Platelet Glycoprotein IIb-IIIa Research Articles

High Prevalence of a Rare Bleeding Disorder: A Report from Glanzmann Thrombasthenia Registry of Al-Madinah, Saudi Arabia

Introduction Glanzmann thrombasthenia (GT) is a hereditary bleeding disorder caused by a defect or deficiency of platelet glycoprotein IIbIIIa. Though GT is considered a rare bleeding disorder, some countries in the Middle East report a high prevalence of GT. Aim Description of GT patients' prevalence, demographic, clinical, and genetic features in AlMadinah, Saudi Arabia. Methods This is a 20-year report from the AlMadinah GT registry in the Madinah Hemophilia Comprehensive Care Center. The registry has included all patients with a confirmed diagnosis of GT. To label a patient as GT, He must have absence or abnormal response in all agonists except ristocetin plus absence or reduced expression of platelet glycoprotein IIbIIIa on Flowcytometry. Next-generation sequencing is used to target sequencing for Molecular testing. The extracted data underwent an analysis process, where 136 GT patients were identified. Only 125 cases have complete data in records. Statistical analysis made via a statistical package of social sciences (SPSS) version 23. The research and ethical committee approved the study. Results The prevalence of GT among Saudis in AlMadinah, Saudi Arabia, based on the Saudi population in 2022, is 1:10,000. One hundred twenty-five GT patients were studied: 67 (54%) females and 58 (46%) males. The median age at the time of the report was 16.5 (10 days to 56 years). Ninety-nine (79%) patients have type 1 GT. Out of 108 (86%) whose Consanguinity status was recorded, 106 (98%) GT patients are products of consanguineous marriage. Seventy (65%) patients were products of first-degree cousin parents. Family history of GT was reported in 107 (86%) cases. Twenty-five families have more than one sibling (2-5) with GT. A single tripe has 50 members with GT. The median age at presentation is 2 years (7 days to 11 years). The most common symptoms are gum bleeds, Epistaxis, and Bruises. Eighteen (16%) patients suffered from a severe bleeding course (defined as bleeding continued for more than 3 days, drop in hemoglobin for more than 2 g / L from the baseline, or needed admission to the intensive care unit). Forty (35%) patients had a moderate course (bleeding requiring hospital admission and not filling the criteria for severe one). Fifty-seven (50%) patients had a mild clinical course (bleeding can be stopped at home). Eighteen (17%) patients had bleeding episodes more than once a month, 34 (32%) had six or fewer bleeds per year, and 57 (52%) patients had one bleeding episode per year. Menorrhagia affected 35 (86%) ladies and girls. Hormonal therapy has been used at least once in their lives to interfere with the menstrual cycle in 39 (95 %) patients. Twenty-eight pregnancies were reported, producing 24 babies (13 girls and 11 boys). A postpartum hemorrhage took place after 18 deliveries (75%). Mild to moderate pregnancy-related bleeding occurred in 7 pregnancies. Iron deficiency anemia was found in 66 (66%) patients out of 100 whose iron status was reported. Eighteen mutations have been identified: 15 mutations in the ITGA2B gene and 3 mutations in the ITGB3 gene (table 1). The mutation ITGA2B:exon13:c.1210+5G>A is a novel mutation found only in one tripe, where all 12 GT patients in this tripe carry this mutation. The mutation ITGA2B: exon20:c.1879-2A>G is the most frequent mutation affecting one tribe only, and all 42 GT patients who descend from this tribe carry this mutation. The second frequent mutation is ITGB3:exon13:c.2112delC, involving 28 GT patients. Though we could not find a correlation between genotype and phenotype, we noticed GT patients carrying mutations exon13:c.2112delC and exon13:c.1210+5G>A running a more severe clinical course compared to patients affected by the mutation ITGA2B: exon20:c.1879-2A>G. Anti-platelet antibodies have been detected in 11 (9%) patients, 2 of them during pregnancy. Apart from Menorrhagia, 10 out of 15 GT patients running severe clinical courses improved when they reached puberty. Conclusions GT is the commonest bleeding disorder in AlMadinah, Saudi Arabia, with a prevalence of 1:10,000. With a high rate of menorrhagia and IDA. The high rate of consanguineous marriage could be a reason for the high prevalence of GT.

Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation

AbstractActivation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin αIIbβ3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the β3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with β3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1–Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating αIIbβ3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1R35E/R35E mice showed similar GPIIb-IIIa activation to those from wild-type littermates in response to multiple agonists. Tln1R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1–talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.

Inherited Bleeding Disorders in the Obstetric Patient.

Inherited bleeding disorders increase the risk of bleeding in the obstetric patient. Randomized controlled trials to compare prophylactic or therapeutic interventions are rare, and guidance documents rely heavily on expert opinion. Here we report the results of a systematic review of the literature for the treatment and prevention of peripartum bleeding in women with an inherited bleeding disorder. The highest-quality evidence is for the use of tranexamic acid in postpartum hemorrhage, which has been shown to decrease bleeding-related mortality in women without bleeding disorders. There is limited evidence for prophylactic use of this agent in women with inherited bleeding disorders. Desmopressin has also been used in observational studies of patients with von Willebrand disease and carriers of hemophilia A with some success, although concerns about the risk of hyponatremia persist. In patients with deficiencies of specific factors, replacement is generally the preferred approach, and concentrates have been studied in deficiencies of VWF and factors VII, VIII, IX, XI, and XIII as well as in patients with fibrinogen deficiency. Because of the small size of these studies, neither safety nor efficacy is well established, although the literature suggests that bleeding history may be more predictive of outcomes than factor levels in many cases. Goal factor levels have not been studied or systematically established in any of these diseases, although observational data suggest that achieving normal levels may be inadequate, particularly for VWF and factor VIII, which are physiologically elevated in pregnancy. For factor deficiencies in which no specific concentrate is available, such as factors II (prothrombin) and V, prothrombin complex concentrate or fresh frozen plasma may be used, and for platelet defects or deficiencies, such as Glanzmann thrombasthenia or Bernard-Soulier syndrome, platelet transfusion is generally first line, although use of recombinant FVIIa has been reported in patients with Glanzmann thrombasthenia to avoid development of, or treat patients with, antibodies to platelet glycoprotein IIbIIIa. Ultimately, data are lacking to definitively support an evidence-based approach to management in any of these disorders, and prospective, controlled studies are desperately needed.

Remote Ischaemic Preconditioning Causes Pathway Specific Attenuation of Platelet Glycoprotein IIb-IIIa Activation in Response to Potent In-Vitro Agonist Stimulation in Spite of Dual Antiplatelet Therapy

Introduction: Remote ischaemic preconditioning (RIPC) leads to reduced myocardial injury after elective and primary PCI, with the mechanism unclear. Dual antiplatelet therapy (DAPT) is important during PCI and reduces thrombotic events. Our aim was to investigate the effect of RIPC on circulating platelet activation. Methods: Patients referred for cardiac catheterisation were invited to participate prior to their procedure and were randomised to RIPC (3 × 5 min 200 mmHg sphygmomanometer inflations, left arm, separated by 5 min deflations) or sham. Venous blood was collected from the contralateral cubital fossa before and immediately after RIPC/sham. Whole blood was incubated with antibodies against CD62P, CD63 and active GPIIb-IIIa (PAC-1) and stimulated with ADP, TRAP6, thrombin + - collagen before analysis by flow-cytometry. Results: Fifty-five patients (29 RIPC, 26 sham) were recruited, mean age 62.7 years, 87% male. There were no differences between the groups with respect to baseline characteristics and medications. DAPT amongst RIPC and sham groups was similar (72% vs 81%, p = 0.54). Compared to aspirin alone, DAPT was effective at reducing GPIIb-IIIa activation in response to ADP (65.5% vs 41.5%, p = 0.001) with less effect against thrombin + collagen (81.1% vs 75.0%, p = 0.31). RIPC reduced GPIIb-IIIa activation in response to TRAP6 (48.4% vs 44.2%, p = 0.003) and thrombin + collagen (78.9% vs 68.8%, p < 0.001) in-spite of dual antiplatelet therapy. There were no changes with other agonists, other platelet markers or with sham treatment. Conclusions: RIPC causes rapid attenuation of platelet GPIIb-IIIa activation to potent agonists, against which DAPT is relatively ineffective. This effect may contribute to the benefit during PCI.

Anticoagulation during Cardiopulmonary bypass in Patients with Heparin-Induced Thrombocytopenia using Heparin and the Platelet Glycoprotein IIb-IIIa Antagonist

Anticoagulation during Cardiopulmonary bypass in Patients with Heparin-Induced Thrombocytopenia using Heparin and the Platelet Glycoprotein IIb-IIIa Antagonist

What have we learned from large population studies of von Willebrand disease?

Von Willebrand factor (VWF) is a critical regulator of hemostatic processes, including collagen binding, platelet adhesion, and platelet aggregation. It also serves as a carrier protein to normalize plasma factor VIII synthesis, release, and survival. While VWF protein measurements by immunoassay are reasonably comparable between institutions, the measurement of VWF ristocetin cofactor activity (VWF:RCo) has significant variability. Other tests of VWF function, including collagen binding or platelet glycoprotein IIb-IIIa binding, are not universally available, yet these functional defects may cause major bleeding even with normal VWF antigen (VWF:Ag) and VWF:RCo assays. This results in both the overdiagnosis and underdiagnosis of VWD. Newer assays of VWF function (using recombinant glycoprotein Ib rather than whole platelets) have been developed that may improve interlaboratory variability. Some of these tests are not uniformly available and may not be licensed in the United States. Large longitudinal studies of VWF in von Willebrand disease (VWD) patients are not available. Patients are sometimes diagnosed with a single diagnostic VWF panel. Plasma VWF levels increase with age, but it is not clear if this results in less bleeding or whether different normal ranges should be used to identify age-related decreases in VWF. In order to quantitatively compare bleeding symptoms in VWD patients and normal individuals, recent studies in the European Union, Canada, United Kingdom, Holland, and the United States have used semiquantitative bleeding assessment tools (BATs). Even with careful centralized testing, including functional assays of VWF, addition of a BAT does not solve all of the problems with VWD diagnosis. No matter where the line is drawn for diagnosis of VWD, VWF is still a continuous variable. Thus, VWD can be a severe hemorrhagic disease requiring frequent treatment or a mild condition that may not be clinically relevant. As will be discussed by Dr. Goodeve in her presentation, genetics has helped us to diagnose type 2 functional variants of VWD but has not been helpful for the many patients who are at the interface of normal and low VWF and carry the possible diagnosis of type 1 VWD. The hematologist's management of patients with reduced levels of VWF still requires both the art and science of clinical medicine.

Remote Ischaemic Preconditioning Attenuates Platelet Glycoprotein IIb-IIIa Conformational Activation in Patients with Coronary Artery Disease on Dual Antiplatelet Therapy

Remote Ischaemic Preconditioning Attenuates Platelet Glycoprotein IIb-IIIa Conformational Activation in Patients with Coronary Artery Disease on Dual Antiplatelet Therapy

Abstract 19565: The Safety and Efficacy of Platelet Glycoprotein Iib Iiia Antagonist by Intracoronary Administration versus Intravenous Administration in Patients With St Segment Elevation Myocardial Infarction: A Meta-analysis

Objective: Evaluate the administration (IC) and intravenous(IV) administration of platelet glycoprotein IIb / IIIa antagonist in STEMI patients. Methods: We searched PubMed, EMBASE, Web of Science, the Chinese Biomedical Medical Literature database, and the Cochrane Library, the retrieval time is May 2015. The papers which compared the safety and efficacy of IC and IV STEMI patients with platelet glycoprotein IIb / IIIa antagonist were included.Two researchers evaluated the trails and collected data according to 5.0.2 Cochrane Handbook. The data were merged by RevMan 5.0 software. Results: Finally we included 17 RCTs(n=8279, including 4260 IC administration patients, and 4019 IV administration patients). The GP IIb / IIIa antagonist was injected in the coronary after stent releasing during PCI in the IC group. Meta analysis showed: In the STEMI trials, intracoronary group had better clinical outcomes than intravenous group in the following index: short time MACE (one month after PCI) [OR=0.47,95%CI:0.23-0.97], congestive heart failure [OR=0.59,95%CI:0.41-0.84], left ventricular ejection fraction [MD=1.11, 95%CI: 0.48-1.74], infarct size [MD=-1.00, 95%CI: -1.65~ -0.35], myocardial blush grade 3 [OR=1.60, 95%CI: 1.12-2.27], drug occupational rate [OR=4.16, 95%CI: 1.45-11.92], wall motion score index[MD=-0.27,95%CI: -0.51~ -0.03]. And the IC and IV group were similar in the death, long time MACE(1 year after PCI), stent thrombosis, sever bleeding, ST-segment resolution -1 month after PCI, target vessel revascularization, myocardial infarction recurrence, TIMI grade 3 after PCI, left ventricular end systolic volume, left ventricular end diastolic volume, intervention success rate, life threatening arrhythmia, stroke after PCI, CK-MB, operation time, E/ A, cardiac death, minor bleeding, with no significant difference (P&gt; 0.05). Conclusions: This updated meta-analysis shows, compared to the IV, IC administration of platelet glycoprotein IIb / IIIa antagonist could reduce the risk of short time MACE (one month after PCI), congestive heart failure, and improve left ventricular ejection fraction, so IC administration of platelet glycoprotein IIb / IIIa antagonist could be a considerable way to be used in STEMI patients.

Prediction of Angiographic Extent of Coronary Artery Disease on the Basis of Clinical Risk Scores in Patients of Unstable Angina.

The correlation of clinical risk predictors and clinical risk scores: Thrombolysis in Myocardial Infarction (TIMI), Platelet Glycoprotein IIb-IIIa in Unstable Angina, Receptor Suppression Using Integrilin Therapy (PURSUIT) and Global Registry of Acute Coronary Events (GRACE) scores in Unstable Angina with angiographic extent of Coronary Artery Disease (CAD) is not known. To know the correlation of clinical risk scores with angiographic extent of coronary artery disease. This was a hospital based single centre, cross-sectional, observational, descriptive study conducted at a tertiary care teaching institute. One hundred and sixty patients with acute unstable angina were evaluated for presence of 9 clinical predictors and their 3 risk scores were calculated. All patients underwent coronary angiography. Correlation with Modified Gensini score and percentage stenosis in culprit artery was done. Data were summarized in the form of Mean, Standard Deviation and Proportions. Multiple linear regressions, Student's t-test and Pearson's coefficient 'r' were also used. Use of aspirin, age >= 65 years & presence of Congestive Heart Failure (CHF) were stronger predictors of Modified Gensini score. Presence of elevated enzymes and age >65 years were more significant predictors of percentage stenosis of culprit artery. GRACE score had better correlation with Modified Gensini score, PURSUIT score had more correlation with percentage stenosis in culprit artery. Use of Aspirin, age >= 65 years, presence of CHF and presence of elevated enzymes are stronger predictors of extent of CAD. Hence we recommend that these factors be given more importance. GRACE and PURSUIT risk scores had more correlation with angiographic extent of CAD.

Mean platelet volume: Interrelation with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels

Mean platelet volume: Interrelation with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels

Association of autoantibody specificity and response to intravenous immunoglobulin G therapy in immune thrombocytopenia: a multicenter cohort study

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder, in which platelet glycoprotein (GP)IIb-IIIa and GPIb-IX are the two most frequently targeted autoantigens. Our previous studies in animal models of ITP demonstrated that intravenous immunoglobulinG (IVIG) could protect against anti-GPIIb-IIIa autoantibody-mediated thrombocytopenia but failed to ameliorate ITP induced by most anti-GPIb-IX autoantibodies. The objective of this human study was to evaluate the association between the specificity of antiplatelet autoantibodies and response to IVIG treatment. In this retrospective study, a cohort of 156 previously untreated adults with severe ITP who underwent IVIG therapy (0.4gkg(-1) day(-1) for 5days) was analyzed. The primary outcome was response defined as platelet counts of ≥30×10(9) L(-1) and a doubling of baseline counts within 7days of dosing, and an absence of bleeding. Among the 66 patients with anti-GPIb-IX autoantibodies, only 24 (36.4%) achieved a response, as compared with 72 of 90 patients (80.0%) who were negative for anti-GPIb-IX autoantibodies (relative risk 2.2; 95% confidence interval 1.6-3.1). This study found no difference in response between patients with anti-GPIIb-IIIa autoantibodies (61.7%) and those without anti-GPIIb-IIIa autoantibodies (61.3%). Logistic regressions, including main effects and the interaction between these two autoantibodies, showed no influence of anti-GPIIb-IIIa autoantibodies on the effects of anti-GPIb-IX autoantibodies with regard to their association with IVIG response. Thus, in adults with ITP, the presence of anti-GPIb-IX autoantibodies is a predictive factor for poor response to IVIG treatment. ClinicalTrials.gov NCT01666795.

THE CONSEQUENCES OF HEPARIN USE IN PRIMARY PERCUTANEOUS CORONARY INTERVENTION: INSIGHTS FROM THE BLUE CROSS BLUE SHIELD OF MICHIGAN CARDIOVASCULAR CONSORTIUM (BMC2)

Heparin is commonly used as the sole procedural anticoagulant in patients undergoing primary PCI (PPCI). Use of Bivalirudin compared with platelet glycoprotein IIbIIIa inhibitors (GPI) has been associated with improved survival in this cohort. There are no data on the comparative effectiveness of

THE COMPARATIVE EFFECTIVENESS OF BIVALIRUDIN AND PLATELET GLYCOPROTEIN IIBIIIA INHIBITORS AMONG PATIENTS UNDERGOING CONTEMPORARY PRIMARY PCI: INSIGHTS FROM THE BLUE CROSS BLUE SHIELD OF MICHIGAN CARDIOVASCULAR COLLABORATIVE (BMC2)

Bivalirudin compared with platelet glycoprotein IIbIIIa inhibitors (GPI) was associated with reduced bleeding, increased stent thrombosis and improved survival in patients undergoing primary PCI (PPCI) in the HORIZONS AMI trial. These findings have not been externally corroborated. Our study cohort

Platelet degranulation and glycoprotein IIbIIIa opening are not related to bleeding phenotype in severe haemophilia A patients

Recently we reported data suggesting that platelets could compensate for the bleeding phenotype in severe haemophilia A (HA). The aim of this study was to confirm these results in a larger population with a detailed characterisation of clinical phenotype. Patients with diagnostic severe HA (FVIII:C <1%) were scored for clinical phenotype by integrating data on age at first joint bleed, joint damage, bleeding frequency and FVIII consumption. Phenotype was defined as onset of joint bleeding-score + arthropathy-score + joint bleeding-score + (2* treatment intensity-score). After a washout period of three days, blood was collected for measurement of basal level of platelet activation, platelet reactivity, endothelial cell activation and presence of procoagulant phospholipids in plasma. Thirty-three patients with severe HA were included, 13 patients with a mild, 12 patients with an average and eight patients with a severe clinical phenotype. No relevant differences in basal level of platelet activation, platelet reactivity, endothelial cell activation and procoagulant phospholipids between all three groups were observed. The mean annual FVIII consumption per kg did not correlate with the platelet P-selectin expression and glycoprotein (GP)IIbIIIa activation on platelets. In conclusion, variability in clinical phenotype in patients with diagnostic severe HA is not related to platelet activation or reactivity, measured as platelet degranulation and platelet GPIIbIIIa opening.

Mean platelet volume: interactions with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels

Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) were investigated in this study. Investigation was performed in a group of healthy volunteers (n = 38) and in a group of patients with acute coronary syndrome (ACS). In patients blood was collected at days 1, 3-5 and 8-12 after ACS development. As an antiaggregant therapy all patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) and most of them--clopidogrel (ADP receptor antagonist) with the exception of part of the patients (n = 44) at day 1 who had not taken clopidogrel before first blood collection. In volunteers platelet aggregation was stimulated by 1.25, 2.5, 5 and 20 M ADP, and in patients--by 5 and 20 M ADP. GP IIb-IIIa and GP Ib content on platelet surface was measured using 125I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels were revealed at 1.25 and 2.5 M ADP (coefficients of correlation (r)--0.396 and 0.373, p < 0.05) and at 5 and 20 those interactions did not reach significant level (r--0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients who received ASA but had not started clopidogrel treatment (r--0.526, p < 0.01 and 0.368, p < 0.05 for 5 and 20 M ADP respectively). Interactions between these parameters were not registered upon combined treatment with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in healthy donors and ACS patients (at all time points) -r from 0.439 to 0.647 (p < or = 0.001 for all correlations). Genetic polymorphisms of GP IIb-IIIa (GP IIIa Leu33Pro) and GP Ib ((-5)T/C (Kozak) and Thr145Met) identified in ACS patients did not affect expression levels of corresponding glycoproteins. The data obtained indicated that increased MPV values correlate with increased platelet aggregation activity and enhanced GP IIb-IIIa and GP Ib expression.

CD8+ T Cells Are Predominantly Protective In a Murine Model Of ITP and Is Required For Effective Steroid Therapy

BackgroundImmune thrombocytopenia (ITP) is a common bleeding disorder characterized by autoantibody mediated destruction of autologous platelets. The predominant autoantibody detected in ITP patients target the platelet Glycoprotein (GP)IIbIIIa, however autoantibodies can only be detected in 50-70% of patients. Cytotoxic CD8+ T-cells (CTL) may therefore also be a significant contributing factor for thrombocytopenia, either through direct cytotoxicity against platelets, or decreasing platelet production through interaction with megakaryocytes in the bone marrow. Nevertheless, whether CD8+ T cell mediated cytotoxicity significantly contributes to thrombocytopenia in ITP is controversial. Interestingly, CD8+ regulatory T cells have been demonstrated to play significant roles in other autoimmune diseases, but their function in the context of ITP has not been adequately examined. Methods and ResultsWe developed both passive and active murine models to investigate mechanisms of pathogenesis and steroid treatment of ITP. In the passive model, we injected anti-b3 or anti-GPIb antibodies to induce thrombocytopenia; this causes transient antibody-mediated thrombocytopenia. We found that a single intraperitoneal (IP) injection of steroids post-antibody injection was effective at rescuing platelet counts. We also adapted an active model of ITP whereby wild-type (WT) BALB/c mice were transfused with splenocytes from WT platelet immunized β3-/- mice to induce thrombocytopenia. This model encompasses both antibody and cell-mediated ITP and causes sustained thrombocytopenia in the mice. In this model, we found steroid treatment administered either orally or through IP-injection were equally efficacious at ameliorating thrombocytopenia. The successful use of steroids to treat thrombocytopenia in these animal models is representative of the therapeutic effects of steroid treatment seen in human ITP patients.To study the role of CD8+ T-cells in the response to steroid treatments in ITP, we depleted CD8+ T-cells from splenocytes prior to transfusion into WT mice. Unexpectedly, we found CD8+ T cell depleted splenocyte (lacking in CTL cells), engrafted mice had lower, but not higher, platelet counts. They were also less responsive to dexamethasone treatment compared to non-depleted engrafted mice. Furthermore, transfusion of splenocytes from immunized β3-/- mice in conjunction with antigen-primed CD8+ T-cells (isolated from immunized β3-/- splenocytes) was able to rescue platelet counts in WT mice. Co-transfusion of non-primed CD8+ T cells from β3-/- mice could not rescue platelet counts. These results indicate that platelet-antigen specific CD8+ Tregs play a dominant protective role in attenuating platelet clearance. In further support of our observations, we detected significantly increased CD8+CD25+Foxp3+ Treg percentages in the blood, thymus and spleen of immunized β3-/- mice. Further in vitro splenocyte cultures demonstrated putative regulatory mechanisms of CD8+CD25+ Tregs from immunized β3-/- splenocytes. We found CD8+CD25+ Tregs significantly inhibited CD4+ T and CD19+ B cell proliferation, platelet apoptosis, and platelet associated IgG production in the presence of platelet-antigens, but increased the secretion of the anti-inflammatory cytokine IL-10. ConclusionTo the best of our knowledge, these are the first reported animal models of steroid treatment of ITP. We demonstrated that steroid therapy was effective and CD8+ T cells are required for this efficacy. We further unveiled a population of CD8+ regulatory T-cells in the CD8+ T cell populations, which were able to rescue platelet counts. This suggests that CD8+CD25+Foxp3+Treg may impart a predominantly protective role, overcoming the cytotoxic function of CD8+ T-cells in ITP. These data provides significant insights into the understanding of immunopathogenesis of ITP, which may be important in designing effective therapy including the potential usage of CD8+ Tregs as a cellular therapeutic method against ITP. Disclosures:No relevant conflicts of interest to declare.

Mutations in the A3 domain of Von Willebrand factor inducing combined qualitative and quantitative defects in the protein

AbstractTwo unrelated families were recruited in the French Reference Center for von Willebrand Disease with moderate bleeding symptoms associated with low von Willebrand factor (VWF) antigen levels, decreased collagen binding assay, and no or partial response to desmopressin. Genetic analysis showed the presence of heterozygous mutations in the A3 domain away from the collagen-binding surface: 1 never reported previously (p.L1696R) and another (p.P1824H) described in a Spanish family. The mutations were reproduced by site-directed mutagenesis and mutant VWF was expressed in different expression systems, COS-7 cells, baby hamster kidney cells, and in VWF-deficient mice through hydrodynamic injection. p.L1696R and p.P1824H were associated with very low expression levels both in vitro and in vivo, with intracellular retention for p.P1824H. Both homozygous mutants displayed decreased binding to collagen types I and III but also decreased binding to platelet glycoproteins Ib and IIbIIIa. Co-transfections with wild-type VWF partially corrected these defects, except that collagen binding remained abnormal. The in vivo thrombosis response was severely reduced for both heterozygous mutants. In conclusion, we report 2 VWF A3 domain mutations that induce a combined qualitative and quantitative defect.

Pharmacogenetics of the Antiplatelet Effect of Aspirin

The concept of "pharmacogenetics" addresses genetically determined variation in how individuals respond to drugs. Accordingly, specific genetic variants have been suggested as contributors to a reduced response to various antiplatelet drugs. Aspirin is a cornerstone in secondary cardiovascular prevention and has been thoroughly investigated. The efficacy of aspirin is well documented, although with considerable interindividual variation. According to meta-analyses, a reduced antiplatelet effect of aspirin confers an increased risk of cardiovascular events. The platelet response to aspirin is assessed by in vitro evaluation of thromboxane-dependent platelet function. A reduced antiplatelet effect of aspirin can be explained by several mechanisms, which are largely determined by clinical, pharmacodynamic, biological and genetic factors. During the past decade, numerous studies have identified genetic polymorphisms significantly associated with cardiovascular events and modulating the antiplatelet effect of aspirin. However, results have been contradictory allowing only few firm conclusions to be drawn. Polymorphisms in genes encoding glycoproteins (IIb/IIIa, Ia/IIa, VI and Ibα), cyclooxygenases (1 and 2), adenosine diphosphate receptors (P2Y1 and P2Y12) and proteins of importance for haemostasis (thromboxane A2 receptor, coagulation factor XIII, etc.) have been investigated. In particular, a polymorphism in the gene encoding glycoprotein IIb/IIIa has been associated with a reduced antiplatelet effect of aspirin. The additive value of an individual's genetic makeup in predicting the antiplatelet effect of aspirin and the risk of cardiovascular events remains controversial. The present review outlines the pharmacology of aspirin and provides an overview of specific genetic variations considered to influence the antiplatelet effect of aspirin.

Abstract 4017: Safety of Intra-Arterial Bolus and Intravenous Infusion of Eptifibatide as an Adjunct to Intravenous Alteplase in Acute Ischemic Stroke: A Prospective Registry

Background and Purpose: Eptifibatide, a competitive platelet glycoprotein IIb-IIIa receptor inhibitor with high selectivity for platelet glycoprotein IIb-IIIa receptors and a short half-life, has been shown to reduce the risk of ischemic events associated with coronary interventions. However, its role in conjunction with full dose intravenous alteplase in neurointerventional procedures needs to be determined. We report the results of an open-label prospective registry to evaluate the safety (in terms of avoiding hemorrhagic complications) and effectiveness (in terms of recannalization rates and clinical improvement) of administering eptifibatide. Method: Patients data was prospectively maintained at a University-affiliated community hospital from January 2010 to June 2011. Consecutive patients with moderate to severe stroke who received intravenous Alteplase and failed to improve were studied. Femoral artery access was established and site of cerebral occlusion crossed with microcatheter. Each patient was administered 135 mcg/kg intraarterial bolus followed by intravenous eptifibatide infusion (0.5-microg/kg/min) for 20 hours. The outcome analysis was done to look at recannalization rates, clinical improvement within 24 hours and mRS at discharge. The primary safety end point was symptomatic intracranial or major extra-cranial bleeding. Result: Sixteen patients (Mean Age 72.7, Range 30-93, Male-5(31.25%), Female-11(68.75%)) underwent treatment with this strategy. Recannalization was achieved in 93.75%(n=16)(Complete Recannalization in 43.75%, Partial Recannalization in 50%). There was immediate clinical improvement in 68.75% and good outcomes in 31.25%. One symptomatic and one asymptomatic intracerebral hemorrhage occurred during the follow-up period. Minor femoral site bleeding without requiring transfusion occurred in one patient. Conclusions: In conclusion, eptifibatide administered as an adjunct to thrombectomy for bridging after intravenous Alteplase appears to be safe and promotes recannalization in patients with acute ischemic stroke. Further studies are required to analyze eptifibatide efficacy after intravenous alteplase and its role in neurointerventional procedures.

Whole Blood Impedance Aggregometry: A New Tool for Severe Inherited Platelet Disorder Diagnosis?

Abstract 5266Diagnosis and characterization of platelet function disorders may be challenging. It requires multiple laboratory data including the assessment of platelet functions. Platelet function analysis is most commonly performed using light transmission aggregometry (LTA). LTA is a time-consuming method requiring centrifugation steps and large blood volumes. It is difficult to perform in children and in cases of thrombocytopenia. In contrast, platelet aggregation in whole blood using impedancemetry (WBI) is a fast method, allows omission of centrifugation steps and performance of platelet function studies under more physiological conditions with small samples size. It is based on the change of resistance proportional to the amount of platelets sticking to two electrodes where an alternating current is applied. Multiplate® (for “multiple electrode aggregometry”, Dynabite Medical) is a new generation of WBI aggregometer using diluted blood and single-use test cells containing twin electrodes that reduce the variation of results. We have already showed the good Multiplate® performance concerning ristocetin-induced platelet aggregation in a population of 30 patients with characterized von Willebrand disease (Valarche et al, 2011). Our aim in this ongoing study was to assess the performance of WBI in patients with inherited platelet function disorders. We tested 8 patients including 2 unrelated patients with Glanzmann Thrombasthenia (GT), 2 unrelated patients with Bernard-Soulier Syndrome (BSS), 1 patient with Gray Platelet Syndrome (GPS) and 3 patients from the same family with a platelet type von Willebrand disease (PTVWD). GT, BSS, and PTVWD diagnosis were confirmed using genotyping. BSS and GPS patients had chronic thrombocytopenia. GT, BSS, GPS and 1/3 PTVWD had platelet function tests with LTA in parallel. WBI was performed on heparinized whole blood diluted at ½ in NaCl at 37°C and triggered using high (0.77 mg/mL, WBI RH) and low (0.5 mg/mL, WBI RL) final ristocetin concentrations, ADP (6.5 Âμ Mol, WBI ADP) and collagen (3.2 Âμg/mL, WBI Coll). Results were expressed in arbitrary unit (AU) corresponding to the area under the aggregation curve observed during 6 min. Normal ranges indicated in brackets were based on the mean +/− 2 SD of 30 healthy volunteers’ results. Results highlighted in grey are those out of the normal ranges (Table 1).Table 1:Results of the 8 patients with inherited platelet disorders.PatientsPlatelet count (109/L)WBI RH (AU) [>500]WBI RL (AU) [<150]WBI ADP (AU) [>550]WBI Coll (AU) [>500]GT 116923441443GT 224955417ND7BSS 134371119129BSS 230254733582GPS7916217ND42PTVWD22099493ND338PTVWD231116560ND1092PTVWD2341174168ND852All patients except those with PTVWD had decreased results with WBI. However, as expected, patients with GT had flat traces using WBI ADP and WBI Coll but normal or only decreased curves (234 – 554 AU) using WBI RH. On the opposite, BSS patients had flat traces using WBI RH but detectable curves using WBI ADP (191 – 335 AU) despite decreased platelet count. The thrombocytopenic GPS patient has a flat trace using WBI Coll and decreased WBI RH (162 AU). Members of the PTVWD family had normal results except a slightly increased result with WBI RH in 1/3 patients. Finally, LTA results performed in 6/8 patients were all in accordance with those of the WBI. In conclusion, in 8 patients with well characterized inherited platelet disorders, WBI was able to detect all abnormalities except PTVWD. In such cases, different ristocetin concentrations use might be critical to increase sensitivity. In our hands, WBI was able to discriminate disorders involving platelet glycoprotein (GP) IIb-IIIa from GP Ib-IX-V: GT patients exhibited flat traces using WBI ADP and WBI Coll, whereas patients with BSS exhibited flat traces with ristocetin. These preliminary results need to be confirmed on a larger population of patients with various characterized platelet function disorders. They suggest that WBI using the Multiplate® analyzer, which is a fast, easy and blood-preserving test, could be a valuable extra step before or in addition to the classic LTA for the diagnosis of severe inherited platelet disorders. Disclosures:No relevant conflicts of interest to declare.