RESUMEN
INTRODUCTION: The 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. OBJECTIVES: This in vitro study describes the combined effects of selected platelet antagonists on ADP-induced platelet activation in different regression models. METHODS: The 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. RESULTS: MRS 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. CONCLUSIONS: These 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.
Asunto(s)
Activación Plaquetaria , Agregación Plaquetaria , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Plaquetas/metabolismo , Humanos , Inhibidores de Agregación Plaquetaria/farmacología , Clorhidrato de Prasugrel/farmacología , Clorhidrato de Prasugrel/uso terapéutico , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y12/metabolismoRESUMEN
Concurrent administration of two drugs may complicate the management of acute coronary syndromes: competitive drug displacement diminishes drug binding and alters drug pharmacodynamics. We investigated the interaction of two antiplatelet compounds (PSB 0777 and cangrelor) with human serum albumin (HSA) to determine whether they compete with one another for the binding to albumin. Both examined compounds have been earlier claimed to bind to HSA (PSB 0777) or plasma proteins (cangrelor). Fluorescence spectroscopy, surface plasmon resonance spectroscopy and molecular modeling indicated that PSB 0777 and cangrelor interacted with HSA with moderate affinity (KDâ¼10-5 M). The binding of cangrelor to HSA involved primarily hydrophobic interactions, while the interaction of PSB 0777 with HSA was driven by hydrophobic and electrostatic forces. It was found that PSB 0777 and cangrelor do not share the same binding site on the protein. Our findings highlight the importance of albumin in the transport of PSB 0777 and cangrelor and suggest that the antiplatelet activity of the examined compounds used in combination is not affected by competition-induced changes in drug binding to HSA.
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The data in this article focus on the F11 Receptor (F11R/JAM-A; Junctional Adhesion Molecule-A; JAM-A, F11R), a cell adhesion protein constitutively expressed on the membrane surface of circulating platelets and localized within the tight junctions of healthy endothelial cells (ECs). Previous reports have shown that F11R/JAM-A plays a critical role in the adhesion of platelets to an inflamed endothelium due to its' pathological expression on the luminal surface of the cytokine-inflamed endothelium. Since platelet adhesion to an inflamed endothelium is an early step in the development of atherosclerotic plaque formation, and with time, resulting in heart attacks and stroke, we conducted a long-term, study utilizing the atherosclerosis-prone ApoE -/- mice to attempt a blockade of the formation of atherosclerotic plaques by preventing the adhesion of platelets to the inflamed vasculature in vivo. Utilizing a nonhydrolyzable peptide derived from an amino acid sequence of F11R/JAM-A, peptide 4D, we have shown in culture that the adhesion of platelets to the inflamed endothelial cells could be blocked by peptide 4D. The present data demonstrate the positive health benefits of chronic peptide 4D administration to the atherosclerosis-prone ApoE-/- mice, and provides new information for potential use of this F11R derived peptide in the prevention of atherosclerosis. The data presented in this article provide further experimental support for the study presented in Babinska et al., Atherosclerosis 284 (2019) 92-101.
RESUMEN
Adenosine analogues have high affinity and selectivity for adenosine receptors (AR), and exhibit anti-platelet activity. Plasma proteins play an important role in the regulation of platelet function and may influence the action of anti-platelet compounds. Little is known about the interactions of AR agonists with plasma proteins. This study investigates the interplay between AR agonists and plasma proteins and the consequences of those interactions. Surface plasmon resonance was employed together with molecular docking study to determine the binding kinetics of four selected ARagonists (PSB0777, Cl-Ado, MRE0094, UK432097) to several carrier proteins and to clarify the nature of these interactions. The influence of a whole plasma and of some plasma components on the effectiveness of ARagonists in the inhibition of platelet function was assessed by flow cytometry (platelet activation) and ELISA (platelet adhesion). Plasma proteins remarkably diminished the effectiveness of ARagonists in inhibiting platelet activation and adhesion in vitro. ARagonists were found to strongly bind to human serum albumin (HSA) and the protein components of lipoproteins - apolipoproteins; HSA was essential for the binding of water-soluble PSB0777, whereas apolipoproteins were needed for interactions with poorly-water soluble compounds such as UK432097 and MRE0094. In addition, HSA was shown to significantly reduce the effectiveness of PSB0777 in inhibiting ADP-induced platelet activation. In conclusion, HSA and lipoproteins are important carriers for ARagonists, which can affect pharmacodynamics of ARagonists used as platelet inhibitors.
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Agonistas del Receptor de Adenosina A2/farmacología , Adenosina/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Adenosina/análogos & derivados , Adenosina/química , Agonistas del Receptor de Adenosina A2/química , Adulto , Femenino , Furanos/química , Furanos/farmacología , Humanos , Masculino , Simulación del Acoplamiento Molecular/métodos , Activación Plaquetaria/fisiología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Receptores Purinérgicos P1/metabolismo , Adulto JovenRESUMEN
BACKGROUND AND AIMS: The F11 Receptor (F11R), AKA Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A), is an adhesion protein constitutively expressed on the membrane surface of circulating platelets and the luminal surface of inflamed endothelial cells (EC). Platelet adhesion to an inflamed endothelium is one of the early steps of atherosclerotic plaque formation. Our previous studies, conducted with cultured EC in vitro, have demonstrated the expression of F11R/JAM-A on the luminal surface of inflamed EC, platelet adhesion to inflamed EC through F11R/JAM-A interactions, and inhibition of this interaction by the presence of F11R/JAM-A antagonistic peptide (F11Rpeptide 4D). In the present study, we examined in vivo the overall health-benefits and cardiovascular effects of long-term treatment of animals prone to atherosclerosis, ApoE-/- mice, with F11R-peptide 4D. METHODS: Twenty ApoE-/- mice were assigned to daily treatment with peptide 4D and compared to their counterparts control untreated mice. Mice were observed for wellness and survival. Plaque size in the aorta and heart was measured using histological analysis. Effects of peptide 4D (or scramble control) on platelet adhesion to inflamed endothelium were measured using intravital microscopy. RESULTS: Significant reductions in atherosclerotic plaques number and size, an overall robust health with longer survival were found in the peptide 4D treated group of ApoE-/- mice. Intravital microscopic studies conducted in exposed vessels of ApoE-/- mice demonstrated significant inhibition by peptide 4D of platelet adhesion to the cytokine-inflamed endothelium. CONCLUSIONS: Our results demonstrate that peptide 4D significantly reduces atherosclerotic plaque formation in ApoE-/- mice and inhibits platelet adhesion to the inflamed arterial endothelium.
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Aterosclerosis/prevención & control , Molécula A de Adhesión de Unión/antagonistas & inhibidores , Péptidos/farmacología , Péptidos/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Adhesividad Plaquetaria/efectos de los fármacosRESUMEN
Several adenosine receptor (AR) agonists have been shown in the past to possess anti-platelet potential; however, the adjunctive role of AR agonists in anti-platelet therapy with the use of P2Y12 receptor inhibitors has not been elucidated so far. This in vitro aggregation-based study investigates whether the inhibition of platelet function mediated by cangrelor or prasugrel metabolite can be potentiated by AR agonists. It evaluates the effect of non-selective (2-chloroadenosine), A2A-selective (UK 432097, MRE 0094, PSB 0777) and A2B-selective AR agonists (BAY 60-6583) on platelet function in relation to their toxicity, specificity towards adenosine receptor subtypes, structure and solubility. UK 432097, 2-chloroadenosine, MRE 0094 and PSB 0777 were found to be more or less potent inhibitors of ADP-induced platelet aggregation when acting alone, and that they remained non-cytotoxic to the cells. These AR agonists were also effective in the potentiation of the effects exerted by P2Y12 antagonists. Considering the estimated IC50 value, UK 432097, showing a relatively high binding affinity to the A2A adenosine receptor, has been identified as the most potent anti-aggregatory agent. This compound diminished platelet aggregation at nanomolar concentrations and further augmented platelet inhibition by P2Y12 antagonists by approx. 60% (Pâ¯<â¯.01). Our results indicate the importance of adenosine receptors as therapeutic targets and point out challenges and potential benefits of therapeutic use of a combined therapy of P2Y12 antagonist and AR agonist in cardioprotection. Our comparative analysis of the effects of AR agonists on platelet response in plasma and whole blood may indirectly suggest that other blood morphology elements contribute little to the inhibition of platelet function by AR agonists.
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Agonistas del Receptor de Adenosina A2/farmacología , Plaquetas/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y12/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adulto , Plaquetas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Furanos/farmacología , Humanos , Masculino , Clorhidrato de Prasugrel/farmacología , Receptores Purinérgicos P2Y12/sangre , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y12 antagonists were similar in high- and low-responders to P2Y12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.
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Adenosina Monofosfato/análogos & derivados , Resistencia a Medicamentos/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Clorhidrato de Prasugrel/farmacología , Agonistas del Receptor Purinérgico P1/farmacología , Receptores Purinérgicos P2Y12/metabolismo , Adenosina Monofosfato/farmacología , Femenino , Humanos , Masculino , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
: Mutations in the genes encoding vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) largely contribute to the inter-individual variations in vitamin K antagonists (VKAs) dose requirements. Up to 50% of the dosage variability can be explained by genetic polymorphisms in these genes. We sought to identify the mutations responsible for VKA resistance in a series of Polish patients. Of the 607 patients treated with VKA, 35 (6%) individuals with the VKA resistance defined as a daily dose of acenocoumarol more than 8âmg (nâ=â15, 43%) or warfarin more than 10âmg (nâ=â20, 57%) were selected for further mutational analysis using Sanger sequencing (VKORC1) or real-time PCR genotyping (CYP2C9). The indications for anticoagulant treatment were venous thromboembolism (nâ=â28, 80%), atrial fibrillation (nâ=â6, 17%), or artificial heart valve (nâ=â1, 3%). Patients taking medication interfering with VKA were ineligible. Almost all of VKA-resistant patients (nâ=â34, 97%) possessed at least one VKORC1*3 (nâ=â29, 83%) or VKORC1*4 (nâ=â15, 43%) haplotypes. In a 70-year-old man atrial fibrillation patient on the daily acenocoumarol dose of 16âmg, a novel p.Ile123Met (c.369C>G) VKORC1 mutation was found. In-silico analysis showed that the p.Ile123Met can functionally underlie the acenocoumarol resistance, presumably by altering VKA binding. To our knowledge this is the first cohort of Polish patients resistant to VKA evaluated for the causal genetic background. We found one new detrimental mutation underlying VKA resistance. Our study highlights a key role of unidentified environmental factors in VKA resistance in daily clinical practice.
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Citocromo P-450 CYP2C9/genética , Vitamina K Epóxido Reductasas/genética , Vitamina K/antagonistas & inhibidores , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , PoloniaRESUMEN
OBJECTIVE: Genetic background of cryptogenic ischemic stroke (IS) and transient ischemic attack (TIA) remains uncertain. Alpha-2-antiplasmin (α2AP) Arg407Lys polymorphism has been shown to be less common in patients with abdominal aortic aneurysm (AAA) compared with healthy controls. We investigated associations of α2AP Arg407Lys polymorphism with cryptogenic IS and TIA. METHODS: We studied 165 consecutive Caucasian patients who experienced cryptogenic IS (n=123) or TIA (n=42). Neurological outcomes were assessed using the modified Rankin Scale (mRS) in the acute phase of cerebral ischemia and 8 (6-12) months after the index episode. Patients were genotyped for α2AP Arg407Lys polymorphism (rs1057335) using real time PCR technique. RESULTS: The allele frequency of Arg407Lys polymorphism was: 0.82/0.18. The 407Lys allele was more frequent in TIA patients compared to the IS group (0.29 vs. 0.14, p=0.003). In the whole group, as well as in IS and TIA patients analyzed separately, possession of the 407Lys allele was associated with excellent outcome (mRS 0-1) during follow-up (p<0.05) but not in the acute phase of ischemic events both in thrombolyzed and nonthrombolyzed IS patients. The multivariate logistic regression model showed that the excellent outcome (mRS 0-1) assessed after 8 (6-12) months since the index cerebral ischemia was predicted by the occurrence of Lys407 allele (OR 6.18, 95% CI, 2.01-18.98, p=0.001). CONCLUSION: The presence of 407Lys allele is associated with better prognosis in cryptogenic cerebrovascular events. Our findings suggest that the α2AP Arg407Lys polymorphism could be involved in the pathogenesis of cerebral ischemia and its outcomes.