Whole Blood Aggregation Research Articles

Synthesis, characterization and thrombolytic activity evaluation of novel pyrazolo[3,4-b]pyridine-4-carbohydrazide derivatives

Thrombosis, a leading cause of numerous fatalities, particularly in developed nations, accounts for thousands of deaths. The contemporary lifestyle inevitably leads to occurrences of venous thrombosis, including deep vein thrombosis and thromboembolism. This article presents a groundbreaking study where a novel series of N′-(arylmethylidene)-6-(p-methoxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b] pyridine-4-carbohydrazide derivatives were efficiently synthesized and screened for thrombolytic activities. Compounds 4a, 4c, 4d, and 4h demonstrated the best antiplatelet activities when using different agonists such as arachidonic acid and collagen. These compounds showed >50 % activity by using agonist collagen and inhibited >50 % platelet aggregation in whole human blood. Compounds 4c and 4d showed the greatest inhibitory effect of 97.98 % and 98.97 %, respectively as compared to other synthesized compounds and aspirin. For platelet aggregation using arachidonic acid, all the synthesized compounds 4a, 4c, 4d and 4h shown >93 % inhibition than that of aspirin. In computational studies, ligands 4c(ap) and 4d(ap) exhibited potent interaction with the COX-1 active site, suggesting inhibitory potential against thrombolytic activities. Ligand 4c(ap) scored 8896 and ACE of -418.31 kcal/mol, while 4d(ap) scored 8742 and ACE of -417.43 kcal/mol. Similarly, ligands 4c(ap) and 4h(ap) displayed outstanding docking scores at the COX-2 active site, with 4c(ap) scoring 8574 (ACE:301.49 kcal/mol) and 4h(ap) scoring 8424 (ACE:276.16 kcal/mol). Synthesized compounds particularly 4c and 4d have the potential to be used for clinical trials and could be beneficial for the designing and development of novel therapeutics for cardiovascular and thromboembolic ailments.

The differential formation and composition of leukocyte-platelet aggregates induced by various cellular stimulants

BackgroundLeukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood. AimsTo quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators. MethodsPhorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy. ResultsIn the presence of PMA, ADP or TRAP-6, about 17–38 % of neutrophils and 61–77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1. ConclusionsFormation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step.

Advances in D-dimer testing: progress in harmonization of clinical assays and innovative detection methods.

The D-dimer is a sensitive indicator of coagulation and fibrinolysis activation, especially valuable as a biomarker of intravascular thrombosis. Measurement of plasma D-dimer levels plays a crucial role in the diagnosis and monitoring of conditions such as deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. A variety of immunoassays, including enzyme-linked immunosorbent assays, latex-enhanced immunoturbidimetric assays, whole-blood aggregation analysis, and immunochromatography assays, are widely used in clinical settings to determine D-dimer levels. However, the results obtained from different D-dimer assays vary significantly. These assays exhibit intra-method coefficients of variation ranging from 6.4% to 17.7%, and the measurement discrepancies among different assays can be as high as 20-fold. The accuracy and reliability of D-dimer testing cannot be guaranteed due to the lack of an internationally endorsed reference measurement system (including reference materials and reference measurement procedures), which may lead to misdiagnosis and underdiagnosis, limiting its full clinical application. In this review, we present an in-depth analysis of clinical D-dimer testing, summarizing the existing challenges, the current state of metrology, and progress towards harmonization. We also review the latest advancements in D-dimer detection techniques, which include mass spectrometry and electrochemical and optical immunoassays. By comparing the basic principles, the definition of the measurand, and analytical performance of these methods, we provide an outlook on the potential improvements in D-dimer clinical testing.

Convolutional neuronal network for identifying single-cell-platelet-platelet-aggregates in human whole blood using imaging flow cytometry.

Imaging flow cytometry is an attractive method to investigate individual cells by optical properties. However, imaging flow cytometry applications with clinical relevance are scarce so far. Platelet aggregation naturally occurs during blood coagulation to form a clot. However, aberrant platelet aggregation is associated with cardiovascular disease under steady-state conditions in the blood. Several types of so-called antiplatelet drugs are frequently described to reduce the risk of stroke or cardiovascular diseases. However, an efficient monitoring method is missing to identify the presence and frequency of platelet-platelet aggregates in whole blood on a single cell level. In this work, we employed imaging flow cytometry to identify fluorescently labeled platelets in whole blood with a conditional gating strategy. Images were post-processed and aligned. A convolutional neural network was designed to identify platelet-platelet aggregates of two, three, and more than three platelets, and results were validated against various data set properties. In addition, the neural network excluded erythrocyte-platelet aggregates from the results. Based on the results, a parameter for detecting platelet-platelet aggregates, the weighted platelet aggregation, was developed. If employed on a broad scale with proband and patient samples, our method could aid in building a future diagnostic marker for cardiovascular disease and monitoring parameters to optimize drug prescriptions in such patient groups.

Adhesion and Activation of Blood Platelets on Laser-Structured Surfaces of Biomedical Metal Alloys.

The laser surface modification of metallic implants presents a promising alternative to other surface modification techniques. A total of four alloyed metallic biomaterials were used for this study: medical steel (AISI 316L), cobalt-chromium-molybdenum alloy (CoCrMo) and titanium alloys (Ti6Al4V and Ti6Al7Nb). Samples of metallic biomaterials after machining were subjected to polishing or laser modification in two different versions. The results of surface modification were documented using SEM imaging and roughness measurement. After modification, the samples were sterilized with dry hot air, then exposed to citrate blood, washed with PBS buffer, fixed with glutaraldehyde, sputtered with a layer of gold and imaged using SEM to enable the quantification of adhered, activated and aggregated platelets on the surface of biomaterial samples. The average total number, counted in the field of view, of adhered platelets on the surfaces of the four tested biomaterials, regardless of the type of modification, did not differ statistically significantly (66 ± 81, 67 ± 75, 61 ± 70 and 57 ± 61 for AISI 316L, CoCrMo, Ti6Al4V and Ti6Al7Nb, respectively) and the average number of platelet aggregates was statistically significantly higher (p < 0.01) on the surfaces of AISI 316L medical steel (42 ± 53) and of the CoCrMo alloy (42 ± 52) compared to the surfaces of the titanium alloys Ti6Al4V (33 ± 39) and Ti6Al7Nb (32 ± 37). Remaining blood after contact was used to assess spontaneous platelet activation and aggregation in whole blood by flow cytometry. An in-depth analysis conducted on the obtained results as a function of the type of modification indicates small but statistically significant differences in the interaction of platelets with the tested surfaces of metallic biomaterials.

138 Escherichia coli-induced platelet aggregation in human whole blood is partly dependent on complement C3b

138 Escherichia coli-induced platelet aggregation in human whole blood is partly dependent on complement C3b

Comprehensive Approach to Developing Baseline Reference Ranges for Platelet Function

Background. Thrombocytopathies are topical issues of pediatrics. Platelet dysfunction clinically manifests as thrombotic and hemorrhagic events of various severity and location. Platelet function can be evaluated via aggregatometry. Specified parameters for aggregatometry can be used in pediatrics as a standard for evaluating platelet function in peripheral blood. There were no similar studies in children in Russian Federation.Objective. The aim of the study is — to justify the significance of reference ranges (RR) development for platelet aggregatometry in pediatrics.Methods. The study included 30 relatively healthy patients aged from 1 to 18 years. Laboratory tests included complete blood count via automatic hematological analyzer; platelet aggregation in whole blood was evaluated via impedance aggregometer; plasmic hemostasis component (Quick prothrombin), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen were evaluated on automatic coagulation analyzer; molecular genetic study of platelet genes polymorphisms was performed via real-time PCR. Several inducers were used to study platelet aggregation: thrombin receptor activating peptide (TRAP), adenosine diphosphate (ADP), arachidonic acid (ASPI). Instrumental methods included electrocardiography, ultrasound of abdomen, urinary system, and thyroid gland.Results. The study included 16 girls and 14 boys. The median age was 9.4, interquartile range (IQR) was 6.5; 14,1. RR for platelet aggregation indicators were determined for all aggregation inducers (thrombin, ADP, arachidonic acid) as a result of the study.Conclusion. The obtained data will allow to improve the diagnosis approach for platelet dysfunction regarding the established reference ranges. The results of further research may contribute in the development of the algorithm for controlling antiplatelet therapy in patients with contraindications or with genetically determined low sensitivity to thrombocyte aggregation inhibitors based on acetylsalicylic acid.

Effects of uridine and nucleotides on hemostasis parameters

Several purinergic receptors have been identified on platelets which are involved in hemostatic and thrombotic processes. The aim of the present study was to investigate the effects of uridine and its nucleotides on platelet aggregation and hemostasis in platelet-rich plasma (PRP) and whole blood. The effects of uridine, UMP, UDP, and UTP at different final concentrations (1 to 1000 µM) on platelet aggregation were studied using an aggregometer. In PRP samples, platelet aggregation was induced by ADP, collagen and epinephrine 3min after addition of uridine, UMP, UDP, UTP and saline (as a control). All thromboelastogram experiments were performed at 1000 µM final concentrations of uridine and its nucleotides in whole blood. UDP and UTP were also tested in thromboelastogram with PRP. Our results showed that UDP, and especially UTP, inhibited ADP- and collagen-induced aggregation in a concentration-dependent manner. In whole blood thromboelastogram experiments, UDP stimulated clot formation while UTP suppressed clot formation. When thromboelastogram experiments were repeated with PRP, UTP's inhibitory effect on platelets was confirmed, while UDP's stimulated clot forming effect disappeared. Collectively, our data showed that UTP inhibited platelet aggregation in a concentration-dependent manner and suppressed clot formation. On the other hand, UDP exhibited distinct effects on whole blood or PRP in thromboelastogram. These data suggest that the difference on effects of UTP and UDP might have arisen from the different receptors that they stimulate and warrant further investigation with regard to their in vivo actions on platelet aggregation and hemostasis.

Deletion of Annexin A1 in Mice Upregulates the Expression of Its Receptor, Fpr2/3, and Reactivity to the AnxA1 Mimetic Peptide in Platelets.

Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings.

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood.

Platelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy. In this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy. We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process.

Characterization of the impact of immune checkpoint inhibitors on platelet activation and aggregation

Immune checkpoint inhibitors (ICIs) are effective oncological drugs which block cellular check-point receptors typically targeted by tumor immune evasion strategies. Despite their benefits, clinicians have reported treatment-associated thromboembolism during ICI therapy in recent years. Though several theories on this ICI-associated pathogenesis exist, the direct effects of ICIs on platelets remains unknown. We therefore investigated the potential direct and indirect effect of PD-1, PD-L1 and CTLA-4-targeting ICIs on platelet functionality in multifaceted in vitro experiments. Interestingly, we could not observe a clear effect of ICI on platelet aggregation and primary hemostasis in whole blood and platelet concentrate-based assays. Furthermore, the presence of ICIs in toll-like receptor stimulation had no significant impact on platelet surface marker expression. In a second approach, we investigated the indirect immunological impact of ICIs on platelet activation by exposing platelets to supernatants from ICI- and Staphylococcal enterotoxin B-exposed PBMCs. Whereas ICIs affected IL-2 levels in supernatants, we could not detect clear differences in the secretion of pro-thrombogenic factors and platelet responses. The obtained data suggest that the direct influence of ICIs on platelet activation or the influence of altered T cell function on platelet activation cannot be considered a major factor in the development of thrombotic events.

Can Isoquinoline Alkaloids Affect Platelet Aggregation in Whole Human Blood?

Isoquinoline alkaloids have multiple biological activities, which might be associated with positive pharmacological effects as well as negative adverse reactions. As bleeding was suggested to be a side effect of the isoquinoline alkaloid berberine, we decided to ascertain if different isoquinoline alkaloids could influence hemocoagulation through the inhibition of either platelet aggregation or blood coagulation. Initially, a total of 14 compounds were screened for antiplatelet activity in whole human blood by impedance aggregometry. Eight of them demonstrated an antiplatelet effect against arachidonic acid-induced aggregation. Papaverine and bulbocapnine were the most potent compounds with biologically relevant IC50 values of 26.9 ± 12.2 μM and 30.7 ± 5.4 μM, respectively. Further testing with the same approach confirmed their antiplatelet effects by employing the most physiologically relevant inducer of platelet aggregation, collagen, and demonstrated that bulbocapnine acted at the level of thromboxane receptors. None of the alkaloids tested had an effect on blood coagulation measured by a mechanical coagulometer. In conclusion, the observed antiplatelet effects of isoquinoline alkaloids were found mostly at quite high concentrations, which means that their clinical impact is most likely low. Bulbocapnine was an exception. It proved to be a promising antiplatelet molecule, which may have biologically relevant effects.

Risks in Induction of Platelet Aggregation and Enhanced Blood Clot Formation in Platelet Lysate Therapy: A Pilot Study.

Platelet concentrates (PCs) are widely used in regenerative medicine; as it is produced from freeze–thawing PC, platelet lysate (PL) has a longer shelf life. The thrombotic risk of PL therapy needs to be explored since PL and PC contain cytokines that contribute to platelet aggregation and thrombus formation. Whole blood samples of 20 healthy subjects were collected; PL was produced from PCs with expired shelf life through freeze–thawing. The direct mixing of PL with platelet-rich plasma (PRP) or whole blood was performed. In addition, rotational thromboelastometry (ROTEM) was used to investigate whether PL enhanced coagulation in vitro; the effects of fibrinogen depletion and anticoagulants were evaluated to prevent hypercoagulation. The results showed that PL induced platelet aggregation in both PRP and whole blood. In ROTEM assays, PL was shown to cause a significantly lower clotting onset time (COT) and clot formation time (CFT), and a significantly greater α angle and maximum clot firmness (MCF). Compared with the controls, which were 1:1 mixtures of normal saline and whole blood, fibrinogen depletion of PL showed no significant difference in CFT, α angle and MCF. Moreover, heparin- and rivaroxaban-added PL groups demonstrated no clot formation in ROTEM assays. Platelet lysate-induced hypercoagulability was demonstrated in vitro in the present study, which could be prevented by fibrinogen depletion or the addition of an anticoagulant.

Prenylcysteine Oxidase 1 (PCYOX1), a New Player in Thrombosis.

Prenylcysteine Oxidase 1 (PCYOX1) is an enzyme involved in the degradation of prenylated proteins. It is expressed in different tissues including vascular and blood cells. We recently showed that the secretome from Pcyox1-silenced cells reduced platelet adhesion both to fibrinogen and endothelial cells, suggesting a potential contribution of PCYOX1 into thrombus formation. Here, we show that in vivo thrombus formation after FeCl3 injury of the carotid artery was delayed in Pcyox1−/− mice, which were also protected from collagen/epinephrine induced thromboembolism. The Pcyox1−/− mice displayed normal blood cells count, vascular procoagulant activity and plasma fibrinogen levels. Deletion of Pcyox1 reduced the platelet/leukocyte aggregates in whole blood, as well as the platelet aggregation, the alpha granules release, and the αIIbβ3 integrin activation in platelet-rich plasma, in response to adenosine diphosphate (ADP) or thrombin receptor agonist peptide (TRAP). Washed platelets from the Pcyox1−/− and WT animals showed similar phosphorylation pathway activation, adhesion ability and aggregation. The presence of Pcyox1−/− plasma impaired agonist-induced WT platelet aggregation. Our findings show that the absence of PCYOX1 results in platelet hypo-reactivity and impaired arterial thrombosis, and indicates that PCYOX1 could be a novel target for antithrombotic drugs.

Exacerbation of Thrombotic Responses to Silver Nanoparticles in Hypertensive Mouse Model.

With advent of nanotechnology, silver nanoparticles, AgNPs owing majorly to their antibacterial properties, are used widely in food industry and biomedical applications implying human exposure by various routes including inhalation. Several reports have suggested AgNPs induced pathophysiological effects in a cardiovascular system. However, cardiovascular diseases such as hypertension may interfere with AgNPs-induced response, yet majority of them are understudied. The aim of this work was to evaluate the thrombotic complications in response to polyethylene glycol- (PEG-) coated AgNPs using an experimental hypertensive (HT) mouse model. Saline (control) or PEG-AgNPs (0.5 mg/kg) were intratracheally (i.t.) instilled four times, i.e., on days 7, 14, 21, and 28 post-angiotensin II-induced HT, or vehicle (saline) infusion. On day 29, various parameters were assessed including thrombosis in pial arterioles and venules, platelet aggregation in whole blood in vitro, plasma markers of coagulation, and fibrinolysis and systemic oxidative stress. Pulmonary exposure to PEG-AgNPs in HT mice induced an aggravation of in vivo thrombosis in pial arterioles and venules compared to normotensive (NT) mice exposed to PEG-AgNPs or HT mice given saline. The prothrombin time, activated partial thromboplastin time, and platelet aggregation in vitro were exacerbated after exposure to PEG-AgNPs in HT mice compared with either NT mice exposed to nanoparticles or HT mice exposed to saline. Elevated concentrations of fibrinogen, plasminogen activator inhibitor-1, and von Willebrand factor were seen after the exposure to PEG-AgNPs in HT mice compared with either PEG-AgNPs exposed NT mice or HT mice given with saline. Likewise, the plasma levels of superoxide dismutase and nitric oxide were augmented by PEG-AgNPs in HT mice compared with either NT mice exposed to nanoparticles or HT mice exposed to saline. Collectively, these results demonstrate that PEG-AgNPs can potentially exacerbate the in vivo and in vitro procoagulatory and oxidative stress effect in HT mice and suggest that population with hypertension are at higher risk of the toxicity of PEG-AgNPs.

Effect of lactic acid addition to equine whole blood on platelet aggregation measured by impedance aggregometry

Acidemia in sick or injured horses is often due to lactic acid accumulation. Alterations in platelet function and hemostasis are among numerous deleterious effects caused by decreased physiologic pH. We aimed to evaluate the effect of hyperlactatemia and resultant acidemia on platelet aggregation in equine whole blood using impedance aggregometry. Platelet aggregation was measured using the Multiplate analyzer in whole blood from 34 healthy horses at baseline and after in vitro addition of lactic acid to adjust the pH. Platelet aggregation of each sample was quantified by the area under the curve measurement reported by the Multiplate system. The association between platelet aggregation and pH was analyzed using a linear mixed-effects model. The association of baseline platelet aggregation with hematocrits (Hcts), white blood cell (WBC) counts, and platelet counts was evaluated using Pearson's correlations. There was a significant association between acidemia and decreased platelet aggregation. No significant correlations were detected between platelet aggregation and Hct, WBC count, or platelet count. Platelet aggregation measured in healthy horses using the Multiplate analyzer showed substantial variation between animals. Acidemia caused by the addition of lactic acid to equine whole blood was associated with a mild though statistically significant decrease in platelet aggregation. In conjunction with other factors, this change may contribute to morbidity-related disorders of hemostasis, although its precise clinical relevance is uncertain.

Characterization of Two Anti-P-Selectin Monoclonal Antibodies (mAbs): Crizanlizumab Shows Comparable or Stronger Effects Versus Inclacumab across Cell Adhesion Assays in Vitro

Background: Vaso-occlusive crises (VOCs) are the hallmark of sickle cell disease (SCD) and are associated with significant morbidity and mortality. Crizanlizumab, a first-in-class humanized anti-P-selectin IgG2 mAb, is approved in >40 countries to reduce/prevent VOCs in SCD patients aged ≥16 yrs. Inclacumab, a fully human anti-P-selectin IgG4 mAb, is in clinical development for SCD. In vitro data suggest that inclacumab may show stronger affinity to P-selectin and greater maximal inhibition of cell-cell interactions vs crizanlizumab (Geng et al ASH 2020).Aim: We investigated in blinded experiments whether crizanlizumab and inclacumab can be differentiated in terms of P-selectin binding and inhibition of P-selectin-mediated cell-cell interactions, via 5 different in vitro assays, including testing of blood samples from healthy volunteers and SCD patients.Methods: For both mAbs, we assessed: P-selectin affinity (surface plasmon resonance [SPR]/Biacore assay); inhibition of adhesion of P-selectin-expressing cells to P-selectin glycoprotein ligand-1 (PSGL-1; cell adhesion bioassay); inhibition of platelet aggregation in native blood from healthy volunteers (whole blood impedance aggregometry [WBA] and flow cytometry-based platelet-leukocyte aggregate [PLA] assays); and inhibition of adhesion of SCD patient whole blood or isolated leukocytes to P-selectin-coated substrate under physiologically relevant shear rates in microfluidic systems (flow adhesion bioassays).Results: The SPR/Biacore assay indicated that both mAbs recognize P-selectin with binding affinities of the same order of magnitude, although there was a trend towards higher binding affinity with inclacumab (mean equilibrium dissociation constant [K D ± standard deviation]: 12.4 ± 1.0 nM [crizanlizumab] vs 6.7 ± 0.7 nM [inclacumab]). However, the function-related cell adhesion bioassay revealed a significant difference between the mAbs in their ability to inhibit adhesion of P-selectin-expressing cells to PSGL-1. Crizanlizumab showed stronger inhibition than inclacumab (potency vs crizanlizumab reference standard: 98.0% for the crizanlizumab control sample [95% CI 85.2‒112.7%] and 26.2% for the inclacumab sample [95% CI 20.7%‒32.6%]).Native whole blood samples for WBA and PLA assays were provided by 12 and 10 healthy volunteers, respectively. Blocking of platelet (WBA assay) or platelet-leukocyte aggregation (PLA assay) in whole blood was comparable for crizanlizumab and inclacumab, with a clear trend towards stronger inhibition by crizanlizumab. In the WBA assay, the half maximal inhibitory concentration (IC 50) was twice as high with inclacumab (10.36 μg/mL) as crizanlizumab (5.11 μg/mL); similar data were seen in the PLA assay (Figure 1; IC 50: 4.11 vs 2.81 μg/mL for inclacumab vs crizanlizumab).Blood samples were provided by 9 SCD patients for the flow adhesion bioassays. Dose-dependent inhibition of adhesion of whole blood and isolated leukocytes to P-selectin was seen with both crizanlizumab and inclacumab, with no difference observed between them (Figure 2). A mAb concentration of 10 μg/mL resulted in statistically significant inhibition of adhesion by 60‒75%; further increasing the mAb concentration did not result in stronger inhibition of adhesion beyond this threshold. In relation to the clinically approved crizanlizumab dose (5.0 mg/kg), a concentration of 100 μg/mL corresponds with the maximum plasma concentration (C max) and ~10-15 μg/mL corresponds with the trough concentration (C trough).Conclusions: While the SPR/Biacore data suggested higher P-selectin binding affinity for inclacumab compared with crizanlizumab, there was a stronger inhibition of P-selectin-mediated cell adhesion with crizanlizumab vs inclacumab in other well-characterized functional in vitro assays. In microfluidic flow adhesion bioassays, blockage of cell adhesion from SCD whole blood and leukocytes was comparable for both mAbs. In summary, these data indicate that comparable or stronger blockage of cell adhesion with crizanlizumab vs inclacumab does not require superior P-selectin binding affinity. The data from healthy volunteers will be complemented by data from SCD patients for the WBA and PLA assays. Ultimately, clinical data are required to evaluate potential differences in the profiles and efficacy of crizanlizumab and inclacumab as treatments for SCD patients. [Display omitted] DisclosuresRubic-Schneider: Novartis Pharma AG: Current Employment. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ledieu: Novartis Pharma AG: Current Employment, Current holder of stock options in a privately-held company. Garcia: Novartis Pharma AG: Current Employment. Hehlen: Novartis Pharma AG: Current Employment. Burnet-Merlin: Light Chain Bioscience - Novimmune SA: Current Employment; Novartis Pharma AG: Ended employment in the past 24 months. Cochin de Billy: Novartis Pharma AG: Current Employment. Greutmann: Novartis Pharma AG: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Krӧner: Novartis Pharma AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: WO2021087050A1. Verneret: Novartis Pharma AG: Current Employment, Current holder of stock options in a privately-held company. Bruederle: Novartis Pharma AG: Current Employment. Gao: Functional Fluidics: Current Employment. Dajee: Novartis: Current Employment.

Platelet aggregation in whole blood is not impaired by a platelet‐sparing leukoreduction filter and instead depends upon the presence of leukocytes

Recent studies characterizing in vitro hemostatic properties of whole blood (WB) leukoreduced (LR) with a platelet-sparing filter have described subtle, if any, changes to viscoelastic clotting; however, reductions in platelet (PLT) content and impedance aggregometry (IA) responses have been noted. The effects of filtration of WB (i.e., filter-contact effects, reduction in platelet and leukocyte count) have not been rigorously investigated as to their individual impacts on platelet IA responses. WB units from healthy donors were collected and characterized to assess the effects of platelet-sparing leukoreduction (LR) upon the in vitro hemostatic measures of platelet IA and thromboelastometry. Further characterization of platelet IA responses was carried out in WB samples to delineate the effects of platelet count and leukocyte presence/absence upon the response. WB filtration reduced the platelet count and IA responses but had no impact on viscoelastic clotting measures in fresh WB. Experiments revealed that IA responses have a linear correlation with platelet count in both apheresis platelets and WB and that passage of platelets through the WB-LR filter has no impact upon the strength of this response. Further experiments in LR WB showed that addition of autologous leukocytes back to the platelets fully restored the platelet aggregation response to pre-filtration levels. WB filtration results in platelet count reduction and leukocyte removal; however, platelet IA is not degraded by passage through the filter. Apparent declines in platelet IA responses can be fully attributed to the reduction in platelet count and the removal of leukocytes.

Colchicine inhibits ROS generation in response to glycoprotein VI stimulation

Colchicine inhibits coronary and cerebrovascular events in patients with coronary artery disease (CAD), and although known to have anti-inflammatory properties, its mechanisms of action are incompletely understood. In this study, we investigated the effects of colchicine on platelet activation with a particular focus on its effects on activation via the collagen glycoprotein (GP)VI receptor, P2Y12 receptor, and procoagulant platelet formation. Therapeutic concentrations of colchicine in vitro (equivalent to plasma levels) significantly decreased platelet aggregation in whole blood and in platelet rich plasma in response to collagen (multiplate aggregometry) and reduced reactive oxygen species (ROS) generation (H2DCF-DA, flow cytometry) in response to GPVI stimulation with collagen related peptide-XL (CRP-XL, GPVI specific agonist). Other platelet activation pathways including P-selectin expression, GPIIb/IIIa conformational change and procoagulant platelet formation (GSAO+/CD62P+) (flow cytometry) were inhibited with higher concentrations of colchicine known to inhibit microtubule depolymerization. Pathway specific mechanisms of action of colchicine on platelets, including modulation of the GPVI receptor pathway at low concentrations, may contribute to its protective role in CAD.

Electrical impedance vs. light transmission aggregometry: Testing platelet reactivity to antiplatelet drugs using the MICELI POC impedance aggregometer as compared to a commercial predecessor

BackgroundPatients' responses to antiplatelet therapy significantly vary, with individuals showing high residual platelet reactivity associated with thrombosis. To personalize thrombosis management, platelet function testing has been suggested as a promising tool able to monitor the antithrombotic effect of antiplatelet agents in real-time. We have prototyped the MICELI, a miniature and easy-to-use electrical impedance aggregometer (EIA), measuring platelet aggregation in whole blood. Here, we tested the capability of the MICELI aggregometer to quantify platelet reactivity on antiplatelet agents, as compared with conventional light-transmission aggregometry (LTA). MethodsPlatelet aggregation in ACD-anticoagulated whole blood and platelet-rich plasma of healthy donors (n = 30) was evaluated. The effect of clopidogrel, ticagrelor, cangrelor, cilostazol, and tirofiban on ADP-induced aggregation was tested, while aspirin was evaluated with arachidonic acid and collagen. Platelet aggregation was recorded using the MICELI or BioData PAP-8E (Bio/Data Corp.) aggregometers. ResultsThe MICELI aggregometer detected an adequate and comparable dose-dependent decrease of platelet aggregation in response to increments of drugs' concentrations, as compared to LTA (the inter-device R2 = 0.79–0.93). Platelet aggregation in platelet-rich plasma recorded by LTA showed higher sensitivity to antiplatelet agents, but it couldn't distinguish between different drug doses as indicated by saturation of the aggregatory response. ConclusionPlatelet aggregation in whole blood as recorded by EIA represents a better model system for evaluation of platelet reactivity as compared with platelet aggregation in platelet-rich plasma as recorded by LTA, since EIA takes into consideration the modulatory effect of other blood cells on platelet hemostatic function and pharmacodynamics of antiplatelet drugs in vivo. As such, the MICELI impedance aggregometer could be potentially employed for the point-of-care monitoring of platelet function in patients on-treatment for personalized tailoring of their antiplatelet regimen.