Augmented thrombin formation is related to circulating levels of extracellular vesicles exposing tissue factor and citrullinated histone-3 in anti-neutrophil cytoplasmic antibody-associated vasculitides

Publisher Copyright: Copyright © 2023 Jonasdottir, Manojlovic, Vojinovic, Nordin, Bruchfeld, Gunnarsson, Mobarrez and Antovic.Objectives: To study circulating myeloperoxidase (MPO)-positive extracellular vesicles (MPO+EVs) exposing citrullinated histone-3 (H3Cit), tissue factor (TF), and plasminogen (Plg) in association to thrombin generation in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV). Methods: We have involved well-characterized patients with AAV together with population-based controls. Flow cytometry was used to assess the levels of MPO+EVs in citrated plasma. MPO+EVs were phenotyped by anti-MPO-antibodies together with anti-CD142 (anti-TF), anti-H3Cit, and anti-Plg antibodies. A modified Calibrated Automated Thrombogram (CAT) assay was utilized to measure thrombin generation in plasma initiated by EVs-enriched pellets. The activity of AAV was evaluated with the Birmingham Vasculitis Activity Score (BVAS). Results: This study comprised 46 AAV patients, 23 in the active stage of the disease and 23 in remission, as well as 23 age- and sex matched population-based controls. Augmented levels of all investigated MPO+ EVs were found in active AAV patients in comparison to the subgroup of patients in remission and controls. Thrombin generation, measured by endogenous thrombin potential (ETP) and peak of thrombin formation, was higher in plasma when triggered by EVs-enriched pellet from AAV patients. ETP and peak were associated with the levels of MPO+TF+ and MPO+H3Cit+ EVs. Additionally, MPO+TF+ EVs correlated with the disease activity evaluated with BVAS. Conclusion: Augmented thrombin generation is found in AAV patients regardless of disease activity and is associated with higher exposure of TF and H3Cit on MPO+EVs. This may contribute to the increased risk of thrombosis seen in AAV patients.Peer reviewe

Determination of the overall haemostasis potential and fibrin gel permeability : Method development and application in research and in clinical materials

We have previously developed a laboratory method that may screen the Overall Haemostasis Potential (OHP) in plasma based on spectophotometric measurement of an area under the fibrin aggregation curve in citrated plasma samples to which tiny amounts of exogenous thrombin and tissue-plasminogen activator (t-PA) are added. When the fibrin aggregation curve is created, the fibrinogen originally present in plasma is gradually converted to fibrin by the generated thrombin. At the same time, plasminogen activation produces plasmin that in turn digests fibrin. Each absorbance value (Abs) represents the fibrin level at the corresponding time point, and the area under the curve, should reflect a balance between the generation and proteolysis of fibrin throughout the measurement period. To improve the assay sensitivity for the application in clinic or research work, various modifications were introduced. Thrombin in a decreased dose (0.04 IU/mL, compared to 0.2 IU/mL previously used) with or without t-PA was added to plasma. Areas under the two fibrin-aggregation curves i.e OHP and Overall Coagulation Potential (OCP) were thus created. A difference between the two parameters reflects the Overall Fibrinolysis Potential (OFP), calculated by (OCP-OHP)/OCP) x 100%. The modified method has shown its usefulness in detecting hypercoagulation in normal pregnancy, preeclampsia and coronary heart disease. Increased levels of OHP were also found in women with previous thromboembolim especially related to the presence of FV Leiden mutation. Moreover, assay of this parameter can screen immediate changes in the haemostatic system after the injection of low molecular mass heparin (dalteparin) and may be used for monitoring the anticoagulant effects. To ensure the sensitivity of the assay for determining different severity of hypocoagulation, further modifications were performed by introducing tissue factor and phospholipids to the reaction system. All the factors belonging to the two pathways of coagulation cascade, apart from FXII, affected the OHP outcome. This indicates that the modified assay system is similar to the haemostasis balance in circulating blood, and may thus become a laboratory too] to estimate bleeding tendency in haemophilic patients and distinguish prothrombotic cases among patients with FXII deficiency. OHP assay is thus a quantitative method to determine the fibrin level associated with combined potential of coagulation and fibrinolysis. However studies on fibrin gel porosity may give information about quality of the fibrin network which is important e.g. in atherosclerosis. We made modifications in a flow measurement previously established by B Blombäck et al and evaluated the resultant advantages. The essential equipment was simplified and the sample volume minimized which rendered the assay easier to apply in any clinical or research laboratory settings. By using different concentrations of thrombin with, or without phospholipids, it was possible to asses whether the fibrin gel porosity depends on both thrombin generation potential and fibrinogen clotting properties or only on the latter respectively. The modified flow measurement technique was used to determine the effects of acetylsalicylic acid (ASA, aspirin) effects on haemostasis. Fibrin gel porosity was more markedly increased during treatment with lower doses of ASA, compared to medium- / high-doses. These results are further confirmed by the findings in three-dimensional confocal microscopy where thicker fibrin fibers and larger network pores with irregular structure were observed during the low dose treatment. The greater increase in fibrin gel permeability and alterations in the structure of the fibrin network support the clinical findings of better prevention of arterial thrombosis such as in stroke and cardiovascular disease during treatment with low ASA doses such as 75mg daily, than with the higher doses

Medicolegal characteristics of domestic violence

Introduction/Objective. Domestic violence is a phenomenon as old as the history of human civilization, present in all cultures, epochs and social systems. Despite the fact that domestic violence represents a dangerous and unacceptable social phenomenon, as well as a significant medical problem, there are still no precise data on the prevalence of this phenomenon in our country. This study aims to determine the elementary forensic characteristics of domestic violence that would represented the basis for future medical research in this field. Methods. A total of 4,593 records of forensic autopsy (n = 3,120) and clinical forensic medical examinations (n = 1,473) were analyzed in the 1996–2005 period in order to determine the cases of domestic violence. Results. The analysis encompassed 300 cases (6.5%) of clinically examined (n = 211; 70.3%) and autopsied (n = 89; 29.7%) victims of domestic violence. A statistically significant increase in domestic violence cases (χ2 = 12.74; p = 0.00036) was determined in the observed period. The victims were mostly females (78%), with the mean age of 45.8 years (min = 0.3; max = 85; SD = 17.7), married (45%), with personal income (74.4%), and urban residence (66.3%). The majority of abusers were males (89.3%). Intimate partner violence was present in 58.3% of the cases. Physical abuse was the most common form of violence (97.7%), while sexual violence (2.3%) and child abuse (4.3%) were rarely recorded. Conclusion. The results of this research indicate that forensic medicine can be of great help in designing appropriate standards for conducting clinical medical examination, preventive programs, and strategies in fighting domestic violence

FAMILY VIOLENCE – MARRIAGE VIOLENCE WITH A FATAL OUTCOME

Family violence represents an especially dangerous social form of violence by means of which the rights of an individual – a member of a family – to live, to have psychic, physical and sexual integrity, freedom, security and human dignity, have been violated. The term marriage violence entails every form of physical, sexual, psychic and economic abuse of women by husbands or illegitimate partner. The family violence represents a widespread form of crime, and since it has become dramatic and dynamic during the recent years, the need for a direct forensic processing of the violence consequences within this specific and sensitive social group has arisen. In accordance with what has been outlined above, three cases of extreme, systematic and continuous marriage violence with fatal consequences of the abused women, whose bodies have been abducted at the Forensics Institute in Nis, have been presented in this paper

Diagnostic Accuracy in Acute Venous Thromboembolism: Comparing D-Dimer, Thrombin Generation, Overall Hemostatic Potential, and Fibrin Monomers

Introduction For acute venous thromboembolism (VTE), a biomarker with higher specificity than D-dimer would be of great clinical use. Thrombin generation and overall hemostatic potential (OHP) reflect the hemostatic balance by globally assessing multiple coagulation factors and inhibitors. These tests discriminate between healthy controls and patients with a prothrombotic tendency but have yet to be established as clinical biomarkers of VTE. Objective This study compares endogenous thrombin potential (ETP) and OHP to D-dimer and fibrin monomers (FM) in outpatients with suspected VTE. Methods A cross-sectional diagnostic study where 954 patients with suspected pulmonary embolism or deep venous thrombosis were recruited consecutively from the medical emergency department at Karolinska University Hospital. D-dimer, FM, OHP, and ETP were analyzed in a subpopulation of 60 patients with VTE and 98 matched controls without VTE. VTE was verified either by ultrasonography or computed tomography and clinical data were collected from medical records. Results Compared with healthy controls, both VTE and non-VTE patients displayed prothrombotic profiles in OHP and ETP. D-dimer, FM, ETP area under the curve (AUC), and ETP T lag were significantly different between patients with VTE and non-VTE. The largest receiver-operating characteristic AUCs for discrimination between VTE and non-VTE, were found in D-dimer with 0.94, FM 0.77, and ETP AUC 0.65. No useful cutoff could be identified for the ETP or the OHP assay. Conclusion Compared with D-dimer, neither ETP nor OHP were clinically viable biomarkers of acute venous thrombosis. The data indicated that a large portion of the emergency patients with suspected VTE were in a prothrombotic state.Funding agencies: Foundation for Coagulation Research at Karolinska Institutet, the ScandinavianResearch Foundation for Varicose Veins and other Venous Diseases, FoU Region Stockholm, the Swedish Society onThrombosis and Haemostasis with Leo Pharma.</p

Increased Expression of Extracellular Vesicles Is Associated With the Procoagulant State in Patients With Established Rheumatoid Arthritis

This study sought to identify different subpopulations of extracellular vesicles (EVs) in plasma from female patients with established rheumatoid arthritis (RA) in relation to the activation of coagulation and fibrin formation in these patients. Forty women were included in the study, 20 patients and 20 age-matched healthy controls. The mean disease duration in patients was 13.0 (5.0–25.0) years, with medium to high disease activity despite ongoing treatment with low-dose prednisolone and methotrexate. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. The concentration of phosphatidylserine-positive (PS+) EVs; platelet (CD42a+), leucocyte (CD45+), monocyte (CD14+), and endothelial (CD144+)-derived EVs; and EVs-expressing tissue factor (CD142+), P-selectin (CD62P+), and E-selectin (CD62E+) were determined by flow cytometry analysis. Overall hemostasis potential (OHP) was assessed to follow the hemostatic disturbances, including the parameters for overall coagulation potential (OCP) and overall fibrinolytic potential (OFP). Fibrin clot turbidity was measured together with clot lysis time, and scanning electron microscopy was performed. Increased concentrations of PS+, CD42a+, CD142+, CD45+, CD14+, and CD62P+ EVs were found in plasma from patients with RA compared to healthy controls, and the concentrations of PS+, CD42a+, CD14+, and CD62P+ EVs were positively correlated with the inflammatory parameters in RA patients. Positive correlations were also found between the levels of PS+ and CD42a+ EVs and OCP as well as between the levels of PS+, CD42a+, and CD62P+EVs and OHP. The levels of PS+, CD42a+, CD14+, CD62P+, and CD62E+ EVs were negatively correlated with OFP. Elevated levels of circulating EVs of different cell origins were found in patients with established RA, in relation to the inflammatory burden and coagulation activation in the disease

Pancreatic Polypeptide-Secreting Tumour of the Proximal Pancreas (PPoma)—Ultra Rare Pancreatic Tumour: Clinically Malign, Histologically Benign

Background and objectives: Here we report a rare case of a pancreatic polypeptide-secreting tumour (PPoma) discovered by accident during an autopsy. These PPomas occur in less than 2% of all pancreatic neoplasms and are almost exclusively silent, i.e., they are non-functional. Symptoms arising from PPoma are due to its compression of surrounding tissue. Materials and methods: The autopsy was performed on a 68-year-old male diagnosed with multiple endocrine neoplasm type 1 (MEN1) due to the patient&rsquo;s sudden death. Results: A solitary, densely fibrotic, pink-brown tumour, 18 mm in size tumorous mass, was localised in the head of the pancreas. Microscopically, the tumour had a glandular structure with a tubuloacinar arrangement of the cells. Immunohistochemically, we detected strong PP (pancreatic polypeptide) intracytoplasmic activity and negative glucagon activity. The PPoma was located in the head of the pancreas, likely resulting in the obstruction of the main pancreatic and common bile duct. Conclusions: To the best of our knowledge, this is the first report suggesting the association of PPomas with MEN1. Also, the PPoma could be the cause of acute hemorrhagic pancreatitis due to its location

Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models

Circulating microparticles (MPs) are procoagulant due to the surface containing phosphatidylserine (PS), which facilitates coagulation. We investigated if MPs improve hemostasis in HA plasma models. MPs isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (STED) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of MPs. MPs partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. In summary, our results demonstrate that PS positive MPs could improve hemostasis in HA plasma models