Comprehensive laboratory assessment of lonoctocog alfa versus octocog alfa in severe haemophilia A.

INTRODUCTION: Lonoctocog alfa is a single-chain factor VIII (FVIII) molecule with high binding affinity to von-Willebrand-factor. While it is well known that its plasma activity is underestimated by one-stage clotting assays (OSCA), there is a lack of knowledge on the post-infusion performance of lonoctocog alfa in global coagulation assays or its potential impact on the haemostatic balance in vivo. AIM: To characterize lonoctocog alfa versus octocog alfa in pre- and post-infusion samples obtained from patients undergoing repeated investigation of incremental recovery (IR). METHODS: Eighteen patients with severe haemophilia A (lonoctocog alfa: 10, octocog alfa: 8) were included. A panel of factor-specific and global coagulation assays was applied, comprising a FVIII OSCA, two FVIII chromogenic substrate assays (CSA), rotational thrombelastography and thrombin generation (TG). Potential activation of coagulation was assessed by measuring plasma thrombin markers and levels of activated protein C. RESULTS: Comparable IRs were found for lonoctocog alfa and octocog alfa (2.36 [IU/dL]/[IU/kg] vs. 2.55 [IU/dL]/[IU/kg], respectively). Lonoctocog alfa activities were found to be underestimated by the FVIII OSCA while also the two FVIII CSAs showed statistically significant assay discrepancies on lonoctocog alfa. Effects of both FVIII products on rotational thrombelastography were less distinct than those on TG parameters. No elevated pre- or significantly shifting post-infusion plasma levels of coagulation biomarkers were detected. CONCLUSION: Lonoctocog alfa and octocog alfa showed comparable recovery and safety in vivo as well as similar impacts on TG in vitro. Observed assay discrepancies on lonoctocog alfa demonstrated variability of results also between different FVIII CSAs.

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.

Genetic Landscape of Factor VII Deficiency: Insights from a Comprehensive Analysis of Pathogenic Variants and Their Impact on Coagulation Activity.

Congenital factor VII (FVII) deficiency is a rare genetic bleeding disorder characterized by deficient or reduced activity of coagulation FVII. It is caused by genetic variants in the F7 gene. We aimed to evaluate the rate of detection of pathogenic variants in the F7 gene in a large group of patients with FVII deficiency and investigate the correlations between the F7 genotype and FVII activity (FVII:C). Moreover, the influence of the common genetic variant rs6046: c.1238G>A; p.(Arg413Gln), designated as the M2 allele, on FVII:C was investigated. Genetic analysis of the F7 gene was performed on 704 index patients (IPs) using either direct Sanger- or next-generation sequencing. Genetic variants were detected in 390 IPs, yielding a variant detection rate (VDR) of 55%. Notably, the VDR exhibited a linear decline with increasing FVII:C levels. We identified 124 genetic variants, of which 48 were not previously reported. Overall, the frequency of the M2 allele was considerably higher in patients with mild deficiency (FVII:C > 20 IU/dl). Furthermore, IPs lacking an identified pathogenic variant exhibited a significantly higher prevalence of the M2 allele (69%) compared to IPs with a disease-causing variant (47%). These results strongly support the association of the M2 allele with decreased FVII:C levels. This study shows the utility of FVII:C as a predictive marker for identifying pathogenic variants in patients with FVII deficiency. The M2 allele contributes to the reduction of FVII:C levels, particularly in cases of mild deficiency.

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.

Variable resistance to freezing and thawing of CD34-positive stem cells and lymphocyte subpopulations in leukapheresis products.

BACKGROUND AIMS: Leukapheresis products for hematopoietic stem cell transplantation can be cryopreserved for various indications. Although it is known that CD34(+) cells tolerate cryopreservation well, a significant loss of CD3(+) cells has been observed, which has been ascribed to several factors, including transport, storage conditions and granulocyte-colony stimulating factor (G-CSF) administration. METHODS: To assess the tolerance of CD34(+) cells and lymphocyte subpopulations for cryopreservation and thawing, the post-thaw recoveries of CD34(+) cells, CD3(+)CD4(+) cells, CD3(+)CD8(+) cells, CD19(+) cells and CD16(+)CD56(+) cells were determined in 90 cryopreserved apheresis products, among which 65 were from G-CSF-mobilized donors, and 34 from unrelated donors that underwent transport before cryopreservation at our center. A controlled rate freezer and 5% dimethyl sulfoxide were used for cryopreservation. RESULTS: We could detect statistically significant differences for CD34(+) cell recovery (93.0 ± 20.7%) when compared to CD3(+)CD4(+) cell (83.1 ± 15.4%, P = 0.014), and CD3(+)CD8(+) cell recovery (83.3 ± 13.9%, P = 0.001). Similarly, CD19(+) cell recovery (98.6 ± 15.1%) was higher than CD3(+)CD4(+) cell (P = 2.5 × 10(-7)) and CD3(+)CD8(+) cell recovery (P = 1.2 × 10(-8)). Post-thaw recovery rates of all cell populations were not impaired in G-CSF-mobilized products compared with non-mobilized products nor in unrelated compared with related donor products. DISCUSSION: Our data suggest a lower tolerance of CD3(+) cells for cryopreservation and demonstrate that freezing-thawing resistance thawing is cell-specific and independent from other factors that affect post-thaw recovery of cryopreserved cells. Thus, a clinical consequence may be the monitoring of post-thaw CD3(+) cell doses of cryopreserved products, such as donor lymphocyte infusions.

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.

Decreased Protein C Pathway Activity in COVID-19 Compared to Non-COVID Sepsis: An Observational and Comparative Cohort Study.

Sepsis-associated coagulopathy increases risk of mortality. Impairment of the anticoagulant protein C (PC) pathway may contribute to the thrombotic phenotype in coronavirus disease 2019 (COVID-19) sepsis. This study assessed the functionality of this pathway in COVID-19 and non-COVID sepsis by measuring its key enzymes, thrombin and activated PC (APC). The study population included 30 patients with COVID-19, 47 patients with non-COVID sepsis, and 40 healthy controls. In healthy controls, coagulation activation and subsequent APC formation was induced by 15 µg/kg recombinant activated factor VII one hour before blood sampling. APC and thrombin in plasma were measured using oligonucleotide-based enzyme capture assays. The indirect thrombin markers prothrombin-fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) were also measured. Compared with stimulated healthy controls, median thrombin, F1+2, and TAT levels were higher in patients with COVID-19 (up to 6-fold, p < 2 × 10-6) and non-COVID sepsis (up to 4.7-fold, p < 0.010). APC levels were 2.4-fold higher in patients with COVID-19 (7.44 pmol/L, p = 0.011) and 3.4-fold higher in non-COVID sepsis patients (10.45 pmol/L, p = 2 × 10-4) than in controls (3.08 pmol/L). Thrombin markers and APC showed correlation in both COVID-19 (r = 0.364-0.661) and non-COVID sepsis patients (r = 0.535-0.711). After adjustment for PC levels, median APC/thrombin, APC/F1+2, and APC/TAT ratios were 2-fold (p = 0.036), 6-fold (p = 3 × 10-7) and 3-fold (p = 8 × 10-4) lower in the COVID-19 group than in the non-COVID sepsis group, and the latter two were also lower in the COVID-19 group than in stimulated healthy controls. In conclusion, it was found that a comparatively lower anticoagulant APC response in COVID-19 patients as compared to non-COVID sepsis patients, potentially linked to endothelial dysfunction, contributes to the prothrombotic phenotype of COVID-19 sepsis.

Animal and Cellular Models in Thrombosis and Hemostasis.

STANDARDIZED IN VITRO AND IN VIVO MODEL SYSTEMS TO SIMPLIFY COMPLEXITY-THAT'S HOW WE LEARN: The discovery of new target molecules and translational progress in the development and refinement of antithrombotic therapies as well as the improved treatment of bleeding disorders strongly relies on standardized ex vivo and in vivo models that closely resemble the respective human pathologies. The standardization of these models requires sound training in specialized hemostasis and thrombosis research laboratories as well as a consistent daily routine. In this theme issue of Hämostaseologie-Progress in Haemostasis, four review articles cover key models that have proven instrumental to gain mechanistic insights on thrombogenesis and hemostatic processes. In recent decades, these models have moved our field forward and enabled translation across scales, from cell-based research to isolated flow chamber systems, to mouse thrombosis models reflecting the pathologic situations as observed in patients, to large animal models.

Functional determination of emicizumab in presence of factor VIII activity.

BACKGROUND: Accurate measurement of emicizumab in the presence of factor (F) VIII is required in patients with severe hemophilia A treated with emicizumab, as well as additional need for FVIII substitution or emicizumab prophylaxis in patients with acquired or moderate to mild hemophilia A. However, the presence of FVIII potentially biases the results. OBJECTIVES: To assess the impact of plasma FVIII activity on determined emicizumab levels and evaluate different strategies for correction for or preanalytical inhibition of FVIII. METHODS: Evaluated strategies comprised of the following: (1) calculation of actual emicizumab plasma levels based on measured FVIII activities and FVIII-affected emicizumab values, (2) preanalytical heat treatment (56 °C for 40 minutes), and (3) neutralization of FVIII activity using FVIII inhibitors. Emicizumab levels and FVIII activities were measured using a modified FVIII one-stage clotting assay and a chromogenic FVIII assay based on bovine factors, respectively. RESULTS: Spiking experiments revealed a consistent linear association between FVIII activities and determined (FVIII-affected) emicizumab results at different emicizumab input levels (∼0.12 µg/mL per IU/dL of FVIII). This principally allowed for mathematical correction of measured emicizumab levels in the presence of FVIII. While a 40% to 50% activity loss of intrinsic plasma emicizumab through heat treatment was observed in patient samples, emicizumab spiked into FVIII-deficient plasma was not or only marginally affected. Application of inhibitor-based FVIII neutralization led to good agreement of results when compared with direct quantification of emicizumab by liquid chromatography-tandem mass spectrometry. CONCLUSION: Inhibitor-based FVIII neutralization appears to be a feasible strategy for accurate measurement of plasma emicizumab levels in the presence of FVIII activity.

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.

Assay for ADAMTS-13 Activity with Flow Cytometric Readout.

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) is a metalloprotease that regulates the size of circulating von Willebrand factor (vWF) multimers. Severe lack of ADAMTS-13 activity [<10% of normal (0.1 IU/mL)] leads to thrombotic thrombocytopenic purpura (TTP), a specific type of thrombotic microangiopathy (TMA). Timely determination of plasma ADAMTS-13 activity is essential to discriminate TTP from other types of TMA with respect to adequate treatment. Identification of the minimal substrate motif for ADAMTS-13 within the A2 domain of vWF (vWF73) as well as the generation of monoclonal antibodies (mAbs) that specifically recognize the ADAMTS-13 cleavage site enabled the development of a variety of methods for determination of plasma ADAMTS-13 activity. In order to further extend the range of analytical platforms applicable for quantitative determination of plasma ADAMTS-13 activity, a specific, vWF/mAb-based assay with flow cytometric readout was developed and validated. Basic assay characteristics include a total assay time of 80 to 90 min, a near linear dynamic range from 0.005 (lower limit of quantification) to 0.2 IU/mL, and intra- and interassay coefficients of variation below 5 and 30% at input plasma ADAMTS-13 activities of 0.015 and ≤0.050 IU/mL, respectively. When compared to the results obtained with a commercially available quantitative ADAMTS-13 activity ELISA, analysis of 18 plasma samples obtained from patients with suspected TTP revealed full agreement of results with respect to the clinical 0.1 IU/mL TTP threshold. Based on these data, it is assumed that the described assay principle can be successfully transferred to virtually all laboratories that have a flow cytometer available.

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.

Aptamer loaded superparamagnetic beads for selective capturing and gentle release of activated protein C.

Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic applications. In this work, we present one-step activation of APC from a commercial source of protein C (PC, Ceprotin) followed by rapid and efficient purification using an APC-specific aptamer, HS02-52G, loaded on MyOne superparamagnetic beads. Due to the Ca2+-dependent binding of APC to HS02-52G, an efficient capturing of APC was applied in the presence of Ca2+ ions, while a gentle release of captured APC was achieved in the elution buffer containing low EDTA concentration (5 mM). The captured and eluted APC showed more than 95% purity according to SDS-PAGE gel analysis and an enzyme-linked fluorescent assay (VIDAS Protein C). The purification yield of 45% was calculated when 4.2 µg APC was used, however this yield reduced to 21% if the starting amount of APC increased to 28.5 µg. Altogether, this method is recommended for rapid and efficient PC activation and APC purification. The purified APC can be used directly for downstream processes where high concentration of pure and active APC is needed.

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.