Thrombin Receptor-activating Peptide Research Articles

Reticulated platelet mass cytometry reveals unexplored therapeutic targets in patients with chronic coronary syndrome

Abstract Background Reticulated platelets (RPs) are young, hyper-reactive thrombocytes that contain more RNA compared with mature platelets (MPs). The measurement of RPs level in peripheral blood with point-of-care systems is fast, reproducible, and inexpensive. Elevated RPs in peripheral blood predict adverse events in patients with acute and chronic coronary syndrome through unknown mechanisms. Preliminary transcriptome analyses reported an enrichment of pro-thrombotic transcripts. However, proteomic analyses are not available, and the biological features of RPs are largely unknown. Purpose We aimed to perform the largest proteomic characterization of RPs using mass cytometry with single-cell resolution in patients with chronic coronary syndrome (CCS) undergoing dual antiplatelet therapy (DAPT). Methods Thrombocytes from peripheral blood of CCS patients were isolated, prepared for mass cytometry (CyTOF) and stained with a custom-made CyTOF-panel of 20 antibodies targeting important transmembrane proteins (anti-CD9, anti-CD29, anti-CD31, anti-CD36-, anti-CD40, anti-CD41, anti-CD42a, anti-CD42b-, anti-CD47, anti-CD61, anti-CD62P-, anti-CD63, anti-CD69, anti-CD107a, anti-CD154, anti-GPVI, antiGPIIb/GPIIIa complex, anti-Par1, anti-PEAR-1 and the negative control anti-CD3 coupled with different metal isotopes). Two samples were prepared from each donor: one baseline sample (non-stimulated platelets) and one sample stimulated with 10 μM thrombin receptor-activating peptide (TRAP). According to previous experiences and common practice, we detected RPs and MPs based on their RNA content. We analyzed the results with a custom bioinformatic pipeline. Results 13 patients with CCS on DAPT were included in this study. Mass cytometry highlighted an expression heterogeneity of relevant transmembrane proteins in thrombocytes of CCS patients (Figure 1A-B colored according to expression level: from blue-low to red-high). CyTOF detected an upregulation of important transmembrane receptors in RPs compared to MPs in quiescent platelets: GPVI (p<0.0001), PAR-1 (p<0.0001), GPIX (p<0.0001), and GPIbα (p<0.0001, Figure 1C). After TRAP-stimulation, RPs expressed higher levels of the activation markers P-Selectin (p=0.0016) and LAMP-3 (CD63, p<0.0001) compared to MPs confirming RPs hyperactivity (Figure 1D). Conclusion We here describe the first biological proteomic characterization with single-cell resolution of RPs biology in CCS patients. The upregulation of the activation markers P-Selectin and LAMP-3 as well as of specific transmembrane proteins as the collagen receptor GPVI and the thrombin receptor PAR-1 in patients treated with DAPT (schematic overview in Figure 2) provides the first solid biomolecular explanation of RPs hyper-reactivity and involvement in cardiovascular disease. Moreover, these results offer unexplored therapeutic targets to tailor antiplatelet therapy based on platelet protein expression in patients with elevated RPs Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Center for Cardiovascular Research (DZHK) Figure 1. Platelet expressionFigure 2. Schematic overview

The Impact of Dabigatran and Rivaroxaban on Variation of Platelet Activation Biomarkers and DRT Following Percutaneous Left Atrial Appendage Closure.

Background: The current post-procedure antithrombotic recommendation for left atrial appendage closure (LAAC) remains empiric. This study was designed to compare variations in platelet activation biomarkers and device-related thrombosis (DRT) under different antithrombotic regimens following LAAC.Methods: This study enrolled 105 consecutive patients with atrial fibrillation who underwent LAAC successfully and received post-procedure anticoagulation with either dabigatran (N = 33) or rivaroxaban (N = 72). After 3 months of anticoagulation treatment, thromboelastogram was used to evaluate thrombin receptor–activating peptide (TRAP)–induced platelet aggregation (PA). Measurements of platelet activation biomarkers, including thrombin–antithrombin complex (TAT), P-selectin, von Willebrand disease (vWF), and CD40L, were performed immediately before the LAAC procedure and after 3 months of post-procedure anticoagulation. Repeated transesophageal echocardiography was performed to evaluate DRT during follow-ups.Results: Three (4.2%) patients in the rivaroxaban and 4 (12.1%) patients in the dabigatran group experienced DRT events (odds ratio (OR) = 0.315, 95% confidence interval (95%CI): 0.066–1.489, p = 0.129) during follow-ups. The TRAP-induced PA was statistically significantly higher in the dabigatran group (62.9% vs 59.7%, p = 0.028*). Statistically significant increases in plasma concentration of TAT, P-selectin, and vWF were observed after 3 months of exposure to dabigatran when compared with rivaroxaban. An increased expression of platelet activation biomarkers was observed in DRT subjects compared with non–DRT subjects in terms of P-selectin and vWF (65.28 ± 13.93 ng/L vs 32.14 ± 12.11 ng/L, p = 0.037; 501.92 ± 106.48 U/L vs 280.98 ± 54.10 U/L, p = 0.045; respectively). Multivariate regression analysis indicated that the use of dabigatran might be an independent predictor of DRT (p = 0.022; OR = 4.366, 95%CI: 0.434–10.839). Furthermore, the CHA2DS2-VASc score (OR = 2.076, p = 0.016) and CD40L levels (OR = 1.015, p = 0.021) were independent predictors of increased D-dimer levels.Conclusions: Post-LAAC anticoagulation with dabigatran may increase the risk of DRT by enhancing platelet reactivity. In light of this potential increased risk in DRT, the authors recommend against using dabigatran for post-procedural anticoagulation in patients who have undergone LAAC.

Antiplatelet Activity of Cucurbita maxima.

Natural extracts constitute an important source in the prevention of noncommunicable diseases, such as cardiovascular diseases. The pumpkin, Cucurbita maxima, is widely consumed in Chile. Pumpkin seeds, despite having crude protein, lipids, and carbohydrates, are regarded as agro-industrial waste. In this work, we correlated the antiplatelet activity of aqueous, ethanolic, and methanolic extracts from pumpkin seeds with their bioactive compounds. In vitro platelet aggregation and activation studies were performed by turbidimetry and flow cytometry, respectively. Results reveal that the extracts inhibited, in a dose-dependent manner, platelet aggregation induced by adenosine diphosphate, thrombin receptor activator peptide 6 (TRAP-6), and collagen. Pumpkin seed extracts inhibited P-selectin secretion and glycoprotein IIb/IIIa activation on TRAP-6-activated platelets. They were found to be rich in fatty acids and a powerful source of plant-based protein, which could be related to the high antiplatelet potential identified in extracts. This research demonstrated that pumpkin seed extracts could be a candidate in the prevention of thrombotic events.

Platelet Surface Protein Expression and Reactivity upon TRAP Stimulation after BNT162b2 Vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a coagulopathy characterized by platelet activation and a hypercoagulable state with an increased incidence of cardiovascular events. The viral spike protein S has been reported to enhance thrombosis formation, stimulate platelets to release procoagulant factors, and promote the formation of platelet-leukocyte aggregates even in absence of the virus. Although SARS-CoV-2 vaccines induce spike protein overexpression to trigger SARS-CoV-2-specific immune protection, thrombocyte activity has not been investigated in this context. Here, we provide the first phenotypic platelet characterization of healthy human subjects undergoing BNT162b2 vaccination. Using mass cytometry, we analyzed the expression of constitutive transmembrane receptors, adhesion proteins, and platelet activation markers in 12 healthy donors before and at five different time points within 4 weeks after the first BNT162b2 administration. We measured platelet reactivity by stimulating thrombocyte activation with thrombin receptor-activating peptide. Activation marker expression (P-selectin, LAMP-3, LAMP-1, CD40L, and PAC-1) did not change after vaccination. All investigated constitutive transmembrane proteins showed similar expressions over time. Platelet reactivity was not altered after BNT162b2 administration. Activation marker expression was significantly lower compared with an independent cohort of mild symptomatic COVID-19 patients analyzed with the same platform. This study reveals that BNT162b2 administration does not alter platelet protein expression and reactivity.

Investigation of platelet function in patients with chronic kidney disease stages IV-V.

Patients with renal failure are at increased risk of both bleeding and thrombosis. Further descriptions of laboratory investigations in these patients are required. Investigation of 24 patients with chronic kidney disease (CKD) stages IV-V with light transmission aggregometry, platelet secretion assays and platelet nucleotide analysis. Patients were in a nonbleeding state and not on antiplatelet medication. Results were compared with our local reference range used within the clinical haematology service. Of the 24 patients, two had decreased responses to arachidonic acid, adenosine diphosphate, collagen, thrombin receptor activator peptide-6 and one had decreased responses to high dose ristocetin, and one had increased response to low dose ristocetin. 11 and 13 out of 24 had abnormal platelet secretion release to collagen and thrombin, respectively. Platelet nucleotide analysis in patients was normal with the exception of a reduction in ADP content in one patient and ATP/ADP ratio in one patient. In our collection of patients with CKD investigated for platelet function in the nonbleeding state, they generally had normal light transmission aggregometry and nucleotide analysis but around 50% had decreased platelet secretion assays. These results could be important in determining the significance of platelet function tests in patients with bleeding symptoms and renal failure. Further characterization of platelet function tests in future will help characterize haemostasis in renal failure further.

Macrostachyols A-D, oligostilbenes from Gnetum macrostachyum inhibited in vitro human platelet aggregation

Introduction: Gnetum macrostachyum is a known Thai medicinal plant as a source of bioactive oligostilbenes, which possess platelet inhibitory activities. The study aimed to evaluate the in vitro human platelet aggregation inhibitory activities of macrostachyols A-D (compounds 1-4) isolated from the roots of G. macrostachyum. Methods: The in vitro human platelet aggregation assay was assayed with a 96-well microtiter plate format. The well-known aggregating agents were used to investigate the possible mechanism of inhibition, including adenosine diphosphate (ADP), arachidonic acid (AA), thromboxane A2 analog (U-46619), collagen, thrombin, and thrombin receptor-activating peptide-6 (TRAP-6). Results: Compound 1 was more potent than ibuprofen (positive control) on the adenosine diphosphate- induced platelet aggregation assay (P < 0.05). Compound 3 was more potent than 1, 2, and 4 (P < 0.05), but all active oligostilbenes were less potent than the positive control (P < 0.05) on the arachidonic acid-induced platelet aggregation assay. The oligostilbenes 1, 2, 3, and 4 also displayed the inhibitory effects on the U-46619-induced platelet aggregation. The tetrameric stilbenes 1 was the only compound that exhibited inhibitory effects on thrombin-induced platelet aggregation without TRAP-6 mediated platelet aggregation. Conclusion: The findings revealed the inhibitory effects of oligostilbenes on human platelet aggregation through a target-specific experimental design. It suggests that oligostilbenes from this plant might be applied as antiplatelet aggregation agents in platelet hyperreactivity- related diseases.

Role of Platelet Function Test in Predicting Postoperative Bleeding Risk after Coronary Artery Bypass Grafting: A Prospective Observational Study

AbstractPatients undergoing cardiac surgery are at risk of excessive bleeding and its associated complications. Excessive bleeding during and after cardiac surgery has an incidence of ~20%. Massive bleeding and subsequent requirement for blood product administration and mediastinal reexploration are associated with significant morbidity and mortality. Postoperative, nonsurgical bleeding in cardiac surgical patients is often multifactorial. Platelet dysfunction, excessive fibrinolysis, hypothermia, preoperative anemia, and deficiency of coagulation factors or their dilution are all suggested etiologies of postoperative bleeding. In the Arachidonic Acid Thromboelastometry (ARATEM) test, platelets are activated with arachidonic acid; in Adenosine diphosphate Thromboelastometry (ADPTEM) test, platelets are activated with adenosine diphosphate; and in TRAPTEM test, platelets are activated with thrombin receptor-activating peptide 6. Measurement time is 6 minutes, and results are expressed in three different parameters: A6 (amplitude at 6 minutes, in Ohm); MS (maximum slope of the aggregation curve in Ohm/min), and AUC (area under the curve in Ohm.min). Algorithm-based point-of-care platelet function testing helped us to preemptively give the right blood component therapy, avoiding fibrinolytic bleeding in the postoperative period.

Avatrombopag, a Novel Thrombopoietin Receptor Agonist, Increases Platelet Counts without Increasing Platelet Activation in Patients with Thrombocytopenia Due to Chronic Liver Disease

Background: The thrombopoietin (TPO) receptor c-mpl is expressed on the surface of megakaryocytes and platelets. In addition to its effect on increasing megakaryocyte growth and maturation, TPO also lowers the threshold for platelet activation in vivo and in vitro . TPO receptor agonists (TPO-RA) may, perhaps to differing extents, also lead to platelet activation. Indeed, in a recent study of patients with chronic liver disease (CLD), the TPO-RA eltrombopag reduced the need for platelet transfusions, but was associated with an increased incidence of thrombosis. Therefore, the goal of this study was to evaluate the effect of avatrombopag, a novel TPO-RA, on in vivo platelet activation and in vitro platelet reactivity in response to agonists in patients with CLD.Methods: CLD patients participating in the Phase 3 E5501-G000-310 and -311 studies (Randomized, Global, Double-blind, Placebo-controlled, Parallel-group Studies to Evaluate the Efficacy and Safety of Once-daily Oral Avatrombopag for the Treatment of Adults with Thrombocytopenia Associated with Liver Disease Prior to an Elective Procedure) could participate in a substudy to evaluate the effects of avatrombopag on platelet function. IRB-approved informed consent was obtained prior to sample collection. Patients (n = 30) were randomized to treatment on Days 1 - 5 with placebo or avatrombopag: 60 mg daily in patients with platelet counts <40 x 109/L, and 40 mg daily in patients with platelet counts 40 to ≤50 x 109/L. Samples were collected at Baseline (Day 1, pre-drug) and after treatment (Day 4 and Day 10). No patient received a platelet transfusion within the 10 days prior to these blood draws.Platelet activation was evaluated by whole blood flow cytometry (which, unlike other methods, is accurate in samples with low platelet counts) with and without in vitro stimulation with adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP). Endpoints included platelet surface activated glycoprotein (GP) IIb-IIIa (as reported by the activation-dependent monoclonal antibody PAC1), platelet surface P-selectin, platelet surface GPIb, platelet phosphatidylserine expression and generation of platelet-derived microparticles. Citrate-anticoagulated whole blood was incubated (15 min, room temperature) with an antibody cocktail containing fluorescein isothiocyanate (FITC)-conjugated PAC1, phycoerythrin (PE)-conjugated anti-P-selectin, and PE-Cy5-conjugated anti-CD42b (GPIb) with or without ADP 0.5 µM or 20 µM or TRAP 1.5 µM or 20 µM. Samples were fixed with 1% formaldehyde, then shipped cold together with the remaining blood sample (for analysis of phosphatidylserine expression and microparticles) to the Center for Platelet Research Studies for analysis. Phosphatidylserine expression on platelets was determined by staining with FITC-conjugated annexin V. Platelet-derived microparticles were identified by light scattering properties and positive staining for CD41 and CD42b. Changes from Baseline for drug-treated (60 mg and 40 mg avatrombopag cohorts combined, n=20) vs. placebo-treated patients (n=10) were compared using a mixed effects model with p<0.05 being considered significant.Results: Avatrombopag, 60 mg and 40 mg treatment, but not placebo treatment, resulted in significant increases in platelet counts at Day 10 (Table). Circulating activated platelets were not increased in avatrombopag-treated patients compared with placebo-treated patients (Table, “no agonist” data), as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. Platelet reactivity, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin after low and high concentrations of ADP and TRAP, was also not increased in avatrombopag-treated patients compared with placebo-treated patients (Table). Similarly, there was no evidence of increased platelet activation in avatrombopag-treated patients compared with placebo-treated patients as assessed by platelet surface GPIb, platelet phosphatidylserine expression and generation of platelet-derived microparticles.Conclusions: In this randomized, double-blind, placebo-controlled, parallel-group study of patients with thrombocytopenia due to CLD, treatment with avatrombopag resulted in increased platelet counts, but did not increase either platelet activation in vivo or platelet reactivity in vitro . [Display omitted] DisclosuresFrelinger:Ionis: Research Funding; Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding; GLSynthesis: Research Funding; Baxalta: Research Funding. Michelson:Elsevier: Patents & Royalties; Eisai: Research Funding; GLSynthesis: Research Funding; Instrumentation Laboratory: Consultancy; Baxalta: Research Funding; Ionis: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding.

Brain-Derived Neurotrophic Factor Induces Platelet Aggregation

Background: A large extra-cerebral pool of Brain-Derived Neurotrophic Factor (BDNF) is found in platelets. With concentrations reaching 100-1000 fold those of neurons, platelet-stored BDNF is hypothesized to play an important role in the vasculature.Aims: To investigate the effect of BDNF on platelets, alone and synergistically with traditional agonists.Methods: The presence of BDNF in platelets was confirmed by Western Blotting and confocal microscopy. The ability of platelets to release BDNF upon stimulation was assessed by ELISA on platelet releasates following stimulation by maximal concentrations of arachidonic acid, adenosine diphosphate (ADP), epinephrine, collagen, or thrombin-receptor activating peptide (TRAP). To investigate the effect of BDNF on platelet function, we used a recombinant BDNF protein (rBDNF, Peprotech) added exogenously to a preparation of washed platelets obtained from healthy volunteers. Aggregation was recorded by light transmission aggregometry over 20 minutes. Synergistic assays were used with priming (sub-threshold) concentrations of either collagen or TRAP. The dependence on secondary mediators was assessed by blocking either COX-1-dependent thromboxane formation with aspirin or the ADP-P2Y12 receptor with AR-C66096. Platelet aggregation was blocked with a GPIIbIIIa inhibitor (abciximab) to assess the contribution of outside-in signalling.Results: Western blotting showed a 14kD band for the washed platelet protein preparations and for rBDNF used as a positive control. BDNF appeared stored in granules on confocal microscopy, consistent with previous reports. BDNF in the platelet releasate was increased 5-fold following platelet aggregation induced by traditional agonists, as compared with baseline. Exogenous rBDNF had no effect on platelet aggregation at low doses (1 and 3 µg/ml), but induced complete biphasic platelet aggregation at a high dose (10 µg/ml). The secondary wave of platelet aggregation was abrogated in the presence of aspirin and AR-C66096, but they had no effect on the primary wave. Platelet aggregation was completely abolished in the presence of the GPIIbIIIa inhibitor abciximab. Synergistic studies were conducted with priming concentrations of rBDNF, collagen and TRAP which were insufficient to elicit platelet aggregation on their own. Incubation of platelets with rBDNF (3 µg/ml) potentiated the platelet responses to priming concentrations of both collagen and TRAP. Indeed, in the absence of BDNF, no platelet aggregation was seen at the concentrations used, whereas incubation with BDNF led to complete and irreversible platelet aggregation to priming concentrations of collagen and TRAP.Conclusion: Platelets contain BDNF within their granules, which they release upon platelet activation with traditional agonists. Stimulation of washed platelets with high concentrations of rBDNF induced platelets to aggregate; in the presence of low concentrations of rBDNF, platelet aggregation was potentiated in response to priming concentrations of traditional agonists. Platelet responses to rBDNF were partly dependent on secondary mediators, as inhibition of COX-1 and the ADP-P2Y12 receptor inhibited the secondary wave of aggregation. However, the primary wave of aggregation was unaltered, thereby suggesting that platelets either possess a BDNF receptor or a traditional receptor that can signal in the presence of BDNF. Further studies are needed to uncover which receptor is implicated in platelet responses to BDNF, and the physiological role of BDNF-induced platelet responses. DisclosuresNo relevant conflicts of interest to declare.

Mass Cytometry of Platelets: Simultaneous Functional and Phenotypic Analysis of Multiple Surface Markers in Healthy Subjects and Patients with Inherited Platelet Disorders Reveals Novel Platelet Sub-Populations

Introduction: Mass cytometry (MC) is a next generation flow cytometry (FC) platform that enables high-dimensional functional and phenotypic analysis of single cells. The technique uses mass spectrometry to detect metal-conjugated antibodies on single cells to evaluate intra- and extra-cellular targets. A key advantage of MC over standard fluorescence-based FC is that compensation for spectral overlap is not required due to the use of heavy metal ion tags in place of fluorescent tags. This allows simultaneous measurement of ≥ 40 different parameters per single cell in comparison to the 3-5 parameters that comprise a typical FC panel for platelet analysis. This is the first study to demonstrate the power of MC for the detailed analysis of platelet surface markers in healthy subjects and in patients with inherited platelet disorders.Methods: We compared MC and FC in the analysis of healthy donor platelets and patients with Hermansky-Pudlak syndrome (HPS) Type II/III and Glanzmann thrombasthenia (GT). IRB-approved informed consent was obtained prior to blood collection. Citrate anticoagulated whole blood was stained with commercially sourced fluorescently-labeled antibodies and, in parallel, with a single MC cocktail containing 14 metal-tagged antibodies directed towards an array of platelet surface markers. The MC antibody panel comprised a collection of commercially available and custom in-house metal-tagged antibodies (Table). Samples were stained in the presence or absence of thrombin receptor activating peptide (TRAP) or adenosine diphosphate (ADP), fixed and analyzed using a Helios Mass Cytometer or BD FACSCalibur flow cytometer. Data were analyzed using FlowJo/CytoBank and presented as mean metal intensity (MMI; MC) or mean fluorescence intensity (MFI; FC).Results: To compare the use of MC and FC for analysis of platelet function, TRAP and ADP dose-response curves were constructed using PAC-1-FITC/159Tb and CD62P-PE/172Yb binding as functional readouts of healthy donor platelet activation. The sigmoidal curves obtained using both platforms were comparable, and results highly correlated (R2 ≥ 0.9; Figure A). MC enabled simultaneous quantitation of additional platelet surface markers (CD9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD63, CD107a, CD154 and GPVI). As measured by MC, ADP and TRAP stimulation increased the platelet membrane surface expression of CD9, CD41, CD61, CD63, CD107a, CD154 and GPVI and decreased CD42a and CD42b. Surface expression of CD29, CD31 and CD36 as determined by MC remained constant following stimulation. HPS platelets demonstrated reduced PAC-1 binding and normal P-selectin (CD62P) surface expression following TRAP stimulation as compared to healthy donor platelets (Figure B). Interestingly, the type II HPS patient had a significantly elevated platelet surface level of CD63 compared to the healthy donor and HPS Type III donor; a feature that may differentiate the two forms of HPS. GT platelets, as expected, lacked surface CD41 and CD61 and this corresponded with a lack of PAC-1 binding following TRAP stimulation. MC revealed the surface level expression of CD29, CD154 and GPVI to be reduced, CD36 to be significantly elevated, and CD42a, CD63 and CD107a to be similar on GT platelets compared to healthy donor platelets. To take advantage of the high number of parameters simultaneously measured on each cell using MC, results were analyzed using visual stochastic neighbor embedding (viSNE), a tool to generate an optimized 2-dimensional representation of high-dimension data. This analysis revealed platelet sub-populations which were common between healthy subjects (Figure C, healthy donors 1, 2), as well as sub-populations that were unique to particular donors (Figure C, healthy donor 3 vs. 1, 2).Conclusions: Simultaneous analysis of multiple platelet surface markers by MC reveals previously unrecognized platelet sub-populations in healthy donors and provides unique insights into the platelet populations that exist in patients with inherited platelet disorders. MC can be used as a more powerful and efficient tool than FC to assess the heterogeneity of platelets across donors and to characterize platelet populations that exist in patients with inherited platelet disorders. Rigorous identification of these platelet sub-populations may enable future determination of their role in health and their possible utility as biomarkers in disease. [Display omitted] DisclosuresMichelson:Pfizer: Research Funding; Eisai: Research Funding; Sysmex: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Instrumentation Laboratory: Consultancy; Baxalta: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; GLSynthesis: Research Funding; Ionis: Research Funding; Ironwood: Research Funding; Elsevier: Patents & Royalties. Frelinger:Baxalta: Research Funding; GLSynthesis: Research Funding; Ironwood: Research Funding; Ionis: Research Funding; Pfizer: Research Funding; Sysmex: Research Funding.

Absence of hypercoagulability after nCoV-19 vaccination: An observational pilot study

BackgroundIt is still unknown whether COVID-19 vaccines induce a prothrombotic state or increase the hypercoagulable condition in subjects with a predisposition to thrombosis. ObjectivesWe evaluated the coagulation profile in a series of healthy subjects who received the first dose of the BNT162b2 or the ChAdOx1 vaccines and assessed whether hypercoagulability developed. Patients/methodsVolunteers among the staff of the University of Padua or health care professionals in the Padua University Hospital who had received either the ChAdOx1 or BNT162b2 vaccine in the previous 10 ± 2 days were eligible. A cohort of unvaccinated volunteers among family members of the University staff acted as control group. Global coagulation monitoring was assessed by whole blood rotational thromboelastometry, whole blood impedance aggregometry and thrombin generation. Platelet count was also obtained. ResultsOne hundred and ninety subjects were enrolled: 101 (53.2%) received the ChAdOx1 vaccine and 89 (46.8%) the BNT162b2 vaccine. Twenty-eight non-vaccinated subjects acted as controls. Thromboelastometry parameters were all comparable among groups. Thrombin receptor activating peptide (TRAP)-, ADP- and ASPI-induced platelet aggregation were similar among groups, as well as platelet count. Endogenous thrombin potential (ETP) was comparable among groups. The results were confirmed after controlling for age, gender and hormonal. Considering women taking combined oral contraceptives or thrombophilia carriers, no differences were detected in thromboelastometry or thrombin generation parameters between subjects who received ChAdOx1 vs. BNT162b2 vaccines. ConclusionsNo significant activation of fibrinogen-driven coagulation, plasma thrombin generation or clinically meaningful platelet aggregation after ChAdOx1 or BNT162b2 vaccination was observed.

Impaired Contraction (Retraction) of Blood Clots Is a Possible Pathogenic Mechanism in Venous Thromboembolism

Introduction: Deep venous thrombosis of the lower limbs, alone or associated with pulmonary embolism, complicates many diseases and remains as one of the most challenging medical and social problems due to a relatively high incidence and life-threatening risk.Blood clots undergo volume shrinkage induced by the contractile forces of activated platelets and propagated through the clot volume due to platelet-fibrin interactions and elasticity of the fibrin network. This process is known as clot contraction (retraction) which remains one of the least studied steps of blood clotting. Moreover, this phenomenon of clot contraction has been shown to occur not only in vitro but also in in vivo, but its (patho)physiological importance remains unclear.Aims: This work was designed to examine the potential pathogenic role of clot contraction in venous thromboembolism (VTE).Methods: We investigated contraction of clots formed from the blood of 55 patients with VTE compared with those of 60 healthy donors. Patients with suspected pulmonary embolism underwent a CT scan of the chest with contrast, which was performed in 32 (58%) patients, of which 23 (71%) were diagnosed with pulmonary embolism. VTE patients were excluded from this study if they were given anticoagulants, thrombolytics or antiplatelet drugs at least 7 days prior to examination. Determination of the kinetics and the extent of clot contraction was carried out using an original method based on the optical detection of clot size over time with the Thrombodynamics Analyzer System (HemaCore, Russia). Functionality of isolated platelets from 11 patients with VTE and 11 control samples of healthy donors was assessed by expression of P-selectin (CD62p) and active integrin αIIbβ3 (determined by its fibrinogen-binding capacity) before and after activation with thrombin-receptor activation peptide (TRAP). Isolated platelets from 4 healthy donors and 4 patients with VTE were prepared for scanning electron microscopy.Results: The main finding of this work was that the degree of clot contraction in blood from patients with VTE was significantly reduced (32±2%) when compared to that of healthy subjects (51±1%, p<0.0001). The average velocity of clot contraction in VTE patients (0.3±0.01%/sec) was also remarkably lower than in healthy donors (0.4±0.01%/sec, p<0.001). In addition, the contraction of clots from deep vein thrombosis patients associated with pulmonary embolism was significantly impaired compared to the isolated vein thrombosis (29±2% and 35±2%, respectively, p<0.05), suggesting that clot compaction could lead to a decrease of the risk of embolization. In patients with acute thrombosis lasting <21 days, the initiation time (lag time) of clot contraction was significantly suppressed compared to VTE patients with subacute thrombosis lasting >21 days (198±15 sec and 128±19 sec, respectively, p<0.05). The reduced ability of clots to contract correlated with adverse changes in the blood composition and pronounced platelet dysfunction. In VTE, platelets were initially activated as revealed by the frequently observed shape change and formation of filopodia in platelets from VTE patients (average 75%, n=4211), compared to the less common morphologically altered platelets from healthy subjects (average 41%, n=480, p<0.001). In contrast to the morphological changes, there was no significant difference in the background platelet activation assessed by the levels of P-selectin expression and αIIbβ3 activation in unstimulated cells. However, unlike in resting untreated platelets, in response to TRAP-induced stimulation, platelets from the blood of VTE patients had a significantly lower expression of P-selectin compared to TRAP-activated normal platelets (30±6.6% and 43±5.8%, respectively, p<0.05). TRAP-induced stimulation also resulted in a reduced fibrinogen-binding capacity compared to the TRAP-activated normal platelets (41±8% and 60±7%, respectively, p<0.05).Conclusions: The results obtained suggest that contraction of clots and thrombi is an underappreciated pathogenic mechanism that may affect the course and outcome of VTE. The clinical importance of the reduced clot contraction as well as the diagnostic and prognostic value of the clot contraction assay in VTE is worth further exploration.The work was supported by the Program for Competitive Growth at Kazan Federal University. DisclosuresNo relevant conflicts of interest to declare.

Dapagliflozin reduces thrombin generation and platelet activation: implications for cardiovascular risk reduction in type 2 diabetes mellitus

Aims/hypothesisPeople with diabetes have an increased cardiovascular risk with an accelerated development of atherosclerosis and an elevated mortality rate after myocardial infarction. Therefore, cardioprotective effects of glucose-lowering therapies are of major importance for the pharmacotherapy of individuals with type 2 diabetes. For sodium–glucose cotransporter 2 inhibitors (SGLT2is), in addition to a reduction in blood glucose, beneficial effects on atherosclerosis, obesity, renal function and blood pressure have been observed. Recent results showed a reduced risk of worsening heart failure and cardiovascular deaths under dapagliflozin treatment irrespective of the diabetic state. However, the underlying mechanisms are yet unknown. Platelets are known drivers of atherosclerosis and atherothrombosis and disturbed platelet activation has also been suggested to occur in type 2 diabetes. Therefore, the present study investigates the impact of the SGLT2i dapagliflozin on the interplay between platelets and inflammation in atherogenesis.MethodsMale, 8-week-old LDL-receptor-deficient (Ldlr−/−) mice received a high-fat, high-sucrose diabetogenic diet supplemented without (control) or with dapagliflozin (5 mg/kg body weight per day) for two time periods: 8 and 25 weeks. In a first translational approach, eight healthy volunteers received 10 mg dapagliflozin/day for 4 weeks.ResultsDapagliflozin treatment ameliorated atherosclerotic lesion development, reduced circulating platelet–leucocyte aggregates (glycoprotein [GP]Ib+CD45+: 29.40 ± 5.94 vs 17.00 ± 5.69 cells, p < 0.01; GPIb+lymphocyte antigen 6 complex, locus G+ (Ly6G): 8.00 ± 2.45 vs 4.33 ± 1.75 cells, p < 0.05) and decreased aortic macrophage infiltration (1.31 ± 0.62 vs 0.70 ± 0.58 ×103 cells/aorta, p < 0.01). Deeper analysis revealed that dapagliflozin decreased activated CD62P-positive platelets in Ldlr−/− mice fed a diabetogenic diet (3.78 ± 1.20% vs 2.83 ± 1.06%, p < 0.01) without affecting bleeding time (85.29 ± 37.27 vs 89.25 ± 16.26 s, p = 0.78). While blood glucose was only moderately affected, dapagliflozin further reduced endogenous thrombin generation (581.4 ± 194.6 nmol/l × min) × 10−9 thrombin vs 254.1 ± 106.4 (nmol/l × min) × 10−9 thrombin), thereby decreasing one of the most important platelet activators. We observed a direct inhibitory effect of dapagliflozin on isolated platelets. In addition, dapagliflozin increased HDL-cholesterol levels. Importantly, higher HDL-cholesterol levels (1.70 ± 0.58 vs 3.15 ± 1.67 mmol/l, p < 0.01) likely contribute to dapagliflozin-mediated inhibition of platelet activation and thrombin generation. Accordingly, in line with the results in mice, treatment with dapagliflozin lowered CD62P-positive platelet counts in humans after stimulation by collagen-related peptide (CRP; 88.13 ± 5.37% of platelets vs 77.59 ± 10.70%, p < 0.05) or thrombin receptor activator peptide-6 (TRAP-6; 44.23 ± 15.54% vs 28.96 ± 11.41%, p < 0.01) without affecting haemostasis.Conclusions/interpretationWe demonstrate that dapagliflozin-mediated atheroprotection in mice is driven by elevated HDL-cholesterol and ameliorated thrombin–platelet-mediated inflammation without interfering with haemostasis. This glucose-independent mechanism likely contributes to dapagliflozin’s beneficial cardiovascular risk profile.Graphical abstract

Platelet Microparticles Enriched in miR-223 Reduce ICAM-1-Dependent Vascular Inflammation in Septic Conditions.

In the process of sepsis, activated platelets shed microvesicles containing microRNAs (miRNAs), which can be internalized by distinct recipient cells in circulation, consequently eliciting a potent capability to regulate their cellular functions in different diseases. In the present study, activated human platelets transferring miR-223 into endothelial cells via platelet-derived microparticles (PMPs) was investigated in vitro during septic conditions with a proposed mechanism involving in downregulation of the enhanced expression of intercellular adhesion molecule-1 (ICAM-1). The uptake of PMPs encasing miR-223 and the adhesion of peripheral blood mononuclear cells (PBMCs) on human coronary artery endothelial cells (HCAECs) were observed by immunofluorescence microscopy upon co-culture with PMPs isolated from sepsis or control plasma. The expression of miR-223-3p and its gene target ICAM1 in HCAECs were quantified by RT-qPCR and ELISA after the cells were incubated with septic or control PMPs, whose levels were induced with thrombin-receptor activating peptide (TRAP). Leukocyte-depleted platelets (LDPs) from septic patients showed a decreased miR-223 level, while septic plasma and PMPs revealed an elevated miRNA level compared to control samples. Similarly, TRAP-activated LDPs demonstrated a reduced intracellular miR-223 expression, while increased levels in the supernatant and PMP isolates were observed vs. untreated samples. Furthermore, TNF-α alone resulted in decreased miR-223 and elevated ICAM1 levels in HCAECs, while PMPs raised the miRNA level that was associated with downregulated ICAM1 expression at both mRNA and protein levels under TNF-α treatment. Importantly, miR-223 was turned out not to be newly synthesized as shown in unchanged pre-miR-223 level, and mature miR-223 expression was also elevated in the presence of PMPs in HCAECs after transfection with Dicer1 siRNA. In addition, septic PMPs containing miR-223 decreased ICAM1 with a reduction of PBMC binding to HCAECs. In conclusion, septic platelets released PMPs carrying functional miR-223 lower ICAM1 expression in endothelial cells, which may be a protective role against excessive sepsis-induced vascular inflammation.

Platelet reticulated forms, size indexes and functional activity. Interactions in healthy volunteers.

Reticulated platelets (RP) are young, functionally active platelet forms which are detected by RNA staining. Their content in the circulation reflects the intensity of bone marrow thrombocytopoesis. The aim of this study was to assess in healthy volunteers the relationship between RP percentage and platelet size and activity. RP were quantitated by thiazole orange staining using flow cytometry. Platelet size indexes included mean platelet volume (MPV), platelet large cell ratio (P-LCR) measured in a Coulter type hematological analyzer and forward scattering (FSC) measured in a flow cytometer. Platelet functional activity was evaluated by expression of activated glycoprotein (GP) IIb-IIIa (PAC-1 antibody binding) and P-selectin with the use of flow cytometry. Platelets were activated by thrombin receptor activating peptide (TRAP) (10 and 1 µM) and ADP (20 and 2.5 µM). The percentage of RP in healthy volunteers varied from 2.9% to 23.8% (mean ± SD ‒ 11.7 ± 4.7%, n = 99) and correlated with all platelet size indexes: MPV, P-LCR and FCS (r from 0.452 to 0.529, p < .001, n = 87–99). On average, RP were distributed at a ratio of 9:1 between 50% subpopulations of large and small platelets according to their FSC index. Expression of GP IIb-IIIa activated form correlated with RP percentage and platelet size indexes when platelets were activated by TRAP and ADP at both applied concentrations (r from 0.309 to 0.560, p from 0.014 to < 0.001, n = 50–62). P-selectin expression correlated with RP percentage and platelet size indexes when platelets were activated by 10 µM TRAP inducing maximum expression of this activation marker (r from 0.332 to 0.556, p from 0.008 to < 0.001, n = 65), but not by weaker agonists: 1 µM TRAP, 20 and 2.5 µM ADP (r < 0.3, n = 54–66). Thus, high RP content in healthy volunteers is associated with increased platelet size and activity in the whole platelet population.

Antiplatelet Activity of Isorhamnetin via Mitochondrial Regulation.

With the diet, we ingest nutrients capable of modulating platelet function, which plays a crucial role in developing cardiovascular events, one of the leading causes of mortality worldwide. Studies that demonstrate the antiplatelet and antithrombotic potential of bioactive compounds are vital to maintaining good cardiovascular health. In this work, we evaluate the flavonol isorhamnetin’s antiplatelet effect on human platelets, using collagen, thrombin receptor activator peptide 6 (TRAP-6), and phorbol myristate acetate (PMA) as agonists. Isorhamnetin induced a significant inhibition on collagen- and TRAP-6-induced platelet aggregation, with half-maximum inhibitory concentration (IC50) values of 8.1 ± 2.6 and 16.1 ± 11.1 µM, respectively; while it did not show cytotoxic effect. Isorhamnetin reduced adenosine triphosphate levels (ATP) in platelets stimulated by collagen and TRAP-6. We also evidenced that isorhamnetin’s antiplatelet activity was related to the inhibition of mitochondrial function without effect on reactive oxygen species (ROS) levels. Additionally, we investigated isorhamnetin’s effect on thrombus formation in vitro under flow conditions on the damaged vessel wall. In this context, we demonstrate that isorhamnetin at 20 µM induced a significant inhibition on platelet deposition, confirming its antithrombotic effect. Our findings corroborate the antiplatelet and antithrombotic potential of isorhamnetin present in many foods of daily consumption.

Impact of electric cardioversion on platelet activation.

IntroductionAtrial fibrillation (AF) comes along with high risk of stroke. This risk continues even after re-establishing sinus rhythm with cardioversion. Aim of this study is to evaluate the contribution of electric cardioversion (EC) to platelet activation and procoagulatory tendency.MethodsExtent of platelet activation before and after electric cardioversion was quantified using flow cytometry, impedance aggregation measurements with Multiplate®, and quantification of serum levels of platelet factor 4 (PF4) and ß-thromboglobulin (ß-TG) in patients with AF (N = 10).ResultsNo significant differences were observed in any of the measured parameters comparing the values from before and after cardioversion. Geometric means of P-selectin expression and integrin αIIbβ3 activation were 0.27 (+/- 0.07) and 2.30 (+/- 2.61) before EC and 0.28 (+/- 0.17) and 1.67 (+/- 1.82) after EC. Levels of ß-TG were 110.11 ng/ml (+/- 3.78) before and 110.51 ng/ml (+/- 2.56) after EC, levels of PF4 were 35.64 ng/ml (+/- 12.94) before and 32.40 ng/ml (+/- 4.95) after EC. Platelet aggregation triggered with adenosine diphosphate (ADP), arachidonic acid, collagen, Ristocetin, or thrombin receptor activating peptide (TRAP) revealed results within the normally expected ranges without significant changes before and after EC.DiscussionElectric cardioversion has no influence on platelet activation markers which is in agreement with other studies reporting electrical cardioversion to be safe.

Platelet reactivity in patients with coronary artery disease on treatment with ivabradine and clopidogrel: The PLATIVA study

The combination of aspirin and clopidogrel remains the most frequent dual antiplatelet therapy in patients with acute coronary syndrome or undergoing percutaneous coronary intervention. Clopidogrel, a P2Y12 adenosine diphosphate (ADP) platelet receptor inhibitor requires transformation by cytochrome P450 (CYP) in the liver to produce an active metabolite. On the other hand, ivabradine is an effective antianginal drug that is also metabolized by the CYP enzyme, acting as a weak inhibitor. Our aim is to evaluate the impact of ivabradine on platelet reactivity in patients receiving clopidogrel and to evaluate the possible pharmacodynamic interactions between them. This is a prospective, observational, post-authorization pharmacodynamic study including patients treated with aspirin and clopidogrel and initiating ivabradine. Patients with platelet abnormalities and those treated with anticoagulants or other CYP inhibitors were excluded. A platelet aggregation test was performed at baseline before the first dose of ivabradine and repeated after 4 weeks of treatment. The primary endpoint was inhibition of platelet aggregation (IPA) after 20 mM ADP stimulation. A total of 34 patients were initially recruited; eight patients were excluded, leaving 25 patients for final analysis. All patients had chronic ischemic heart disease. The maximum ivabradine dosage of 7.5 mg twice daily was achieved in seven patients (28%). After 4 weeks of treatment with ivabradine, no significant differences were found in maximal platelet aggregation from baseline, as assessed with 20 μM ADP stimulation. In addition, no significant difference in IPA from baseline was detected after stimulation with ADP 5 μM, arachidonic acid, thrombin receptor-activated peptide 25 μM, and platelet reactivity index. Ivabradine did not alter antiplatelet response to clopidogrel in patients treated concomitantly with both drugs.

Prostacyclin Analogues Inhibit Platelet Reactivity, Extracellular Vesicle Release and Thrombus Formation in Patients with Pulmonary Arterial Hypertension.

(1) Background: Prostacyclin analogues (epoprostenol, treprostinil, and iloprost) induce vasodilation in pulmonary arterial hypertension (PAH) but also inhibit platelet function. (2) Objectives: We assessed platelet function in PAH patients treated with prostacyclin analogues and not receiving prostacyclin analogues. (3) Methods: Venous blood was collected from 42 patients treated with prostacyclin analogues (49.5 ± 15.9 years, 81% female) and 38 patients not receiving prostacyclin analogues (55.5 ± 15.6 years, 74% female). Platelet reactivity was analyzed by impedance aggregometry using arachidonic acid (AA; 0.5 mM), adenosine diphosphate (ADP; 6.5 µM), and thrombin receptor-activating peptide (TRAP; 32 µM) as agonists. In a subset of patients, concentrations of extracellular vesicles (EVs) from all platelets (CD61+), activated platelets (CD61+/CD62P+), leukocytes (CD45+), and endothelial cells (CD146+) were analyzed by flow cytometry. Platelet-rich thrombus formation was measured using a whole blood perfusion system. (4) Results: Compared to controls, PAH patients treated with prostacyclin analogues had lower platelet reactivity in response to AA and ADP (p = 0.01 for both), lower concentrations of platelet and leukocyte EVs (p ≤ 0.04), delayed thrombus formation (p ≤ 0.003), and decreased thrombus size (p = 0.008). Epoprostenol did not affect platelet reactivity but decreased the concentrations of platelet and leukocyte EVs (p ≤ 0.04). Treprostinil decreased platelet reactivity in response to AA and ADP (p ≤ 0.02) but had no effect on the concentrations of EVs. All prostacyclin analogues delayed thrombus formation and decreased thrombus size (p ≤ 0.04). (5) Conclusions: PAH patients treated with prostacyclin analogues had impaired platelet reactivity, EV release, and thrombus formation, compared to patients not receiving prostacyclin analogues.

Effect of Prolonged Hypothermic Cardiopulmonary Bypass, Heparin, and Protamine on Platelet: A Small-Group Study.

Cardiopulmonary bypass (CPB) is associated with platelet dysfunction (PD), an important cause of postoperative bleeding. The etiology of PD is not completely understood. We mapped the platelets' function during CPB to determine the etiology of PD. Platelets activation, measured by procaspase activating compound-1 and P-selectin expression (CD62P), after activation by adenosine diphosphate and thrombin receptor activator peptide, were decreased by protamine. Changes during CPB were insignificant. Platelet-leukocyte aggregation was increased by CPB but not by protamine. Platelet apoptosis marker, annexin V, was increased by protamine. Changes during CPB were insignificant. Our findings demonstrate that protamine given after CPB plays a central role in PD and count decrease.