ADP-induced Platelet Activation Research Articles

Inhibition of mitochondrial calcium transporters alters adp-induced platelet responses.

ADP-stimulated elevation of cytosolic Ca2+ is an important effector mechanism for platelet activation. The rapidly elevating cytosolic Ca2+ is also transported to mitochondrial matrix via Mitochondrial Ca2+ Uniporter (MCU) and extruded via Na+/Ca2+/Li+ Exchanger (NCLX). However, the exact contribution of MCU and NCLX in ADP-mediated platelet responses remains incompletely understood. The present study aimed to elucidate the role of mitochondrial Ca2+ transport in ADP-stimulated platelet responses by inhibition of MCU and NCLX with mitoxantrone (MTX) and CGP37157 (CGP), respectively. As these inhibitory strategies are reported to cause distinct effects on matrix Ca2+ concentration, we hypothesized to observe opposite impact of MTX and CGP on ADP-induced platelet responses. Platelet aggregation profiling was performed by microplate-based spectrophotometery while p-selectin externalization and integrin αIIbβ3 activation were analyzed by fluorescent immunolabeling using flow cytometery. Our results confirmed the expression of both MCU and NCLX mRNAs with relatively low abundance of NCLX in human platelets. In line with our hypothesis, MTX caused a dose-dependent inhibition of ADP-induced platelet aggregation without displaying any cytotoxicity. Likewise, ADP-induced p-selectin externalization and integrin αIIbβ3 activation was also significantly attenuated in MTX-treated platelets. Concordantly, inhibition of NCLX with CGP yielded an accelerated ADP-stimulated platelet aggregation which was associated with an elevation of p-selectin surface expression and αIIbβ3 activation. Together, these findings uncover a vital and hitherto poorly characterized role of mitochondrial Ca2+ transporters in ADP-induced platelet activation.

GDF-15 Inhibits ADP-Induced Human Platelet Aggregation through the GFRAL/RET Signaling Complex.

Growth differentiation factor-15 (GDF-15) is proposed to be strongly associated with several cardiovascular diseases, such as heart failure and atherosclerosis. Moreover, some recent studies have reported an association between GDF-15 and platelet activation. In this study, we isolated peripheral blood platelets from healthy volunteers and evaluated the effect of GDF-15 on adenosine diphosphate (ADP)-induced platelet activation using the platelet aggregation assay. Subsequently, we detected the expression of GDF-15-related receptors on platelets, including the epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), transforming growth factor-beta receptor I (TGF-βRI), transforming growth factor-beta receptor II (TGF-βRII), glial-cell-line-derived neurotrophic factor family receptor α-like (GFRAL), and those rearranged during transfection (RET). Then, we screened for GDF-15 receptors using the GDF-15-related receptor microarray comprising these recombinant proteins. We also performed the immunoprecipitation assay to investigate the interaction between GDF-15 and the receptors on platelets. For the further exploration of signaling pathways, we investigated the effects of GDF-15 on the extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and Janus kinase 2 (JAK2) pathways. We also investigated the effects of GDF-15 on the ERK and AKT pathways and platelet aggregation in the presence or absence of RET agonists or inhibition. Our study revealed that GDF-15 can dose-independently inhibit ADP-induced human platelet aggregation and that the binding partner of GDF-15 on platelets is GFRAL. We also found that GDF-15 inhibits ADP-induced AKT and ERK activation in platelets. Meanwhile, our results revealed that the inhibitory effects of GDF-15 can be mediated by the GFRAL/RET complex. These findings reveal the novel inhibitory mechanism of ADP-induced platelet activation by GDF-15.

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

Enhanced metabolic activation of and platelet response to clopidogrel in T cell-deficient mice through induction of Cyp2c and Cyp3a and inhibition of Ces1

BackgroundT cells and platelets reciprocally coordinate mutual functions through crosstalk or interaction. However, it is not known whether metabolic activation of and platelet response to clopidogrel could be changed if T cells were deficient or impaired in some cases and, if any, how it would work. ObjectivesThe objective of this study was to dissect the potential changes in platelet responses to and metabolic activation of clopidogrel in the case of T cell deficiency and to elucidate their mechanisms involved. MethodsBALB/c athymic nude mice or euthymic mice (controls) pretreated with cyclosporine A (CsA), thymosin α1 (Tα1), or their combination were used to investigate the changes in ADP-induced platelet activation and aggregation, systemic exposure of clopidogrel and its metabolites, and mRNA/protein expression and activity levels of clopidogrel-metabolizing enzymes in the liver, respectively. ResultsNude mice exhibited significantly enhanced antiplatelet effects of clopidogrel due to increased formation of clopidogrel active metabolite in the liver, where the enzyme activity levels of Cyp2c and Cyp3a were significantly elevated compared with control mice. Furthermore, the effects of CsA pretreatment on the metabolism of clopidogrel in euthymic mice were identical to those seen in athymic mice. As expected, concomitant use of Tα1 reversed all the observed effects of CsA on clopidogrel metabolism and relevant metabolic enzymes. ConclusionsT cell deficiency or suppression enhances the antiplatelet effects of clopidogrel due to the boosted metabolic activation of clopidogrel in the liver through a dramatic induction of Cyp2c and Cyp3a in mice, suggesting that the metabolism of substrate drugs of Cyp2c and Cyp3a may be enhanced by T cell impairment.

Platelet dysfunction after trauma: From mechanisms to targeted treatment.

Platelet dysfunction after trauma: From mechanisms to targeted treatment.

Coumarin Derivatives Inhibit ADP-Induced Platelet Activation and Aggregation

Coumarin was first discovered in Tonka bean and then widely in other plants. Coumarin has an anticoagulant effect, and its derivative, warfarin, is a vitamin K analogue that inhibits the synthesis of clotting factors and is more widely used in the clinical treatment of endovascular embolism. At present, many artificial chemical synthesis methods can be used to modify the structure of coumarin to develop many effective drugs with low toxicity. In this study, we investigated the effects of six coumarin derivatives on the platelet aggregation induced by adenosine diphosphate (ADP). We found that the six coumarin derivatives inhibited the active form of GPIIb/IIIa on platelets and hence inhibit platelet aggregation. We found that 7-hydroxy-3-phenyl 4H-chromen-4-one (7-hydroxyflavone) had the most severe effect. In addition, we further analyzed the downstream signal transduction of the ADP receptor, including the release of calcium ions and the regulation of cAMP, which were inhibited by the six coumarin derivatives selected in this study. These results suggest that coumarin derivatives inhibit coagulation by inhibiting the synthesis of coagulation factors and they may also inhibit platelet aggregation.

Palmatine Derivatives as Potential Antiplatelet Aggregation Agents via Protein Kinase G/Vasodilator-Stimulated Phosphoprotein and Phosphatidylinositol 3-Kinase/Akt Phosphorylation.

Sixty palmatine (PMT) derivatives were synthesized and evaluated for antiplatelet aggregation taking berberine as the lead, and the structure-activity relationship was first systematically described. Among them, compound 2v showed the best potency in reducing adenosine diphosphate (ADP)-induced platelet aggregation in a dose-dependent manner. It greatly suppressed ADP-induced platelet aggregation, activation, and Akt phosphorylation in vitro and ex vivo after oral administration to mice. It also effectively inhibited carrageenan-induced thrombus formation in the mouse tail and lung, as well as reduced the serum P-selectin level. Compound 2v might simultaneously bind to protein kinase G to improve vasodilator-stimulated phosphoprotein phosphorylation and bind to phosphatidylinositol 3-kinase to inhibit Akt phosphorylation, which synergically reduced platelet aggregation, thereby achieving antithrombotic efficacy. Therefore, PMT derivatives constituted a novel family of antiplatelet aggregation agents with the advantage of a good safety profile, worthy of further investigation.

Platelet Activation and Platelet Indices as Markers for Disease Progression in Women with Breast Cancer

Background: Several studies reported the role platelet activation, platelet volume (MPV) and other Indices in breast cancer but the data is inconsistent and/or diverse. The aim of this study was to systematically evaluate the role of platelet activation and platelet volume indices in women with BC as predictors for cancer progression and poor prognosis. Methods: Patients were recruited from our local oncology center between 2019 to 2020 following ethics approval. 80 patients with locally invasive BC, 20 metastatic and 100 controls were recruited. ADP-induced platelet activation was assessed by light-transmission aggregometry. Platelet P-selectin (CD62P) expression with and without ADP stimulation was assessed by flow cytometry. A comprehensive analysis of platelet count and platelet volume indices (PVIs) (MPV, PDW, MPV/P and PDW/P) was conducted. Data were analyzed in relation to tumor pathology, hormone receptors (ER, PR, HER-2) and proliferation index Ki-67. Regression analyses were conducted for the prediction of poor prognosis, tumor aggression and metastatic potential. Results: We found a significant increase in platelet aggregation (MA), CD62P expression, CD62P+ADP, MPV, PDW, MPV/P and PDW/P in the metastatic group compared to the locally invasive group. Univariate regression analysis showed significance for ADP, MA, CD62P+ADP, MPV and PDW/P. Conclusion: MPV/P and PDW/P can be used as simple low-cost predictors for cancer progression and poor prognosis. We conclude platelet activation and specific platelet indices can help predict prognosis in females with BC.

Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

Background Tetramethylpyrazine (TMP) is an alkaloid in Chinese herbal medicine, which possesses antiplatelet activity. TMP inhibits platelet activation in many ways. The platelet P2Y12 receptor for adenosine 5′ diphosphate (ADP) plays a central role in platelet function, hemostasis, and thrombosis. Here, we investigated the inhibitory effect of TMP on P2Y12 receptor-related platelet function. Methods The inhibitory potential of TMP was assessed using agonist-induced platelet aggregation, flow cytometric analysis of CD62p expression, PAC-1 activation, and fibrin clot retraction. After the P2Y12 receptor-related signaling pathway was inhibited using the blocker, platelet activation was studied by platelet aggregation, CD62p expression, and PAC-1 activation. The secretion of cyclic adenosine monophosphate (cAMP) was measured using enzyme-linked immunosorbent assay (ELISA), and the expression of signaling pathway protein, phosphorylation of vasodilator-stimulated phosphoprotein, and phosphorylation of Akt were investigated using western blotting. The release of platelet inflammatory mediators was measured using ELISA. Results TMP had an antiplatelet effect by inhibiting ADP-induced aggregation, P-selectin secretion, and glycoprotein (GP) IIb/IIIa expression and reducing the release of atherosclerotic-related inflammatory mediators (sCD40L and IL-1β). TMP decreased the area of clot retraction, reflecting inhibition of GPIIb/IIIa activation. TMP inhibited adenosine diphosphate-induced platelet activation via increased cAMP production, VASPser157 phosphorylation, and Akt dephosphorylation. Conclusion TMP selectively inhibits ADP-induced platelet activation via P2Y12 receptor-related signaling pathways.

A comparison of different regression models for the quantitative analysis of the combined effect of P2Y12 and P2Y1 receptor antagonists on ADP-induced platelet activation

IntroductionThe combination index (CI), a common quantitative indicator of the degree of synergy/antagonism, may be determined using different regression methods. However, any analysis with constraints has the potential for underestimating the combined effect of multiple drugs. ObjectivesThis in vitro study describes the combined effects of selected platelet antagonists on ADP-induced platelet activation in different regression models. MethodsThe inhibitory effects of P2Y12 receptor antagonists in combination with P2Y1 receptor antagonists (i.e. cangrelor with MRS 2279, prasugrel metabolite with MRS 2179 and PSB 0739 with MRS 2179) were characterized with the aid of three software packages: CompuSyn (for linear regression with constraints), CISNE (for non-linear regression with constraints) and GraphPad Prism (for non-linear regression without constraints). The synergism between P2Y12 and P2Y1 inhibitors was quantified by CI and synergy area. ResultsMRS 2279 and MRS 2179 were found to act synergistically with selected P2Y12 receptor antagonists to potentiate their antiplatelet effect. The models of regression with constraints, linear regression in particular, demonstrated a worse fit for experimental data than non-linear regression without constraints; this resulted in an incorrect estimation of the combined effects of two antiplatelet drugs, i.e., underestimating the CI and overestimating the synergy area. Also, the synergy area was found to better reflect the differences among models than the CI. ConclusionsThese findings suggest that non-linear regression without constraints offers more precise quantitative determination of combined effects between two drugs compared to the regression models with constraints.

Antiplatelet and Antithrombotic Effects of Isaridin E Isolated from the Marine-Derived Fungus via Downregulating the PI3K/Akt Signaling Pathway.

Isaridin E, a cyclodepsipeptide isolated from the marine-derived fungus Amphichorda felina (syn. Beauveria felina) SYSU-MS7908, has been demonstrated to possess anti-inflammatory and insecticidal activities. Here, we first found that isaridin E concentration-dependently inhibited ADP-induced platelet aggregation, activation, and secretion in vitro, but did not affect collagen- or thrombin-induced platelet aggregation. Furthermore, isaridin E dose-dependently reduced thrombosis formation in an FeCl3-induced mouse carotid model without increasing the bleeding time. Mechanistically, isaridin E significantly decreased the ADP-mediated phosphorylation of PI3K and Akt. In conclusion, these results suggest that isaridin E exerts potent antithrombotic effects in vivo without increasing the risk of bleeding, which may be due to its important role in inhibiting ADP-induced platelet activation, secretion and aggregation via the PI3K/Akt pathways.

Is platelet responsiveness to clopidogrel attenuated in overweight or obese patients and why? A reverse translational study in mice

Overweight or obese patients exhibit poorer platelet responses to clopidogrel. However, the mechanisms behind this phenotype remain to be elucidated. Here, we sought to discover whether and why obesity could affect the metabolic activation of and/or platelet response to clopidogrel in obese patients and high-fat diet-induced obese mice. A post hoc stratified analysis of an observational clinical study was performed to investigate changes in residual platelet reactivity with increasing body weight in patients taking clopidogrel. Furthermore, high-fat diet-induced obese mice were used to reveal alterations in systemic exposure of clopidogrel thiol active metabolite H4, ADP-induced platelet activation and aggregation, the expression of genes involved in the metabolic activation of clopidogrel, count of circulating reticulated and mature platelets, and proliferation profiles of megakaryocytes in bone marrow. The relevant genes and potential signalling pathways were predicted and enriched according to the GEO datasets available from obese patients. Obese patients exhibited significantly attenuated antiplatelet effects of clopidogrel. In diet-induced obese mice, systemic exposure of clopidogrel active metabolite H4 was reduced but that of its hydrolytic metabolite was increased due to down-regulation of certain P450s but up-regulation of carboxylesterase-1 in the liver. Moreover, enhanced proliferation of megakaryocytes and elevated platelet count also contributed. Obesity attenuated metabolic activation of clopidogrel and increased counts of circulating reticulated and mature platelets, leading to impaired platelet responsiveness to the drug in mice, suggesting that clopidogrel dosage may need to be adjusted adequately in overweight or obese patients.

Berberine and Its Main Metabolite Berberrubine Inhibit Platelet Activation Through Suppressing the Class I PI3Kβ/Rasa3/Rap1 Pathway.

Background: Berberine (BBR), a natural product, was reported to inhibit platelet aggregation; however, the molecular mechanisms remain unclear. This study aims to investigate the effects and mechanisms of BBR in inhibiting platelet activation and thrombus formation. Methods: Flow cytometry, immunofluorescence, and Western blot were used to determine the inhibitory effects and mechanisms of BBR and its main metabolite berberrubine (M2) on platelet activation in vitro and ex vivo. Purified integrin αIIbβ3, class I PI3K kit, and molecular docking were used to identify the possible targets of BBR and M2. A carrageenan-induced mouse thrombosis model was used to evaluate the effects of BBR on thrombus formation in vivo. Results: In vitro, BBR and M2 significantly inhibited ADP-induced integrin αIIbβ3 activation, reduced the level of P-selectin on the platelet membrane, and suppressed the binding of fibrinogen to the platelets. In this process, BBR and M2 greatly suppressed the PI3K/Akt pathway and inhibited Rasa3 membrane translocation and Rap1 activation. Furthermore, BBR and M2 selectively inhibited class I PI3Kβ, perhaps through binding to its active site. The activities of BBR were stronger than those of M2. After oral administration, BBR significantly inhibited the PI3K/Akt pathway and Rap1 activation and suppressed ADP-induced platelet activation and carrageenan-induced thrombosis in mice without prolonging bleeding time. Conclusions: We reveal for the first time the possible targets and mechanisms of BBR and M2 in inhibiting platelet activation. Our research may support the future clinical application of BBR as an antiplatelet drug in the prevention or treatment of thrombotic diseases.

Protein Disulfide Isomerase Negatively Regulates Platelet Function through Modulation of the Thromboxane A2 Receptor

Protein disulfide isomerase (PDI) is an intracellular oxidoreductase that cleaves and forms disulfide bonds in substrate proteins to facilitate their proper folding. However, platelets and endothelium release PDI upon vascular injury. This extracellular PDI serves a critical function in platelet activation and thrombus formation. A PDI antagonist is currently in phase II/III clinical trials for thrombosis prevention. Yet the mechanisms of how PDI enables platelets to respond to the local redox environment remain poorly understood. In particular, the substrates on which PDI acts to modify platelet function have yet to be elucidated.To identify platelet receptors that are controlled by PDI, we evaluated the ability of PDI to modify the effect of different platelet agonists on a platelet aggregation. Recombinant PDI (rPDI) had no effect on platelet aggregation induced by a dose curve of a PAR1 activating peptide, a PAR4 activating peptide, thrombin, collagen, or epinephrine. Unexpectedly, rPDI markedly inhibited aggregation induced by the stable thromboxane A2 analog U46619 (13.8±5.6% of control). In contrast, ERp57, a related thiol isomerase, had little effect and a mutant PDI that lacks the active site cysteines responsible for disulfide bond rearrangement failed to inhibit U46619-induced platelet aggregation, implicating disulfide bond rearrangement. rPDI also blocked U46619-induced dense granule secretion (15.0±5.5% of control), α-granule secretion (24.4±0.1% of control) and α2β3 activation (65.0±0.1% of control). Conversely, inhibition of endogenous PDI augmented U46619-mediated platelet activation. To confirm that PDI was acting upstream in the U46619 activation pathway, we evaluated proximal signaling events. rPDI significantly decreased U46619-induced p38 MAPK phosphorylation in both platelets and HEK cells overexpressing the thromboxane prostanoid (TP) receptor, indicating inhibition of Gα12/13 pathways. In contrast, there was little inhibition of Ca2+ flux, indicating lack of inhibition of Gαq-mediated signaling. These results suggest the presence of a functional disulfide bond within the TP receptor that is sensitive to PDI and required for Gα12/13 signaling.Evaluation of the amino sequence of the TP receptor demonstrates two cysteines, Cys-105 in the extracellular loop I and Cys-183 in the extracellular loop II, that form a disulfide bond and may be modified by redox environment. To determine if the platelet TP receptor is sensitive to modification of redox environment, we evaluated U46619-induced signaling in the presence of GSH and GSSG. Incubation with >0.25 mM GSH reduced U46619-induced platelet aggregation to 11.0±0.6% of control, whereas GSSG did not alter platelet aggregation in response to U46619. These results indicate a functional disulfide bond in the TP receptor that responds to changes in redox environment.Evaluation of additional agonists demonstrated that ADP-induced aggregation was sensitive to rPDI. rPDI did not affect the initial response to 3.5 μM ADP, but completely abrogated the secondary wave of aggregation. This defect in the secondary wave could be overcome by using 5 μM ADP, thus suggesting impaired endogenous ADP release. Indeed, rPDI blocked dense granule release even in response to 5 μM ADP. This result is consistent with the known reliance of dense granule release on TP stimulation and suggests that the negative regulation of PDI on ADP-induced platelet activation is secondary to the inhibition of PDI on the TP receptor.These are the first studies to our knowledge that demonstrate the ability of a thiol isomerase to modulate the function of a G-protein coupled receptor via disulfide bond rearrangement. This mechanism could provide a means to modify signaling through the TP receptor in respond to changes in redox environment. DisclosuresNo relevant conflicts of interest to declare.

Short-term standard alcohol consumption enhances platelet response to clopidogrel through inhibition of Nrf2/Ces1 pathway and induction of Cyp2c in mice

AimsDrinking alcohol is prevalent worldwide; however, it is unknown whether alcohol could affect the antiplatelet effects of clopidogrel in patients when taking both concomitantly. This study was designed to investigate the influence of short-term standard alcohol consumption on the metabolic activation of and platelet response to clopidogrel in mice as well as the mechanisms involved. Main methodsMale C57BL/6J mice were administered with normal saline (vehicle control) or alcohol at 2 g/kg/day for 7 days, and then gavaged with vehicle control or a single dose of clopidogrel at 10 mg/kg. Inhibition of ADP-induced platelet aggregation and activation by clopidogrel, plasma concentrations of clopidogrel and its active metabolite H4, and changes in mRNA and protein expression of genes related to clopidogrel metabolism and its regulation were measured in mice pretreated with or without alcohol. Key findingsCompared with vehicle control, alcohol pretreatment significantly reduced hydrolysis of clopidogrel as a result of significant down-regulation of Nrf2-mediated Ces1 expression (responsible for the formation of clopidogrel carboxylate), increased metabolic activation of clopidogrel due to significant up-regulation of Cyp2c (for the formation of active thiol metabolite H4), and consequently enhanced inhibition of ADP-induced platelet aggregation and activation by clopidogrel. SignificanceShort-term standard alcohol consumption would significantly enhance suppression of ADP-induced platelet aggregation and activation by clopidogrel through significant inhibition of Nrf2/Ces1 signaling pathway and induction of Cyp2c, suggesting that alcohol may interact with drugs that are predominantly metabolized by CES1 or CYP2C in patient care, including clopidogrel.

The potency of selatogrel, a reversible antagonist of the P2Y12 receptor, is affected by calcium concentration

Here, we report the in vitro characterization of the P2Y12 receptor antagonist selatogrel (ACT-246475). Binding studies with radiolabeled selatogrel demonstrated that selatogrel is a competitive antagonist of ADP binding to the P2Y12 receptor with a fast onset of action. Consequently, selatogrel was confirmed to be a potent inhibitor of P2Y12-mediated intra-platelet signaling and ADP-induced platelet activation. Characterization of selatogrel in platelet-rich plasma in vitro demonstrated that the mode of anti-coagulation affected the anti-platelet potency. Specifically, in platelet-rich plasma containing physiological calcium concentration (anticoagulated with a direct thrombin inhibitor), selatogrel achieved half-maximal inhibition of ADP-induced platelet aggregation at a 3-fold lower concentration than in conditions with low calcium concentration (anticoagulated with citrate). Furthermore, calcium-dependent reduction in selatogrel potency was observed in whole blood platelet aggregation using the VerifyNow™ system with a 3.7-fold potency loss in low calcium conditions. A comparable potency loss was also observed with the reversible P2Y12 receptor antagonists ticagrelor, cangrelor and elinogrel. Furthermore, receptor-binding experiments using radiolabeled selatogrel confirmed a 3-fold lowering of selatogrel binding affinity to the P2Y12 receptor in low calcium conditions. In conclusion, our data suggest that in low calcium conditions (i.e., citrate-anticoagulated blood), there is a risk of underestimating the potency of reversible P2Y12 receptor antagonists. To avoid overdosing, and a potential increase in bleeding risk, we propose that the ex vivo evaluation of reversible P2Y12 receptor antagonists should be performed with platelet assay systems containing physiological calcium concentration.

Рутинные лабораторные показатели в стадировании рака шейки матки. Новые возможности

Aim: to identify and assess novel potentialities of routine lab tests to improve the accuracy of preoperative staging (spread) of high-grade squamous intraepithelial lesions (HSIL) and cervical cancer (CC). Patients and Methods: 410 women aged 18–82 (median age 42 [34; 51] years) with verified HSIL or CC were enrolled. Women were selected through medical records. Test results were monitored during in-person examinations since the formulation of clinical diagnosis and after surgical staging. If no surgical staging was performed, histopathology and disease stage as established by protocol examinations using instrumental and lab tests were considered. Women were subdivided into groups based on disease stage and method of treatment. Several parameters (complete blood count, hemostasis, biochemical markers of iron metabolism) were measured. In HSIL, minimal mandatory instrumental tests included pelvic and regional lymph node ultrasound. In CC stage 1–4, pelvic and abdominal ultrasound, pelvic MRI, abdominal CT with contrast, and (sometimes) PET CT. Results: several routine parameters of peripheral blood (hemoglobin, RBCs, ESR, fibrinogen, soluble fibrin monomer complexes/SFMC, D-dimer, ADP-induced platelet activation) were identified. Changes in the levels of these parameters within/above/below reference ranges illustrate the systemic effect of malignancy on the body and are a significant marker of regional metastasis of CC undiagnosed during a clinical examination. Conclusion: complex assessment of these parameters in CC allows for suspecting metastasis and requires more careful and reasonable examinations to improve the accuracy of prehospital diagnosis and determine management strategy. KEYWORDS: HSIL, cervical cancer, diagnosis, metastases, hematological markers, hemostasis, peripheral blood parameters, disease stage. FOR CITATION: Stuklov N.I., Sushinskaya T.V., Epifanova S.V. et al. Routine laboratory tests for staging of cervical cancer. Novel potentialities. Russian Journal of Woman and Child Health. 2021;4(3):229–237 (in Russ.). DOI: 10.32364/2618-8430-2021-4-3-229-237.

Abstract 16292: Selective Platelet Protease-Activated-Receptor (PAR)1 and PAR4 Mediated Thrombin Desensitisation in the Elderly is Correlated With Inflammation and Chronic Thrombin Receptor Stimulation

Introduction: Platelet activation, by adenosine diphosphate (ADP) via P2Y 12 receptors and thrombin via PAR1 and PAR4, is a key therapeutic target in cardiovascular disease (CVD). The efficacy of antiplatelet agents diminishes in the elderly, but it is unknown whether these pathways change with aging. Hypothesis: Platelet activation pathways change with aging. Methods: Platelet activity was evaluated in young (20-30yrs), middle-aged (40-55yrs) and elderly (≥70yrs) healthy volunteers (n=174). Whole blood aggregometry and flow cytometry (P-selectin: α-granule release; CD63: dense granule release; PAC1 binding: activated GPIIb/IIIa) were performed under basal conditions and post ex vivo stimulation with ADP, thrombin, PAR1 agonist or PAR4 agonist. EC 50 and E max values were derived for each agonist. Receptor cleavage and quantification (P2Y 12 ; PAR1; PAR4; GPIbα) were assessed with flow cytometry. Thrombin generation (D-Dimer) and inflammation (interleukin [IL]-1β; tumour necrosis factor [TNF]-α) were assessed via ELISA. Results: The elderly had higher basal platelet activation markers (P-selectin, CD63, activated GPIIb/IIIa) than the young, with higher basal activity correlating with increasing IL-1β. P2Y 12 receptor density was higher in the elderly and associated with greater ADP-induced platelet aggregation and activation. Elderly subjects had less platelet activation in response to thrombin (higher EC 50 ), demonstrating hyporeactivity to selective stimulation of PAR1 or PAR4, more basal PAR1/PAR4 cleavage, and less inducible PAR1/PAR4 cleavage. This was associated with reduced thrombin binding receptor GPIbα and reduced secondary ADP contribution to thrombin-mediated activation. D-Dimer and TNF-α levels were elevated in the elderly, and inversely correlated with platelet thrombin sensitivity, implying a role of desensitization from chronic thrombin receptor stimulation. Conclusion: Aging is associated with increased basal platelet activation and hyperreactivity to ADP, but selective desensitization to thrombin. The latter appears mediated by chronic thrombin receptor stimulation and inflammation. Age-specific antiplatelet strategies may require selective targeting of these pathways to treat CVD in the elderly.

MRP4 over-expression has a role on both reducing nitric oxide-dependent antiplatelet effect and enhancing ADP induced platelet activation

The impact of inhibition of multidrug resistance protein 4 (MRP4) on nitric oxide (NO) resistance and on ADP-induced platelet aggregation is unknown. The aim of this investigation was to verify whether platelet NO resistance correlates with MRP4 expression and evaluate whether this can be reduced by in vitro MRP4 inhibition mediated by cilostazol. Moreover, we assessed if inhibition of MRP4-mediated transport reduces ADP-induced platelet reactivity. The inhibitory effect of sodium nitroprusside (SNP), a NO-donor that enhances cyclic guanosine monophosphate (cGMP) cytosolic concentration, was assessed in platelets obtained from aspirin treated patients and in a control population. The inhibitory effect of SNP was evaluated by ADP-induced aggregation in SNP-treated platelets. The impact of MRP4 on ADP-induced platelet aggregation was performed in high on aspirin residual platelet reactivity (HARPR) patients and compared to healthy volunteers (HV), and a control cohort (CTR). In aspirin-treated patients with high levels of MRP4, reduced SNP inhibition was found compared to those with low levels of MRP4. MRP4 inhibition by cilostazol significantly reduced ADP-induced platelet aggregation in HARPR population, and to a lesser extent in HV and CTR populations. In conclusion, cilostazol can mitigate the hyper-reactive platelet phenotype of HARPR patients by reducing residual ADP-induced platelet aggregation and increasing NO-dependent endothelial antiplatelet effects.

Anthocyanins in berries exhibited anti-atherogenicity and antiplatelet activities in a metabolic syndrome population

Metabolic syndrome (MetS) is a global challenge for atherosclerosis. It was hypothesized that a four-week consumption of anthocyanin supplements by MetS patients who had three or more risk factors linked with metabolic syndrome would have a greater improvement in cardiometabolic biomarkers and would also reduce the risk of thrombosis. A total of 55 participants in two groups of Normal healthy and MetS (age 25-75y) were given 320 mg anthocyanin supplements twice daily for 4 weeks. Platelet coagulant activities, lipid profiles, fasting blood glucose, and inflammatory and oxidative stress biomarkers were measured before and after supplementation to evaluate the atheroprotective effects of anthocyanins in the study subjects. Four weeks of anthocyanin supplementation significantly decreased cardiometabolic risk factors including the average serum fasting blood glucose (FBG) (by 13.3%, P < .05) and lipid profiles by significant reduction in triglyceride (by 24.9%, P < .05) and LDL-C (by 33.1%, P < .05) in the MetS group. Anthocyanin supplementation also decreased high sensitivity C-reactive protein (hs-CRP) level (by 28%, P < .05) in females. However, no significant differences in serum UA (uric acid) and HDL-C were observed between anthocyanin pre- and post-treatment in both groups. Moreover, Anthocyanin supplements decreased ADP-induced platelet activation configuration expressed as P-selectin by 40% (P < .05). There was a positive correlation between decreased hs-CRP values and the levels of LDL-C and FBG in the MetS group (P < .05). These results support the hypothesis that anthocyanin supplementation exerts anti-atherogenicity effects by improving cardiometabolic risk factors and reducing thrombogenicity in the MetS population.