Abstract Hemophilia is a rare inherited bleeding disorder caused by a deficiency of factor VIII or IX, leading to insufficient thrombin generation and impaired clot formation. While advances in replacement and nonfactor therapies have improved patient outcomes, challenges such as inhibitor development, joint disease, and breakthrough bleeding persist. Extracellular vesicles (EVs) are small membrane-bound particles released from all kinds of cells and have emerged as key mediators of coagulation, vascular integrity, and immune responses. In hemophilia, EVs provide procoagulant surfaces, carry tissue factor and phosphatidylserine, and may stabilize clot structure, partially compensating for deficient coagulation. Clinical studies further suggest that EV levels and composition are altered by factor replacement or bypassing agents, indicating their role as biomarkers of treatment response. Beyond their diagnostic potential, engineered EVs are being explored as innovative therapeutic tools capable of delivering clotting factors and modulating joint inflammation. This review outlines current knowledge on circulating EVs in hemophilia, emphasizing their mechanistic roles, clinical evidence, and therapeutic potential.

PURPOSE OF REVIEW:SARS-CoV-2 disease (COVID-19) is increasingly recognized as a thromboinflammatory vascular disorder characterized by dysregulated complement activation, endothelial injury, and sustained hypercoagulability. This review examines emerging evidence that extracellular vesicles act as key intermediaries linking complement activation to coagulation in acute and postacute COVID-19 infection. RECENT FINDINGS:Recent studies demonstrate that extracellular vesicles released from platelets, endothelial cells, and neutrophils are markedly increased in COVID-19 and exhibit a combined procoagulant and complement-active phenotype. Sub-lytic complement attack, particularly membrane attack complex (MAC) deposition, triggers phosphatidylserine exposure and extracellular vesicle shedding, generating vesicles that support thrombin generation and propagate complement activity in the circulation. Extracellular vesicle-associated complement components, including C1q, C3 fragments, MASP2, and preassembled MACs, promote tissue factor decryption, platelet activation, and assembly of the prothrombinase complex, establishing a self-amplifying thromboinflammatory loop. Proteomic profiling further reveals compartment-specific extracellular vesicle signatures, with systemic extracellular vesicles enriched in complement and coagulation pathways. Importantly, complement-bearing and tissue factor-bearing extracellular vesicles persist beyond acute infection and are increasingly implicated in postacute sequelae of COVID-19. SUMMARY:Extracellular vesicles serve as mobile platforms integrating complement activation with coagulation, providing a mechanistic framework for acute and chronic immunothrombosis in COVID-19. Targeting extracellular vesicle-mediated complement-coagulation crosstalk may offer novel diagnostic and therapeutic opportunities.

OBJECTIVE:Uncontrolled hemorrhage with hemostatic derangement still represents a major preventable cause of death following severe traumatic injury. The purpose of this experimental study was to further investigate the overall hemostasis potential and clot formation after severe traumatic hemorrhage. METHODS:Male New Zealand white rabbits ( n  = 12) were subjected to traumatic injury with 40% hemorrhage of their estimated blood volume. The rate of thrombin generation was measured by calibrated automated thrombogram assay, overall hemostatic potential (OHP) by fibrin-aggregation on spectrophotometer, and microvesicles using flow cytometry. The clot structure was analyzed using scanning electron microscopy (SEM). RESULTS:After traumatic hemorrhage, standard laboratory coagulation parameters showed decreased platelet count and fibrinogen, and increased INR and lactate. The OHP and overall coagulation potential (OCP) declined by 59% ( P  < 0.001) and 51% ( P  < 0,001), respectively. Overall fibrinolytic potential increased from 82% to 86% ( P  = 0.008). Platelet derived CD9 positive microvesicles decreased by 84% ( P  = 0.002). Endogenous thrombin potential decreased from 483 RFU/min to 383 RFU/min ( P  = 0.005). SEM analysis of the fibrin clot structure showed a more porous clot with a decrease in fiber diameter from 72 μm to 56 μm ( P  = 0.002). CONCLUSIONS:This experimental study provides important aspects on coagulopathy and clot structure after traumatic hemorrhage including reduced thrombin generation and hyperfibrinolysis resulting in a more porous fibrin clot with lower density and thinner fibers. The methods used may be applied in the clinical research setting to gain further insights about hemostasis and clot formation for targeted therapy after traumatic hemorrhage.

Background The complement system is crucial in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) pathogenesis. Extracellular vesicles (EVs) serve as carriers of bioactive substances, influencing the immune system. We aimed to investigate myeloperoxidase-positive EVs (MPO(+)EVs) exposing complement components (C3a, C4d), terminal complement complex (TCC) and complement factor B (CFB) in relation to disease activity and kidney involvement. Methods Plasma MPO(+)EVs carrying complement products, C3a, C4d, TCC or CFB, were analysed by flow cytometry. Clinical data, including kidney biopsy findings, were retrieved. Disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS). Results Eighty-one patients with AAV with granulomatosis with polyangiitis (n=59) or microscopic polyangiitis (n=22) were included. Active disease was noted in 73 (90.1%) patients and 50 (68.5%) had kidney involvement. Patients with kidney involvement had higher concentrations of MPO+, MPO(+)C3a(+), MPO(+)C4d(+) and MPO(+)TCC(+)EVs compared to patients without, and concentrations correlated positively with BVAS (p<0.05), respectively. Levels of EVs showed a negative correlation with estimated glomerular filtration rate, and a positive correlation with the proportions of necrosis and crescents in kidney biopsies. ROC curve analysis indicated that MPO(+)EVs could distinguish patients with kidney involvement from non-renal AAV. A binary multivariable logistic regression confirmed an independent association between levels of EV subsets and kidney involvement. Conclusions Elevated levels of MPO(+)EVs exposing complement components in patients with kidney involvement indicate activation of the classical or lectin and common complement pathways in renal AAV. Furthermore, the association between levels of MPO(+)EVs with the presence of crescents and necrotic lesions in kidney tissue supports a role in renal inflammatory activity.

Key PointsThrombin injures podocytes through its cognate receptors , and thrombin generation increases during proteinuric glomerular disease.in vitroIn this study, plasma prothrombin levels modulated podocyte health and function in a rat model of glomerular disease.in vivoThrombin antagonism may simultaneously prevent thrombosis and CKD progression due to glomerular disease.BackgroundCKD is a leading cause of death; its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes loss of coagulation zymogens, cofactors, and inhibitors resulting in a hypercoagulable state characterized by enhanced thrombin generation. Both CKD and proteinuria significantly increase the risk of thromboembolic disease. Meanwhile, anticoagulant medications (which antagonize thrombin and thereby prevent thromboembolism) have been shown to reduce proteinuria in rats, and thrombin has been shown to injure cultured human and rat podocytes. We thus aimed to directly determine the influence of circulating prothrombin, the zymogen precursor of thrombin, on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria.MethodsGlomerular proteinuria was induced with puromycin aminonucleoside in rats. Prothrombin was either knocked down using an antisense oligonucleotide-targeting prothrombin mRNA or elevated by serial intravenous prothrombin protein infusions, previously established methods to model hypoprothrombinemia and hyperprothrombinemia, respectively. After 10 days, plasma prothrombin levels were determined and kidneys were examined for (pro)thrombin colocalization to podocytes, histology, and electron microscopy. Podocytopathy, podocytopenia, proteinuria, and plasma albumin were measured.ResultsAntisense oligonucleotide-mediated prothrombin knockdown significantly reduced prothrombin colocalization to podocytes, tubular injury, podocyte foot process effacement, podocytopathy, and proteinuria, along with improved plasma albumin in the puromycin aminonucleoside glomerular disease model. By contrast, elevated prothrombin levels significantly increased podocytopathy and proteinuria. Podocytopenia was significantly improved in hypoprothrombinemic versus hyperprothrombinemic rats.ConclusionsThrombin generation is enhanced by glomerular proteinuria, and thrombin injures conditionally immortalized podocytes in vitro. In this study, prothrombin knockdown ameliorated in vivo podocyte injury and improved podocyte function in the rat puromycin aminonucleoside-induced glomerular disease model, whereas hyperprothrombinemia exacerbated podocyte injury and diminished podocyte function.

Von Willebrand disease (vWD) is a common bleeding disorder with different subtypes. Laboratory diagnosis is challenging, involving several expensive and complex assays. The von Willebrand factor (vWF) collagen binding assay (VWF:CB) has been described to improve the diagnosis of vWD, but there is a lack of consensus and its implementation into guidelines and diagnostic algorithms is incomplete. A cohort of 88 patients with inherited vWD and 10 patients investigated for vWD due to a bleeding phenotype were recruited and underwent analysis of vWF multimers, vWF antigen (VWF:Ag) and VWF:CB. The total bleeding score (BS) was calculated by using the bleeding assessment tool recommended by the International Society on Thrombosis and Haemostasis. An optimal VWF:CB/VWF:Ag ratio cutoff for differentiating between patients with all multimer sizes and those lacking high molecular weight multimers (HMWM) was established, and the findings were validated in a separate retrospective clinical data cohort. The association between VWF:CB/VWF:Ag ratio and BS was also assessed. VWF:CB/VWF:Ag ratio was a very good discriminator of HMWM presence, yielding a sensitivity of 1.0 and a specificity of 0.79 at the optimal cutoff in the validation dataset. VWF:CB/VWF:Ag ratio was a significant predictor of BS (R2 = 0.39). VWF:CB/VWF:Ag ratio is a significant predictor of BS and multimer analysis can be safely omitted in patients with vWD and a VWF:CB/VWF:Ag ratio above 0.6, confirming the VWF:CB assay as an important contributor to vWD diagnosis.

For patients with hemophilia A and high-titer inhibitors treated with bypassing agents there are no reliable methods to assess treatment effect. We investigated the utility of global hemostatic methods in assessing treatment with bypassing agents (rFVIIa or activated prothrombin complex [aPCC]). All patients with hemophilia A and inhibitors followed at the Coagulation Unit or the Pediatric Coagulation Unit at Karolinska University Hospital aged 6 years and above were eligible for this noninterventional study. Baseline plasma samples were spiked with bypassing agents in increasing concentrations (aPCC 50 U/kg, 100 U/kg, 150 U/kg, and rFVIIa 90 μg/kg and 270 μg/kg) in vitro. For patients treated with factor concentrates or bypassing agents follow-up samples were collected (in vivo tests). The samples were analyzed using overall hemostatic potential (OHP), and calibrated automated thrombogram, Calibrated Automated Thrombogram (CAT). Nine patients with hemophilia A with inhibitors were included. Spiking with rFVIIa normalized the coagulation potential in 6/8 samples, in 3 only with high dose. Only one sample did not improve adequately after spiking with aPCC. The improvement in hemostasis was reliably shown by both CAT and OHP. The baseline potential was, however, more often measurable by OHP compared to CAT. Factor concentrate had been administered to 5 patients normalizing the hemostatic potential in vivo in 2 (without spiking). The hemostatic improvement induced by spiking with rFVIIa or aPCC is shown by OHP and CAT, but the results have to be evaluated in larger cohorts.

ABSTRACT Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.

INTRODUCTION:Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis.METHODS:Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers.RESULTS:No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness.CONCLUSIONS:Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.

Background Extracellular vesicles (EVs) are membrane-coated vesicles derived from virtually any cell type under different stimuli. Interstitial lung disease (ILD) is the leading cause of death in SSc. So far, only one study demonstrated elevated levels of EVs in patients with SSc-ILD (1), however the association between EVs and progressive ILD (pILD) was not previously investigated. Objectives To evaluate the concentration of different subpopulations of EVs in plasma from SSc patients in relation to the occurrence of pILD associated to SSc. Methods This was a prospective cohort study, including 59 SSc patients without any comorbidity (31 (52.5%) with ILD and 28 (47.5%) without).The median disease duration in ILD group was 3 years and 6 years in those without. ILD was defined either as X-ray positive findings or ground-glass opacification on HRCT. EVs were analyzed with flow cytometry after staining of platelet-poor plasma with fluorescent cell-specific monoclonal antibodies. The concentration of the following phosphatidylserine-positive EVs was analyzed at baseline: endothelial EVs (EEVs; CD144+), platelet EVs (PEVs;CD42b+), leucocyte EVs (LEVs; CD45+), EVs expressing ICAM1 (CD54+), TF (CD142+) and HMGB1. The serum concentration of ICAM1,VEGF,and IL6 were measured by ELISA. Lung functional tests were done every 6-12 months over a 3-years period of follow-up (FU). pILD was defined by the decline of forced vital capacity (FVC) from baseline visit of ≥10%, or an FVC decline of 5-9% along with a DLCO decline of >15% [2]. Results An inverse relation was noticed in SSc patients between EVs and FVC (EEVs r=-0.3, p<0.05; LEVs r=-0.4, p<0.01; EVsICAM1r=-0.4, p<0.01; EVsTF r=-0.4, p<0.01; EVS HMGB1r=-0.3, p<0.05). Significantly increased levels of VEGF, ICAM1, IL6, PEVs, and EVs expressing ICAM1, TF or HMGB1 were found in SSc ILD cohort compared to those without (p<0.05, respectively). There was a positive association between all explored EVs and VEGF in ILD group (p<0.05). Multivariable (Ml) logistic regression (R)‚ adjusted for VEGF raveled that PEVs(OR 1.004, 95%CI 1.001-1.01) and EVs ICAM1 (OR 1.3, 95%CI 1.1-1.5) were independently associated with ILD.Over a FU period, 12/31ILD patients developed pILD, of which 58% had early diffuse subtype with < 1-year disease duration and were anti Scl 70 positive. Only 4/12 pILD patients have ever been treated with imunossupresive therapy. pILD occurred within 1st FU year in 50% of patients. All analyzed EVs (Figure 1), IL6 and VEGF, were found elevated in pILD (p<0.05, respectively). Moreover, the levels of PEVs, EVs expressing ICAM1 (AUC 0.9, p<0.001, respectively),TF, HMGB1, EEVs and LEVs (AUC 0.7, p<0.05, respectively) showed good validity in identifying pILD. There was only a positive correlation between VEGF and both EVs ICAM1 (r=0.8, p<0.01) and EVs HMGB1 (r=0.7, p<0.01) within pILD group. Applying Ml Cox R controlling for VEGF, EVs ICAM1 were confirmed independently significantly associated with pILD (OR 1.1, 95% CI 1.02-1.1). Conclusion Our results provide evidence that different circulating EV subpopulations might play a role not only in SSc ILD pathogenesis but also in the genesis of pILD. EVs expressing ICAM1 could be a novel predictive biomarker of SSc-related ILD progression, identifying patients who would potentially benefit from early aggressive treatment. References [1]D Leleu et al. Elevated Circulatory Levels of Microparticles Are Associated to Lung Fibrosis and Vasculopathy During Systemic Sclerosis. Front Immunol. 2020 Oct 23;11:532177.[2]O Distler et al. Predictors of progression in systemic sclerosis patients with interstitial lung disease.Eur Respir J. 2020 May; 55(5): 1902026. Acknowledgements: NIL. Disclosure of Interests None Declared.Figure 1The levels of circulating (A) EEVs, (B) PEVs, (C) LEVs, (D) EVs ICAM1, (E) EVs TF,(F) EVs HMGB1 in patients without ILD, progressive ILD (pILD) and non-progressive ILD (non-pILD) over follow-up. *p < 0.05; **p < 0.01

Objective.To assess thrombin generation, fibrin formation, and structure together with the fibrinolytic status in patients with systemic sclerosis (SSc) in relation to the occurrence of digital ulcers (DUs) during the course of disease.Methods.We studied variables of endothelial dysfunction, thrombin generation, overall hemostatic potential, and fibrin clot turbidity in plasma from 58 patients with SSc (39 with DU history and 19 DU-naïve) and 46 matched healthy controls (HCs). Fibrin structure was visualized using scanning electron microscopy (SEM). Finally, 39 patients with a history of DUs were followed for 1.5 years and the predictive value of all investigated markers for new DU onset was explored.Results.Significantly enhanced endogenous thrombin potential (ETP) and prolonged clot lysis time (CLT) were found in patients with DUs compared to HCs. CLT was prolonged in patients with DUs compared to those without, showing good validity in identifying DUs with an area under the curve of 0.7 (95% CI 0.6–0.8). The levels of ETP and intercellular adhesion molecule 1 were independently associated with CLT. Over the follow-up period, 20 patients developed new DUs. CLT was prolonged (P < 0.001) in patients with new DU episodes, especially those with recurrent DUs. Regression analysis showed that the Raynaud phenomenon visual analog scale and CLT were predictors of new DUs (OR 1.1, 95% CI 1.0–1.1 and OR 1.2, 95% CI 1.1–1.3, respectively). SEM confirmed denser fibrin clots in patients with new DUs.Conclusion.Our results suggest that impaired fibrinolysis might have an emerging role in underlying digital vasculopathy and its progression in SSc.

Introduction. Ischemic stroke (IS) is a heterogeneous dis-order caused by several genetic and environmental risk factors. It was suggested that coagulation disorders cause 1-4% of cases with IS, especially in patients with early onset of IS. Case report. We describe a case of a young adult male who developed an unprovoked IS. Biochemical, immunological, and thrombophilia screening, as well as DNA sequencing, were performed in order to reveal molecular pathology underlying the stroke of the patient. Thrombophilia testing showed that patient was a homozygous carrier for PAI-1 4G/5G and MTHFR C677T mutations. Additional genetic analysis revealed the presence of the recently reported F2 c.1824C>T gene variant, located in the last exon of the pro-thrombin gene and has previously been shown to cause hy-perprothrombinemia, hypofibrinolysis, and altered fibrin clot phenotype. Conclusion. Our results suggest that the newly reported F2 c.1824C>T gene variant might have a synergistic effect with PAI 4G/4G and MTHFR 677TT genotype in the formation of altered fibrin clot phenotype characterized by thin, densely packed fibrin fibers, which makes clot less susceptible to fibrinolysis and greatly in-creases the risk for early ischemic stroke onset.

Background Extracellular vesicles (EVs) are membrane-coated vesicles most often generated from the vasculature and circulating blood cells upon cell activation and during the early phase of apoptosis (1). Objectives To evaluate the concentration of different subpopulations of EVs in plasma from patients with SSc in relation to the disease duration and to the markers of endothelial injury. Methods Our study included 59 SSc patients (36 limited (lSSc) and 23 diffuse cutaneous subset (dSSc)) and 46 healthy age and gender matched controls subjects. Disease duration less than 3 years in patients with dSSc and less than 5 years in those with lSSc was considered as early disease stage. Clinical evaluation of patients was performed. EVs were analysed with flow cytometry after staining platelet poor plasma with fluorescent cell-specific monoclonal antibodies. The concentration of following phosphatidylserine-positive EVs (PS+ EVs) were analyzed: total EVs, endothelial EVs (EEVs; CD144+), platelet EVs (PEVs; CD42b+), monocytes EVs (LEVs; CD14+), EVs expressing ICAM1 (CD54+), VCAM1 (CD106+) and P selectin (CD62p+). Serum levels of ICAM1, VCAM1 and P selectin were determined with ELISA. Results Median disease duration of our cohort was 4 (0-29) years (early lSSc [20/36]: 2.5 (0-4.5) years; early dSSc [11/23]: 10 (1-30) months). All types of investigated EVs were significantly elevated in SSc patients compared to controls (p<0.05). Patients with early disease stage had significantly increased levels of all PS+EVs compared to HC (p<0.05). Moreover, the levels of EVs expressing ICAM1 and VCAM1 showed good validity in identifying patients with early disease stage (AUC 0.7, p<0.01). PEVs were increased in early dSSc compared to early lSSc, but the difference did not reach statistical significance (p=0.07). There was a correlation between serum levels and the levels of EVs expressing specific adhesion molecules (ICAM1: r=0.7, p<0.01; VCAM1: r=0.7, p<0.01; P selectin: r=-0.7, p<0.01), only within the group with early dSSc subtype of the disease. Further correlations were detected between ICAM1+EVs with either mRSS (r=0.07, p< 0.01) or EUSTAR activity index (r=0.07, p= 0.02) among patients with early dSSc. Conclusion Increased levels of circulating EVs of different cell origin were present in patients with early SSc. EVs expressing either ICAM1 or VCAM1 could be novel biomarkers of early disease. EVs expressing ICAM1 showed association with severity of skin involvement and disease activity in patients with early dSSc giving insight into their role in the pathogenesis of SSc. References [1]J Colic, M Matucci-Cerinic, S Guiducci and N Damjanov. Review: Microparticles in systemic sclerosis, targets or tools to control fibrosis: This is the question!. Journal of Scleroderma and Related Disorders. 2019; 5(34):239719831985735 Disclosure of Interests None declared

Normal pregnancy associated with complex changes of hemostasis, leading to hypercoagulability states. The presence of acquired or genetic prothrombotic risk factors might affect the proper maternal-fetal circulation and result in pregnancy loss. Hence, the screening for the novel prothrombotic variants associated with pregnancy loss would be beneficial. Our aim was to investigate the potential association of recently reported c.*64_*66del variant in prothrombin gene with the etiology of pregnancy loss. Study included 105 women with pregnancy loss and 155 controls. Analyses in patients? plasma samples, as well as in vitro analyses on transfected Cos-7 cell line were performed in order to investigate the mechanism by which this variant could perturb the coagulation and lead to pregnancy loss. Analyses in patients' DNA and plasma samples involved: DNA sequencing and PCR-RFLP assay for detection of FII c.*64_*66del variant, routine thrombophilia screening, thrombin generation assay and Western blot analysis of prothrombin plasma level. In vitro analyses included transient transfections of Cos-7 cell line with wild-type and c.*64_*66del mutated constructs of pCIneo?SV40 expression vector. Real-Time PCR and Western blot analysis were used to determine the effect of FII c.*64_*66del variant on mRNA and protein level in constructs. Three women in patients group (2.9%) were detected as heterozygous carriers of FII c.*64_*66del, while none was found among controls. The carriers routine thrombophilia parameters were in reference range and similar prothrombin plasma level in FII c.*64_*66del carriers and non-carriers were detected. The endogenous thrombin potential was slightly increased in FII c.*64_*66del carriers compared to control plasma, but this difference was not statistically significant. Results of in vitro analyses showed significantly decreased prothrombin mRNA and protein level for c.*64_*66del variant compared to wild-type. Results of our pilot study have shown a trend of higher prevalence of FII c.*64_*66del variant in women with pregnancy loss. However, further studies are needed to completely elucidate whether FII c.*64_*66del variant affects prothrombin expression during pregnancy and to account its potential role in etiology of pregnancy loss.

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.

Background: Digital ulcers (DU) are a common visible manifestation of vasculopathy in systemic sclerosis (SSc), which could be recurrent and associated with disability and mortality (1). Although vasculopathy is connected with impaired coagulation/fibrinolysis system and aberrant expression of adhesion molecules, there are few data about their role in developing recurrent DU. Objectives: To evaluate the possible role of Fibrin generation/Fibrinolysis parameters and adhesion molecules in the prediction of new ischaemic DU oncet during a 1-year follow-up and their impact on the time to new DU onset (TD). Methods: From 58 consecutive patients with SSc who fulfilled the 2013 ACR/EULAR SSc criteria and have never been treated with endothelin receptor antagonist, phosphodiesterase 5 inhibitors or prostanoids, a total of 38 patients with ever had DU, either active at inclusion or in past, were enrolled in a prospective cohort study. Each patient was given a “DU diary”. Demografic, clinical and serologycal data were recorded. The serum concentration of ICAM1 and E selectin were measured by ELISA. Haemostatic potential parameters: overall haemostasis (OHP), overall coagulation (OCP) and overall fibrinolysis (OFP) potential were assessed. Maximum absorbance (Cmax), reflects the fibrin clot density and clot lysis time (Lys50t0, time from initiation of clot formation to the time at which a 50% fall in absorbance from Cmax in the lysis assay), reflects fibrinolytic susceptibility, were calculated (2). Fibrin structure was visualised using scanning electron microscopy (SEM). Results: Over the follow-up period,18 patients (45.5%) developed new DU with the average TD of 7.4±2.9 months. There was no differance in ASA and CCB treatment among two groups (p>0.05). The OFP value was significantly decreased (p<0.01), Lys50t0 prolonged (p<0.05), while OHP was increased (p<0.05) in patients experienced new DU. Lys50t0 showed good validity in identifying patients with new DU oncet (AUC 0.683 95% CI 0.5 - 0.9). By multivariate analysis including clinical data in model the Lys50t0 (HR 1.2, 95% CI 1.1-1.3, p=0.018) and active DU at enrollment (HR 9.6, 95% CI 1.4-66.8, p=0.022) were identified as independent risk factors for the occurrence of new DU. TD was inversly correlated with ICAM1(p<0.001), E selectin (p<0.05), Cmax (p <0.05) and Lys50t0 (p<0.001). Model explaining 81.6% of the TD variability included Lys50t0 (β=- 0.55,p=0.003), E selectin (β=- 0.44,p=0.014) and fibrinogen (β=- 0.49,p=0.017). SEM revealed denser fibrin clots with thinner fibres in group experienced new DU compared to clots formed in plasma of patient without DU. Conclusion: Our results provide evidence that impaired fibrinolysis has critical role in the progression of microvascular disease, identifing clot lysis time as a strong predictor in the oncet of new digital ulcers in Systemic sclerosis. The higher level of E selectin and the longer lysis time are, the shorter is time until the onset of new digital ulcer. These results could be used in selection of patients at high risk of developing recurrent digital ulcer and therefore allow earlier therapeutic intervention. References: [1]Allanore Y, Distler O, Matucci-Cerinic M, Denton CP. Review: Defining a Unified Vascular Phenotype in Systemic Sclerosis. Arthritis Rheumatol. 2018 Feb;70(2):162-170 [2]Carter AM, et al. Heritability of clot formation, morphology, and lysis: the EuroCLOT study. Arterioscler Thromb Vasc Biol. 2007 Dec;27(12):2783-9. Figure 1. SEM images of fibrin network in 1 representative sample from a SSc patient with (A) and 1from patient without (B) new DU oncet Disclosure of Interests: None declared

Background:Vasculopathy in Systemic sclerosis (SSc) is connected with the activation of coagulation. However, the fibrinolytic activity still remains unclear since the most preliminary evidences are discordant (1, 2).Objectives:To assess the haemostatic function, fibrin clot density and clot lysis time in SSc patients and healthy controls (HC) to determine their relation to disease findings.Methods:Patients who fulfilled the 2013 ACR/EULAR SSc criteria and have never been treated with endothelin receptor antagonist, phosphodiesterase 5 inhibitors or prostanoids were eligibile. Our study included 58 SSc patients [36 limited (lcSSc) and 22 diffuse cutaneous SSc (dcSSc)] and 46 sex/age-matched HC. Clinical evaluation of patients was performed, including high-resolution CT (HRCT), pulmonary function tests and the revised EUSTAR activity index. The interstitial lung disease (ILD) group (n = 15) was defined as moderate or severe changes on HRCT, with a forced vital capacity (FVC) < 85% predicted, without evidence of significant pulmonary arterial hypertension. The serum concentration of ICAM1 and von Willebrand factor antigen (VWF) were measured by ELISA. Haemostatic potential parameters; including overall haemostasis (OHP), overall coagulation (OCP) and overall fibrinolysis (OFP) potential, were assessed and endogenous thrombin potential (ETP) was determined. Maximum absorbance (Cmax), reflects the fibrin clot density and clot lysis time (Lys50t0), reflects fibrinolytic susceptibility, were calculated from OHP and OCP curves (3). Fibrin structure was visualised using scanning electron microscopy (SEM).Results:The OFP value was significantly decreased, Lys50t0 prolonged (p<0.05), while OHP and ETP were increased (p<0.05) in patients. In dSSc group ETP, OHP, Cmax and Lys50t0 were higher compared to HC (p<0.05). In SSc group, a positive association was found between coagulation parameters (OCP, OHP, Cmax) and the erythrocyte sedimentation rate (ESR), fibrinogen and ICAM1 (respectively p<0.05). Lys50t0 was positively correlated with ICAM1, ESR and VWF (respectively p<0.001, p<0.05, p<0.05). An inverse correlation was found between Cmax and both the diffusing capacity of the lungs for carbon monoxide (r=-0.408, p<0.01) and FVC (r=-0.318, p<0.01). Increased Cmax was found in ILD respect to HC (p<0.01). Denser plasma clot was associated with active disease (p<0.01). Longer Lys50t0 was observed in pitting scars group (p<0.01). Prolonged Lys50t0 was independently predicted by ICAM1 (OR 1.12, 95% CI 1.03–1.2, p<0.01).Conclusion:Our results provide evidences of denser plasma fibrin clot among patients with lung involvement and impaired fibrinolysis, selectively presented among SSc patients with piting scars. Thus, these patients might be at risk for thrombotic complications. Raised ICAM-1 levels could reflect impaired fibrinolysis, giving insight the important role of this molecule in endothelial homeostasis.References:[1]Cerinic MM, et al. Blood coagulation, fibrinolysis, and markers of endothelial dysfunction in systemic sclerosis. Semin Arthritis Rheum. 2003;32:285–95[2]Lippi G, et al. Plasma D-dimer concentration in patients with systemic sclerosis.Thromb J. 2006;4:2.[3]Carter AM, et al. Heritability of clot formation, morphology, and lysis: the EuroCLOT study. Arterioscler Thromb Vasc Biol. 2007 Dec;27(12):2783-9Figure 1.SEM images of fibrin network in 1 representative sample from a SSc (A) and 1from HC sample (B).Disclosure of Interests:Jelena Colic: None declared, Aleksandra Antovic: None declared, Iva Pruner: None declared, Jelena Vojinovic Consultant of: Roche, Abbvie, Pfizer, MSD, Speakers bureau: Roche, Abbvie, Pfizer, MSD, Mirjana Sefik Bukilica: None declared, Nemanja Damjanov Grant/research support from: from AbbVie, Pfizer, and Roche, Consultant of: AbbVie, Gedeon Richter, Merck, Novartis, Pfizer, and Roche, Speakers bureau: AbbVie, Gedeon Richter, Merck, Novartis, Pfizer, and Roche

Development of inhibitors to factor VIII (FVIII) occurs in approximately 30% of severe hemophilia A (HA) patients. These patients are treated with bypassing agents (activated prothrombin complex concentrate [aPCC] and recombinant activated FVII-rFVIIa). Recently, a bispecific FIX/FIXa- and FX/FXa-directed antibody (emicizumab) has been approved for the treatment of HA patients with inhibitors. However, the data from clinical studies imply that coadministration of emicizumab and bypassing agents, especially aPCC, could have a thrombotic effect. This study was aimed to address the question of potential hypercoagulability of emicizumab and bypassing agents' coadministration, we have investigated fibrin clot formation and structure in the in vitro model of severe HA after adding sequence-identical analogue (SIA) of emicizumab and bypassing agents. Combined overall hemostasis potential (OHP) and fibrin clot turbidity assay was performed in FVIII-deficient plasma after addition of different concentrations of SIA, rFVIIa, and aPCC. Pooled normal plasma was used as control. The fibrin clots were analyzed by scanning electron microscopy (SEM). OHP and turbidity parameters improved with the addition of aPCC, while therapeutic concentrations of rFVIIa did not show substantial improvement. SIA alone and in combination with rFVIIa or low aPCC concentration improved OHP and turbidity parameters and stabilized fibrin network, while in combination with higher concentrations of aPCC expressed hypercoagulable pattern and generated denser clots. Our in vitro model suggests that combination of SIA and aPCC could potentially be prothrombotic, due to hypercoagulable changes in fibrin clot turbidity and morphology. Additionally, combination of SIA and rFVIIa leads to the formation of stable clots similar to normal fibrin clots.

BACKGROUND Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. METHODS To determine the frequency of the FII c.1824C>T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. RESULTS Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. CONCLUSION Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.