Platelet Fibrinogen Receptor Research Articles

Treatment of Primary Immune Thrombocytopenia with Thrombopoietin Receptor Agonists: Effect On Platelet Function and Plasma Thrombin Generation

Abstract 1089 Introduction:Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by mild to severe thrombocytopenia caused by autoantibodies against platelet proteins that lead to platelets destruction and suboptimal platelet production. Platelet count and bleeding phenotype vary widely between ITP patients. In spite of the low platelet number, some thrombocytopenic ITP patients seldom bleed which might indicate the presence of additional mechanisms that contribute to the haemostasis in such patients. Objective:Thrombopoietin receptor agonists (TPO-RA) have recently been introduced for the treatment of patients with ITP and can noticeably increase platelet count in most of the ITP patients, even in those with severe refractory thrombocytopenia. The aim of this work was to study platelet function and thrombin generation in thrombocytopenic ITP patients before treatment with TPO-RA and once they had recovered the normal platelet count as consequence of the treatment. Methods:Fourteen ITP patients with thrombocytopenia (TP-ITP, platelet count less than 100,000 platelets/microliter) were studied before starting TPO-RA treatment (4 patients with romiplostim and 10 patients with eltrombopag) and after reaching a platelet count higher than 100,000 platelets/microliter (NP-ITP). Thirty-three healthy subjects were included as the control group.Platelet activation was determined by flow cytometry through binding of FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 microM TRAP activated platelets. Immature platelet fraction was determined labeling platelets with thiazole orange. Expression of fibrinogen receptor was determined by flow cytometry with specific mAbs. Apoptosis was determined by flow cytometry through FITC-annexin V binding to phosphatidylserine (PS).Thrombin generation was measured in platelet-free plasma by the method of Hemker (Calibrated Automated Thrombography, CAT). Activation was performed with a final concentration of 4 microM of phospholipids and 1 pM of tissue factor.Comparisons of quantitative variables were made with ANOVA and Dunn test. Results were expressed as mean±SD. Values of p≤0.05 were considered statistically significant. Results:Platelets from TP- and NP-ITP patients showed a basal expression of activated fibrinogen receptor similar to controls. Platelets from TP-ITP patients presented a reduced ability for being activated by TRAP (binding of PAC-1 expressed as % of positive platelets: 89±19 % in controls and 56±22 % in TP-ITP, p<0.01). TPO-RA treatment did not improve platelet activation despite increasing platelet count (binding of PAC-1: 47±19 % in NP-ITP, p<0.01). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients. Platelets from either TP- or NP-ITP patients, expressed more PS than controls under basal conditions. Percentage of platelets that bound FITC-annexin V were: 47±12 % in controls, 63±13 % in TP-ITP and 60±15 % in NP-ITP, p<0.001). Percentage of immature platelets in TP-ITP patients were higher than in controls (0.8±0.9 % in controls and 6.6 ±4.0 % in TP-ITP, p<0.05) and was reduced by TPO-RA treatment (in NP-ITP patients: 2.2 ±1.7 %).CAT experiments showed that TP-ITP patients had an increased endogenous thrombin potential (ETP, p<0.001) and reached higher maximum levels of thrombin (peak height, p <0.001) than controls. This procoagulant characteristic of plasma from TP-ITP patients was unchanged after treatment with TPO-RA and the recovery of a normal platelet count. Conclusions:Our results showed that treatment of ITP patients with TPO-RA is effective for increasing platelet production but did not ameliorate platelet function or procoagulant conditions of the disease which may indicate that treatment with TPO-RA do not interfere with the mechanisms involved in impairment of platelet function and generation of a procoagulant state. This increased thrombin generation of plasma from ITP patients has to be taken under consideration when evaluating thrombotic risk of therapeutic treatments, and we would like to point out the need of performing more studies to elucidate causes of the increased thrombogenic potential observed in ITP patients. Disclosures:No relevant conflicts of interest to declare.

Clinical utility gene card for: Glanzmann thrombasthenia

Glanzmann thrombasthenia (Glanzmann–Naegeli syndrome, platelet αIIbβ3 deficiency, platelet fibrinogen receptor deficiency). OMIM# of the disease 273800. Analyzed genes or DNA/chromosome segments ITGA2B (αIIb) and ITGB3 (β3) – chromosome 17q21.32. OMIM# of the gene(s) 607759 (ITGA2B) and 173470 (ITGB3). Review of the analytical and clinical validity as well as of the clinical utility of DNA-based testing for mutations in diagnostic and prenatal settings, and for risk assessment in relatives.

Preventing Platelet Thrombosis With a PAR1 Pepducin

Platelet-mediated arterial thrombosis, such as occurs after atherosclerotic plaque rupture, erosion, or percutaneous coronary interventions (PCI), is the underlying cause for most myocardial infarctions and many ischemic strokes.1 Platelets adhere to damaged blood vessels, aggregate with one another, and facilitate the generation of thrombin, which in turn makes fibrin. Thrombin is also a highly potent stimulator of platelets, and shear stress in settings such as PCI may lead to periprocedural complications due to thrombin-dependent platelet activation.2,3 Human platelets possess 2 main thrombin G-protein–coupled receptors, protease-activated receptor (PAR) 1 and 4 (Figure), which, when cleaved by thrombin, trigger a host of intracellular signaling events resulting in secretion of granule contents including ADP, production of thromboxane A2, and activation of the platelet fibrinogen receptor integrin αIIbβ3 (GPIIb/IIIa). In the setting of acute coronary syndrome and PCI, antiplatelet therapy for secondary prevention of vascular events consists of aspirin to reduce thromboxane A2 production, P2Y12 antagonists to block the effects of ADP, and GPIIb/IIIa inhibitors. However, despite the use of these therapies, the rate of ischemic events remains high. Furthermore, this approach is estimated to prevent only ≈15% to 17% of lethal cardiovascular events with a ceiling effect.4,5 The ability of thrombin to activate platelets in the presence of aspirin and P2Y12 antagonists may explain some of the residual risk. Thus, strategies that target thrombin signaling in platelets have been the focus of considerable attention. Figure. Platelet activation pathways, the role of thrombin receptors. In humans, protease-activated receptors (PAR)-1 and PAR-4 …

Ectonucleotide Triphosphate Diphosphohydrolase-1 (CD39) Mediates Resistance to Occlusive Arterial Thrombus Formation after Vascular Injury in Mice

Modulation of purinergic signaling, which is critical for vascular homeostasis and the response to vascular injury, is regulated by hydrolysis of proinflammatory ATP and/or ADP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) to AMP, which then is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine. We report here that compared with littermate controls (wild type), transgenic mice expressing human ENTPDase-1 were resistant to the formation of an occlusive thrombus after FeCl(3)-induced carotid artery injury. Treatment of mice with the nonhydrolyzable ADP analog, adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S], negated the protection from thrombosis, consistent with a role for ADP in platelet recruitment and thrombus formation. ENTPD-1 expression decreased whole-blood aggregation after stimulation by ADP, an effect negated by adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S] stimulation, and limited the ability to maintain the platelet fibrinogen receptor, glycoprotein α(IIb)/β(3), in a fully activated state, which is critical for thrombus formation. In vivo treatment with a CD73 antagonist, a nonselective adenosine-receptor antagonist, or a selective A(2A) or A(2B) adenosine-receptor antagonist, negated the resistance to thrombosis in transgenic mice expressing human ENTPD-1, suggesting a role for adenosine generation and engagement of adenosine receptors in conferring in vivo resistance to occlusive thrombosis in this model. In summary, our findings identify ENTPDase-1 modulation of purinergic signaling as a key determinant of the formation of an occlusive thrombus after vascular injury.

The platelet fibrinogen receptor: from megakaryocyte to the mortuary

Platelets are integral to normal haemostatic function and act to control vascular haemorrhage with the formation of a stable clot. The fibrinogen receptor (glycoprotein IIb/IIIa [GPIIb/IIIa]) is the most abundant platelet integrin and, by binding fibrinogen, facilitates irreversible binding of platelets to the exposed extracellular matrix and enables the cross-linking of adjacent platelets. The vital role of GPIIb/IIIa requires tight control of both its synthesis and function. After transcription from distinct domains on chromosome 17, the two subunits of the heterodimer are carefully directed through organelles with intricate regulatory steps designed to prevent the cellular expression of a dysfunctional receptor. Similarly, exquisite control of platelet activation via bidirectional signalling acts to limit the inappropriate and excessive formation of platelet-mediated thrombus. However, the enormous diversity of genetic mutations in the fibrinogen receptor has resulted in a number of allelic variants becoming established. The Pro33 polymorphism in GPIIIa is associated with increased cardiovascular risk due to a pathological persistence of outside-in signalling once fibrinogen has dissociated from the receptor. The polymorphism has also been associated with the phenomenon of aspirin resistance, although larger epidemiological studies are required to establish this conclusively. A failure of appropriate receptor function due to a diverse range of mutations in both structural and signalling domains, results in the bleeding diathesis Glanzmann's thrombasthaenia. GPIIb/IIIa inhibitors were the first rationally designed anti-platelet drugs and have proven to be a successful therapeutic option in high-risk primary coronary intervention. As our understanding of bidirectional signalling improves, more subtle and directed therapeutic strategies may be developed.

Platelet Functions and Risk Prognosis in Myelodysplastic Syndromes,

Abstract 3806▪▪This icon denotes a clinically relevant abstractThe myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem cell disorders with peripheral cytopenias and increased incidence of leukemic transformation.The prognosis of MDS is determined by several factors, including the presence of specific cytogenetic abnormalities, the percentage of blastoid cells in bone marrow and peripheral blood, the number of affected cell lineages, and transfusion dependency. The most commonly used risk stratification system is the International Prognostic Scoring System (IPSS). This score divides patients into a lower risk subset (low and intermediate-1) and a higher risk subset (intermediate-2 and high).Patients with MDS may have hemorrhagic complications with serious outcomes that are among the major causes of death in this population. These bleeding episodes that are often related to thrombocytopenia also occur in MDS patients with normal platelet count.The aim of this work was to study functional characteristics of platelets in MDS patients and their relationship to risk evaluated as indicated by IPSS.Eighty diagnosed MDS patients risk-stratified according to IPSS were included: 40 with low-risk, 29 with intermediate-1-risk (I-1), 8 with intermediate-2-risk (I-2) and 3 with high-risk. Eighty healthy donors were included as control group.Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-μm aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time, CT), which is expressed in seconds.Platelet activation was determined through FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 μM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (αIIb and β3 subunits) was determined by flow cytometry with specific mAbs.Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane phosphatidylserine (PS) exposed in basal conditions.I-2 and high-risk patients were gathered together in a high-risk group in order to analyze experimental results. Statistical analysis was performed with one-way ANOVA and Tukey test.CTs obtained with COL-EPI and COL-ADP cartridges in controls and low risk patients were similar and significantly shorter than CTs observed in I-1-risk and high-risk MDS patients (p<0.05).Platelets from all MDS patients showed a reduced capability for being activated by 100 μM TRAP. This impairment was more evident in I-1-risk and high-risk patients: PAC-1 binding, in arbitrary units (AU), was 11368±1017 in controls; 7849±789 in low-risk MDS (p<0.05); 4161±591 in I-1-risk MDS (p<0.01 versus control and p<0.05 versus low-risk) and 492±184 in high-risk MDS (p<0.01 versus control and p<0.05 versus low-risk). The platelet surface expression of P-selectin induced by 100 μM TRAP was also reduced: 5102±340 AU in controls, 3318±400 AU in low-risk MDS (p<0.05); 1880 ±197 AU in I-1-risk MDS (p<0.05 versus control and versus low-risk), and 1211±130 AU in high-risk MDS (p<0.05 versus control and versus low-risk). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients.Platelets from MDS patients expressed more PS than controls under basal conditions. Mean fluorescence values for FITC-annexin binding were: 383±16 in controls; 444±21 in low-risk (p<0.05); 575±52 in I-1-risk MDS (p<0.05 versus control and versus low-risk); 611±17 in high-risk MDS (p<0.05 versus control and versus low-risk).Our results indicated that platelets from MDS patients had less ability to be activated and were more apoptotic than control ones. These dysfunctions were more pronounced when the risk of the disease was higher according to IPSS. Disclosures:No relevant conflicts of interest to declare.

Role of Thiol Isomerase ERp5 in Thrombus Formation

Abstract 370Protein disulfide isomerase is required for thrombus formation in various in vivo models of thrombosis. Another member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5), is released from activated platelets and co-immunoprecipitates with beta 3 integrin (Jordan et al, 2005). We further investigated the association of ERp5 with the platelet fibrinogen receptor alpha IIb beta 3 and the significance of ERp5 release in thrombus formation in vivo. Recombinant purified ERp5 was labeled with Alexa 488 and used in direct binding assays to CHO cells expressing wild type (WT) alpha IIb beta 3, CHO cells expressing mutant alpha IIb beta 3 (containing an Asp119Tyr substitution in the beta 3 subunit) and to control CHO cells. The mutant alpha IIb beta 3 does not bind fibrinogen. ERp5 bound to CHO cells expressing wild type (WT) alpha IIb beta 3 in a dose-dependent manner but did not bind to CHO cells expressing mutant alpha IIb beta 3 or to control CHO cells. The relative increase in the geomean of Alexa 488-labeled ERp5 binding to 0.5 ×106 WT alpha IIb beta 3 CHO cells over that bound to control CHO cells was 20, 45 and 85% for ERp5 concentrations of 80, 160 and 400 nM respectively. Binding of ERp5 (160 nM) to WT alpha IIb beta 3 expressing CHO cells was further increased by 75% when the integrin was activated with 2 mM Mn2+ compared to non-activated WT alpha IIb beta 3 CHO cells. A role for ERp5 in thrombus formation was studied in the laser injury model of thrombosis in mouse cremaster arterioles using a rabbit polyclonal anti-ERp5 antibody, immunoaffinity purified against recombinant ERp5. This antibody detected ERp5 in the releasate of thrombin-activated mouse platelets in vitro by Western blot and on the surface of thrombin-activated mouse platelets by flow cytometry. Dylight 649-labeled anti-CD42b was infused into the mouse circulation to detect platelet accumulation and Alexa 488-labeled anti-ERp5 antibody at 0.05 ug/g, a dose that does not inhibit thrombus formation, was infused to detect ERp5. The fluorescent anti-ERp5 signal detected at the thrombus site was compared to the signal produced by a non-specific IgG labeled with Alexa 488 infused into a control mouse. Anti-ERp5 fluorescence was detected in the thrombus with kinetics that followed platelet accumulation whereas there was minimal signal from the control IgG. We examined whether higher doses of anti-ERp5 affect thrombus formation. Platelet and fibrin accumulation were detected using fluorescently labeled anti-CD42b antibody and monoclonal anti-fibrin-specific antibody respectively before or after the injection of unlabeled anti-ERp5 antibody or pre-immune IgG at 2.5 ug/g. Platelet and fibrin accumulation, expressed as area under the curve of the median integrated fluorescence over time, was obtained from 14 thrombi in 6 mice formed before infusion of antibody, 18 thrombi in 2 mice formed after infusion of control IgG and 29 thrombi in 3 mice formed after infusion of anti-ERp5. Anti-ERp5 infusion caused a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (p<0.01). There was no difference in platelet and fibrin accumulation before infusion of antibody and after infusion of control antibody. These results provide evidence for a role of a second thiol isomerase, ERp5, in thrombus formation, a function which may be mediated through its association with alpha IIb beta 3. Disclosures:No relevant conflicts of interest to declare.

Fibrinogen replacement therapy: a critical review of the literature.

Fibrinogen is a plasma glycoprotein with a molecular weight of 340 kDa; it is synthesised by the liver. The conversion of fibrinogen to fibrin is catalysed by thrombin and plays a key role in clot formation and stabilisation. In addition, fibrinogen induces platelet activation and aggregation by binding to the platelet fibrinogen receptor glycoprotein GPIIb/IIIa1. Fibrinogen replacement therapy is currently indicated as prophylaxis and therapy of haemorrhage in congenital and acquired fibrinogen deficiency, this latter being associated with liver failure, disseminated intravascular coagulation, massive transfusion and cardiac surgery)2,3. Hypofibrinogenaemia is defined by a decreased level of normal fibrinogen between 0.5 g/L and the lower limit of the normal range for the local laboratory (usually 1.5 g/L)4. Fibrinogen supplementation can be provided by transfusion of fresh-frozen plasma (FFP), cryoprecipitate and fibrinogen concentrate5,6. However, as FFP has several limitations including a low fibrinogen content, which means that large volumes must be given, and the risk of transfusion-related complications (e.g., transfusion-related acute lung injury [TRALI] and viral transmission), this critical analysis is focused on the role of cryoprecipitate and fibrinogen concentrate.

Platelet Apoptosis and Agonist Mediated Activation In Myelodysplastic Syndromes

AbstractAbstract 1866Myelodysplastic syndromes (MDS) comprise distinct disorders characterized by dysplastic and ineffective hematopoiesis that seems to be related to an increased apoptosis of bone marrow cells (Nimer, Blood 111: 4841, 2008). Clinical manifestations in MDS range from a diverse degree of anemias, leuko- or thrombocytopenias to severe transfusion-dependent peripheral pancytopenias. Thrombocytopenia and platelet dysfunction contribute to hemorrhagic complications observed in MDS. Many of the features of apoptosis such as membrane fragmentation, microvesiculation and phosphatidylserine (PS) exposure are observed during platelet activation to a procoagulant state, raising the possibility that apoptosis may regulate platelet function.The aim of this work was to determine whether a correlation exists between apoptosis and activation processes in platelets from MDS patients.Twenty six patients diagnosed MDS and classified according to WHO-2008 were included: 6 with refractory anemia (RA), 7 with RA with ringed sideroblasts (RARS), 6 with refractory RA with excess blasts-1 (RAEB-1) and 7 with cytopenia with multilineage dysplasia (RCMD) associated with isolated 5q deletion. Twenty six healthy donors were included as control group.Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane PS exposed under basal conditions and after stimulation with a PAR-1 receptor agonist (TRAP, SFLLRN, thrombin receptor-activating peptide 6). Levels of pro- apoptotic Bax and anti-apoptotic Bcl-2 proteins were determined by densitometric analysis of western blots performed with platelet lysates.Platelet activation was determined through FITC-fibrinogen, FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 mM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (aIIb and b3 subunits) was determined by flow cytometry with specific mAbs.Platelets from RA, RARS and RCMD patients expressed more PS than control ones under basal conditions (p<0.05) as well as after 100 mM TRAP stimulation (p<0.05). Moreover, platelets from these MDS patients expressed more Bax protein than control group (p<0.05).On the other hand, PS exposure and Bax content in platelets from RAEB-1 patients were similar to controls, but they expressed a higher amount of Bcl-2 (p<0.05). No correlation was observed between PS exposure or Bax expression and platelet number.Platelets from all MDS patients showed an impaired activation by TRAP, even when PS exposure was higher than in control group. This diminished response to TRAP was not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients.Our results suggest that platelets from RA, RARS and RCMD patients are more apoptotic than control ones and that a correlation between platelet surface PS and activation does not seem to exist. Moreover, dissimilarity in expression pattern of apoptotic proteins among MDS types indicates differences in the intracellular mechanisms underlying these pathologies. Disclosures:No relevant conflicts of interest to declare.

Platelet Activation and Biofilm Formation by Aerococcus urinae , an Endocarditis-Causing Pathogen

The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons. Biofilm formation and platelet aggregation are believed to contribute to bacterial virulence in IE. Five A. urinae isolates from human blood were shown to form biofilms in vitro, and biofilm formation was enhanced by the presence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet-rich plasma from healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets, as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and on platelet activation since it was sensitive to both trypsin and prostaglandin E(1). Plasma proteins at the bacterial surface were needed for platelet aggregation; and roles of the complement system, fibrinogen, and immunoglobulin G were demonstrated. Complement-depleted serum was unable to support platelet aggregation by Au3 and complement blockade using compstatin-inhibited platelet activation. Platelet activation by Au3 was inhibited by blocking of the platelet fibrinogen receptor, and this isolate was also shown to bind to radiolabeled fibrinogen. Removal of IgG from platelet-rich plasma by a specific protease inhibited the platelet aggregation induced by A. urinae, and blockade of the platelet FcRγIIa hindered platelet activation induced by Au3. Convalescent-phase serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregate platelets in an otherwise nonresponsive donor. Our results show that A. urinae exhibits virulence strategies of importance for IE.

The Critical Function of c-Myb and CD9 On Megakaryopoiesis.

Abstract 4587The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival and differentiation of hematopoietic cells. We previously reported that the c-Myb knock down (KD) mice exhibited anemia and thrombocytosis. To better understand the transcriptional regulatory program that accompanies the decrease of c-Myb, we performed a transcriptome analysis of megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs). We found that CD9 expression was dramatically increased in the MEPs from c-Myb KD mice. CD9 belongs to a tetraspanin family and regulates cell motility and adhesion. Several reports described that CD9 is involved in platelet function by forming a complex with integrin family members including platelet fibrinogen receptor integrin aIIb-b III. To elucidate the functional contribution of CD9 to the thrombogenesis within the regulatory hierarchy mediated by c-Myb, we first examine the relation between c-Myb and CD9. We found the recruitment of c-Myb to the promoter region of CD9 by chromatin immunoprecipitation assay. A reporter assay showed that c-Myb represses CD9 promoter activity. These results indicate that CD9 is directly repressed by c-Myb. Since an agonistic antibody against CD9 stimulated megakaryocytic colony formation, we investigated the role of CD9 during megakaryopoiesis in vivo by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Consistent with this result, the recovery phase of platelet counts were delayed in the CD9-null mice after the bone marrow suppression with 5-fluorouracil, indicating the impaired platelet production. In CD9-null mice, the proliferation of megakaryocytes was promoted and circulating megakaryocytes in the peripheral blood were increased, which might compensate the PPF impairment of CD9-null megakaryocytes. Our study suggests that c-Myb suppresses CD9 expression under the steady state, while, in the stress megakaryopoiesis, CD9 is derepressed and acts to enhance platelet production. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis. Disclosures:Ninomiya:Alexion: Research Funding.

Glanzmann Mutations : New Allelic Variants and Role of the β3 Lys253 in the αIIb/β3 Interaction Highlighted by the Lys253Met Substitution.

Abstract 1312Poster Board I-336 IntroductionGlanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation. GT results from defects of the platelet fibrinogen receptor αIIbβ3. GT mutations provide useful tools for structure-function relationship studies of αIIbβ3. Patient and methodsGenomic DNA from 6 patients has been amplified for αIIb and β3 promoters and exons sequences. PCR products were directly sequenced. Potential RNA processing alterations have been studied in silico by using Genscan, NNSPLICE and ESEFinder online tools. When no RNA splicing anomaly was predicted, the effect of single point mutation on αIIbβ3 expression has been studied by using transiently transfected Cos cells. Finally, structural consequences of amino acid substitutions has been studied using the published model of αIIbβ3 (code PDB 2VDL) and structural modelling. Results7 new mutations have been characterized. 1 deletion / insertion, 2 single point mutations inducing stop codon and 1 resulting in splicing site disruption were identified. The 3 last identified single point mutations were not predicted to affect normal RNA processing but has been shown to prevent normal expression of mutant αIIbβ3 at the surface of Cos cells. The p.Meth118Arg and p.Gly221Asp substitutions that induce both important steric hindrance and charge modifications, are located inside the β-I domain of β3. So they should deeply alter the proper folding of the β-I domain, preventing the complex expression at the platelet surface. On the other hand, the p.Lys253Met protrudes from the β-I domain toward the αIIb β-propeller. A structural model of the Met253 β-I mutant has been done. An estimation of the direct electrostatic and desolvation free energies of interaction between the β-I domain surface and the αIIb β-propeller indicated that rather than the presence of a methionine, it is the lost of the Lys253 which is responsible for the complex expression defect. ConclusionSeven new GT mutations have been identified and the p.Lys253Met substitution helped to define a key role of the Lys253 in the αIIb β-propeller / β3 β-I domains interaction. DisclosuresNo relevant conflicts of interest to declare.

Human platelets recognize a novel surface protein, PadA, on Streptococcus gordonii through a unique interaction involving fibrinogen receptor GPIIbIIIa.

The concept of an infectious agent playing a role in cardiovascular disease is slowly gaining attention. Among several pathogens identified, the oral bacterium Streptococcus gordonii has been implicated as a plausible agent. Platelet adhesion and subsequent aggregation are critical events in the pathogenesis and dissemination of the infective process. Here we describe the identification and characterization of a novel cell wall-anchored surface protein, PadA (397 kDa), of S. gordonii DL1 that binds to the platelet fibrinogen receptor GPIIbIIIa. Wild-type S. gordonii cells induced platelet aggregation and supported platelet adhesion in a GPIIbIIIa-dependent manner. Deletion of the padA gene had no effect on platelet aggregation by S. gordonii but significantly reduced (>75%) platelet adhesion to S. gordonii. Purified N-terminal PadA recombinant polypeptide adhered to platelets. The padA mutant was unaffected in production of other platelet-interactive surface proteins (Hsa, SspA, and SspB), and levels of adherence of the mutant to fetuin or platelet receptor GPIb were unaffected. Wild-type S. gordonii, but not the padA mutant, bound to Chinese hamster ovary cells stably transfected with GPIIbIIIa, and this interaction was ablated by addition of GPIIbIIIa inhibitor Abciximab. These results highlight the growing complexity of interactions between S. gordonii and platelets and demonstrate a new mechanism by which the bacterium could contribute to unwanted thrombosis.

Overexpression of the partially activated alpha(IIb)beta3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet-like extensions in Chinese hamster ovary cells.

We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. Here we demonstrate that the partially activated alpha(IIb)beta3D723H or alpha(IIb)beta3D723A salt bridge mutants, but not fully activated alpha(IIb)beta3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of alpha(IIb)beta3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant alpha(IIb)beta3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type alpha(IIb)beta3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO alpha(IIb)beta3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. CHO alpha(IIb)beta3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions.

Protein Kinase Cδ Differentially Regulates Platelet Functional Responses

Protein Kinase C delta (PKCdelta) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. The purpose of this study was to investigate the role of PKCdelta in platelets. We evaluated the role of PKCdelta in platelets using two approaches--pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (delta V1-1)TAT, and in the murine platelets lacking PKCdelta, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCdelta. However, PKCdelta had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCdelta null mouse platelets and in human platelets pretreated with (delta V1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCdelta-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCdelta differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCdelta deficiency does not affect the thrombus formation in vivo.

Prolonged platelet activation in individuals with elevated blood pressure in response to a moderate exercise challenge

We examined the magnitude of 20-min moderate exercise-induced platelet activation in 50 volunteers with normal (n=31) or elevated blood pressure (EBP; n=19). Blood was drawn before, immediately after, and 25 min after exercise. Antibody-staining for platelet activation markers, P-selectin, and fibrinogen receptors was done with and without adenosine diphosphate (ADP) stimulation in whole blood for flow cytometric analyses. Exercise led to increases in percent aggregated platelets and percent platelets expressing P-selectin or PAC-1 binding (ps< or =.001). This increase in percent platelets expressing P-selectin continued even after a 25-min rest only in the EBP group (p< or =.01) accompanied by an increase in percent of aggregated platelets (p< or =.05). Although ADP stimulation led to increased platelet activation at rest, it was attenuated following exercise, even among EBP individuals. A moderate exercise challenge induced prolonged platelet activation in individuals with EBP but attenuation in activation to further stimulation by an agonist. Findings suggest that a recovery period after physical stress appears critical in individuals with high BP regarding platelet activation and aggregation, which can lead to an acute coronary syndrome in vulnerable individuals.

Functional Analysis of CD9 during Megakaryopoiesis

CD9 is a four transmembrane protein belonging to a tetraspanin family and regulates cell motility and adhesion. Several reports have indicated that CD9 form complexes with integrin including platelet fibrinogen receptor integrin aIIb-b III and involve in platelet function. We have previously reported that the c-myb knock down (KD) mice exhibited anemia and thrombocytopenia, and the expression level of CD9 mRNA was markdly increased in the c-myb KD mice. Reverse correlation of c-Myb expression with the CD9 gene expression was verified and agonistic antibody of CD9 stimulated megakaryocytic colony formation. These observations suggested that the CD9 expression was downregulated by c-myb. In our current study, we investigated the role of CD9 during megakaryopoiesis and platelet function by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Furthermore, after the bone marrow suppression with 5-fluorouracil (5-Fu), the recovery phase of platelet counts were delayed in the CD9-null mice. To clarify the reason of this platelet- recover delay, the number of bone marrow megakaryocyte was investigated using anti-vWF antibody staining, but the serial measurement of megakaryocyte number in CD9-null mice was not changed compared with wild-type mice. And also megakaryocyte ploidy was not changed in CD9-null mice compared with wild-type mice. Previous reports revealed that the cytoskeleton reorganization has a key role for the PPF formation, and Rac1-PAK1 signaling is important for actin restructure and aggregation prior to the cytoskeleton reorganization. The PPF formation was suppressed by Rac1 inhibitor. Although the mRNA expression levels of Rac1 in CD9-null mice were almost same as that in wild-type mice, PAK1 activation, major target of Rac1, was delayed in CD9-null mice compared with wild-type mice using thrombin-stimulating platelets. These results suggested that the delay of PAK1 activation cause the suppression of PPF formation in CD9-null mice.Our previous and current studies demonstrate that c-Myb suppresses the CD9 expression in a steady-state condition, while in the stress megakaryocytosis, CD9 is upregulated and acts to induce megakaryopoiesis and platelets production through Rac1-PAK1 signaling pathway. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis.

Platelet and endothelial adhesion on fluorosurfactant polymers designed for vascular graft modification

A prominent failure mechanism of small diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts is platelet-mediated thrombosis. We have designed a surface modification for ePTFE consisting of a self-assembling fluorosurfactant polymer (FSP) bearing biologically active ligands, including adhesive peptides and polysaccharide moieties. The goal of this biomimetic construct is to improve graft hemocompatibility by promoting rapid surface endothelialization, whereas minimizing platelet adhesion. Here we present a direct comparison of platelet and endothelial cell (EC) adhesion to FSPs containing one of three cell-adhesion peptides: cyclic Arg-Gly-Asp-D-Phe-Glu (cRGD), cyclic *Cys-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys* (cRRE, *denotes disulfide bond cyclization), linear Gly-Arg-Gly-Asp-Ser-Pro-Ala (RGD), or a polysaccharide moiety: oligomaltose (M-7), later designed to prevent nonspecific protein adhesion. Measurements of soluble peptide-integrin binding indicated that cRRE exhibits very low affinity for the alpha(IIb)beta(3) platelet fibrinogen receptor. Static and dynamic adhesion of washed, activated platelets on FSP-modified surfaces revealed that M-7 and cRRE promote significantly less platelet adhesion compared to RGD and cRGD FSPs, whereas EC adhesion was similar on all peptide FSPs and minimal on M-7 FSP. These results illustrate the potential for ligands presented in a FSP surface modification to selectively adhere ECs with limited platelet attachment.

αIIbG236E causes Glanzmann thrombasthenia by impairing association with β3

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin αIIbβ3. The N-terminal domain of the αIIb subunit is folded in a β-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of β3. Analysing the mutations of Italian GT patients we found that a patient had a αIIb G236E missense substitution that substitutes a glycine from the highly conserved ΦΦGΦ motif of blade 4 of the β-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated αIIb in conjunction with normal β3. Using flow cytometry analysis we found the percentage of HEK cells transfected with αIIbG236Eβ3 that reacted with anti αIIbβ3 was very low. In HEK cells transfected with either αIIbβ3 or αIIbG236Eβ3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against αIIb was present in both lysates, although less intense in cells transfected with αIIbG236Eβ3. In reducing condition αIIb from cells transfected with αIIbβ3 was nearly all mature, while in cells transfected with αIIbG236Eβ3 the ratio pro-αIIb: αIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against αIIb and immunoblotted with an antibody reacting with β3. While in immunoblots from cells transfected with αIIbβ3 a band corresponding to β3 was strongly detectable, in immunoblots originating from cells transfected with αIIbG236Eβ3 no band at the same level of normal β3 was detected. Immunofluorescence studies showed accumulation of αIIbG236Eβ3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the αIIbG236E mutation causes GT by impairing the association with β3 during biogenesis of the receptor.

Abstract 450: Phosphorylation Of Platelet αIIbβ3 Is Crucial For Arterial Thrombosis In Vivo And Microparticle Generation

Functional activity of platelet fibrinogen receptor αIIbβ3 is crucial for hemostasis and thrombosis. The process of αIIbβ3 activation in platelet aggregation is tightly regulated. It has been previously shown that β3 subunit of the complex undergo tyrosine phosphorylation, which, in turn, is believed to control recruitment of several intracellular adaptors. Mutations of Tyr747/ Tyr759 within the cytoplasmic domain of αIIbβ3 (DiYF substitution) were found to result in reversible platelet aggregation. To assess whether αIIbβ3 tyrosine phosphorylation is critical for arterial thrombosis, we utilized intravital microscopy to monitor thrombus formation in vivo in WT and DiYF mice. Compared to WT, DiYF mice exhibited delayed platelet adhesion and reduced rate of thrombus formation at the initial stages of thrombosis. Likewise, isolated DiYF platelets exhibited reduced adhesion to collagen under in vitro sheer conditions compared to WT. The progression phase of thrombosis in vivo was similar in WT and DiYF mice. The most dramatic difference was observed at the final phase of thrombus formation since it took 3-times longer for blood vessels in DiYF mice to occlude compared to WT. To specifically address the role if β3 phosphorylation in platelet αIIbβ3 vs αvβ3 on leukocytes and vascular cells, we transfused labeled WT and DiYF platelets into irradiated WT mice with low blood cells and platelet counts. It was found that transfusion of DiYF but not WT platelets resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion time in vivo. Similar differences were observed in tail bleeding test. Importantly, we have found that the lack of β3 phosphorylation impaired an ability of platelets to generate microparticles in response to activation, an event believed to be critical for the final stages of thrombosis. When stimulated with thrombin and PMA, DiYF platelets shed ~50% less Annexin V-positive microparticles as compared to WT platelets. Thus, β3 tyrosine phosphorylation of αIIbβ3 in platelets is crucial for the microparticles generation by activated platelets and the overall process of arterial thrombosis in vivo.