon PAR4-AP activation.Conclusion All the results confirm different roles of PI3-K and MLCK in the course of thrombin receptors activation. PI3-K accelerates the short course of GPIb centralisation for two PARs signal pathways, while MLCK inhibites the restoration of GPIbα in PAR1 pathway

Influence of Protein Kinase C and Calcium in the Course of Thrombin Receptors Activation.

Object In order to compare the functions of protein kinase C (PKC) and calcium (Ca2+) in platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptors activation, then to investigate the role of Gq signal transmission pathway in the course of thrombin receptors activation.Methods Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet at different time point (0, 1, 2, 5, 10, 30min), then the alterations of platelet aggregation and GPIb were analyzed in the involvement of Ro-31-2220 (inhibitor of PKC) and BAPTA/AM (calcium chelator).Results Either PAR1 or PAR4 peptide can induce absolute platelet aggregation, together with a reversible internalization of GPIb. Platelet aggregation was inhibited by Ro-31-2220 or BAPTA/AM while the shape change curve still occurred upon PARs activation. In addition, Ro-31-2220 decreases GPIb centralisation upon PAR1 stimulation (P <0.05 at 1, 2 min), though it blocks the pool of GPIb inside platelet in PAR4 activation (P <0.05 at 10, 30 min). Meanwhile, GPIb internalization was blocked by BAPTA for both peptides (P <0.05 at 1∼10 min).Conclusion All the results confirm a critical role of Gq pathway in thrombin signal transmission through the involvement of protein kinase C and calcium. Calcium is closely correlated with the thrombin receptors activation, seemed to be similar for two PARs signal pathways. Protein kinase C urges GPIb centralisation in PAR1 pathway and accelerates GPIbα return in PAR4 pathway.

Antiplatelet Properties of Escitalopram in Patients with the Metabolic Syndrome: A Dose-Ranging In Vitro Study

There is an increasing body of evidence suggesting that selective serotonin reuptake inhibitors exhibit clinical benefit beyond treating depression, by simultaneously inhibiting platelet activity. We recently demonstrated that escitalopram (ESC), but not its major metabolites, inhibits multiple platelet biomarkers in healthy volunteers. Considering that the metabolic syndrome represents one of the major risk factors for vascular disease, we here determined how ESC affects platelet activity in such patients. We assessed the in vitro effects of preincubation with escalating (50-200 nM/l) concentrations of ESC on platelet aggregation, expression of major surface receptors by flow cytometry, and quantitatively by platelet function analyzers. Blood samples were obtained from 20 aspirin-naïve patients with documented metabolic syndrome. Pretreatment of blood samples with medium (150 nM/l), or high (200 nM/l) doses of ESC resulted in a significant inhibition of platelet aggregation induced by ADP (p=0.007) and by collagen (p=0.004). Surface platelet expression of GPIb (CD42, p=0.03), LAMP-3 (CD63, p=0.04), and GP37 (CD165, p=0.03) was decreased in the ESC-pretreated samples. Closure time by the PFA-100 analyzer was prolonged after the 200 nM/l dose (p=0.02), indicating platelet inhibition under high shear conditions. On the other hand, the lowest tested concentration of ESC (50 nM/l) did not affect platelet activity in these patients. The in vitro antiplatelet characteristics of ESC in patients with the metabolic syndrome are similar to those in healthy volunteers. However, higher ESC doses are required to induce equally potent platelet inhibition. These data justify prospective ex vivo studies with the highest therapeutic dose to determine the potential clinical advantage of ESC in high-risk patients with vascular disease.

Schwere Thrombozytopenie und sieben problemlose Geburten

Summary Severe thrombocytopenia and seven problem-free births We present four patients from two Swiss families with a rare thrombocytopathy, Bernard-Soulier syndrome (BSS), with varying haemorrhagic diatheses. The diagnosis of BSS was suspected on the basis of a low platelet count and morphological changes to the thrombocytes. The platelets showed an isolated defect on ristocetin-induced agglutination. Immunophenotyping of the thrombocytes demonstrated lack of GP Ib expression of varying degree in comparison to normal controls. The combination of these findings confirmed the diagnosis of BSS. Differential diagnosis and treatment strategies are discussed. A congenital thrombocytopathy should always be considered in patients with thrombocytopenia of unknown origin and abnormal platelet morphology.

Metabolic energy reduction by glucose deprivation and low gas exchange preserves platelet function after 48 h storage at 4 °C

We showed earlier that metabolically suppressed platelets (MSP) prepared by incubation in glucose-free, antimycin A medium at 37 degrees C better sustained storage at 4 degrees C than untreated controls at 22 degrees C. However, the use of the mitochondrial inhibitor antimycin A is incompatible with platelet transfusion. The aim of this study was to investigate how energy-reduced (ER) platelets could be prepared in the absence of antimycin A. Platelets in gas-impermeable bags in glucose-free medium were kept at 22 degrees C for 4 h to reduce energy stores and thereafter stored at 4 degrees C (ER22-4). Controls were energy-reduced platelets without prior incubation at 22 degrees C (ER4), and MSPs in test tubes and untreated platelets in gas-permeable bags with glucose and stored at 22 degrees C (C22) and 4 degrees C (C4). After 48 h storage, ER22-4 were superior to C22 with respect to pH preservation (6 x 4 +/- 0 x 4 vs. 5 x 0 +/- 0 x 4, n= 4), platelet count (800 +/- 225 vs. 650 +/- 150 x 10(9)), thrombin receptor-activating peptide-induced aggregation (50 +/- 15 vs. 10 +/- 5%) and glycoprotein (GP)Ib alpha expression (60 +/- 15% vs. 28 +/- 15). GPIb alpha expression was higher in ER22-4 than in ER4, indicating that energy suppression preserved GPIb alpha during cold storage. Metabolic suppression without the use of antimycin A could be mimicked by storage of platelets in glucose-free medium in gas-impermeable bags. Energy suppression preserved GPIb alpha expression during storage at 4 degrees C.

Role of adenosine diphosphate in the course of thrombin signal transmission

To study the effects adenosine diphosphate (ADP) on platelet aggregation and expression of glycoprotein (GP) on the surface of platelet membrane after activation of thrombin receptors, so as to investigate its role in thrombin signal transmission. Peripheral blood samples were collected from from 10 healthy volunteers. Platelets were extracted. The thrombin receptor activating peptides (TRAP), protease-activated receptor 1 activated peptide (PAR1-AP, SFLLRN, 25 micromol/L) and PAR4-AP (AYPGKF, 250 micromol/L) were added into the suspension of platelets respectively to induce platelet aggregation. In apyrase inhibition test apyrase II was added into the suspension of platelets for 2 hours and then PAR1-AP or PAR4-AP was added respectively to observe the the expression of GPIb and P-selectin with flow cytometry. Either PAR1 and PAR4 induced platelet aggregation. After apyrase II stimulation the PAR4-AP induced platelet aggregation was not influenced and PAR1-AP induced platelet aggregation was partially inhibited with a reversible aggregation curve. Stimulated by PAR1-AP and PAR4-AP the GPIb decreased firstly and then gradually returned to normal. Apyrase VII have not significant influence on the GPIb expression, but accelerated the return of GPIb to the platelet surface after PAR1 stimulation so that the lowest point was accelerated to 2 min, compared to that of the control group (5 min) and there were significantly differences 10 and 30 min later between these 2 groups (all P < 0.05). The P-selectin expression was remarkably increased 2 min after the PAR1-AP and PAR4-AP induction and peaked 2 min later. Apyrase VII did not significantly influence the P-selectin expression in these 2 activation ways. ADP plays an important role in the thrombin signal transmission, especially in the PAR1 pathway.

Metalloproteinase Regulation Determines Integrin αIIbβ3 Function and Enhances the Capacity of ES Cell-Derived Platelets.

To resolve the lack of sources for products used in blood transfusion therapy, the utilization of embryonic stem cells (ESCs) has been proposed. However, the use of ESC-derived cells has potential drawbacks including, immunological rejection and teratoma formation. Being fully differentiated cells lacking a nucleus, platelets can be irradiated to eliminate passenger ESCs and other lineages before transfusion. We previously established in vitro differentiation system whereby proplatelet-producing megakaryocytes can be derived from murine ESCs on OP9 stroma, but the platelets were generated with a low efficiency and exhibited weak agonist-induced integrin αIIbβ3 activation and spreading on immobilized fibrinogen. Here, we describe an improved method to obtain ESC-derived platelets of potential use for hemostasis and thrombosis in vivo. We generated a novel murine ESC line wherein the GPIbα promoter regulates EGFP expression as a marker (GP-G ESCs). The combination of flow cytometry with GP-G-derived embryoid bodies and OP9 co-culture enabled us to enrich integrin αIIb + c-kit + CD9 + Sca-1 − progenitors on day 5–6 of culture as a major fraction capable of differentiating into GFP+ mature megakaryocytes on day 8–10 in the presence of TPO. This strategy increased platelet production by 10-fold (P<0.01) compared to our previous report (PNAS, 2002, 99:12819). Interestingly, αIIb+platelets included 35–40% GPIbα-negative population besides GPIbα-positive on day 13–15, which was caused by an increased activity of matrix metalloproteinase (MMP) and TACE (ADAM17) to shed GPIbα in culture. Inhibition of MMP and TACE with GM6001 or TAP1 delayed the clearance rate on post transfusion recovery in vivo as transfused into a platelet-depleted mouse model. Maintaining GPIbα expression in the presence of MMP/TACE inhibitors restored the specific fibrinogen-bound to integrin αIIbβ3 and platelet spreading on fibrinogen as evidenced by the generation of filopodia and lamellipodia. Collectively, these results suggest that 1) ESC-derived αIIb+ c-kit + CD9+ Sca-1 − progenitors may contribute to enrich megakaryocytopoiesis at high efficiency; 2) inhibition of metalloproteinase activity to retain GPIbα expression is required for αIIbβ 3 integrin inside-out and outside-in signaling in ESC-derived platelets; and 3) these platelets may be useful alternatives for future clinical application.

Deletion of Two Contiguous Genes, Platelet GPIbβ (Glycoprotein Ibβ) and Septin SEPT5, in a Boy with Bernard-Soulier Syndrome and Developmental Delay: A Possible New Contiguous Gene Syndrome.

Mutations or deletions in the genes for platelet GPIb/V/IX (consisting of GPIbα, GPIbβ, GPV, GPIX) cause the bleeding disorder Bernard-Soulier syndrome. Septins are GTP-binding proteins essential for active membrane movement such as vesicle trafficking. In non-dividing cells (i.e. platelets) septins are implicated in exocytosis. Platelets from a SEPT5 knockout mouse show altered serotonin secretion and platelet aggregation suggesting that SEPT5 is involved in secretion in platelets.We report on a 4 year old boy born to consanguineous parents who developed several life-threatening mucosal bleeding episodes (when he suffered from upper respiratory infections) and showed developmental delay. Giant platelets were present in the blood smear, and ristocetin-induced platelet aggregation was reduced indicating that the boy suffered from Bernard-Soulier syndrome. Immune transmission electron microscopy of the platelets showed reduced expression of GPIb. PCR- and Southern analysis demonstrated that exon 1 and partly exon 2 of GPIbβ was deleted on both alleles. Further experiments showed that the deletion extended 5′ from exon 1 of GPIbβ to include the whole gene coding for SEPT5 located 5′ of GPIbβ. After bone marrow transplantation from a HLA-identical unrelated donor the boy developed no further bleeding symptoms.Conclusions: We identified a patient with Bernard-Soulier syndrome and mental retardation who had not only a homozygous deletion of exon 1 and partly exon 2 of GPIbβ but also a deletion of the whole adjacent SEPT5 gene, possibly indicating a novel contiguous gene syndrome. Whether absence of SEPT5 contributed to particularly severe bleeding symptoms and to developmental delay remains to be proven.

Impaired Viability, Platelet Survival, Morphology and Function in Mice Lacking Filamin A.

Platelet filamin A (FlnA), encoded by the FLNA gene on the X-chromosome, cross-links actin filaments, links them to the GPIbα subunit of the von Willebrand factor (vWf) receptor in the plasma membrane, and serves as a scaffold for signaling proteins. Mice lacking FlnA were generated. FlnA deficiency is embryonic lethal in hemizygous males due to pericardial and visceral hemorrhage. Heterozygous females are born, but experience premature mortality. The distribution of FlnA-expressing hematopoietic cells in FlnA+/− females was investigated by intracellular flow cytometry. About half of all hematopoietic cells investigated express FlnA, including bone marrow megakaryocytes, suggesting a random X-chromosome inactivation in these cells. However, about 90% of all blood platelets express FlnA. Mice lacking platelet FlnA were further generated by breeding FlnA loxP females with GATA-1 Cre males. FlnA loxP/+ GATA-1 Cre female offspring outnumber FlnA loxP/y GATA-1 Cre males by a ratio of 7:1. FlnA loxP/y GATA-1 Cre males have about 25% of normal blood platelet counts and their platelets are large, lack a normal actin cytoskeleton, and have decreased GPIbα expression, which has an altered distribution by electron microscopy. FlnA-null platelets are cleared rapidly from the circulation when transfused into GATA-1 Cre recipients and have severely impaired functional and signaling responses downstream of the collagen receptor GPVI, i.e. α-granule secretion, integrin αIibβ3 activation and protein tyrosine phosphorylation. Surprisingly, thrombin-mediated GPIbα down-regulation and botrocetin-mediated vWf binding are minimally affected in FlnA-null platelets. The data show the critical nature of FlnA for platelet survival, morphology and function.

Platelet inhibition with prasugrel (CS-747) compared with clopidogrel in patients undergoing coronary stenting: the subset from the JUMBO study

Background: Based on the preclinical and phase 1 studies, prasugrel, a novel platelet ADP P2Y12 receptor blocker, may be a more potent platelet inhibitor than clopidogrel. This study compared the...

Two Candidate Genes for Low Platelet Count Identified by Genome-Wide Linkage Analysis: Glycoprotein IX and Thrombopoietin.

Although the 'normal' range of platelet count in a healthy person is quite broad (150 to 400 ×109/L), an individual's platelet count is maintained within a much narrower range, constantly balanced between thrombopoiesis and senescence. Platelet counts within the normal range have been shown to be largely determined by genetic factors with heritability estimates from twin studies ranging from 0.57 to 0.80. A genome-wide linkage analysis was carried out in a large Asian Indian kindred with β thalassemia to search for genes influencing variation in normal platelet count. Significant linkage is shown at two locations on chromosome 3q with age, sex and β thalassemia adjusted platelet counts. Linkage analysis showed one marker (D3S1309) on chromosome 3q with a lod score of 3.26 and another marker (D3S1282) approximately 30 cM centromeric, with a lod score of 2.52. Multipoint linkage analysis across 90 cM of chromosome 3q identified two peaks across the region with maximum multipoint lod scores of 3.52 and 4.11 under markers D3S1309 and D3S1282, respectively. Two strong candidate genes for platelet variation were identified in the linked region; thrombopoietin (THPO) and glycoprotein IX (GPIX). Resequencing of four individuals revealed five single nucleotide polymorphisms (SNPs) in THPO and one mutation in the transmembrane region of GPIX. Analysis of variance showed that the GPIX mutation and one THPO SNP accounted for 6% and 4% of the variation in platelet count in the kindred, respectively. The THPO SNP lies in the 3′ untranslated region (3′ UTR) of the gene and has not been previously reported.Although no function could be attributed to the THPO SNP it could potentially influence post-transcriptional control of gene expression. The G to A transition in nucleotide 653 (Ref seq NM_000174) resulted in an Ala 156 (GCC) to Thr (ACC) replacement in the GPIX protein. The GPIX mutation was recently identified in a Chinese patient with Bernard-Soulier syndrome (BSS), a rare bleeding disorder characterized by severe thrombocytopenia and giant platelets. The Chinese patient was homozygous for the mutation; however, heterozygous relatives did not show the characteristic BSS symptoms, leading the authors to conclude that the mutation acted under a recessive model of inheritance. Transient transfection studies by the same group confirmed that the mutation prevents GP Ib/IX complex insertion in the cytoplasmic membrane of platelets, and suppresses GPIbα and GPIX expression, but does not cause its intracellular degradation. One copy of the GPIX mutation was found in 300 European individuals with platelet counts within the normal clinical range.Conclusion: Although the genetic determinants of many thrombocythemias and congenital thrombocytopenias have been identified, very little is known about the genetic determinants of normal platelet count variation. The results suggest that two QTLs on chromosome 3q influence platelet count variation in the Asian Indian kindred, with the GPIX transmembrane mutation and the 3′ UTR SNP in THPO, being strong candidates.

Redistribution of Activation-Dependent Antigens on the Platelet Surface In Vivo and In Vitro.

Platelets play a crucial role in thrombosis, platelet activation induces an alteration of platelet membrane glycoproteins between the platelet surface and surface connected canalicular system (SCCS). In an attempt to better understand this process and assess its significance in the study of thrombosis disease, we used flowcytometry for detecting the redistribution of antigens on the platelet surface following activation. The expression of platelet membrane glycoproteins (GP) Ib, IIIa and P-selectin as well as the platelet agglutination induced by ristocetin were measured in 20 normal subjects, whose platelets were stimulated by the peptide SFLLRNP(TRAP, thrombin receptor activated peptide) at different time points (0~30minute). The same antigens were also analyzed in whole blood in 30 patients with cerebral infarction. As expected, a stable increase of P-selectin was obtained from 2 minute upon TRAP activation, while a reversible reduction of GPIbα and agglutinability by ristocetin were showed at the same time, with a maximum decrease at 2 minute, then followed by a return of GP Ibα to platelet surface. In addition, the platelet membrane GPIIIa was not significantly changed in the initial stage after platelet stimulation and increased by degrees 10 minutes later. Compared with those in normal group, there was an apparent increase of P-selectin and a lower expression of GPIb in the patients with cerebral infarction, while GPIIIa was not significantly changed in all the samples. In summary, TRAP can mediate platelet activation, which leads to the redistribution of GPIb and GPIIIa in a time-dependent manner, together with the release of P-selectin. Meanwhile, the change of activation-dependent antigens on the platelet surface could be an important index reflecting the platelet activation in vivo.

Platelet glycoprotein I(b)alpha and integrin alpha2 beta1 polymorphisms: gene frequencies and linkage disequilibrium in a population diversity panel.

Genetic variants in the GP1BA and ITGA2 genes have been proposed as potential modifiers for arterial vascular disease and bleeding disorders. Since ancestry may play an important role in the prevalence of these variants, we sought to determine their allele frequency and linkage disequilibrium in a collection of 1064 DNA samples from 51 ethnic groups. We studied haplotypes of ITGA2 defined by single nucleotide substitutions at positions -52, 807, and 1648, and GP1BA variants defined by sequence changes in positions -5 (Kozak), 1018 (T145M, HPA-2) and 1285 (VNTR A, B, C and D). Frequency of haplotypes of ITGA2 showed considerable variation across the different groups, with a higher prevalence of the haplotype -52C or T/807C/1648A observed in African compared with caucasian and Asian populations. The haplotypes 52C/807T/1648A and -52T/807T/1648A were not observed in caucasians or South Americans. While relative frequencies of the GP1BA Kozak alleles were comparable across groups, the methionine allele (HPA-2b) showed a higher frequency in Africa (0.26) than in the other groups. We also observed a high prevalence of the VNTR B allele in the African and Israeli populations. Haplotype analysis revealed incomplete linkage disequilibrium between the HPA-2 and VNTR alleles. Incorporation of GP1BA variants into the set of SNPs already genotyped by the HapMap project disrupted the pre-existing haplotype block. These data provide a valuable resource for optimal selection of variants best tailored for association studies of vascular disease or bleeding disorders when examining individuals of different ancestral origins.