Acute ischemic STROKE - from laboratory to the Patient's BED (STROKELABED): A translational approach to reperfusion injury. Study Protocol.

Cerebral edema (CE) and hemorrhagic transformation (HT) are frequent and unpredictable events in patients with acute ischemic stroke (AIS), even when an effective vessel recanalization has been achieved. These complications, related to blood-brain barrier (BBB) disruption, remain difficult to prevent or treat and may offset the beneficial effect of recanalization, and lead to poor outcomes. The aim of this translational study is to evaluate the association of circulating and imaging biomarkers with subsequent CE and HT in stroke patients with the dual purpose of investigating possible predictors as well as molecular dynamics underpinning those events and functional outcomes. Concurrently, the preclinical study will develop a new mouse model of middle cerebral artery (MCA) occlusion and recanalization to explore BBB alterations and their potentially harmful effects on tissue. The clinical section of the study is based on a single-center observational design enrolling consecutive patients with AIS in the anterior circulation territory, treated with recanalization therapies from October 1, 2015 to May 31, 2020. The study will employ an innovative evaluation of routine CT scans: in fact, we will assess and quantify the presence of CE and HT after stroke in CT scans at 24 h, through the quantification of anatomical distortion (AD), a measure of CE and HT. We will investigate the relationship of AD and several blood biomarkers of inflammation and extracellular matrix, with functional outcomes at 3 months. In parallel, we will employ a newly developed mouse model of stroke and recanalization, to investigate the emergence of BBB changes 24 h after the stroke onset. The close interaction between clinical and preclinical research can enhance our understanding of findings from each branch of research, enabling a deeper interpretation of the underlying mechanisms of reperfusion injury following recanalization treatment for AIS.

Whole Exome Sequencing in Vaccine-Induced Thrombotic Thrombocytopenia (VITT).

Background: In February 2021, a few cases of unusual, severe thrombotic events associated with thrombocytopenia reported after vaccination with ChAdOx1 nCoV-19 (Vaxzevria) or with Johnson & Johnson's Janssen vaccine raise concern about safety. The vaccine-induced thrombotic thrombocytopenia (VITT) has been related to the presence of platelet-activating antibodies directed against platelet Factor 4. Objectives: We investigated VITT subject genetic background by a high-throughput whole exome sequencing (WES) approach in order to investigate VITT genetic predisposition. Methods: Six consecutive patients (females of Caucasian origin with a mean age of 64 years) were referred to the Atherothrombotic Diseases Center (Department of Experimental and Clinical Medicine, Azienda Ospedaliero-Universitaria Careggi, Florence) with a diagnosis of definite VITT underwent WES analysis. WES analysis was performed on the Illumina NextSeq500 platform. Results:WES analysis revealed a total of 140,563 genetic variants. Due to VITT's rare occurrence, we focused attention on rare variants. The global analysis of all high-quality rare variants did not reveal a significant enrichment of mutated genes in biological/functional pathways common to patients analyzed. Afterwards, we focused on rare variants in genes associated with blood coagulation and fibrinolysis, platelet activation and aggregation, integrin-mediated signaling pathway, and inflammation with particular attention to those involved in vascular damage, as well as autoimmune thrombocytopenia. According to ACMG criteria, 47/194 (24.2%) rare variants were classified as uncertain significance variants (VUS), whereas the remaining were likely benign/benign. Conclusion: WES analysis identifies rare variants possibly favoring the prothrombotic state triggered by the exposure to the vaccine. Functional studies and/or extensions to a larger number of patients might allow a more comprehensive definition of these molecular pathways.

Heparin-induced thrombocytopenia after cardiac surgery. A single-center, retrospective cohort study.

Background: Cardiac surgery is a high-risk setting for heparin-induced thrombocytopenia (HIT). However, large differences in its incidence, rate of thrombotic complications, and mortality have been reported in this context. Few studies address the pharmacologic management of HIT specifically in this setting. Objectives: To describe the incidence, outcomes, and management of patients with HIT in our cohort and to compare them with patients presenting platelet factor 4/heparin antibodies but without platelet-activating capacity. Methods: A retrospective observational study was conducted over a period of 10 years and 6 months on 13,178 cardiac operations in a single high-volume cardiac surgery center. Results: HIT was diagnosed in 0.22% of patients. HIT with associated thromboembolic complications occurred in 0.04% of cases. Two deaths at 30 days were registered, both in patients with associated thrombosis. The 4T score showed a 99.9% negative predictive value. The immunoglobulin G-specific chemiluminescence test positivity rate was highly predictive of HIT. Warfarin was often started early after surgery, and although it was rarely stopped when the diagnosis of HIT was made, no new thromboembolic complications subsequently occurred. Thrombocytopenia appeared to be a poor prognostic sign, whatever the cause. Conclusion: Although rare, HIT is characterized by high mortality in this setting, especially if thrombotic complications occur. Large multicentric studies or an international registry should be created to enhance the scientific evidence on HIT diagnosis and management in this context.

The serum metabolomic profiles of atrial fibrillation patients treated with direct oral anticoagulants or vitamin K antagonists.

AIMS: Long-term oral anticoagulation is the primary therapy for preventing ischemic stroke in patients with atrial fibrillation (AF). Different types of oral anticoagulant drugs can have specific effects on the metabolism of patients. Here we characterize, for the first time, the serum metabolomic and lipoproteomic profiles of AF patients treated with anticoagulants: vitamin K antagonists (VKAs) or direct oral anticoagulants (DOACs). MATERIALS AND METHODS: Serum samples of 167 AF patients (median age 78 years, 62 % males, 70 % on DOACs treatment) were analyzed via high resolution 1H nuclear magnetic resonance (NMR) spectroscopy. Data on 25 metabolites and 112 lipoprotein-related fractions were quantified and analyzed with multivariate and univariate statistical approaches. KEY FINDINGS: Our data provide evidence that patients treated with VKAs and DOACs present significant differences in their profiles: lower levels of alanine and lactate (odds ratio: 1.72 and 1.84), free cholesterol VLDL-4 subfraction (OR: 1.75), triglycerides LDL-1 subfraction (OR: 1.80) and 4 IDL cholesterol fractions (ORs âˆ¼ 1.80), as well as higher levels of HDL cholesterol (OR: 0.48), apolipoprotein A1 (OR: 0.42) and 7 HDL cholesterol fractions/subfractions (ORs: 0.40-0.51) are characteristic of serum profile of patients on DOACs' therapy. SIGNIFICANCE: Our results support the usefulness of NMR-based metabolomics for the description of the effects of oral anticoagulants on AF patient circulating metabolites and lipoproteins. The higher serum levels of HDL cholesterol observed in patients on DOACs could contribute to explaining their reduced cardiovascular risk, suggesting the need of further studies in this direction to fully understand possible clinical implications.

Machine learning approach for prediction of outcomes in anticoagulated patients with atrial fibrillation.

BACKGROUND: The accuracy of available prediction tools for clinical outcomes in patients with atrial fibrillation (AF) remains modest. Machine Learning (ML) has been used to predict outcomes in the AF population, but not in a population entirely on anticoagulant therapy. METHODS AND AIMS: Different supervised ML models were applied to predict all-cause death, cardiovascular (CV) death, major bleeding and stroke in anticoagulated patients with AF, processing data from the multicenter START-2 Register. RESULTS: 11078 AF patients (male n = 6029, 54.3%) were enrolled with a median follow-up period of 1.5 years [IQR 1.0-2.6]. Patients on Vitamin K Antagonists (VKA) were 5135 (46.4%) and 5943 (53.6%) were on Direct Oral Anticoagulants (DOAC). Using Multi-Gate Mixture of Experts, a cross-validated AUC of 0.779 ± 0.016 and 0.745 ± 0.022 were obtained, respectively, for the prediction of all-cause death and CV-death in the overall population. The best ML model outperformed CHA2DSVA2SC and HAS-BLED for all-cause death prediction (p < 0.001 for both). When compared to HAS-BLED, Gradient Boosting improved major bleeding prediction in DOACs patients (0.711 vs. 0.586, p < 0.001). A very low number of events during follow-up (52) resulted in a suboptimal ischemic stroke prediction (best AUC of 0.606 ± 0.117 in overall population). Body mass index, age, renal function, platelet count and hemoglobin levels resulted the most important variables for ML prediction. CONCLUSIONS: In AF patients, ML models showed good discriminative ability to predict all-cause death, regardless of the type of anticoagulation strategy, and major bleeding on DOAC therapy, outperforming CHA2DS2VASC and the HAS-BLED scores for risk prediction in these populations.

Ex Vivo Antiplatelet Effects of Oral Anticoagulants.

BACKGROUND: The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. METHODS: We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. RESULTS: All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. CONCLUSIONS: Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Digital droplet PCR versus quantitative PCR for lipoprotein (a) kringle IV type 2 repeat polymorphism genetic characterization.

BACKGROUND: Lipoprotein(a) [Lp(a)] level variability, related to atherothrombotic risk increase, is mainly attributed to LPA gene, encoding apolipoprotein(a), with kringle IV type 2 (KIV2) copy number variation (CNV) acting as the primary genetic determinant. Genetic characterization of Lp(a) is in continuous growth; nevertheless, the peculiar structural characteristics of this variant constitute a significant challenge to the development of effective detection methods. The aim of the study was to compare quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR) in the evaluation of KIV2 repeat polymorphism. METHODS: We analysed 100 subjects tested for cardiovascular risk in which Lp(a) plasma levels were assessed. RESULTS: Correlation analysis between CNV values obtained with the two methods was slightly significant (R = 0.413, p = 0.00002), because of the wider data dispersion in qPCR compared with ddPCR. Internal controls C1, C2 and C3 measurements throughout different experimental sessions revealed the superior stability of ddPCR, which was supported by a reduced intra/inter-assay coefficient of variation determined in this method compared to qPCR. A significant inverse correlation between Lp(a) levels and CNV values was confirmed for both techniques, but it was higher when evaluated by ddPCR than qPCR (R = -0.393, p = 0.000053 vs R = -0.220, p = 0.028, respectively). When dividing subjects into two groups according to 500 mg/L Lp(a) cut-off value, a significantly lower number of KIV2 repeats emerged among subjects with greater Lp(a) levels, with stronger evidence in ddPCR than in qPCR (p = 0.000013 and p = 0.001, respectively). CONCLUSIONS: Data obtained support a better performance of ddPCR in the evaluation of KIV2 repeat polymorphism.

Effects of a dietary intervention with lacto-ovo-vegetarian and Mediterranean diets on apolipoproteins and inflammatory cytokines: results from the CARDIVEG study.

BACKGROUND: Apolipoproteins have been recently proposed as novel markers of cardiovascular disease (CVD) risk. However, evidence regarding effects of diet on apolipoproteins is limited. AIM: To compare the effects of Mediterranean diet (MD) and lacto-ovo vegetarian diet (VD) on apolipoproteins and traditional CVD risk factors in participants with low-to-moderate CVD risk. METHODS: Fifty-two participants (39 women; 49.1 ± 12.4 years), followed MD and VD for 3 months each. Medical and dietary information was collected at the baseline. Anthropometric parameters and blood samples were obtained at the beginning and the end of interventions. RESULTS: MD and VD resulted in significant improvement in anthropometric and lipid profiles. Both diets led to a reduction in most of the inflammatory parameters. As for apolipoproteins, a significant change was observed for ApoC-I after VD (+ 24.4%; p = 0.020). MD led to a negative correlation between ApoC-III and carbohydrates (R = - 0.29; p = 0.039) whereas VD between ApoD and saturated fats (R = - 0.38; p = 0.006). A positive correlation emerged after VD between HDL and ApoD (R = 0.33; p = 0.017) and after MD between plasma triglycerides and ApoC-I (R = 0.32; p = 0.020) and ApoD (R = 0.30; p = 0.031). IL-17 resulted to be positively correlated with ApoB after MD (R = 0.31; p = 0.028) and with ApoC-III after VD (R = 0.32; p = 0.019). Subgroup analysis revealed positive effects on apolipoproteins from both diets, especially in women, individuals older than 50 years-old or with < 3 CVD risk factors. CONCLUSIONS: Both diets seem to improve CVD risk, however, MD showed a greater positive effect on apolipoproteins in some subgroups, thus suggesting how diet may influence new potential markers of CVD risk. TRIAL REGISTRATION: registered at clinicaltrials.gov (identifier: NCT02641834) on December 2015.

The Role of Extracellular Matrix and Inflammation in the Stratification of Bleeding and Thrombotic Risk of Atrial Fibrillation on Oral Anticoagulant Therapy: Insights from Strat-Af Study.

In anticoagulated atrial fibrillation (AF) patients, the validity of models recommended for the stratification of the risk ratio between benefits and hemorrhage risk is limited. We hypothesize that both circulating and neuroimaging-based markers might improve the prediction of bleeding and thrombotic risk in anticoagulated AF patients. The Strat-AF study is an observational, prospective, single-center study enrolling 170 patients with AF; recruited patients are evaluated by means of a comprehensive protocol, with clinical, cerebral magnetic resonance imaging and circulating biomarkers assessment. The main outcome is the evaluation of cerebral microangiopathy related to the levels of circulating biomarkers of inflammation and extracellular matrix (ECM) remodeling. At multivariate logistic regression analysis adjusted for age, sex, CHA2DS2-VASc, HAS-BLED and type of anticoagulant, matrix metalloproteinases (MMP)-2 levels were significantly and positively associated with the presence of cerebral microbleeds (CMBs). A significant association between MMP-2, tissue inhibitor of metalloproteinases (TIMP)-1,-2,-4 levels and white matter hyperintensity was also found. Concerning the small vessel disease (SVD) score, MMP-2 and TIMP-1,-2 levels were associated with the presence of two and three or more signs of SVD, whereas TIMP-4 levels were associated with the presence of three signs of SVD with respect to patients with no instrumental signs of SVD. As regarding the presence of enlarged perivascular spaces (EPVS), a significant association was found for high levels of interleukin (IL)-8 and TIMP 1-2-3. These results demonstrate that patients with AF have evidence of impaired ECM degradation, which is an independent risk factor for thrombotic complications of AF patients on oral anticoagulant therapy. The incorporation of these markers in the prognostic schemes might improve their clinical capability in predicting stroke risk and thrombotic complications.

Cognitive phenotypes and factors associated with cognitive decline in a cohort of older patients with atrial fibrillation: The Strat-AF study.

BACKGROUND AND PURPOSE: The multifactorial relationship between atrial fibrillation (AF) and cognitive impairment needs to be elucidated. The aim of this study was to assess, in AF patients on oral anticoagulants (OACs), the prevalence of cognitive impairment, defined according to clinical criteria or data-driven phenotypes, the prevalence of cognitive worsening, and factors associated with cognitive outcomes. METHODS: The observational prospective Strat-AF study enrolled AF patients aged ≥ 65 years who were receiving OACs. The baseline and 18-month protocol included clinical, functional, and cognitive assessment, and brain magnetic resonance imaging. Cognitive outcomes were: empirically derived cognitive phenotypes; clinical diagnosis of cognitive impairment; and longitudinal cognitive worsening. RESULTS: Out of 182 patients (mean age 77.7 ± 6.7 years, 63% males), 82 (45%) received a cognitive impairment diagnosis, which was associated with lower education level and functional status, and higher level of atrophy. Cluster analysis identified three cognitive profiles: dysexecutive (17%); amnestic (25%); and normal (58%). Compared to the normal group, the dysexecutive group was older, and had higher CHA2 DS2 -VASc scores, while the amnestic group had worse cognitive and functional abilities, and medial temporal lobe atrophy (MTA). Out of 128 followed-up patients, 35 (27%) had cognitive worsening that was associated with lower education level, worse cognitive efficiency, CHA2 DS2 -VASc score, timing of OAC intake, history of stroke, diabetes, non-lacunar infarcts, white matter hyperintensities and MTA. In multivariate models, belonging to the dysexecutive or amnestic group was a main predictor of cognitive worsening. CONCLUSIONS: In our cohort of older AF patients, CHA2 DS2 -VASc score, timing of OAC intake, and history of stroke influenced presence, type and progression of cognitive impairment. Empirically derived cognitive classification identified three groups with different clinical profiles and better predictive ability for cognitive worsening compared to conventional clinical diagnosis.

Nuclear Magnetic Resonance-Based Metabolomics to Predict Early and Late Adverse Outcomes in Ischemic Stroke Treated with Intravenous Thrombolysis.

Metabolic perturbations and inflammatory mediators play a fundamental role in both early and late adverse post-acute ischemic stroke outcomes. Using data from the observational MAGIC (MArker bioloGici nell'Ictus Cerebrale) study, we evaluated the effect of 130 serum metabolic features, using a nuclear magnetic spectroscopy approach, on the following outcomes: hemorrhagic transformation at 24 h after stroke, non-response to intravenous thrombolytic treatment with the recombinant tissue plasminogen activator (rt-PA), and the 3 month functional outcome. Blood circulating metabolites, lipoproteins, and inflammatory markers were assessed at the baseline and 24 h after rt-PA treatment. Adjusting for the major determinants for unfavorable outcomes (i.e., age, sex, time onset-to-treatment, etc.), we found that acetone and 3-hydroxybutyrate were associated with symptomatic hemorrhagic transformation and with non-response to rt-PA; while 24 h after rt-PA, levels of triglycerides high-density lipoprotein (HDL) and triglycerides low-density lipoprotein (LDL) were associated with 3 month mortality. Cholesterol and phospholipids levels, mainly related to smaller and denser very low-density lipoprotein (VLDL) and LDL subfractions were associated with 3 month poor functional outcomes. We also reported associations between baseline 24 h relative variation (Δ) in VLDL subfractions and ΔC-reactive protein, Δinterleukin-10 levels with hemorrhagic transformation. All observed metabolic changes reflect a general condition of energy failure, oxidative stress, and systemic inflammation that characterize the development of adverse outcomes.

Infection with SARS-CoV-2 Variants Is Associated with Different Long COVID Phenotypes.

COVID-19 has been associated with a broad range of long-term sequelae, commonly referred to as "long-COVID" or "post-COVID-19" syndrome. Despite an increasing body of literature, long COVID remains poorly characterized. We retrospectively analysed data from electronic medical records of patients admitted to the post-COVID-19 outpatient service of the Infectious and Tropical Diseases Unit, Careggi University Hospital, Florence, Italy, between June 2020 and June 2021, 4-12 weeks after hospital discharge. A total of 428 patients, 41% women, median age 64 years, underwent a follow-up visit a median 53 days after hospital discharge. Overall, 76% patients reported at least one persistent symptom, including dyspnoea (37%), chronic fatigue (36%), insomnia (16%), visual disorders (13%) and brain fog (13%). Increasing oxygen support (OR 1.4, 95% CI 1.1-1.8), use of immunosuppressants (OR 6.4, 95% CI 1.5-28) and female sex (OR 1.8, 95% CI 1.1-2.9) were associated with a higher risk of long COVID symptoms. Comparison between symptomatic patients infected in the period March-December 2020 (prevalent circulation of wild-type SARS-CoV-2) with those infected in the period January-April 2021 (prevalent circulation of B.1.1.7 Alpha variant) showed a significant modification in the pattern of symptoms belonging to the neurological and cognitive/emotional categories. Our findings confirmed shortness of breath and chronic fatigue as the most frequent long COVID manifestations, while female sex and severe COVID-19 course were the main risk factors for developing lingering symptoms. SARS-CoV-2 variants may induce different long COVID phenotypes, possibly due to changes in cell tropism and differences in viral-host interaction.

Tracking an Elusive Killer: State of the Art of Molecular-Genetic Knowledge and Laboratory Role in Diagnosis and Risk Stratification of Thoracic Aortic Aneurysm and Dissection.

The main challenge in diagnosing and managing thoracic aortic aneurysm and dissection (TAA/D) is represented by the early detection of a disease that is both deadly and "elusive", as it generally grows asymptomatically prior to rupture, leading to death in the majority of cases. Gender differences exist in aortic dissection in terms of incidence and treatment options. Efforts have been made to identify biomarkers that may help in early diagnosis and in detecting those patients at a higher risk of developing life-threatening complications. As soon as the hereditability of the TAA/D was demonstrated, several genetic factors were found to be associated with both the syndromic and non-syndromic forms of the disease, and they currently play a role in patient diagnosis/prognosis and management-guidance purposes. Likewise, circulating biomarker could represent a valuable resource in assisting the diagnosis, and several studies have attempted to identify specific molecules that may help with risk stratification outside the emergency department. Even if promising, those data lack specificity/sensitivity, and, in most cases, they need more testing before entering the "clinical arena". This review summarizes the state of the art of the laboratory in TAA/D diagnostics, with particular reference to the current and future role of molecular-genetic testing.

Can CHA2DS2-VASc and HAS-BLED Foresee the Presence of Cerebral Microbleeds, Lacunar and Non-Lacunar Infarcts in Elderly Patients With Atrial Fibrillation? Data From Strat-AF Study.

Anticoagulants reduce embolic risk in atrial fibrillation (AF), despite increasing hemorrhagic risk. In this context, validity of congestive heart failure, hypertension, age ≥ 75 years, diabetes, stroke, vascular disease, age 65-74 years and sex category (CHA2DS2-VASc) and hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly, drugs/alcohol concomitantly (HAS-BLED) scales, used to respectively evaluate thrombotic and hemorrhagic risks, is incomplete. In patients with AF, brain MRI has led to the increased detection of "asymptomatic" brain changes, particularly those related to small vessel disease, which also represent the pathologic substrate of intracranial hemorrhage, and silent brain infarcts, which are considered risk factors for ischemic stroke. Routine brain MRI in asymptomatic patients with AF is not yet recommended. Our aim was to test predictive ability of risk stratification scales on the presence of cerebral microbleeds, lacunar, and non-lacunar infarcts in 170 elderly patients with AF on oral anticoagulants. Ad hoc developed R algorithms were used to evaluate CHA2DS2-VASc and HAS-BLED sensitivity and specificity on the prediction of cerebrovascular lesions: (1) Maintaining original items' weights; (2) augmenting weights' range; (3) adding cognitive, motor, and depressive scores. Accuracy was poor for each outcome considering both scales either in phase 1 or phase 2. Accuracy was never improved by the addition of cognitive scores. The addition of motor and depressive scores to CHA2DS2-VASc improved accuracy for non-lacunar infarcts (sensitivity = 0.70, specificity = 0.85), and sensitivity for lacunar-infarcts (sensitivity = 0.74, specificity = 0.61). Our results are a very first step toward the attempt to identify those elderly patients with AF who would benefit most from brain MRI in risk stratification.

Detection of Platelet-Activating Antibodies Associated with Vaccine-Induced Thrombotic Thrombocytopenia by Flow Cytometry: An Italian Experience.

Rare cases of thrombocytopenia and thrombosis after anti-COVID-19 adenovirus-associated mRNA vaccines (VITT) due to platelet-activating anti-platelet-factor 4 (PF4)/polyanion antibodies have been reported. VITT laboratory diagnosis, similarly to heparin-induced thrombocytopenia (HIT) diagnosis, requires immunoassays for anti-PF4/polyanion antibodies identification, such as ELISA assays and platelet-activating functional tests, such as heparin-induced platelet activation test (HIPA), to confirm their pathogenicity. We compared the flow cytometry (FC) measurement of platelet p-selectin exposure to the gold standard functional test HIPA for diagnosis confirmation in 13 patients with a clinical VITT syndrome (6M/7F; median age 56 (33-78)) who resulted positive to anti-PF4/polyanion antibodies ELISA assays (12/13). FC and HIPA similarly identified three different patterns: (1) a typical non-heparin-dependent VITT pattern (seven and six patients by FC and HIPA, respectively); (2) low/no platelet activation in patients under IvIg therapy (five out of five and two out of four patients by FC and HIPA, respectively); (3) a HIT pattern. Antibodies investigated by FC became negative after 7, 17, and 24 days of therapy in three patients. FC measurement of P-selectin exposure was as sensitive as HIPA but simpler to detect anti-PF4/polyanion antibodies in VITT patients. FC could reliably discriminate VITT from HIT, thus helping for the choice of the anticoagulant.

The p.P1127S pathogenic variant lowers von Willebrand factor levels through higher affinity for the macrophagic scavenger receptor LRP1: Clinical phenotype and pathogenic mechanisms.

BACKGROUND: The index case is a 21-year-old Italian woman with a mild hemorrhagic syndrome and von Willebrand factor antigen (VWF:Ag) = 34.3 U/dl, VWF recombinant glycoprotein Ib (VWF:GpIbR) = 32.8 U/dl, and factor VIII (FVIII) = 55.3 IU/dl. AIMS: The aim of this study is to characterize from a genetic and biochemical standpoint this low VWF phenotype. METHODS: Coagulation and biochemical methods were used to study the structural and functional pattern of VWF multimers in the index case's plasma. Recombinant wild-type and p.P1127S VWF variants were produced using human embryonic kidney (HEK)-293 cells. In addition, genetic screening was carried out to detect single nucleotide variants of some scavenger VWF/FVIII receptor genes such as CLEC4M, STAB2, and ASGR2. RESULTS: Genetic investigation revealed that the index case inherited from her mother the heterozygous missense mutation c.3379C > T (VWF exon 25), causing the p.P1127S substitution in the VWF D'D3 domain. The index case was also homozygous for the scavenger receptor ASGR2 c.-95 CC-genotype. Desmopressin normalized the VWF level of the patient, although its clearance was faster (t1/2  = 6.7 h) than in normal subjects (t1/2  = 12 ± 0.7 h). FVIII-VWF interaction, A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif-13 levels, ristocetin-induced-platelet-aggregation, and VWF multimeric pattern were normal. The p.P1127S variant was normally synthesized and secreted by HEK-293 cells, and molecular modeling predicts a conformational change showing higher affinity for the macrophagic scavenger receptor lipoprotein receptor-related protein 1 (LRP1), as also experimentally verified. CONCLUSIONS: The p.P1127S variant may cause a low VWF phenotype, stemming from an increased VWF affinity for the scavenger receptor LRP1 and, consequently, an accelerated clearance of VWF.

Lipid and metabolite correlation networks specific to clinical and biochemical covariate show differences associated with sexual dimorphism in a cohort of nonagenarians.

This study defines and estimates the metabolite-lipidic component association networks constructed from an array of 20 metabolites and 114 lipids identified and quantified via NMR spectroscopy in the serum of a cohort of 355 Italian nonagenarians and ultra-nonagenarian. Metabolite-lipid association networks were built for men and women and related to an array of 101 clinical and biochemical parameters, including the presence of diseases, bio-humoral parameters, familiarity diseases, drugs treatments, and risk factors. Different connectivity patterns were observed in lipids, branched chains amino acids, alanine, and ketone bodies, suggesting their association with the sex-related and sex-clinical condition-related intrinsic metabolic changes. Furthermore, our results demonstrate, using a holistic system biology approach, that the characterization of metabolic structures and their dynamic inter-connections is a promising tool to shed light on the dimorphic pathophysiological mechanisms of aging at the molecular level.

Differential Diagnosis between Marfan Syndrome and Loeys-Dietz Syndrome Type 4: A Novel Chromosomal Deletion Covering TGFB2.

Marfan syndrome (MFS) and Loeys-Dietz syndrome type 4 (LDS4) are two hereditary connective tissue disorders. MFS displays ectopia lentis as a distinguishing, characterising feature, and thoracic aortic ectasia, aneurysm, dissection, and systemic features as manifestations overlapping with LDS4. LDS4 is characterised by the presence of hypertelorism, cleft palate and/or bifid uvula, with possible ectasia or aneurysms in other arteries. The variable age of onset of clinical manifestations makes clinical diagnosis more difficult. In this study, we report the case of a patient with Marfan syndrome diagnosed at our centre at the age of 33 on the basis of typical clinical manifestations of this syndrome. At the age of 38, the appearance of ectasia of the left common iliac artery and tortuosity of the iliac arteries suggested the presence of LDS4. Next Generation Sequencing (NGS) analysis, followed by Array-CGH, allowed the detection of a novel chromosomal deletion including the entire TGFB2 gene, confirming not only the clinical suspicion of LDS4, but also the clinical phenotype associated with the haploinsufficiency mechanism, which is, in turn, associated with the deletion of the entire gene. The same mutation was detected in the two young sons. This emblematic case confirms that we must be very careful in the differential diagnosis of these two pathologies, especially before the age of 40, and that, in young subjects suspected to be affected by MFS in particular, we must verify the diagnosis, extending genetic analysis, when necessary, to the search for chromosomal alterations. Recently, ectopia lentis has been reported in a patient with LDS4, confirming the tight overlap between the two syndromes. An accurate revision of the clinical parameters both characterising and overlapping the two pathologies is highly desirable.