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Background: Von Willebrand disease (VWD), the most prevalent hereditary bleeding disorder, results from deficiency of von Willebrand factor (VWF)., Objectives: This large cohort study aims to offer a comprehensive exploration of mutation spectra and laboratory features in quantitative VWF deficiencies, shedding light on genetic underpinnings and genotype-phenotype associations., Methods: Our cohort consisted of 221 Caucasian index patients with quantitative VWD, along with 47 individuals whose plasma VWF levels fell within the lower normal boundaries (50-70 IU/dL). We conducted comprehensive VWF assays and genetic analyses, encompassing VWF gene sequencing, copy number variation investigations, and bioinformatic assessments., Results: Following International Society on Thrombosis and Haemostasis-Scientific and Standardization Committee VWF guidelines, 77 index patients were characterized as having type 1 VWD (VWF antigen [VWF:Ag] < 30 IU/dL), 111 as having type 1 VWD (VWF:Ag, 30-50 IU/dL), and 33 as having type 3 VWD. Mutation detection rates were 88%, 65%, and 92%, respectively. Notably, blood group O overrepresentation was evident in type 1 with VWF:Ag of 30 to 50 IU/dL, particularly among mutation-negative patients, suggesting a potential causal role of blood group O. A total of 223 VWF variants, comprising 147 distinct variations, were identified in quantitative VWD patients, of which 57 were novel variants (39%). Additionally, approximately 70% of individuals with VWF levels within the lower normal boundaries (50-70 IU/dL) displayed VWF variants., Conclusion: Our data advance our understanding of the molecular mechanisms underlying quantitative VWD, offering valuable insights for future research and clinical management. Distinct mutation patterns were observed among subgroups, particularly the contrast between type 1 VWD (VWF:Ag < 30 IU/dL) and type 1 VWD (VWF:Ag, 30-50 IU/dL), an area with limited prior investigation., Competing Interests: Declaration of competing interests The authors declare that they have no conflicts of interest and do not have anything to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024 The Author(s).)

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., Competing Interests: Declaration of competing interests J.M. has received honoraria from Octapharma and Siemens Healthineers. J.O. has received research funding from Bayer, Biotest, CSL Behring, Octapharma, Pfizer, Swedish Orphan Biovitrum, and Takeda; consultancy, speakers bureau, honoraria, scientific advisory board, and travel expenses from Bayer, Biogen Idec, BioMarin, Biotest, Chugai Pharmaceutical Co, Ltd, CSL Behring, Freeline, Grifols, LFB, Novo Nordisk, Octapharma, Pfizer, F. Hoffmann-La Roche Ltd, Sanofi, Spark Therapeutics, Swedish Orphan Biovitrum, and Takeda. N.S.H., A.D., M.v.L., S.G., H.R., C.K., T.A., M.A., and B.P. report no conflicts of interest., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Thrombocytopenia after transcatheter aortic valve implantation (TAVI) has been evaluated in several studies, but the pathomechanisms behind this phenomenon are not well described. Here, we assess the influence of TAVI on hemostasis-related biomarkers and their potential role in post-TAVI thrombocytopenia., Methods and Results: We assessed perioperative platelet counts in 307 patients undergoing transfemoral TAVI. Additionally, hemostasis-related biomarkers including thrombin-anti-thrombin complex (TAT), prothrombin activation fragment 1+2 (F1+2), plasmin-α₂-antiplasmin complex (PAP), and d-dimer were assessed in a subcohort of 35 patients. The course of these parameters was correlated with platelet counts and compared in patients with and without a relevant thrombocytopenia, i.e., a decline in platelet count of ≥50% compared to baseline. Levels of TAT (8.6±8.7μg/l), PAP (992±597μg/l), and d-dimer (1.43±1.66mg/l) were elevated above normal range before TAVI. TAT, F1+2, and d-dimer significantly increased after TAVI, and d-dimer as well as PAP remained elevated until day 7, indicating TAVI-induced increased thrombin formation associated with secondary fibrinolysis. Post-TAVI thrombocytopenia occurred in 17.9% of patients and was associated with a significantly higher incidence of post-TAVI complications, e.g. acute kidney injury and vascular complications. No impact of activated coagulation on thrombocytopenia was observed., Conclusions: TAVI induces an increase in hemostasis-related biomarkers, which persists until 1week after the procedure. The frequent finding of post-TAVI thrombocytopenia appears not to be linked to increased thrombin formation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Human-activated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory and cytoprotective functions. This feature renders APC to be a promising vascular-inflammatory biomarker., Objective: The aim of the present study was the development and validation of a technique that allows the measurement of APC plasma levels under practical laboratory conditions., Methods/patients: Based on the APC-binding ssDNA aptamer HS02-52G we developed an oligonucleotide-based enzyme capture assay (OECA) that quantifies aptamer-captured APC through hydrolysis rates of a fluorogenic peptide substrate. After optimization of pre-analytical conditions, plasma APC levels were measured in healthy individuals and patients undergoing hip replacement surgery., Results and Conclusion: A combination of APC-OECA with an aprotinin-based quenching strategy allowed APC analysis with a limit of detection as low as 0.022 ± 0.005 ng mL(-1) (0.39 ± 0.10 pmol L(-1)) and a limit of quantification of 0.116 ± 0.055 ng mL(-1) (2.06 ± 0.98 pmol L(-1)). While APC plasma levels in healthy individuals fell below the quantifiable range of the APC-OECA platform, levels substantially increased in patients undergoing hip replacement surgery reaching peak values of up to 12 ng mL(-1) (214 pmol L(-1)). When normalized to the amount of thrombin generated, interindividual variabilities in the APC generating capacity were observed. In general, with a turn-around time from blood sampling to generation of test results of < 7 h, the APC-OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions., (© 2012 International Society on Thrombosis and Haemostasis.)

Activated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory, and cytoprotective properties. Using recombinant APC, we identified a class of single-stranded DNA aptamers (HS02) that selectively bind to APC with high affinity. Interaction of HS02 with APC modulates the protease activity in a way such that the anticoagulant functions of APC are inhibited and its reactivity toward the protein C inhibitor is augmented in a glysoaminoglycan-like fashion, whereas APC's antiapoptotic and cytoprotective functions remain unaffected. Based on these data, the binding site of HS02 was localized to the basic exosite of APC. These characteristics render the exosite-specific aptamers a promising tool for the development of APC assays and a potential therapeutic agent applicable for the selective control of APC's anticoagulant activity.

Background: HD1-22 is a bivalent aptamer that binds to thrombin with high affinity (K(d) = 0.65 nm) and occupies both anion binding exosites without blocking the active centre of the enzyme. HD1-22 has been developed by connecting the exosite 1 binding aptamer HD1 and the exosite 2 binding aptamer HD22 through a poly-dA linker., Objectives: To characterize the anticoagulant profile of HD1-22 in comparison to the clinically established direct acting thrombin inhibitors bivalirudin and argatroban, and to test the efficacy of antidote-oligodeoxynucleotides., Methods and Results: HD1-22 prolongs clotting times of the thrombin time, activated partial thromboplastin time, ecarin clotting time, and lag-time of the tissue factor triggered thrombin generation assay in a dose-dependent manner. On a molar basis, its anticoagulant activity was nearly identical to bivalirudin and superior to argatroban. Thrombin-induced platelet aggregation was more effectively inhibited by HD1-22 than by bivalirudin. The HD1-22 aptamer retains the ability of the HD1-moiety to bind to (pro)exosite 1 of prothrombin and inhibits the prothrombinase activity nearly 2-fold better than HD1. The anticoagulant activities of HD1-22 are fully reversed by addition of antidote-oligodeoxynucleotides., Conclusions: The strong thrombin-inhibiting activity, together with the availability of a rapid acting antidote strategy, makes HD1-22 an interesting anticoagulant candidate, especially for use in clinical situations where effective anticoagulation and rapid reversal of the anticoagulant effect are required. The data obtained warrant further clinical studies.

Recombinant hirudin has been found to be immunogenic in patients treated with lepirudin following heparin-induced thrombocytopenia (HIT). We assessed the incidence of immunoglobulin G (IgG) antihirudin antibodies by enzyme-linked immunosorbent assay in 112 patients enrolled in a dose-finding study with desirudin. Patients received desirudin subcutaneously following orthopedic hip surgery at 10 mg twice a day (n = 17), 15 mg twice a day (n = 75), and 20 mg twice a day (n = 20). Of 112 patients, 11 (9.8%) developed antihirudin antibodies independently of the dose. The rate of immunization did not differ from that observed in HIT patients treated with lepirudin (P =.113). Plasma concentrations of desirudin did not differ between antihirudin antibody-positive and -negative patients. Antihirudin antibodies had no impact on incidences of deep vein thrombosis and/or pulmonary embolism, allergic reactions, and hemorrhage. However, the total number of immunized patients observed was low and so infrequent (but severe) effects of antihirudin antibodies cannot be excluded.

Lupus anticoagulants (LAs) are acquired antiphospholipid antibodies, and the occurrence of LA is associated with an increased risk of developing thrombosis. In a population of 46 patients with LA with or without LA-associated thrombophilia, it was analyzed whether the concentration of LA could be correlated to the individual thrombotic risk in patients with LA. No significant difference was found in the concentrations of LA measured by routinely used functional and immunologic assays in patients with LA with thrombophilia when compared with patients with LA without thrombophilia. Inhibition of thrombomodulin (TM) activity by LA has been postulated to be one of the major pathogenic mechanisms causing thrombophilia in LA. Therefore the inhibition of endothelial cell-dependent TM activity by LA was analyzed by using a protein C (PC) activation assay. Reduced rates of PC activation were found in only 2 out of the 46 cases, ruling out that inhibition of TM activity is a common phenomenon in patients with LA. However, anionic phospholipids are necessary to ascertain the anticoagulant activity of activated PC (APC). To prove the hypothesis that the anticoagulant activity of APC is inhibited by LA, the anticoagulant response of purified APC added to LA-containing plasma was measured through the amount of factor VIII inactivation. Thirteen out of 14 patients with recurrent thrombotic events and 10 out of 19 patients with one single episode of thrombosis showed an APC response outside the mean--2 SD range of normal human controls. In contrast, among 13 patients with LA without symptoms, only one showed an abnormal APC response. From these data it is concluded that LA inhibits the APC anticoagulant activity and that this type of acquired APC dysfunction may contribute to the pathogenesis of LA-associated thrombophilia. Moreover, the APC anticoagulant response assay may prove to be a useful marker to identify patients with LA with a high thrombotic risk.