Inhibitory effects of resveratrol on platelet activation and thrombosis in colon cancer through regulation of the MAPK and cGMP/VASP pathways.

Thrombosis is the primary cause of death in patients with cancer. Resveratrol inhibits platelet activation, a crucial pathophysiological basis of thrombosis, in healthy individuals. However, its effects and mechanisms of action in patients with colon cancer remain unknown. Here, we investigated the effect of resveratrol on platelet adhesion and aggregation in patients with colon cancer. Through numerous in vitro and in vivo analyses, including flow cytometry, western blotting, ELISA, and immunofluorescence and colon cancer rat models, we demonstrated that resveratrol reduced thrombosis in patients with colon cancer by inhibiting the phosphorylation of the MAPK and activating the cyclic-GMP/vasodilator-stimulated phosphoprotein pathway. These findings demonstrate the potential of resveratrol in reducing thrombosis in patients with colon cancer and could be used to develop novel therapeutic strategies for this condition.

Flow Cytometry and Platelets.

Clinical assessment of platelet activation by flow cytometry is useful in the characterization and diagnosis of platelet-specific disorders and as a measure of risk for thrombosis or bleeding. Platelets circulate in a resting, "unactivated" state, but when activated they undergo alterations in surface glycoprotein function and/or expression level, exposure of granule membrane proteins, and exposure of procoagulant phospholipids. Flow cytometry provides the means to detect these changes and, unlike other platelet tests, is appropriate for measuring platelet function in samples from patients with low platelet counts. The present review will focus on flow cytometric tests for platelet activation markers.

Comprehensive analysis of L-PRF exudate components and their impact on whole blood platelets.

OBJECTIVE: This study assessed the cellular composition and effects of leukocyte-platelet-rich fibrin (L-PRF) exudate on whole blood platelets from healthy volunteers. Key objectives included evaluating leukocyte subpopulations, platelet activation markers, platelet-leukocyte interactions and quantifying inflammatory cytokines within the L-PRF exudate. MATERIALS AND METHODS: L-PRF was obtained from 20 healthy donors. Flow cytometry methodologies were used to assess intracellular calcium kinetics and activated GPIIbIIIa, and P-selectin expression. Leukocyte subpopulations and platelet-leukocyte interactions were characterized using monoclonal antibodies. Inflammatory cytokines (IL-8, IL-1ß, IL-6, IL-10, TNF, IL-12p70) within L-PRF exudate were quantified using a cytometric bead array. RESULTS: The expression of activated GPIIbIIIa, and P-selectin exhibited a significant increase (p < 0.001) when L-PRF exudate was added to platelets of whole blood. Regarding intracellular Ca2+ mobilization, the L-PRF exudate elicited significant responses (p < 0.001). L-PRF exudate contained different leukocytes populations, being TCD4 + the most representative of T cells. It was possible to stablish a profile of cytokines produced by the L-PRF exudate, with human IL-8 cytokine exhibiting the highest average (16.90 pg/mL). CONCLUSIONS: Despite the study limitations, the research yielded important insights: 1- L-PRF exudate can stimulate platelet activation, essential in healing, tissue inflammation and remodeling. 2-The presence of leukocyte subpopulations within L-PRF exudate reflexes its complexity and potential to enhance immune responses. 3-The analysis of inflammatory cytokines within L-PRF exudate revealed its immunomodulatory potential. These findings are valuable evidences for understanding the potential role of L-PRF exudate in regenerative dentistry and medicine, offering innovative therapeutic strategies. CLINICAL RELEVANCE: This research highlights crucial aspects that could significantly influence the clinical use of L-PRF exudate in the oral cavity. The findings support the application of L-PRF exudate in both surgical and regenerative dentistry, facilitating the development of innovative therapeutic strategies to enhance patient outcomes.

Effect of Diminazene Aceturate, an ACE2 activator, on platelet CD40L signaling induced glial activation in rat model of hypertension.

Hypertension causes platelet activation and adhesion in the brain resulting in glial activation and neuroinflammation. Further, activation of Angiotensin-Converting Enzyme 2/Angiotensin (1-7)/Mas Receptor (ACE2/Ang (1-7)/MasR) axis of central Renin-Angiotensin System (RAS), is known to reduce glial activation and neuroinflammation, thereby exhibiting anti-hypertensive and anti-neuroinflammatory properties. Therefore, in the present study, the role of ACE2/Ang (1-7)/MasR axis was studied on platelet-induced glial activation and neuroinflammation using Diminazene Aceturate (DIZE), an ACE2 activator, in astrocytes and microglial cells as well as in rat model of hypertension. We found that the ACE2 activator DIZE, independently of its BP-lowering properties, efficiently prevented hypertension-induced glial activation, neuroinflammation, and platelet CD40-CD40L signaling via upregulation of ACE2/Ang (1-7)/MasR axis. Further, DIZE decreased platelet deposition in the brain by reducing the expression of adhesion molecules on the brain endothelium. Activation of ACE2 also reduced hypertension-induced endothelial dysfunction by increasing eNOS bioavailability. Interestingly, platelets isolated from hypertensive rats or activated with ADP had significantly increased sCD40L levels and induced significantly more glial activation than platelets from DIZE treated group. Therefore, injection of DIZE pre-treated ADP-activated platelets into normotensive rats strongly reduced glial activation compared to ADP-treated platelets. Moreover, CD40L-induced glial activation, CD40 expression, and NFкB-NLRP3 inflammatory signaling are reversed by DIZE. Furthermore, the beneficial effects of ACE2 activation, DIZE was found to be significantly blocked by MLN4760 (ACE2 inhibitor) as well as A779 (MasR antagonist) treatments. Hence, our study demonstrated that ACE2 activation reduced the platelet CD40-CD40L induced glial activation and neuroinflammation, hence imparted neuroprotection.

Marijuana may lead to increased platelet activity in trauma patients.

INTRODUCTION: Delta-9-tetraydrocannabinol (THC) usage is associated with venous thromboembolic events (VTE) in trauma patients. We hypothesized that THC â€‹+ â€‹trauma patients would have less platelet inhibition than THC - patients using thromboelastography with platelet mapping (TEG-PM). METHODS: Results from initial TEG- PM assays and patient's UDS were reviewed between 2019 and 2023. Mean levels of arachidonic acid (AA) and adenosine diphosphate (ADP) percent inhibition were compared by THC status. RESULTS: 793 patients had TEG-PM and UDS data. Mean levels of arachidonic acid (AA) percentage inhibition were 32.6 â€‹± â€‹34.2. AA inhibition was lower for THC â€‹+ â€‹vs THC- patients (THC+ 23.9 â€‹± â€‹27.0 vs THC- 34.3 â€‹± â€‹35.3, P â€‹< â€‹0.001). There was no association between THC status and ADP inhibition (THC+ 32.5 â€‹± â€‹27.1 vs THC- 30.8 â€‹± â€‹28.4, P â€‹= â€‹0.536). DISCUSSION: To our knowledge, our data are the first to suggest a clinically measurable increase in platelet reactivity in THC â€‹+ â€‹trauma patients. More work is needed to determine if addition of aspirin to the chemoprophylaxis strategy for THC â€‹+ â€‹patients would mitigate the known association of THC with VTE.

Whole Blood Aggregometry in Mice.

Aggregometry plays a crucial role in both clinical diagnostics and research within hematology, serving as a fundamental tool for understanding platelet function and its implications in physiological and pathological processes. In research, aggregometry provides insights into platelet aggregation dynamics and aids in understanding the underlying mechanisms of hemostasis, thrombosis, and related disorders. Light transmission aggregometry (LTA) and lumi-aggregometry, as well as whole blood aggregometry, are commonly employed methods. While LTA and lumi-aggregometry allow for specific platelet function assessment under controlled conditions, whole blood aggregometry provides a more physiologically relevant approach by evaluating platelet aggregation within the context of whole blood. Although both methodologies offer unique advantages, whole blood aggregometry allows for preservation of the native cellular environment, simplicity, and potential for better clinical correlation. In a clinical setting, with human blood samples, protocols are established for both LTA and whole blood aggregometry as they are frequently used diagnostic tools. A protocol for LTA and lumi-aggregometry in murine models has been described; however, to date, there is no standardized protocol for whole blood aggregometry in murine models accessible to hematology researchers. This article aims to outline a simple, basic protocol for murine whole blood aggregometry, offering an alternative method to the commonly used LTA aggregometry in research settings. Standardizing whole blood aggregometry protocols in murine models could enhance experimental reliability and facilitate translational research efforts in hematology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Whole blood aggregometry in mice Support Protocol: Phenylhydrazine-induced anemia in wild-type mice Basic Protocol 2: Hematocrit percentage in mice.

Preferential and sustained platelet activation in COVID-19 survivors with mental disorders.

Pre-existing mental disorders are considered a risk factor for severe COVID-19 outcomes, possibly because of higher vascular burden. Moreover, an unconventional platelet activation characterizes COVID-19 and contributes to inflammatory and thrombotic manifestations. In the light of the inflammation theory of mental disorders, we hypothesized that patients with mental disorders could be sensitive to the SARS-CoV-2 elicited platelet activation. We investigated platelet activation in 141 COVID-19 survivors at one month after clearance of the virus, comparing subjects with or without an established pre-existing diagnosis of mental disorder according to the DSM-5. We found that platelets from patients with a positive history of psychiatric disorder underwent unconventional activation more frequently than conventional activation or no activation at all. Such preferential activation was not detected when platelets from patients without a previous psychiatric diagnosis were studied. When testing the effects of age, sex, and psychiatric history on the platelet activation, GLZM multivariate analysis confirmed the significant effect of diagnosis only. These findings suggest a preferential platelet activation during acute COVID-19 in patients with a pre-existing psychiatric disorder, mediated by mechanisms associated with thromboinflammation. This event could have contributed to the higher risk of severe outcome in the psychiatric population.

Reduced platelet activation and thrombus formation in male transgenic model mice of Alzheimer's disease suggests early sex-specific differences in platelet pathophysiology.

Alzheimer's disease (AD) is the most common form of dementia and characterized by extracellular amyloid-ß (Aß) plaques, intracellular neurofibrillary tau tangles and neurodegeneration. Over 80 % of AD patients also exhibit cerebral amyloid angiopathy (CAA). CAA is a cerebrovascular disease caused by deposition of Aß in the walls of cerebral blood vessels leading to vessel damage and impairment of normal blood flow. To date, different studies suggest that platelet function, including activation, adhesion and aggregation, is altered in AD due to vascular Aß deposition. For example, the transgenic AD model mice APP23 mice that exhibit CAA and parenchymal Aß plaques, show pre-activated platelets in the blood circulation and increased platelet integrin activation leading to a pro-thrombotic phenotype in these mice late stages of AD. However, it is still an open question whether or not platelets exhibit changes in their activation profile before they are exposed to vascular Aß deposits. Therefore, the present study examined platelets from middle-aged transgenic APP23 mice at the age of 8-10 months. At this age, APP23 mice show amyloid plaques in the brain parenchyma but not in the vasculature. Our analyses show that these APP23 mice have unaltered platelet numbers and size, and unaltered surface expression of glycoproteins. However, the number of dense granules in transgenic platelets was increased while the release was unaltered. Male, but not female APP23 mice, exhibited reduced platelet activation after stimulation of the thrombin receptor PAR4 and decreased thrombus stability on collagen under flow conditions ex vivo compared to control mice. In an arterial thrombosis model in vivo, male APP23 mice showed attenuated occlusion of the injured artery compared to controls. These findings provide clear evidence for early changes in platelet activation and thrombus formation in male mice before development of overt CAA. Furthermore, reduced platelet activation and thrombus formation suggest sex-specific differences in platelet physiology in AD that has to be considered in future studies of platelets and their role in AD.

Red ginseng extract inhibits lipopolysaccharide-induced platelet-leukocyte aggregates in mice.

Background: Platelet-leukocyte aggregates (PLAs) play important roles in cardiovascular disease and sepsis. Red ginseng extract (RGE) has been well-studied for its antiplatelet and anti-inflammatory activities. However, the potential inhibitory effects of RGE on PLA have not been investigated. Methods: Six-week-old ICR mice were given oral gavage of RGE for 7 days, followed by an intraperitoneal injection of 15 mg/kg of lipopolysaccharide. Mice were euthanized 24 h later, and blood samples were collected for further analysis. Flow cytometry was utilized to sort populations of PLAs and platelet-neutrophil aggregates (PNAs). By using confocal microscopy, PNAs were validated. Morphological changes in platelets and leukocytes were visualized with scanning electron microscopy. Expressions of tissue factor (TF) and platelet factor 4 (PF4) were investigated using enzyme-linked immunosorbent assay. Results: Populations of activated platelets, PLAs and PNAs, were significantly increased with LPS-induction. Treatment with 200 and 400 mg/kg of RGE decreased platelet activation. Moreover, the populations of PLAs and PNAs were reduced. PNAs were visible in the blood of septic mice, and this was attenuated by treatment with 400 mg/kg of RGE. Morphologically, sepsisinduced platelet activation and fibrin formation in the blood. This was reduced with RGE treatment. Sepsis-induced increase in the plasma levels of TF and PF4 was also reduced with RGE treatment. Conclusion: This study shows that RGE is a potential therapeutic that reduces the activation of platelets and targets PLA and PNA formation. Detailed inhibitory mechanisms of RGE should be studied.

Flow cytometry vs conventional methods for the evaluation of anti-PF4/heparin antibodies: a single center study.

Aim: Heparin-induced thrombocytopenia (HIT) is a rare, life-threatening, immune-mediated adverse effect of heparin administration. This study compares frequently used laboratory assays in terms of their effectiveness in HIT diagnosis. Materials & methods: Fifty patients with suspected HIT were tested by gel immunoassay and solid phase PF4/heparin antibody ELISA. On positive results, platelet activation markers P-selectin and Annexin V were assayed using flow cytometry. Results: Thirty/50 patients were negative for both immunoassays. Flow cytometry was performed in the 20 immunoassay positive patients. Platelet activation was observed in 7/20 in the presence of low heparin concentration (0.2 IU/ml). Conclusion: The results are in accordance with the currently available literature and flow cytometry seems a promising alternative in HIT laboratory investigation.

[Box: see text].

Noninvasive Biomarkers for Alcohol-Related Liver Disease-A Proteomic Related Preliminary Report.

Increased alcohol intake over decades leads to progressive alcohol-related liver disease (ALD) and contributes to increased mortality. It is characterized by reduced platelet count. Platelets have a role in protecting vascular integrity and involved in liver regeneration. Alcohol affects the platelet count and its function. Platelet function is regulated by their proteins, released during pathophysiological conditions. Therefore, platelet proteome plays a vital role during ALD. This preliminary study consists of 10 patients with ALD. It includes the preparation of human platelets for the proteomic approach. We performed liquid chromatography-mass spectrometry for the samples. A total of 536 proteins were identified in patients with ALD of which 31 proteins were mentioned as a candidate based on their clinical significance. The advancement of diagnostic or therapeutic tools based on the application of platelet proteins in ALD is still far off. Platform for platelet and its proteome research may give diagnostic and prognostic insights into ALD. Platelet proteomes could possibly be concluded as therapeutic and potential diagnostic or prognostic markers in ALD. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-023-01120-9.

Designing an experimental method for assessing biocompatibility of circuit coatings using biomarkers for platelet activation during cardiopulmonary bypass.

INTRODUCTION: Cardiopulmonary bypass is an essential component of cardiothoracic surgeries. However, significant complications such as systemic inflammatory response syndrome (SIRS) resulting from cardiopulmonary bypass (CPB) are a common occurrence due to contact between circulating blood and foreign surfaces that leads to platelet activation. It is suggested that different available CPB circuit coatings can potentially reduce platelet activation. However, there have been no published evidence-based reports confirming these claims. In addition, there is no well-established protocol for studying platelet activation biomarkers during CPB in vitro in a laboratory setting. METHODS: CPB was simulated in the laboratory using bovine blood in two different types of coated CPB circuits: Trillium® Biosurface by Medtronic, and XcoatingTM Surface by Terumo. Fresh bovine blood samples were collected and circulated through the CPB circuit following the standard protocol used in the operation rooms. Blood samples were then collected at 5 min, 30 min, and 55 min during the circulation. Blood plasmas were separated and subjected to enzyme-linked immunosorbent assay to measure most established platelet activation markers P-selectin, Platelet Factor 4 (PF4), Glycoprotein IIb/IIIa (GPIIb/IIIa), and ß-thromboglobulin (ß-TG) at different time points. RESULTS: The biomarker values at 30 min and 55 min were compared to the base values at 5 min for each type of CPB circuit. The results of the means from all measured biomarkers showed data measurements that indicated no significant variability within each coating. All collected data points fell within ±2 SD of the means, which was considered acceptable variations across technical replicates.  Conclusion: In this study, we were able to establish an in vitro protocol in the laboratory setting that is precise and reliable with minimum intra-variability. This established protocol will allow for future studies in which different coated CPB circuits can be compared for their effectiveness in blocking platelet activation during the CPB.

Thrombotic events associated with immune checkpoint inhibitors and novel antithrombotic strategies to mitigate bleeding risk.

Although immunotherapy is expanding treatment options for cancer patients, the prognosis of advanced cancer remains poor, and these patients must contend with both cancers and cancer-related thrombotic events. In particular, immune checkpoint inhibitors are associated with an increased risk of atherosclerotic thrombotic events. Given the fundamental role of platelets in atherothrombosis, co-administration of antiplatelet agents is always indicated. Platelets are also involved in all steps of cancer progression. Classical antithrombotic drugs can cause inevitable hemorrhagic side effects due to blocking integrin ß3 bidirectional signaling, which regulates simultaneously thrombosis and hemostasis. Meanwhile, many promising new targets are emerging with minimal bleeding risk and desirable anti-tumor effects. This review will focus on the issue of thrombosis during immune checkpoint inhibitor treatment and the role of platelet activation in cancer progression as well as explore the mechanisms by which novel antiplatelet therapies may exert both antithrombotic and antitumor effects without excessive bleeding risk.

Xuefu Zhuyu decoction alleviates deep vein thrombosis through inhibiting the activation of platelets and neutrophils via sirtuin 1/nuclear factor kappa-B pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuefu Zhuyu Decoction (XZD), a renowned traditional Chinese medicine prescription, is widely employed for the management of conditions characterized by qi-stagnation and blood stasis. Although its anti-thrombotic effect on deep vein thrombosis (DVT) patients has been clinically observed, the underlying mechanism remains largely unexplored. AIM OF THE STUDY: Our aim was to investigate the mechanisms by which XZD exerted its effect on DVT. MATERIALS AND METHODS: The ultra performance liquid chromatography (UPLC) technique was employed to evaluate quality of XZD. To examine the effect of XZD on DVT, a DVT rat model with inferior vena cava (IVC) stenosis was established. The 4D-label-free proteomics approach was then utilized to uncover the possible mechanisms of XZD against DVT. Based on proteomics, citrullinated histone H3 (CitH3), along with serum levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) were observed the inhibitory activity of XZD on neutrophil activation. Subsequently, the marker of platelet activation, specifically glycoprotein IIb (CD41) and glycoprotein IIIa (CD61), were assessed along with the secretion of von Willebrand factor (vWF) to investigate the inhibitory activity of XZD on platelet activation. Finally, we explored the impact of XZD on the sirtuin 1 (SIRT1)/nuclear factor kappa-B (NF-κB) pathway, which was associated with the activation of platelets and neutrophils. RESULTS: Eight distinct components were identified for the quality control of XZD. XZD effectively reduced thrombus weight and length in DVT rats, without affecting the coagulation function or hematological parameters in the systemic circulation. Proteomics analysis revealed that XZD alleviated DVT by inhibiting the activation of platelets and neutrophils. The protein expression of CitH3, along with serum levels of TNF-α and IL-1ß, were reduced in XZD-treated DVT rats. Similarly, protein expressions of CD41 and CD61, along with the release of vWF, were markedly down-regulated in XZD-treated DVT rats. Finally, treatment with XZD resulted in an up-regulation of SIRT1 protein expression and a down-regulation of both acetylated NF-κB/p65 and phosphorylated NF-κB/p65 protein expressions in endothelium. CONCLUSIONS: XZD alleviates DVT by inhibiting the activation of platelets and neutrophils at the injured endothelium via the regulation of SIRT1/NF-κB pathway.

1.8-cineole prevents platelet activation and aggregation by activating the cAMP pathway via the adenosine A2A receptor.

AIMS: Dysregulated platelet aggregation is a fatal condition in many bacterial- and virus-induced diseases. However, classical antithrombotics cannot completely prevent immunothrombosis, due to the unaddressed mechanisms towards inflammation. Thus, targeting platelet hyperactivation together with inflammation might provide new treatment options in diseases, characterized by immunothrombosis, such as COVID-19 and sepsis. The aim of this study was to investigate the antiaggregatory effect and mode of action of 1.8-cineole, a monoterpene derived from the essential oil of eucalyptus leaves, known for its anti-inflammatory proprieties. MAIN METHODS: Platelet activity was monitored by measuring the expression and release of platelet activation markers, i.e., P-selectin, CD63 and CCL5, as well as platelet aggregation, upon treatment with 1.8-cineole and stimulation with several classical stimuli and bacteria. A kinase activity assay was used to elucidate the mode of action, followed by a detailed analysis of the involvement of the adenylyl-cyclase (AC)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway by Western blot and ELISA. KEY FINDINGS: 1.8-cineole prevented the expression and release of platelet activation markers, as well as platelet aggregation, upon induction of aggregation with classical stimuli and immunological agonists. Mechanistically, 1.8- cineole influences the activation of the AC-cAMP-PKA pathway, leading to higher cAMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Finally, blocking the adenosine A2A receptor reversed the antithrombotic effect of 1.8-cineole. SIGNIFICANCE: Given the recognized anti-inflammatory attributes of 1.8-cineole, coupled with our findings, 1.8-cineole might emerge as a promising candidate for treating conditions marked by platelet activation and abnormal inflammation.

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

Background: Assessment 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. Objectives: Our aim was to compare well-used platelet reactivity assays. Methods: Blood 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. Results: A 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 (ß = -1.735; P = 4.59 × 10-780; variance proportion = 46.2%) and P2Y12 antagonists blocking adenosine diphosphate responses (ß = -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. Conclusion: Caution 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.

Platelet Biorheology and Mechanobiology in Thrombosis and Hemostasis: Perspectives from Multiscale Computation.

Thrombosis is the pathological clot formation under abnormal hemodynamic conditions, which can result in vascular obstruction, causing ischemic strokes and myocardial infarction. Thrombus growth under moderate to low shear (<1000 s-1) relies on platelet activation and coagulation. Thrombosis at elevated high shear rates (>10,000 s-1) is predominantly driven by unactivated platelet binding and aggregating mediated by von Willebrand factor (VWF), while platelet activation and coagulation are secondary in supporting and reinforcing the thrombus. Given the molecular and cellular level information it can access, multiscale computational modeling informed by biology can provide new pathophysiological mechanisms that are otherwise not accessible experimentally, holding promise for novel first-principle-based therapeutics. In this review, we summarize the key aspects of platelet biorheology and mechanobiology, focusing on the molecular and cellular scale events and how they build up to thrombosis through platelet adhesion and aggregation in the presence or absence of platelet activation. In particular, we highlight recent advancements in multiscale modeling of platelet biorheology and mechanobiology and how they can lead to the better prediction and quantification of thrombus formation, exemplifying the exciting paradigm of digital medicine.

Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

BACKGROUND: ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. METHODS AND RESULTS: We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the ex vivo thromboelastometric parameters. CONCLUSIONS: AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in ex vivo thromboelastometric parameters. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

Platelet Aggregation Studies and Coagulation Profile in Sickle Cell Disease in Symptomatic and Steady State Patients.

To determine whether there is higher degree of platelet and/ or coagulation activation in sickle cell anaemia (SS) patients in complications and with clinical risk factors. A cross sectional study was conducted at a tertiary health care centre in central India with study groups: sickle cell disease (SCD): sickle cell anaemia (SS) and sickle cell trait (AS) consisting of 100 subjects each and controls (AA) with 40 subjects. Platelet aggregation (PA) with ADP, collagen and epinephrine, PT and aPTT were performed in all subjects and PA with ristocetin in ten candidates of each group. ANOVA and student's unpaired t test were used to compare PA and coagulation profile of the three groups with respect to age groups, gender, present diagnosis, history of complications, frequency of hospital admissions (high ≥ 3/year) and frequency of blood transfusion (high > 2/year). The max PA% with ADP was significantly less in SS patients in steady state, which was even lesser in those having symptoms, complications in past/ present, high-frequency hospital admission and > 2 blood transfusions per year subgroups, as compared to all other groups and subgroups, but not consistently with collagen and epinephrine. The max PA % with ristocetin was least in SS with complications. No statistically significant difference in PT and aPTT values among the various clinical risk subgroups and groups was found. SCD patients can be monitored by using PA with ADP for their timely and better management. PA with ADP, PT and aPTT should be added to the workup of these patients for improved prognostication.