The Effects of Aspirin dose in Children with Congenital and Acquired Heart Disease. Results from the Paediatric Study of Aspirin Efficacy using Diagnostic and Monitoring Tools (PAED-M).

The optimal dose of aspirin required in children with congenital and acquired heart disease is not known. The primary aim of this prospective observational study was to evaluate the effects of aspirin dose on platelet inhibition. The secondary aim was to determine the prevalence and clinical predictors of aspirin non-responsiveness. Measurements were by Thromboelastography with Platelet Mapping (TEGPM) only in children less than 2 years (y) of age with particular emphasis on the parameter known as maximum amplitude with arachidonic acid (MAAA) and using both TEGPM, and light transmission aggregometry (LTA) in children greater than 2 y. We prospectively studied 101 patients with congenital and acquired cardiac disease who were receiving empirical doses of aspirin for a minimum of 4 weeks but no other antiplatelet agents. Patients were stratified according to dose concentration and age. There was a trend toward lower age in patients with no response or semi-response to aspirin. All patients were considered responsive to aspirin in the higher-dose quartile (Q4) with a median dose of 4.72 (4.18-6.05) mg/kg/day suggesting that patients in this age group may require 5 mg/kg/day as an empirical dose. In children > 2 y, there was no significant difference in inhibition found in patients dosed at higher doses in Q3 versus Q4 suggesting that patients in this cohort are responsive with 3 mg/kg/day dose. The current practices may lead to reduced platelet inhibition in some children due to under-dosing or overdosing in others. In conclusion, younger children require higher doses of aspirin. Laboratory assessment is warranted in this population to mitigate against under and overdosing.

Aspirin Responsiveness in a Cohort of Pediatric Patients with Right Ventricle to Pulmonary Artery Conduits and Transcatheter Valve Replacement Systems.

The aim of this study was to determine the rate of aspirin responsiveness in a cohort of pediatric patients with in situ xenograft valved right ventricle to pulmonary artery (RV-PA) conduits and/or transcatheter valve replacements (TVR). Aspirin is routinely prescribed to these patients. Optimizing anti-platelet therapy could promote valve longevity and reduce the risk of infective endocarditis in this at-risk group. This was a prospective, observational study. Patients were recruited from both ward and outpatient settings. Patients were eligible if under 18 years and taking aspirin. Non-response to aspirin was defined as > 20% platelet aggregation using light transmission platelet aggregometry (LTA) and < 50% platelet inhibition by thromboelastography with platelet mapping (TEGPM). Participants were invited to provide a confirmatory sample in cases of aspirin resistance and dose adjustments were made. Thirty patients participated. Median age was 9 years (2 months to 18 years). The majority (93%) had complex right ventricular outflow tract pathology. 13 (43%) had an RV-PA conduit and 24 (80%) had a TVR, with valve situated in conduit in 7 (23%) cases. Rate of aspirin non-response on initial testing was 23% (n = 7/30) with median LTA 74.55% (60-76%) and TEG 13.25% (0-44%) in non-responders. Non-responders were more likely to be under 1 year. Two patients required dose increases and one patient non-adherence to dose was identified. Four patients on repeat testing were responsive to aspirin by laboratory tests. The rate of aspirin non-response on laboratory testing in this cohort of patients was 23% and resulted in therapeutic intervention in 10%.

Personalized Dual Antiplatelet Therapy in Acute Coronary Syndromes: Striking a Balance Between Bleeding and Thrombosis.

PURPOSE OF REVIEW: Dual antiplatelet therapy (DAPT)-aspirin in conjunction with a P2Y12 inhibitor-is the cornerstone of managing patients with acute coronary syndromes post-revascularization, but the clinical response is highly variable, with potentially devastating consequences. Herein, we review the mechanisms underpinning said variability and explore emerging approaches to normalizing therapeutic benefit. RECENT FINDINGS: The potent P2Y12 inhibitors, prasugrel and ticagrelor, exhibit minimal inter-individual variability, replacing clopidogrel in DAPT and achieving greater rates of therapeutic response. However, these benefits decline in later phases when bleeding risk begins to supersede that of ischemia. Guided de-escalation of P2Y12 inhibition as well as shortening DAPT duration have emerged as strategies that retain antithrombotic efficacy while reducing bleeding risk. Aspirin is the other component of DAPT but is also used in isolation for secondary prevention of thrombotic disease. In contrast to the P2Y12 inhibitors, genetic influences on aspirin non-response appear to be outweighed by a triad of clinical factors: non-adherence, enteric aspirin use, and inappropriate dosing according to bodyweight and BMI. Multiple de-escalation strategies for DAPT have been shown to mitigate bleeding risk, but it remains unclear which approach is ideal, necessitating head-to-head investigations to determine which exhibits the most favorable cost-to-benefit ratio. However, there is likely a role for more than one approach in clinical practice, depending on patient risk profile. Our approach to aspirin use is also in need of reassessment: strategies to improve adherence, avoidance of enteric aspirin in cardiac patients, and dose adjustment according to bodyweight and/or BMI are all likely to improve rates of therapeutic response. Moreover, platelet function testing may have a role in identifying patients expected to benefit from primary prophylactic aspirin.

Weight as an assay-independent predictor of poor response to enteric aspirin in cardiovascular patients.

Aspirin non-response is associated with poor outcome but there is no agreement between the different methods to asses it. Weight has been shown to be a predictor of poor response but only using one method. In this study, we determine the effects of weight on different assays of platelet function. The response to aspirin was determined in 138 cardiology patients using serum thromboxane, arachidonic acid-induced platelet aggregation and VerifyNow©. Twenty-five percent of patients showed an inadequate response to aspirin in at least one assay on the initial test. After ensuring patient compliance only 5% of patients were considered to be non-responders. Only 9% of non-responders were non-responsive in all three assays. When switched to plain aspirin, only 2% of patients were non-responsive. All patients responded adequately to 150 mg aspirin. The non-responders were significantly heavier than responders (78.5 kg ± 14.0 (SD); BMI: 28.4 kg/m2± 4.4 v's 102.6 kg ± 20.6, P = .0016; BMI: 38.3 kg/m2 ± 7.6, P= .0015). A rule-based approach of using plain aspirin in patients over 90 kg or BMI 32 along with patient education to ensure compliance will ensure that all patients respond to their aspirin without the need for testing.

Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αIIbß3, and platelet factor 4.

Bacterial adhesion to platelets is mediated via a range of strain-specific bacterial surface proteins that bind to a variety of platelet receptors. It is unclear how these interactions lead to platelet activation. We demonstrate a critical role for the immune receptor FcγRIIA, αIIbß3, and Src and Syk tyrosine kinases in platelet activation by Staphylococcus aureus, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus pneumoniae. FcγRIIA activation is dependent on immunoglobulin G (IgG) and αIIbß3 engagement. Moreover, feedback agonists adenosine 5'-diphosphate and thromboxane A2 are mandatory for platelet aggregation. Additionally, platelet factor 4 (PF4) binds to bacteria and reduces the lag time for aggregation, and gray platelet syndrome α-granule-deficient platelets do not aggregate to 4 of 5 bacterial strains. We propose that FcγRIIA-mediated activation is a common response mechanism used against a wide range of bacteria, and that release of secondary mediators and PF4 serve as a positive feedback mechanism for activation through an IgG-dependent pathway.

Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbß3.

Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet-bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbß3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbß3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5'-diphosphate (ADP) and thromboxane A2 (TxA2) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria.

Platelet interactions with viruses and parasites.

While the interactions between Gram-positive bacteria and platelets have been well characterized, there is a paucity of data on the interaction between other pathogens and platelets. However, thrombocytopenia is a common feature with many infections especially viral hemorrhagic fever. The little available data on these interactions indicate a similarity with bacteria-platelet interactions with receptors such as FcγRIIa and Toll-Like Receptors (TLR) playing key roles with many pathogens. This review summarizes the known interactions between platelets and pathogens such as viruses, fungi and parasites.

The therapeutic potential of I-domain integrins.

Due to their role in processes central to cancer and autoimmune disease I-domain integrins are an attractive drug target. Both antibodies and small molecule antagonists have been discovered and tested in the clinic. Much of the effort has focused on αLß2 antagonists. Maybe the most successful was the monoclonal antibody efalizumab, which was approved for the treatment of psoriasis but subsequently withdrawn from the market due to the occurrence of a serious adverse effect (progressive multifocal leukoencephalopathy). Other monoclonal antibodies were tested for the treatment of reperfusion injury, post-myocardial infarction, but failed to progress due to lack of efficacy. New potent small molecule inhibitors of αv integrins are promising reagents for treating fibrotic disease. Small molecule inhibitors targeting collagen-binding integrins have been discovered and future work will focus on identifying molecules selectively targeting each of the collagen receptors and identifying appropriate target diseases for future clinical studies.

Towards a greater understanding of reduced response to aspirin in children with congenital heart disease post-cardiac surgery using immature platelet fraction.

OBJECTIVE: A high platelet turnover rate may produce a population of platelets that confers an inadequate response to aspirin. We aimed to investigate the relationship between residual platelet aggregation and platelet turnover in paediatric cardiology patients on aspirin monotherapy by evaluating the fraction of immature platelets as a marker for turnover and secondly to test the predictive value of the immature platelet fraction (IPF) to classify patients as responsive or non-responsive to aspirin. METHODS: Sixty patients divided into two age categories (≤90 days, >90 days of age) were included in this prospective observational study. Patients were then stratified into tertiles using their IPF level. Platelet studies included thromboelastography with platelet mapping (TEGPM). RESULTS: The overall incidence of 'inadequate response to aspirin' was 38 % in our patient cohort recently post-cardiac surgery a consequence that warrants further study. The frequency of inadequate response to aspirin was higher in the upper tertile of IPF when compared to the lower tertile, (88 %) versus (4 %) respectively (p < 0.05). The 'cut off' for IPF was determined to be 3.9 % with a sensitivity of 95.7 %, and a specificity of 92.9 % (area under the curve of 0.955 [CI 0.896-1.014, p < 0.05]). CONCLUSION: This study demonstrates that inadequate response to aspirin occurs in approximately 38 % of patients undergoing specific high-risk congenital cardiac procedures using the dosing practice of a national centre. This study supports the hypothesis that an elevated platelet turnover may result in aspirin being less effective in patients who are recently post cardiac surgery. These data are of direct translational relevance.

Prevalence of ex vivo high on-treatment platelet reactivity on antiplatelet therapy after transient ischemic attack or ischemic stroke on the PFA-100(®) and VerifyNow(®).

BACKGROUND: The prevalence of ex vivo high on-treatment platelet reactivity (HTPR) to commonly prescribed antiplatelet regimens after transient ischemic attack (TIA) or ischemic stroke is uncertain. METHODS: Platelet function inhibition was simultaneously assessed with modified light transmission aggregometry (VerifyNow; Accumetrics Inc, San Diego, CA) and with a moderately high shear stress platelet function analyzer (PFA-100; Siemens Medical Solutions USA, Inc, Malvern, PA) in a pilot, cross-sectional study of TIA or ischemic stroke patients. Patients were assessed on aspirin-dipyridamole combination therapy (n = 51) or clopidogrel monotherapy (n = 25). RESULTS: On the VerifyNow, HTPR on aspirin was identified in 4 of 51 patients (8%) on aspirin-dipyridamole combination therapy (≥ 550 aspirin reaction units on the aspirin cartridge). Eleven of 25 (44%) patients had HTPR on clopidogrel (≥ 194 P2Y12 reaction units on the P2Y12 cartridge). On the PFA-100, 21 of 51 patients (41%) on aspirin-dipyridamole combination therapy had HTPR on the collagen-epinephrine (C-EPI) cartridge. Twenty-three of 25 patients (92%) on clopidogrel had HTPR on the collagen-adenosine diphosphate (C-ADP) cartridge. The proportion of patients with antiplatelet HTPR was lower on the VerifyNow than PFA-100 in patients on both regimens (P < .001). CONCLUSIONS: The prevalence of ex vivo antiplatelet HTPR after TIA or ischemic stroke is markedly influenced by the method used to assess platelet reactivity. The PFA-100 C-ADP cartridge is not sensitive at detecting the antiplatelet effects of clopidogrel ex vivo. Larger prospective studies with the VerifyNow and with the PFA-100 C-EPI and recently released Innovance PFA P2Y cartridges (Siemens Medical Solutions USA, Inc) in addition to newer tests of platelet function are warranted to assess whether platelet function monitoring predicts clinical outcome in ischemic cerebrovascular disease.

Sepsis - it is all about the platelets.

Sepsis is accompanied by thrombocytopenia and the severity of the thrombocytopenia is associated with mortality. This thrombocytopenia is characteristic of disseminated intravascular coagulation (DIC), the sepsis-associated coagulopathy. Many of the pathogens, both bacterial and viral, that cause sepsis also directly activate platelets, which suggests that pathogen-induced platelet activation leads to systemic thrombosis and drives the multi-organ failure of DIC. In this paper we review the mechanisms of platelet activation by pathogens and the evidence for a role for anti-platelet agents in the management of sepsis.

Mechanism of outside-in {alpha}IIb{beta}3-mediated activation of human platelets by the colonizing Bacterium, Streptococcus gordonii.

OBJECTIVE: To better understand the mechanism of platelet recruitment and activation by Streptococcus gordonii. The oral bacterium Streptococcus gordonii, is amongst the most common pathogens isolated from infective endocarditis patients, and has the property of being able to activate platelets, leading to thrombotic complications. The mechanism of platelet recruitment and activation by S. gordonii is poorly understood. METHODS AND RESULTS: Infective endocarditis is a bacterial infection of the heart valves that carries a high risk of morbidity and mortality. The oral bacterium, S gordonii, is among the most common pathogens isolated from patients with infective endocarditis and is able to activate platelets, leading to thrombotic complications. Platelets interact with S gordonii via glycoprotein Ibα- and α(IIb)ß(3)-recognizing S gordonii surface proteins haemaglutitin salivary antigen (Hsa) and platelet adherence protein A, respectively. The inhibition of glycoprotein Ibα or α(IIb)ß(3) using blocking antibodies or deletion of S gordonii Hsa or platelet adherence protein A significantly reduces platelet adhesion. Immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins have recently played a role in transmitting activating signals into platelets. Platelet adhesion to immobilized S gordonii resulted in tyrosine phosphorylation of the ITAM-bearing receptor, FcγRIIa, and phosphorylation of downstream effectors (ie, spleen tyrosine kinase [Syk] and phospholipase C [PLC]-γ2). Tyrosine phosphorylation of FcγRIIa resulted in platelet-dense granule secretion, filopodial and lamellipodial extension, and platelet spreading. Inhibition of FcγRIIa ablated both dense granule release and platelet spreading. CONCLUSIONS: Streptococcus gordonii binding to the α(IIb)ß(3)/FcγRIIa integrin/ITAM signaling complex results in platelet activation that likely contributes to the thrombotic complications of infective endocarditis.

TIN-a combinatorial compound collection of synthetically feasible multicomponent synthesis products.

The synthetic feasibility of any compound library used for virtual screening is critical to the drug discovery process. TIN, a recursive acronym for 'TIN Is Not commercial', is a virtual combinatorial database enumeration of diversity-orientated multicomponent syntheses (MCR). Using a 'one-pot' synthetic technique, 12 unique small molecule scaffolds were developed, predominantly styrylisoxazoles and bis-acetylenic ketones, with extensive derivatization potential. Importantly, the scaffolds were accessible in a single operation from commercially available sources containing R-groups which were then linked combinatorially. This resulted in a combinatorial database of over 28 million product structures, each of which is synthetically feasible. These structures can be accessed through a free Web-based 2D structure search engine or downloaded in SMILES, MOL2, and SDF formats. Subsets include a 10% diversity subset, a drug-like subset, and a lead-like subset that are also freely available for download and virtual screening ( http://mmg.rcsi.ie:8080/tin ).

The role of platelets in the pathogenesis of cerebral malaria.

Malaria is a major cause of morbidity and mortality in the developing world and cerebral malaria is responsible for the majority of malaria-associated deaths. There is a strong association between thrombocytopenia and outcome in malaria, suggesting a role for platelets in the pathogenesis of malaria. This thrombocytopenia is likely due to platelet activation possibly through an interaction between PfEMP1 on plasmodium and CD36 on platelets. Platelet activation by plasmodium has two potential consequences. It can lead to the formation of micro-aggregates of infected red blood cells and platelets which can occlude blood vessels and it also leads to binding to and activation of the endothelium.

Platelet-bacterial interactions.

Many bacteria are capable of interacting with platelets and inducing platelet aggregation. This interaction may be a direct interaction between a bacterial surface protein and a platelet receptor or may be an indirect interaction where plasma proteins bind to the bacterial surface and subsequently bind to a platelet receptor. However, these interactions usually do not trigger platelet activation as a secondary co-signal is also required. This is usually due to specific antibody bound to the bacteria interacting with FcgammaRIIa on the platelet surface. Secreted bacterial products such as gingipains and lipopolysaccharide may also be capable of triggering platelet activation.

How not to discover a drug - integrins.

INTRODUCTION: Integrins are a family of 24 cell adhesion receptors that play a role in the biggest unmet needs in medicine - cardiovascular disease, immunology and cancer. Their discovery promised huge potential for the pharmaceutical industry. Areas covered. Over 35-years since their discovery, there is little to show for the hundreds of billions of dollars of investment in anti-integrin drug discovery programmes. In this review the author discusses the reasons for the failure of this promising class of drugs and the future for this class of drugs. Expert opinion. Within 10-years, there was a plethora of potent, specific anti-integrin molecules and since their discovery, many of these agents have entered clinical trials. The success in discovering these agents was due to recently discovered monoclonal antibody technology. The integrin-recognition domain Arg-Gly-Asp (RGD) provided the basis for discovering small molecule inhibitors to integrins - both cyclic peptides and peptidomimetics. Most agents failed in the Phase III clinical trials and those agents that did make it to the market were plagued with issues of toxicity and limited efficacy and were soon replaced with non-integrin targeting agents. Their failure was due to a combination of poor pharmacokinetics and pharmacodynamics, complicated by the complex pathophysiology of integrins.

Targeting SARS-CoV-2-Platelet Interactions in COVID-19 and Vaccine-Related Thrombosis.

It is clear that COVID-19 is more than a pneumonia and is associated with a coagulopathy and multi-organ failure. While the use of anti-coagulants does reduce the incidence of pulmonary emboli, it does not help with survival. This suggests that the coagulopathy is more likely to be platelet-driven rather than thrombin-driven. There is significant evidence to suggest that SARS-CoV-2 virions directly interact with platelets to trigger activation leading to thrombocytopenia and thrombosis. I propose a model of multiple interactions between SARS-CoV-2 and platelets that has many similarities to that with Staphylococcus aureus and Dengue virus. As platelet activation and thrombosis are major factors in poor prognosis, therapeutics that target the platelet-SARS-CoV-2 interaction have potential in treating COVID-19 and other virus infections.

Human platelets recognize a novel surface protein, PadA, on Streptococcus gordonii through a unique interaction involving fibrinogen receptor GPIIbIIIa.

The concept of an infectious agent playing a role in cardiovascular disease is slowly gaining attention. Among several pathogens identified, the oral bacterium Streptococcus gordonii has been implicated as a plausible agent. Platelet adhesion and subsequent aggregation are critical events in the pathogenesis and dissemination of the infective process. Here we describe the identification and characterization of a novel cell wall-anchored surface protein, PadA (397 kDa), of S. gordonii DL1 that binds to the platelet fibrinogen receptor GPIIbIIIa. Wild-type S. gordonii cells induced platelet aggregation and supported platelet adhesion in a GPIIbIIIa-dependent manner. Deletion of the padA gene had no effect on platelet aggregation by S. gordonii but significantly reduced (>75%) platelet adhesion to S. gordonii. Purified N-terminal PadA recombinant polypeptide adhered to platelets. The padA mutant was unaffected in production of other platelet-interactive surface proteins (Hsa, SspA, and SspB), and levels of adherence of the mutant to fetuin or platelet receptor GPIb were unaffected. Wild-type S. gordonii, but not the padA mutant, bound to Chinese hamster ovary cells stably transfected with GPIIbIIIa, and this interaction was ablated by addition of GPIIbIIIa inhibitor Abciximab. These results highlight the growing complexity of interactions between S. gordonii and platelets and demonstrate a new mechanism by which the bacterium could contribute to unwanted thrombosis.

Platelet function/reactivity testing and prediction of risk of recurrent vascular events and outcomes after TIA or ischaemic stroke: systematic review and meta-analysis.

BACKGROUND: The prevalence of ex vivo 'high on-treatment platelet reactivity (HTPR)' and its relationship with recurrent vascular events/outcomes in patients with ischaemic cerebrovascular disease (CVD) is unclear. METHODS: A systematic review and meta-analysis was performed in accordance with the PRISMA statement. MEDLINE, EMBASE and Cochrane Library were searched for completed manuscripts until May 2019 on TIA/ischaemic stroke patients, ≥ 18 years, treated with commonly-prescribed antiplatelet therapy, who had platelet function/reactivity testing and prospective follow-up data on recurrent stroke/TIA, myocardial infarction, vascular death or other cerebrovascular outcomes. Data were pooled using random-effects meta-analysis. Primary outcome was the composite risk of recurrent stroke/TIA, myocardial infarction or vascular death. Secondary outcomes were recurrent stroke/TIA, severe stroke (NIHSS > 16) or disability/impairment (modified Rankin scale ≥ 3) during follow-up. RESULTS: Antiplatelet-HTPR prevalence was 3-65% with aspirin, 8-56% with clopidogrel and 1.8-35% with aspirin-clopidogrel therapy. Twenty studies (4989 patients) were included in our meta-analysis. There was a higher risk of the composite primary outcome (OR 2.93, 95% CI 1.90-4.51) and recurrent ischaemic stroke/TIA (OR 2.43, 95% CI 1.51-3.91) in patients with vs. those without 'antiplatelet-HTPR' on any antiplatelet regimen. These risks were also more than twofold higher in patients with vs. those without 'aspirin-HTPR' and 'dual antiplatelet-HTPR', respectively. Clopidogrel-HTPR status did not significantly predict outcomes, but the number of eligible studies was small. The risk of severe stroke was higher in those with vs. without antiplatelet-HTPR (OR 2.65, 95% CI 1.00-7.01). DISCUSSION: Antiplatelet-HTPR may predict risks of recurrent vascular events/outcomes in CVD patients. Given the heterogeneity between studies, further prospective, multi-centre studies are warranted.

The interaction of bacterial pathogens with platelets.

In recent years, the frequency of serious cardiovascular infections such as endocarditis has increased, particularly in association with nosocomially acquired antibiotic-resistant pathogens. Growing evidence suggests a crucial role for the interaction of bacteria with human platelets in the pathogenesis of cardiovascular infections. Here, we review the nature of the interactions between platelets and bacteria, and the role of these interactions in the pathogenesis of endocarditis and other cardiovascular diseases.