Impedance Aggregometry Research Articles

Rapid method for screening of both calcium and magnesium chelation with comparison of 21 known metal chelators.

Chelation is the rational treatment modality in metal overload conditions, but chelators are often non-selective and can, hence, cause an imbalance in the homeostasis of physiological metals including calcium and magnesium. The aim of this study was to develop an affordable, rapid but sensitive and precise method for determining the degree of chelation of calcium and magnesium ions and to employ this method for comparison on a panel of known metal chelators. Spectrophotometric method using o-cresolphthalein complexone (o-CC) was developed and its biological relevance was confirmed in human platelets by impedance aggregometry. The lowest detectable concentration of calcium and magnesium ions by o-CC was 2.5μM and 2μM, respectively. The indicator was stable for at least 110days. Four and seven out of twenty-one chelators strongly chelated calcium and magnesium ions, respectively. Importantly, the chelation effect of clinically used chelators was not negligible. Structure-activity relationships for eight quinolin-8-ols showed improvements in chelation particularly in the cases of dihalogen substitution, and a negative linear relationship between pKa and magnesium chelation was observed. Calcium chelation led to inhibition of platelet aggregation in concentrations corresponding to the complex formation. A novel method for screening of efficacy and safety of calcium and magnesium ion chelation was developed and validated.

Impact of Dipyrone Administration on Postoperative Analgesia and Aspirin Effect in Patients Undergoing Coronary Artery Bypass Grafting: The Prospective Randomized DipASA Study

ObjectiveThe aim of the study was to investigate the impact of dipyrone administration on postoperative analgesia and acetylsalicylic acid (ASA) effect in patients undergoing coronary artery bypass grafting (CABG). DesignA prospective randomized study. SettingSingle-university hospital setting. ParticipantsNinety-eight patients who underwent CABG between April 2022 and May 2023. InterventionsASA effect were measured at six time points with impedance aggregometry (Multiplate) and thromboelastography (TEG6sPlateletMapping). Patients were randomized to one of three groups: intravenous ASA and dipyrone at the same time (group 1), intravenous ASA and dipyrone with a 2-hour delay (group 2), and intravenous ASA alone (group 3). Postoperative analgesic effects (numeric rating scale, NRS) and the prevalence of potential ASA non-response (defined as ASPI>40U and TEG-ASA-inhibition<50%) were recorded. Measurements and Main Results80 of 90 analyzed patients took ASA preoperatively. All patients received intravenous ASA 100mg from postoperative day 1. The effect of ASA did not significantly differ between the study groups at any time for either platelet function test. NRS values did not differ between the study groups at any time (p=0.469). Patients in group 3 received significantly more additional co-analgesics than patients who received dipyrone (p=0.005). ASA non-response was detected in 38.9% and 67.8% on 7th postoperative day, respectively. ConclusionsDipyrone given after CABG seems to be safe and did not show any significant effect on platelet inhibition after ASA administration. Patients taking dipyrone postoperatively need significantly less additional co-analgesics. The ASA effect on platelet function should be checked at least once after surgery.

Peculiarities of Hemostasis and Proteomics in Patients With Acute Myocardial Infarction and Healthy Volunteers After SARS-CоV-2 Infection.

To identify the features of plasma, platelet hemostasis, and proteomic composition of the blood plasma in patients with acute myocardial infarction (AMI) and healthy volunteers after COVID-19. The study included patients with AMI who have recently had COVID-19 (AMI-post-COVID, n=56) and patients with AMI who have not recently had COVID-19 (AMI-control, n=141). Healthy volunteers constituted the control groups and were also divided into control-post-COVID (n=32) and control-control (n=71) groups. Previous SARS-CoV-2 infection was determined by anti-N IgG in the blood serum, the level of which persists for 6-10 months after the disease. Hemostasis was evaluated by thromboelastometry (on whole blood), thrombodynamics (on platelet-poor plasma), fibrinolysis, impedance aggregometry, and proteomic analysis. The AMI-post-COVID and AMI-control groups had higher values of thrombus growth rate, size and density based on the data of thromboelastometry and thrombodynamics, as well as increased concentrations of the complement system components, proteins regulating the state of the endothelium, and a number of acute-phase and procoagulant proteins compared to the control groups. Furthermore, in the AMI-post-COVID group, compared to the AMI-control group, the thrombus density was lower, and its lysis rates were higher when measured by the thrombodynamics method on platelet-poor plasma, while the platelet aggregation induced by ADP and thrombin was higher. Also, in the control-post-COVID group, compared to the control-control group, the thrombus formation rate was lower, whereas, in contrast, the thrombus size as measured by the thrombodynamics method and the platelet aggregation induced by arachidonic acid and thrombin were higher. In addition, in the AMI-post-COVID group, compared to the AMI-control group, the concentrations of proteins involved in inflammation and hemostasis were lower. Patients with AMI who have recently had COVID-19 are characterized by a less pronounced activation of the immune response compared to patients with AMI who have not had COVID-19. This may be due to long-term chronic inflammation and depletion of components of the immune activation system after SARS-CoV-2 infection. Long-term activation of the hemostasis system in both patients with AMI and healthy volunteers after COVID-19 is primarily due to the platelet component of hemostasis.

Dynamics of Thrombogenicity and Platelet Function and Correlation with Bleeding Risk in Patients Undergoing M-TEER Using the PASCAL System.

Transcatheter mitral valve repair is performed in a patient population at risk for thrombotic and bleeding events. The effects on platelet function and reactivity and their association with bleeding events after mitral transcatheter edge-to-edge therapy (M-TEER) have not been systematically examined. We sought to investigate the association of different parameters of platelet function and thrombogenicity with bleeding events post M-TEER. In this single-center study, 100 consecutive patients with mitral regurgitation receiving TEER were analyzed. Blood was taken directly from the guide-catheter in the left atrium before and after placing the device. Blood samples were analyzed using impedance aggregometry (Multiplate) and TEG6s. The results were compared pre- and postprocedural. The primary outcome was any bleeding complication according to the Bleeding Academic Research Consortium classification within 6 months. A total of 41 patients experienced bleeding events. TEG analysis showed a significant decrease in ADP aggregation and increase in ADP inhibition. In ROC-analysis, TEG ADP aggregation and inhibition and Multiplate ADP aggregation showed moderate predictive values for bleeding events. The delta-ADP-Test (Multiplate) showed the strongest prediction of bleeding (area under the curve: 0.69). Adding platelet function and TEG markers to a model of clinical bleeding risk factors improved the prediction for bleeding events. This study indicates that thrombogenicity might be affected immediately after M-TEER probably due to changes in flow conditions. In particular, platelet aggregation involving the ADP receptor pathway significantly correlated with postprocedural bleeding events. Whether these results could guide peri-interventional antithrombotic therapy and improve peri- and postprocedural outcome requires further investigation.

Quantitative and functional changes in platelets and fibrinogen following cardiopulmonary by-pass in children

IntroductionCardiopulmonary bypass (CPB) causes coagulopathy, increasing the risk of postoperative bleeding and mortality. The underlying causes of post-CPB coagulopathy and the factors associated with its occurrence are not yet fully understood. This study assesses platelet and fibrinogen concentration and function following CPB in children with congenital heart diseases (CHD).MethodsWe analyzed prospective data from 104 patients aged 0–16 years who underwent CPB surgery for CHD. Blood samples were collected before surgery and within 30 min of CPB completion. In addition to usual coagulation tests, functional analyses were performed using point of care systems with thromboelastometry and impedance aggregometry.ResultsPlatelet count, fibrinogen concentration, and platelet and fibrinogen activities significantly decreased after CPB. The duration of CPB was directly associated with a reduction in platelet count and fibrinogen level (r = −0.38, p &amp;lt; 0.001; r = −0.21, p = 0.03, respectively), but not with their measured activity. Postoperative percentages of baseline values for platelet count (58.36% [43.34–74.44] vs. 37.44% [29.81–54.17], p &amp;lt; 0.001) and fibrinogen concentration (73.68% [66.67–82.35] vs. 65.22% [57.89–70.83], p &amp;lt; 0.001) were significantly higher in patients who did not experience hypothermia during surgery. Age was inversely associated with the decrease in platelet count (r = 0.63, p &amp;lt; 0.001), TRAPTEM AUC (r = 0.43, p &amp;lt; 0.001), fibrinogen concentration (r = 0.44, p &amp;lt; 0.001) and FIBTEM MCF (r = 0.57, p &amp;lt; 0.001).ConclusionPost-CPB coagulopathy is multifactorial and not solely attributed to hemodilution. It also involves functional changes in coagulation cascade components, which can be demonstrated by thromboelastometry and impedance aggregometry. Young children, patients requiring prolonged CPB surgery, or those experiencing hypothermia are particularly affected.

A novel miniaturized roller pump circuit for simulation of extracorporeal circulation.

Extracorporeal circulation induces pronounced effects on haemostasis and rheology. To study these, an ex vivo simulation model is an attractive alternative but often requires large amounts of blood. We sought to create a miniaturized roller pump circuit requiring minimal amounts of blood and to test if the circuit could be used to compare coagulation, platelet function and blood rheology between a dextran-based and a crystalloid-based priming solution. A miniaturized roller pump circuit requiring only 27 ml of blood was created. Blood samples from 8 cardiac surgery patients were mixed with either a dextran-based or a crystalloid-based solution and circulated for 60 min. Coagulation was assessed by rotational thromboelastometry, and platelet function by impedance aggregometry and flow cytometry, before and at 5 and 60 min of circulation. A time-dependent impairment of coagulation was observed in both groups. Maximum clot firmness was lower with dextran-based than with crystalloid-based priming at 5 min (HEPTEM 37 ± 4 vs 43 ± 4 mm, P < 0.001; EXTEM 37 ± 4 vs 43 ± 4 mm, P < 0.001; FIBTEM 3 ± 2 vs 9 ± 2 mm, P < 0.001) and at 60 min (HEPTEM 29 ± 9 vs 38 ± 5 mm, P < 0.001; EXTEM 30 ± 7 vs 39 ± 5 mm, P < 0.001; FIBTEM 3 ± 2 vs 8 ± 3 mm, P = 0.002). The EXTEM clotting time was longer with dextran-based solution at 5 (109 ± 19 vs 63 ± 7 sec, P < 0.001) and at 60 min (176 ± 72 vs 73 ± 7 sec, P = 0.004). The novel miniaturized roller pump circuit can be used to mimic extracorporeal circulation for selected research questions. Dextran-based priming caused a significant impairment in haemostasis compared with a standard crystalloid solution.

Antiplatelet Effect of Low-Dose Prasugrel in Elderly Patients Undergoing Percutaneous Coronary Interventions.

Low-dose prasugrel (5 mg) has been proposed for patients with Acute Coronary Syndrome (ACS) and advanced age or low body weight. However, the routine use of dose-adjusted prasugrel in this high-risk subset of patients is still debated. This study aimed to assess the prevalence and predictors of HRPR among elderly patients treated with low-dose (5 mg) prasugrel to evaluate the routine use of dose-adjusted prasugrel in this high-risk subset of patients. We included 59 elderly patients (≥75 years) treated with Dual Antiplatelet Therapy (DAPT: acetylsalicylic acid (ASA) 100-160 mg + prasugrel 5 mg) after Percutaneous Coronary Interventions (PCI) and undergoing platelet function assessment (by whole blood impedance aggregometry) 30-90 days post-discharge. At a median follow-up of 43 days (interquartile range-IQR: 32-54), high-on treatment residual platelet reactivity (HRPR) occurred in 25 patients (42.4%), who displayed a greater body mass index (BMI) (p=0.02), lower levels of vitamin D (p=0.05) and were more frequently treated with nitrates (p=0.03). After multivariate analysis, BMI was the only independent predictor of prasugrel HRPR, and a BMI >26 was the best cut-off for predicting HRPR (adjusted Odds Ratio - OR=8.6, 95%CI: 2.2-33.9, p=0.002). Among elderly patients receiving DAPT after PCI, HRPR is common with low-dose prasugrel. A greater BMI, especially for values ≥26, is the only independent predictor of HRPR with prasugrel 5 mg.

Clusters of patients with non-ST-segment elevation acute coronary syndrome depending on the laboratory data

Aim. To identify clusters of patients with non-ST segment elevation acute coronary syndrome (NSTE-ACS) depending on the laboratory data, including platelet function tests.Material and methods. The study included 109 patients hospitalized in the cardiology department with NSTE-ACS. All patients underwent a standard examination, including a complete blood count. In addition, the platelet function was assessed using impedance aggregometry with various inducers (adenosine diphosphate (5 and 10 μmol/L), collagen (2 μmol/L)). We assessed the P-selectin expression level using flow cytometry. Patients were followed up for 6 months. As a statistical method, we performed сluster analysis by K-means algorithm.Results. We registered 18 adverse outcomes (myocardial infarction, unstable angina) during 6-month follow-up. Cluster analysis revealed 3 clusters. The first cluster included 39 (36%) patients with higher monocyte values and lower hemoglobin values, probably indicating chronic inflammation and a tendency towards anemia. The second cluster included 13 older patients (12%) with high comorbidity and high platelet function. Both the first and second clusters recorded 23% adverse outcomes. Third cluster, on the contrary, included mostly younger patients with fewer comorbidities, which had more favorable course of ACS (no signs of platelet hyperaggregation, chronic inflammation and anemia). This cluster included more than half (n=57, 52%) of all patients; adverse outcomes at 6 months in these patients developed 2-fold less frequently than in the other clusters (11% vs 23%).Conclusion. Among patients with NSTE-ACS, groups differing in laboratory parameters were identified, based on which clusters were formed using the k-means method. Clusters with high levels of platelet functional activity, monocyte count and low hemoglobin values included older patients with severe comorbidity and showed a tendency towards more frequent adverse outcomes of the disease.

Platelet aggregation response to cyclooxygenase inhibition and thromboxane receptor antagonism using impedance aggregometry: A pilot study.

Impedance aggregometry is an alternative to light transmission aggregometry that allows analysis of platelet function in whole blood samples. We hypothesized (1) impedance aggregometry would produce repeatable results, (2) inhibition of cyclooxygenase with aspirin would attenuate aggregation responses to collagen and abolish the aggregation response to arachidonic acid (AA), and (3) thromboxane receptor antagonism (terutroban) would attenuate the aggregation response to AA. Venous blood was obtained from 11 participants three times separated by at least 2 weeks. One sample followed 7-day-aspirin intervention (81 mg once daily; ASA), the others no intervention (control). Aggregation was induced using 1 μg/mL collagen ([col 1]), 5 μg/mL collagen ([col 5]), and 50 mM AA via impedance aggregometry to determine total aggregation (AUC) analyzed for intra-test repeatability, inter-test repeatability, intervention (ASA or control), and incubation (saline or terutroban). [col 1] showed high intra-test (p ≤ 0.03 visit 1 and 2) and inter-test repeatability (p < 0.01). [col 5] and AA showed intra- ([col 5] p < 0.01 visit 1 and 2; AA p < 0.001 visit 1 and 2) but not inter-test repeatability ([col 5] p = 0.48; AA p = 0.06). ASA attenuated AUC responses to [col 1] (p < 0.01), [col 5] (p = 0.03), and AA (p < 0.01). Terutroban attenuated AUC in response to AA (p < 0.01). [col 1] shows sufficient repeatability for longitudinal investigations of platelet function. [col 5] and AA may be used to investigate mechanisms of platelet function and metabolism at a single time point.

Anti-Platelet Activity of Sea Buckthorn Seeds and Its Relationship with Thermal Processing.

Sea buckthorn (Hippophae rhamnoides L.) is a tree or shrub with small, orange berries. Sea buckthorn seeds have shown many properties beneficial to human health, including antioxidant, anti-hypertensive, anti-hyperlipidemic, and retinoprotective activities. Seeds, as a component of food, are often exposed to high temperatures, which can increase or decrease their biological activity. In our previous study, we showed that both raw and roasted sea buckthorn seeds had significant antioxidant activity, which was measured in human plasma in vitro. In this paper, we evaluated the effect of extracts from raw and roasted sea buckthorn seeds on several parameters of hemostasis in vitro, including thrombus formation in full blood (measured by the Total Thrombus formation Analysis System-T-TAS), blood platelet activation (based on the exposition of P-selectin, the active form of GPIIb/IIIa on their surface and platelet-derived microparticles formation), aggregation (measured with impedance aggregometry), adhesion to fibrinogen and collagen, arachidonic acid metabolism in washed platelets stimulated by thrombin, and COX-1 activity. We also measured the levels of free 8-isoprostane in plasma and the total non-enzymatic antioxidant status of plasma. The extract from roasted seeds (50 µg/mL) significantly prolonged the time of occlusion measured by T-TAS-the AUC10 (area under the curve) value was decreased by approximately 18%. Both extracts decreased the exposition of the active form of GPIIb/IIIa on the surface of platelets activated with 10 μM ADP (by 38.4-62.2%) and 20 μM ADP (by 39.7-51.3%). Moreover, the extract from raw seeds decreased the exposition of P-selectin on the surface of platelets stimulated with 20 μM ADP (by 31.2-34.9%). The adhesion of thrombin-stimulated platelets to fibrinogen and collagen was inhibited only by the extract from roasted sea buckthorn seeds (by 20-30%). Moreover, the extract from raw seeds inhibited the level of TBARS (thiobarbituric acid-reactive substances, an indicator of enzymatic peroxidation of arachidonic acid) in washed platelets stimulated with thrombin; the activity of COX-1 was inhibited by both extracts, although the effect of the extract from raw seeds was stronger. These results indicate that sea buckthorn seeds have anti-platelet activity that is not decreased by thermal processing, but more research is needed to determine which exact chemical compounds and mechanisms are responsible for this phenomenon.

Repeatability of Platelet Aggregation Characteristics Using Impedance Aggregometry With and Without Aspirin Intervention

Nearly 40% of adults over 50-years-old regularly take aspirin for primary or secondary prevention of cardiovascular disease and/or adverse cardiovascular events. The effcacy of aspirin therapy is commonly assessed with light transmission aggregometry. However, this method requires removal of large platelets as well as the mechanical disturbance of platelets remaining to be tested in the sample. Impedance aggregometry presents a solution to this problem, but the repeatability of impedance aggregometry analysis remains to be tested. We hypothesized repeated impedance aggregometry analysis would show decreased platelet aggregation activity following aspirin intervention compared with control visits. Venous blood samples were obtained from ten participants on three occasions each, separated by at least two weeks. One sample followed seven days of aspirin intervention (81 mg once daily; ASA) and the other two with no intervention (control). Platelet aggregation was induced using 1 μg/ml collagen (Col1), 5 μg/ml collagen (Col5), and 0.5 mM arachidonic acid (AA) via impedance aggregometry to determine area under the aggregation curve (AUC, au), time to aggregation initiation following addition of reagent (Lag, s), the rate of aggregation (Slope), and the maximal aggregation (Amp, Ohms). These characteristics were statistically analyzed for an impact of aggregometer channel (intra-test repeatability), visit (inter-test repeatability), intervention (ASA or control), and incubation (samples were incubated with either saline or Terutroban solution to block thromboxane receptors). Results were analyzed by multiple paired t-tests and represented with lowest relevant p-value if insignificant or highest relevant p-value if significant. Impedance aggregometry revealed no difference in any aggregation characteristics (AUC, Lag, Slope, or Amp) for duplicate samples (all p ≥ 0.10) or visits (all p ≥ 0.06). ASA decreased AUC, Slope, and Amp while increasing or completely inhibiting Lag response to the addition of all reagents (all p ≤ 0.01). Terutroban incubation had no effect on aggregation characteristics following the ASA intervention (all p ≥ 0.09), but attenuated the AUC, Slope, and Amp (all p ≤ 0.01), and entirely prevented or substantially increased the Lag response to AA (p ≤ 0.01) without impacting the responses to Col1 or Col5 (all p ≥ 0.24). These findings suggest the impedance aggregometry approach is a repeatable method for platelet aggregometry analysis for the characteristics of AUC, Lag, Slope, and Amp. NIH R01 Grant 1R01AG067471-01 NIH F31 Grant 1F31HL170616-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Platelet activity, coagulation, and fibrinolysis in long-term users of anabolic-androgenic steroids compared to strength-trained athletes

IntroductionUse of anabolic-androgenic steroids (AAS) is associated with adverse cardiovascular (CV) effects, including potential prothrombotic effects. This study aimed to assess platelet activation and aggregation, coagulation, and fibrinolysis, in long-term AAS users compared to non-using strength-trained athletes. Materials and methodsThirty-seven strength-trained men using AAS were compared to seventeen non-using professional strength-trained athletes at similar age (median 33 years). AAS use was verified by blood and urine analyses. Platelet Function Analyzer 100 (PFA-100) and whole blood impedance aggregometry with thrombin, arachidonic acid, and ADP as agonists, were performed to evaluate platelet aggregation. ELISA methods were used for markers of platelet activation. Fibrinogen, D-dimer, the coagulation inhibitors protein S and C activity, and antithrombin were measured by routine. Fibrinolysis was evaluated by Plasminogen Activator Inhibitor-1 (PAI-1) activity. ResultsThere were no significant differences in platelet aggregation between the two groups. Von Willebrand factor was lower among the AAS users (p < 0.01), and P-Selectin was slightly higher (p = 0.05), whereas CD40 Ligand, β-thromboglobulin, and thrombospondin did not differ significantly. No differences were found in the assessed coagulation inhibitors. Higher D-dimer levels (p < 0.01) and lower PAI-1 activity (p < 0.01) were found among the AAS users. ConclusionsThe investigated long-term users of AAS did not exhibit elevated platelet activity compared to strength-trained non-using athletes. However, AAS use was associated with higher D-dimer levels and lower PAI-1 activity. These findings suggest that any prothrombotic effect of long-term AAS use may predominantly involve other aspects of the hemostatic system than blood platelets.

Direct red blood cell effect on thrombosis is dependent on the interaction of tissue factor and calcium with membrane phosphatidylserine.

Prior literature has implicated red blood cells (RBCs) in the initiation of thrombosis and suggests that posttransfusion hypercoagulability may occur secondary to the effects of RBCs. Elevated serum tissue factor is a known sequelae of acute trauma. Phosphatidylserine (PS) is a prothrombotic phospholipid present within the RBC cell membrane. We hypothesized that RBC aggregation is dependent on the interaction between RBC membrane bound (exposed) PS, extracellular calcium, and tissue factor. Human whole blood (WB) was separated into components, including RBCs and platelet-rich plasma (PRP). Whole blood, PRP, and RBCs underwent impedance aggregometry utilizing arachidonic acid (AA), ADP, collagen, calcium, and tissue factor (TF)-based agonists. Red blood cells then underwent impedance aggregometry utilizing combined calcium and TF agonists. Red blood cells were pretreated with Annexin V, a known PS blocking agent, and underwent impedance aggregometry with combined calcium and TF agonists to determine if the mechanism of calcium/TF-induced RBC aggregability is dependent on PS. Red blood cells treated with calcium, TF, calcium+TF, and pre-treated with Annexin V followed by calcium+TF were perfused through an in vitro model of pulmonary microcirculatory flow. Red blood cell aggregation was significantly higher than that of WB and PRP when utilizing a TF agonist, an effect unique to TF. The combination of calcium and TF demonstrated significantly higher RBC aggregation than either agonist alone. Pretreatment with Annexin V resulted in a significantly reduced aggregability of RBC following treatment with TF + calcium. Red blood cells aged to 42 days did not exhibit significant change in aggregation. Exposure to calcium and TF significantly reduced time to thrombosis of RBCs perfused through a pulmonary microcirculatory model. Treatment with both TF and calcium synergistically induces RBC aggregation. Phosphatidylserine appears to play an integral role in the TF/calcium-based, age-independent RBC aggregation response. Red blood cells treated with TF + calcium exhibit more rapid thrombus formation in an in vitro model of pulmonary microcirculatory perfusion.

Aspirin resistance in patients with ventricular assist devices-A follow-up study.

Despite combined anticoagulation therapy consisting of a vitamin K antagonist and an antiplatelet agent, thromboembolic complications often occur in patients with a left ventricular assist device (LVAD). In addition, bleeding events are also common. Resistance to antiplatelet drugs is a well-known phenomenon; however, the utilization of laboratory chemistry testing for the presence of such resistance, and then switching therapy, is controversial. We tested 132 patients with LVAD (HeartWare n = 57, HeartMate II n = 22, HeartMate 3 n = 53) on acetylsalicylic acid (ASA) therapy for resistance and followed them for a maximum of 7 years regarding pump thrombosis. Light transmission aggregometry (LTA) and impedance aggregometry (IPA) were performed for testing platelet function. We could show that patients with ASA resistance displayed an increased risk of pump thrombosis, regardless of the test used (LTA: OR = 6.20, CI [1.86-20.64], p = 0.003; IPA: OR = 12.14, CI [3.00-49.07], p < 0.001). In patients with a HeartMate 3, we could not detect any pump thrombosis associated with aspirin resistance. Furthermore, there was no significant difference in bleeding events between patients with ASA resistance and ASA responders. Laboratory testing of ASA resistance seems to be a good tool to detect an increased risk of pump thrombosis, at least for patients with a HeartWare or HeartMate II. The extent to which these thromboses can be prevented with a change of medication has to be investigated in further studies. No pump thrombosis was detected in patients with a HeartMate 3, and the question should be asked as to what constellation of underlying and concomitant diseases must be present to justify ASA therapy for these patients.

Assessment of hemostasis in hyperthyroid and euthyroid cats using two viscoelastic assays and platelet aggregometry.

Hyperthyroidism in humans is associated with a hypercoagulable state and an increased risk of thromboembolism. To evaluate hemostatic variables in hyperthyroid and euthyroid cats with the hypothesis that hyperthyroid cats will have evidence of altered hemostasis consistent with a potential hypercoagulable state. Client-owned hyperthyroid (n = 16) and euthyroid (n = 15) cats over 8 years of age. Prospective observational study. Hyperthyroid and euthyroid cats were enrolled. Rotational thromboelastometry (ROTEM), whole-blood platelet impedance aggregometry (WBPIA) and a point-of-care viscoelastic coagulation monitor (VCM-Vet) were performed immediately after minimally traumatic venipuncture under sedation. Hyperthyroid cats had significantly higher values for variables as assessed by VCM-Vet: A10 (34 [17-47] vs 25 [17-38], P = .003); A20 (39.5 [23-55] vs 31 [21-45], P = .003); and MCF (41 [24-58] vs 35 [22-49], P = .03). Hyperthyroid cats had significantly different values versus the euthyroid cohort as assessed by different ROTEM channels: increased A10, INTEM (61.5 [39-75] vs 54 [23-66], P = .007) and FIBTEM (18 [10-35] vs 13 [2-27], P = .01); increased A20, INTEM (68 [45-78] vs 61 [30-70], P = .006) and FIBTEM (17 [10-34] vs 11 [2-25], P = .002); increased MCF, EXTEM (72 [65-81] vs 69 [34-78], P = .04), INTEM (70 [45-85] vs 62 [35-71], P = .01) and FIBTEM (18 [13-37] vs 14 [3-27], P = .02); increased alpha angle, EXTEM (80 [68-85] vs 76 [41-84], P = .01); shortened CT, EXTEM (52.5 [29-73] vs 60 [52-92], P = .003) and FIBTEM (52.5 [16-75] vs 65 [53-165], P = .001); and decreased ML, FIBTEM (20 [1-36] vs 33 [19-59], P <.001). No significant differences were found with WBPIA. The hyperthyroid cats in this study had evidence of altered hemostasis as assessed by 2 viscoelastic methodologies, and characterized by increased clot amplitude, firmness, and faster coagulation times vs euthyroid controls.

Factors that modulate platelet reactivity as measured by 5 assay platforms in 3429 individuals

BackgroundAssessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing “one-size-fits-all” approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses. ObjectivesOur aim was to compare well-used platelet reactivity assays. MethodsBlood and platelet-rich plasma obtained from the Framingham Heart Study (N = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry (Roche Diagnostics), Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits. ResultsA strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity (r = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed (r = 0.375; adenosine diphosphate Optimul Emax vs light transmission aggregometry large area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid–induced aggregation, and explained substantial variance (β = −1.735; P = 4.59 × 10−780; variance proportion = 46.2%) and P2Y12 antagonists blocking adenosine diphosphate responses (β = −1.612; P = 6.75 × 10−27; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity. ConclusionCaution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.

Prospective evaluation of platelet function and fibrinolysis in 20 dogs with trauma.

To determine platelet function and assess fibrinolysis in dogs following trauma using multiple electrical impedance aggregometry and a modified thromboelastographic (TEG) technique. To determine if the severity of trauma, as assessed by the Animal Trauma Triage (ATT) score and clinicopathological markers of shock, is associated with a greater degree of platelet dysfunction and fibrinolysis. University teaching hospital. Twenty client-owned dogs with trauma (occurring <24h prior to admission and blood sampling) and ATT score of >4 were prospectively recruited. A control group of 10 healthy dogs was included. None. Platelet function was measured using multiple electrode platelet aggregometry (MEPA) utilizing arachidonic acid, ADP, and collagen agonists. Fibrinolysis was assessed in citrated whole blood with the addition of tissue plasminogen activator (tPA; 50U/mL) using kaolin-activated TEG. Conventional statistical analysis was performed to compare coagulation parameters between the groups and assess linear correlations. Median (interquartile range) ATT score was 5 (5-7), and 65% (n=13) of dogs suffered polytrauma. Mean(±SD) time from trauma to blood sampling was 9hours (±6). Median (interquartile range) shock index and plasma lactate concentration were 1.1 (0.7-2.0, n=16) and 2.9mmol/L (0.9-16.0, n=18), respectively. Four dogs did not survive to discharge (20%). There were no differences between the trauma and control group coagulation variables. A moderate negative correlation between ATT score and area under the curve for ADP was found (P=0.043, r2 =-0.496). Preliminary evaluation of platelet function measured by MEPA, and fibrinolysis measured by tPA-modified TEG, is not significantly different in this population of dogs with traumatic injury compared to healthy dogs.

Soluble urokinase plasminogen activator receptor, platelet aggregation, and carotid plaque thickness in diabetes: A cross-sectional analysis

BackgroundSoluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker strongly linked with cardiovascular disease in diabetes. By investigating its association with platelet aggregation levels and carotid plaque thickness, we can potentially improve the characterization of cardiovascular pathophysiology in type 1 (T1D) and type 2 diabetes (T2D). MethodsSuPAR was measured post-hoc in plasma collected in two cross-sectional studies including 1) individuals with T1D and 2) individuals with T2D without known cardiovascular disease. Platelet aggregation was analyzed employing impedance aggregometry using the agonists arachidonic acid (ASPI), adenosine diphosphate (ADP), and thrombin receptor activating peptide-6 (TRAP). Carotid plaque thickness was assessed bilaterally, and the thickest plaque (cPTmax) was analyzed. Correlations and associations of suPAR with platelet aggregation and cPTmax were assessed with Pearson's R and linear regressions adjusted for sex, age, C-reactive protein, estimated glomerular filtration rate, and platelet levels (only for aggregation analysis). ASPI analyses were only performed in participant not receiving aspirin treatment. Standardized estimates with 95 % confidence intervals are presented. ResultsTwo-hundred eighty-three with T1D (mean age 55 years, 44 % women) and 493 individuals with T2D (mean age 65 years, 33 % women) were included. SuPAR levels were positively correlated to platelet aggregation in both T1D (ADP: R = 0.18, p = 0.003; ASPI R = 0.13, p = 0.026) and T2D (ADP R = 0.14, p = 0.002; ASPI R = 0.14, p = 0.017). In adjusted linear regressions higher SuPAR levels were associated with higher ADP induced platelet aggregation in both T1D (26 (8, 44) AU/min, p = 0.004) and T2D (22 (3, 42) AU/min, p = 0.025), while associations to the other agonists were not conclusive. Higher suPAR levels were associated with thicker cPTmax in T1D (0.19 (0.07, 0.32) mm, p = 0.003), but not in T2D (p = 0.94). ConclusionHigher levels of suPAR, an inflammatory generalized disease biomarker, were associated with increased platelet aggregation and thicker cPTmax, in type 1 diabetes. Similar associations with platelet aggregation were found in type 2 diabetes, but not for carotid plaque thickness. These results suggest distinct cardiovascular pathomechanisms in T1D and T2D, although prospective studies are needed to confirm these findings.

NLRP3 Inflammasome-Mediated Platelet Activation and Thrombus Formation in Sickle Cell Mice Can be Targeted By the BTK Inhibitor Ibrutinib

Introduction: Platelets are abnormally activated in sickle cell disease (SCD) and are involved in promoting vascular occlusions and thrombosis. The NOD like receptor protein 3 (NLRP3) inflammasome is a critical inflammatory mediator that has recently been found to be upregulated in platelets in SCD. Our recent discovery of Bruton Tyrosine Kinase (BTK) as a regulator of the NLRP3 inflammasome has provided an opportunity to suppress NLRP3 inflammasome activation in platelets via the BTK inhibitor ibrutinib. The role of the NLRP3 inflammasome in regulating platelet activation in SCD and its potential targetability through BTK inhibition is poorly understood. Methods: Townes SCD mice, which express human sickle hemoglobin (HbSS), and control mice (HbAA) at age 16 to 28 weeks were used for this study. Where indicated, mice were injected with the NLRP3 inhibitor MCC950, the BTK inhibitor ibrutinib or vehicle control 4 hours prior to blood collection. NLRP3 inflammasome activation was assessed in isolated platelets with fluorescent labeled inhibitor of caspase-1 (FLICA) assay and immunofluorescence staining of intraplatelet NLRP3 and the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) with confocal microscopy. We measured platelet surface expression of P-selectin (CD62P), platelet ATP secretion, platelet spreading, platelet aggregation, and in vitro thrombus formation using flow cytometry, luminescence, scanning electron microscopy, whole blood impedance aggregometry, and a flow chamber with blood perfused at shear rates of 1,700 s -1, respectively. Isolated platelets or blood samples were treated with the NLRP3 activator nigericin where indicated. Collagen was used as platelet agonist. Data are shown as mean ± SD. Results: We found increased platelet P-selectin expression (CD62P%: 74.57 ± 3.03 vs 35.18 ± 0.73, p&amp;lt;0.01), platelet ATP secretion (AUC: 16.24 ± 2.17 vs 3.16 ± 0.94, p&amp;lt;0.01), platelet spreading (spreading area, µm 2: 5.47 ± 1.97 vs 2.35 ± 0.23, p&amp;lt;0.01), platelet aggregation (AUC: 68.18 ± 6.84 vs 36.28 ± 3.29, p&amp;lt;0.01), and thrombus formation (thrombus area %: 64.24 ± 14.64 vs 27.44 ± 5.99, p&amp;lt;0.05) in blood from SCD mice as compared to control mice. NLRP3:ASC speck formation (Pearson coefficient: 0.62 ± 0.03 vs 0.38 ± 0.02, p&amp;lt;0.001) and caspase-1 activation levels (RFUs: 476.6 ± 54.84 vs 237.8 ± 50.09, p&amp;lt;0.001) were elevated in platelets from SCD mice as compared to control mice, indicating platelet NLRP3 inflammasome activation in SCD mice. Next, we investigated the role of the NLRP3 inflammasome in regulating platelet function in SCD mice. Treatment of mice with the NLRP3 inhibitor MCC950 resulted in a reduction of upregulated platelet P-selectin expression (CD62P%: 33.55 ± 17.21 vs 74.23 ± 9.95, p&amp;lt;0.001), platelet ATP secretion (AUC: 5.98 ± 1.2 vs 13 ± 2.91, p&amp;lt;0.001), platelet spreading (spreading area, µm 2: 2.42 ± 0.75 vs 5.47 ± 1.97, p&amp;lt;0.001), platelet aggregation (AUC: 39.85 ± 2.5 vs 64.65 ± 6.5, p&amp;lt;0.001), and thrombus formation (thrombus area %: 41.63 ± 7.78 vs 70.22 ± 5.5, p&amp;lt;0.001) (MCC950 vs vehicle) in SCD mice but had no effect in control mice. We next investigated the effect of the BTK inhibitor ibrutinib. Injection of ibrutinib interfered with elevated platelet NLRP3 inflammasome activation in SCD mice, as shown by quantification of intraplatelet NLRP3:ASC specks (Pearson coefficient: 0.56 ± 0.04 vs 0.72 ± 0.03, p&amp;lt;0.001) and platelet caspase-1 activity (RFUs: 193 ± 55.24 vs 386.8 ± 88.08, p&amp;lt;0.001) (ibrutinib vs vehicle) but had no effect in control mice. Ibrutinib treatment decreased upregulated platelet P-selectin (CD62P%: 37.81 ± 10.65 vs 64.74 ± 17.73, p&amp;lt;0.01), platelet ATP secretion (AUC: 6.78 ± 0.24 vs 13.9 ± 1.12, p&amp;lt;0.001), platelet spreading (spreading area, µm 2: 2.32 ± 0.79 vs 6.45 ± 0.99, p&amp;lt;0.001), platelet aggregation (AUC: 40.08 ± 3.56 vs 62.13 ± 3.99, p&amp;lt;0.001), and thrombus formation (thrombus area, %: 44.42 ± 8.24 vs 66.97 ± 11.83, p&amp;lt;0.001) (ibrutinib vs vehicle) in SCD mice. The NLRP3 activator nigericin reversed these inhibitory effects of ibrutinib in SCD mice, indicating that ibrutinib interferes with platelet activation and thrombus formation in SCD mice through the NLRP3 inflammasome. Conclusion: The NLRP3 inflammasome is a critical mediator of platelet activation and thrombus formation in SCD mice, which is targetable by the BTK inhibitor ibrutinib, making it a potentially useful therapeutic approach for managing the prothrombotic state in SCD.

Antiplatelet effect of acetylsalicylic acid in patients early after cardiac valve surgery: comparative investigation of the impairment of platelet inhibition by dipyrone versus acetaminophen

Abstract Background Low-dose acetylsalicylic acid (ASA) is recommended in current guidelines for a period of 3 months after surgical implantation of a bioprosthesis in aortic position or after surgical repair of the mitral valve. Dipyrone (D; metamizole) and acetaminophen (A; paracetamol) are frequently used non-opioid analgesics after surgery. The active metabolite of dipyrone (4-methylaminoantipyrine) is a reversible inhibitor of cyclooxygenase-1 and an interaction with the antiplatelet effect of ASA in more than 50 % of patients has been reported. Material and methods We enrolled 162 patients scheduled for cardiac surgery in this prospective investigator-initiated clinical study. Patients were randomized at day 1 after surgery to either 4 x 1g dipyrone iv/day or 4 x 1g acetaminophen iv/day. All patients were on ASA 100 mg daily (200 mg at day 1). The primary endpoint was the proportion of patients with adequate antiplatelet response to ASA assessed via determination of thromboxane B2 in platelet rich plasma after stimulation with arachidonic acid (AA) at day 4/5 after surgery (cutpoint ≤209.8 ng/ml thromboxane B2). Key secondary endpoints comprised platelet reactivity assessed by light transmission aggregometry [LTA] and by multiple electrode impedance aggregometry [MEIA]. Results 15 patients dropped out of the study. Thus, data from 71 patients randomized to dipyrone and 76 patients randomized to acetaminophen were available for data analysis. Patients had a mean age of 66.5±7.6 years and 97 were male and 50 female. Aortic valve was replaced in 92 patients, 54 underwent mitral valve reconstruction and combined surgery was performed in a single patient. The primary endpoint analyzed at day 4/5 after surgery is shown in the Figure. The proportion of non-responders was 12.5% (D) and 3.0% (A), respectively. Platelet reactivity at day 4/5 after surgery was assessed after stimulation with arachidonic acid by LTA or MEIA. No statistically significant difference between the treatment strata was determined in both assays: Maximum aggregation in LTA was 5.0% (median; 95% confidence interval [95% CI]: 4.0 - 8.0%) in D+ASA and 6.0% (95% CI: 5.0 - 11.0%) in A+ASA (p=0.528) and MEIA AUC was 345.5 AU*min (95% CI: 268.0 - 419.0) in D+ASA and 276.5 AU*min (95% CI: 241.0 - 330.0) in A+ASA (p=0.098). Plasma concentrations of MAA in patients treated with dipyrone were higher in patients identified as responders to ASA (86.6 µM; 95% CI: 67.9 - 102.4 µM) compared to non-responders (31.0 µM; 95% CI: 2.9 – 146.7 µM; p=0.0147). Summary and conclusion The proportion of non-responders to ASA is higher in patients treated with dipyrone compared to acetaminophen. The proportion of non-responders in the dipyrone-treated group in our study is substantially lower than reported in previous studies. High plasma concentrations of MAA in responders may contribute to enhanced inhibition of COX-1 in patients treated with dipyrone.