Effect of toxins from different periodontitis-associated bacteria on human platelet function.

BACKGROUND: Periodontitis is caused by a dysbiosis of oral bacteria resulting in alveolar bone destruction and teeth loss. The role of platelets in pathogenesis of periodontitis is a subject of research. The release of toxins from periodontitis-associated bacteria may influence platelet function and contribute to the modulation of hemostatic or inflammatory responses. Therefore, we explored platelet function upon exposure to defined toxins: leukotoxin A from Aggregatibacter actinomycetemcomitans (LtxA), a synthetic version of the C14-Tri-LAN-Gly peptide from Fusobacterium nucleatum (C14), and lipopolysaccharides from Porphyromonas gingivalis (LPS). METHODS: Light transmission aggregometry was performed after the addition of toxins to platelet-rich plasma in different doses. Flow cytometry was used to identify inhibitory effects of toxins by measuring phosphorylation of the vaso-dilator-stimulated phosphoprotein or to identify activating effects by the detection of CD62P expression. The release of chemokines derived from washed platelets was determined by immunoassays. RESULTS: Collagen-induced threshold aggregation values were diminished upon incubation with LtxA and C14, accompanied with an increase of vaso-dilator-stimulated phosphoprotein (VASP) phosphorylation, indicating platelet inhibition. In contrast, LPS did not affect aggregation but slightly enhanced CD62P expression under co-stimulation with low-dose thrombin pointing to slight platelet activation. The three toxins did not relevantly influence the secretion of chemokines. CONCLUSIONS: Although weak, the investigated toxins differently influenced human platelet function. LtxA and C14 mediated inhibitory effects, whereas LPS contributed to a slight activation of platelets. Further analysis of specific cellular responses mediated by bacterial toxins may render novel targets and suggestions for the treatment of periodontitis.

Prospective, international, multisite comparison of platelet isolation techniques for genome-wide transcriptomics: communication from the SSC of the ISTH.

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbß3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.

Physiological Changes Associated with Aging: Identification of Novel Biomarkers for Frailty Syndrome in Women.

This study explores the physiological changes associated with aging that lead to frailty syndrome, characterized by reduced vitality and degeneration across multiple bodily systems, increasing susceptibility to various pathologies. While established scales like the Fried Phenotype and Frailty Trait Scale (FTS) are commonly used for assessing frailty, incorporating biomarkers is crucial for accurate diagnosis and prognosis. Our research examines plasma oxylipin levels in frail elderly individuals to identify novel biomarkers. Diagnostic criteria for frailty included assessments using the Fried Phenotype and FTS-5, with blood samples collected from 71 elderly participants (50 women and 21 men) with mean ages of 73.6 ± 5.9 and 76.2 ± 6.2 years, respectively. Women exhibited elevated platelet counts (p-value 0.0035). The significant differences in oxylipin concentrations associated with the Fried Phenotype were particularly noteworthy, predominantly observed in women. Specifically, in women, decreased grip strength (<15 kg) and slow gait speed (<0.8 m/s) correlated with increased levels of thromboxane B2 (TxB2) and 7-HDoHE (p-values 0.0404, 0.0300, 0.0033, and 0.0033, respectively). Additionally, elevated 7-HDoHE levels correlated with a BMI exceeding 28 kg/m2 (p-value 0.0123) and Physical Activity Scale for the Elderly (PASE) scores surpassing 5 points (p-value 0.0134) in women. In summary, our findings emphasize that frail older individuals, particularly women, exhibit higher levels of TxB2 and 7-HDoHE compared to their non-frail counterparts, aligning with established frailty classification and scale parameters, suggesting their potential as indicative biomarkers.

Platelet-Rich Plasma (PRP) Mitigates Kidney Dysfunction in Alloxan-Induced Diabetic Mice via Modulation of Renal Iron Regulatory Genes.

Kidney dysfunction is a prevalent complication of diabetes mellitus, contributing significantly to diabetes-related morbidity and mortality. We aim to explore whether platelet-rich plasma administration can modulate iron regulation mechanism within the kidney, thereby mitigating renal dysfunction associated with diabetes. Albino mice with an average body weight of 20 ± 5 g were randomly divided into five groups (N = 50; n = 10): Control Group, PRP Group, diabetic group (DG), treated group A (TA), and treated group B (TB). A single intraperitoneal dose of alloxan (160 mg/kg of body weight) was administered to mice in the DG and in both treated groups. Upon confirmation of diabetes, the DG was left untreated, while PRP treatment (0.5 ml/kg of body weight) was administered to the TA and TB groups for two and four weeks, respectively. Histological examinations of kidney tissues revealed notable signs of damage in DG, which were subsequently improved upon PRP treatment. Likewise, PRP treatment restored the changes in liver enzymes, oxidative stress biomarkers and serum electrolytes in both treated groups. Furthermore, there was an observed upregulation of iron regulatory genes, such as Renin, Epo, Hepc, Kim1, and Hfe, in the DG, accompanied by a downregulation of Tfr1 and Fpn; however, Dmt1 and Dcytb1 expression remained unaltered. Treatment with PRP restored the expression of iron regulatory genes in both treated groups. This study concluded that PRP treatment effectively restored the renal histochemistry and the expression of renal iron regulatory genes in an alloxan-induced diabetic mice model.

Mechanisms and management of thrombosis in cancer: focus on gastrointestinal malignancies.

Cancer patients have an increased risk of venous thromboembolism (VTE) which is their second cause of death after disease progression itself. Several thrombotic risk factors coexist in cancer patients, including the ability of both cancer and tumoral microenvironment's cells to directly or indirectly activate platelets and the enzymes of the coagulation cascade, resulting in a hyper-coagulable state of blood. This narrative review gives an overview of the main mechanisms leading to VTE in cancer patients, including the role that platelets and the clotting proteins may have in tumor growth and metastasis. Noteworthy, the haemostatic balance is altered in cancer patients who may, next to a thrombosis tendency, also have an increased risk of bleeding. To highlight the complexity and the precariousness of the haemostatic balance of these patients, we discuss two specific gastrointestinal malignancies: hepatocellular carcinoma, which is frequently associated with liver cirrhosis, a condition that causes profound alterations of haemostasis, and colorectal cancer, which is characterized by a fragile mucosa that is prone to bleeding. Understanding the molecular mechanisms of cancer-associated thrombosis may give a unique opportunity to develop new innovative drugs, acting differently on distinct pathways and potentially allowing to reduce the risk of bleeding related to antithrombotic therapies. Significance Statement The topic is significant because understanding the molecular mechanisms leading to cancer associated thrombosis and bleeding, focusing on gastrointestinal malignancies, enables the development of more rationale and innovative antithrombotic strategies for cancer associated thrombosis. Eventually, this will support an improved and patient-tailored antithrombotic management in vulnerable oncologic patients.

Interrogating a Compound Library in Search of an Inhibitor for TREM-like Transcript-1 to Fibrinogen Binding.

Cardiovascular disease (CVD) remains one of leading causes of death worldwide. Aberrant platelet function mediate fibrin(ogen) rich thrombi that lead to occlusive thrombi associated with mortality. The receptor, TREM-like transcript-1 (TLT-1), stored in the platelet a-granules and released upon platelet activation, binds fibrinogen and von Willebrand factor. Once it is released from platelets TLT-1 is a potential therapeutic target to prevent the thrombosis associated with CVD. Here we design an assay to screen a compound library of small molecules inhibitors. HEK-293 cells stably transfected with a full length human treml-1 construct were used to screen library of 800 compounds, for inhibition of TLT-1 to fibrinogen binding in an attachment assay using crystal violet staining. The possible cytotoxicity of the best compounds was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide MTT and calcein AM staining assays. Here we demonstrate that the addition of TLT-1 to HEK-293 cells increases cell adhesion by more than 2-fold. We identified ~80 compounds that inhibit binding by more than 80%. We further tested the top compounds and confirmed that reduction of hTLT-1 to fibrinogen bound in the top compounds was not caused by cytotoxicity, as per colorimetric and fluorescent viability assays. Four compounds were identified as potential small molecule inhibitors one of which, BM-8372, demonstrated significant effect in platelet aggregation assays. Significance Statement TLT-1 is a key platelet receptor that binds fibrinogen and mediates clot formation The developed assay successfully screens 800 small molecules, pinpointing ~80 potent inhibitors that reduce TLT-1 binding by over 80%. Importantly, the study rigorously rules out cytotoxicity concerns, affirming the therapeutic potential of the identified compounds. By elucidating TLT-1's role and presenting promising inhibitors, this research offers a significant stride toward developing novel strategies to combat CVD-related thrombosis.

IC Regimen: Delaying Resistance to Lorlatinib in ALK Driven Cancers by Adding Repurposed Itraconazole and Cilostazol.

Lorlatinib is a pharmaceutical ALK kinase inhibitor used to treat ALK driven non-small cell lung cancers. This paper analyses the intersection of past published data on the physiological consequences of two unrelated drugs from general medical practice-itraconazole and cilostazol-with the pathophysiology of ALK positive non-small cell lung cancer. A conclusion from that data analysis is that adding itraconazole and cilostazol may make lorlatinib more effective. Itraconazole, although marketed worldwide as a generic antifungal drug, also inhibits Hedgehog signaling, Wnt signaling, hepatic CYP3A4, and the p-gp efflux pump. Cilostazol, marketed worldwide as a generic thrombosis preventative drug, acts by inhibiting phosphodiesterase 3, and, by so doing, lowers platelets' adhesion, thereby partially depriving malignant cells of the many tumor trophic growth factors supplied by platelets. Itraconazole may enhance lorlatinib effectiveness by (i) reducing or stopping a Hedgehog-ALK amplifying feedback loop, by (ii) increasing lorlatinib's brain levels by p-gp inhibition, and by (iii) inhibiting growth drive from Wnt signaling. Cilostazol, surprisingly, carries minimal bleeding risk, lower than that of aspirin. Risk/benefit assessment of the combination of metastatic ALK positive lung cancer being a low-survival disease with the predicted safety of itraconazole-cilostazol augmentation of lorlatinib favors a trial of this drug trio in ALK positive lung cancer.

Current Advances in Nanomaterials Affecting Functions and Morphology of Platelets.

Nanomaterials have been extensively used in the biomedical field due to their unique physical and chemical properties. They promise wide applications in the diagnosis, prevention, and treatment of diseases. Nanodrugs are generally transported to target tissues or organs by coupling targeting molecules or enhanced permeability and retention effect (EPR) passively. As intravenous injection is the most common means of administration of nanomedicine, the transport process inevitably involves the interactions between nanoparticles (NPs) and blood cells. Platelets are known to not only play a critical role in normal coagulation by performing adhesion, aggregation, release, and contraction functions, but also be associated with pathological thrombosis, tumor metastasis, inflammation, and immune reactions, making it necessary to investigate the effects of NPs on platelet function during transport, particularly the way in which their physical and chemical properties determine their interaction with platelets and the underlying mechanisms by which they activate and induce platelet aggregation. However, such data are lacking. This review is intended to summarize the effects of NPs on platelet activation, aggregation, release, and apoptosis, as well as their effects on membrane proteins and morphology in order to shed light on such key issues as how to reduce their adverse reactions in the blood system, which should be taken into consideration in NP engineering.

GATA1 in Normal and Pathologic Megakaryopoiesis and Platelet Development.

GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.

Time-Dependent Changes in Pulmonary Turnover of Thrombocytes During Critical COVID-19.

OBJECTIVES BACKGROUND: Under normal conditions, pulmonary megakaryocytes are an important source of circulating thrombocytes, causing thrombocyte counts to be higher in arterial than venous blood. In critical COVID-19, thrombocytes may be removed from the circulation by the lungs because of immunothrombosis, possibly causing venous thrombocyte counts to be higher than arterial thrombocyte counts. In the present study, we investigated time-dependent changes in pulmonary turnover of thrombocytes during critical COVID-19 by measuring arteriovenous thrombocyte differences. We hypothesized that the early stages of the disease would be characterized by a net pulmonary removal of circulating thrombocytes because of immunothrombosis and that later stages would be characterized by a net pulmonary release of thrombocytes as normal pulmonary function is restored. DESIGN: Cohort study with repeated measurements of arterial and central venous thrombocyte counts. SETTING: ICU in a large university hospital. PATIENTS: Thirty-one patients with critical COVID-19 that were admitted to the ICU and received invasive or noninvasive mechanical ventilation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We found a significant positive association between the arteriovenous thrombocyte difference and time since symptom debut. This finding indicates a negative arteriovenous thrombocyte difference and hence pulmonary removal of thrombocytes in the early stages of the disease and a positive arteriovenous thrombocyte difference and hence pulmonary release of thrombocytes in later stages. Most individual arteriovenous thrombocyte differences were smaller than the variance coefficient of the analysis. CONCLUSIONS: The results of this study support our hypothesis that early stages of critical COVID-19 are characterized by pulmonary removal of circulating thrombocytes because of immunothrombosis and that later stages are characterized by the return of normal pulmonary release of thrombocytes. However, in most cases, the arteriovenous thrombocyte difference was too small to say anything about pulmonary thrombocyte removal and release on an individual level.

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

BACKGROUND: Leukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood. AIMS: To quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators. METHODS: Phorbol-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. RESULTS: In 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. CONCLUSIONS: Formation 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.

Utility of thromboelastography with platelet mapping (TEG-PM) for monitoring platelet transfusion in qualitative platelet disorders.

BACKGROUND: Patients with pathogenic variants in RASGRP2 (inherited platelet disorder (IPD)-18) have normal platelet counts but show impaired platelet aggregation due to diminished activation of αIIbß3 integrin. This defect results in moderate to severe bleeding episodes, especially following surgical procedures, which require patients to be transfused with platelets and/or pro-hemostatic agents. We recently demonstrated that hemostatic efficacy of transfused platelets is limited by dysfunctional endogenous platelets in a mouse model of IPD-18 (Rasgrp2-/- mice), as dysfunctional platelets were recruited to the forming hemostatic plug but did not participate in clot contraction. Consequently, higher amounts of transfused platelets were required to outcompete these dysfunctional cells and to reverse bleeding. OBJECTIVE: We here studied the usefulness of thromboelastography with platelet mapping (TEG-PM), a method to evaluate platelet-dependent clot contraction, for ex vivo monitoring of the hemostatic potential in Rasgrp2-/- mice transfused with various amounts of wild-type (WT) platelets. RESULTS: Rasgrp2-/- whole blood samples did not contract in TEG-PM, consistent with a critical role of this protein in αIIbß3 activation. Addition of WT platelets improved TEG parameters (K time, α-angle, MA) in a ratio dependent manner, consistent with our recent in vivo studies showing impaired hemostasis at a 5:1, but not at a 2:1 ratio of mutant to WT platelets. K and α values were identified as better predictors of transfusion efficacy than MA, the most platelet-dependent TEG parameter. CONCLUSION: This proof-of-concept study supports the use of TEG-PM to monitor platelet transfusion ratios and hemostatic potential in IPD-18 and potentially other platelet disorders.

Management of massive haemorrhage in transfusion medicine services in the Middle East and North Africa.

BACKGROUND AND OBJECTIVES: Massive transfusion protocols (MTPs) are critical in managing haemorrhage, yet their utilization varies. There is lack of data on the utilization of MTPs in the Middle East and North Africa (MENA) region. This study aims to assess the degree of utilization of MTPs in the region. MATERIALS AND METHODS: We conducted a survey to collect data on MTP use, inviting medical directors of transfusion services from various hospitals. Data were analysed to determine the prevalence of MTP utilization, their compositions, challenges in application and areas of future need. RESULTS: Eighteen respondents participated, representing 11 countries in the region. Thirteen hospitals implemented MTP, and eight included paediatrics. Eleven institutions used more than one definition of massive haemorrhage, with the most common being ≥10 red blood cell (RBC) units transfused for adults and replacement of >50% total blood volume in paediatrics. The majority of sites with MTPs utilized 1:1:1 RBCs:platelets:plasma ratio (70%). Variations were observed in the types and blood groups of components used. Two sites utilized whole blood, while six are considering it for future use. Utilization of adjunctive agents and frequency of laboratory testing varied among the sites. Challenges included the lack of medical expertise in protocol development, adherence and paediatric application. The need assessment emphasized the need for developing regional guidelines, standardized protocols and training initiatives. CONCLUSION: Although several hospitals have adopted MTPs, variations exist in activation criteria, blood product ratios and monitoring. Challenges include the lack of medical expertise, protocol adherence and addressing paediatric needs. Standardizing protocols, enhancing training and paediatric application are crucial for improving massive transfusion management in the region.

Surface topography modulates initial platelet adhesion to titanium substrata.

The clinical success of implanted biomaterials such as dental implants is largely determined by the molecular signaling that occurs at the tissue-implant interface. The modification of surface topography is a widely-employed strategy for optimizing tissue integration with dental implants. However, little is known regarding the direct, cellular-level effects of substratum topography on platelet signaling and adhesion, despite these cells being the first to encounter the implant surface during surgical placement. Here we compared platelet adhesion and secretion on four (4) different titanium surfaces, notably, the modifications applied to commercially available dental implants: smooth (S) titanium; acid-etched (AE), sandblasted (SB) and a combined acid-etching/sandblasting procedure (SLA). Platelets were isolated from human blood, washed, and seeded on to the 4 test surfaces; platelet adhesion was quantified by microscopy. In addition, the secretion of critical molecules stored in platelet granules (platelet factor 4, PF4; soluble P-selectin, sCD62P; transforming growth factor-beta1, TGF-ß1; platelet-derived growth factor-AB, PDGF-AB) was measured by enzyme-linked immunosorbent assay (ELISA) analysis of the supernatants. There was greater platelet adhesion to the rougher AE and SB surfaces, however, the concentration of the secreted growth factors was comparable on all surfaces. We conclude that while surface topography can be engineered to modulate initial platelet adhesion, granule secretion is likely regulated as a separate and independent process.

A case study of a patient with platelet transfusion refractoriness (PTR) combined with human leucocyte antigen (HLA) antibody positivity during hepatic arterial infusion chemotherapy in conjunction with the 'atezolizumab plus bevacizumab' regimen.

Hepatic arterial infusion chemotherapy in conjunction with the combination therapy of atezolizumab (T) and bevacizumab (A) is widely used in hepatocellular carcinoma. Some adverse events such as hypertension, weakness and elevated transaminase levels occurred during treatment, while there is currently no reported case about thrombocytopenia with concomitant HLA antibody-positive PTR. We summarize the critical care nursing experience of a patient with PTR because of HLA antibody positivity during hepatic arterial infusion chemotherapy in conjunction with atezolizumab plus bevacizumab (T + A) regimen. This paper explains the nursing measures for patients with severe thrombocytopenia and proposes nursing measures for situations where conventional treatments are ineffective. Key nursing points include the administration of intravenous immunoglobulin (IVIG) and HLA-compatible platelets, prevention of complications, psychological care, oral care, and skin management. Through systematic treatment and targeted nursing care, the patient's platelet count rebounded after 9 days, leading to a successful recovery and discharge. Subsequent follow-up assessments revealed the patient's sustained well-being. Thrombocytopenia is a potential adverse reaction during the treatment of liver cancer. When platelet transfusion is ineffective, vigilance is necessary for the possibility of HLA positivity, and prompt symptomatic management is warranted.

RUNX1 Isoforms Regulate RUNX1 and Target-Genes Differentially in Platelets-Megakaryocytes: Association with Clinical Cardiovascular Events.

Background: Hematopoietic transcription factor RUNX1 is expressed from proximal P2 and distal P1 promoter to yield isoforms RUNX1 B and C, respectively. The roles of these isoforms in RUNX1 autoregulation and downstream-gene regulation in megakaryocytes and platelets are unknown. Objectives: To understand the regulation of RUNX1 and its target genes by RUNX1 isoforms. Methods: We performed studies on RUNX1 isoforms in megakaryocytic HEL cells and HeLa cells (lack endogenous RUNX1), in platelets from 85 healthy volunteers administered aspirin or ticagrelor, and on the association of RUNX1 target genes with acute events in 587 patients with cardiovascular disease (CVD). Results: In chromatin immunoprecipitation and luciferase promoter assays, RUNX1 isoforms B and C bound and regulated P1 and P2 promoters. In HeLa cells RUNX1B decreased and RUNX1C increased P1 and P2 activities, respectively. In HEL cells, RUNX1B overexpression decreased RUNX1C and RUNX1A expression; RUNX1C increased RUNX1B and RUNX1A. RUNX1B and RUNX1C regulated target genes (MYL9, F13A1, PCTP, PDE5A and others) differentially in HEL cells. In platelets RUNX1B transcripts (by RNAseq) correlated negatively with RUNX1C and RUNX1A; RUNX1C correlated positively with RUNX1A. RUNX1B correlated positively with F13A1, PCTP, PDE5A, RAB1B, and others, and negatively with MYL9. In our previous studies, RUNX1C transcripts in whole blood were protective against acute events in CVD patients. We found that higher expression of RUNX1 targets F13A1 and RAB31 associated with acute events. Conclusions: RUNX1 isoforms B and C autoregulate RUNX1 and regulate downstream genes in a differential manner and this associates with acute events in CVD.

Macrothrombocytopenia with leukocyte inclusions in a patient with Wilson disease: a case report and literature review.

BACKGROUND: Wilson disease (WD) is an autosomal recessive disorder caused by homozygous or compound heterozygous mutations in ATP7B. Clinical manifestations primarily involve liver and nervous system lesions, with rarely observed hematologic manifestations. CASE PRESENTATION: In the present case, a patient with WD presented with thrombocytopenia, giant platelets, and Döhle-like cytoplasmic inclusions in the leukocytes. Initially, the May-Hegglin anomaly was considered; however, whole-exome sequencing did not reveal any mutation in the MYH9 gene but a heterozygous mutation was found in (C.2804 C > T, p.T935M) in the ATP7B gene. After two years, the patient developed tremors in his hands, lower limb stiffness, and foreign body sensation in the eyes. Additionally, Kayser-Fleischer rings in the corneal limbus were detected by slit-lamp examination. Copper metabolism test indicated a slight decrease in serum ceruloplasmin. Transmission electron microscopy revealed that the inclusion bodies of leukocytes were swollen mitochondria. Mass spectrometry analysis showed that the copper levels were almost 20-fold higher in the leukocytes of the patient than in those of the control group. Based on the Leipzig scoring system, a diagnosis of WD was confirmed. Zinc sulfate treatment ameliorated the patient's symptoms and enhanced platelet, serum ceruloplasmin, and albumin levels. CONCLUSIONS: In conclusion, this case represents the first documented instance of WD presenting as thrombocytopenia, giant platelets, and Döhle-like cytoplasmic inclusions in the leukocytes. Excessive cellular copper accumulation likely underlies these findings; however, understanding precise mechanisms warrants further investigation.

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.

Kinetics and toxicity of nanoplastics in ex vivo exposed human whole blood as a model to understand their impact on human health.

The ubiquitous presence of nanoplastics (NPLs) in the environment is considered of great health concern. Due to their size, NPLs can cross both the intestinal and pulmonary barriers and, consequently, their presence in the blood compartment is expected. Understanding the interactions between NPLs and human blood components is required. In this study, to simulate more adequate real exposure conditions, the whole blood of healthy donors was exposed to five different NPLs: three polystyrene NPLs of approximately 50 nm (aminated PS-NH2, carboxylated PS-COOH, and pristine PS- forms), together with two true-to-life NPLs from polyethylene terephthalate (PET) and polylactic acid (PLA) of about 150 nm. Internalization was determined in white blood cells (WBCs) by confocal microscopy, once the different main cell subtypes (monocytes, polymorphonucleated cells, and lymphocytes) were sorted by flow cytometry. Intracellular reactive oxygen species (iROS) induction was determined in WBCs and cytokine release in plasma. In addition, hemolysis, coagulation, and platelet activation were also determined. Results showed a differential uptake between WBC subtypes, with monocytes showing a higher internalization. Regarding iROS, lymphocytes were those with higher levels, which was observed for different NPLs. Changes in cytokine release were also detected, with higher effects observed after PLA- and PS-NH2-NPL exposure. Hemolysis induction was observed after PS- and PS-COOH-NPL exposure, but no effects on platelet functionality were observed after any of the treatments. To our knowledge, this is the first study comprehensively evaluating the bloodstream kinetics and toxicity of NPL from different polymeric types on human whole blood, considering the role played by the cell subtype and the NPLs physicochemical characteristics in the effects observed after the exposures.

Clinical application of liquid biopsy in colorectal cancer: detection, prediction, and treatment monitoring.

Colorectal cancer (CRC) is one of the most prevalent malignancies affecting the gastrointestinal tract and is ranked third among cancers with the highest incidence and second-highest mortality rate worldwide. CRC exhibits a slow progression providing a wide treatment window. The currently employed CRC screening methods have shown great potential to prevent CRC and reduce CRC-related morbidity and mortality. The diagnosis of CRC is achieved by colonoscopy and tissue biopsy, with studies showing that liquid biopsy is more effective in detecting and diagnosing early CRC patients. Increasing number of studies have shown that the tumor components shed into circulating blood can be detected in liquid form, and can be applied in the clinical management of CRC. Analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or tumor-associated platelets (TEPs) in the blood can be used for early screening and diagnosis of CRC, aid tumor staging, treatment response monitoring, and prediction of CRC recurrence and metastasis in a minimally invasive manner. This chapter provides an updated review of CTCs, ctDNA, and TEPs as novel biomarkers for CRC, highlighting their strengths and limitations.