Light Transmission Aggregation Research Articles

Abstract 3097: Platelet COX-1 Dependent And Independent Heterogeneity In Response To Low-Dose Aspirin

Background: Platelets are key mediators of thrombosis and hemostasis. While aspirin decreases cardiovascular events in high-risk populations, its pharmacological and therapeutic effect is limited by heterogeneity in response and its effect on COX-1 dependent and independent pathways. We sought to investigate the heterogeneity of response of low-dose (81mg/day) aspirin. Methods: Following an overnight fast, 39 healthy controls had 4 weekly phlebotomies and received 81 mg aspirin daily between weeks 3 and 4. At each collection, platelet activity was measured using light transmission aggregation (LTA) in response to arachidonic acid (AA [1.5 mM]; COX-1 dependent ) and epinephrine (2.0 μM), ADP (2.0 μM), and collagen (1.0 μM; COX-1 independent pathways). Results: Median age was 27 (21, 59) years, 48.7% were female, 53.9% were white, and 7.7% were Hispanic. Median platelet aggregation in response to AA (1.5 mM) was 90.9%, 91.6%, and 91.4% in weeks 1, 2, and 3, demonstrating a robust reproducible response. Baseline AA-induced platelet aggregation was modestly correlated with ADP- (r=0.28, p=0.054), collagen- (r=0.42, p=0.0036), and epinephrine-induced (r=0.32, p=0.029) platelet aggregation. Following 1 week of aspirin, AA-induced platelet aggregation decreased to 11.8% (p<0.0001). The median (interquartile range) platelet inhibition in response to AA was 87.3% (78.5%, 92.5%) demonstrating a robust COX-1 dependent response. Platelet inhibition in response to epinephrine (52.1% [46.4, 62.6]), collagen (79.5% [70.3, 87.4]), and ADP (29.8% [22.0, 36.0]), albeit statistically significant, was less robust. While platelet inhibition in response to AA was correlated to platelet inhibition in response to collagen (r=0.64 p=1.12E-5) and epinephrine (r=0.44, p=0.0052), there was no correlation in response to ADP (r=-0.04, p=0.81) induced platelet inhibition. Conclusions: Our findings demonstrate a robust COX-1 dependent aggregation response and a more heterogeneous COX-1 independent aggregation response to low-dose aspirin. The mechanism and clinical significance of these findings require further study.

Platelet Functional Testing Via High-Throughput Microtiter Plate-Based Assays.

Platelets play a critical role in hemostasis and thrombosis; therefore, in vitro assays that measure platelet reactivity are fundamental tools to gain insight into these physiologic processes, to diagnose platelet disorders, and to develop antithrombotic therapies. However, conventional platelet assays such as aggregometry, the clinical gold standard for assessing platelet function, are low throughput and require specialized equipment. Since platelets have a finite life span ex vivo, processes to miniaturize and multiplex assays allow a much broader overview of platelet function in significantly less time than conventional assays. Several groups have developed simplified, high-throughput approaches to quantify platelet activation with standard laboratory equipment to lower the barrier of entry to study platelet biology. This article describes a panel of optimized and validated high-throughput microplate assays to comprehensively assess platelet functionality, independently or in combination, to increase throughput and reduce costs. Specifically, following stimulation of platelets, a plate reader can be used to measure light transmission aggregation via absorbance; dense-granule secretion based on ATP-dependent luminescence generation; and cytosolic calcium levels with a cell-permeant, fluorescent Ca2+ -sensitive dye. Additionally, platelets are an easily accessible component of the blood that share signaling pathways with other cells, making them ideal for high-throughput drug screens. The highly adaptable and complementary assays presented in this article can be used to decipher the molecular mechanism underlying platelet activation or to identify novel inhibitors. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microtiter plate-based light transmission aggregometry Basic Protocol 2: Measuring dense-granule secretion in high-throughput microplate assays Basic Protocol 3: Microtiter plate-based calcium mobilization Support Protocol: Platelet isolation and enumeration.

Implication of inflammation on Coxsackie virus and Adenovirus receptor expression on cardiomyocytes and the role of platelets in patients with dilated cardiomyopathy

BackgroundCoxsackie Virus and Adenovirus Receptor (CXADR or CAR) is involved in the pathogenesis of inflammatory dilated cardiomyopathy (DCM). We aimed to examine the relationship of CAR expression on platelets and cardiomyocytes with virus persistence, local and systemic inflammation and platelet activity in patients with DCM. MethodsEndomyocardial biopsy (EMB) samples of 38 patients (mean age 39.5±11.3 years, 20 male) with DCM were analyzed for CAR expression, local inflammation grade by immunohistochemistry and virus persistence by real-time PCR. Platelet morphology was analyzed in all patients and 30 healthy subjects (HS) using scanning electron microscopy, platelet activity by light transmission aggregation, and CAR persistence by immunofluorescence. Platelets of 20 patients were analyzed for cytomegalovirus and herpes simplex virus 1-2 by immunofluorescence. Serum levels of tumor necrosis factor alpha (TNF α) and Interleukin-6 were assessed using ELISA in all studied subjects. ResultsCAR expression in EMB samples was related to the heart failure functional class and the level of IL-6. Platelets from DCM patients showed enhanced spontaneous and ADP induced aggregation. Platelets’ CAR expression was >4 fold higher in DCM than HS and was observed predominantly at sites of intercellular communications in microaggregates and leukocyte-platelet aggregates. CAR-positive patients showed significantly higher TNF-α and IL-6 serum levels in CAR-negative patients. Platelets of 6 (30%) DCM patients revealed the mature cytomegalovirus and herpes simplex viruses particles. ConclusionTight junction protein CAR may serve as a docking pin creating a new type of contact structure that could be responsible for signaling between neighboring cells in pathological conditions.

Diagnosis of Platelet Function Disorders: A Challenge for Laboratories.

In patients with normal plasmatic coagulation and bleeding tendency, platelet function defect can be assumed. Congenital platelet function defects are rare. Much more commonly they are acquired. The clinical bleeding tendency of platelet function defects is heterogeneous, which makes diagnostic approaches difficult. During the years, a large variety of tests for morphological phenotyping and functional analysis have been developed. The diagnosis of platelet function defects is based on standardized bleeding assessment tools followed by a profound morphological evaluation of the platelets. Platelet function assays like light transmission aggregation, luminoaggregometry, and impedance aggregometry followed by flow cytometry are commonly used to establish the diagnosis in these patients. Nevertheless, despite great efforts, standardization of these tests is poor and in most cases, quality control is lacking. In addition, these tests are still limited to specialized laboratories. This review summarizes the approaches to morphologic phenotyping and platelet testing in patients with suspected platelet dysfunction, beginning with a standardized bleeding score and ending with flow cytometry testing. The diagnosis of a functional defect requires a good collaboration between the laboratory and the clinician.

Intensified antiplatelet therapy in patients after percutaneous coronary intervention with high on-treatment platelet reactivity: the OPTImal Management of Antithrombotic Agents (OPTIMA)-2 Trial.

High on-treatment platelet reactivity (HOPR) is associated with increased risk of cardiovascular events in patients undergoing percutaneous coronary intervention (PCI). We randomised post-PCI patients with HOPR after 5days of standard dual antiplatelet therapy (DAPT) to intensified therapy with aspirin 100mg once daily in combination with either clopidogrel 150mg once daily, clopidogrel 75mg once daily plus cilostazol 100mg twice daily, ticagrelor 90mg twice daily, or standard therapy with clopidogrel 75mg once daily (STD) for 1month, after which all patients were switched to standard DAPT for a further 11months. The primary outcome was residual HOPR rate at 1month. We screened 1724 patients with light transmission aggregation studies and randomised 434 with HOPR. At 1month the proportion of patients with persistent HOPR was significantly lower in the intensified therapy groups compared with STD group. Compared to the group receiving STD therapy, those receiving intensified therapy had significantly lower rate of major adverse cardiovascular events (MACE) at both 1month and 12months with no significant increase in bleeding. In patients with post-PCI HOPR, 1month of intensified antiplatelet therapy provides greater platelet inhibition and improves outcomes without increasing bleeding. Clinical Trial Registration URL: http://www.clinicaltrials.gov; Unique Identifier: NCT01955200.

Glanzmann Thrombasthenia: Perspectives from Clinical Practice on Accurate Diagnosis and Optimal Treatment Strategies.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive disorder of fibrinogen-mediated platelet aggregation due to a quantitative or qualitative deficit of the αIIbβ3 integrin at the platelet surface membrane resulting from mutation(s) in ITGA2B and/or ITGB3. Patients tend to present in early childhood with easy bruising and mucocutaneous bleeding. The diagnostic process requires consideration of more common disorders of haemostasis and coagulation prior to confirming the disorder with platelet light transmission aggregation, flow cytometry of CD41 and CD61 expression, and/or exon sequencing of ITGA2B and ITGB3. Antifibrinolytic therapy, recombinant activated factor VII, and platelet transfusions are the mainstay of therapy, although the latter may trigger formation of anti-platelet antibodies in GT patients and inadvertent platelet-refractory disease. The management of these patients therefore remains complex, particularly in the context of trauma, labour and delivery, and perioperative care. Bone marrow transplantation remains the sole curative option, although the venue of gene therapy is being increasingly explored as a future alternative for definitive treatment of GT.

Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti‐platelet therapy

BackgroundEndothelium‐derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO‐sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache. ObjectiveWe investigated using GC‐activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti‐thrombotic effect of both drugs. MethodsIn vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride‐induced arterial thrombosis model were also performed. ResultsThe GC‐activator BAY‐70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra‐platelet cyclic nucleotide levels. Furthermore, mice administered sub‐maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow. ConclusionsUsing in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti‐platelet effects without altering blood flow. Therefore, modulation of intra‐platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti‐thrombotic regimen while minimizing vasodilator side effects.

Platelet preparation for function testing in the laboratory and clinic: Historical and practical aspects

Laboratory tests of platelet function are instrumental in studying platelet physiology and inherited or acquired platelet abnormalities. Light transmission aggregometry, developed in the early 1960s, is still considered the gold standard for the identification and diagnosis of platelet function disorders. Since then, novel techniques have been developed, including flow‐based assays and flow cytometry. In this tutorial, we describe the basic methodologies for the preparation of citrated platelet‐rich plasma and washed platelet suspensions and discuss their respective advantages and limitations as well as important factors to consider to perform high‐quality tests of platelet function. In addition, the methodologies of the main platelet function tests (light transmission aggregation, flow‐based assays, and flow cytometric assays) are described, and their respective strengths and limitations are discussed to assess various aspects of platelet biology.

Comparison of platelet function tests for the in vitro quality assessment of platelet concentrates produced under real-life conditions

Platelet quality in different platelet concentrates (PCs) has been the subject of several studies. Nonetheless, there is a lack of robust data on the correlation and agreement among platelet function tests as a prerequisite for the association of PC functionality in vitro with platelet function in vivo post PC transfusion. The purpose of our study was to correlate a larger panel of platelet function assays in PCs and to assess whether the methods agree sufficiently and can be used interchangeably. Twelve apheresis platelet concentrates in plasma (APC), 16 pooled platelet concentrates in plasma (PPC), and 12 PPC in T-sol (PPCA) were examined on days 1 and 4 after production. PCs were tested for platelet count, light transmission aggregation (LTA) induced by ADP, collagen, or TRAP; platelet ATP release induced by collagen; and spontaneous and ADP and TRAP-induced increase in CD62P and PAC1 expression measured by flow cytometry. All tests were performed in undiluted platelet-rich plasma, recalcified and mixed with an inhibitor of factor Xa and thrombin. Most platelet function parameters correlated significantly with each other, but agreement among methods was insufficient. A proper inverse correlation was observed between ADP-induced LTA and spontaneous platelet activation assessed by CD62P expression (r = −0.61, p < 0.0001). Spontaneous CD62P correlated also significantly with spontaneous PAC1 (r = 0.69, p < 0.0001) and inversely with TRAP-induced CD62P expression (r = −0.86, p < 0.0001). We found significant correlations among all flow cytometric assays measuring platelet CD62P and PAC1 expression induced by ADP or TRAP. Subsequent Bland Altman analysis revealed insufficient agreement between methods. With one exception (collagen-induced LTA compared with TRAP-induced LTA, percentage error = 16%) the limits of agreement expressed as percentage error exceeded the chosen acceptable difference of 30%.In APC, platelet count was 41% and 44% higher, respectively, than in PPC and PPCA (p < 0.0001). Spontaneous CD62P and PAC1 expression were significantly greater, and ADP-induced aggregation and agonist-induced increase in CD62P and PAC1 were significantly lower in PPCA compared to APC and PPC on day 4 of storage. ADP and TRAP-induced CD62P and PAC1 activatability fell significantly during storage between day 1 and day 4 in APC and PPCA, but not in PPC. In conclusion, different platelet function tests capture different aspects of platelet function and do not correlate and agree sufficiently to be used interchangeably.

Comparison of four methods to assess high-on platelet reactivity under P2Y12 receptor inhibitor

P2Y12 receptor inhibitors are antiplatelet agents commonly prescribed in the treatment of coronary artery disease. Their efficacy can be limited by high on-treatment platelet reactivity (HPR), which can be evaluated by different biological assays. Most commonly, HPR is evaluated by flow cytometric vasodilator-stimulated phosphoprotein-phosphorylation (VASP-P) assay, which can be time consuming. To evaluate the potential interest of novel technologies, we compared four different assays.Ninety patients receiving P2Y12 inhibitors were included. Four technologies were evaluated: the current standard test measuring VASP-P by flow cytometry, the historical reference test based on light transmittance aggregation (LTA), and two relatively novel techniques: whole blood multiple electrode aggregometry (MEA) and platelet function analyzer (PFA), which are less time consuming.The three latter tests were compared with the VASP-P assay as a reference using receiver operating characteristics (ROC) analysis: LTA has an excellent comparability with the VASP test (ROC AUC > 0.9); the other two tests (multiplate and PFA) have only satisfactory comparability (ROC AUC around 0.7) and therefore may not replace the VASP “gold standard” test, if importance is attached to a quantitative assessment of the substitution parameter of VASP. Nevertheless, if a binary approach of the anti-aggregation result is sought, then one can conclude that the three tests are equivalent since Cohen’s kappa coefficients are very close for the three tests (k = 0.548 for LTA; k = 0.554 for MEA; k = 0.570 for PFA/P2Y), and a similar proportion of patients are misclassified (15% for LTA, 14% for MEA, and 13.6% for PFA). Discriminant factor analysis using all the parameters provided by each test did not improve the diagnostic performance of MEA or PFA.In conclusion, only LTA shows a good comparability to the VASP assay using ROC curve analysis, probably because misclassified patients have results close to the cutoff values. All three tests have moderate agreement regarding the classification of patients as responders to P2Y12 inhibition.

Factors associated with high probability of target blood pressure non-achievement in hypertensive patients

One of the topic issue of modern cardiology is factors of target blood pressure level non-achievement clarifying due to a better understanding how we can reduce cardiovascular complications. The aim of the study is to determine the factors of poor blood pressure control using the ambulatory blood pressure monitoring parameters and adenosine 5'-diphosphate-induced platelet aggregation parameters in patients with arterial hypertension. Material and methods. The study involved 153 patients with essential hypertension (EH) stage II, II degree. The ambulatory blood pressure monitoring (ABPM) was performed in patients during at least two of first-line antihypertensive drugs in optimal daily doses usage by the ABPM bifunctional device (Incart, S.-P., R.F.). Platelet aggregation was carried out using light transmittance aggregation by optical analyzer (Solar, R.B.) with adenosine 5'-diphosphate (Sigma-Aldrich) at final concentration of 10.0 × 10 -6 mol / L. The first group were inadequately controlled essential hypertensive individuals with high systolic or/and diastolic BP level according to the ABPM results, and the second one were patients with adequately controlled EH. Groups of patients were comparable in age (60.39 ± 10.74 years vs. 62.80 ± 9.63; p = 0.181, respectively). In the group of EH patients who reached the target level of blood pressure, women predominated (60% vs. 39.81%; p = 0.021, respectively). We used the binary logistic regression analysis to determine the predictors of target blood pressure level poor reaching using ABPM and platelet aggregation parameters. Results According to the univariate logistic regression analysis, the dependent factors influencing the target blood pressure level poor reaching are the average diurnal diastolic blood pressure (DBP) (OR = 44.8); diurnal variability of systolic blood pressure (SBP) (OR = 4.4); square index of hypertension for diurnal periods SBP (OR = 318.9); square index of hypertension for diurnal periods DBP (HS = 36,6); the average pulse blood pressure (OR = 65.9), the rate of morning rise of SBP (OR = 3.6), and the maximum aggregation time of 5-minute ADP-induced platelet aggregation (OR = 3.5). Independent factors influencing the target blood pressure level pure reaching are the average diurnal DBP (OR = 47.8); variability of diurnal SBP (HS = 4.6); the maximum aggregation time of ADP-induced platelet aggregation (OR = 3.4). Female sex is associated with the best prognosis for the target level of BP reaching (OR = 0.4; 95% CI = 0.2‒0.8; p = 0.012). Conclusions . The independent factors influencing the target blood pressure level poor reaching in patients with EH are two ABPM parameters (the average diurnal diastolic blood pressure of more than 76 mm Hg and the variability of SBP diurnal more than 17 mm Hg); and one ADP-induced platelet aggregation parameter (the maximum aggregation time more than 143 seconds). The female sex is associated with better prediction of achieving the target level of blood pressure.

Screening for platelet function disorders with Multiplate and platelet function analyzer

Light transmission aggregation (LTA) is the gold standard for the diagnosis of platelet function disorders (PFDs), but it is time-consuming and limited to specialized laboratories. Whole-blood impedance aggregometry (Multiplate) and platelet function analyzer (PFA) may be used as rapid screening tools to exclude PFDs. The aim of this study is to assess the diagnostic performance of Multiplate and PFA for PFDs, as detected by LTA.Data from preoperative patients, patients referred to the hematologist for bleeding evaluation, and patients with a diagnosed bleeding disorder were used. PFDs were defined as ≥2 abnormal LTA curves. Diagnostic performance of Multiplate and PFA for detecting PFDs was expressed as sensitivity and specificity. The ability of Multiplate agonists and PFA kits to detect corresponding LTA curve abnormalities was expressed as area under the receiver operating characteristic curve.Prevalence of PFDs was 16/335 (4.8%) in preoperative patients, 10/54 (18.5%) in referred patients, and 3/25 (12%) in patients with a diagnosed bleeding disorder. In preoperative and referred patients, the sensitivity of Multiplate and PFA for detecting mild PFDs varied between 0% and 40% and AUCs for detecting corresponding LTA curve abnormalities were close to 0.50. In patients with a diagnosed bleeding disorder, both assays could detect Glanzmann thrombasthenia (GT) with sensitivity of 100% and AUCs of 0.70–1.00.Multiplate and PFA cannot discriminate between preoperative and referred patients with and without mild PFDs, meaning that they cannot be used as screening tests to rule out mild PFDs in these populations. Both Multiplate and PFA can detect GT in previously diagnosed patients.

Platelet inhibition during ticagrelor monotherapy versus ticagrelor plus aspirin in patients with coronary artery disease (TEMPLATE study): study protocol for a randomised controlled trial

BackgroundDual antiplatelet therapy (DAPT) with aspirin (ASP) and a P2Y12 blocker is currently standard care after percutaneous coronary intervention (PCI) with stent insertion, and aims to inhibit platelet function in order to prevent stent thrombosis. The P2Y12 blocker ticagrelor (TIC) has greater antiplatelet effect than the previously used members of this class, such as clopidogrel. In healthy volunteers, TIC is sufficient to cause strong platelet inhibition, with little additional effect from ASP. Omission of ASP may improve the safety of antiplatelet regimes by reducing bleeding. However, the effect of single antiplatelet treatment with TIC, compared to DAPT with TIC + ASP, has not been studied in detail in patients with coronary artery disease.MethodsTo compare TIC with TIC + ASP, we have initiated a single centre, open-label randomised controlled trial (TEMPLATE study) in adults receiving DAPT following PCI with a sample size of 110 patients. Patients are invited to join the study when, as part of standard care, they are due to switch from DAPT (ASP + any P2Y12 blocker) to single antiplatelet treatment with ASP alone after 6–12 months. Patients are randomised to receive either TIC or TIC + ASP for 4 weeks. All patients then revert to standard care with ASP alone. Blood samples and clinical data are collected at three study visits: at baseline during treatment with ASP + any P2Y12 blocker (visit 1); approximately 4 weeks after visit 1 during treatment with either TIC or TIC + ASP (visit 2); and approximately 8 weeks after visit 1 when treatment has reverted to ASP alone (visit 3). The primary outcome is the extent of platelet inhibition, measured by light transmission aggregation, flow cytometry, flow chamber and plasma biomarker tests. The primary analysis will compare the extent of platelet inhibition between the TIC and TIC + ASP groups at visit 2, adjusted for baseline platelet reactivity. Secondary analyses will compare the extent of platelet inhibition at visit 2 with that at visit 3.DiscussionThis is the first study to compare in detail the extent of platelet inhibition in patients who are receiving TIC compared with TIC + ASP. The study findings will complement larger-scale trials of the clinical efficacy and safety of TIC compared to TIC + ASP.Trial registrationISRCTN registry, identifier ISRCTN84335288. Registered on 23 June 2014.

Indicators of ADP-induced platelet aggregation in patients with hypertension complicated by ischemic hemisphere stroke

Arterial hypertension is one of the most significant factor in the development of cardio-vascular complications in patients due to elevated blood pressure, maintenance of disorders of endothelial function and coagulation system, as well as changes in platelets function. The purpose of the work was to determine the characteristics of ADP-induced platelet aggregation in hypertension patients with ischemic hemisphere stroke. Material and methods. The enrolled patients (n =152) were divided into 3 groups. The first one included 38 patients (an average age of 64 [57−72] years, (male 35%)) with arterial hypertension (AH) who reached the target level of blood pressure (according to the «office» measurements and ambulatory blood pressure monitoring). The second group included 96 AH patients aged 60 [64−71] years, (62% men) who did not reach the target level of blood pressure. And the third group involved 18 patients aged 66 [66−72] years (male 56%) with AH complicated by ischemic hemisphere stroke. Platelet aggregation was performed using light transmittance aggregation by optical analyzer (Solar, R.B.) with adenosine 5'-diphosphate (Sigma-Aldrich) at final concentration of 10.0 × 10 6 mol / L. Results . There were significantly higher rates in hypertensive patients with ischemic hemisphere stroke of the maximum degree of platelet aggregation in 10 minutes (T max, 10min ) by 18 % (78 [78−100] % vs. 66 [57−83] %, respectively, U = 554.0; p = 0.015), the maximum aggregation time (t Tmax, 10min ) by 34 % (78 [78−100] s vs. 66 [57−83] s, respectively, U = 554.0, p = 0.015). The degree of platelet aggregation at the end of the recording time (T 10min ) and the area under the curve of the end of the 10 min. (AUC 10min ), respectively, by 19.4 % and 15 % than in AH patients who have not reached the target level of blood pressure. Conclusions . Hypertensive patients who did and did not reach the target level of blood pressure showed no significant difference in analyzed parameters of ADP-induced platelet aggregation. However, there was approximately twofold increase in deaggregation (K desagg, 10min ) among patients who did not reach the target level of blood pressure compared with those who reached it. Meanwhile, hypertensive individuals with ischemic hemisphere stroke had significantly higher ADP−induced aggregation parametrs as percentage of aggregation during 10 min. (T 10min ), the area under the aggregation curve at the end of 5th (AUC 5 min ) and 10th min. (AUC 10min ) in comparison to hypertensives who did not reach the target blood pressure level. The influence of drugs on the studied parameters of ADP-induced platelet aggregation in patients is not established.

Comprehensive Platelet Phenotypic Laboratory Testing and Bleeding History Scoring for Diagnosis of Suspected Hereditary Platelet Disorders: A Single-Institution Experience.

Patients with hereditary/congenital platelet disorders (HPDs) have a broad range of clinical manifestations and laboratory phenotypes. We assessed the performance characteristics of the International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) and clinically validated platelet laboratory tests for diagnosis of HPDs. The records of 61 patients with suspected HPDs were reviewed and ISTH-BAT scores calculated. Nineteen (31%) patients had thrombocytopenia, and 46 (75%) had positive ISTH-BAT scores. Thirteen and 17 patients had prolonged PFA-100 (Dade Behring, Miami, FL) adenosine diphosphate and epinephrine closure times, respectively. Twenty-two had abnormal platelet light transmission aggregation. Twenty-four had platelet transmission electron microscopy (PTEM) abnormalities (10 dense granule deficiency, 14 other ultrastructural abnormalities). Positive ISTH-BAT scores were associated with thrombocytopenia (P < .0001) and abnormal PTEM (P = .002). Twenty-three patients had normal results. ISTH-BAT identified patients with suspected HPDs but lacked a robust association with laboratory abnormalities. Despite comprehensive laboratory testing, some patients may have normal results.

Adrenalin-induced platelet aggregation in essential hypertensive patients in the acute phase of hemispheric ischaemic stroke: LTA results

Acquired platelet disorders caused by pathological state or medical induced have pure knowledge according to the experts, especially in essential hypertensive (EH) patients with high and very high cardiovascular risk due to the association with an increased risk of death as stroke and myocardial infarction.The aim of the study was to determine adrenalin-induced platelet aggregation parameters in essential hypertensive patients in acute phase of hemispheric ischaemic stroke.Materials and methods. A total of 98 study participants were analyzed. The first group (n=46; aged 63 (56−71); 38 % man) were well-controlled essential hypertensives according to the «office» and ABMP results, and the second one (n=52; aged 63 (56−71); 66 % man) were EH patients with an acute hemispheric ischaemic stroke (HIS). Light transmittance aggregation (LTA) was conducted with adrenaline (5 μmol/l)).We analyzed the percentage and time of maximal aggregation monitored for 5 and 10 min (Tmax,5minand tTmax,5min, respectively); the percentage of aggregation in 1 min, 2 min, 3 min, 5 min, 10 min (T1min, T2min, T3min, T5min and T10min, respectively); area under the curve at the end of 1, 5 and 10 min periods (AUC1min, AUC5min and AUC10min, respectively); the slope of aggregation (∆T, %/min.); deaggregation at the end of 5 and 10 min periods (Kdesagg,5min andKdesagg,10min,respectively); LTA tracings.Results. There was a decrease of Tmax,5min by 57 % (р=0,028); T1min by 71 % (р=0,002), T2min by 65 % (р=0,002), T3 min by 60 % (р=0,004) and T5min by 59 % (р=0,027); ∆T by 62 % (р=0,002); AUC1min by 84 % (р=0,002), AUC5min by 62 % (р=0,004), AUC10min by 60 % (р=0,018) in group of EH patients with an acute HIS in comparison with the group of well-controlled EH patients. Moreover, as analyzed LTA tracing only so called «considerably decreased» (P=0,020) and «fused» patterns of aggregation (P=0,008) achieved significance in groups.Conclusion. Light transmission aggregometry (optical) with adrenalin (μmol/л) provides the quantitative and qualitative information about the platelet function ability to aggregate in EH patients with and without acute complications. In EHs with an acute HIS there is 57-84% decrease of quantitative parameters in comparison to the same data in well-controlled EHs. Furthermore, they have aggregation patterns which show the different level of the platelet aggregation process reduction, and also there are less so called «fused» patterns of aggregation. We suggested that there is because of treatment which influences substantially the platelet function.

Laboratory Testing Protocols for Heparin-Induced Thrombocytopenia (HIT) Testing.

Heparin-induced thrombocytopenia (HIT) represents a significant high morbidity complication of heparin therapy. The clinicopathological diagnosis of HIT remains challenging for many reasons; thus, laboratory testing represents an important component of an accurate diagnosis. Although there are many assays available to assess HIT, these essentially fall into two categories-(a) immunological assays, and (b) functional assays. The current chapter presents protocols for several HIT assays, being those that are most commonly performed in laboratory practice and have the widest geographic distribution. These comprise a manual lateral flow-based system (STiC), a fully automated latex immunoturbidimetric assay, a fully automated chemiluminescent assay (CLIA), light transmission aggregation (LTA), and whole blood aggregation (Multiplate).

Abstract 38: Integration of Different Platelet Activity Assays Into a Reproducible Platelet Score

Background: While platelets play a key role in the pathogenesis of atherothrombosis, measuring platelet activity remains a research tool. We sought to 1) develop an integrated platelet score (PS) combining multiple platelet activity measurements, 2) assess its reproducibility over time, 3) investigate its correlation pre- and post-aspirin, and 4) compare subjects with cardiovascular disease (CVD) versus healthy controls. Methods: Following an overnight fast, 48 healthy controls had 4 weekly phlebotomies and received 81 mg aspirin daily between weeks 3 and 4. At each collection, platelet activity was measured using light transmission aggregation (LTA) in response to submaximal epinephrine, ADP, collagen, arachidonic acid (AA) and saline, and whole blood impedance aggregation in response to collagen, ADP and AA. The PS was derived using principal component analysis from healthy controls off antiplatelet therapy. A cohort of 124 subjects with CVD on aspirin was evaluated as a comparator group. Results: A PS was constructed using 12 measurements from LTA and whole blood aggregation, which accounted for 75% of the variation. The PS significantly correlated with all the platelet measurements used to construct the score (P&lt;0.001 for each measurement). During weeks 1 to 3, the weekly correlation and reproducibility were excellent (r&gt;0.77, ICC 0.82, P&lt;.0001). After 1-week of aspirin therapy, the post-PS score shifted to lower values (P&lt;0.001) and significantly correlated with the pre-PS score (r=0.64, p&lt;.001). Among controls, female sex was the only variable associated with higher PS after multivariable adjustment (P=0.01). The PS was significantly higher in subjects with CVD (-123.8 [-151.5, -83.23]) versus healthy controls (-101.4 [-140.3, -62.03]; P=0.04). Conclusion: Platelet activity can be summarized into a PS that is reproducible over time, significantly associated with platelet activity measured on aspirin, and higher in women and subjects with CVD. Future studies are needed to investigate this score in other high-risk phenotypes and to determine its association with incident cardiovascular events.

Value of Platelet Esoteric Testing in Laboratory Diagnosis of Platelet Disorders: A Single Center Experience

Introduction Patients with inherited and acquired platelet disorders (PD) frequently present with thrombocytopenia and/or mucocutaneous bleeding. Diagnosis of PDs relies on both clinical and laboratory investigation, which encompasses both routine and esoteric platelet testing. The former includes platelet indices, light transmission aggregation (LTA) and platelet function analyzer (PFA-100). The latter includes platelet glycoprotein (GP) assessment by flow cytometry and platelet transmission electron microscopy (PTEM). Several recent studies have demonstrated limited sensitivities of routine platelet testing. It is uncertain if a combination of both routine and esoteric testing can vastly improve the sensitivity. Our goal is to assess the value of platelet esoteric testing and the ultimate sensitivity of laboratory platelet testing in diagnosing PDs. Methods Patients (between year 2012 and 2015) with a known or suspected inherited or acquired platelet disorders were recruited for platelet routine and esoteric testing. Patients' clinical features including ISTH bleeding assessment tool (BAT) scores, platelet count, LTA, PFA-100 ADP (CADP) and epinephrine (CEPI) cartridge closure times, GP expression by flow cytometry and PTEM were performed. All clinical and laboratory variables were collected and statistically analyzed. Results A total of 69 patients (51 females; median age 40 years; range: 8 months to 76 years) were enrolled to this cohort study. By retrospective chart review, 34 patients (49%) had a positive ISTH-BAT score (range 0 to 16). The most common bleeding manifestations (on any degree of severity) were cutaneous bleeding present in 53% of patients (n=37), menorrhagia in 47% of females (n=24), epistaxis in 36% (n=25) and postoperative bleeding in 35% (n=24). Joint, muscle and CNS hemorrhage were the least frequent with 7%, 6% and 1% of patients reporting bleeding in these sites. A total of 22 patients (32%) had thrombocytopenia at the time of their evaluation. Fifty-eight patients (84%) had platelet aggregation studies and of those 15 (26%) had an abnormal study. PFA-100 was performed in 57 patients (83%) of whom 42% (n=24) had prolonged closure times with either ADP or epinephrine. PTEM was performed on all samples. Twenty-seven patients (39%) had abnormal platelet structure including 17 patients who had dense granule deficiency. GP by flow cytometry was performed in 41 patients (59%) and abnormal glycoprotein expression was detected in 6 patients (15%). Of the 55 patients who had all tests performed, 22 patients (40%) had completely normal results. Of the routine tests, only PFA-CEPI or -CADP prolongation is associated with the likelihood of a PTEM abnormality (P&lt;0.005). With the assistance of molecular testing, there were 2 cases of MYH-9 mutation-related platelet disorders, one of RUNX1 mutation-associated thrombocytopenia, one of York platelet syndrome, two of Bernard Soulier variants, one of Quebec syndrome and three of gray platelet syndrome or variants. Bleeding scores showed no statistically significant association with severity of any laboratory abnormalities. Conclusion In this cohort, the sensitivity of routine platelet testing in clinically suspected PDs is about 30-40%. Addition of platelet esoteric testing improves the sensitivity to 60%. However, the degree of platelet laboratory abnormalities do not predict severity of bleeding. The findings of this cohort underscore the importance of clinical assessment and both platelet routine and esoteric testing in investigation of patients with suspected PDs. Disclosures No relevant conflicts of interest to declare.

213 Synergy between Endothelial Mediators and P2Y12 Receptor Blockade as a Potential Determinant in Tailoring Anti-Platelet Therapy

<h3>Introduction</h3> Following ACS or PCI, P2Y12 receptor blockers such as clopidogrel or prasugrel are standardly prescribed alongside aspirin as dual anti-platelet therapy in a “one size fits all” approach, however recurrent thrombotic events occur. Despite the logical hypothesis that thrombotic risk should be associated with level of P2Y12 platelet inhibition, randomised control trials have repeatedly failed to show any clinical benefit of guiding anti-platelet therapy by platelet function tests (PFT). Whilst traditional PFT measure reactivity to ADP, they crucially ignore that P2Y12 receptor antagonists also produce powerful anti-thrombotic effects by potentiating the actions of the inhibitory endothelial mediators prostacyclin (PGI2) and nitric oxide (NO). <h3>Methods</h3> In vitro, IC50 curves produced from light transmission aggregation (LTA) traces for PGI2, NO and prasugrel active metabolite (PAM: 3 μM) were used to generate isobolograms. <i>Ex vivo</i>, healthy male volunteers (n = 8 each) received either prasugrel (10 mg) or aspirin (75 mg) for 7 days. Platelet-rich plasma (PRP) was obtained by centrifugation before and after treatment. Platelet responses to TRAP-6 amide (25 μM) or collagen (4 μg/ml) in the presence of PGI2 (1 nM) and/or the NO donor, DEA/NONOate (100 nM), or vehicle, were assessed by LTA, p-selectin binding by flow cytometry, and downstream cyclic nucleotide and VASP phosphorylation by immunoassay. <h3>Results</h3> Isobolographic analysis of <i>in vitro</i> studies showed P2Y12 blockade powerfully enhanced the synergy between NO and PGI2 (10 fold for TRAP-6). Ex-vivo platelet aggregation responses to TRAP-6 were unaffected by the addition of PGI2+NO prior to therapy. Aspirin monotherapy produced minimal inhibition of TRAP-6, though these became stronger in the presence of PGI2+NO (58%±8 to 28%±9; p &lt; 0.05). Inhibitory effects were observed with prasugrel in the presence of PGI2 and NO individually (control, 63 ± 3%; PGI2, 43 ± 6%; NO, 50 ± 5%), and these became much greater with the combination (PGI2+NO, 7%±3). P-selectin expression was reduced from pre-treatment (vehicle, 32 ± 6%) to (PGI2+NO, 11 ± 4%) post aspirin, and from (vehicle, 25 ± 6%) to (PGI2+NO, 2 ± 4%) following prasugrel. cAMP levels increased with PGI2+NO in the aspirin group (0.97 ± 0.06 vs. 1.50 ± 0.19) compared to (1.41 ± 0.07 vs. 3.4 ± 0.58) in the prasugrel group. <h3>Conclusions</h3> PGI2 and NO synergise with P2Y12 blockade to cause powerful platelet inhibition suggesting that an individual’s endothelial function and production of NO and PGI2 are central determents of the anti-thrombotic protection from P2Y12 receptor blocker therapy. Whilst a therapeutic window of platelet reactivity is an attractive concept our research suggests that endothelial function testing alongside PFT would permit better risk stratification. Our research also suggests therapeutically that enhancing endothelial-derived mediators or optimising downstream cyclic nucleotide signalling could reduce both thrombotic and bleeding risk.