Expression Of Fibrinogen Receptor Research Articles

Abstract 2079: Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression

Abstract Background: Hypercoagulability is a common clinical manifestation in patients with cancers, especially in pancreatic ductal adenocarcinoma (PDAC). Over 20% of PDAC patients with advanced-stage and worse prognosis undergo a venous thromboembolism (VTE) incidence. Fibrinogen (FG) may contribute to VTE due to its high expression in advanced PDAC tumors. Here, we set to determine the biological roles of tumor-derived FG in PDAC. In addition, our previous study showed that Von Willebrand Factor (VWF) A2 domain can effectively bind to FG and reduce platelet clot formation. Therefore, the functional roles of A2 on FG in PDAC were also studied. Methods: The correlation between survival rate and FG expression in PDAC was analyzed using RNAseq profiles of 149 PDAC patient samples from the TCGA database. PDAC subtyping of Baylor College of Medicine (BCM) PDAC patient-derived xenograft (PDX) cohort was performed using RNAseq data according to Moffitt’s criteria. The FG (three chains FGA, FGB, FGG) overexpression (OE) and knock-out (KO) PDAC cell lines were established. The gene expression and biological functions of FG were performed in PDX and cell lines using IHC and immunoblot assays. The FG effect on PDAC progression in vivo was studied in mouse models. Results: TCGA analysis showed that elevated tumor-derived FG gene expression is significantly correlated with worse overall survival and disease-free survival rates in PDAC patients. PDX lines RNAseq analysis clearly separated BCM PDAC PDXs into two subtypes: basal-like and classical PDACs. Basal-like PDXs showed significantly stronger FG staining than that in classical ones. Compared to the classical subtype, the basal-like subtype showed significantly upregulated genes that are associated with the epithelial-mesenchymal transition (EMT), IL6/JAK/STAT3, and TNFα signaling pathways. Accordingly, basal-like subtype PDAC exposed to IL6 or TNFα significantly increased FG and FG receptor expression with activated STAT3 and AKT pathways and elevated EMT markers, whereas classical subtype cells have minimal changes. In addition, we found that the FG-OE cells exhibit enhanced cell proliferation and migration properties whereas FG-KO cells reduced the effects. Consistent with in vitro data, FG-OE PDAC showed significantly higher tumor burdens than that of control PDAC in both orthotopic and subcutaneous PDAC mouse models. Furthermore, A2 protein treatment can effectively block the FG function and dramatically inactivate the FG-induced EMT process and AKT signaling pathway. Conclusions: Tumor-derived fibrinogen is upregulated in a basal-like subtype of PDAC cells and may contribute to tumor growth and metastasis by activating aberrant signaling pathways and promoting EMT. Our findings imply that elevated fibrinogen in PDAC could contribute to tumor progression and the A2 protein which target to fibrinogen may be a promising therapy for basal-like subtype PDAC. Citation Format: Dongliang Liu, Lisa Brubaker, Yichi Niu, Emily LaPlante, George Van Buren, Aleksandar Milosavljevic, Changyi Chen, Chenghang Zong, Miguel Cruz, Qizhi Yao. Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2079.

Microfluidic Chip and Flow Cytometry for Examination of the Antiplatelet Effect of Ticagrelor

Objective To examine the antiplatelet effect of ticagrelor by microfluidic chip and flow cytometry under shear stress in vitro. Methods Microfluidic chip was used to examine the effect of ticagrelor on platelet aggregation at the shear rates of 300/s and 1500/s.We adopted the surface coverage of platelet aggregation to calculate the half inhibition rate of ticagrelor.The inhibitory effect of ticagrelor on ADP-induced platelet aggregation was verified by optical turbidimetry.Microfluidic chip was used to construct an in vitro vascular stenosis model,with which the platelet reactivity under high shear rate was determined.Furthermore,the effect of ticagrelor on the expression of fibrinogen receptor (PAC-1) and P-selectin (CD62P) on platelet membrane activated by high shear rate was analyzed by flow cytometry. Results At the shear rates of 300/s and 1500/s,ticagrelor inhibited platelet aggregation in a concentration-dependent manner,and the inhibition at 300/s was stronger than that at 1500/s (both P<0.001).Ticagrelor at a concentration ≥4 μmol/L almost completely inhibited platelet aggregation.The inhibition of ADP-induced platelet aggregation by ticagrelor was similar to the results under flow conditions and also in a concentration-dependent manner.Ticagrelor inhibited the expression of PAC-1 and CD62P. Conclusion We employed microfluidic chip to analyze platelet aggregation and flow cytometry to detect platelet activation,which can reveal the responses of different patients to ticagrelor.

Platelet Phenotype and Function Changes With Increasing Duration of Extracorporeal Membrane Oxygenation.

To investigate platelet pathophysiology associated with pediatric extracorporeal membrane oxygenation (ECMO). Prospective observational study of neonatal and pediatric ECMO patients from September 1, 2016, to December 31, 2019. The PICU in a large tertiary referral pediatric ECMO center. Eighty-seven neonates and children (< 18 yr) supported by ECMO. None. Arterial blood samples were collected on days 1, 2, and 5 of ECMO and were analyzed by whole blood flow cytometry. Corresponding clinical data for each patient was also recorded. A total of 87 patients were recruited (median age, 65 d; interquartile range [IQR], 7 d to 4 yr). The median duration of ECMO was 5 days (IQR, 3-8 d) with a median length of stay in PICU and hospital of 18 days (IQR, 10-29 d) and 35 days (IQR, 19-75 d), respectively. Forty-two patients (48%) had at least one major bleed according to a priori determined definitions, and 12 patients (14%) had at least one thrombotic event during ECMO. Platelet fibrinogen receptor expression decreased (median fluorescence intensity [MFI], 29,256 vs 26,544; p = 0.0005), while von Willebrand Factor expression increased (MFI: 7,620 vs 8,829; p = 0.0459) from day 2 to day 5 of ECMO. Platelet response to agonist, Thrombin Receptor Activator Peptide 6, also decreased from day 2 to day 5 of ECMO, as measured by binding with anti-P-selectin, PAC-1 (binds activated GPIIb/IIIa), and anti-CD63 monoclonal antibodies (P-selectin area under the curve [AUC]: 63.46 vs 42.82, respectively, p = 0.0022; PAC-1 AUC: 93.75 vs 74.46, p = 0.0191; CD63 AUC: 55.69 vs 41.76, p = 0.0020). The loss of platelet response over time may contribute to bleeding during ECMO. These novel insights may be useful in understanding mechanisms of bleeding in pediatric ECMO and monitoring platelet markers clinically could allow for prediction or early detection of bleeding and thrombosis.

Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Platelet Features from Immune Thrombocytopenia Patients Refractory to Therapeutic Treatments

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder in which patients have wide variations in the number, activity and characteristics of their platelets. The goal of treatment of ITP is to raise platelet count to a level that will decrease or stop bleeding. Depending on the platelet count and the bleeding symptoms of each patient, therapeutic management is different. Pharmacological approaches employed for ITP treatment mainly aim to boost platelet count or to suppress immune system. Nevertheless, their single or combined application has been tried with a limited outcome in some ITP patients. These refractory patients constitute a clinical challenge. Identifying features of platelets from refractory ITP patients might be useful for a better understanding and management of this special clinical situation.Objective: This work aimed to determine whether platelets from refractory ITP patients have some distinguishing characteristic different from those from ITP patients responders to treatments (corticoids and thrombopoietin receptor-agonists).Methods: Citrated blood samples from 71 patients with primary chronic ITP responders to first or second line treatments and from 10 refractory ITP patients were included.Platelet surface receptors were analysed by flow cytometry (FCM, FACScan, BD Biosciences) with specific monoclonal antibodies against CD41/αIIb and CD61/β3, CD42a and CD42b.Platelet activation was assessed by FCM through PAC1-FITC binding (a mAb that recognizes activated conformation of fibrinogen receptor) after stimulation with 100 µM TRAP (agonist of the PAR-1 receptor) and with 10 µM ADP.Platelet apoptosis was analysed by FCM through FITC-annexin V (BD Pharmingen) binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions and by determination of caspases -3, -8, and -9 activities (BD Millipore).Platelet surface exposure of lectins were analyzed, respectively, with 1 µg/ml FITC-conjugated Wheat germ agglutinin lectin (WGA) or 1 µg/ml FITC-conjugated Erythrina cristagalli lectin (ECL). WGA binds to sialic acid and N-acetylglucosaminyl residues and ECL is a galactose specific legume lectin.Comparisons of quantitative variables were made with SPSS.22 software.Results: Platelets from refractory ITP patients showed a lower ability for being activated by TRAP or ADP agonists than those from ITP patients responders to treatments. Diminished responses to activation might be due to the reduced surface expression of fibrinogen receptor observed in platelets from refractory ITP patients (TABLE 1).Platelets from refractory ITP patients expressed more PS than those from responders-ITP patients under basal conditions. Moreover, platelets from refractory ITP patients showed higher activity of their caspases -3, -8 and -9 (TABLE 1).Platelets are known to contain sialic acid on glycoprotein carbohydrate side-chains. Abnormal activation of diverse sialidases causes a loss of sialic acid bound to the carbohydrate side chains of glycoproteins from platelets, leading to increased exposure of β-galactose which favors platelet capture through Ashwell-Morel receptors. This fact provides insights into the potential role of the analysis of the binding of lectins to glycoproteins of platelets from ITP patients.No differences were observed between groups for binding of ECL (responders ITP patients: 26.71±14.66, refractory ITP patients: 43.70 ± 26.16). In contrast, binding of WGA was significantly higher in the group of patients with refractory ITP (FIGURE 1).Conclusion: Platelets of refractory ITP patients had less capacity of activation by agonist stimulation and an enhanced apoptosis than platelets from ITP patients responders to treatments. Moreover, adhesion receptors expressed in their platelet surface were reduced and their glycosylation pattern seemed to differ from those of ITP patients responders to treatments. This observation poses the need of studying glycosylation processes and their consequence in platelet function and half-life.Work supported by grant from FIS-FEDER PI15/01457. NVB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresÁlvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy. Jimenez-Yuste:Novo Nordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy.

MiR-204-5p and Platelet Function Regulation: Insight into a Mechanism Mediated by CDC42 and GPIIbIIIa.

Background Several platelet-derived microRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking. Aims To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues. Methods Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor, and a fibrinogen-binding assay. Quantitative polymerase chain reaction and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy. Results miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet–fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation. Conclusion We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.

Platelet Integrin αIIbβ3 Activation is Associated with 25-Hydroxyvitamin D Concentrations in Healthy Adults.

Cardiovascular events are associated with low circulating vitamin D concentrations, although the underlying mechanisms are poorly understood. This study investigated associations between 25-hydroxyvitamin D concentrations, platelet function, and single-nucleotide polymorphisms (SNPs) in genes influencing vitamin D biology in the 500 Functional Genomics (500FG) cohort. In this observational study, platelet activation and function were measured by flow cytometry by binding of fibrinogen to the activated fibrinogen receptor integrin αIIbβ3 and expression of P-selectin, markers of platelet aggregation and degranulation, respectively. These parameters were correlated to serum 25-hydroxyvitamin D and genotyping was performed to investigate SNPs in genes important for vitamin D biology. Circulating 25-hydroxyvitamin D concentrations correlated inversely with baseline platelet binding of fibrinogen to integrin αIIbβ3 (Pearson's r= -0.172, p = 0.002) and platelet responses to platelet agonist cross-linked collagen-related peptide (CRP-XL) (Pearson's r= -0.196,p = 0.002). This effect was due to circulating vitamin D levels ≤50nmol/L, since no differences in platelet fibrinogen binding were observed between subjects with normal 25-hydroxyvitamin D concentrations (>75nmol/L) and a 25-hydroxyvitamin D insufficiency (50-75 nmol/L). No correlations between 25-hydroxyvitamin D concentrations and platelet P-selectin expression were found. Several SNPs in the GC region of the vitamin D binding proteingene were associated with platelet responses to CRP-XL. Low circulating vitamin D concentrations are associated with increased platelet fibrinogen binding to integrin αIIbβ3 in unstimulated samples and after stimulation with CRP-XL. These findings may contribute to the increased incidence of cardiovascular events in vitamin D deficient adults and its seasonal variation. Further studies are needed to investigate causality.

Unexpected low expression of platelet fibrinogen receptor in patients with chronic myeloproliferative neoplasms: how does it change with aspirin?

SummaryThis study was conducted to evaluate the expression of fibrinogen receptors on platelets of Philadelphia‐negative chronic myeloproliferative neoplasm (MPN) patients. We collected blood samples from 40 consecutive MPN patients and healthy volunteers. We performed flow cytometry analysis of P‐selectin expression and integrin beta‐3, activation of glycoprotein (GP) IIb/IIIa and fibrinogen receptor exposure (PAC‐1 binding). Surprisingly, we found a very low PAC‐1 binding capacity in MPN patients; however, the expression of PAC‐1 was almost completely recovered with aspirin intake. We hypothesize that the hypercoagulable states observed in MPN patients could depend on a primarily plasma‐driven impairment of fibrin turnover and thrombin generation.

Unexpected Low Expression of Platelet Fibrinogen Receptor in Patients with Chronic Myeloproliferative Neoplasms: How Does It Change with Aspirin?

INTRODUCTIONPatients affected by Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered at high risk of thrombo-haemorrhagic events, but the role of the platelet count in the assessment of the risk of vascular events is still controversial. A tight correlation was found between the platelet count and plasma sCD40L, which appears to be required for thrombus formation in vivo. However sCD40L is increased both in MPNs and reactive thrombocytosis. Intravascular aggregates of platelets and leukocytes, mediated by P-selectin and CD11b on the former and the latter, respectively, have been observed.Both of this processes should imply a perpetual - and measurable - platelet activation. Several studies on platelet function have been already proposed, nevertheless the mechanisms through which platelets are able to trigger vascular events, are not yet adequately clarified.A refined method for the determination of platelet activation appears to be the use of platelet PAC-1 antibody, able to identify the expression of the fibrinogen receptor of platelet glycoprotein IIb/IIIa. This expression is indeed unique in the process of platelet activation, and yet rarely analyzed. Moreover, since the platelet fibrinogen receptor exposure seems to be influenced by turbulence in blood flow, we have thought that its evaluation could provide important biological evidences to explain some clinical manifestations, such as microvascular disturbances.METHODSBlood samples from 40 consecutive MPNs patients who never received cytoreductive agents, were obtained. 28/40 patients were receiving a continuative antiplatelet prophylaxis with low dose aspirin (ASA, 75-100 mg) at the time of collection, while 12/40 of them were not on such therapy. Our aim was to verify the expression of platelet fibrinogen receptors (PFRs) in the two different groups of patients compared to healthy volunteers, using whole blood flow cytometry. In each experiment sodium citrate and heparin (positive control of platelet activation) tubes were collected from the same patient. Within 10 minutes from blood sampling, 5 ml of whole blood from each tube was incubated for 20 minutes at room temperature in the dark with saturating concentration of CD61 PerCP, CD62P PE and PAC-1 FITC. Positive control was also incubated with PAC-1 in the presence of Arg-Gly-Asp-Ser (RGDS) in order to test the specific antibody binding. Samples were fixed with paraformaldehyde 1% for 30 minutes at 4°C in the dark and analyzed on a flow cytometer.RESULTSSurprisingly, we have been able to verify a very low PAC-1 binding to platelets in patients with MPNs not receiving cytoreduction nor antiplatelet agents (33%) if compared to that observed in healthy subjects (61%; p<0.0001). The use of aspirin seems conversely to restore the expression of platelet fibrinogen receptor, as PAC-1 binding capacity is comparable to that of healthy volunteers (56%). No difference was found with respect to the JAK2 Val617Phe mutation and its allele burden. Interestingly, by focusing on the group of patients under antiplatelet prophylaxis and with no history of thrombosis, it was found that subjects with persistent microcirculatory disorders show a higher PAC-1 binding capacity if compared to the asymptomatic ones (67% vs 52%, p= 0.04).CONCLUSIONIn untreated MPNs, a large amount of platelets are resting in a conformation that is unable to bind fibrinogen, as demonstrated by the low PAC-1 expression in cytofluorimetry. This lack of activity can be reversed by administering ASA, which is also known for its fibrinolytic and hypoprothrombinemic effects. We hypotesize that the hypercoagulable states observed in these patient could depend on a primarly plasma-driven impairment of fibrin turnover and thrombin generation. Further investigations are going to be promoted by our Group. PAC-1 could be at the same time a good marker of aspirin resistance in patients experiencing microcirculatory disorders. [Display omitted] DisclosuresMartinelli:Janssen: Consultancy; Jazz Pharmaceuticals: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Novartis: Speakers Bureau.

Increased Platelet-Monocyte Interaction in Stable COPD in the Absence of Platelet Hyper-Reactivity

Background: Chronic obstructive pulmonary disease (COPD) is well known for its cardiovascular co-morbidities. Increased platelet-monocyte interaction is found in COPD and may reflect altered platelet function and a potential role for anti-platelet therapy. Objectives: The objectives were to investigate platelet-monocyte interaction, platelet activation and reactivity and plasmatic coagulation in stable COPD. Methods: Platelet-monocyte interaction and platelet activation were determined by flow cytometry in 30 stable COPD patients and 25 controls. Platelet activation was measured by binding of fibrinogen to the activated fibrinogen receptor and platelet P-selectin expression at baseline and after platelet stimulation with platelet agonists. Plasmatic coagulation was measured by D-dimer and thrombin generation. Results: Platelet-monocyte interaction was increased in stable COPD (median fluorescence intensity of platelet CD61 was 19.8 [IQR 14.0-33.2] vs. 10.0 [IQR 8.7-16.7], p = 0.002). In contrast, platelet activation and reactivity, reflected by fibrinogen binding and P-selectin expression, were the same in both groups. Plasma P-selectin and interleukin-6 were increased in COPD (p = 0.01 and p = 0.02, respectively), whereas soluble fibrinogen, D-dimer and thrombin generation were similar. Conclusions: Increased platelet-monocyte interaction was found in the absence of platelet hyper-reactivity and activation of plasmatic coagulation in stable COPD. Future clinical evaluation of the effects of different anti-platelet drugs in COPD is warranted, as anti-platelet therapy may interfere with platelet-monocyte interaction.

Opposite effects of Agrimonia pilosa Ledeb aqueous extracts on blood coagulation function

Agrimonia pilosa Ledeb (APL) has showed anticoagulant and antithrombotic activities in some studies, whereas its actual effects on blood coagulation are still unclear. This study was designed to observe the in vitro effects of APL aqueous extracts on blood coagulation, as well as to investigate the underlying mechanisms. Studies were divided into four groups: 0, 4, 20, and 80 g/L of APL aqueous extracts mixed with plasma or whole blood samples. Clotting time of whole blood, plasma coagulation tests, activities of plasma coagulation factors, plasma calcium ion, platelet aggregation test, and platelet fibrinogen receptor as well as the blood viscosity were measured. It was observed that the APL aqueous extracts in 4 g/L significantly prolonged the whole blood clotting time and activated partial thromboplastin time, shortened prothrombin time, decreased activities of coagulation factor VIII, IX and XI, and levels of platelet aggregation and fibrinogen receptor expression. However, coagulation factor VII activity, and blood viscosity were increased after the extracts treatment. And the effects of APL extracts were in a concentration-dependent manner (0-80 g/L). The results suggest that APL aqueous extracts have a total anticoagulant activity, whereas they exhibit opposite effects of greater anticoagulant activity than pro-coagulant activity.

Procoagulant Profile of Platelets from Immune Thrombocytopenia Patients

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder in which both increased platelet destruction and insufficient platelet production are involved. Patients can have a range of bleeding manifestations from none to severe at a similar platelet count. In some cases, patients have fewer bleeding symptoms than expected considering the low platelet count that they might have.Objective: The aim of this study was to determine the procoagulant profile of platelets from ITP patients in order to determine whether any of their features may explain this observation.Methods: Twenty-five patients with chronic ITP [(68±100)x109 platelets/L, mean age: 59.6 ± 16.1 years old, 56% female)] and thirty-five healthy controls [(256±36)x109 platelets/L, mean age: 41.6 ± 13.5 years old, 51% female) were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP).To obtain washed platelets, the top two-thirds volumes of PRP were collected and centrifuged (650 g for 10 min at 23°C) after the addition of acid-citrate-dextrose (ACD, 1:10) and the pellet was resuspended in an equal volume of HEPES buffer.Platelet activation was determined by flow cytometry through binding of FITC-PAC1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 micromol/L thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) or 20 micromol/L ADP.Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions.To characterize platelet ability to bind coagulation factors, washed platelets (1x108/mL) were activated with 100 micromol/L TRAP and then incubated with FVa and/or FXa (5nM each, 10 min, ambient temperature). After fixation with 2% paraformaldehyde to cross-link the platelet-bound factors Va and Xa, platelets were washed two times with Hepes Buffer. Non-specific binding sites were blocked with 8% bovine serum albumin (30 min, room temperature). Following centrifugation, platelets were first incubated with anti-CD41-PE, anti-FVa and/or anti-FXa and then with a secondary FITC-goat anti-mouse IgG and stored at 4°C until flow cytometry analyses.Results: Platelets from ITP patients showed a basal expression of activated fibrinogen receptor similar to controls and a reduced ability for being activated by agonists (% of positive platelets for TRAP-induced PAC1 binding: 60±20 % in controls and 35±23 % in ITP, p<0.01; ADP-induced PAC1 binding: 63±14 % in controls and 50±23 % in ITP, p<0.05). Diminished responses to activation were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients.Platelets from ITP patients expressed more PS than controls under basal conditions [mean fluorescence (MF) for FITC-annexin V binding was: 336±128 in controls, 588±25 in ITP, p<0.05].Since the PS is the anchor site of the prothrombinase complex, we studied the binding of FVa and FXa at baseline and after activating platelets with TRAP. The binding of these factors in both conditions was higher in the group of patients with ITP (MF for basal FVa binding: 41.4±14.4 in controls, 58.1±24 in ITP, p <0.02; MF for TRAP-induced FVa binding: 44.1±11.4 in controls, 81.4±38 in ITP, p<0.001; MF for basal FXa binding: 45.7±18.4 in controls, 58.1±24 in ITP, p <0.005; MF for TRAP-induced FXa binding: 46.1±16.4 in controls, 72.0±24 in ITP, p<0.05). The lower the platelet count the higher increase in PS exposure (Spearman r =-0,518, p <0.001) and the union of FVa (Spearman r = -0.8571, p <0.001) and FXa (Spearman r = -0.7455, p<0.05).Conclusions: Platelets from ITP patients, despite having less capacity of activation by agonist stimulation, have an increased procoagulant surface with greater ability to bind prothrombinase complex (FXaVa) than those from healthy controls. This feature might be a procoagulant compensatory mechanism that could reduce the risk of bleeding in patients with ITP.This work was supported by a grants from the FIS-FEDER, PI12/01831 and PI15/01457 DisclosuresNo relevant conflicts of interest to declare.

Xanthohumol from hop cones (Humulus lupulus L.) prevents ADP-induced platelet reactivity

Hop cones (Humulus lupulus L.), very rich source of phenolic compounds, possessing anticancer, antioxidant and anti-inflammatory activities, are considered as beneficial diet ingredients improving human health. In this study, the antiplatelet action of xanthohumol (XN), the principal flavonoid in hop cones, was investigated. XN significantly attenuated ADP-induced blood platelet aggregation (97.2 ± 35.7 AU for 6 μg/ml of XN vs. 120.4 ± 30.1 AU for 0.17% dimethyl sulfoxide (DMSO), p < 0.001) and significantly reduced the expression of fibrinogen receptor (activated form of GPIIbIIIa) on platelets’ surface (47.6 ± 15.8 for 1.5 μg/ml XN, 44.6 ± 17.3% for 3 μg/ml XN vs. 54.5 ± 19.2% for control or 43.3 ± 18.4% for 6 μg/ml XN vs. 49.7 ± 19.4% for 0.17% DMSO, p < 0.05 or less). These findings suggest that the phenolic compounds originating from hops (XN) have a novel role as antiplatelet agents and can likely be used as dietary supplements in prophylactic approaches.

Specific cell-derived microvesicles: Linking endothelial function to carotid artery intima-media thickness in low cardiovascular risk menopausal women

BackgroundDecreases in endothelial function measured by reactive hyperemic index (RHI) correlated with increases in carotid intima-media thickness (CIMT) in recently menopausal women with a low risk cardiovascular profile. Factors linking this association are unknown. ObjectiveAssess, longitudinally, markers of platelet activation and cell-derived, blood-borne microvesicles (MV) in relationship to RHI and CIMT in asymptomatic, low risk menopausal women. MethodsRHI by digital pulse tonometry (n = 93), CIMT by ultrasound (n = 113), measures of platelet activation and specific cell-derived, blood-borne MV were evaluated in women throughout the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic. ResultsCIMT, but not RHI, increased significantly over 4 years. The average change in CIMT correlated significantly with the average follow-up values of MV positive for common leukocyte antigen [CD45; ρ = 0.285 (P = 0.002)] and VCAM-1 [ρ = 0.270 (P = 0.0040)]. Using principal components analysis (PC) on the aggregate set of average follow-up measures, the first derived PC representing numbers of MV positive for markers of vascular endothelium, inflammatory cells (leukocyte and monocytes), pro-coagulant (tissue factor), and cell adhesion molecules (ICAM-1 and VCAM-1) associated with changes in RHI and CIMT. Changes in RHI associated with another PC defined by measures of platelet activation (dense granular ATP secretion, surface expression of P-selectin and fibrinogen receptors). ConclusionsMV derived from activated endothelial and inflammatory cells, and those expressing cell adhesion and pro-coagulant molecules may reflect early vascular dysfunction in low risk menopausal women. Assays of MV as non-conventional measures to assess cardiovascular risk in asymptomatic women remain to be developed.

Longitudinal effects of menopausal hormone treatments on platelet characteristics and cell-derived microvesicles

Activated platelets serve as a catalyst for thrombin generation and a source of vasoactive and mitogenic factors affecting vascular remodeling. Oral menopausal hormone treatments (MHT) may carry greater thrombotic risk than transdermal products. This study compared effects of oral and transdermal MHT on platelet characteristics, platelet proteins, and platelet-derived microvesicles (MV) in recently menopausal women. Platelets and MV were prepared from blood of a subset of women (n = 117) enrolled in the Kronos Early Estrogen Prevention Study prior to and after 48 months of treatment with either oral conjugated equine estrogen (0.45 mg/day), transdermal 17β-estradiol (50 µg/day), each with intermittent progesterone (200 mg/day for 12 days a month), or placebo pills and patch. Platelet count and expression of platelet P-selectin and fibrinogen receptors were similar across groups. An aggregate measure of 4-year change in vasoactive and mitogenic factors in platelet lysate, by principle component analysis, indicated significantly lower values in both MHT groups compared to placebo. Increases in numbers of tissue factor positive and platelet-derived MV were significantly greater in the transdermal compared to placebo group. MHT was associated with significantly reduced platelet content of vasoactive and mitogenic factors representing a potential mechanism by which MHT may affect vascular remodeling. Various hormonal compositions and doses of MHT could differentially regulate nuclear transcription in bone marrow megakaryocytes and non-genomic pathways in circulating platelets thus determining numbers and characteristics of circulating MV. Thrombotic risk associated with oral MHT most likely involves liver-derived inflammatory/coagulation proteins rather than circulating platelets per se.

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.

The disulfide isomerase ERp57 mediates platelet aggregation, hemostasis, and thrombosis

AbstractA close homologue to protein disulfide isomerase (PDI) called ERp57 forms disulfide bonds in glycoproteins in the endoplasmic reticulum and is expressed on the platelet surface. We generated 2 rabbit Abs to ERp57. One Ab strongly inhibited ERp57 in a functional assay and strongly inhibited platelet aggregation. There was minimal cross-reactivity of this Ab with PDI by Western blot or in the functional assay. This Ab substantially inhibited activation of the αIIbβ3 fibrinogen receptor and P-selectin expression. Furthermore, adding ERp57 to platelets potentiated aggregation. In contrast, adding a catalytically inactive ERp57 inhibited platelet aggregation. When infused into mice the inactive ERp57 prolonged the tail bleeding times. We generated 2 IgG2a mAbs that reacted with ERp57 by immunoblot. One of these Abs inhibited both ERp57 activity and platelet aggregation. The other Ab did not inhibit ERp57 activity or platelet aggregation. The inhibitory Ab inhibited activation of αIIbβ3 and P-selectin expression, prolonged tail bleeding times, and inhibited FeCl3-induced thrombosis in mice. Finally, we found that a commonly used mAb to PDI also inhibited ERp57 activity. We conclude that a glycoprotein-specific member of the PDI family, ERp57, is required for platelet aggregation, hemostasis, and thrombosis.

Effect of pre-infarction angina on platelet reactivity in acute myocardial infarction

BackgroundPrevious studies demonstrated that patients with PIA have a smaller infarct and better in-hospital outcome after acute myocardial infarction, than those without angina. This protective effects has been attributed to ischemic preconditioning (PC), to earlier reperfusion after fibrinolysis or its better collateral circulation development. In this study we aimed at assessing platelet reactivity in patients with history of pre-infarction angina (PIA) in the acute phase of ST segment elevation myocardial infarction (STEMI) and 1month later. Methods85 consecutive patients (63±10.5years, 60 male) with a first STEMI treated by primary percutaneous coronary intervention (PCI) were studied at admission and 1month later. Platelet reactivity was evaluated by flow cyometry with and without adenosine diphosphate (ADP) stimulation, by measuring monocyte-platelet aggregates (MPAs) and glycoprotein IIb/IIIa (CD41) expression in the MPA gate, and CD41 and fibrinogen receptor (PAC-1) expression in the platelet gate. ResultsMPAs and expression of platelet receptors CD41 and PAC-1 were significantly lower in patients with than patients without PIA, both with and without ADP stimulation. After 1month, all cytometry variables both with and without ADP stimulation were similar in the two groups. ConclusionsThis study shows, for the first time, that patients with a first STEMI who experience PIA show a lower platelet reactivity as compared with those without history of PIA.

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.

Evidence of increased platelet reactivity in the first six months after acute ST segment elevation myocardial infarction

Introduction Platelets play a crucial role in the pathogenesis of acute coronary syndromes. Accordingly, previous studies showed increased platelet reactivity on admission in these patients. In this study we assessed platelet reactivity at short-medium term follow-up in patients with ST-segment elevation acute myocardial infarction (STEMI). Materials and methods Fifty-nine patients (58 ± 11 years, 45 men), treated with primary angioplasty, were studied 1 month after STEMI. Thirty-five patients were retested at 6 months. Twenty matched patients with stable coronary artery disease served as controls. Platelet reactivity was assessed by flow cyometry at rest and at peak exercise, with and without adenosine diphosphate (ADP) stimulation, by measuring monocyte-platelet aggregates (MPAs) and glycoprotein IIb/IIIa (CD41) expression in the MPA gate, and CD41 and fibrinogen receptor (PAC-1) expression in the platelet gate. Results Compared to controls, basal MPAs and CD41 in the MPA gate were higher in STEMI patients both at 1 month (p = 0.001 and p = 0.002, respectively) and at 6 months (p = 0.03 and p = 0.01, respectively). Basal CD41 and PAC-1 expression was also higher in STEMI patients at the two assessments compared to controls (P < 0.001 for both). Exercise induced a similar increase in platelet reactivity in patients and controls. ADP induced a higher increase in CD41 platelet expression in STEMI patients compared to controls both at 1 and 6 months (P < 0.001). Conclusion Platelet reactivity is increased in the first 6 months after STEMI. The persistence of increased platelet reactivity in this time period may play a role in the early recurrence of coronary events after STEMI.