Light Transmission Platelet Aggregometry Research Articles

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%.

Classic Light Transmission Platelet Aggregometry: Do We Still Need it?

For more than 50 years, light transmission aggregometry has been accepted as the gold standard test for diagnosing inherited platelet disorders in platelet-rich plasma, although there are other functional approaches performed in whole blood. In this article, several advantages and disadvantages of this technique over other laboratory approaches are discussed in the view of recent guidelines, and the necessity of functional assays, such as light transmission aggregometry in the era of molecular genetic testing, is highlighted.

An assessment of platelet response to ticagrelor in post-percutaneous coronary intervention patients using light transmission platelet aggregometry (LTA)

Background: Low on-treatment platelet reactivity (LTPR) or High on-treatment platelet reactivity (HTPR) with P2Y12 inhibitors is associated with bleeding (LTPR) or ischemic events (HTPR) in patients on dual antiplatelet therapy (DAPT) after percutaneous coronary intervention (PCI). Objective: Mapping of Indonesian patients’ platelet response secondary to ticagrelor and thus identifying either LTPR or HTPR. Method: During May – June 2019, 20 post-PCI patients on aspirin–ticagrelor combination were included. Light transmission aggregometry (LTA) for monitoring platelet function was used. Ticagrelor’s LTA &lt;40% is LTPR while &gt;70% is HTPR. Result: Patients were mostly male (18 patients) and aged between 40-73 years old with a history of diabetes and/or hypertension and smoking. About eight patients (40%) were LTPR and one (5%) patient was HTPR. Conclusion: A personalised DAPT strategy for preventing bleeding events, a de-escalation, might be valuable for Indonesian patients.

P1071: IMPACT OF ACETYLSALICYLIC ACID DOSAGE ON INCIDENCE OF THROMBOSIS AND MORTALITY IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS.

Background: Bcr/abl – negative myeloproliferative neoplasms (MPN) are associated with a higher risk of thrombosis and higher vascular mortality. Acetylsalicylic acid (ASA) used in prevention decreases risk of vascular events in patients with MPN. There is some evidence that ASA in dose 100 mg once daily results in incomplete inhibition of a platelet function over the whole 24-hour interval in most patients with essential thrombocythemia (ET). High on-treatment reactivity (HOTR) was corrected with dosing ASA twice daily. Evidence of clinical consequences of these findings has been missing. Aims: Authors compared thrombosis incidence, mortality and safety among cohorts of MPN patients with adequate on-treatment reactivity (AOTR) on ASA dosed once daily, twice daily and patients with HOTR. Methods: Response to ASA in patients with MPN was evaluated by a platelet light transmission aggregometry induced by arachidonic acid. AOTR was defined as maximal amplitude up to 20% just before the next dose of ASA. In patients with HOTR, ASA dosing was changed to twice daily with a dose from 50 mg step by step up to 150 mg till AOTR was reached. A few patients didn´t change their doses at their own decision despite HOTR. The patients were regularly followed-up. Incidence of thrombosis, adverse events and mortality were compared among the cohorts. Results: The platelet aggregation was tested in 69 patients with MPN on ASA. Patients with ET, polycythemia vera (PV), primary myelofibrosis, post-PV plus post-ET myelofibrosis and MPN not otherwise specified presented 16, 42, 36, 5 and 1%, respectively. The adequate responses (AOTR) were found in 57 (83%), cohort 1. The insufficient responses were found in 12 persons (17%), in 30% of patients on an enterosolvent form of ASA. In these 12 patients the dose of ASA was changed to 2x50mg in 4 and to 2x100mg in 1 patient (cohort 2) and remained unchanged in seven (cohort 3). There were no differences in frequency of risk factors besides higher frequency of diabetes mellitus in group 3 compared to group 1 (p=0,021). Non-adherence to regular administration of ASA was found in 10% of patients. The follow-up was 36,8 months. All-cause mortality was higher in the cohort 3 compared to the cohort 1 (p=0,009), see fig. 1, but thrombotic episodes incidence not. The cohort 2 had not different incidence of both thrombotic episodes and deaths compared to the cohort 1. No proximal gastrointestinal bleeding and other gastropathies occurred. There was no bleeding episode on ASA in dose of 2x50mg daily. A non-severe bleeding dependant on any ASA dose were found. Repeated measurement of platelet aggregation was done in 29 patients in median 49,5 months after previous measurements. There was no statistically significant difference of adequate/high platelet reactivity on ASA between the first and the last measurement. Image:Summary/Conclusion: The frequency of MPN patients with higher platelet reactivity on ASA was lower than in the most data in literature. Correction of an ASA dosage to twice daily reaching AOTR resulted in lower mortality. Other studies are required to confirm these data. Initial dose 2x50mg seems to be effective and safer than higher dosage. The long-term reproducibility of platelet aggregation testing results was preserved.

Platelet Aggregation Assays Do Not Reliably Diagnose Platelet Delta Granule Storage Pool Deficiency.

BackgroundPatients with platelet dysfunction disorders present with a variety of mucocutaneous bleeding symptoms including easy bruising, frequent epistaxis, bleeding gums upon tooth brushing and for women, heavy menstrual bleeding. Available laboratory assays to evaluate platelet function include the platelet function analyzer (PFA) and in larger centers with coagulation laboratories, light transmission platelet aggregometry (LTA) analyses. Both assays are known to have a number of limitations, especially in the diagnosis of platelet delta granule storage pool deficiency (δ-SPD). δ-SPD is an underdiagnosed condition caused by decreased numbers of platelet dense granules (DGs) and is best diagnosed by electron microscopy (EM). Patients with platelet δ-SPD have a decreased response to low levels of the agonist adenosine diphosphate (ADP) in the second wave of light transmittance with LTA or decreased ADP secretion by fluorescence lumiaggregometry. There are few reports that have evaluated patients with δ-SPD and their respective LTA results. One report published in 1987 described normal LTA assays in 23% of patients with δ-SPD; a more recent report described LTA as having the sensitivity to detect only about 52% of patients with δ-SPD. The purpose of our study was intended to review the LTA and EM results of patients suspected of having a platelet function disorder at our institution for comparison with previously published studies.MethodsOur study included 344 patients who had been evaluated by both LTA and whole mount EM. Aggregometry utilized five agonists: ADP, epinephrine, collagen, arachidonic acid, and ristocetin. DGs were enumerated in 100 whole-mounted platelets to determine a mean number of dense granules per platelet (DGs/PL).ResultsSeventy-seven percent of our patients were found to have δ-SPD (264/344); 68% (179/264) of these subjects had an abnormal platelet LTA. Thirty-two percent (85/264) of our patients had normal LTA results but were found to have δ-SPD with a mean of 2.54 ± 0.15 DG/PL (normal = 4 - 6 DG/PL).ConclusionThese data confirm previous reports suggesting the utilization of LTA alone in patients with histories of unexplained bleeding may miss the diagnosis of platelet δ-SPD. It is, therefore, prudent to assess platelet DG number by EM, especially if platelet LTA assessment is normal.

Platelet Function Tests and Inflammatory Markers for the Differentiation of Primary Thrombocytosis and Secondary Thrombocytosis

BACKGROUNDPrimary thrombocytosis (PT) that results from clonal bone marrow abnormality such as myeloproliferative neoplasm (MPN) can induce abnormal platelet function and acquired von Willebrand disease. Secondary thrombocytosis (ST) that may accompany inflammations, infections, malignancies, or stress is associated with elevated markers of the acute phase reactants, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). The aim of this study is to evaluate role of platelet function tests by light transmission platelet aggregometry (LTA), plasma von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity (vWF:RCo) and inflammatory markers (ESR, CRP) for the differentiation of PT and ST.METHODSThis was a prospective study in patients age 18 years or older with platelet counts higher than 450 x 109/L who admitted or attended the out-patient clinics in Chiang Mai University Hospital during November 2016 to June 2017. They were classified to PT from MPN according to WHO 2016 Classification and ST. Primary outcomes were sensitivity (Se) and specificity (Sp) of platelet function tests by LTA for the differentiation of PT and ST. Secondary outcomes were Se and Sp of ESR, CRP, vWF, and vWF:RCo for the differentiation of PT and ST as well as correlation between these markers and thrombohemorrhagic complications in patients with PT and ST. Categorical variables were compared using the Chi-squared test or Fisher exact test as appropriate. Continuous variables were compared by t-test or Wilcoxon rank-sum test (Mann-Whitney test). Level of significance was defined as p-value < 0.05.RESULTSThere were 49 patients enrolled in the study, mean age was 50.26 ± 18.26 years. Twenty-four patients (49%) had PT including 13 chronic myeloid leukemia (54%), 9 essential thrombocythemia (38%), 1 primary myelofibrosis (4%), and 1 polycythemia vera (4%). Twenty-five patients (51%) had ST including 10 malignancies (40%), 8 chronic infections (32%), 4 post-splenectomy (16%), and 3 autoimmune diseases (12%). Mean platelet count was higher in PT compared with ST (1083.79 ± 469.46 x 109/L and 716.28 ± 200.01 x 109/L respectively, p = 0.006). There was no difference in age and sex between groups. Abnormal LTA induced by collagen, arachidonic acid (AA), and epinephrine (Epi) were significantly presented in PT compared with ST (29%, 25%, and 50% respectively in PT; and 0%, 0%, 3% respectively in ST; p = 0.004, 0.010, and 0.004 respectively). The Se and Sp of collagen vs. AA vs. Epi-induced abnormal platelet aggregation for the differentiation of PT from ST were Se of 29.2% vs. 25% vs. 50%, and Sp of 100% vs. 100% vs. 88%, respectively. Mean ESR and CRP were significantly higher in ST (ESR 51.08 ± 22.38 mm/hr and CRP 56 ± 61.74 mg/L) compared with PT (ESR 16.30 ± 18.17 mm/hr and CRP 17.39 ± 37.96 mg/L; p < 0.001 and p = 0.006, respectively). The Se and Sp of ESR > 48 mm/hr in the differentiation of ST from PT were 76% and 91.7%, respectively while Se and Sp of CRP > 14.8 mg/L were 60% and 83.3%, respectively. No significant difference in proportion of patients with low vWF:Ag between groups (8.3% in PT and 0% in ST; p = 0.141) whereas PT group had significant higher proportion of patients with low vWF:RCo (45.8% in PT and 12.0% in ST; p = 0.009). The Se and Sp of abnormal vWF:RCo to differentiation of PT from ST were 45.8% and 88%, respectively. Thrombohemorrhagic complications at the time of diagnosis was 21% in PT while no complication in ST (p = 0.006). Risk factors of thrombohemorrhagic complications were platelet counts more than 1,569.8 x 109/L (p = 0.03) and Epi-induced abnormal platelet aggregometry (p = 0.02).CONCLUSIONAbnormal platelet function determined by LTA with collagen, AA, epinephrine had high specificity to differentiate between PT and ST. vWF:RCo, ESR and CRP levels might be helpful for the differentiation of PT and ST. PT has higher incidence of thrombohemorrhagic complication compare to ST and is correlated with high platelet counts and Epi-induced abnormal platelet aggregometry. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Compound Heterozygous ITGB3 Pathogenic Variants In A Patient With Glanzmann Thrombasthenia

Glanzmann Thrombasthenia is a rare platelet function disorder which is characterized by decreased expression and/or dysfunction of the platelet receptor αIIbβ3 (GPIIb/IIIa). The disease is due to alterations in ITGA2B or ITGB3 the genes encoding for the receptor subunits αIIb (GPIIb) and β3 (GPIIIa) and mostly inherited autosomal recessively. We report about a one-year old girl presenting with petechiae and hematomas shortly after birth. Platelet light transmission aggregometry was impaired after stimulation with ADP, arachidonic acid and collagen. Stimulation with ristocetin reached normal values, but showed an undulating course. Flow cytometry revealed severely decreased expression of CD41 (αIIbβ3). Molecular genetic analysis of the candidate genes and family genotyping identified two compound heterozygous variants in Exon 10 of ITGB3: c.1552C>T (p.Gln518*) and c.1639T>G (p.Cys547Gly). According to the guidelines of ACMG the variants were classified as pathogenic (Class 5). The nonsense variant c.1552C>T has not been reported before.

Time course of platelet activation following platelet-rich plasma preparation on automated platelet light transmission aggregometry using CS-2000i

Time course of platelet activation following platelet-rich plasma preparation on automated platelet light transmission aggregometry using CS-2000i

Platelet CD34 Expression and a Congenital Collar Bone Malformation Associated with a Partial CBFB Deletion in a Case with a Bleeding Disorder

Mutations in the transcription factor Growth Factor Independence 1B (GFI1B) and Runt-related transcription factor 1 (RUNX1) are causal to familial bleeding disorders. Mutant RUNX1 and GFI1B affect megakarocyte development, resulting in decreased numbers of platelets with significantly reduced α-granules and platelets that express the hematopoietic stem and progenitor cell marker CD34. A case that presented in our clinic with a bleeding disorder (Tosetto bleeding score of 11) was diagnosed with a δ-granule secretion defect. Platelet light transmission aggregometry revealed an aggregation defect upon stimulation with epinephrine and low concentrations of collagen and ADP. ATP release from δ-granules was normal after stimulation with a high concentration of collagen, however, no ATP was released upon stimulation with epinephrine and low levels were measured after stimulation with the thromboxane analogue U46619. Whole mount electron microscopy showed increased numbers of δ-granules in platelets derived from the index case compared to controls (average 5.84 δ-granules vs 3.66, per platelet respectively). To identify a genetic cause for the disease, a bleeding disorder gene panel was screened following whole exome sequencing. No disease-causing variants were called. Open exome analysis detected a ~100Kb heterozygous chromosome 16q22.1 deletion that was confirmed by SNP array. The observed deletion covers nine genes, including the last exon of CBFB. To date, none of the deleted genes have been implicated in inherited bleeding or platelet disorders. However, conditional knockout of Cbfb in mice results in a plethora of hematopoietic abnormalities including disturbed megakaryopoiesis and thrombocytopenia. CBFB is part of a heterodimeric transcription factor complex with either RUNX1, -2 or -3. Because of the CBFB-RUNX1 interaction and the fact that bleeding disorders caused by RUNX1 mutations result in platelet CD34 expression, we tested the platelets of the case reported here. Using flow cytometry, we clearly detected platelet CD34 expression compared to healthy controls, albeit to a lower level compared to RUNX1 and GFI1B affected cases. Mutations in RUNX2 may cause cleidocranial dysplasia and entire CBFB deletions have been reported in rare cases with congenital bone abnormalities. The medical record of the case presented here indicated that she had undergone surgery at the age of 13 for a congenital malformed collar bone. These data suggest a relation between the 16q/CBFB deletion and the observed abnormalities. To address this, we determined whether the abnormalities segregated with the 16q deletion. The mother and son of the index case had no history of clinical bleedings, no signs of bone malformations and did not have the 16q deletion. The father who was deceased was reported by the family not to have a bleeding propensity nor bone abnormalities. Platelets from the son did not express CD34. Remarkably, platelets from the son showed an aggregation defect upon stimulation with epinephrine and ADP, similar to the index patient. No ATP was released from δ-granules following stimulation with epinephrine. Thus, the 16q deletion associates with platelet CD34 expression and a congenital bone abnormality but is not responsible for the δ -granule secretion defect as the latter was observed in the son who did not have the 16q deletion, nor bleedings. Defective downmodulation of CD34 during platelet formation in the index case reported here suggests abnormal megakaryocyte development that might contribute in a multifactorial manner to bleedings. Recently, we reported CD34 expression on platelets from patients with rare GFI1B variants that may predispose to a bleeding propensity, but do not cause bleedings on their own. Thus, it will be important to determine whether the 16q deletion (and C-terminal truncation of CBFB) reported here affects megakaryocyte development and if so, contributes to the bleeding disorder in the index case. In contrast to Cbfb null mice, Cbfb heterozygous mice do not exhibit discernable hematopoietic phenotypes. Thus, if the C-terminal CBFB truncation contributes to the disease characteristics, it is likely that this occurs through a dominant-negative mechanism rather than haploinsufficiency. The findings reported here warrant further studies into the role of 16q (and CBFB) deletions in megakaryocyte development. Disclosures No relevant conflicts of interest to declare.

Platelet Function Tests and Inflammatory Markers for the Differentiation of Primary Thrombocytosis and Secondary Thrombocytosis.

Background:The prognosis and management of primary thrombocytosis (PT) and secondary thrombocytosis (ST) are different. This study aims to evaluate the role of platelet function tests by light transmission platelet aggregometry (LTA), plasma von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity (vWF:RCo) and inflammatory markers [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP)] for the differentiation between PT and ST. Methods:This prospective study was carried out in patients with platelet counts greater than 450 x 109/L. Primary outcomes were the sensitivity and specificity of platelet function tests by LTA for the differentiation of PT and ST. Secondary outcomes were sensitivity and specificity of ESR, CRP, vWF:Ag, and vWF:RCo for the differentiation of PT and ST. Results:Fifty-two patients were enrolled onto the study of which 26 (50%) had PT. The sensitivity and specificity of epinephrine, collagen, and arachidonic acid (AA) induced abnormal LTA for the differentiation of PT from ST were sensitivity of 50%, 38.5%, 26.9% and specificity of 88.5%, 100%, 100% respectively. The sensitivity and specificity of abnormal ESR, CRP, and either abnormal ESR or CRP in the differentiation of ST from PT were sensitivity of 88.5%, 80.8%, 100% and specificity of 65.4%, 61.5%, 46.2% respectively. The sensitivity and specificity of low vWF:Ag and vWF:RCo in the differentiation of PT from ST were sensitivity of 7.69%, 42.3% and specificity of 100%, 88.5% respectively. Conclusions:Abnormal platelet function determined by LTA with collagen, AA, epinephrine had high specificity ratings enabling the differentiation between PT and ST. vWF:RCo, ESR and CRP levels could be helpful in differentiating between PT and ST.

Quantitative dynamics of reversible platelet aggregation: mathematical modelling and experiments

Although reversible platelet aggregation observed in response to ADP stimulation in the presence of calcium is a well-known phenomenon, its mechanisms are not entirely clear. To study them, we developed a simple kinetic mass-action-law-based mathematical model to use it in combination with experiments. Light transmission platelet aggregometry (LTA) induced by ADP was performed for platelet-rich plasma or washed platelets using both conventional light transmission and aggregate size monitoring method based on optical density fluctuations. Parameter values of the model were determined by means of parameter estimation techniques implemented in COPASI software. The mathematical model was able to describe reversible platelet aggregation LTA curves without assuming changes in platelet aggregation parameters over time, but with the assumption that platelet can enter the aggregate only once. In the model, the mean size of platelet aggregates correlated with the solution transparency. This corresponded with flow cytometry analysis and with optical density fluctuations data on aggregate size. The predicted values of model parameters correlated with ADP concentration used in experiments. These data suggest that, at the start of the aggregation, when platelet integrins switch “on”, large unstable platelet aggregates are rapidly formed, which leads to an increase in light transmission. However, upon fragmentation of these aggregates, the probability of the post-aggregate platelets’ attachment to each other decreases preventing new aggregation and resulting in the reversible aggregation phenomenon.

Platelet aggregation response in immune thrombocytopenia patients treated with romiplostim.

The thrombopoietin receptor agonist romiplostim is used for the long-term treatment of chronic immune thrombocytopenia (ITP). ITP patients have an increased thrombotic risk, which could be exacerbated if romiplostim increased platelet hyperreactivity or caused spontaneous platelet aggregation. To investigate this possibility, this study examined platelet function in romiplostim-treated ITP patients and healthy subjects. Light transmission platelet aggregometry utilizing arachidonic acid, collagen, epinephrine, ristocetin, ADP, and saline (to assess spontaneous aggregation) was performed for each subject. In addition, the ADP AC50 (ADP concentration that induced half-maximal aggregation) was determined for each patient as a sensitive measurement of altered platelet reactivity. Fifteen ITP patients and 7 healthy subjects entered the study. All ITP patients had active disease and were receiving weekly romiplostim as the sole ITP-directed therapy. Platelet aggregation in response to the strong agonists arachidonic acid, collagen, and ristocetin was not significantly different between ITP patients and healthy subjects (P = 0.2442, P = 0.0548, and P = 0.0879, respectively). Platelet aggregation in response to weak agonists was significantly reduced in ITP patients compared with that in healthy subjects: median (range) aggregation to ADP, 45% (15-84%) versus 89% (70-95%) (P = 0.0010), and epinephrine, 21% (1.6-90%) versus 88% (79-94%) (P = 0.0085). The median AC50 of ADP was threefold higher in ITP patients versus that in healthy subjects (6.3μM vs 2.1μM) (P = 0.0049). Significant spontaneous aggregation was not observed in any patient. Platelets from romiplostim-treated ITP patients do not show evidence for spontaneous aggregation or hyperreactivity, but instead have a modestly reduced aggregation response to ADP and epinephrine.

Fentanyl Delays the Platelet Inhibition Effects of Oral Ticagrelor: Full Report of the PACIFY Randomized Clinical Trial.

Morphine delays oral P2Y 12 platelet inhibitor absorption and is associated with adverse outcomes after myocardial infarction. Consequently, many physicians and first responders are now considering fentanyl as an alternative. We conducted a single-centre trial randomizing cardiac patients undergoing coronary angiography to intravenous fentanyl or not. All participants received local anaesthetic and intravenous midazolam. Those requiring percutaneous coronary intervention (PCI) with stenting received 180 mg oral ticagrelor intra-procedurally. The primary outcome was area under the ticagrelor plasma concentration–time curve (AUC 0–24 hours ). The secondary outcomes were platelet function assessed at 2 hours after loading, measured by P2Y 12 reaction units (PRUs) and light transmission platelet aggregometry. Troponin-I was measured post-PCI using a high-sensitivity troponin-I assay (hs-TnI). All participants completed a survey of pain and anxiety. Of the 212 randomized, 70 patients required coronary stenting and were loaded with ticagrelor. Two participants in the no-fentanyl arm crossed over to receive fentanyl for pain. In as-treated analyses, ticagrelor concentrations were higher in the no-fentanyl arm (AUC 0–24 hours 70% larger, p = 0.03). Platelets were more inhibited by 2 hours in the no-fentanyl arm (71 vs. 113 by PRU, p = 0.03, and 25% vs. 41% for adenosine diphosphate response by platelet aggregation, p < 0.01). Mean hs-TnI was higher with fentanyl at 2 hours post-PCI (11.9 vs. 7.0 ng/L, p = 0.04) with a rate of enzymatic myocardial infarction of 11% for fentanyl and 0% for no-fentanyl ( p = 0.08). No statistical differences in self-reported pain or anxiety were found. In conclusion, fentanyl administration can impair ticagrelor absorption and delay platelet inhibition, resulting in mild excess of myocardial damage. This newly described drug interaction should be recognized by physicians and suggests that the interaction between opioids and oral P2Y 12 platelet inhibitors is a drug class effect associated with all opioids. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT02683707 ( NCT02683707).

Effect of intravenous fentanyl on ticagrelor absorption and platelet inhibition among patients undergoing percutaneous coronary intervention: Design, rationale, and sample characteristics of the PACIFY randomized trial

IntroductionMorphine reduces and delays the absorption of oral P2Y12 platelet inhibitors. Fentanyl is another opiate often administered during percutaneous coronary interventions (PCI). The PACIFY study will test whether intravenous fentanyl also impairs the absorption and action of oral P2Y12 inhibitors. MethodsPACIFY is a single-center trial randomizing adults undergoing clinically-indicated coronary angiography to have the procedure with or without fentanyl. All patients will receive local anesthetic and intravenous midazolam. Doses of all drugs are at the discretion of treating providers. Patients that require PCI receive 180mg of oral ticagrelor during the angiography procedure. The primary outcome is area under the ticagrelor plasma concentration-time curve. Secondary outcomes include platelet inhibition 2-h after loading, measured both by VerifyNow® (Accriva Diagnostics, San Diego, California) and by light-transmission platelet aggregometry. We will also survey patient comfort and measure high-sensitivity troponin levels. Patients and outcomes assessors are blinded, treating providers are not. ResultsA total of 212 patients are participating, 70 of whom required PCI. Baseline characteristics are numerically well balanced in both study arms. Mean age is 63years and 27% are female. There were no differences in total midazolam dose during the index PCI procedure, whereas mean total fentanyl dose was 9 mcg in the no-fentanyl arm (2 participants in this arm required fentanyl for bailout treatment of pain) versus 96 mcg in the fentanyl arm. ConclusionsThis study will provide important information on the impact of fentanyl administered during PCI on the absorption of ticagrelor and its antiplatelet activity.Trial Registration: clinicaltrials.gov Identifier: NCT02683707.

Ferro reports robust 1Q 2017 performance

Morphine reduces and delays the absorption of oral P2Y12 platelet inhibitors. Fentanyl is another opiate often administered during percutaneous coronary interventions (PCI). The PACIFY study will test whether intravenous fentanyl also impairs the absorption and action of oral P2Y12 inhibitors.PACIFY is a single-center trial randomizing adults undergoing clinically-indicated coronary angiography to have the procedure with or without fentanyl. All patients will receive local anesthetic and intravenous midazolam. Doses of all drugs are at the discretion of treating providers. Patients that require PCI receive 180 mg of oral ticagrelor during the angiography procedure. The primary outcome is area under the ticagrelor plasma concentration-time curve. Secondary outcomes include platelet inhibition 2-h after loading, measured both by VerifyNow® (Accriva Diagnostics, San Diego, California) and by light-transmission platelet aggregometry. We will also survey patient comfort and measure high-sensitivity troponin levels. Patients and outcomes assessors are blinded, treating providers are not.A total of 212 patients are participating, 70 of whom required PCI. Baseline characteristics are numerically well balanced in both study arms. Mean age is 63 years and 27% are female. There were no differences in total midazolam dose during the index PCI procedure, whereas mean total fentanyl dose was 9 mcg in the no-fentanyl arm (2 participants in this arm required fentanyl for bailout treatment of pain) versus 96 mcg in the fentanyl arm.This study will provide important information on the impact of fentanyl administered during PCI on the absorption of ticagrelor and its antiplatelet activity.Trial Registration: clinicaltrials.gov Identifier: NCT02683707.

Assessment of three point-of-care platelet function assays in adult trauma patients

BackgroundAntiplatelet (AP) medication use is common among trauma patients and is associated with poor outcomes. Management options for platelet dysfunction in trauma patients are controversial, expensive, and potentially harmful. Although light transmission platelet aggregometry is considered the standard test to assess platelet function, it is cumbersome and not generally available. Currently, there are no widely accepted platelet function point-of-care tests for acute trauma. Study designProspective observational study from 2014 to 2015. Baseline Multiplate aggregometry aspirin area under the platelet aggregation curve (ASPI AUC), Thrombelastography Platelet Mapping percent inhibition of arachidonic acid (TEG-PM AA), and VerifyNow Aspirin Test (ARU) were compared for ability to detect any AP medication use (aspirin or clopidogrel), platelet dysfunction, and identify patients at risk for intracranial hemorrhage (ICH) progression by calculating the area under receiver operating characteristic curves (AUC), sensitivity, specificity, and positive and negative predictive values. Adenosine diphosphate assays were similarly evaluated. ResultsSixty-four patients were enrolled, 25 were taking AP medications. AP patients were older (71.6 versus 35.0 y, P < 0.001) and received more platelet transfusions, but other baseline characteristics were similar. Median ASPI AUC (22.0 versus 53.5 P < 0.001) and VerifyNow ARU (503.5 versus 629.0, P < 0.001) were lower, whereas TEG-PM AA (51.8% versus 18.3%, P < 0.001) was higher in AP patients. Multiplate ASPI AUC, TEG-PM AA percent inhibition, and VerifyNow ARU could identify AP medication use (AUC: 0.90, 0.77, and 0.90, respectively). Adenosine diphosphate assays did not correlate with AP medication use in this population. TEG-PM AA percent inhibition and VerifyNow ARU correlated well with Multiplate ASPI AUC to identify platelet dysfunction (AUC: 0.78, 0.89, respectively). ICH occurred in 29 patients; 12 of which had progression of their injury. ASPI AUC (AUC: 0.50) and VerifyNow ARU (AUC: 0.59) did not correlate, and TEG-PM AA percent inhibition (AUC: 0.66) minimally correlated with progression. ConclusionsMultiplate, TEG-PM, and VerifyNow are useful point-of-care tests which identify AP medication use and platelet dysfunction in trauma patients. Initial TEG-PM AA percent inhibition may be associated with risk for ICH progression. However, additional large, prospective studies are needed.

The Nitric Oxide-Generating Prodrug Js-K Is Selectively Toxic Towards AML Compared to Normal Human Hematopoietic Cells

Nitric oxide (NO) induces apoptosis and differentiation in acute myeloid leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a lead agent of the arylated diazeniumdiolate class. JS-K is active in vitro and in vivo against AML, multiple myeloma, and several solid tumors. JS-K is directly cytotoxic to malignant cells and inhibits angiogenesis in vitro and in vivo. We have previously shown that JS-K is not toxic towards normal murine hematopoietic cells.Aiming at its clinical application to treat AML, we have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic®P123 polymers. The formulation (P123/DMSO) consists of 2.25% P123 and 2% dimethyl sulfoxide in phosphate buffered saline. P123/JS-K is currently at an advanced stage of pre-clinical development.Here, we compared the cytotoxicity of JS-K and its formulation between normal hematopoietic and AML cells. HL-60 AML cells or normal human CD34+ cells isolated from 2 different cord blood units were treated for 24 hours with P123/DMSO or P123/JS-K before plating in semi-solid media. Colonies were scored at 10 days. P123/DMSO alone had no effect on colony formation by HL-60 or normal CD34+ cells. P123/JS-K at concentrations of 0.1, 0.25, and 0.5 μM inhibited HL-60 colony growth by 27, 74, and 100%, respectively (P < 0.05 for the comparison between 0.25, 0.5 μM and controls).At the same concentrations, P123/JS-K inhibited the growth of erythroid colonies from the first cord blood sample, by 26, 43, and 61%, respectively. At a concentration of 0.1 μM, P123/JS-K did not inhibit the growth of erythroid colonies from the second cord blood sample but inhibited erythroid colony growth by 18, and 27% at concentrations of 0.25 and 0.5 μM, respectively. However, the differences with untreated controls were not statistically significant for either cord blood sample.At the same concentrations P123/JS-K inhibited the growth of myeloid colonies from the first cord blood sample, by 8, 3, and 37%, respectively. At a concentration of 0.1 μM, P123/JS-K did not inhibit the growth of myeloid colonies from the second cord blood sample but inhibited myeloid colony growth by 2, and 6% at concentrations of 0.25 and 0.5 μM, respectively. However, the differences with untreated controls were not statistically significant for either cord blood sample.With clinical development in mind, we also sought to determine whether the P123/DMSO formulation affects platelet function. P123/DMSO was added to normal donor blood obtained in either EDTA or citrate anticoagulant. With either anticoagulant, P123/DMSO did not affect the measured hemoglobin, white blood cell, or platelet counts. Inspection of peripheral blood smears (Wright stain) obtained with either anticoagulant revealed no platelet clumping. We then studied the effect of P123/DMSO on platelet aggregation in vitro in response to ADP, collagen, ristocetin, or arachidonic acid. Light transmission platelet aggregometry was performed using platelet-rich plasma from a healthy volunteer with normal platelet counts and function. In order to determine whether P123/DMSO could enhance or inhibit platelet aggregation, we chose a range of agonist concentrations from sub-threshold to concentrations expected to induce complete aggregation. P123/DMSO was added at a concentration expected to be equal to peak in vivolevels based on a prior dog toxicology study. Normal aggregation responses were observed in the saline control. There were no significant differences between this control and responses observed in the presence of P123/DMSO. Specifically, significantly decreased aggregation in P123/DMSO-treated platelets was not observed in response to agonists expected to elicit complete aggregation responses. Likewise, enhanced aggregation was not observed in response to sub-threshold agonist concentrations.Our results confirm the potent anti-leukemic activity and limited toxicity against normal human hematopoietic cells of JS-K formulated in P123 micelles. Furthermore, the formulation does not affect platelet aggregation. We therefore expect that P123/JS-K will have a favorable hematologic toxicity profile and as such, will constitute an important addition to our armamentarium for the treatment of AML. DisclosuresDeininger:Gilead: Research Funding; BMS: Consultancy, Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Shami:JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Multi-Targeted Antithrombotic Therapy for Total Artificial Heart Device Patients

To prevent thrombotic or bleeding events in patients receiving a total artificial heart (TAH), agents have been used to avoid adverse events. The purpose of this article is to outline the adoption and results of a multi-targeted antithrombotic clinical procedure guideline (CPG) for TAH patients. Based on literature review of TAH anticoagulation and multiple case series, a CPG was designed to prescribe the use of multiple pharmacological agents. Total blood loss, Thromboelastograph® (TEG), and platelet light-transmission aggregometry (LTA) measurements were conducted on 13 TAH patients during the first 2 weeks of support in our institution. Target values and actual medians for postimplant days 1, 3, 7, and 14 were calculated for kaolinheparinase TEG, kaolin TEG, LTA, and estimated blood loss. Protocol guidelines were followed and anticoagulation management reduced bleeding and prevented thrombus formation as well as thromboembolic events in TAH patients postimplantation. The patients in this study were susceptible to a variety of possible complications such as mechanical device issues, thrombotic events, infection, and bleeding. Among them all it was clear that patients were at most risk for bleeding, particularly on postoperative days 1 through 3. However, bleeding was reduced into postoperative days 3 and 7, indicating that acceptable hemostasis was achieved with the anticoagulation protocol. The multidisciplinary, multi-targeted anticoagulation clinical procedure guideline was successful to maintain adequate antithrombotic therapy for TAH patients.

Comparison of common platelet receptors between the chacma baboon (Papio ursinus) and human for use in pre-clinical human-targeted anti-platelet studies

Anti-platelet agents play a central part in the treatment and prevention of acute thrombotic events. Discriminating animal models are needed for the development of novel agents. The chacma baboon has been extensively used as a model to evaluate anti-platelet agents. However, limited data exist to prove the translatability of this species to humans. We aimed to determine the suitability of the chacma baboon in preclinical human targeted GPIIb/IIIa, GPIbα and P2Y12 studies. Light-transmission platelet aggregometry (LTA), whole blood impedance aggregometry, receptor number quantification and genomic DNA sequencing were performed. Baboon ADP and arachidonic acid-induced LTA aggregation results differed significantly from human values, even at increased concentrations. LTA ristocetin-induced agglutination was comparable between species, but baboon platelets needed twice the concentration of ristocetin to elicit a similar response. Citrated baboon blood had significantly less aggregation than humans when evaluated with impedance aggregometry. However, hirudinised baboon whole blood gave similar aggregation as humans at the same agonist concentrations. GPIIb, GPIIIa and GPIbα numbers were significantly more on the baboon platelets. None of the amino acids deemed vital for receptor function, ligand binding or receptor inhibition, were radically different between the species. However, a conservative change in a calcium-binding region of GPIIb may render the baboon platelets more sensitive to calcium-binding agents. The chacma baboon may be used for the evaluation of human-targeted GPIIb/IIIa-, GPIbα- and P2Y12-inhibiting agents. However, the best anticoagulant, optimal agonist concentrations, increase in receptor number and sequence differences must be considered for any future studies.

Comparison between urinary 11-dehydrothromboxane B2 detection and platelet Light Transmission Aggregometry (LTA) assays for evaluating aspirin response in elderly patients with coronary artery disease

Aspirin is widely used in the primary and secondary prevention of cardiovascular diseases. The aim of our study was to compare between two established methods of aspirin response, urinary 11-dehydrothromboxane B2 (11dhTXB2) and platelet Light Transmission Aggregometry (LTA) assays in elderly Chinese patients with coronary artery disease (CAD), and to investigate the clinical significance of both methods in predicting cardiovascular events. Urinary 11dhTxB2 assay and arachidonic acid-induced (AA, 0.5mg/ml) platelet aggregation by Light Transmission Aggregometry (LTAAA) assay were measured to evaluate aspirin responses. High-on aspirin platelet reactivity (HAPR) was defined as urinary 11dhTxB2>1500pg/mg or AA-induced platelet aggregation≥15.22%—the upper quartile of our enrolled population. The two tests showed a poor correlation for aspirin inhibition (r=0.063) and a poor agreement in classifying HAPR (kappa=0.053). With a mean follow-up time of 12months, cardiovascular events occurred more frequently in HAPR patients who were diagnosed by LTA assay as compared with no-HAPR patients (22.5% versus 10.6%, P=0.039, OR=2.45, 95% CI=1.06–5.63). However, the HAPR status, as determined by urinary 11dTXB2 measurement, did not show a significant correlation with outcomes.