Light Transmission Aggregometry Research Articles

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.

Low COAT platelets are frequent in patients with bleeding disorders of unknown cause (BDUC) and can be enhanced by DDAVP

Low COAT platelets are frequent in patients with bleeding disorders of unknown cause (BDUC) and can be enhanced by DDAVP

Abstract 485: Platelet Aggregation Is Increased In Psoriasis And Associated With Biomarkers Of Vascular Health

Background: Psoriasis is associated with vascular inflammation and increased cardiovascular (CV) risk. Platelet hyperactivity is implicated in impaired vascular health and CV disease (CVD). The association between platelet activity, psoriasis, and vascular health has not been fully explored. Objective: To measure platelet aggregation in patients with psoriasis and controls and explore the association with biomarkers of inflammation and vascular stiffness. Methods: Participants with psoriasis (n=33, age 51 ± 16 years, 61% male), affecting 7% ± 14 of their body surface area (BSA), were compared to healthy controls (n=15) (Table 1A). Platelet aggregation in response to adenosine diphosphate (ADP) agonists, epinephrine (Epi), and arachidonic acid (AA) was measured via light transmission aggregometry (LTA). Vascular stiffness was assessed by pulse wave velocity (PWV), a metrics of arterial stiffness and biomarker of CV risk. High-sensitivity C-reactive protein (hs-CRP) was measured in a clinical laboratory. Results: Enrolled psoriasis participants were older, were more frequently white, and had higher body mass index and elevated hs-CRP and PWV compared to controls (Table 1A). Platelet aggregation was higher in psoriasis patients after stimulation with ADP (p=0.01), Epi (p=0.05), and AA with ex vivo aspirin co-incubation (p=0.04) when compared with controls in multivariable models. Platelet aggregation to ADP trended to positive association with PWV (r=0.25, p=0.08), and hs-CRP (r=0.29, p=0.06) after adjustment for age and sex (Figure 1C). Conclusion: Platelet aggregation was increased in participants with psoriasis and trended towards positive association with biomarkers of vascular stiffness and inflammation. These findings have important implications for future clinical trials of targeting platelet activity to reduce CV risk in psoriasis.

Human platelet‐mediated transfer of cardioprotection by remote ischemic conditioning is abrogated by aspirin, but not by ticagrelor

BackgroundRepeated ischemia/reperfusion in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) protects against injury from sustained myocardial ischemia/reperfusion (I/R). Platelets are considered as mediators/targets of RIC´s protection. We have recently reported that infusion of washed platelets, taken from healthy volunteers after RIC, reduced infarct size (IS) in isolated rat hearts (FASEB Journal 2021; 35; S1; DOI: 10.1096/fasebj.2021.35.S1.03444). Anti‐platelet drugs are potential confounders of RIC.AimTo study the impact of anti‐platelet drugs, the cyclooxygenase inhibitor aspirin and the P2Y12 inhibitor ticagrelor, on the platelet‐mediated transfer of cardioprotection.MethodsWe included 18 healthy volunteers. As a contemporary control, volunteers were subjected to RIC through 3 x 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/ 5 min deflation. After at least 2 weeks, these volunteers were again subjected to RIC 3 hours after pretreatment with oral aspirin (500‐1000 mg) and again at least another 2 weeks later 2 hours after pretreatment with oral ticagrelor (180 mg). Venous blood samples were taken before RIC and 60 min after RIC, respectively. Washed platelets were prepared using apyrase, prostaglandin E1 and citrated Tyrode‐buffer. The platelet count was adjusted to 2.5 x 105platelets/µL. The functionality of washed platelets was tested in response to thrombin (1U/ml), aggregation measured with turbodimetric light transmission aggregometry and expressed as the increase in light transmission in percent of control. Platelet preparations with an aggregation <60% were discarded. Male Lewis rats were sacrificed, their hearts isolated and perfused at constant pressure with buffer. Washed platelets before and after RIC, respectively, were infused for 8 min (2.5 x 104 platelets/µL) into rat hearts before global 30 min/ 120 min I/R. IS was demarcated by triphenyl tetrazolium chloride staining and calculated in percent of ventricular mass.ResultsWe confirm, that infusion of platelets after RIC reduces IS in isolated hearts with global I/R (Figure). This platelet‐mediated transfer of cardioprotection was abrogated by aspirin‐pretreatment of the volunteers (Figure). Platelets before RIC from ticagrelor‐pretreated volunteers reduced IS per se; nevertheless, platelets after RIC further reduced IS (Figure).ConclusionHuman platelets serve as carriers for RIC’s cardioprotective signal through an aspirin‐sensitive and thus cyclooxygenase‐dependent mechanism. The P2Y12 inhibitor ticagrelor per se induces a cardioprotective platelet‐dependent signal, which is further enhanced by RIC. The precise signal transfer from the platelets to the myocardium and the nature of the platelet‐derived factors remain to be further identified.

Platelet Function Is Associated With Dementia Risk in the Framingham Heart Study.

BackgroundVascular function is compromised in Alzheimer disease (AD) years before amyloid and tau pathology are detected and a substantial body of work shows abnormal platelet activation states in patients with AD. The aim of our study was to investigate whether platelet function in middle age is independently associated with future risk of AD.Methods and ResultsWe examined associations of baseline platelet function with incident dementia risk in the community‐based FHS (Framingham Heart Study) longitudinal cohorts. The association between platelet function and risk of dementia was evaluated using the cumulative incidence function and inverse probability weighted Cox proportional cause‐specific hazards regression models, with adjustment for demographic and clinical covariates. Platelet aggregation response was measured by light transmission aggregometry. The final study sample included 1847 FHS participants (average age, 53.0 years; 57.5% women). During follow‐up (median, 20.5 years), we observed 154 cases of incident dementia, of which 121 were AD cases. Results from weighted models indicated that platelet aggregation response to adenosine diphosphate 1.0 µmol/L was independently and positively associated with dementia risk, and it was preceded in importance only by age and hypertension. Sensitivity analyses showed associations with the same directionality for participants defined as adenosine diphosphate hyper‐responders, as well as the platelet response to 0.1 µmol/L epinephrine.ConclusionsOur study shows individuals free of antiplatelet therapy with a higher platelet response are at higher risk of dementia in late life during a 20‐year follow‐up, reinforcing the role of platelet function in AD risk. This suggests that platelet phenotypes may be associated with the rate of dementia and potentially have prognostic value.

Ibrutinib Inhibits BMX-Dependent Endothelial VCAM-1 Expression In Vitro and Pro-Atherosclerotic Endothelial Activation and Platelet Adhesion In Vivo.

Inflammatoryactivation of the vascular endothelium leads to overexpression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), contributing to the pro-thromboticstate underpinning atherogenesis. While the role of TEC family kinases (TFKs) in mediating inflammatory cell and platelet activation is well defined, the role of TFKs in vascular endothelial activation remains unclear. We investigated the role of TFKs in endothelial cell activation in vitro and in a nonhuman primate model of diet-induced atherosclerosis in vivo. In vitro, we found that ibrutinib blocked activation of the TFK member, BMX, by vascular endothelial growth factors (VEGF)-A in human aortic endothelial cells (HAECs). Blockade of BMX activation with ibrutinib or pharmacologically distinct BMX inhibitors eliminated the ability of VEGF-A to stimulate VCAM-1 expression in HAECs. We validated that treatment with ibrutinib inhibited TFK-mediated platelet activation and aggregation in both human and primate samples as measured using flow cytometry and light transmission aggregometry. We utilized contrast-enhancedultrasound molecular imaging to measure platelet GPIbα and endothelialVCAM-1 expression in atherosclerosis-prone carotid arteries of obese nonhuman primates. We observed that the TFK inhibitor, ibrutinib, inhibited platelet deposition and endothelial cell activation in vivo. Herein we found that VEGF-A signals through BMX to induce VCAM-1 expression in endothelial cells, and that VCAM-1 expression is sensitive to ibrutinib in vitro and in atherosclerosis-prone carotid arteries in vivo. These findings suggest that TFKs may contribute to the pathogenesis of atherosclerosis and could represent a novel therapeutic target.

Triple apheresis platelet concentrate quality after pneumatic tube system, conveyor box, and courier transport: An observational study.

Background and AimsPlatelets are prone to activation from handling; they are therefore transported as gently as possible, most commonly by courier. Speedier methods like pneumatic tube system (PTS) transport could improve patient care but may subject platelets to mechanical stress. To test the impact of mechanical stress caused by transport, we compared a PTS with a conveyor box and courier transport on apheresis platelet function.MethodsFourteen apheresis platelet concentrate triple donations were analyzed by light transmission aggregometry (LTA), rotational thrombelastometry (ROTEM), and flow cytometry before and after indoor transport over 800 m by PTS, conveyor, and courier, respectively, while recording shocks and vibrations with a high‐frequency acceleration data logger. Shock index scores were calculated as shock intensity (g‐force) times frequency.ResultsThe shock index was 81 for courier, 6279 for conveyor, and 9075 for PTS. Flow cytometry revealed no significant difference in platelet surface expression of CD62p before (16%) and after transport via courier (15%), conveyor (14%), or PTS (16%). LTA with adenosine phosphate and thrombin receptor‐activating peptide‐6 resulted in comparable platelet aggregation for courier, conveyor, and PTS. ROTEM assays showed no relevant differences in coagulation time, clot formation time, and maximum clot firmness between transport modes.ConclusionThough the mechanical challenge was smallest with courier transport, there were no significant differences in platelet activation or aggregation between the three transport modes. These data contradict restrictions on the use of PTSs for platelet concentrate transport.

Galectin‐9 activates platelet ITAM receptors glycoprotein VI and C‐type lectin‐like receptor‐2

BackgroundPlatelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate‐binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets. ObjectiveIn this study, we investigated the role of galectin‐9 (Gal‐9) as a novel ligand for platelet glycoprotein VI (GPVI) and C‐type lectin‐like receptor 2 (CLEC‐2). MethodsPlatelet spreading, aggregation, and P‐selectin expression in response to Gal‐9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid‐phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal‐9 and GPVI, and immunoprecipitation for detecting CLEC‐2 phosphorylation. Wild‐type (WT), GPVI‐knockout (Gp6−/−), and GPVI and CLEC‐2‐double knockout (Gp6−/−/Gp1ba‐Cre‐Clec1bfl/fl) mice were used. ResultsWe have shown that recombinant Gal‐9 stimulates aggregation in human and mouse washed platelets dose‐dependently. Platelets from both species adhere and spread on immobilized Gal‐9 and express P‐selectin. Gal‐9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal‐9 stimulated tyrosine phosphorylation of CLEC‐2 and proteins known to lie downstream of GPVI and CLEC‐2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI‐deficient murine platelets exhibited significantly impaired aggregation in response to Gal‐9, which was further abrogated in GPVI and CLEC‐2‐double‐deficient platelets. ConclusionsWe have identified Gal‐9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC‐2.

Association of Genetic Variability in Selected Genes with Platelet Hyperaggregability and Arterial Thrombosis

Abstract Introduction: Inherited platelet hyperaggregability, so called “Sticky platelet syndrome” (SPS), is a prothrombotic platelet disorder. The syndrome contributes more often to arterial than venous thrombosis. The most common localization of arterial occlusion involves cerebral or coronary arteries. However, SPS may also lead to thrombosis in the atypical sites of the circulation. This qualitative platelet alteration causes platelet hyperaggregability after a very low concentration of platelet inducers – adenosine diphosphate (ADP) and/or epinephrine (EPI). The precise genetic background of the syndrome has not been defined. In the present study we aimed to determine the association between selected single nucleotide polymorphisms (SNPs) within genes for platelet endothelial aggregation receptor 1 (PEAR1) and murine retrovirus integration site 1 (MRVI1) and the risk for arterial thrombosis in patients with SPS. The products of these selected genes play an important role in platelet aggregation. Patients and methods: We examined 69 patients with SPS and a history of arterial thrombosis and 69 healthy blood donors who served as controls. SPS was confirmed by a light transmission aggregometry (LTA) according to the method and criteria described by Mammen and Bick. We assessed two SNPs within PEAR1 gene (rs12041331, rs1256888) and two SNPs within MRVI1 gene (rs1874445, rs7940646). Results: Selected PEAR1 and MRVI1 polymorphisms seem not to be a risk factor for the development of SPS as the syndrome with an arterial thrombosis phenotype. However, in the subgroup of SPS1 patients there was found a decreased frequency of the minor A allele of SNP rs12041331 in PEAR1 gene (borderline p value, p=0.061) that can be hypothesized as protective against arterial thrombosis. In the same SPS1 subgroup the haplotype TA in PEAR1 gene also showed a decreased frequency with a borderline insignificance (p=0.056). We can theorize also about its protective role in SPS1 patients. We did not confirm the protective effect of polymorphism (T/T of rs 12566888) in PEAR1 against arterial thrombosis in SPS patients and SPS subgroups. Conclusion: Our results support the idea that examined genetic variability of the selected SNPs in PEAR1 and MRVI1 genes is not associated with platelet hyperaggregability manifested as arterial thrombosis. The possible protective role of the minor A allele of SNP rs12041331 as well as a role of haplotype TA in PEAR1 gene related to the arterial thrombosis found in the subgroup of SPS1 patients needs to be verified in further research.

An optimized agonist peptide of protease-activated receptor 4 and its use in a validated platelet-aggregation assay

Protease-activated receptor 4 (PAR4) is a promising drug target to improve the efficacy/safety window of antiplatelet agents. The native peptide GYPGQV and the more potent peptide AYPGKF, are PAR4-specific activators. However, these PAR4 agonist peptides (APs) elicit an agonist response, for example, platelet aggregation, at concentrations of 50 to 1000 µM in platelet-function assays, thereby limiting their utility to monitor the pharmacodynamic effects of PAR4 antagonists over a wide concentration range. Improved pharmacodynamic assays are needed for clinical development of PAR4 antagonists. We attempted to identify potent PAR4 APs to aid development of robust assays for optimization of PAR4 antagonists. Using an AYPG-based biased phage display peptide library approach followed by chemical peptide optimization, A-Phe(4-F)-PGWLVKNG was identified. This peptide demonstrated an EC50 value of 3.4 µM in a platelet-aggregation assay, which is 16-fold more potent than AYPGKF. Using this new PAR4 AP, a platelet-rich plasma-aggregation assay using light-transmission aggregometry was developed and validated in a series of precision and reproducibility tests. PAR4 antagonist responses to PAR4 AP A-Phe(4-F)-PGWLVKNG (12.5 µM to 100 µM) were subsequently evaluated in this assay in vitro and ex vivo in a human study using BMS-986120, a PAR4 antagonist that entered clinical studies.

Effects of ticagrelor monotherapy vs. clopidogrel monotherapy on platelet reactivity: a randomized, crossover clinical study (SINGLE study)

Increasing clinical trials demonstrated that the discontinuation of aspirin while maintaining a P2Y12 inhibitor monotherapy could decrease the risk of bleeding without losing the antithrombotic effect. However, no data are available on the platelet reactivity of patients undergoing ticagrelor monotherapy vs. clopidogrel. Therefore, we performed this study to observe the efficacy of ticagrelor monotherapy vs. clopidogrel in Chinese patients with chronic coronary syndrome. This randomized, single-blinded, crossover trial enrolled 50 patients who were administered with ticagrelor (90 mg twice daily for 2 weeks) or clopidogrel (75 mg once daily for 2 weeks). Followed by a 2-week washout period, the two groups of patients underwent a crossover trial. Light transmission aggregometry (LTA) and thromboelastography (TEG) assays were used to test platelet reactivity. The platelet aggregation rate (PAgR) of ADP and AA was significantly lower with ticagrelor than clopidogrel (PAgR of ADP, 27.30% (7.30%-42.635%) vs. 35.55% (12.03%-69.25%), P = .0254; PAgR of AA, 77.80% (21.60%-86.43%) vs. 83.10% (67.13%-87.20%), P = .0400). There was no significant difference in PAgR of collagen and epinephrine between the two groups. The TEG assay showed that ADP and AA, which induced the inhibition of platelet aggregation, were significantly higher in the ticagrelor group than those in the clopidogrel group [ADP%, 69.00% (59.68%–88.95%) vs. 60.55% (35.98%–78.35%), P = .0020; AA%, 53.65% (22.75%–79.28%) vs. 15.15% (5.75%–70.25%), P = .0127]. High on-treatment platelet reactivity (HTPR) on ADP was 2.17% with ticagrelor and 19.57% with clopidogrel. HTPR on AA was 50.00% with ticagrelor and 69.57% with clopidogrel. Ticagrelor and clopidogrel caused the inhibition of ADP and AA-induced platelet aggregation. Moreover, ticagrelor monotherapy had a stronger inhibitory effect than clopidogrel monotherapy (except on collagen and epinephrine).

Chronic venous disease, platelet and haemostatic abnormalities contribute to the pathogenesis of pigmented purpuric dermatoses.

To investigate the aetiology of pigmented purpuric dermatoses (PPD). 63 patients with a provisional diagnosis of PPD were assessed. Skin biopsies were performed to confirm the clinical diagnosis. Haemostasis was assessed using platelet function analyser-100 (PFA-100), light transmission aggregometry (LTA), impedance aggregometry (Multiplate) and measurement of clotting times and clotting factors. Chronic venous disease (CVD) was assessed by duplex ultrasound. When not contraindicated, patients were advised to discontinue haemostatic-modifying drugs or supplements for 4weeks after which the laboratory measurements were repeated and the clinical resolution of PPD was assessed. Subsequently, a cohort of patients identified with CVD underwent endovenous interventions and further resolution of PPD was assessed. CVD was found in 48 patients (76.2%) while haemostatic abnormalities were found in 36 (57.1%). 30 patients (47.6%) had concurrent CVD and haemostatic abnormalities. Modifiable risk factors such as the intake of platelet inhibitors or other drugs and supplements such as fish oil were identified in 53 patients (84.1%). These could be ceased in 35 patients of whom 28 (80.0%) achieved either complete or partial resolution of PPD. Treatment of the underlying CVD was performed in 18 patients resulting in complete or partial resolution in 17 (94.4%). In seven patients (11.1%), no CVD or haemostatic abnormalities were identified, and the risk factors included dietary factors such as excessive caffeine or soft drink consumption. Haemostatic abnormalities and CVD contribute to the pathogenesis of PPD. Resolution of PPD in the vast majority of patients may be achieved by cessation of modifiable risk factors and in particular platelet-modifying drugs or supplements and treatment of the underlying venous disease.

Influence of cardiopulmonary exercise test on platelet function in patients with coronary artery diseases on antiplatelet therapy

BackgroundCardiac rehabilitation reduces mortality and morbidity rate of patients with coronary artery diseases (CAD); however, acute exercise stimulation may also increase the thrombotic risk through platelet activation. Studies on the effects of cardiac rehabilitation on platelet function have been sparse.MethodsA total of 28 patients (24 men and 4 women; average age = 54.6 ± 8 years old) with stable CAD were enrolled in this study and divided into Aspirin-treated (n = 11; Aspirin group) and dual-antiplatelet-treated group (DAPT group; n = 17). Symptom-limited cardiopulmonary exercise test (CPET) with a cycle ergometer was performed on all the patients. Before and after CPET, platelet function was evaluated using light transmission aggregometry and whole blood flow cytometry.ResultsAll patients completed the CPET without provoked cardiac events, and the mean value of peak oxygen uptake (Peak Vo2) was 19.3 ± 3 ml/(kg min). Prior to CPET, platelet aggregation was significantly suppressed in DAPT group compared to Aspirin group (43.0 ± 21.5 vs. 72.9 ± 7.5, p < 0.001). CPET promoted platelet aggregation in Aspirin group (72.9 ± 7.5 vs. 80.9 ± 7.6, p = 0.005) and DAPT group (43.0 ± 21.5 vs. 50.1 ± 20.9, p = 0.010), and platelet count was increased in Aspirin (210.9 ± 54.6 vs. 227.5 ± 58.1, p = 0.001) and DAPT group (217.5 ± 63.8 vs. 229.7 ± 63.7, p = 0.001). However, the expression levels of CD62p and PAC-1 were not affected by CPET in both groups.ConclusionSymptom-limited CPET enhanced platelet aggregation in patients with CAD despite treatment with antiplatelet, mainly via platelet count augmentation, but not through single platelet activation.Trial registration: Effects of high intensity interval training versus moderate intensity continue training in cardiac rehabilitation on platelet function of patients with coronary heart diseases: a exploratory randomized controlled trial.ChiCTR-INR-17010717. Registered 23 February 2017, https://www.chictr.org.cn/edit.aspx?pid=18206&htm=4.

Variation of platelet function in clinical phenotypes of acute venous thromboembolism – Results from the GMP‐VTE project

BackgroundThe role of platelets in the pathogenesis of venous thromboembolism (VTE) is receiving increasing attention; however, limited information is available on platelet function in the acute phase of the disease. ObjectiveTo characterize platelet function according to VTE phenotypes. Patients/MethodsIn total, 154 subjects (isolated pulmonary embolism [iPE], n = 28; isolated deep vein thrombosis [iDVT], n = 35; DVT+PE, n = 91) were included. In this study platelet function analyzer (PFA)‐200, light transmission aggregometry (LTA), thrombin generation (TG) in presence (PRP) and absence (PFP) of platelets and platelet flow cytometry were investigated. LASSO regression was used to select clinical and platelet biomarkers that distinguish between VTE phenotypes. ResultsPFA‐200 results did not differ between VTE phenotypes. LTA from DVT+PE subjects showed lowest maximum aggregation after epinephrine and adenosine diphosphate compared to iPE and iDVT. Lower % of PAC‐1‐positive platelets after in‐vitro trigger were present in DVT+PE and iPE compared to iDVT. TG in PRP had lower peak height and velocity in DVT+PE and iPE against iDVT. The results of LASSO regression for the distinction between DVT+PE vs iDVT identified 18 variables (AUC =0.93) of which 72% were platelet biomarkers. For distinction between iPE and iDVT, 10 variables were selected (AUC = 0.96) of which 50% were platelet‐related. Obesity was the only variable weakly discriminating between DVT+PE vs iPE (AUC = 0.66). ConclusionThis explorative study suggests an important distinction between PE‐related phenotypes and iDVT when considering clinical and platelet function data. Lower platelet‐dependent TG along with reduced platelet reactivity suggest higher platelet degranulation in PE‐dependent phenotypes compared to iDVT.

Stability and utility of flow cytometric platelet activation tests: A modality to bridge the gap between diagnostic demand and supply

Light transmission aggregometry (LTA) is the gold standard for the diagnosis of platelet function disorders (PFDs). The requirement of customized aggregometer, large blood volume, normal platelet count and processing within 4 hours of venipuncture for LTA makes platelet function testing inaccessible to wider population. Flow cytometric platelet activation test (PACT) may overcome these limitations. This study compares the performance of PACT with LTA, characterizes diagnostic patterns of PFDs on PACT and assesses the stability of PACT beyond 4 hours of venipuncture in controls (n = 5) at different temperature conditions. LTA and PACT were performed in 121 healthy controls and 66 patients with suspected PFD. PACT had excellent agreement (kappa = 0.93) with LTA and 94.1% sensitivity, 98.5% specificity. PACT had distinct patterns in Bernard Soulier Syndrome (n = 10), Glanzmann Thrombasthenia (n = 24), δ-granule disorder (n = 7), and other PFDs (n = 12). PACT could assess platelet function in patients (14%) with thrombocytopenia/lipemia wherein LTA was inconclusive. PACT was stable up to 24 hours in samples stored/transported at 2–8◦C. The results of utility and stability are only valid for the specific markers, agonist concentrations, and conditions investigated in this paper. PACT is a useful modality for the diagnosis of PFD, especially in children, thrombocytopenia cases or in the setup where an aggregometer is not readily available.

Canine Cancer Cells Activate Platelets via the Platelet P2Y12 Receptor

In addition to their well-known functions in haemostasis, anucleated platelets have a critical role in cancer biology. Many human and non-human cancer types can directly interact with and activate platelets, promoting cancer malignancy and progression. Although naturally occurring canine neoplastic diseases mimic the biologically complex conditions of human cancers more closely than laboratory-bred mice, studies evaluating the relationship between cancer cells and platelets in dogs are scarce, and the effects of tumour cells on platelets in these animals are unknown. To evaluate whether cancer cells could activate canine platelets, we assessed the response of platelet-rich plasma to cultured canine cancer cells using light transmittance aggregometry. Similar to human and murine cancer cell research, we demonstrated that both canine osteosarcoma and mammary carcinoma cells activated canine platelets invitro, resulting in platelet aggregation. The degree of aggregation was most pronounced at a cancer cell to platelet ratio of 1:200 for most cell lines. Mechanistic studies revealed that the platelet adenosine diphosphate (ADP) receptor P2Y12 is essential for canine platelet aggregation induced by canine cancer. ADP receptor blockage on platelets inhibited >50% of cancer cell-induced maximum platelet aggregation in all cell lines evaluated. As in other species, our results suggest that canine cancers may activate canine platelets invivo. This mechanism is likely relevant for the biology and progression of cancer in the dog.

Pharmacodynamic Profiles of Dual-Pathway Inhibition with or without Clopidogrel versus Dual Antiplatelet Therapy in Patients with Atherosclerotic Disease

Inhibition of thrombin-mediated signaling processes using a vascular dose of rivaroxaban in adjunct to antiplatelet therapy, known as dual-pathway inhibition (DPI), reduces atherothrombotic events in patients with stable atherosclerotic disease. However, there are limited data on the pharmacodynamic (PD) effects of this strategy and how it compares to standard dual antiplatelet therapy (DAPT). This investigation was conducted in selected cohorts of patients (n = 40) with stable atherosclerotic disease-enrolled within a larger prospective, open-label, parallel-group PD study-who were treated with either aspirin plus clopidogrel (DAPT), aspirin plus rivaroxaban 2.5 mg/bid (DPI), or DAPT plus rivaroxaban 2.5 mg/bid. Multiple PD assays providing a comprehensive assessment of markers of thrombosis were used. PD endpoints included platelet-mediated global thrombogenicity measured by light transmittance aggregometry (LTA) following stimuli with CATF (collagen-related peptide + adenosine diphosphate [ADP] + tissue factor [TF]), markers of P2Y12 reactivity, markers of platelet aggregation using LTA following several stimuli (arachidonic acid, ADP, collagen, TF, and thrombin receptor-activating peptide [TRAP]), thrombin generation, and thrombus formation. There was no difference in platelet-mediated global thrombogenicity between groups. Rivaroxaban significantly reduced thrombin generation and was associated with a trend toward reduced TF-induced platelet aggregation. Clopidogrel-based treatments reduced markers of P2Y12 signaling and TRAP-induced platelet aggregation. There were no differences between groups on markers of cyclooxygenase-1-mediated activity. Compared with DAPT, DPI does not result in any differences in platelet-mediated global thrombogenicity, but reduces thrombin generation. These PD observations suggest that modulating thrombin generation-by means of factor Xa inhibition-in adjunct to antiplatelet therapy provides effective antithrombotic effects, supporting the efficacy and safety findings of a DPI strategy observed in clinical trials.

A Comparative Assessment Study of Known Small-molecule GPVI Modulators.

The GPVI platelet receptor was recently validated as a safe antiplatelet target for the treatment of thrombosis using several peptidic modulators. In contrast, few weakly potent small-molecule GPVI antagonists have been reported. Those that have been published often lack evidence for target engagement, and their biological efficacy cannot be compared because of the natural donor variability associated with the assays implemented. Herein, we present the first side-by-side assessment of the reported GPVI small-molecule modulators. We have characterized their functional activities on platelet activation and aggregation using flow cytometry as well as light transmission and electrical impedance aggregometry. We also utilized microscale thermophoresis (MST) and saturation transfer difference (STD) NMR to validate GPVI binding and have used this along with molecular modeling to suggest potential binding interactions. We conclude that of the compounds examined, losartan and compound 5 are currently the most viable GPVI modulators.

Serotonin-Affecting Antidepressant Use in Relation to Platelet Reactivity.

Depression is an independent risk factor of cardiovascular disease morbidity. Serotonin is a key neurotransmitter in depressive pathology, contained within platelets, and is a weak activator of platelets. Our study assessed the link between platelet reactivity traits, depression, and antidepressant (AD) use in a large population sample. Our study was conducted in the Framingham Heart Study (n = 3,140), and AD use (n = 563) and aspirin use (n = 681) were noted. Depression was measured using the Center for Epidemiological Studies‐Depression (CES‐D) survey. Platelet reactivity traits were measured across multiple agonists using five distinct assays. We utilized a linear mixed effects model to test associations between platelet traits and depression, adjusting for age, sex, aspirin use, and AD use. Similarly, we analyzed trait associations with any AD use, serotonin‐affecting ADs, and norepinephrine‐affecting ADs, respectively. There were strong associations with reduced platelet function and AD use, particularly with serotonin‐affecting medications. This included lower Optimul epinephrine maximal aggregation (P = 4.87E‐13), higher U46619 half maximal effective concentration (P = 9.09E‐11), lower light transmission aggregometry (LTA) adenosine diphosphate (ADP) final aggregation (P = 1.03E‐05), and higher LTA ADP disaggregation (P = 2.28E‐05). We found similar associations with serotonin‐affecting ADs in an aspirin‐taking subset of our sample. There were no significant associations between platelet traits and depression. In the largest study yet of AD use and platelet function we show that antidepressants, particularly serotonin‐affecting ADs, inhibit platelets. We did not find evidence that depressive symptomatology in the absence of medication is associated with altered platelet function. Our results are consistent with AD use leading to platelet serotonin depletions, decreased stability of platelet aggregates, and overall decreased aggregation to multiple agonists, which may be a mechanism by which ADs increase risk of bleeding and decrease risk of thrombosis.

A new decade awaits sticky platelet syndrome: where are we now, how do we manage and what are the complications?

ABSTRACT Introduction Sticky platelet syndrome is a less known platelet function disorder with a familiar occurrence and likely genetic background. Clinically, it is characterized by an increased risk of venous and arterial thromboembolic events and obstetric placenta-mediated complications. The increased aggregation after low-dose ADP and/or epinephrine is its distinctive laboratory feature. Though described for almost 40 years, several issues regarding its etiology, involved pathomechanisms, genetic background, optimal diagnostic and treatment approach remain controversial. Areas covered The work aims to summarize published studies, the actual definition of the syndrome, and point out its drawbacks. A literature search on Medline, Embase, and archives from EHA congresses was performed (terms: ‘sticky platelet syndrome’ – ‘platelet hyperreactivity’ – ‘platelet hyperaggregability’). The authors added in their unpublished data. The introductory overview of the present understanding is followed by the discussion of the pathophysiologic, diagnostic, and therapeutic problems. Expert Opinion Despite the growing evidence provided by case reports and series, the lack of robust studies limits the decision-making on diagnostics and management. The diagnostic issues, particularly the standardization of light transmission aggregometry, represent the crucial problem for the broader acceptance of the syndrome.