Platelet Physiology.

Platelets are the smallest blood cells, numbering 150 to 350 × 109/L in healthy individuals. The ability of activated platelets to adhere to an injured vessel wall and form aggregates was first described in the 19th century. Besides their long-established roles in thrombosis and hemostasis, platelets are increasingly recognized as pivotal players in numerous other pathophysiological processes including inflammation and atherogenesis, antimicrobial host defense, and tumor growth and metastasis. Consequently, profound knowledge of platelet structure and function is becoming more important in research and in many fields of modern medicine. This review provides an overview of platelet physiology focusing particularly on the structure, granules, surface glycoproteins, and activation pathways of platelets.

Platelet surface GPIbα, activated GPIIb-IIIa, and P-selectin levels in adult veno-arterial extracorporeal membrane oxygenation patients.

Our objective was to characterize platelet surface glycoprotein (GP)Ibα, activated GPIIb-IIIa, and P-selectin levels during and after extracorporeal membrane oxygenation (ECMO). We performed a single center cohort study of 10 adult patients on ECMO for cardiogenic shock. Patients had blood samples drawn on ECMO day 1 or 2, day 3, day 5, and 48-72 hours after ECMO decannulation. Platelets from untreated blood samples and samples treated with either adenosine diphosphate (ADP) or thrombin receptor agonist peptide (TRAP) had surface GPIbα, activated GPIIb-IIIa, and P-selectin levels measured using flow cytometry. Platelet surface GPIbα levels varied significantly by time on ECMO (p = .002) and were significantly higher on ECMO day 5 compared to ECMO day 1 (p = .01). GPIbα levels during ECMO did not differ significantly from levels after ECMO decannulation (p = .14). Activated GPIIb-IIIa levels did not change significantly during ECMO, but were significantly higher after ECMO decannulation (p = .04). There were no significant differences in P-selectin levels during ECMO (p = .87) or after ECMO decannulation (p = .41). Platelet surface GPIbα and P-selectin levels were similar during and after ECMO whereas activated GPIIb-IIIa levels were lower during ECMO, particularly in response to TRAP stimulation, potentially contributing to ECMO-induced coagulopathy.

Real-World Anticoagulant Use and Incidence of Venous Thromboembolism and Major Bleeding in Children.

PURPOSE: Children generally have a lower risk of venous thromboembolism (VTE) than adults, but those with acute and chronic conditions requiring hospitalization and surgical procedures are at increased risk. Anticoagulant use in children has not been systematically studied, and limited data exist. This study aimed to provide data on the conditions associated with use of anticoagulants, the type of anticoagulant used in children, and the incidence of thromboembolism and major bleeding events reported in this population. METHODS: To increase understanding of the use of anticoagulant therapies in children with at-risk conditions, 3 health claims databases in the United States were analyzed to describe the characteristics of use of heparins, warfarin, and direct oral anticoagulants (DOACs). Cumulative drug exposure was determined for continuous exposure, defined as >30 days. Unadjusted event rates of VTE and major bleeding after exposure to these therapies were reported. The data were presented descriptively and are not intended for comparison or to imply any causation. FINDINGS: Anticoagulants were infrequently used in the pediatric population, including at any time point after Fontan surgery for congenital heart disease. Heparins were used most frequently in the population overall and especially for patients aged <12 years. DOACs were used least often and primarily for patients ages 12 to <18 years. Among pediatric patients exposed to anticoagulants, unadjusted incidence rates of VTE per 1000 person-years of exposure ranged from 30.8 to 34.0 for all DOACs, 21.6 to 46.2 for warfarin, and 6.0 to 7.3 for heparins. Rates per 1000 person-years for major bleeding ranged from 0 to 4.9 for all DOACs, 4.3 to 6.7 for warfarin, and 3.7 to 4.6 for heparins. IMPLICATIONS: With results from clinical trials evaluating DOACs in the pediatric population expected in the next 2 years, these descriptive real-world data may provide a baseline understanding of current prescribing patterns and outcomes associated with the use of DOACs and other anticoagulants in routine pediatric clinical practice. This information represents the use of real-world evidence and may function as the benchmark for evaluating changes in prescription practices and potential outcomes in the future.

Familial thrombocytopenia due to a complex structural variant resulting in a WAC-ANKRD26 fusion transcript.

Advances in genome sequencing have resulted in the identification of the causes for numerous rare diseases. However, many cases remain unsolved with standard molecular analyses. We describe a family presenting with a phenotype resembling inherited thrombocytopenia 2 (THC2). THC2 is generally caused by single nucleotide variants that prevent silencing of ANKRD26 expression during hematopoietic differentiation. Short-read whole-exome and genome sequencing approaches were unable to identify a causal variant in this family. Using long-read whole-genome sequencing, a large complex structural variant involving a paired-duplication inversion was identified. Through functional studies, we show that this structural variant results in a pathogenic gain-of-function WAC-ANKRD26 fusion transcript. Our findings illustrate how complex structural variants that may be missed by conventional genome sequencing approaches can cause human disease.

Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers.

Clinical trials in sickle cell disease (SCD) often focus on health care utilization for painful vaso-occlusive crises (VOCs). However, no objective, quantifiable pain biomarkers exist, pain is not specific to VOCs, health care utilization varies between patients, unreported at-home VOCs likely contribute to long-term outcomes, and patient-reported outcomes are seldom considered. This noninterventional, longitudinal, 6-month study aimed to develop tools to identify VOCs in SCD patients with or without health care utilization. Participants wore an actigraph device, tracking sleep and activity. Patients with SCD used an electronic patient-reported outcome (ePRO) tool to collect data on pain, medication, fatigue, and daily function. Patients self-reported when they experienced VOC pain (VOC day). Biomarkers were collected every 3 weeks (non-VOC). Self-reported VOCs triggered at-home or in-hospital blood collection. The study enrolled 37 participants with SCD; 35 completed the study. Participants reported 114 VOC events and 346 VOC days, of which 62.3% and 78.3%, respectively, were self-treated at home. The ePRO and actigraphy captured end points of pain, functionality, fatigue, activity, and sleep; each was significantly altered on VOC days compared with non-VOC days. Biomarkers collected at home or in the hospital on VOC days were significantly altered compared with non-VOC baseline values, including leukocyte-platelet aggregates, microfluidic-based blood cell adhesion, interleukin-6, C-reactive protein, interleukin-10, tumor necrosis factor-α, and thrombin-antithrombin. The Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) trial shows the feasibility of accurately monitoring out-of-hospital pain by using patient-reported VOC days as potential end points for clinical trials in SCD; it describes the changes in biomarkers and activity measured by actigraphy that may enable improved identification and assessment of VOCs.

Biomarkers of platelet activation and cardiovascular risk in the DAPT trial.

Prolonged use of dual antiplatelet therapy (DAPT) post-percutaneous coronary intervention (PCI) has been shown to reduce the risk of major adverse cardiovascular events (MACE), but with increased bleeding. It remains unknown whether biomarkers of platelet activation may be useful for identifying patients at increased risk of MACE. The DAPT study was a randomized trial of 12 versus 30 months of DAPT in patients who underwent PCI. Serum biomarkers [myeloid-related protein (MRP)-8/14, P-selectin, soluble CD-40 ligand (sCD40L)] were assessed in 1399 patients early post-PCI. On-treatment platelet reactivity index (PRI) using VASP phosphorylation was assessed in 443 patients randomized to continued DAPT at 1 year. MACE was defined as CV death, MI, or ischemic stroke. Multivariable models were adjusted for baseline characteristics, index event, and stent type. A stepwise increase in the risk of MACE was observed with increasing tertiles of both MRP-8/14 and P-selectin (p-trend = 0.04 for both). After multivariable adjustment, the adjusted HR (95% CI) for MACE in patients in the top tertile was 1.94 (1.14-3.30) for MRP-8/14 and 1.62 (0.99-2.64) for P-selectin. In contrast, baseline sCD40L was not associated with CV risk. Among patients randomized to continued DAPT, higher on-treatment platelet reactivity was not significantly associated with risk of MACE (p-trend = 0.32; adj-HR T3 vs. T1 1.54, 95% CI 0.20-12.18) or bleeding (P-trend = 0.17; adj-HR 0.25, 95% CI 0.05-1.21). MRP-8/14 and soluble P-selectin may be useful for identifying patients at increased risk of MACE after PCI. The utility of on-treatment platelet function testing requires further study.Clinical Trial Registration https://www.clinicaltrials.gov . Unique identifier NCT00977938.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Illustrated State-of-the-Art Capsules of the ISTH 2020 Congress.

The 2020 Congress of the International Society of Thrombosis and Haemostasis (ISTH) was held virtually July 12-15, 2019, due to the coronavirus disease 2019 pandemic. The congress convenes annually to discuss clinical and basic topics in hemostasis and thrombosis. Each year, the program includes State of Art (SOA) lectures given by prominent scientists. Presenters are asked to create Illustrated Capsules of their talks, which are concise illustrations with minimal explanatory text. Capsules cover major themes of the presentation, and these undergo formal peer review for inclusion in this article. Owing to the shift to a virtual congress this year, organizers reduced the program size. There were 39 SOA lectures virtually presented, and 29 capsules (9 from talks omitted from the virtual congress) were both submitted and successful in peer review, and are included in this article. Topics include the roles of the hemostatic system in inflammation, infection, immunity, and cancer, platelet function and signaling, platelet function disorders, megakaryocyte biology, hemophilia including gene therapy, phenotype tests in hemostasis, von Willebrand factor, anticoagulant factor V, computational driven discovery, endothelium, clinical and basic aspects of thrombotic microangiopathies, fibrinolysis and thrombolysis, antithrombotics in pediatrics, direct oral anticoagulant management, and thrombosis and hemostasis in pregnancy. Capsule authors invite virtual congress attendees to refer to these capsules during the live presentations and participate on Twitter in discussion. Research and Practice in Haemostasis and Thrombosis will release 2 tweets from @RPTHJournal during each presentation, using #IllustratedReview, #CoagCapsule and #ISTH2020. Readers are also welcome to utilize capsules for teaching and ongoing education.

Novel Antiplatelet Agents in Cardiovascular Disease.

Antiplatelet therapy reduces atherothrombotic risk and has therefore become a cornerstone in the treatment of cardiovascular disease. Aspirin, adenosine diphosphate P2Y12 receptor antagonists, glycoprotein IIb/IIIa inhibitors, and the thrombin receptor blocker vorapaxar are effective antiplatelet agents but significantly increase the risk of bleeding. Moreover, atherothrombotic events still impair the prognosis of many patients with cardiovascular disease despite established antiplatelet therapy. Over the last years, advances in the understanding of thrombus formation and hemostasis led to the discovery of various new receptors and signaling pathways of platelet activation. As a consequence, many new antiplatelet agents with high antithrombotic efficacy and supposedly only moderate effects on regular hemostasis have been developed and yielded promising results in preclinical and early clinical studies. Although their long journey from animal studies to randomized clinical trials and finally administration in daily clinical routine has just begun, some of the new agents may in the future become meaningful additions to the pharmacological armamentarium in cardiovascular disease.

Changes in neurocognitive function and central nervous system structure in childhood acute lymphoblastic leukaemia survivors after treatment: a meta-analysis.

Acute lymphoblastic leukaemia (ALL) is the most common malignancy in children. Although the survival rate has increased dramatically over the last decades, patients struggle with the adverse side effects of treatment. Treatment for ALL includes chemotherapy and irradiation - both of which are linked to cognitive impairments and alterations in central nervous system (CNS) structure and function detected by neuroimaging and in neurocognitive studies. The present article is a meta-analysis of the existing evidence for the mechanisms underlying changes in the CNS and neurocognitive function in ALL survivors after treatment. We found that compared with controls, ALL survivors develop: (i) cognitive sequelae in intelligence, academics, attention, memory, processing speed and executive function domains; (ii) decreased grey and white matter volume in cortical and several subcortical brain regions, with functional changes particularly in frontal regions and the hippocampus; (iii) neurocognitive impairments related to CNS changes; and (iv) reduction, but not resolution, of late neurocognitive sequelae in patients in whom prophylactic irradiation was replaced by systemic/intrathecal chemotherapy. Continued work with advanced functional magnetic resonance imaging techniques will hopefully allow the detection of early CNS changes as biomarkers to help guide early diagnosis and intervention for neurocognitive defects in patients with childhood ALL.

Ticagrelor versus placebo for the reduction of vaso-occlusive crises in pediatric sickle cell disease: Rationale and design of a randomized, double-blind, parallel-group, multicenter phase 3 study (HESTIA3).

BACKGROUND: An unmet need for therapies exists to reduce sickle cell disease (SCD) complications in pediatric patients. Activated platelets contribute to the formation of cellular aggregates during sickling and vaso-occlusive crises (VOCs). Ticagrelor is an oral, direct-acting, and reversible adenosine diphosphate P2Y12 receptor antagonist that inhibits platelet activation and aggregation. Although ticagrelor was well tolerated in two phase 2 studies in children and young adults with SCD, larger and longer-term treatment studies are needed to assess ticagrelor's efficacy to reduce VOCs. HESTIA3 will evaluate the efficacy, safety, and tolerability of ticagrelor versus placebo over a minimum of 1 year (maximum 2 years) in pediatric patients with SCD. METHODS: Approximately 180 patients (aged ≥ 2 to <18 years) with SCD (≥ 2 VOCs in the prior year) from 18 countries will be randomized 1:1 to ticagrelor or placebo. Primary endpoint: number of VOCs (a composite endpoint of painful crises and/or acute chest syndrome); key secondary endpoints: hospitalizations, pain intensity and analgesic use during VOCs, acceptability of formulation, and health-related quality of life. The weight-based doses of ticagrelor are set by modeling and simulation. Platelet inhibition data, measured by the vasodilator-stimulated phosphoprotein assay, will be collected for exploratory purposes. CONCLUSIONS: HESTIA3 aims to demonstrate that using greater target platelet inhibition than previous studies on SCD, ticagrelor will decrease the frequency of VOC in pediatric patients. Trial Identifier: NCT03615924; EudraCT2017-002421-38.

Rivaroxaban, a direct Factor Xa inhibitor, versus acetylsalicylic acid as thromboprophylaxis in children post-Fontan procedure: Rationale and design of a prospective, randomized trial (the UNIVERSE study).

BACKGROUND: The Fontan procedure is the final step of the 3-stage palliative procedure commonly performed in children with single ventricle physiology. Thrombosis remains an important complication in children after this procedure. To date, guideline recommendations for the type and duration of thromboprophylaxis after Fontan surgery are mainly based on extrapolation of knowledge gained from adults at risk for thrombosis in other clinical settings. Warfarin is being used off-label, and because of its multiple interactions with other drugs and food, a new alternative is highly desirable. Rivaroxaban, a direct Factor Xa inhibitor with a predictable pharmacokinetic profile, is a candidate to address this medical need. STUDY DESIGN: The UNIVERSE study is a prospective, open-label, active-controlled, multicenter study in children 2 to 8 years of age who have single ventricle physiology and had the Fontan procedure within the 4 months preceding enrollment. This study consists of 2 parts. In Part A, rivaroxaban pharmacokinetics, pharmacodynamics, safety, and tolerability are assessed to validate the pediatric dosing selected. In Part B, safety and efficacy of rivaroxaban versus acetylsalicylic acid are evaluated for thromboprophylaxis in children post-Fontan procedure. Children in each part will receive study drug for 12 months. Part A has been completed with 12 children enrolled. Enrollment into Part B is currently ongoing. CONCLUSIONS: The UNIVERSE study aims to provide dosing, pharmacokinetics/pharmacodynamics, safety, and efficacy information on the use of rivaroxaban, an oral anticoagulant, versus acetylsalicylic acid, an antiplatelet agent, in children with single ventricle physiology after the Fontan procedure.

GLS-409, an Antagonist of Both P2Y1 and P2Y12, Potently Inhibits Canine Coronary Artery Thrombosis and Reversibly Inhibits Human Platelet Activation.

Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y12 receptor antagonist reduces ischemic events in patients with acute coronary syndrome. Previous evidence from our group, obtained in a preclinical model of recurrent platelet-mediated thrombosis, demonstrated that GLS-409, a diadenosine tetraphosphate derivative that inhibits both P2Y1 and P2Y12 ADP receptors, may be a novel and promising antiplatelet drug candidate. However, the salutary antiplatelet effects of GLS-409 were accompanied by a trend toward an unfavorable increase in bleeding. The goals of this study were to: 1) provide proof-of-concept that the efficacy of GLS-409 may be maintained at lower dose(s), not accompanied by an increased propensity to bleeding; and 2) establish the extent and kinetics of the reversibility of human platelet inhibition by the agent. Lower doses of GLS-409 were identified that inhibited in vivo recurrent coronary thrombosis with no increase in bleeding time. Human platelet inhibition by GLS-409 was reversible, with rapid recovery of platelet reactivity to ADP, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. These data support the concept that GLS-409 warrants further, larger-scale investigation as a novel, potential therapy in acute coronary syndromes.

Tunable activation of therapeutic platelet-rich plasma by pulse electric field: Differential effects on clot formation, growth factor release, and platelet morphology.

BACKGROUND: Activation of platelet-rich plasma (PRP) by pulse electric field (PEF) releases growth factors which promote wound healing (e.g., PDGF, VEGF for granulation, EGF for epithelialization). AIMS: To determine after PEF activation of therapeutic PRP: 1) platelet gel strength; 2) profile of released growth factors; 3) alpha- and T-granule release; 4) platelet morphology. METHODS: Concentrated normal donor PRP was activated by 5 µsec (long) monopolar pulse, ~4000 V/cm (PEF A) or 150 nsec (short) bipolar pulse, ~3000 V/cm (PEF B) in the presence of 2.5 mM (low) or 20 mM (high) added CaCl2. Clot formation was evaluated by thromboelastography (TEG). Surface exposure of alpha granule (P-selectin) and T-granule (TLR9 and protein disulfide isomerase [PDI]) markers were assessed by flow cytometry. Factors in supernatants of activated PRP were measured by ELISA. Platelet morphology was evaluated by transmission electron microscopy (TEM). RESULTS: Time to initial clot formation was shorter with thrombin (<1 min) than with PEF A and B (4.4-8.7 min) but clot strength (elastic modulus, derived from TEG maximum amplitude) was greater with PEF B than with either thrombin or PEF A (p<0.05). Supernatants of PRP activated with PEF A had higher EGF levels than supernatants from all other conditions. In contrast, levels of PF4, PDGF, and VEGF in supernatants were not significantly different after PEF A, PEF B, and thrombin activation. T-granule markers (TLR9 and PDI) were higher after thrombin than after PEF A or B with low or high CaCl2. By TEM, platelets in PEF-treated samples retained a subset of granules suggesting regulated granule release. CONCLUSION: Pulse length and polarity can be modulated to produce therapeutic platelet gels as strong or stronger than those produced by thrombin, and this is tunable to produce growth factor profiles enhanced in specific factors important for different stages of wound healing.

Novel aspects of antiplatelet therapy in cardiovascular disease.

Antiplatelet therapy is a cornerstone in the secondary prophylaxis of adverse cardiovascular events such as myocardial infarction and stroke. The cyclooxygenase inhibitor aspirin remains the most frequently prescribed antiplatelet drug, followed by adenosine diphosphate P2Y12 receptor blockers. Glycoprotein IIb-IIIa antagonists are intravenously available antiplatelet agents preventing platelet-to-platelet aggregation via the fibrinogen receptor. The thrombin receptor inhibitor vorapaxar allows the targeting of yet a third pathway of platelet activation. Despite the advent of novel agents and major advances in antiplatelet treatment over the last decade, atherothrombotic events still impair the prognosis of many patients with cardiovascular disease. Consequently, antiplatelet therapy remains a field of intense research and a large number of studies on its various aspects are published each year. This review article summarizes recent developments in antiplatelet therapy in cardiovascular disease focusing particularly on the duration of dual antiplatelet therapy, new treatment regimens, the role of platelet function testing, and potential future targets of antiplatelet agents.

Assessment of whole blood thrombosis in a microfluidic device lined by fixed human endothelium.

The vascular endothelium and shear stress are critical determinants of physiological hemostasis and platelet function in vivo, yet current diagnostic and monitoring devices do not fully incorporate endothelial function under flow in their assessment and, therefore, they can be unreliable and inaccurate. It is challenging to include the endothelium in assays for clinical laboratories or point-of-care settings because living cell cultures are not sufficiently robust. Here, we describe a microfluidic device that is lined by a human endothelium that is chemically fixed, but still retains its ability to modulate hemostasis under continuous flow in vitro even after few days of storage. This device lined with a fixed endothelium supports formation of platelet-rich thrombi in the presence of physiological shear, similar to a living arterial vessel. We demonstrate the potential clinical value of this device by showing that thrombus formation and platelet function can be measured within minutes using a small volume (0.5 mL) of whole blood taken from subjects receiving antiplatelet medications. The inclusion of a fixed endothelial microvessel will lead to biomimetic analytical devices that can potentially be used for diagnostics and point-of-care applications.

Platelet Physiology.

Platelets are the smallest blood cells, numbering 150 to 350 × 10(9)/L in healthy individuals. The ability of activated platelets to adhere to an injured vessel wall and form aggregates was first described in the 19th century. Besides their long-established roles in thrombosis and hemostasis, platelets are increasingly recognized as pivotal players in numerous other pathophysiological processes including inflammation and atherogenesis, antimicrobial host defense, and tumor growth and metastasis. Consequently, profound knowledge of platelet structure and function is becoming more important in research and in many fields of modern medicine. This review provides an overview of platelet physiology focusing particularly on the structure, granules, surface glycoproteins, and activation pathways of platelets.

Synergistic Inhibition of Both P2Y1 and P2Y12 Adenosine Diphosphate Receptors As Novel Approach to Rapidly Attenuate Platelet-Mediated Thrombosis.

OBJECTIVE: Unlike currently approved adenosine diphosphate receptor antagonists, the new diadenosine tetraphosphate derivative GLS-409 targets not only P2Y12 but also the second human platelet adenosine diphosphate receptor P2Y1 and may, therefore, be a promising antiplatelet drug candidate. The current study is the first to investigate the in vivo antithrombotic effects of GLS-409. APPROACH AND RESULTS: We studied (1) the in vivo effects of GLS-409 on agonist-stimulated platelet aggregation in anesthetized rats, (2) the antithrombotic activity of GLS-409 and the associated effect on the bleeding time in a canine model of platelet-mediated coronary artery thrombosis, and (3) the inhibition of agonist-stimulated platelet aggregation by GLS-409 versus selective P2Y1 and P2Y12 inhibition in vitro in samples from healthy human subjects before and 2 hours after aspirin intake. In vivo treatment with GLS-409 significantly inhibited adenosine diphosphate- and collagen-stimulated platelet aggregation in rats. Further, GLS-409 attenuated cyclic flow variation, that is, platelet-mediated thrombosis, in vivo in our canine model of unstable angina. The improvement in coronary patency was accompanied by a nonsignificant 30% increase in bleeding time. Of note, GLS-409 exerted its effects without affecting rat and canine hemodynamics. Finally, in vitro treatment with GLS-409 showed effects similar to that of cangrelor and the combination of cangrelor with the selective P2Y1 inhibitor MRS 2179 on agonist-stimulated platelet aggregation in human platelet-rich plasma and whole blood before and 2 hours after aspirin intake. CONCLUSIONS: Synergistic inhibition of both P2Y1 and P2Y12 adenosine diphosphate receptors by GLS-409 immediately attenuates platelet-mediated thrombosis and effectively blocks agonist-stimulated platelet aggregation irrespective of concomitant aspirin therapy.

New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors.

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P(1),P(4)-di(adenosine-5') tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure-activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents.