Glycoprotein IIb Research Articles

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

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

Comparative efficacy and safety of reperfusion therapy with fibrinolytic agents in patients with ST-segment elevation myocardial infarction: a systematic review and network meta-analysis

Fibrinolytic therapy offers an alternative to mechanical reperfusion for ST-segment elevation myocardial infarction (STEMI) in settings where health-care resources are scarce. Comprehensive evidence comparing different agents is still unavailable. In this study, we examined the effects of various fibrinolytic drugs on clinical outcomes. We did a network meta-analysis based on a systematic review of randomised controlled trials comparing fibrinolytic drugs in patients with STEMI. Several databases were searched from inception up to Feb 28, 2017. We included only randomised controlled trials that compared fibrinolytic agents as a reperfusion therapy in adult patients with STEMI, whether given alone or in combination with adjunctive antithrombotic therapy, against other fibrinolytic agents, a placebo, or no treatment. Only trials investigating agents with an approved indication of reperfusion therapy in STEMI (streptokinase, tenecteplase, alteplase, and reteplase) were included. The primary efficacy outcome was all-cause mortality within 30-35 days and the primary safety outcome was major bleeding. This study is registered with PROSPERO (CRD42016042131). A total of 40 eligible studies involving 128 071 patients treated with 12 different fibrinolytic regimens were assessed. Compared with accelerated infusion of alteplase with parenteral anticoagulants as background therapy, streptokinase and non-accelerated infusion of alteplase were significantly associated with an increased risk of all-cause mortality (risk ratio [RR] 1·14 [95% CI 1·05-1·24] for streptokinase plus parenteral anticoagulants; RR 1·26 [1·10-1·45] for non-accelerated alteplase plus parenteral anticoagulants). No significant difference in mortality risk was recorded between accelerated infusion of alteplase, tenecteplase, and reteplase with parenteral anticoagulants as background therapy. For major bleeding, a tenecteplase-based regimen tended to be associated with lower risk of bleeding compared with other regimens (RR 0·79 [95% CI 0·63-1·00]). The addition of glycoprotein IIb or IIIa inhibitors to fibrinolytic therapy increased the risk of major bleeding by 1·27-8·82-times compared with accelerated infusion alteplase plus parenteral anticoagulants (RR 1·47 [95% CI 1·10-1·98] for tenecteplase plus parenteral anticoagulants plus glycoprotein inhibitors; RR 1·88 [1·24-2·86] for reteplase plus parenteral anticoagulants plus glycoprotein inhibitors). Significant differences exist among various fibrinolytic regimens as reperfusion therapy in STEMI and alteplase (accelerated infusion), tenecteplase, and reteplase should be considered over streptokinase and non-accelerated infusion of alteplase. The addition of glycoprotein IIb or IIIa inhibitors to fibrinolytic therapy should be discouraged. None.

Effects of combination therapy of tirofiban and reteplase on endothelial function, coagulation function and inflammatory factors in elderly patients with acute ST-segment elevation myocardial infarction

Objective To investigate the effect of the combination therapy of tirofiban and reteplase on endothelial function, coagulation function and plaque inflammation in elderly patients with ST-elevation acute myocardial infarction (STEMI). Methods 100 patients with STEMI were treated with percutaneous transluminal coronary intervention (PCI) from January 2014 to June 2016 in our hospital.39 cases in the control group used conventional oral aspirin, clopidogrel and statins and other treatment.61 cases in the observation group received tirofiban and reteplase on the basis of the control group.The expression of endothelial microparticles (EMP) was detected by flow cytometry (FCM), and the ICAM-1, high sensitivity C-reactive protein (hs-CRP), tumor necrosis factor α(TNF-α) and interleukin-6 (IL-6), endothelin-1(ET-1) were measured by enzyme-linked immunosorbent assay (ELISA). The thrombin time (TT), activated partial thrombin time (APTT), prothrombin time (PT) and other indicators were measured by PUN-2048A coagulation instrument, then statistical analysis was performed. Results The postoperative levels of EMP, ICAM-1 and ET-1 of control group were (693.46±90.72), (768.58±20.46)μg/L and (31.27±8.18)ng/L, which were significantly higher than those in the observation group [(652.36±67.39), (752.37±25.0)μg/L, (28.22±5.05)ng/L], the differences were statistically significant (t=2.41, 2.67, 2.68, all P<0.05). After operation, the hs-CRP, TNF-α, IL-6 levels in the control group were (4.16±2.35)mg/L, (4.32±2.02)ng/L, (10.59±3.16)ng/mL, which were significantly higher than those in the observation group [(2.22±1.47)mg/L, (2.74±1.52)ng/L, (6.33±2.24)ng/mL], the differences were statistically significant(t=2.65, 2.67, 3.42, all P<0.05). The postoperative TT, PT, APTT in the observation group were (26.31±3.18)s, (14.34±1.67)s, (27.20±4.12)s, which were significantly longer than those in the control group [(24.03±2.84)s, (12.56±1.43)s, (24.55±3.62)s], the differences were statistically significant(t=2.15, 2.31, 2.65, all P<0.05). Conclusion Tirofiban combined with reteplase can improve endothelial function, inhibit inflammatory reaction and regulate coagulation function. Key words: Myocardial infarction; Platelet membrane glycoprotein IIb; Tirofiban hydrochloride; Reteplase

Evaluation of platelet surface glycoproteins in patients with Glanzmann thrombasthenia: Association with bleeding symptoms.

Background & objectives:Glanzmann thrombasthenia (GT) is a rare, inherited autosomal recessive disorder characterized by qualitative or quantitative deficiency of integrin αIIbβ3 [glycoprotein IIb (GPIIb)/IIIa, CD41/CD61] diagnosed by absent or reduced platelet aggregation to physiological agonists, namely, collagen, adenosine-di-phosphate, epinephrine and arachidonic acid. The objective of this study was to quantitate platelet surface GPs, classify GT patients and relate the results with the severity of bleeding and platelet aggregation studies.Methods:Fifty one patients of GT diagnosed by platelet aggregation studies were evaluated for the expression of CD41, CD61, CD42a and CD42b on platelet surface by flow cytometry. The association between the clinical phenotype based on bleeding score and GT subtype on flow cytometric evaluation was assessed.Results:Twenty four (47%) patients of GT were classified as type I (as CD41/CD61 were virtually absent, <5%), six (11.8%) patients as type II (5-20% CD41/CD61) and 21 (41.2%) as type III or GT variants as they had near normal levels of CD41 and CD61. Type III GT patients had significantly lower numbers of severe bleeders (P=0.034), but the severity of bleeding did not vary significantly in type I and II GT patients. In all GT patients, mean CD41 expression was found to be lower than mean CD61 expression (P=0.002).Interpretation & conclusions:Type I GT was found most common in our patients and with lowered mean CD41 expression in comparison with CD61. Type III GT patients had significantly lower numbers of severe bleeders, but the severity of bleeding did not vary significantly in type I and II GT patients.

Local Intracoronary Infusion of Glycoprotein IIb/IIIa Inhibitors via a Perfusion Catheter versus Intracoronary Guiding Catheter Injection during Primary Percutaneous Coronary Intervention: A Pilot Observational Study.

Glycoprotein IIb IIIa inhibitors improved short- and long-term outcome when added to primary percutaneous coronary intervention (PPCI) in patients with ST-segment-elevation myocardial infarction (STEMI). We hypothesized that intracoronary eptifibatide infusion via a perfusion catheter improves angiographic and clinical outcome of patients with STEMI undergoing PPCI, versus conventional intracoronary bolus injection. Prospectively, we enrolled 80 patients with acute STEMI and thrombolysis in myocardial infarction (TIMI) thrombus grade ≥ 2. Patients were assigned to receive eptifibatide (180 μg) either via a dedicated coronary perfusion catheter (ClearWayTM) during PPCI (group I), or guiding catheter (group II). Assessment of TIMI thrombus grade, TIMI flow grade, and TIMI myocardial perfusion (TMP) grade was performed both at baseline and post- procedurally. The primary 'angiographic' endpoint was final TMP grade 0/1. The primary 'clinical' endpoint was a composite of cardiac death, non-fatal re-infarction, target vessel revascularization, and recurrent ischemia at 30-day follow-up. Mean age was 52.3 ± 8.9 years (17.5% females). Clearance of visible thrombus (TIMI thrombus grade 0) at final angiogram was more frequent in group I. Additionally, both final TIMI flow grade 3 and final TMP grade 3 occurred more frequently in group I. The primary angiographic endpoint was more frequent in group II versus group I (17.5% versus 0%, respectively, p = 0.001). The primary clinical endpoint was more frequent in group II (20% versus 0%, respectively, p = 0.003). In patients with STEMI, intracoronary eptifibatide infusion via a perfusion catheter during PPCI improved immediate angiographic outcome, and reduced clinical events at 30-day follow-up, versus bolus injection via the guiding catheter.

Platelet microparticles: a biomarker for recanalization in rtPA-treated ischemic stroke patients.

ObjectivesIdentification of a biomarker for acute recanalization could have significant clinical impact.MethodsWe prospectively collected baseline, 24‐h, and 90‐day clinical and imaging data from consecutive ischemic stroke patients who fulfilled standard clinical eligibility criteria for treatment with intravenous recombinant tissue plasminogen activator (rtPA). Computed tomography angiography was acquired at 24 h and assessed using the thrombolysis is myocardial infarction (TIMI) scale with a score of 2b/3 indicating recanalization. Blood samples collected at 24 h after stroke symptom onset were used to measure the inflammatory biomarkers of glycoprotein IIb (CD41) expressing microparticles (MP), C‐reactive protein (CRP), COX 2, APOE, and Angiopoietin 1. Analysis was performed using linear regression and Pearson's correlation coefficient.ResultsA total of 57 patients met study eligibility criteria and had sufficient data and sample quality to be analyzed. Circulating levels of platelet derived CD41 + MP were significantly related to reperfusion (Pearson correlation, PC: 0.554, P < 0.001) and recanalization (PC: 0.495, P < 0.001) as well as related with 3‐month modified Rankin Score (PC: 0.483, P < 0.001). CRP was significantly negatively correlated with recanalization on 24 h CTA (PC: −0.292, P = 0.041). Backward logistic regression with CRP and CD41 + MP increased the association with reperfusion (r 2 = 0.357 P < 0.001).InterpretationThere is a significant relationship between the inflammatory biomarkers CD41 + MP and CRP and recanalization.

GG Polymorphism of Platelet ITGA2B Gene Increases the Magnitude of Interleukin-6 Release after Cardiopulmonary Bypass

Objective: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response which is thought to be a significant cause of postoperative organ dysfunction and mortality. In this study we aimed to investigate the effect of ITGA2B (integrin alpha 2b, platelet glycoprotein IIb of IIb/IIIa complex) gene polymorphism on the magnitude of inflammatory response after CPB

Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals

Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 109/L) than healthy individuals (240 × 109/L, p < 0.01), but a higher MPV (p = 0.03) and IPF (p < 0.01). IPC was similar for the two groups (p = 0.74). PRP had significantly lower MPV (p < 0.01) and significantly higher platelet count and IPC (both p-values <0.03) when compared with whole blood. IPF was similar for PRP and whole blood (p = 0.18). COX-1 expression was 10 times higher and COX-2 expression was 50% higher in PRP than in whole blood (pCOX-1 < 0.01, pCOX-2 < 0.01). Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (pCOX-1 < 0.01) and PRP, though this was nonsignificant in PRP (pCOX-1 = 0.17). In ITP patients, positive correlations were found between platelet turnover and COX-1 expression (all p-values <0.01, rho = 0.80–0.94), whereas healthy individuals showed significant though weaker correlations between platelet turnover and COX-1 and COX-2 expressions (all p-values <0.03, rho = 0.44–0.71). GPIIb, IX, and Ib expression was increased in ITP patients compared with healthy individuals (all p-values < 0.03). GPIIb, IX, Ib, and IIIa showed positive correlations with platelet turnover in ITP patients (all p-values <0.02, rho = 0.71–0.94), but weak and nonsignificant correlations in healthy individuals (all p-values >0.14, rho = 0.11–0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in platelets.

Dealing with prognostic signature instability: a strategy illustrated for cardiovascular events in patients with end-stage renal disease.

BackgroundIdentification of prognostic gene expression markers from clinical cohorts might help to better understand disease etiology. A set of potentially important markers can be automatically selected when linking gene expression covariates to a clinical endpoint by multivariable regression models and regularized parameter estimation. However, this is hampered by instability due to selection from many measurements. Stability can be assessed by resampling techniques, which might guide modeling decisions, such as choice of the model class or the specific endpoint definition.MethodsWe specifically propose a strategy for judging the impact of different endpoint definitions, endpoint updates, different approaches for marker selection, and exclusion of outliers. This strategy is illustrated for a study with end-stage renal disease patients, who experience a yearly mortality of more than 20 %, with almost 50 % sudden cardiac death or myocardial infarction. The underlying etiology is poorly understood, and we specifically point out how our strategy can help to identify novel prognostic markers and targets for therapeutic interventions.ResultsFor markers such as the potentially prognostic platelet glycoprotein IIb, the endpoint definition, in combination with the signature building approach is seen to have the largest impact. Removal of outliers, as identified by the proposed strategy, is also seen to considerably improve stability.ConclusionsAs the proposed strategy allowed us to precisely quantify the impact of modeling choices on the stability of marker identification, we suggest routine use also in other applications to prevent analysis-specific results, which are unstable, i.e. not reproducible.

The Current Role of Glycoprotein IIb/ IIIa Inhibitors in Percutaneous Coronary Intervention*

The central role of platelets in acute coronary syndromes (ACS) and percutaneous coronary interventions (PCI) is well appreciated. The various platelet activation mechanisms finally lead to the expression and activation of surface glycoprotein IIb/IIIa receptors that mediate platelet aggregation and thrombus formation. Glycoprotein IIb/IIIa inhibitors (GPIs) are the most potent antiplatelent agents and their role in ACS treatment and PCI has been dominant in the recent past. The advent of stents and thienopyridines minimized ischemic complications and in parallel the role of GPIs in low risk PCI. Despite being effective in decreasing PCI-related ischemic complications, the major drawback of GPI use is a relative increase of hemorrhagic complications that can unfavorably affect prognosis. The availability of bivalirudin, which is regarded as an equally effective but safer antithrombotic agent when compared to the combination of heparin and GPIs, despite an ongoing controversy, has also led to a decrease of GPI use in PCI for ACS. Finally the advent of novel potent antiplatelet agents (prasugrel, ticagrelor and soon cangrelor) further contained GPI use in patients with ischemic – thrombotic risk that clearly exceeds bleeding risk and mainly for bail-out in case of a thrombotic event during PCI. A concise overview of accumulated data regarding optimal use of GPIs as determined by large clinical trials and recent guidelines is herein attempted.

Gene mutations of platelet glycoproteins and response to tirofiban in acute coronary syndrome.

Glycoprotein inhibitors (abciximab, eptifibatide and tirofiban) are used in patients with unstable angina and non-ST-segment elevation myocardial infarction before percutaneous coronary intervention. Of these, tirofiban is the least effective. We hypothesized that the response to tirofiban might be associated with glycoprotein gene mutations. Prospective study at Emergency Unit, Heart Institute (InCor), University of São Paulo. Intrahospital evolution and platelet aggregation in response to tirofiban were analyzed in relation to four glycoprotein mutations in 50 patients indicated for percutaneous coronary intervention: 17 (34%) with unstable angina and 33 (66%) with non-ST-segment elevation myocardial infarction. Platelet aggregation was analyzed using the Born method. Blood samples were obtained before and one hour after tirofiban infusion. Glycoproteins Ia (807C/T ), Ib (Thr/Met) , IIb (Ile/Ser ) and IIIa (PIA ) were the mutations selected. Hypertension, dyslipidemia, diabetes, smoking, previous coronary artery disease and stroke were similar between the groups. Mutant glycoprotein IIIa genotypes had lower platelet aggregation before tirofiban administration than that of the wild genotype (41.0% ± 22.1% versus 55.9% ± 20.8%; P = 0.035). Mutant glycoprotein IIIa genotypes correlated moderately with lower platelet inhibition (r = -0.31; P = 0.030). After tirofiban administration, platelet glycoprotein Ia, Ib, IIb and IIIa mutations did not influence the degree of inhibition of platelet aggregation or intrahospital mortality. Mutations of glycoproteins Ia, Ib, IIb and IIIa did not influence platelet aggregation in response to tirofiban in patients with unstable angina and non-ST-segment elevation myocardial infarction.

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.

Abstract P120: Improving the Angiogenic Abilities of Mobilized Peripheral Blood Stem Cells Achieved by Priming with Activated Platelet Supernatant for Regenerative Cell Therapy

Platelets play a critical role in hemostasis and also have ability to promote angiogenesis and tissue repair by secreting of numerous cytokine and making angiogenic condition. We investigated whether autologous ‘activated platelet supernatant (APS)’ has effect on enhancing pro-angiogenic potential of peripheral blood stem cells (PBSC) for stem cell-based therapy for ischemic diseases. Granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (mobPBSC) were isolated from healthy volunteers, while APS was collected from platelet rich plasma by thrombin activation. mobPBSCs were primed with APS (APS primed mobPBSCs) for 6 hours, and APS primed mobPBSCs characterized their angiogenic ability. For the safety analysis, we estimated the thrombogenicity of platelets in whole blood mixed with APS primed mobPBSCs by expression of glycoprotein IIb and IIIa on platelets. APS had a higher level of various cytokines, such as IL8, IL17, PDGF and VEGF than naïve platelet supernatants. And APS primed mobPBSCs had more expression of angiogenic factors, surface markers (i.e. CD34, CD31, and CXCR4) and integrins (integrin α5, β1 and β2) than Veh primed and Pre primed mobPBSC. Also APS primed mobPBSCs were polarized toward CD14++/CD16+ pro-angiogenic monocytes. And result in adhesion to endothelial cells and fibronectin which represents cell to cell and cell to extracellular matrix adhesion, respectively. The culture supernatant of APS-primed mobPBSCs contained high levels of IL8, IL10, IL17 and TNFα, and augmented proliferation and capillary network formation of HUVEC. In-vivo transplantation of APS-primed mobPBSC into athymic mice ischemic hindlimbs and Matrigel plugs elicited vessel differentiation and tissue repair. In thrombogenicity test, platelet activity increased after mixing whole blood with mobPBSC regardless of the priming agent. However, this was reduced by pretreatment of aspirin, which is an antiplatelet agent prescribed to patients with ischemic diseases. Our data demonstrate that mobPBSCs primed with APS improve angiogenic potential, and that can be adjunctive strategy to enhance the efficiency of stem cell therapy for ischemic diseases.

Expression of the Recombinant Soluble Glycoprotein IIb: Lessons Learned and Future Directions

Expression of the Recombinant Soluble Glycoprotein IIb: Lessons Learned and Future Directions

Podoplanin and CLEC-2 drive cerebrovascular patterning and integrity during development

AbstractMice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplaninfl/fl mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage.

Platelets are associated with xenograft tumor growth and the clinical malignancy of ovarian cancer through an angiogenesis-dependent mechanism.

Platelets are known to facilitate tumor metastasis and thrombocytosis has been associated with an adverse prognosis in ovarian cancer. However, the role of platelets in primary tumour growth remains to be elucidated. The present study demonstrated that the expression levels of various markers in platelets, endothelial adherence and angiogenesis, including, platelet glycoprotein IIb (CD41), platelet endothelial cell adhesion molecule 1 (CD31), vascular endothelial growth factor (VEGF), lysyl oxidase, focal adhesion kinase and breast cancer anti-estrogen resistance 1, were expressed at higher levels in patients with malignant carcinoma, compared with those with borderline cystadenoma and cystadenoma. In addition, the endothelial markers CD31 and VEGF were found to colocalize with the platelet marker CD41 in the malignant samples. Since mice transplanted with human ovarian cancer cells (SKOV3) demonstrated elevated tumor size and decreased survival rate when treated with thrombin or thrombopoietin (TPO), the platelets appeared to promote primary tumor growth. Depleting platelets using antibodies or by pretreating the cancer cells with hirudin significantly attenuated the transplanted tumor growth. The platelets contributed to late, but not early stages of tumor proliferation, as mice treated with platelet-depleting antibody 1 day prior to and 11 days after tumor transplantation had the same tumor volumes. By contrast, tumor size in the early TPO-injected group was increased significantly compared with the late TPO-injected group. These findings suggested that the interplay between platelets and angiogenesis may contribute to ovarian cancer growth. Therefore, platelets and their associated signaling and adhesive molecules may represent potential therapeutic targets for ovarian cancer.

The effect and duration of prophylactic platelet transfusions before insertion of a central venous catheter in patients with bone marrow failure evaluated with point-of-care methods and flow cytometry.

Patients with bone marrow failure and severe thrombocytopenia are frequently given prophylactic platelet transfusion before interventions. The clinical effects of such transfusions, however, are poorly defined. We performed a prospective observational study on patients with bone marrow failure scheduled for prophylactic platelet transfusion before the insertion of a central venous catheter. The objectives were to evaluate the effect and duration of prophylactic platelet transfusions on central venous catheter insertion in thrombocytopenic patients with bone marrow failure. Thirty-nine adult patients with bone marrow failure and platelet counts below 50 × 10/L were consecutively enrolled before prophylactic platelet transfusion for subclavian central venous catheter insertion. Blood samples were drawn from the patients before platelet transfusion, 1 hour, and 4 hours after completion of the transfusion. The coagulation profile was assessed by conventional hematological tests, thromboelastometry (ROTEM) assays (EXTEM and FIBTEM), multiple electrode aggregometry (Multiplate) assays including adenosine diphosphate, collagen, and thrombin receptor agonist peptide, and by flow cytometry for the platelet expression of P-selectin (CD62P) and activated glycoprotein IIb-IIIa (PAC-1). Bleeding complications were classified with a 5-grade scale, according to the Common Terminology Criteria for Adverse Events. Seventeen women and 22 men were included in the study. Platelet count was increased from 24 × 10/L (18-32) before to 42 × 10/L (31-50) 1 hour after transfusion (P < 0.0001) and was not significantly different 4 hours after transfusion (40 × 10/L (29-50), P = 0.047). Maximal clot firmness EXTEM was increased from 38 mm (32-45) before to 46 mm (41-52) 1 hour after transfusion (P < 0.0001) and did not change 4 hours after transfusion. Clotting time EXTEM was decreased from 58.5 seconds (50-78) beforehand to 53 seconds (45-61) 1 hour after transfusion (P = 0.0006) and was not significantly different 4 hours after transfusion (57 seconds (52-70, P = 0.025). FIBTEM results were all unchanged after transfusion. All Multiplate analyses were significantly increased after 1 hour and were not diminished 4 hours after transfusion. Four grade 1 bleeding episodes occurred, but no grade 2 to 5 bleeding could be detected. Flow cytometry analyses showed mixed results with no overall trend. Prophylactic platelet transfusions in thrombocytopenic patients with bone marrow failure improve hemostatic parameters on ROTEM and Multiplate by increasing the number of platelets, and not through enhancement of platelet function. Improved clotting parameters on ROTEM and platelet aggregation on Multiplate appear to persist between 1 and 4 hours after transfusion.

Effect of erythropoietin on free radical oxidation and glycoprotein expression in platelets under conditions of chronic renal failure.

A short-term open prospective study examined 62 patients at the terminal stage of chronic renal failure. The experimental group received erythropoietin in a total dose of about 40,000 U. The expression of glycoproteins IIb-IIIa, IIb, and Ib was enhanced, the content of LPO products was elevated, and SOD and catalase activities were reduced in platelets from patients with chronic renal failure. Administration of erythropoietin partially restored free radical oxidation and expression of glycoproteins IIb-IIIa, IIb, and Ib in platelets. A significant correlation was revealed between the expression of platelet receptors on the one hand, and content of LPO products and SOD and catalase activities, on the other hand.

Does the Inhibition of the Final Common Pathway of Platelet Aggregation Reduce the No-Reflow Phenomenon During Primary Percutaneous Coronary Intervention? Tirofiban no Infarto Agudo do miocárdio e a não Reperfusão (TIARA)

ABSTRACT Background Primary percutaneous coronary intervention is currently the preferred method to treat patients with STsegment elevation acute myocardial infarction. The no-reflow phenomenon, which is the inability to reperfuse a region of the myocardium after restoration of patency of a previously occluded epicardial coronary artery, is observed in a considerable proportion of these patients. The benefit of IIb/IIIa glycoprotein inhibitors, blocking the final common pathway of platelet aggregation, has been suggested in studies of acute coronary syndromes, but their actual efficacy in the context of no-reflow in patients treated with primary percutaneous coronary intervention remains unclear. Methods The aim of this multicenter, double-blinded, placebo controlled study is to assess the impact of the early administration of the low molecular weight glycoprotein IIb/ IIIa inhibitor tirofiban on the incidence of no-reflow using angiographic and electrocardiographic methods to determine: (1) the epicardial coronary flow, using the TIMI score, and the microcirculatory flow, using the MBG score of opacification and myocardial flow; (2) the resolution of the ST segment elevation, as the final index of the success of reperfusion. Conclusions If the decrease in no-reflow incidence at 90 minutes and 24 hours after primary percutaneous coronary intervention is confirmed, this pilot study should guide the implementation of a larger study to investigate the possible impact of the systematic inhibition of the final common pathway of platelet aggregation on the mortality of ST-segment elevation acute myocardial infarction patients.

Eptifibatide-induced acute profound thrombocytopenia: a case report

BackgroundThe interactions among cells or among cells and components of the extracellular matrix, is a crucial pathophysiological process involving some molecules collectively known as adhesion molecules (CAMs). Glycoprotein IIb / IIIa receptors are only restricted to blood platelets and they bind fibrinogen and adhesion proteins such as fibronectin, vitronectin, von Willebrand factor to form cross bridges between adjacent platelets. IIb/IIIa receptor antagonists are an object of intense research activity for target therapy worldwide during the last decades. Three GPIIb/IIIa inhibitors, abciximab, tirofiban, and eptifibatide, have been approved for clinical use. Profound thrombocytopenia is an uncommon but clinically important complication of glycoprotein IIb/IIIa inhibitors.Case presentationThis case report discusses a forty-four-year-old male patient with acute coronary syndrome who underwent percutaneous coronary intervention and developed profound thrombocytopenia within 4 hours of first administration of eptifibatide.ConclusionThis report adds another case of eptifibatide-induced thrombocytopenia to the medical literature and endorses the importance of platelet count monitoring after initiating therapy with this agent.