Platelet Surface Research Articles

Role of Tumor Microenvironment in the Risk of Thromboembolic Complications in Ovarian Cancer

Introduction. Incidence of ovarian cancer remains high in the overall prevalence of oncological pathology. Adjuvant chemotherapy refers to its treatment options. Patients with oncological pathology are faced with a high risk of thrombosis and thromboembolism, with up to 30% lethal outcome within a month of its development. A number of cancer cells are known to induce platelet aggregation, contributing to thrombosis and metastasis as a result of this interaction. Accordingly, the paper is aimed at presenting a clinical case for demonstrating the role of P-selectin expression in the complications in a patient with ovarian cancer. Materials and methods. The present paper evaluates platelet activation marker in a patient undergoing chemotherapy courses after cytoreductive surgery. Following the case conference and in accordance with the clinical recommendations of the Russian Oncology Association (AOR) and Russian Society of Clinical Oncology (RUSSCO), cytoreduction (CC-0), radical hysterectomy, transverse colectomy, left hemicolectomy with rectum resection were performed. The interventions included ascendostomy, pelvic, lateral right-sided and left-sided peritonectomy, pelvic lymphoadenectomy, total omentectomy, Renape-French HIPEC (hyperthermic intraperitoneal chemotherapy), abdominal and pelvic drainage. Expression of P-selectin on the platelet surface was measured as a marker of platelet activation. Results and discussion. At the time of admission, the patient had high CD62 expression activity compared to healthy volunteers (CD62 ADP- — 11.2%, CD62 ADP — 24.7% vs CD62 ADP- — 1.3%, CD62 ADP — 17.2%). During the complex treatment of ovarian cancer, the platelet activation increased (CD62 ADP- — 21.8 %, CD62 ADP+ — 30.1 %). At discharge, CD62 expression values reached the conditional norm, presumably indicating thrombosis development. Conclusion. Tumor microenvironment influences the hemostasis system. Detailed study into this issue obtains a high potential for the prevention of primary and secondary thromboembolic complications in oncologic patients.

Effects of selective serotonin reuptake inhibitors on platelet functions: a literature review.

Many epidemiological studies have evidenced an increased bleeding risk associated with selective serotonin reuptake inhibitors (SSRIs), yet the underlying mechanisms remain unclear. This review summarizes data on SSRIs' effects on platelet functions assessed with assays used in clinical practice and highlights the areas that deserve further investigation. Conflicting results of SSRI effects on platelet aggregation were observed irrespectively of the agonist used, the antidepressant drug or the study type. Alike, discrepant results were reported with flow-cytometry-based assays assessing either platelet surface glycoprotein levels, integrin activation, agonist-induced secretion of intraplatelet granule content or membrane anionic phospholipid exposure. Other tests may have detected a platelet function defect in SSRIs samples, however, results were largely inconsistent. Critical literature examination unveils very low certainty of evidence on potential SSRI effect on platelet functions. Findings are often inconsistent even when similar methods are used, most likely because of differences in study design, included patients (age, comorbid conditions), SSRIs' type and dose, uncontrolled confounding factors, and statistical analysis power. Further studies are needed to disentangle any intrinsic antiplatelet effect of SSRIs and the multiple confounding factors, mainly the depression control itself and the degree of platelet SERT inhibition.

Soluble Glycoprotein VI Levels Assessed Locally within the Extra- and Intracerebral Circulation in Hyper-Acute Thromboembolic Stroke: A Pilot Study.

Background: Severe acute ischemic stroke (AIS) is mainly caused by thromboembolism originating from symptomatic carotid artery (ICA) stenosis or in the heart due to atrial fibrillation. Glycoprotein VI (GPVI), a principal platelet receptor, facilitates platelet adherence and thrombus formation at sites of vascular injury such as symptomatic ICA stenosis. The shedding of GPVI from the platelet surface releases soluble GPVI (sGPVI) into the circulation. Here, we aimed to determine whether sGPVI can serve as a local biomarker to differentiate between local atherosclerotic and systemic cardiac thromboembolism in AIS. Methods: We conducted a cohort study involving 105 patients undergoing emergency endovascular thrombectomy (EVT) for anterior circulation stroke. First, sGPVI concentrations were measured in systemic arterial plasma samples collected at the ipsilateral ICA level, including groups with significantly (≥50%) stenotic and non-stenotic arteries. A second sample, taken from the intracerebral pial circulation, was used to assess GPVI shedding locally within the ischemic brain. Results: Our analysis revealed no significant increase in systemic sGPVI levels in patients with symptomatic ≥ 50% ICA stenosis (3.2 [95% CI 1.5-5.0] ng/mL; n = 33) compared with stroke patients without significant ICA stenosis (3.2 [95% CI 2.3-4.2] ng/mL; n = 72). Additionally, pial blood samples, reflecting intravascular molecular conditions during collateral flow, showed similar sGPVI levels when compared to the systemic ICA samples in both groups. Conclusions: Our findings indicate that GPVI is not locally cleaved and shed into the bloodstream in significant amounts during hyper-acute ischemic stroke, neither at the level of symptomatic ICA nor intracranially during collateral blood supply. Therefore, sGPVI does not appear to be suitable as a local stroke biomarker despite strong evidence of a major role for GPVI-signaling in stroke pathophysiology.

Exploring the effect of hydroxyapatite nanoparticle shape on red blood cells and blood coagulation.

Aim: In this study, we evaluated the effects of two types of hydroxyapatite (HAP) nanoparticles, sharing the same surface chemistry but differing in shape, on the biological characteristics of plasma, platelets and red blood cells.Materials & methods: Initially, two different shapes (rod-shaped and sphere-shaped) of HAPs were characterized. These HAPs were then co-cultured with plasma and red blood cells to examine their impact on coagulation and hemolysis. The impact of HAPs on white blood cells count in mice were evaluated following gavage and tail vein injection.Results: Sphere-shaped HAP is more likely to adsorb onto platelet surfaces, while rod-shaped HAP is more likely to cause hemolysis. Although there are differences in the in vitro experimental results between sphere-shaped HAP and rod-shaped HAP, both types demonstrate good blood compatibility at a 20mM concentration. Furthermore, in vivo experiments showed that sphere-shaped nano-HAP induced a more pronounced increase in white blood cell count, suggesting that it may exhibit greater toxicity.Conclusion: While differences exist in the blood compatibility test results between the two HAPs, these differences are minimal, with both results falling within a safe range. Overall, HAP demonstrates excellent blood compatibility.

Rosuvastatin inhibit ox-LDL-induced platelet activation by the p38/MAPK pathway.

This study intends to explore the effects of Rosuvastatin on ox-LDL induced platelet activation and its molecular mechanism. Platelet aggregation rate was detected by aggregometer. ELISA kit was used to detect the levels of cAMP. Immunofluorescence staining was used to detect the platelet adhesion. The expression levels of platelet surface markers CD62p and PAC-1 were detected by flow cytometry. The protein levels of p-p38, p-IKKa and p-IKKB in platelets were detected by western blot. We found that rosuvastatin significantly inhibited platelet aggregation and increased the level of cAMP in a dose-dependent manner. Immunofluorescence staining results showed that rosuvastatin could inhibit platelet adhesion. Flow cytometry results showed that rosuvastatin could reduce the expression of platelet activation markers. Western blot results showed that rosuvastatin could down-regulate the expression levels of p-p38, p-IKKa and p-IKKb. Our study revealed the rosuvastatin could inhibit the aggregation, adhesion and activation of platelet induced by ox-LDL, its mechanism may be related to inhibition of p38/MAPK signal pathway.

An Automated pre-Dilution Setup for Von Willebrand Factor Activity Assays.

Accurate quantification of von Willebrand factor ristocetin cofactor activity (VWF:RCo) is critical for the diagnosis and classification of von Willebrand disease, the most common hereditary and acquired bleeding disorder in humans. Moreover, it is important to accurately assess the function of von Willebrand factor (VWF) concentrates within the pharmaceutical industry to provide consistent and high-quality biopharmaceuticals. Although the performance of VWF:RCo assay has been improved by using coagulation analyzers, which are specialized devices for blood and blood plasma samples, scientists still report a high degree of intra- and inter-assay variation in clinical laboratories. Moreover, high, manual sample dilutions are required for VWF:RCo determination of VWF concentrates within the pharmaceutical industry, which are a major source for assay imprecision. For the first time, we present a precise and accurate method to determine VWF:RCo, where all critical pipetting and mixing steps are automated. A pre-dilution setup was established on CyBio FeliX (Analytik-Jena) liquid handling system, and an adapted VWF:RCo method on BCS-XP analyzer (Siemens) is used. The automated pre-dilution method was executed on three different, most frequently used coagulation analyzers and compared to manual pre-dilutions performed by an experienced operator. Comparative sample testing revealed a similar assay precision (coefficient of variation = 5.9% automated, 3.1% manual pre-dilution) and no significant differences between the automated approach and manual dilutions of an expert in this method. While no outliers were generated with the automated procedure, the manual pre-dilution resulted in an error rate of 8.3%. Overall, this operator-independent protocol enables standardization and offers an efficient way of fully automating VWF activity assays, while maintaining the precision and accuracy of an expert analyst. Key features • Automated pre-dilution setup for von Willebrand factor concentrates of various natures. • Combination of a liquid handling system (CyBio FeliX) with a coagulation analyzer (BCS-XP). • Simplifies method transfer to other laboratories. • Basic training for CyBio FeliX and BCS-XP is required. Graphical overview VWF:RCo assay principle and measurement setup. Platelets (yellow ellipsoids) with negative surface charge (- - -) are treated with formaldehyde, which partly denatures the cell surface and thus stabilizes platelets for use as assay reagents. Stabilized platelets (dark-yellow-framed yellow ellipsoids) are then brought in contact with ristocetin A (chemical structure shown; black dots), which binds to the platelet surface and facilitates binding of VWF (green circles). The graphs show an example of quantitative determination of platelet agglutination by measurement of light transmission, where increasing amounts of VWF increase light transmission over time. The photo in the left-bottom corner shows the CyBio FeliX setup for VWF sample dilution and the photo in the right-bottom corner displays the BCS-XP system, which is used for VWF:RCo measurements.

Platelet changes and bleeding symptoms in children, adolescents, and adults with 22q11.2 deletion syndrome.

The deletion region of 22q11.2 deletion syndrome (22q11.2DS) contains a gene encoding glycoprotein Ibβ (GPIbβ), which is required to express the GPIb/IX/V complex on the platelet surface. Therefore, patients with 22q11.2DS may have congenital platelet disorders. However, information is limited on platelets and bleeding symptoms. In this study, we investigated clinical information, including bleeding symptoms, platelet counts, and GPIb expression levels in children and adolescents/adults with 22q11.2DS. Thirty-two patients with 22q11.2DS were enrolled in a prospective cohort study between 2022 and 2023 at outpatient clinics within our institute. The median platelet counts in adolescents/adults with 22q11.2DS were significantly lower than those in children (p<.0001). A gradual decrease was found along with increasing age (p=.0006). Values of median GPIb expression on platelet surfaces (66% in children and 70% in adolescents/adults) were significantly lower than those in healthy controls (p<.0001 and p=.0002). Bleeding symptoms included surgery-related bleeding (52%), purpura (31%), and epistaxis (22%); most of them were minor. The median International Society on Thrombosis and Hemostasis bleeding assessment tool score was not significantly different between children and adolescents/adults (p=.2311). Although there was an age-related decrease in platelet count and a disease-related decrease in GPIb expression, no difference in bleeding symptoms was found between children and adolescents/adults. 22q11.2DS overall had minor bleeding symptoms in daily life, and the disease had little effect on spontaneous bleeding. However, some patients had major bleeding events; further accumulation of data on hemostasis during surgery and trauma is required.

Platelet-Type von Willebrand Disease: Complex Pathophysiology and Insights on Novel Therapeutic and Diagnostic Strategies.

von Willebrand disease (VWD) is the most common well-studied genetic bleeding disorder worldwide. Much less is known about platelet-type VWD (PT-VWD), a rare platelet function defect, and a "nonidentical" twin bleeding phenotype to type 2B VWD (2B-VWD). Rather than a defect in the von Willebrand factor (VWF) gene, PT-VWD is caused by a platelet GP1BA mutation leading to a hyperaffinity of the glycoprotein Ibα (GPIbα) platelet surface receptor for VWF, and thus increased platelet clearing and high-molecular-weight VWF multimer elimination. Nine GP1BA gene mutations are known. It is historically believed that this enhanced binding was enabled by the β-switch region of GPIbα adopting an extended β-hairpin form. Recent evidence suggests the pathological conformation that destabilizes the compact triangular form of the R-loop-the GPIbα protein's region for VWF binding. PT-VWD is often misdiagnosed as 2B-VWD, even the though distinction between the two is crucial for proper treatment, as the former requires platelet transfusions, while the latter requires VWF/FVIII concentrate administration. Nevertheless, these PT-VWD treatments remain unsatisfactory, owing to their high cost, low availability, risk of alloimmunity, and the need to carefully balance platelet administration. Antibodies such as 6B4 remain undependable as an alternative therapy due to their questionable efficacy and high costs for this purpose. On the other hand, synthetic peptide therapeutics developed with In-Silico Protein Synthesizer to disrupt the association between GPIbα and VWF show preliminary promise as a therapy based on in vitro experiments. Such peptides could serve as an effective diagnostic technology for discriminating between 2B-VWD and PT-VWD, or potentially all forms of VWD, based on their high specificity. This field is rapidly growing and the current review sheds light on the complex pathology and some novel potential therapeutic and diagnostic strategies.

High-Dimensional Single-Cell Mass Cytometry Demonstrates Differential Platelet Functional Phenotypes in Infants With Congenital Heart Disease.

Congenital heart disease (CHD) is a group of complex heart defects associated with hematologic abnormalities, including increased risk of thrombotic and bleeding events. Past studies have observed evidence of platelet hyperreactivity, while other studies showed decreased platelet activation in patients with CHD. The goal of this study was to develop a mass spectrometry approach to characterize single platelets in infants with CHD and identify potential etiology for such discrepant results. We enrolled 19 infants with CHD along with 21 non-CHD controls at Yale New Haven Children's Heart Center. A single-cell high-dimensional mass cytometry method was developed to quantitatively interrogate platelet surface markers in whole blood. Additionally, plasma cytokine analysis was performed through a multiplexed panel of 52 vascular and inflammatory markers to assess for platelet releasates. We found that infants with CHD had significant differences in platelet activation and functional markers by mass cytometry compared with non-CHD controls. Based on cell surface markers, we classified the platelets into 8 subpopulations (P0 to P7). Distinct subpopulations of platelets (P1, P4, and P5) exhibiting decreased aggregatory phenotype but altered secretory phenotypes were also identified and found to be more abundant in the blood of infants with CHD. Electron microscopy identified increased proportion of hypogranular platelets in CHD. Moreover, cytokine analysis demonstrated an overall increase in plasma cytokines and biomarkers in CHD, including IL (interleukin)-6, IL-8, IL-27, RANTES, and VWF (von Willebrand factor), which are expressed in platelet granules and can be released upon activation. We developed a robust mass cytometry approach to identify platelet phenotypic heterogeneity. Infants with CHD had alterations in distinct subpopulations of platelets with overall reduced aggregatory phenotype and secretory dysfunction. These findings suggest that platelets in infants with CHD may be exhausted due to persistent stimulation and may explain both bleeding and thrombotic vascular complications associated with CHD.

Endothelial protein disulfide isomerase A1 enhances membrane stiffness and platelet-endothelium interaction in hyperglycemia via SLC3A2 and LAMC1

BackgroundDiabetes carries an increased risk of cardiovascular disease and thromboembolic events. Upon endothelial dysfunction, platelets bind to endothelial cells to precipitate thrombus formation; however, it is unclear which surface proteins regulate platelet-endothelium interaction. We and others have shown that peri/epicellular protein disulfide isomerase A1 (pecPDI) influences the adhesion and migration of vascular cells. ObjectivesWe investigated whether pecPDI regulates adhesion-related molecules on the surface of endothelial cells and platelets that influence the binding of these cells in hyperglycemia. MethodsImmunofluorescence was used to assess platelet-endothelium interaction in vitro, cytoskeleton reorganization, and focal adhesions. Hydrogen peroxide production was assessed via Amplex Red assays (ThermoFisher Scientific). Cell biophysics was assessed using atomic force microscopy. Secreted proteins of interest were identified through proteomics (secretomics), and targets were knocked down using small interfering RNA. Protein disulfide isomerase A1 (PDI) contribution was assessed using whole-cell PDI or pecPDI inhibitors or small interfering RNA. ResultsPlatelets of healthy donors adhered more onto hyperglycemic human umbilical vein endothelial cells (HUVECs). Endothelial, but not platelet, pecPDI regulated this effect. Hyperglycemic HUVECs showed marked cytoskeleton reorganization, increased H2O2 production, and elongated focal adhesions. Indeed, hyperglycemic HUVECs were stiffer compared with normoglycemic cells. PDI and pecPDI inhibition reversed the abovementioned processes in hyperglycemic cells. A secretomics analysis revealed 8 proteins secreted in a PDI-dependent manner by hyperglycemic cells. Among these, we showed that genetic deletion of LAMC1 and SLC3A2 decreased platelet-endothelium interaction and did not potentiate the effects of PDI inhibitors. ConclusionEndothelial pecPDI regulates platelet-endothelium interaction in hyperglycemia through adhesion-related proteins and alterations in endothelial membrane biophysics.

Preparation Method and Quality Evaluation of Novel Frozen Human Platelets

To optimize the technical parameters related to the preparation of novel frozen human platelets and formulate corresponding protocol for its preparation. Novel frozen human platelets were prepared with O-type bagged platelet-rich plasma (PRP), the key technical parameters (DMSO addition, incubation time, centrifugation conditions, etc.) of the preparation process were optimized, and the quality of the frozen platelets was evaluated by routine blood tests, apoptosis rate, platelet activation rate and surface protein expression level. In the preparation protocol of novel frozen human platelets, the operation of centrifugation to remove supernatant was adjusted to before the procedure of platelets freezing, and the effect of centrifugation on platelets was minimal when the centrifugation condition was 800×g for 8 min. In addition, platelets incubated with DMSO for 30 min before centrifugation exhibited better quality after freezing and thawing. The indexes of novel frozen human platelets prepared with this protocol remained stable after long-term cryopreservation. The preparation technique of novel frozen human platelets was established and the protocol was formulated. It was also confirmed that the quality of frozen platelets could be improved by incubating platelets with DMSO for 30 min and then centrifuging them at 800×g for 8 min in the preparation of novel frozen human platelets.

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

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

Physicochemical Features of Thrombin Binding to Platelet Membrane

Thrombin is a key enzyme of the blood coagulation system, which has been actively studied since the beginning of the last century. The formation of thrombin from prothrombin in the area of vessel injury leads not only to the formation of fibrin – an important structural component of the hemostatic clot – but also to the activation of platelets, endothelium and immune system cells. The binding of thrombin to the platelet surface is thought to play a critical role in the process of platelet activation and may also ensure the maintenance of a high concentration of thrombin within the thrombus due to the concentration of protease on the platelet surface. To date, all major thrombin receptors on platelets have been thoroughly characterized: through various experimental methods, the physicochemical parameters of the corresponding intermolecular interactions have been established. Since the interaction of thrombin with platelets leads to their activation, which includes changes in the number of receptors as a result of granule secretion, the interpretation of the observed kinetic binding curves faces a number of difficulties. It is known that some receptors as a result of platelet activation are able to redistribute on the membrane and form dimers and clusters, which makes the kinetics of thrombin binding to platelets an extremely complex process depending on many factors, such as activator concentrations, platelet state, and other local parameters of the system. This review aims to describe the current understanding of the interaction of thrombin with the platelet membrane and to outline important unresolved issues in this area of research. The survey provides not only information on structural and kinetic features of thrombin binding to individual platelet membrane proteins, but also analyzes the relationship between the relevant interaction parameters and previously obtained data on the integral kinetics of protease binding to the platelet surface.

New insights of glycoprotein Ib-IX-V complex organization and glycoprotein Ibα in platelet biogenesis.

Glycoprotein (GP) Ib-IX-V, a platelet surface receptor that plays a critical role in platelet adhesion and platelet-mediated immune responses, consists of GPIbα, GPIbβ, GPIX, and GPV in a stoichiometry of 2 : 4 : 2 : 1. Forming a complex is essential for GPIb-IX-V to function. GPIb-IX-V also plays an important role in platelet biogenesis by regulating the number and size of platelets. Yet how GPIb-IX-V regulates platelet biogenesis remains elusive. This review will summarize recent findings in the complex organization of GPIb-IX-V and its role in platelet biogenesis. Proteomics studies suggest that GPIbα, GPIbβ, GPIX, and GPV form the complex in a ratio of 1 : 2 : 1 : 1, which is supported by analysis of molecular weight of GPIb-IX-V and GPIb-IX and the structure of entire GPIb-IX-V. To activate platelets, GPIbα requires binding of CLEC-2 to trigger signals. Furthermore, disrupting the GPIbα anchorage to filamin A causes defects in platelet budding away from proplatelets leading to giant platelets and a low platelet count. New studies challenge the traditional model for the organization of GPIb-IX-V as a complex and indicate the role of GPIb-IX-V in platelet production. Those studies provide insights for GPIb-IX-V in the regulation of platelet activation and platelet biogenesis.

Friction between a single platelet and fibrinogen

Friction has been considered to mediate physiological activities of cells, however, the biological friction between a single cell and its ligand-bound surface has not been thoroughly explored. Herein, we established a friction model for single cells based on an atomic force microscopy (AFM) combined with an inverted fluorescence microscopy (IFM) to study the friction between a highly sensitive platelet and fibrinogen-coated surface. The study revealed that the friction between the platelet and fibrinogen-coated tip is mainly influenced by specific ligand–receptor interaction. Further, we modeled the biological friction, which consists of specific interaction, non-specific interaction, and mechanical effect. Besides, the results suggested that the velocity can also affect specific ligand–receptor interactions, resulting in the friction change and platelet adhesion to fibrinogen surfaces. The study built a friction model between a single cell and its ligand-bound surface and provided a potential method to study the biological friction by the combination of AFM and IFM.

Soluble glycoprotein VI predicts abdominal aortic aneurysm growth rate and is a novel therapeutic target

AbstractA common feature in patients with abdominal aortic aneurysms (AAAs) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA-associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation affects the pathogenesis of AAA. Using RNA sequencing, we identified that the platelet-associated transcripts are significantly enriched in the ILT compared with the adjacent aneurysm wall and healthy control aortas. We found that the platelet-specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of patients with AAAs. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in 2 independent cohorts of patients with AAAs is highly predictive of an AAA diagnosis and associates more strongly with aneurysm growth rate than D-dimer in humans. Finally, intervention with the anti-GPVI antibody (JAQ1) in mice with established aneurysms blunted the progression of AAA in 2 independent mouse models. In conclusion, we show that the levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, for which none currently exists.

Pathophysiology and Diagnostics of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is due to autoantibodies against platelet surface antigens. ITP is considered as either primary, with no clear etiology, or as secondary ITP (drug-induced; underlying diseases). Autoantibodies lead both to loss of platelets in the spleen and/or liver but simultaneously reduce their production. Contrary to other disorders with thrombocytopenia, ITP has reduced levels of thrombopoetin. ITP remains a diagnosis of exclusion. A single defining laboratory test does not exist. Glycoprotein-specific antibodies can be detected in only about 50% of cases. Ruling out EDTA-induced pseudo thrombocytopenia is of particular relevance. Secondary causes of thrombocytopenia should be excluded through medical history (especially medication history), physical examination and possibly bone-marrow puncture.

Antithrombotic Effect of Oil from the Pulp of Bocaiúva-Acrocomia aculeata (Jacq.) Lodd. ex Mart. (Arecaceae).

The study aimed to evaluate the antithrombotic action of Acrocomia aculeata pulp oil (AAPO) in natura, in an in vitro experimental model. AAPO was obtained by solvent extraction, and its chemical characterization was performed by gas chromatography coupled to a mass spectrometer (GC-MS). In vitro toxicity was evaluated with the Trypan Blue exclusion test and in vivo by the Galleria mellonella model. ADP/epinephrine-induced platelet aggregation after treatment with AAPO (50, 100, 200, 400, and 800 μg/mL) was evaluated by turbidimetry, and coagulation was determined by prothrombin activity time (PT) and activated partial thromboplastin time (aPTT). Platelet activation was measured by expression of P-selectin on the platelet surface by flow cytometry and intraplatelet content of reactive oxygen species (ROS) by fluorimetry. The results showed that AAPO has as major components such as oleic acid, palmitic acid, lauric acid, caprylic acid, and squalene. AAPO showed no toxicity in vitro or in vivo. Platelet aggregation decreased against agonists using treatment with different concentrations of AAPO. Oil did not interfere in PT and aPTT. Moreover, it expressively decreased ROS-induced platelet activation and P-selectin expression. Therefore, AAPO showed antiplatelet action since it decreased platelet activation verified by the decrease in P-selectin expression as well as in ROS production.

Exploring ligands that target von Willebrand factor selectively under oxidizing conditions through docking and molecular dynamics simulations.

The blood protein von Willebrand factor (VWF) is a large multimeric protein that, when activated, binds to blood platelets, tethering them to the site of vascular injury and initiating blood coagulation. This process is critical for the normal hemostatic response, but especially under inflammatory conditions, it is thought to be a major player in pathological thrombus formation. For this reason, VWF has been the target for the development of anti-thrombotic therapeutics. However, it is challenging to prevent pathological thrombus formation while still allowing normal physiological blood coagulation, as currently available anti-thrombotic therapeutics are known to cause unwanted bleeding, in particular intracranial hemorrhage. This work explores the possibility of inhibiting VWF selectively under the inflammatory conditions present during pathological thrombus formation. In particular, the A2 domain of VWF is known to inhibit the neighboring A1 domain from binding to the platelet surface receptor GpIbα, and this auto-inhibitory mechanism has been shown to be removed by oxidizing agents released during inflammation. Hence, finding drug molecules that bind at the interface between A1 and A2 only under oxidizing conditions could restore such an auto-inhibitory mechanism. Here, by using a combination of computational docking, molecular dynamics simulations, and free energy perturbation calculations, a ligand from the ZINC15 database was identified that binds at the A1A2 interface, with the interaction being stronger under oxidizing conditions. The results provide a framework for the discovery of drug molecules that bind to a protein selectively in the presence of inflammatory conditions.

MEASUREMENT OF PLATELET ACTIVATION MARKERS CD62P AND CD63 TO DETERMINE THE EFFICIENCY OF DUAL THERAPY WITH ASPIRIN AND CLOPIDOGREL: A PRELIMINARY STUDY

Background: A dual antiplatelet therapy with aspirin and clopidogrel is advised to prevent stent thrombosis. However, the most appropriate assay for evaluating the antiplatelet effects remains undefined. Objective: This study aimed to assess the efficacy of measuring platelet surface activation markers, CD62P and CD63, using flow cytometry to determine the effectiveness of dual antiplatelet therapy. Methods: Thirty patients who received aspirin plus clopidogrel before cerebral artery stent implantation and thirty unrelated healthy controls were enrolled. The expression of CD62P and CD63 was measured using flow cytometry. The diagnostic performances were evaluated and compared with the conventional light-transmitted aggregometry (LTA) using 5.0 and 20.0 µM of adenosine di-phosphate (ADP). Results: The expression ratios of both markers were significantly lower in the patients receiving dual antiplatelet therapy than controls (p &lt; 0.001). The area under the receiver operating characteristic (ROC) curve (AUC) of CD62P using flow cytometry and the LTA using 5.0 and 20.0 µM of ADP were comparable (0.97 vs. 0.95 and 0.97 vs. 0.96, respectively), whereas those of CD63 was lower than the LTA (0.88 vs. 0.96). The sensitivity of CD62P and CD63 were 71.4% and 60.0%, and the specificity of CD62P and CD63 were 100.0% and 96.7%, respectively. Conclusion: Flow cytometry measurements of CD62P could be utilized to identify the efficacy of dual antiplatelet therapy. Additional studies are suggested to support this issue and its effects on clinical outcomes.