Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach.

BACKGROUND: The endothelial cell-dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation. METHODS: Endothelial colony-forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC (activated PC) formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of TM (thrombomodulin) expression, ECFCs were stimulated with IL-1ß (interleukin 1ß). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII). RESULTS: The median peak APC concentration was 1.12 nmol/L in experiments with IL-1ß stimulated ECFCs and 3.66 nmol/L without IL-1ß. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031, P=0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo. CONCLUSIONS: Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.

The impact of Bis-GMA free and Bis-GMA containing resin composite as posterior restoration on marginal integrity: a randomized controlled clinical trial.

BACKGROUND: There have been concerns surrounding the utilization of Bis-GMA, a type of bisphenol A (BPA) derivative, within the dental industry. The aim of this study was to compare the performance of bulk fill Bis-GMA-free resin composite class II restorations in respect of its marginal integrity in comparison to bulk fill Bis-GMA-containing resin composite class II restorations over a 12-month period in a parallel clinical trial utilizing a split-mouth, double-blind, randomized strategy. METHODS: 20 patients participated in this study. Each patient has received one pair of class II posterior restorations, Bis-GMA-free (Admira fusion x-tra), and Bis-GMA containing (x-tra fil) on each side of the mouth (split-mouth strategy), (n = 40). The restorations' marginal integrity was evaluated based on Ryge's criteria (modified USPHS) at baseline (after 1 week), as well as 1 month, 3 months, 6 months, 9 months, and after 12 months of follow-up by two calibrated examiners. The statistical analyses utilizing the Friedman and Wilcoxon tests, the significance level was adjusted to 0.05. RESULTS: Following the 12-month period, all patients attended the recall visits to evaluate the restorations. The Wilcoxon signed-rank and Friedman tests, revealed that both types of bulk fill had 100% of Alpha (A) scores at baseline and after 1 month with no significant statistical differences. After 3, 6, 9, and 12 months, both tested bulk fill restorations showed Bravo (B) score with Bis-GMA free 10% and 5% for Bis-GMA containing with no statistically significant difference (p ≤ 0.05) for clinical marginal integrity parameter in USPHS criteria. CONCLUSIONS: Bis-GMA-free resin composites demonstrated satisfactory, marginal integrity compared with Bis-GMA-containing resin composites within 12 months. TRIAL REGISTRATION: The protocol of the current study was registered at www. CLINICALTRIALS: gov , with the identification number NCT05480852 on 29/07/2022. All procedures involving human participants were performed in accordance with the ethical standards of the Research Ethics Committee of the Faculty of Dentistry, Minia University, Egypt, under the approval number 419 on 27/06/2020.

Increased Activated Protein C Response Rates Reduce the Thrombotic Risk of Factor V Leiden Carriers But Not of Prothrombin 20210G>A Carriers.

RATIONALE: Carriers of the most common prothrombotic mutations FVL (factor V Leiden) and FII (prothrombin) 20210G>A show a highly variable clinical phenotype. Using standardized in vivo coagulation activation followed by activity pattern analysis we have recently shown, that the FVL mutation accelerates thrombin and APC (activated protein C) formation in carriers without a history of venous thromboembolism (VTE). OBJECTIVE: The aim of this prospective cohort study was to investigate, if the FII 20210G>A mutation induces a similar reaction pattern, and if the response rates differ in FVL and FII 20210G>A mutation carriers with prior VTE (VTE+). METHODS AND RESULTS: We comparatively analyzed 30 FVL carriers, 28 FII 20210G>A carriers (thereof 13 VTE+ each) and 15 healthy controls. Changes in plasma levels of thrombin, prothrombin activation fragment 1+2 (F1+2), TAT (thrombin-antithrombin complex), APC, and D-dimer were monitored over 8 hours after infusion of recombinant factor VIIa (15 µg/kg). An increase of F1+2 and TAT levels was observed, that did neither differ between FVL and FII 20210G>A carriers nor between asymptomatic and VTE+ carriers of these mutations. Median plasma levels of APC increased more (P=0.008) in FVL carriers (from 1.39 to 7.79 pmol/L) than in FII 20210G>A carriers (from 1.03 to 5.79 pmol/L), and more in FII 20210G>A carriers (P=2×10-4) than in healthy controls (from 0.86 to 3.00 pmol/L). Most importantly, however, the APC response was greater (P=0.015) in asymptomatic (n=13) than in VTE+ (n=12) heterozygous FVL carriers, with an increase of APC levels from 1.44 to 8.11 pmol/L versus 1.27 to 5.62 pmol/L. CONCLUSIONS: These in vivo data demonstrate that the FII 20210G>A and FVL mutations share an intermediate phenotype that is characterized by increased thrombin formation after coagulation activation. Furthermore, our data support the conclusion that the APC activating capacity of FVL carriers modifies the thrombotic risk of this common prothrombotic mutation.

Functional Characterization of Antithrombin Mutations by Monitoring of Thrombin Inhibition Kinetics.

Inactivation of thrombin by the endogenous inhibitor antithrombin (AT) is a central mechanism in the regulation of hemostasis. This makes hereditary AT deficiency, which is caused by SERPINC1 gene mutations, a major thrombophilic risk factor. Aim of this study was to assess to what extent AT mutations impair thrombin inhibition kinetics. The study population included 36 thrombophilic patients with 19 different mutations and mean AT levels of 65% in a thrombin-based functional assay, and 26 healthy controls. To assess thrombin inhibition kinetics, thrombin (3.94 mU/mL final concentration) was added to citrated plasma. Subsequently, endogenous thrombin inhibition was stopped by addition of the reversible thrombin inhibitor argatroban and the amount of argatroban-complexed thrombin quantified using an oligonucleotide-based enzyme capture assay. The plasma half-life of human thrombin was significantly longer in patients with AT mutations than in the controls (119.9 versus 55.9 s). Moreover, it was disproportionately prolonged when compared with preparations of wild type AT in plasma, in whom a comparable thrombin half-life of 120.8 s was reached at a distinctly lower AT level of 20%. These findings may help to better understand the increased thrombotic risk of SERPINC1 mutations with near normal AT plasma levels in functional assays.

Prognostic and Therapeutic Implications of Immune Classification by CD8+ Tumor-Infiltrating Lymphocytes and PD-L1 Expression in Sinonasal Squamous Cell Carcinoma.

Sinonasal squamous cell carcinoma (SNSCC) is an aggressive tumor predominantly arising in the maxillary sinus and nasal cavities. Advances in imaging, surgical and radiotherapeutic techniques have reduced complications and morbidity; however, the prognosis generally remains poor, with an overall 5-year survival rate of 30-50%. As immunotherapy may be a new therapeutic option, we analyzed CD8+ tumor-infiltrating lymphocytes (TILs) and the tumor microenvironment immune type (TMIT, combining CD8+ TILs and PD-L1) in a series of 57 SNSCCs. Using immunohistochemistry, tissue samples of 57 SNSCCs were analyzed for expression of CD8 on TILs and of PD-L1 on tumor cells. The results were correlated to the clinical and survival data. In total, 88% (50/57) of the tumors had intratumoral CD8+ TILs; 19% (11/57)-CD8high (>10%); and 39/57 (68%)-CD8low (1-10%). PD-L1 positivity (>5%) was observed in 46% (26/57) of the SNSCCs and significantly co-occurred with CD8+ TILs (p = 0.000). Using univariate analysis, high intratumoral CD8+ TILs and TMIT I (CD8high/PD-L1pos) correlated with a worse survival rate. These results indicate that SNSCCs are immunogenic tumors, similar to head and neck squamous cell carcinomas. Nineteen percent of the cases were both CD8high and PD-L1pos and this subgroup may benefit from therapy with immune checkpoint inhibitors.

Through modulation of cardiac Ca2+ handling, UCP2 affects cardiac electrophysiology and influences the susceptibility for Ca2+ -mediated arrhythmias.

NEW FINDINGS: What is the central question of this study? Knockdown of UCP2 reduces mitochondrial Ca2+ uptake. This suggests that Ucp2 knockout mice need to have additional effects on cytosolic Ca2+ handling to prevent Ca2+ overload. However, the specific mechanisms and their impact on cardiac electrophysiology remain speculative. What is the main finding and its importance? In Ucp2 knockout mice, decreased mitochondrial Ca2+ uptake is compensated for by functional inhibition of L-type Ca2+ channels and resultant shortening of action potential duration. UCP2-dependent modulations have a major impact on cardiac electrophysiology, resulting in alterations of ECG characteristics and a higher susceptibility to Ca2+ -mediated ventricular arrhythmias. Uncoupling protein 2 (mitochondrial, proton carrier) (UCP2) belongs to a superfamily of mitochondrial ion transporters. Owing to its beneficial influence on production of reactive oxygen species, it is suggested to reduce cardiac ischaemia-reperfusion injury. Recent studies have uncovered its ability to regulate mitochondrial Ca2+ uptake and therefore to influence cardiac cytosolic Ca2+ handling, indicating compensatory pathways to avoid toxic Ca2+ overload in Ucp2 knockout (Ucp2-/- ) mice. However, the specific mechanisms and their impact on cardiac electrophysiology remain speculative. Molecular analyses, whole-cell patch clamp in cardiomyocytes and ECG studies were performed in Ucp2-/- and wild-type (WT) control mice. Furthermore, to explore the impact on cardiac arrhythmogenicity, ECG monitoring was performed in basal conditions and during Ca2+ -mediated stress using Bay K 8644. Although cardiac ryanodine receptor 2, NCX1, L-type Ca2+ channel (LTCC) and SERCA2a expression were not altered, Ucp2-/- mice revealed major variations in cardiac electrophysiology. The LTCC current and APD90 were decreased in Ucp2-/- mice, indicating compensatory mechanisms. Furthermore, in Ucp2-/- mice, an increased slope factor of action potential upstrokes and more hyperpolarized resting membrane potential were measured, suggesting variations in cardiac excitability. In agreement with alterations of cellular physiology in Ucp2-/- mice, reductions in PR and QRS as well as shortening of the QTc interval were noted in ECG recordings. Importantly, an increased incidence of cellular after-depolarizations and more pronounced susceptibility to Ca2+ -mediated arrhythmias were observed. Furthermore, although expression of UCP3 was not different, levels of PRMT1 were significantly higher in Ucp2-/- mice. Our observations indicate compensatory mechanisms by which Ucp2-/- mice prevent toxic cytosolic Ca2+ overload. UCP2-dependent modulations have a major impact on cardiac electrophysiology and influence susceptibility to Ca2+ -mediated ventricular arrhythmias.

UCP3 Regulates Single-Channel Activity of the Cardiac mCa1.

Mitochondrial Ca(2+) uptake (mCa(2+) uptake) is thought to be mediated by the mitochondrial Ca(2+) uniporter (MCU). UCP2 and UCP3 belong to a superfamily of mitochondrial ion transporters. Both proteins are expressed in the inner mitochondrial membrane of the heart. Recently, UCP2 was reported to modulate the function of the cardiac MCU related channel mCa1. However, the possible role of UCP3 in modulating cardiac mCa(2+) uptake via the MCU remains inconclusive. To understand the role of UCP3, we analyzed cardiac mCa1 single-channel activity in mitoplast-attached single-channel recordings from isolated murine cardiac mitoplasts, from adult wild-type controls (WT), and from UCP3 knockout mice (UCP3(-/-)). Single-channel registrations in UCP3(-/-) confirmed a murine voltage-gated Ca(2+) channel, i.e., mCa1, which was inhibited by Ru360. Compared to WT, mCa1 in UCP3(-/-) revealed similar single-channel characteristics. However, in UCP3(-/-) the channel exhibited decreased single-channel activity, which was insensitive to adenosine triphosphate (ATP) inhibition. Our results suggest that beyond UCP2, UCP3 also exhibits regulatory effects on cardiac mCa1/MCU function. Furthermore, we speculate that UCP3 might modulate previously described inhibitory effects of ATP on mCa1/MCU activity as well.

Hemostatic imbalance induced by tamoxifen in estrogen receptor-positive breast cancer patients: An observational study.

BACKGROUND: Estrogen receptor (ER)-positive (ER+) breast cancer accounts for approximately 75% of all breast cancers. Tamoxifen, a selective estrogen receptor modulator, is the standard adjuvant treatment. Although better tolerated than aromatase inhibitors, tamoxifen increases the risk of venous thromboembolism (VTE) 1.4-fold. AIM: To assess the hemostatic imbalance induced by tamoxifen in adjuvant treatment of ER+ breast cancer. METHOD: Twenty-five patients in remission from ER+ breast cancer under tamoxifen were included. One hundred and thirty one age- and BMI-matched healthy controls were included to establish reference ranges of thrombin generation assay (TGA) parameters. TGA was performed in the absence and presence of exogenous activated protein C (APC) to calculate the normalized APC sensitivity ratio (nAPCsr), a marker of APC resistance. RESULTS: All TG parameters except the endogenous thrombin potential (ETP) (-APC) were significantly impacted by tamoxifen (p < 0.001). In absence of APC, regardless of TGA parameters, at least 50% of results were outside the reference ranges except for ETP, which was above the upper reference limit in only two individuals. The most impacted parameter was the Peak Height with 52% (-APC) and 80% (+APC) of results above the upper reference range limit, respectively. The nAPCsr was significantly higher in tamoxifen users (mean ± standard deviation = 3.18 ± 0.91) compared to the control group (2.19 ± 0.92, p < 0.0001). CONCLUSION: This observational study showed that patients in remission from ER+ breast cancer taking tamoxifen had altered thrombin generation, as well as an acquired APC resistance. Moreover, this is the first study using the validated ETP-based APC resistance assay in tamoxifen-treated patients.

High Prevalence of F2 20210G > A in Splanchnic Vein Thrombosis and Cerebral Venous Sinus Thrombosis: A Retrospective Cohort Study of Patients with Thrombosis in Atypical Sites.

INTRODUCTION: Atypical sites for thrombosis include deep vein thrombosis (DVT) of the upper extremity (UE-DVT), splanchnic vein thrombosis (SVT), and cerebral venous sinus thrombosis (CVST). In addition to specific pathogenic factors, their underlying mechanisms share similarities with typical venous thromboembolism (VTE), namely, DVT of the lower extremity and/or pulmonary embolism, but are less understood. METHODS: Records of unselected patients with a history of typical VTE (n = 2,011), UE-DVT (n = 117), SVT (n = 83), and CVST (n = 82), who were referred to the Institute in Bonn for ambulatory thrombophilia testing, were retrospectively analyzed. Acquired and hereditary thrombosis risk factors were comparatively assessed. RESULTS: UE-DVT was characterized by a high rate (50.4%) of site-specific acquired risk factors. Compared with typical VTE, SVT was more frequently associated with systemic inflammation, infection, or malignancy (2.2 vs. 12.0%, p = 3·10-8) and the JAK2 V617F mutation was present in 16.9%. In CVST compared with typical VTE, demographics and higher rates of oral contraception (43.2 vs. 57.6%, p = 0.011) and pregnancy (4.2 vs. 10.9%, p = 0.012) suggest a significant hormonal influence on etiology. While the prevalence of inhibitor deficiencies and factor V Leiden mutation did not differ between cohorts, the prevalence of F2 20210G > A was higher in SVT (15.7%, p = 0.003) and CVST (15.9%, p = 0.003) than in typical VTE (7.0%). CONCLUSION: The cohorts with thrombosis in atypical sites showed distinctive patterns of acquired risk factors. Further studies are warranted to provide additional mechanistic insight into the role of hormonal influence in CVST and the contribution of F2 20210G > A to the development of SVT and CVST.

Fibrinolysis biomarker, thrombin, and activated protein C level alterations after coagulation activation depend on type of thrombophilia and clinical phenotype.

Background: Recently, we have shown alterations in the anticoagulant response to recombinant activated factor VII (rFVIIa)-induced coagulation activation in patients with thrombophilia. Objectives: This study aimed to extend this in vivo model to fibrinolysis biomarkers. Methods: This interventional in vivo study included 56 patients with thrombophilia and previous venous thromboembolism (VTE+), 38 without VTE (VTE-), and 35 healthy controls. Plasma levels of D-dimer, plasmin-α2-antiplasmin (PAP) complex, and plasminogen activator inhibitor-1 (PAI-1) were monitored for over 8 hours after rFVIIa infusion (15 µg/kg) along with thrombin markers and activated protein C (APC). Results: Throughout cohorts, median PAP increased by 40% to 52% (P < 3.9 × 10-10) and PAI-1 decreased by 59% to 79% (P < 3.5 × 10-8). In contrast to thrombin-antithrombin (TAT) complex, which also increased temporarily (44% to 115%, P < 3.6 × 10-6), changes in PAP and PAI-1 did not reverse during the observation period. The area under the measurement-time curves (AUCs) of PAP and TAT, which are measures of plasmin and thrombin formation, respectively, were each greater in the VTE+ cohort than in healthy controls (median PAP-AUC = 0.48 vs 0.27 ng·h/L [P = .003], TAT-AUC = 0.12 vs 0.03 nmol·h/L [P = 2.5 × 10-4]) and were correlated with one another (r = 0.554). As evidenced by the respective AUCs, asymptomatic factor (F)V Leiden carriers showed less PAP formation (0.22 vs 0.41 ng·h/L, P = 9 × 10-4), more pronounced PAI-1 decline (0.10 vs 0.18 ng·h/L, P = .01), and increased APC formation (28.7 vs 15.4 pmol·h/L, P = .02) than those within the VTE+ group (n = 19 each). Conclusion: rFVIIa-induced thrombin formation is associated with fibrinolysis parameter changes outlasting the concomitant anticoagulant response. Both correlate with thrombosis history in FV Leiden and might help explain its variable clinical expressivity.

[Systemic hantavirus-infection in a comatose HIV patient]. / Systemische Hantavirus-Infektion bei einem komatösen HIV Patienten.

SYMPTOMS: A 40 year old, disoriented, HIV- and Hepatitis B positive male patient was admitted with 40.3 °C. Clinically he presented a sinustachycardia (160/min) and hypotension (70/60 mmHg). INVESTIGATIONS/DIAGNOSIS: Laboratory analyses showed elevated infection parameters, azotemia, proteinuria and thrombopenia. CD(4+)T-helper cells: 320/µl (32 %), HIV RNA: <40 copies/ml, Hepatitis B DNA: 20800 copies/ml. Hantavirus serology (immunofluorescence antibody assay): 1:2048; serotype Puumala. TREATMENT/COURSE: An early-goal-directed therapy and antibiotic treatment with Piperacillin and Tazobactam was initiated. The patient developed a bipulmonal infiltrate and an acute respiratory distress syndrome (ARDS ) requiring tracheal intubation, as well as a triad of fever, renal failure and profound hemorrhagic symptoms. This led to the diagnosis of the Puumala infection. Due to the parallel HIV- and Hepatits B infection an antiretroviral therapy was initiated. CONCLUSION: In summary the Puumala infection bears the potential for a severe multi-organ failure, which is not typical for this usually benign infection.

Juvenile Recurrent Parotitis: Video-Documented Sialendoscopy.

Juvenile recurrent parotitis (JRP) is characterised by recurrent episodes of painful parotid swelling in children. JRP is the second most common cause of parotitis in childhood, behind only paramyxovirus. The prevention of recurrent attacks represents the most dramatic and serious aspect of this pathology. Since 2004, different authors have evaluated sialendoscopy for the diagnostic and therapeutic management of JRP. In this paper, we share our clinical experience of the use of sialendoscopy for the treatment of JRP. We document with video sialendoscopy the glandular pathology in four children with a mean age of 11.5 years, who had suffered from 3-6 episodes/year of inflammation prior to treatment. The use of sialendoscopy in our patients was effective in preventing recurrences. For the first time, the videosialendoscopy of a series of children diagnosed with JRP is documented in the literature.

Inactive Matrix Gla Protein in Relation to Renal and Cardiac Functions and Cardiac Valvular Calcification Among Type 2 Diabetes Patients.

Background: Matrix Gla protein (MGP) is a robust innate suppressor of the detrimental process of vascular calcification in the human body. Objectives: The interrelationship between circulating MGP levels and renal and cardiac dysfunction, besides echocardiographic calcification score (ECS) was investigated in a sample of type 2 diabetes (T2D) patients. Methods: The study included 130 subjects. They were 95 patients with T2D and 35 age- and sex-matched healthy controls. Patients were further subdivided into 52 T2D patients without DKD (eGFR ⩾ 60 ml/minute/1.73 m²) and 43 T2D persons with DKD (eGFR > 60 ml/minute/1.73 m²). Serum MGP levels, determined by ELISA, renal function tests, lipid profile, and echocardiography were studied in all participants. Results: Significantly elevated circulating inactive MGP level was noted in individuals having T2D compared to controls. It correlated negatively with eGFR and left ventricular (LV) diastolic and systolic functions and positively with indices of LV hypertrophy. ECS was significantly increased in both T2D groups compared to controls and in DKD group compared to the diabetic group without DKD. A significant positive correlation was observed between inactive MGP and ECS. Conclusion: Serum inactive MGP may contribute to the development of DKD and to the associated process of cardiac valvular calcification. It may be a beneficial diagnostic marker for early prediction of cardiac calcification and preclinical LV systolic and diastolic dysfunction in T2D patients, especially in those complicated with DKD.

[Transcatheter aortic valve replacement: when should it be used in aortic stenosis?]. / Kathetergestützter Aortenklappenersatz: eine neue therapeutische Option der Aortenklappenstenose?

30% of patients with significant aortic stenosis are not considered for operative aortic valve replacement because of the high perioperative risk. An alternative catheter based option for these patients is the transcatheter aortic valve replacement (TAVI). In general, there are two approaches for TAVI: transfemoral and transapical. Transfemoral aortic valve replacement is performed by transcatheter replacement of an aortic valve via the femoral arteries. Transapical valve replacement is achieved by transcatheter implantation via the fifth intercostal space. The most common complications are vessel injuries, bleeding complications, new onset of AV-block, development of paravalvular insufficiency, acute kidney injury, stroke and TIA. The first long-term observations suggest positive results. First clinical trials in a high-risk population show a promising outcome. Therefore TAVI offers a reasonable therapy option for patients with high perioperative risk. Further long-term clinical trials are still pending.

Variation in Plasma Levels of Apixaban and Rivaroxaban in Clinical Routine Treatment of Venous Thromboembolism.

Direct oral anticoagulants (DOACs) apixaban and rivaroxaban are broadly used in the management of venous thromboembolism (VTE). Although not routinely required, measurement of their plasma concentration is advised for an increasing number of indications. Due to the lack of therapeutic ranges, current guidelines recommend reporting DOAC plasma levels together with expected levels from previous pivotal studies. The aim of this study was to assess DOAC level variation in a large VTE patient population. Drug concentrations determined by measurement of the anti-Xa-activity using drug-specific calibrators in citrated plasma samples from patients on rivaroxaban (n = 1471) or apixaban (n = 725) were analyzed. Observed 5th-95th percentile ranges of apixaban peak/trough levels (63-299/13-114 ng/mL for 5 mg, 37-161/7-68 ng/mL for 2.5 mg twice daily) were similar to previously reported mass-spectrometry-based reference data, and 10th-90th percentile ranges of rivaroxaban peak/trough levels (98-367/8-55 ng/mL for 20 mg, 51-211/5-27 ng/mL for 10 mg once daily) were even narrower. Age and drug levels correlated weakly (r ≤ 0.330). Drug levels measured repeatedly in subgroups of patients showed a strong correlation (r ≥ 0.773). In conclusion, anti-Xa-activity-based measurement of apixaban and rivaroxaban yields reliable results. However, the paucity of levels off-range underlines the need for evidence-based thresholds to better assist clinical decision making.

Increased Prevalence of Elevated D-Dimer Levels in Patients on Direct Oral Anticoagulants: Results of a Large Retrospective Study.

Elevated D-dimer levels during anticoagulant therapy with vitamin K antagonists (VKA) are associated with an increased risk of thrombosis. It has been hypothesized that elevated D-dimer levels in patients receiving direct oral anticoagulants (DOACs) also indicate an increased risk of thrombosis recurrence, but data on the distribution of D-dimer levels in patients with VTE on DOACs are sparse. In the present study we retrospectively analyzed D-dimer levels in patients taking DOACs after first or recurrent venous thrombosis (n = 1,716, 1,126 thereof rivaroxaban, 481 apixaban, 62 edoxaban, and 47 dabigatran). Patients on VKA (n = 402) served as control group. Thrombotic events in the study population were categorized into distal deep venous thrombosis (DVT, n = 552 patients), distal DVT with pulmonary embolism (PE, n = 166), proximal DVT (n = 685), proximal DVT with PE (n = 462), PE without DVT (n = 522), DVT of the upper extremity (n = 78), cerebral venous sinus thrombosis (CVST, n = 48), and other venous thrombosis (n = 74). In VKA users a median D-dimer level of 0.20 mg/l was observed. In patients on DOACs D-dimer levels were significantly higher, with 0.26 mg/l for rivaroxaban, 0.31 mg/l for apixaban (P < 10-16 each), 0.24 mg/l for edoxaban (P = 2 × 10-5), and 0.25 mg/l for dabigatran (P = 4 × 10-4). These differences in comparison to patients on VKA treatment could not be explained by the patients' age, sex, body mass index, and type of thrombosis as these characteristics did not differ significantly between cohorts. Moreover, the prevalence of D-dimer levels above age-adjusted cut-offs [≥0.50 mg/l in ≤50-year-old patients, ≥(age × 0.01) mg/l in >50-year-old patients] was higher in patients on rivaroxaban (13.9%, RR 1.74, 95% CI 1.21-2.50), apixaban (17.0%, RR 2.14, 95% CI 1.45-3.15) and dabigatran (23.4%, RR 2.94, 95% CI 1.59-5.44) than in patients on VKA (8.0%). In patients on edoxaban D-dimer levels above the reference range were observed in 14.5%, but no statistical significance was reached in comparison to the VKA cohort. In conclusion, the obtained data suggest, that the type of oral anticoagulant should be considered in the clinical assessment of D-dimer levels in thrombosis patients. Further studies are warranted to evaluate a potential association between elevated D-dimer levels and thrombosis risk in patients on DOACs.

Activated Factor XI is Increased in Plasma in Response to Surgical Trauma but not to Recombinant Activated FVII-Induced Thrombin Formation.

AIM: Feedback activation of factor XI (FXI) by thrombin is believed to play a critical role in the amplification phase of thrombin generation and to contribute to thrombosis development and hemostasis. However, the activation of FXI by thrombin has been shown in vitro to require a cofactor. In this study, the role of thrombin in activated FXI (FXIa) formation in vivo is investigated. METHODS: The study population comprised probands in whom coagulation activation was triggered by low-dose (15 µg/kg) recombinant activated factor VII (rFVIIa, n=89), of whom 34 with (VTE+) and 45 without a history of venous thromboembolism (VTE-), and patients undergoing major orthopedic surgeries (n=45). FXIa was quantified via an enzyme capture assay using a monoclonal FXI-specific antibody. Thrombin formation was monitored using an oligonucleotide-based enzyme capture assay and the thrombin activation markers prothrombin fragment 1＋2 (F1＋2) and thrombin-antithrombin complex (TAT). RESULTS: In the rFVIIa cohort, FXIa and thrombin remained below their lower limit of quantification of 3.48 and 1.06 pmol/L, respectively. By contrast, during the surgeries, median FXIa levels increased from 3.69 pmol/L pre-operatively to 9.41 pmol/L mid-operatively (P=4·10-4) and remained significantly elevated 24 h thereafter, with 9.38 pmol/L (P=0.001). Peak levels of F1+2 were comparable in the VTE+, VTE-, and surgery cohort (235, 268, and 253 pmol/L), whereas peak TAT levels were higher in the surgery cohort (53.1, 33.9, and 147.6 pmol/L). CONCLUSIONS: Under in vivo conditions, the activation of FXI requires specific local features that are present at the wounded site including potential cofactors of thrombin.

PC Deficiency Testing: Thrombin-Thrombomodulin as PC Activator and Aptamer-Based Enzyme Capturing Increase Diagnostic Accuracy.

Protein C (PC) activity tests are routinely performed in a thrombophilia workup to screen for PC deficiency. Currently used tests combine conversion of PC to activated PC (APC) by the snake venom Protac with subsequent APC detection through hydrolysis of a chromogenic peptide substrate or prolongation of a clotting time. In this prospective cohort study, we analyzed how different modes of PC activation and subsequent APC determination influence the diagnostic accuracy of PC activity testing in a cohort of 31 patients with genetically confirmed PC deficiency. In addition to chromogenic and clot-based measurement, an oligonucleotide-based enzyme capture assay utilizing a basic exosite-targeting aptamer was used for APC detection. To study the influence of the PC activation step on diagnostic sensitivity, PC activation through Protac and through the thrombin-thrombomodulin (TM) complex were compared. Twenty-six (84%) and 24 (77%) PC deficient patients were identified as true-positive using the chromogenic and the clot-based PC activity assay, respectively. True-positive results increased to 27 (87%) when the basic exosite-targeting aptamer approach was used for APC measurement. Additional replacement of the PC activator Protac by thrombin-TM gave true-positive results in all patients. These data indicate that the mode of PC activation is crucial in determining the accuracy of PC activity testing and that diagnostic sensitivity can be significantly improved by replacing the PC activator Protac with thrombin-TM. APC detection using a basic exosite-targeting aptamer achieves high sensitivity toward mutations outside the active center while being less subject to interfering factors than clot-based PC activity assays.

Role of acquired von Willebrand syndrome in the development of bleeding complications in patients treated with Impella RP devices.

Axial flow pumps are standard treatment in cases of cardiogenic shock and high-risk interventions in cardiology and cardiac surgery, although the optimal anticoagulation strategy remains unclear. We evaluated whether laboratory findings could predict bleeding complications and acquired von Willebrand syndrome (avWS) among patients who were treated using axial flow pumps. We retrospectively evaluated 60 consecutive patients who received Impella devices (Impella RP: n = 20, Impella CP/5.0: n = 40; Abiomed Inc., Danvers, USA) between January 2019 and December 2020. Thirty-two patients (53.3%) experienced major or fatal bleeding complications (Bleeding Academic Research Consortium score of > 3) despite intravenous heparin being used to maintain normal activated partial thromboplastin times (40-50 s). Extensive testing was performed for 28 patients with bleeding complications (87.5%). Relative to patients with left ventricular support, patients with right ventricular support were less likely to develop avWS (87.5% vs. 58.8%, p = 0.035). Bleeding was significantly associated with avWS (odds ratio [OR]: 20.8, 95% confidence interval [CI]: 3.3-128.5; p = 0.001) and treatment duration (OR: 1.3, 95% CI 1.09-1.55; p = 0.003). Patients with avWS had longer Impella treatment than patients without avWS (2 days [1-4.7 days] vs. 7.3 days [3.2-13.0 days]). Bleeding complications during Impella support were associated with avWS in our cohort, while aPTT monitoring was not sufficient to prevent bleeding complications. A more targeted anticoagulation monitoring might be needed for patients who receive Impella devices.