Platelet Aggregation In Blood Samples Research Articles

Effects of ibrutinib on in vitro platelet aggregation in blood samples from healthy donors and donors with platelet dysfunction

ABSTRACT Background: Ibrutinib, a first-in-class, once-daily inhibitor of Bruton's tyrosine kinase (BTK), is approved in the US and EU for the treatment of various B-cell malignancies. In clinical studies, BTK inhibitors have been associated with increased bleeding risk, which may result from BTK inhibition in platelets. Methods: To better understand the mechanism of ibrutinib in bleeding events, we isolated platelet-rich plasma from healthy donors (n = 8) and donors with conditions associated with impaired platelet function or with potentially increased bleeding risk (on hemodialysis, taking aspirin, or taking warfarin; n = 8 each cohort) and used light transmission aggregometry to assess platelet aggregation in vitro after exposure to escalating concentrations of ibrutinib, spanning and exceeding the pharmacologic range of clinical exposure. Results: Platelet aggregation was induced by agonists of 5 major platelet receptors: adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP6), ristocetin, collagen, or arachidonic acid (AA). Platelet aggregation induced by ADP, TRAP6, ristocetin, and AA was not meaningfully inhibited by the maximal concentrations of ibrutinib (10 µM). In contrast, collagen-induced platelet aggregation was dose-dependently inhibited by ibrutinib in all donor cohorts (maximum aggregation % with 10 μM ibrutinib, −64% to −83% of agonist activity compared to control agonist samples but without ibrutinib). Conclusion: These results confirm prior reports and support a mechanistic role for the inhibition of collagen-induced platelet aggregation in bleeding events among susceptible individuals receiving ibrutinib therapy.

Effect of Bruton's tyrosine kinase inhibitors on platelet aggregation in patients with acute myocardial infarction

AimsDespite widespread use of dual antiplatelet therapy in acute myocardial infarction, there remains a residual risk of morbidity and mortality. Bruton's Tyrosine Kinase inhibitors have been found to inhibit platelet aggregation through the Glycoprotein VI collagen-mediated pathway. The Bruton's Tyrosine Kinase inhibitor, Ibrutinib is used in the management of haematological malignancies and another Bruton's Tyrosine Kinase inhibitor, ONO-4059 (also known as tirabrutinib), is in clinical development. This is an observational study to evaluate the effects of Ibrutinib and ONO-4059 on platelet aggregation after acute myocardial infarction. Methods and resultsTwenty patients with a confirmed diagnosis of acute myocardial infarction were enrolled and blood samples obtained within 48 h of hospital admission. All patients were on dual antiplatelet therapy; aspirin plus a P2Y12 inhibitor (clopidogrel or ticagrelor). Blood samples were treated ex vivo with increasing concentrations of Ibrutinib (0, 0.5, 1, 2 μM) and ONO-4059 (0, 0.2, 0.5, 1 μM). Platelet aggregation was measured in response to collagen using a Multiplate analyser to estimate the area under the curve, with lower values indicating lower platelet aggregation. The median age was 63 years and 80% were male. The median area under the curve values for Ibrutinib concentrations 0 (control), 0.5, 1 and 2 μM were 18.5, 8 (P = 0.0004), 4.5 (P < 0.0001) and 2 (P < 0.0001) units and for ONO-4059 concentrations 0 (control), 0.2, 0.5 and1μM, median area under the curve values were 13, 12 (P = 0.7), 6.5 (P = 0.0001) and 5.5 (P = 0.0004 compared to control). ConclusionThe Bruton's Tyrosine Kinase inhibitors, Ibrutinib and ONO-4059, show further inhibition of platelet aggregation in blood samples from patients with acute myocardial infarction, receiving dual antiplatelet therapy in a dose dependent manner. These results provide a rationale for Bruton's Tyrosine Kinase inhibitors to be tested as a potential new antiplatelet strategy for acute myocardial infarction.

Functionalized Nanoparticles: NIR‐Triggered Release of Nitric Oxide with Upconversion Nanoparticles Inhibits Platelet Aggregation in Blood Samples (Part. Part. Syst. Charact. 2/2018)

Functionalized Nanoparticles: NIR‐Triggered Release of Nitric Oxide with Upconversion Nanoparticles Inhibits Platelet Aggregation in Blood Samples (Part. Part. Syst. Charact. 2/2018)

NIR‐Triggered Release of Nitric Oxide with Upconversion Nanoparticles Inhibits Platelet Aggregation in Blood Samples

AbstractThere is a great challenge to overcome the limitation of tissue penetration depth, while maximizing the benefit of light‐triggered biochemical cascades in a well‐defined mode simultaneously. Here, a new method of near‐infrared (NIR) light‐triggered release of nitric oxide (NO) by developing upconversion nanoparticles (UCNPs)‐based conjugate chemistry is reported. As the key nanotransducer in the design, core–shell‐structured UCNPs are encapsulated with a layer of SiO2 and then covalently linked with a potent NO‐releasing donor (S‐nitroso‐N‐acetyl‐dl‐penicillamine, SNAP). It is featured with highly localized breakage of chemical bonds of SNAP molecules by NIR–UV upconversion, enabling simultaneous NO release in a light dosage‐dependent manner. The biological effects of NO releasing are demonstrated by cellular imaging and inhibition of platelet aggregation from blood samples. This work provides a flexible and robust platform to generate cell‐signaling gas molecules trigged by NIR laser with deep tissue penetration.

In Vitro Effects of Pantoprazole on Platelet Aggregation in Blood Samples From Clopidogrel and Aspirin-treated Patients.

Platelet inhibition during treatment with the antiplatelet drug clopidogrel is prone to great interindividual variability and is believed to be affected by several factors such as genetics and drug-drug interactions. Proton pump inhibitors have been shown to interfere with the liver metabolism of clopidogrel. However, there are limited data on any direct effects proton pump inhibitors may have on clopidogrel. The aim of the study was to evaluate whether the in vitro addition of pantoprazole affects platelet aggregation in blood samples from clopidogrel and aspirin-treated patients. Blood samples were drawn from 66 patients on clopidogrel and aspirin who underwent coronary angiography. Platelet aggregation was analyzed using the bed-side Plateletworks assay before and after the addition of 2 different amounts of pantoprazole. The addition of 2.5 μL (4 mg/mL) pantoprazole, final concentration 0.01 mg/mL, was followed by a significant reduction (26%, P ≤ 0.001) of platelet aggregation, which was further reduced (39%, P ≤ 0.001) when a higher dose, 10 μL (4 mg/mL), final concentration 0.04 mg/mL, was added. In conclusion, platelet aggregation was significantly decreased by in vitro addition of pantoprazole. To explore the clinical relevance of this, future studies are needed.

Effects of fibrinogen and platelet supplementation on clot formation and platelet aggregation in blood samples from cardiac surgery patients

IntroductionBleeding after cardiac surgery may be caused by surgical factors, impaired haemostasis, or a combination of both. Transfusion of blood products is used to improve haemostasis, but little is known about what combination is optimal. We hypothesized that addition of both fibrinogen and platelets to blood samples from cardiac surgery patients would improve clot formation and platelet aggregation to a greater extent than if the components were added separately. Materials and MethodsIncreasing doses of fibrinogen concentrate (+0.5, 1.0, and 1.5g·l-1) and/or platelet concentrate (+46, 92, and 138×109 platelets l-1) were added to postoperative blood samples from 15 cardiac surgery patients. Clot formation was assessed with rotational thromboelastometry and platelet aggregation was assessed with multiple-electrode aggregometry before and after addition of the blood products. The effects of the different additives were compared. Results and ConclusionsEx vivo supplementation with fibrinogen or platelet concentrate resulted in significantly shortened clotting time and improved clot strength in a dose-dependent manner. Combination of fibrinogen and platelets further improved the clotting time and strength. Platelet supplementation enhanced platelet aggregation in a dose-dependent manner while fibrinogen had no or reducing effect. Combining fibrinogen and platelets improved platelet aggregation less than the use of platelets alone. In conclusion, combined platelet and fibrinogen supplementation of blood samples from cardiac surgery patients had an additive effect on clot formation compared to the individual components, but it resulted in less platelet aggregation than with platelet supplementation alone. These results may have implications for clinical transfusion protocols.

Ethylenediaminetetraacetic Acid (EDTA) - dependent pseudothrombocytopenia: a case report.

We present a case of 26-year-old female patient who was presented as a case of Isolated Thrombocytopenia and found to have EDTA induced Pseudo-Thrombocytopenia with no associated pathologies. Pseudo-thrombocytopenia (PTP) is defined by falsely low platelet counts on automated analysers and is caused by in vitro phenomena including large platelet aggregates in blood samples. Platelet aggregates on account of their large size are frequently not included in the platelet window of auto-analysers, but are counted as leukocytes, leading to misleadingly low platelet counts and high leukocyte counts [1]. The phenomenon can be suspected on examining the platelet histogram and review of the smear should confirm the presence of aggregates. Ethylenediaminetetraacetic acid (EDTA) is a well-known anticoagulant since early 1950s and it has certain advantages over other anticoagulants [2]. It inhibits clotting by removing or chelating calcium from the blood. EDTA most important advantage is that it does not distort blood cells, making it ideal for the most hematological tests. It is known to cause erroneous results of platelet counts by automated hematological analysers yielding low platelets counts. This relatively rare phenomenon has been found in the normal population as well as being associated with some disease entities. It occurs with an incidence of approximately 0.1% of the normal population [3]. it has never been reported to be associated with bleeding tendency or dysfunction of platelets. Far from being a harmless curiosity, undiagnosed PTP is a pitfall leading to unwarranted investigations, platelet transfusions, and treatment with glucocorticoids and may lead to surgical procedures being cancelled. A 26-year-old female patient was referred to our lab for investigations as a case of newly diagnosed thrombocytopenia. She presented to a local physician with fever and Upper Respiratory Tract Infection (URTI). The CBC revealed normal parameters apart from a low platelet count; 10 x10^9/L. A blood film [Table/Fig-1] was ordered and it revealed platelet aggregations. History, physical and systemic examination was unremarkable. Other laboratory investigations were normal as well as brain CT scan and abdominal ultrasound. The lab asked for CBC to be drawn in a citrate tube as well as a Heparin tube to diagnose if this could be an EDTA induced platelet clumping. The CBC and the blood film results were the same in both tubes and similar to the EDTA tube results except that the count was higher with the Heparin tube; 72x10^9/L. [Table/Fig-1]: Blood film revealing platelet aggregations EDTA induced PTP was first reported by Gowland et al., (1969). It is attributed to EDTA induced alteration of surface glycoproteins (GP) and anionic phospholipids, which enables binding of antiplatelet antibodies, which in turn cause agglutination. Antibodies against the platelet GPIIb/IIIa have been reported in this context [4]. Platelet agglutinins generally belong to the immunoglobulin M class. However, immunoglobulin G and immunoglobulin A are also described. Around 20% of cases with EDTA induced PTP show the phenomenon in citrate anticoagulant as well [5]. Oxalate and heparin have also been implicated in PTP. Normal platelet function seems to be a criterion for platelet aggregation. Some resources state that clumping of platelets in patients with EDTA- induced Pseudo-Thrombocytopenia can be prevented by the use of other anticoagulants such as Sodium Citrate or Heparin. Other resources have found that even these agents can induce platelet clumping as well. This is comparable to the results in our case. We drew blood of the pateint in three tubes containing three different types of anticoagulants; EDTA, Sodium Citrate and Heparin. The three blood samples were run in fully automated Hematology analyser. The platelet count with the EDTA tube was 11.0 x10^9/L, with the Sodium Citrate tube was 22.0 x10^9/L and with the Heparin tube was 72 x10^9/L. The peripheral blood morphology of the three blood samples revealed platelets aggregations. A prospective study of 20,761 routine clinical blood specimens was conducted to evaluate the incidence and causes of thrombocytopenia [6]. It revealed that the incidence was 0.15% and 72% of which had EDTA induced platelet clumping, 28% had spuriously low platelet counts due to large platelets. Another study states that when Theophylline was used as an anticoagulant in blood samples showing aggregation with EDTA, blood film examination revealed no platelet clumping [6]. Kamath et al., [1] reported resolution of Pseudothrombocytopenia using 37°C incubation and Kanamycin. Unrecognized Pseudo-Thrombocytopenia may result in unnecessary laboratory testing and unwarranted interventions. Examination of a well prepared peripheral blood smear is mandatory for every case of thrombocytopenia to rule out platelet clumping (Pseudo-Thrombocytopenia). For a definitive diagnosis of EDTA Induced Thrombocytopenia, the simultaneous collection of blood into EDTA citrate and Heparin containing or other anticoagulant containing tubes may provide a simple, rapid mean of identifying the presence of EDTA- Induced Pseudo-Thrombocytopenia.

In vitro effects of lipid emulsion on platelet function and thromboelastography in canine blood samples

To determine whether soybean oil emulsion has an in vitro effect on platelet aggregation and thromboelastography in blood samples obtained from healthy dogs. 12 healthy adult dogs. Blood samples were collected from each dog into tubes containing EDTA, hirudin, or sodium citrate for a CBC, collagen- and ADP-induced impedance aggregometry, or thromboelastography, respectively. Whole blood platelet aggregation, determined with ADP or collagen agonists, was measured in blood samples containing hirudin and final lipid concentrations of 0, 1, 10, and 30 mg/mL. The thromboelastographic variables R (reaction time), K (clotting time), α angle, and maximum amplitude were evaluated in blood samples containing sodium citrate and final lipid concentrations equivalent to those used for assessment of platelet aggregation. Median maximum ADP- and collagen-induced platelet aggregation in blood samples containing 1, 10, or 30 mg of lipid/mL did not differ significantly from the value for the respective lipid-free blood sample. Maximum amplitude determined via thromboelastography was significantly reduced in blood samples containing 10 and 30 mg of lipid/mL, compared with findings for lipid-free blood samples. Values of other thromboelastographic variables did not differ, regardless of lipid concentrations. Maximum amplitude determined via thromboelastography in canine blood samples was significantly affected by the addition of lipid to final concentrations that are several orders of magnitude higher than clinically relevant lipid concentrations in dogs. Lipid treatment appears to have no significant effect on hemostatic variables in dogs, although clinical studies should be performed to confirm these in vitro findings.

Pseudothrombocytopenia observed with ethylene diamine tetra acetate and citrate anticoagulants, resolved using 37°C incubation and Kanamycin

Pseudothrombocytopenia (PTP) is defined by falsely low platelet counts on automated analyzers caused by in vitro phenomena including large platelet aggregates in blood samples. Diagnosis and resolution of PTP is crucial as it can lead to unwarranted interventions. We discuss a case of PTP in a pre-surgical setting, which was resolved using 37°C incubation and Kanamycin.

Antiplatelet effect of statins is augmented in diabetic rabbits

The effects of various statins on platelet aggregation in blood samples from normal and diabetic rabbits were measured. All of the statins used in our study inhibited platelet aggregation by about 20% at 1μM. Our results show that diabetes increased the rate of platelet aggregation from 48±5% to 72±8%, however, statins inhibited the rate ofplatelet aggregation by about 60% (p<0.01). The addition of leptin (125ng/ml) to blood samples from healthy rabbits increased the aggregation rate to about 64%, but statins decreased this rate to about 26%. Our results indicate that diabetes increases the rate ofplatelet aggregation in rabbits and increases an-tiplatelet efficacy of statins due to interactions with leptin.

Use of the Platelet Reactivity Index by Grotemeyer, Platelet Function Analyzer, and Retention Test Homburg To Monitor Therapy with Antiplatelet Drugs

In 1974, Wu and Hoak described a method for determining circulating platelet aggregates. This method was modified by Grotemeyer in 1983. The platelet reactivity index (PR) is based on the ratio of platelet aggregates in blood samples obtained in different buffer solutions. Platelet aggregates are resolved when blood is sampled in EDTA-buffer, but remain fixed when EDTA-formalin-buffer is used. Generally, the PR is preferred, because in vitro manipulations of platelets are not necessary, and the results are calculated. PR values above 1.05 are suspicious for elevated platelet aggregation. PR values above 1.2 indicate pathological changes in platelet aggregation. The PR is inexpensive (4.0 euro dollars) and rapid to perform. PR values were used successfully to identify nonresponders to secondary prophylaxis with acetylsalicylic acid (ASA), that is, patients suffering from stroke (33%) and patients after cardiac ischemia (18%). Furthermore, elevated PR values correlated significantly with the incidence of arterial thromboembolic complications. The PR correlated well in our prospective study with values received from the retention test Homburg (RT-H) and the platelet function analyzer (PFA-100). The data indicate that the values of the PR seem to be highly predictive for the evaluation of the ASA therapy. However, the PR is not feasible for the determination of the ASA overdosage.

Platelet reactivity index by Grotemeyer

In 1974 Wu and Hoak described a method for determining circulating platelet aggregates. This method was modified by Grotemeyer in 1983. The platelet reactivity index (PR) is based on the ratio of platelet aggregates in blood samples obtained in different buffer solutions. Platelet aggregates are resolved, when blood is sampled in EDTA-buffer, but remain fixed when EDTA-formalin-buffer is used. Generally, the PR is preferred, because in vitro manipulations of platelets are not necessary, and the results are estimated automatically. PR values above 1.05 are suspicious for elevated platelet aggregation. PR values above 1.2 indicate pathological changes in platelet aggregation. The PR is inexpensive (4.0 D ) and rapid to perform. PR-values were used successfully to identify non-responders to secondary prophylaxis with acetylsalicylic acid (ASA), i. e. patients suffering from stroke (33%) and after cardiac ischaemia (18%). Furthermore, elevated PR-values correlated significantly with the incidence of arterial thromboembolic complications. The PR correlated well in a own prospective study (drug monitoring) with values received from the retention test Homburg (RT-H) and the platelet function analyser (PFA-100). These data indicate that the values of the PR seems to be highly predictive for the evaluation of the ASA therapy. However, the PR is not suitable for the determination of ASA overdosage.

Plättchenreaktivitätsindex nach Grotemeyer

ZusammenfassungZur Untersuchung der Plättchenfunktion wurde das von Wu und Hoak 1974 beschriebene Thrombozytenfunktionssystem von Grotemeyer 1983 modifiziert. Dieses System wurde gewählt, weil In-vitro-Manipulationen an den Plättchen nicht mehr notwendig sind und die Testauswertung maschinell (automatische Blutbildbestimmung) erfolgt. Die Testanordnung basiert darauf, dass Plättchenaggregate, die in vivo bestehen oder durch die Blutabnahme entstehen, durch Ethylendiamintetraessigsäure (EDTA) aufgelöst, durch EDTA-Formalin aber sofort fixiert werden. Ein Plättchenreaktivitätsindex nach Grotemeyer (PR) größer als 1,05 ist verdächtig, Werte über 1,2 sicher pathologisch. Der Test ist preiswert pro Messung (4 €) und schnell durchführbar (Blutbildmessungen und Index-Berechnung).Der PR wurde in drei prospektiven Studien zur Identifizierung von Non-Respondern bei der Sekundärprophylaxe (Hirninfarkt, PTCA bei kardialer Ischämie) arterieller Ischämien mit Azetylsalizylsäure (ASS) erfolgreich eingesetzt. So werden bei Hirninfarkt-Patienten bis 33% Non-Responder, bei Patienten mit kardialer Ischämie bis 18% erkannt. Des Weiteren finden sich in diesen Studien in der Gruppe mit pathologischem PR signifikant mehr thromboembolische Endpunkte als in der Gruppe mit normalem PR. In einer eigenen prospektiven Untersuchung zeigten sich signifikante Korrelationen zwischen PR zum RTH (Retentionstest Homburg) und PFA-100 beim therapeutischen Monitoring von ASS. Der PR scheint prädiktiv zur Beurteilung der Effizienz einer Therapie, aber nicht für die Erkennung bekannter unerwünschter Arzneimittelwirkungen von ASS (z. B. Überdosierung).

Monophosphoryl lipid A: a novel nitric oxide-mediated therapy to attenuate platelet thrombosis?

Nitric oxide (NO) is a potent inhibitor of platelet aggregation. However, the benefits of NO-based therapies can be confounded by concomitant hypotension. Monophosphoryl lipid A (MLA) is a nontoxic derivative of endotoxin that purportedly increases nitric oxide synthase (NOS) activity and, presumably, NO production, yet has a hemodynamically benign profile. Thus our aims were to determine whether (a) MLA attenuates in vivo platelet aggregation in damaged and stenotic canine coronary arteries by a NO-mediated mechanism but without reductions in arterial pressure; and (b) the platelet inhibitory effects are manifest in vitro. To address the first aim, anesthetized dogs underwent coronary injury + stenosis, resulting in cyclic variations in coronary blood flow (CFVs) caused by the formation/dislodgement of platelet-rich thrombi. In protocol I, dogs received MLA (100 microg/kg + 40 microg/kg/h) or vehicle beginning 15 min before stenosis. Protocol II was identical, except the NOS inhibitor aminoguanidine was coadministered with MLA/vehicle. Coronary patency was assessed throughout the initial 3 h after injury + stenosis. Infusion of MLA did not result in hypotension. However, in protocol I, the median nadir of the CFVs was higher (2.1 vs. 0.8 ml/min; p < 0.05), median duration of total thrombotic occlusion tended to be reduced (0 vs. 10.4 min; p = 0.1), and mean flow-time area, expressed as a percentage of baseline flow, was increased (53 +/- 9% vs. 33 +/- 3%; p < 0.05) in MLA-treated versus vehicle-treated dogs. In contrast, in protocol II, vessel patency was comparable in both groups. Finally, whole blood impedance aggregometry (protocol HI) revealed a significant reduction in the in vitro platelet aggregation in blood samples receiving exogenous MLA, which was blocked by coadministration of exogenous aminoguanidine. Thus MLA attenuates platelet-mediated thrombosis in both damaged and stenotic canine coronary arteries and in vitro, possibly by an NO-mediated mechanism, but without concomitant hypotension.

Platelet aggregation in aortic and coronary venous blood in patients with and without coronary disease. 3. Role of tachycardia stress and propranolol.

We studied 16 patients with coronary artery disease (CAD) to evaluate platelet aggregation in blood samples withdrawn simultaneously from the aorta and coronary sinus. At rest, mean platelet aggregation in coronary venous blood was significantly lower than that in aortic blood. Platelet counts in coronary venous blood were also lower than in the aortic blood in each of the six CAD patients in whom counts were done. Platelet aggregation was lower in seven patients who were taking propranolol than in the remaining nine who were not taking propranolol. During tachycardia stress, platelet aggregation increased in all patients, but the magnitude of increase was greater in patients not taking propranolol. In four other patients without CAD, platelet aggregation and counts were also studied in the same fashion and were similar in both the aortic and coronary venous blood. These data suggest that in certain CAD patients, platelet consumption or destruction within atherosclerotic vasculature may occur. Propranolol may reduce platelet aggregation at rest and modify excessive aggregation during tachycardia stress in certain CAD patients.