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Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (V L and V H ) are connected via an artificial linker. One particular scFv, iKM33, directed against blood coagulation factor VIII (FVIII) was shown to inhibit major FVIII functions and is useful in FVIII research. We aimed to investigate the properties of iKM33 enabled with protease-dependent disintegration. Three variants of iKM33 bearing thrombin cleavage sites within the linker were expressed using a baculovirus system and purified by two-step chromatography. All proteins retained strong binding to FVIII by surface plasmon resonance, and upon thrombin cleavage, dissociated into V L and V H as shown by size-exclusion chromatography. However, in FVIII activity and low-density lipoprotein receptor-related protein 1 binding assays, the thrombin-cleaved iKM33 variants were still inhibitory. In a pull-down assay using an FVIII-affinity sorbent, the isolated V H , a mixture of V L and V H , and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated V L and V H assembled into scFv-like heterodimer on FVIII, and the isolated V H alone also bound FVIII. We discuss the potential use of both protease-cleavable scFvs and isolated Fv subunits retaining high affinity to the antigens in various practical applications such as therapeutics, diagnostics, and research.

Background: Factor VIII (FVIII) binding to endogenous von Willebrand factor (VWF) has constrained half-life extension of recombinant FVIII (rFVIII) products for hemophilia A. Efanesoctocog alfa (rFVIIIFc-VWF-XTEN; BIVV001) is a novel fusion protein designed to decouple FVIII from VWF in circulation and maximize half-life prolongation by XTEN ® polypeptides and Fc fusion. FVIII, VWF, and platelets interact to achieve normal hemostasis. Thus, bioengineered FVIII replacement products, such as efanesoctocog alfa, require comprehensive assessment of their hemostatic potential., Objectives: We compared functional clot formation and injury-induced platelet accumulation between efanesoctocog alfa and rFVIII., Patients/methods: The hemostatic potential of efanesoctocog alfa and rFVIII were assessed by measuring their dose-dependent effects on in vitro fibrin generation in hemophilic plasma and in vivo injury-induced platelet accumulation using intravital microscopy and repeat saphenous vein laser-induced injuries in hemophilia A mice., Results: Equal concentrations of efanesoctocog alfa or rFVIII (up to 1 IU/ml) added to plasma from patients with hemophilia A elicited similar kinetics for dose-dependent fibrin polymerization between factor products. In the presence of tissue plasminogen activator (tPA), clots formed had similar stability between products. Single intravenous doses (50, 100, or 150 IU/kg) of efanesoctocog alfa or rFVIII shortly before repeat saphenous vein laser-induced injuries increased platelet accumulation over time in a dose-dependent manner in hemophilia A mice. Platelet deposition kinetics were similar between products., Conclusions: Equivalent doses of efanesoctocog alfa and rFVIII had similar efficacy in promoting fibrin clot formation and injury-induced platelet accumulation. The hemostatic potential of efanesoctocog alfa was indistinguishable from that of rFVIII., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background and Objectives: Hemophilia A (HA) is an X-linked blood disorder. It is caused by pathogenic F8 gene variants, among which missense mutations are the most prevalent. The resulting amino acid substitutions may have different impacts on physicochemical properties and, consequently, on protein functionality. Regular prediction tools do not include structural elements and their physiological significance, which hampers our ability to functionally link variants to disease phenotype, opening an ample field for investigation. The present study aims to elucidate how physicochemical changes generated by substitutions in different protein domains relate to HA, and which of these features are more consequential to protein function and its impact on HA phenotype., Methods: An in silico evaluation of 71 F8 variants found in patients with different HA phenotypes (mild, moderate, severe) was performed to understand protein modifications and functional impact. Homology modeling was used for the structural analysis of physicochemical changes including electrostatic potential, hydrophobicity, solvent-accessible/excluded surface areas, disulfide disruptions, and substitutions indexes. These variants and properties were analyzed by hierarchical clustering analysis (HCA) and principal component analysis (PCA), independently and in combination, to investigate their relative contribution., Results: About 69% of variants show electrostatic changes, and almost all show hydrophobicity and surface area modifications. HCA combining all physicochemical properties analyzed was better in reflecting the impact of different variants in disease severity, more so than the single feature analysis. On the other hand, PCA led to the identification of prominent properties involved in the clustering results for variants of different domains., Conclusions: The methodology developed here enables the assessment of structural features not available in other prediction tools (e.g., surface distribution of electrostatic potential), evaluating what kind of physicochemical changes are involved in FVIII functional disruption. HCA results allow distinguishing substitutions according to their properties, and yielded clusters which were more homogeneous in phenotype. All evaluated properties are involved in determining disease severity. The nature, as well as the position of the variants in the protein, were shown to be relevant for physicochemical changes, demonstrating that all these aspects must be collectively considered to fine-tune an approach to predict HA severity., Competing Interests: Declaration of Competing Interest None, (Copyright © 2022. Published by Elsevier B.V.)

Introduction: Hereditary factor (F) XIII-deficiency is a known risk factor for postoperative complications, but data of acquired FXIII-deficiency in malignancies are limited. Therefore, we evaluated the role of acquired FXIII-deficiency in surgery for advanced epithelial ovarian cancer (EOC)., Materials and Methods: We performed a retrospective analysis of patients with known serum FXIII status and treatment between 2011 and 2018 at our center. We defined cohorts according to FXIII with values > 75% as normal (group A), 55-75% as reduced (group B) and < 55% as low (group C). Complications were classified according to the Clavien-Dindo Classification, class III-V complications were defined as severe., Results: 347 patients with EOC were identified. 180 patients (51.2%) were in group A, 82 patients (23.6%) in group B, and 85 patients (24.4%) in group C. Lower levels of FXIII were associated with higher amount of ascites, FIGO IV, high grade serous histology, low albumin, and higher CA-125 levels. Regarding intraoperative variables, low FXIII was associated with longer duration of surgery, higher blood loss, higher surgical complexity score/number of bowel anastomosis and a higher probability for macroscopic residual disease. The risk of severe complications in group A was 12.2%, 24.4% in group B, and 31.8% in group C. In a multivariate model, low FXIII (OR 2.8), > 1 bowel anastomosis (OR 2.7), age-adjusted Charlson comorbidity index ≥ 4 (OR 3.6) and a longer duration of surgery (> 285 min.) were significant predictive factors for severe complications., Conclusion: FXIII is associated with tumor and treatment burden. A low level of FXIII is associated with postoperative complications. The knowledge about the presurgical serum FXIII-level might be helpful to plan the treatment strategy., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Platelet-targeted FVIII gene therapy can efficiently recover bleeding phenotype for hemophilia A (HA), yet characteristics of thrombus formation with this ectopic expression of factor VIII (FVIII) in platelets remain unclear. Here, we generated 2bF8 trans mice restrictively expressing human B-domain-deleted FVIII (hBDD FVIII) in platelets on a hemophilic (FVIII null ) mice background. The results showed no statistical difference in clot strength and stability between wild-type (WT) and 2bF8 trans mice, but with a prolonged reaction time (R-time), by thromboelastography. Fluid dynamics analysis showed that at the shear rates of 500 to 1,500 s -1 , where physiological hemostasis often develops, the thrombi formed in 2bF8 trans mice were more stable than those in FVIII null mice, while at high pathological shear rates (2,500 s -1 ), mimicking atherosclerosis, thrombus size and fibrin deposition in 2bF8 trans mice were less than those in WT mice. Thrombus morphology analysis showed that there was a locally concentrated deposition of fibrin in thrombus at the injured site and fibrin co-localized with activated platelets in 2bF8 trans mice. Moreover, a higher ratio of fibrin to platelets was found in thrombus from 2bF8 trans mice following laser-induced injury in cremaster arterioles, which might be the underlying mechanism of thrombus stability in 2bF8 trans mice at physiological arterial circumstance. These observations suggest that specific morphological features of the thrombi might contribute to the efficacy and safety of platelet-targeted FVIII gene therapy for HA., Competing Interests: None declared., (Thieme. All rights reserved.)

Background & Aims: It has been suggested that the ratio of procoagulant factor VIII to anticoagulant protein C (FVIII/PC) reflects the hemostatic equilibrium. Moreover, FVIII/PC predicted decompensation/death in a small study not accounting for portal hypertension severity. We investigated (i) the prognostic value of FVIII/PC (outcome-cohort) and (ii) whether FVIII/PC reflects the hypercoagulable state (assessed by thrombomodulin-modified thrombin generation assay [TM-TGA]) or the risk of bleeding/thrombotic events in patients undergoing hepatic venous pressure gradient (HVPG) measurement during follow-up., Methods: (i) The outcome-cohort comprised 576 patients with evidence of advanced chronic liver disease (liver stiffness measurement ≥10 kPa and/or HVPG ≥6 mmHg). (ii) TM-TGA-cohort patients (n = 142) were recruited from the prospective VIenna CIrrhosis Study (VICIS: NCT03267615)., Results: (i) FVIII/PC significantly increased across clinical stages (p <0.001) as well as HVPG (p <0.001) and MELD score (p <0.001) strata and remained independently associated with decompensation/liver-related death (adjusted hazard ratio 1.06; 95% CI 1.01-1.11; p = 0.013), even after multivariable adjustment. It was also associated with acute-on-chronic liver failure (ACLF) development (adjusted hazard ratio 1.10; 95% CI 1.02-1.19; p = 0.015) in patients with decompensated cirrhosis. (ii) FVIII/PC showed a weak positive correlation with endogenous thrombin potential (Spearman's ρ = 0.255; p = 0.002), but this association disappeared after adjusting for the severity of liver disease. FVIII/PC was not associated with the development of bleeding (p = 0.272) or thrombotic events (p = 0.269). However, FVIII/PC correlated with biomarkers of different pathophysiological mechanisms that promote liver disease progression., Conclusion: FVIII/PC provides prognostic information regarding hepatic decompensation/death and ACLF, independently of established prognostic indicators. However, this is not evidence that hypercoagulability drives disease progression, as the correlation between FVIII/PC and thrombin generation is confounded by liver disease severity and FVIII/PC was not associated with thrombosis. Therefore, FVIII/PC does not reflect coagulation and results from previous studies on FVIII/PC require re-interpretation., Clinical Trial Number: NCT03267615 (in part)., Lay Summary: A balanced coagulation system is essential for preventing bleeding episodes and blood clot formation (thrombosis). Blood of patients with advanced liver disease may have increased coagulation potential, possibly promoting the worsening of liver disease via thrombosis in the blood vessels of the liver. The ratio between the results of 2 blood tests (procoagulant factor VIII to anticoagulant protein C) has been suggested to reflect these increases in coagulation potential. Our study demonstrates, on the one hand, that this ratio is a versatile predictor of the development of complications of cirrhosis, yet on the other hand, that it is unrelated to coagulation., Competing Interests: Conflicts of interest The authors have nothing to disclose regarding the work under consideration for publication. Conflicts of interests outside the submitted work: L.B., R.J.N., J.W., R.P., L.H., M.J., A.F.S., G.S., P.Q., and T.L. have nothing to disclose. Be.Sc. received travel support from AbbVie, Ipsen and Gilead. Be.Si. received travel support from AbbVie and Gilead. D.B. received travel support from AbbVie and Gilead and speaker fees from AbbVie. M.P. served as a speaker and/or consultant and/or advisory board member for Bayer, Bristol-Myers Squibb, Eisai, Ipsen, Lilly, MSD, and Roche and received travel support from Bayer and Bristol-Myers Squibb. C.A. received honoraria for lectures and advisory boards from Bayer, Boehringer-Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, and Pfizer. M.T. received grant support from Albireo, Alnylam, Cymabay, Falk, Gilead, Intercept, MSD, Takeda, and UltraGenyx, honoraria for consulting from Albireo, Boehringer-Ingelheim, BiomX, Falk, Genfit, Gilead, Intercept, Janssen, MSD, Novartis, Phenex, Regulus, and Shire, speaker fees from Bristol-Myers Squibb, Falk, Gilead, Intercept, and MSD, as well as travel support from AbbVie, Falk, Gilead, and Intercept. T.R. received grant support from AbbVie, Boehringer-Ingelheim, Gilead, Intercept, MSD, Myr Pharmaceuticals, Philips Healthcare, Pliant, Siemens, and W. L. Gore & Associates; speaking honoraria from AbbVie, Gilead, Gore, Intercept, Roche, and MSD; consulting/advisory board fees from AbbVie, Bayer, Boehringer-Ingelheim, Gilead, Intercept, MSD, and Siemens; and travel support from AbbVie, Boehringer-Ingelheim, Gilead, and Roche. M.M. served as a speaker and/or consultant and/or advisory board member for AbbVie, Bristol-Myers Squibb, Collective Acumen, Gilead, and W. L. Gore & Associates and received travel support from AbbVie, Bristol-Myers Squibb, and Gilead. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aim: To identify the von Willebrand factor (VWF) gene variant status in Croatian adult patients diagnosed with von Willebrand disease (VWD), provide differential diagnosis of VWD subtypes, and identify patients with mild hemophilia A (HA) who were earlier misdiagnosed as VWD., Methods: Coagulation testing included determination of VWF gain-of-function mutant glycoprotein Ib binding activity (VWF:GPIbM), VWF antigen, VWF collagen-binding activity, and multimeric analysis. Genetic analysis of VWF and FVIII genes was performed with next-generation sequencing (NGS)., Results: The study enrolled 50 patients (72% women; median age 37 years, range 18-75) from 44 unrelated families. Fourteen patients were heterozygous for VWF gene variants compatible with type-1 VWD. Twelve had variants associated with type 2, of whom seven were classified as type 2A, four as type 2B, and one as type 2N. Six type-3 VWD patients were either homozygotes for null variants or combined heterozygotes. Eleven variants within the VWF gene were novel. Three female patients had variants within the FVIII gene, and were re-classified as mild-HA carriers, of whom one had causative novel variants both within VWF and FVIII genes. Fifteen patients remained without a defined genetic cause of their disorder, of whom five had VWF:GPIbM levels below 50%., Conclusion: Croatian adult patients with VWD have considerable genetic heterogeneity. NGS of both VWF and FVIII genes provided accurate differential diagnosis of VWD subtypes and distinction of VWD from mild HA.

Vascular calcification occurs in arterial aging, atherosclerosis, diabetes mellitus, and chronic kidney disease. Transforming growth factor-β1 (TGF-β1) is a key modulator driving the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs), leading to vascular calcification. We hypothesize that milk fat globule-epidermal growth factor 8 (MFG-E8), a glycoprotein expressed in VSMCs, promotes the osteogenic transdifferentiation of VSMCs through the activation of TGF-β1-mediated signaling. We observe that the genetic deletion of MFG-E8 prevents calcium chloride-induced vascular calcification in common carotid arteries (CCAs). The exogenous application of MFG-E8 to aged CCAs promotes arterial wall calcification. MFG-E8-deficient cultured VSMCs exhibit decreased biomineralization and phenotypic transformation to osteoblast-like cells in response to osteogenic medium. MFG-E8 promotes β1 integrin-dependent MMP2 expression, causing TGF-β1 activation and subsequent VSMC osteogenic transdifferentiation and biomineralization. Thus, the established molecular link between MFG-E8 and vascular calcification suggests that MFG-E8 can be therapeutically targeted to mitigate vascular calcification., (© 2022. The Author(s).)

Pulmonary endarterectomy (PEA) is the main treatment for chronic thromboembolic pulmonary hypertension (CTEPH). Postoperative unfractionated heparin dosing can be monitored by activated partial thromboplastin time (APTT) or by anti-factor Xa activity (anti-Xa). In pseudo heparin resistance, APTT response to heparin is blunted due to elevated Factor VIII (FVIII) which can underestimate anticoagulation. We examined possible pseudo heparin resistance after PEA and assessed the impact of FVIII. APTT response to heparin before and after operation was determined in 13 PEA patients anticoagulated with unfractionated heparin. APTT and anti-Xa concordance was analyzed from paired postoperative samples, and antithrombin, fibrinogen and FVIII levels were measured. Single-cell RNA sequencing was used to characterize FVIII gene expression in PEA specimens of 5 patients. APTT response to heparin was blunted after PEA. APTT and anti-Xa were discordant in 36% of postoperative samples and most common discordant patterns were subtherapeutic APTT with therapeutic (16%) or supratherapeutic (11%) anti-Xa. Overall, APTT underestimated anticoagulation relative to anti-Xa in one-third of the samples. FVIII levels were elevated before surgery, increased substantially 1 and 3 days (median 4.32 IU/mL) after PEA, and were higher in discordant than concordant samples. Single-cell RNA sequencing showed FVIII gene expression in PEA specimen endothelial cells. Pseudo heparin resistance is common after PEA likely due to highly elevated postoperative FVIII levels indicating that anti-Xa reflects postoperative heparinization better than APTT in these patients. FVIII production by the pulmonary artery endothelium may participate in local prothrombotic processes important for CTEPH pathogenesis., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Trousseau sign was the first demonstration of a close relationship between cancer and thrombosis. Currently, venous thromboembolism (VTE) is five to six times more likely to occur in cancer patients, whereas there is a greater risk of cancer diagnoses following thromboses. In considering novel players, factor VIII (FVIII), an essential coagulation cofactor with emerging extracoagulative functions, has been identified as an independent VTE risk factor in cancer; however, the basis of this increase is unknown., Objective: To investigate the possible direct expression and secretion of FVIII by cancer cells., Methods: Bladder cancer, with a high VTE risk, and normal bladder tissue and epithelium, were used to investigate FVIII. Factor VIII protein and secretion were examined in bladder cancer cell lines. Expanding to other cancers, the Cancer Cell line Encyclopedia database was used to analyze FVIII, tissue factor, FV, FVII, FIX, FX, and von Willebrand factor (VWF) mRNA in 811 cell lines subdivided according to origin. Factor VIII protein synthesis, secretion, and bioactivity were investigated in a profile of cancer cell lines of differing origins., Results and Conclusions: Although expressed in the normal bladder epithelium, FVIII mRNA and protein were higher in matched bladder neoplasms, with synthesis and secretion of bioactive FVIII evident in bladder cancer cells. This can be extended to other cancer cell lines, with a pattern reflecting the tumor origin, and that is independent of VWF and other relevant players in the coagulation cascade. Here, evidence is provided of a possible independent role for FVIII in cancer-related pathophysiology., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by deficient activity of blood coagulation factor VIII (FVIII). In addition, hemophilia patients show associated diseases including osteopenia, altered inflammation and vascular fragility which may represent the consequence of recurrent bleeding or may be related to the direct FVIII deficiency. Nowadays, recombinant FVIII is proposed to treat hemophilia patients with no circulating FVIII inhibitor. Initially described as a coenzyme to factor IXa for initiating thrombin generation, there is emerging evidence that FVIII is involved in multiple biological systems, including bone, vascular and immune systems. The present study investigated: (i) the functional activities of recombinant human FVIII (rFVIII) on endothelial cells, and (ii) the impact of rFVIII activities on the functional interactions of human monocytes and endothelial cells. We then investigated whether rFVIII had a direct effect on the adhesion of monocytes to the endothelium under physiological flow conditions. We observed that direct biological activities for rFVIII in endothelial cells were characterized by: (i) a decrease in endothelial cell adhesion to the underlying extracellular matrix; (ii) regulation of the transcriptomic and protein profiles of endothelial cells; (iii) an increase in the vascular tubes formed and vascular permeability in vitro; and (iv) an increase in monocyte adhesion activated endothelium and transendothelial migration. By regulating vascular permeability plus leukocyte adhesion and transendothelial migration, the present work highlights new biological functions for FVIII., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Allosteric disulfide bonds permit highly responsive, transient 'switch-like' properties that are ideal for processes like coagulation and inflammation that require rapid and localised responses to damage or injury. Haemophilia A (HA) is a rare bleeding disorder managed with exogenous coagulation factor(F) VIII products. FVIII has eight disulfide bonds and is known to be redox labile, but it is not known how reduction/oxidation affects the structure-function relationship, or its immunogenicity-a serious complication for 30% severe HA patients. Understanding how redox-mediated changes influence FVIII can inform molecular engineering strategies aimed at improving activity and stability, and reducing immunogenicity. FVIII is a challenging molecule to work with owing to its poor expression and instability so, in a proof-of-concept study, we used molecular dynamics (MD) to identify which disulfide bonds were most likely to be reduced and how this would affect structure/function; results were then experimentally verified. MD identified Cys1899-Cys1903 disulfide as the most likely to undergo reduction based on energy and proximity criteria. Further MD suggested this reduction led to a more open conformation. Here we present our findings and highlight the value of MD approaches., Competing Interests: The authors have declared that no competing interests exist.

Background: Coagulopathic bleeding is common during adult extracorporeal membrane oxygenation (ECMO), and acquired von Willebrand syndrome is a contributing factor. We compared ECMO patient blood samples that were treated in vitro with recombinant von Willebrand Factor concentrate and plasma-derived von Willebrand Factor concentrate. Our hypothesis was that recombinant von Willebrand Factor (vWF) would have greater efficacy in increasing vWF function. Secondarily, we hypothesized that recombinant vWF would have less impact on thrombin generation., Methods: Thirty ECMO patients and 10 cardiac surgical controls were enrolled in the study. ECMO patient blood samples were treated in vitro with low- and high-dose recombinant vWFs and low- and high-dose plasma-derived vWFs. Whole blood ristocetin-induced platelet aggregation (RIPA), plasma ristocetin cofactor activity (RCo), and thrombin generation were compared between ECMO patient blood samples and control blood samples and between vWF-treated ECMO patient blood samples and nontreated samples., Results: ECMO patient blood samples had severely reduced median RIPA compared to control samples 2 ohms (1-12 [25th-75th percentile]) vs 20 ohms (11-42) (P < .001). Treatment of ECMO patient blood samples with high-dose recombinant vWF significantly increased median RIPA to 10 ohms (2-15) (P < .001), while low-dose recombinant vWF and low- and high-dose plasma-derived vWFs did not significantly increase RIPA; 6 ohms (3-14), 4 ohms (1-13), and 6 ohms (2-10), respectively (P = .25, >.99, and >.99). Treatment with high-dose recombinant vWF and low- and high-dose plasma-derived vWFs significantly increased median plasma RCo to 4.7 international units (IU)/mL (3.7-5.9), 3.3 IU/mL (2.7-4.8), and 3.9 IU/mL (3.4-5.3), respectively, compared to controls 1.8 IU/mL (1.5-2.3) (all P < .001). Treatment with low- and high-dose plasma-derived vWFs significantly increased mean endogenous thrombin potential (6270.2 ± 2038.7 and 6313.1 ± 1913.3) compared to nontreated samples (5856.7 ± 1924.6) (P = .04 and .006), whereas treatment with low- and high-dose recombinant vWFs had no significant effect on mean endogenous thrombin potential (5776.1 ± 2087.3 and 5856.2 ± 1946.4) (P > .99 for both comparisons)., Conclusions: In vitro treatment of ECMO patient blood samples with high-dose recombinant vWF was superior to low-dose recombinant vWF and plasma-derived vWF in terms of improving RIPA. In addition, recombinant vWF treatment did not increase endogenous thrombin potential, which may reduce overall thrombotic risk if it used to treat acquired von Willebrand syndrome in ECMO patients., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 International Anesthesia Research Society.)

Established reference intervals (RIs) of coagulation parameters generally based on the general population are not applicable to specific women. In order to accurately evaluate the coagulation status of non-pregnant women and pregnant women, specific RIs should be established. Our study recruited 465 non-pregnant women and 1972 pregnant women aged 20-45 years. Eight tests including antithrombin (AT), protein C (PC), free protein S (fPS), lupus anticoagulant (LA), D-dimer, fibrin/fibrinogen degradation products (FDP), coagulation factor VII (FVII), and factor VIII (FVIII) were performed on ACL TOP automated coagulation instrument. The RIs for these tests were established in non-pregnant and pregnant women at different gestational weeks. Compared to the non-pregnant group, the medians of AT and fPS were lower, while the medians of PC, LA normalized ratio, D-dimer, FDP, FVII, and FVIII were higher. During pregnancy, AT and fPS activity showed a decreasing trend, with the increase of gestational age. PC activity, LA normalized ratio, D-dimer concentrations, FDP concentrations, FVII, and FVIII activity presented an increasing trend, with the increase of gestational age. The non-pregnant women-specific RIs and the gestational age-specific RIs of AT, PC, fPS, LA normalized ratio, D-dimer, FDP, FVII, and FVIII needed to be established for accurate clinical diagnoses., (© 2022. The Author(s).)

Hemophilia A (HA) is caused by mutations in the coagulation factor VIII (FVIII) gene (F8) . Gene therapy is a hopeful cure for HA; however, FVIII inhibitors formation hinders its clinical application. Given that platelets promote coagulation via locally releasing α-granule, FVIII ectopically expressed in platelets has been attempted, with promising results for HA treatment. The B-domain-deleted F8 ( BDDF8 ), driven by a truncated ITGA2B promoter, was targeted at the ribosomal DNA (rDNA) locus of HA patient-specific induced pluripotent stem cells (HA-iPSCs). The F8 -modified, human induced pluripotent stem cells (2bF8-iPSCs) were differentiated into induced hematopoietic progenitor cells (iHPCs), induced megakaryocytes (iMKs), and mesenchymal stem cells (iMSCs), and the FVIII expression was detected. The ITGA2B promoter-driven BDDF8 was site-specifically integrated into the rDNA locus of HA-iPSCs. The 2bF8-iPSCs were efficiently differentiated into 2bF8-iHPCs, 2bF8-iMKs, and 2bF8-iMSCs. FVIII was 10.31 ng/10 6 cells in lysates of 2bF8-iHPCs, compared to 1.56 ng/10 6 cells in HA-iHPCs, and FVIII was 3.64 ng/10 6 cells in 2bF8-iMSCs lysates, while 1.31 ng/10 6 cells in iMSCs with CMV-driven BDDF8 . Our results demonstrated a high expression of FVIII in iHPCs and iMSCs derived from hiPSCs with site-specific integration of ITGA2B promoter-driven BDDF8 , indicating potential clinical prospects of this platelet-targeted strategy for HA gene therapy.

Aim: Both MFGE8 and HMGB1 were vital players for aneurysmal subarachnoid hemorrhage. However, whether HMGB1 was served as the downstream target of MFGE8 was unknown. To test this new mechanism, we performed the SAH model in rats., Method: All treatments were injected intraventricularly into the right lateral ventricles. SAH grade, brain water content, and neurological function scores were evaluated. HMGB1 expression was studied by double immunofluorescence staining. HE and Nissl's staining were performed to observe the pathological change. Inflammatory factors were measured by ELISA method., Results: High expression of MFGE8 could improve neurological function and reduce the brain edema and pro-inflammatory factors. Injection of rhMFGE8 inhibited HMGB1. To further verify the regulation of MFGE8 in HMGB1, we used rhHMGB1 and glycyrrhizin, and the results indicated MFGE8 produced excellent effect on SAH rats via inhibiting HMGB1., Conclusion: In a word, MFGE8 improved EBI caused by SAH, depending on HMGB1 that was the potential mechanism.

Hemophilia A is an X-linked hereditary disorder that results from deficient coagulation factor VIII (FVIII) activity, leading to spontaneous bleeding episodes, particularly in joints and muscles. FVIII deficiency has been associated with altered bone remodeling, dysregulated macrophage polarization, and inflammatory processes that are associated with the neoformation of abnormal blood vessels. Treatment based on FVIII replacement can lead to the development of inhibitors that render FVIII concentrate infusion ineffective. In this context, hemophilia has entered a new therapeutic era with the development of new drugs, such as emicizumab, that seek to restore the hemostatic balance by bypassing pathologically acquired antibodies. We discuss the potential extrahemostatic functions of FVIII that may be crucial for defining future therapies in hemophilia., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background:  Emicizumab, a bispecific monoclonal antibody for hemophilia A (HA), has strong pharmacodynamic effects in several coagulation assays resulting in dosing difficulties with Factor VIII (FVIII) concentrates during bleeding emergencies., Materials and Methods: Single and multiple regression models were studied to estimate FVIII activity using 27 archived plasma samples from three patients with HA without inhibitor under emicizumab treatment. Explanatory variables were FVIII chromogenic assay (CSA), Ad|min1|, Ad|min2|, the number of seconds of APTT, and the FVIII one-stage assay (OSA), which were measured without idiotype antibodies. The response variable was FVIII OSA measured with idiotype antibodies., Results: In the simple linear model, the FVIII CSA regression coefficient was 1.04 and the intercept was -14.55 ( r 2  = 0.95; p < 0.001). In the multiple regression model, FVIII OSA and FVIII CSA were selected based on the Akaike Information Criterion, with regression coefficients of 1.74 and 1.15, respectively, and an intercept of -92.03 ( r 2  = 0.96, p < 0.001)., Conclusions: The regression models can estimate the FVIII:C levels in patients with HA receiving emicizumab and would be useful in a bleeding emergency and/or surgery.

Deep vein thrombosis (DVT) is common in patients following gynecological surgery. Coagulation factor VIII (FVIII) is an important part of the human coagulation system, and FVIII:C is a component of FVIII with anticoagulant activity. 800 patients who underwent gynecological surgery were enrolled. General clinical data were harvested, and pre - and postoperative serum FVIII levels were determined. Lower-extremity ultrasound examination and/or postoperative pulmonary angiography were performed. Related data were analyzed statistically. DVT was the first manifestation of venous thromboembolism in all cases. There were a total of 46 cases, and the incidence of DVT was 5.8%. Progression to pulmonary embolism was confirmed in 16 cases, with an incidence of 2.0%. The independent risk factors for DVT after gynecological surgery were postoperative FVIII:C levels (odds ratio [OR] = 1.01), age (OR = 6.57), and operation time ≥3 hours (OR = 2.90) ( P < 0.05). When the FVIII:C level was greater than the 75th centile (≥150 IU/dL), the risk of DVT was 2.99 times higher than that below the 25th centile (<100 IU/dL) ( P < 0.05). When combined with the risk factor of operation time ≥3 hours, the risk increased to 3.17 times ( P = 0.10). When combined with age ≥60 years, the risk was significantly increased, reaching 12.0 times ( P < 0.05). Serum FVIII:C levels are an independent risk factor for DVT after gynecological surgery. Higher levels increase the risk of DVT after gynecological surgery, and they may have a dose-dependent relationship. A synergistic effect exists in combination with other risk factors, which further increases the risk.

Background: The goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose., Methods: In this phase 1-2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5  × 10 11 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2 × 10 12 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII., Results: The median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti-AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], -2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration)., Conclusions: Sustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.)., (Copyright © 2021 Massachusetts Medical Society.)

Background: The development of factor VIII (FVIII) inhibitor in patients with hemophilia A (PWHA) is a great challenge for hemophilia care. Both genetic and environmental factors led to complications in PWHA. The development of inhibitory antibodies is usually induced by the immune response. Tumor necrosis factor α (TNF- α ), one of the cytokines, might contribute to its polymorphism. In this study, we investigated the clinical factors, level of serum TNF- α , and polymorphism of c.-308G > A TNF - α gene in inhibitor development in severe PWHA., Methods: A cross-sectional study was conducted among all PWHA in West Java province. The clinical parameters, FVIII, FVIII inhibitor, and serum TNF- α level were assessed. The genotyping of -380G > A TNF- α gene polymorphism was performed using polymerase chain reaction and Sanger sequencing., Results: Among the 258 PWHA, 216 (83.7%) were identified as severe PWHA. The FVIII inhibitor was identified in 90/216 (41.6%) of severe PWHA, consisting of 45 high-titer inhibitors (HTI) and 45 low-titer inhibitors (LTI). There was a significant correlation between serum TNF- α level and the development of HTI ( p = 0.043). The cutoff point of serum TNF- α level, which can be used to differentiate between HTI and LTI, was 11.45 pg/mL. The frequency of FVIII replacement therapy was significant only in HTI of severe PWHA regarding serum TNF- α level ( p = 0.028). There is no correlation between polymorphisms of -380G > A TNF- α gene and inhibitor development ( p = 0.645)., Conclusions: The prevalence of FVIII inhibitor in severe PWHA in West Java, Indonesia, was 41.6%. The frequency of replacement therapy is a risk factor for inhibitor development. Serum TNF- α level might be used to differentiate between high and low inhibitor levels in severe hemophilia A, and this might support decision making regarding treatment options for inhibitor in severe hemophilia A., Competing Interests: The authors declare no conflict of interest regarding the publication of this article., (Copyright © 2021 Susi Susanah et al.)

Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg 372 , Arg 740 , and Arg 1689 . Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg 1689 . An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg 1689 . We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54-65 residues). Two synthetic peptides, spanning residues 1659-1669 with sulfated Tyr 1664 and residues 1675-1685 with sulfated Try 1680 , inhibited thrombin-catalyzed FVIII activation and cleavage at Arg 1689 . Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664-1666 and 1683-1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg 1689 in the alanine-substituted rFVIII mutants within 1663-1666 residues were similar to those of wild type (WT). Similar studies of 1680-1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg 1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680-1684 residues containing sulfated Tyr 1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg 1689 ., Competing Interests: None declared., (Thieme. All rights reserved.)

Deficiency of factor V is a congenital autosomal recessive coagulopathy associated with mutations in the F5 gene that results in mild-to-severe bleeding episodes. Factor V is a component of the prothrombinase complex responsible for accelerating conversion of prothrombin to thrombin. At the present time there are no therapeutic factor V concentrates available. This study was designed to lay the preliminary foundations for future cell-based therapy for patients with severe factor V deficiency. The study showed that hepatospheres, which produce coagulation factors VIII, IX, and V, synthetize and store intracellular glycogen and express albumin levels up to 8 times higher than those of undifferentiated cells. Factor IX and factor V gene expression increased significantly in hepatospheres as compared to undifferentiated cells, whereas factor VIII gene expression remained constant. The factor V protein was detected in the hepatospheres´ secretome. Considering the enormous potential of mesenchymal stem cells as therapeutic agents, this study proposes a highly reproducible method to induce differentiation of mesenchymal stem cells from human placenta to factor V-producing hepatospheres. This strategy constitutes a preliminary step towards a curative treatment of factor V deficiency through advanced therapies such as cell therapy., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acquired haemophilia (AH) is a suddenly occurring severe blood diathesis that affects both males and females and is caused by autoantibodies which inhibit coagulation factor VIII. The report describes an unusual case of acquired haemophilia in which an epileptic seizure and haemorrhage into the ventricular system of the brain were the first manifestations of the disease. In addition, APTT was prolonged to 94.6 seconds and the factor VIII level was as low as 1.5%. The level of anti-FVIII antibody was extremely high - 272BU/ml. The patient did not undergo invasive diagnostic procedure or an operation. Recombinant factor VIIa was used to control the bleeding. In order to eradicate the inhibitor, the patient received prednisone and cyclophosphamide. Complete remission was achieved after 5.5 weeks of treatment.

Purpose of Review: To summarize the recent literature related to female hemophilia A carriers with respect to prevalence in the population, the impact of baseline factor VIII levels and other influences on bleeding phenotype, and clinical management needs., Recent Findings: Many female hemophilia A carriers are at risk for abnormal bleeding, yet they are underrecognized by healthcare providers and their bleeding symptoms are underreported. Low FVIII levels are consistently associated with clinically significant bleeding and correlate well with skewed X chromosome inactivation (XCI). Most interestingly, bleeding tendency is also observed in some hemophilia A carriers with normal factor VIII levels and requires further investigation. Well controlled studies investigating peripartum and periprocedural FVIII levels and adequate hemostatic treatment are necessary to inform management guidelines., Summary: Prevalence and bleeding tendency of hemophilia A carriers remain underreported, despite a significant proportion having low FVIII levels. Skewed XCI may explain low FVIII but does not explain the bleeding risk encountered in a larger proportion of hemophilia A carriers with random XCI and borderline/normal FVIII., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on hemophilia A, in which the definition of F8 variants' causative role and association to bleeding phenotypes is crucial for proper classification, genetic counseling, and management of affected individuals. We extensively characterized a large panel of hemophilia A-causing variants (n = 30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed us to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise classification of HA-affected individuals and favored the development of personalized therapeutic approaches., Competing Interests: Declaration of interests M.P. is the inventor of a patent (PCT/IB2011/054573) on modified U1snRNAs. All other authors declare no competing interests., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Hemophilia is a monogenic mutational disease affecting coagulation factor VIII or factor IX genes. The palliative treatment of choice is based on the use of safe and effective recombinant clotting factors. Advanced therapies will be curative, ensuring stable and durable concentrations of the defective circulating factor. Results have so far been encouraging in terms of levels and times of expression using mainly adeno-associated vectors. However, these therapies are associated with immunogenicity and hepatotoxicity. Optimizing the vector serotypes and the transgene (variants) will boost clotting efficacy, thus increasing the viability of these protocols. It is essential that both physicians and patients be informed about the potential benefits and risks of the new therapies, and a register of gene therapy patients be kept with information of the efficacy and long-term adverse events associated with the treatments administered. In the context of hemophilia, gene therapy may result in (particularly indirect) cost savings and in a more equitable allocation of treatments. In the case of hemophilia A, further research is needed into how to effectively package the large factor VIII gene into the vector; and in the case of hemophilia B, the priority should be to optimize both the vector serotype, reducing its immunogenicity and hepatotoxicity, and the transgene, boosting its clotting efficacy so as to minimize the amount of vector administered and decrease the incidence of adverse events without compromising the efficacy of the protein expressed.

Objectives: Patients hospitalized because of community-acquired-pneumonia (CAP) are at risk of cardiovascular diseases. Although plasma procoagulant imbalance play a role, mechanisms are not completely understood. We aimed to investigate whether there is a measurable state of procoagulant imbalance following inflammation determined by CAP., Methods: We analyzed blood from 51 CAP patients at admission and 51 healthy subjects (HS) for (i) pro and anticoagulants, (ii) thrombin generation (TG) with or without thrombomodulin (TM), which is the physiologic activator of the protein C anticoagulant pathway and(iii) by assessing the ratio between von Willebrand-factor (VWF) and its protease ADAMTS13. Thirty patients were re-analyzed one month after discharge when CAP was resolved., Results: Median levels of TG parameters, including the endogenous thrombin potential (ETP), the ETP-TM-ratio (with/without TM), peak-thrombin and velocity index were higher in patients at baseline than HS. In particular, the median (IQR) ETP-TM-ratio in patients vs. HS was 0.88 (0.83-0.91) vs. 0.63 (0.48-0.71), p<0.001. Factor (F)VIII, a potent procoagulant involved in TG was higher in patients at baseline than HS [195 U/dL (100-388) vs. 127(108-145)], p<0.001]. The ratio of VWF/ADAMTS13 was higher at baseline than HS. Cumulatively, the findings indicate a state of pro-coagulant imbalance, which (although reduced), remained high [i.e., ETP-TM-ratio, 0.80 (0.74-0.84); FVIII, 152 U/dL (122-190)] one month after discharge when the infection was resolved., Conclusions: Patients with CAP possess a state of pro-coagulant imbalance, which remains substantially high, even when the infection is resolved. The findings suggest CAP patients as candidates for antithrombotic prophylaxis even after the resolution of infection. Clinical trials are warranted to assess the benefit/risk ratio of prophylaxis extension., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Hemophilia A is an X-linked inherited blood coagulation disorder caused by the production and circulation of defective coagulation factor VIII protein. People living with this condition receive either prophylaxis or on-demand treatment, and approximately 30% of patients develop inhibitor antibodies, a serious complication that limits treatment options. Although previous studies performed targeted mutations to identify important residues of FVIII, a detailed understanding of the role of each amino acid and their neighboring residues is still lacking. Here, we addressed this issue by creating a residue interaction network (RIN) where the nodes are the FVIII residues, and two nodes are connected if their corresponding residues are in close proximity in the FVIII protein structure. We studied the characteristics of all residues in this network and found important properties related to disease severity, interaction to other proteins and structural stability. Importantly, we found that the RIN-derived properties were in close agreement with in vitro and clinical reports, corroborating the observation that the patterns derived from this detailed map of the FVIII protein architecture accurately capture the biological properties of FVIII.

Antibody inhibitor development in hemophilia A represents the most significant complication resulting from factor VIII (fVIII) replacement therapy. Recent studies have demonstrated that epitopes present in the C1 domain contribute to a pathogenic inhibitor response. In this study, we report the structure of a group A anti-C1 domain inhibitor, termed 2A9, in complex with a B domain-deleted, bioengineered fVIII construct (ET3i). The 2A9 epitope forms direct contacts to the C1 domain at 3 different surface loops consisting of Lys2065-Trp2070, Arg2150-Tyr2156, and Lys2110-Trp2112. Additional contacts are observed between 2A9 and the A3 domain, including the Phe1743-Tyr1748 loop and the N-linked glycosylation at Asn1810. Most of the C1 domain loops in the 2A9 epitope also represent a putative interface between fVIII and von Willebrand factor. Lastly, the C2 domain in the ET3i:2A9 complex adopts a large, novel conformational change, translocating outward from the structure of fVIII by 20 Å. This study reports the first structure of an anti-C1 domain antibody inhibitor and the first fVIII:inhibitor complex with a therapeutically active fVIII construct. Further structural understanding of fVIII immunogenicity may result in the development of more effective and safe fVIII replacement therapies., (© 2021 by The American Society of Hematology.)

Objective: To assess support for a causal relationship between hemostatic measures and migraine susceptibility using genetic instrumental analysis., Methods: Two-sample Mendelian randomization instrumental analyses leveraging available genome-wide association study (GWAS) summary statistics were applied to hemostatic measures as potentially causal for migraine and its subtypes, migraine with aura (MA) and migraine without aura (MO). Twelve blood-based measures of hemostasis were examined, including plasma level or activity of 8 hemostatic factors and 2 fibrinopeptides together with 2 hemostasis clinical tests., Results: There were significant instrumental effects between increased coagulation factor VIII activity (FVIII; odds ratio [95% confidence interval] 1.05 [1.03, 1.08]/SD, p = 6.08 × 10 -05 ), von Willebrand factor level (vWF; 1.05 [1.03, 1.08]/SD, p = 2.25 × 10 -06 ), and phosphorylated fibrinopeptide A level (1.13 [1.07, 1.19]/SD, p = 5.44 × 10 -06 ) with migraine susceptibility. When extended to migraine subtypes, FVIII, vWF, and phosphorylated fibrinopeptide A showed slightly stronger effects with MA than overall migraine. Fibrinogen level was inversely linked with MA (0.76 [0.64, 0.91]/SD, p = 2.32 × 10 -03 ) but not overall migraine. None of the hemostatic factors was linked with MO. In sensitivity analysis, effects for fibrinogen and phosphorylated fibrinopeptide A were robust, whereas independent effects of FVIII and vWF could not be distinguished, and FVIII associations were potentially affected by pleiotropy at the ABO locus. Causal effects from migraine to the hemostatic measures were not supported in reverse Mendelian randomization. However, MA was not included due to lack of instruments., Conclusions: The findings support potential causality of increased FVIII, vWF, and phosphorylated fibrinopeptide A and decreased fibrinogen in migraine susceptibility, especially for MA, potentially revealing etiologic relationships between hemostasis and migraine., (© 2021 American Academy of Neurology.)

Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII, and biochemical studies have suggested APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein with an APC-resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately fivefold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, a tail clip assay was performed in homozygous HA/FV Leiden (FVL) mice infused with FVIII-QQ or FVIII-WT in the presence or absence of monoclonal antibody 1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FVL mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, indicating the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics., (© 2021 by The American Society of Hematology.)

It is well known that high von Willebrand factor (VWF) and factor VIII (FVIII) levels are associated with an increased risk of cardiovascular disease. It is still debated whether VWF and FVIII are biomarkers of endothelial dysfunction and atherosclerosis or whether they have a direct causative role. Therefore, we aimed to unravel the pathophysiological pathways of increased VWF and FVIII levels associated with cardiovascular risk factors. First, we performed a randomized controlled trial in 34 Göttingen miniswine. Diabetes mellitus (DM) was induced with streptozotocin and hypercholesterolemia (HC) via a high-fat diet in 18 swine (DM + HC), while 16 healthy swine served as controls. After 5 months of follow-up, FVIII activity (FVIII:C) was significantly higher in DM + HC swine (5.85 IU/mL [5.00-6.81]) compared with controls (4.57 [3.76-5.40], p  = 0.010), whereas VWF antigen (VWF:Ag) was similar (respectively 0.34 IU/mL [0.28-0.39] vs. 0.34 [0.31-0.38], p  = 0.644). DM + HC swine had no endothelial dysfunction or atherosclerosis during this short-term follow-up. Subsequently, we performed a long-term (15 months) longitudinal cohort study in 10 Landrace-Yorkshire swine, in five of which HC and in five combined DM + HC were induced. VWF:Ag was higher at 15 months compared with 9 months in HC (0.37 [0.32-0.42] vs. 0.27 [0.23-0.40], p  = 0.042) and DM + HC (0.33 [0.32-0.37] vs. 0.25 [0.24-0.33], p  = 0.042). Both long-term groups had endothelial dysfunction compared with controls and atherosclerosis after 15 months. In conclusion, short-term hyperglycemia and dyslipidemia increase FVIII, independent of VWF. Long-term DM and HC increase VWF via endothelial dysfunction and atherosclerosis. Therefore, VWF seems to be a biomarker for advanced cardiovascular disease., Competing Interests: F.A. received the CSL Behring-Professor Heimburger Award 2018 and a travel grant from Sobi. F.W.G.L. received research support from CSL Behring and Shire/Takeda for performing the WiN study; is a consultant for uniQure, Novo Nordisk, and Shire/Takeda, the fees of which go to the institution; and has received a travel grant from Sobi. He is also a DSMB member for a study by Roche., (Thieme. All rights reserved.)

Chronic graft-versus-host disease (cGvHD) is an immune mediated late complication of allogeneic hematopoietic stem cell transplantation (alloHSCT). Discovery of adequate biomarkers could identify high-risk patients and provide an effective pre-emptive intervention or early modification of therapeutic strategy, thus reducing prevalence and severity of the disease among long-term survivors of alloHSCT. Inflammation, endothelial injury, and endothelial dysfunction are involved in cGvHD development. Altered levels of acute phase reactants have shown a strong correlation with the activity of several immune mediated disorders and are routinely used in clinical practice. Since elevated von Willebrand factor (VWF) and factor VIII (FVIII) levels have been described as acute phase reactants that may indicate endothelial dysfunction and inflammation in different settings, including chronic autoimmune diseases, they could serve as potential candidate biomarkers of cGvHD. In this review we focused on reported data regarding VWF and FVIII as well as other markers of inflammation and endothelial dysfunction, evaluating their potential role in cGvHD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lelas, Greinix, Wolff, Eissner, Pavletic and Pulanic.)

Genetic risk score (GRS) analysis is a popular approach to derive individual risk prediction models for complex diseases. In venous thrombosis (VT), such type of analysis shall integrate information at the ABO blood group locus, which is one of the major susceptibility loci. However, there is no consensus about which single nucleotide polymorphisms (SNPs) must be investigated when properly assessing association between ABO locus and VT risk. Using comprehensive haplotype analyses of ABO blood group tagging SNPs in 5425 cases and 8445 controls from 6 studies, we demonstrate that using only rs8176719 (tagging O1) to correctly assess the impact of ABO locus on VT risk is suboptimal, because 5% of rs8176719-delG carriers do not have an increased risk of developing VT. Instead, we recommend the use of 4 SNPs, rs2519093 (tagging A1), rs1053878 (A2), rs8176743 (B), and rs41302905 (O2), when assessing the impact of ABO locus on VT risk to avoid any risk misestimation. Compared with the O1 haplotype, the A2 haplotype is associated with a modest increase in VT risk (odds ratio, ∼1.2), the A1 and B haplotypes are associated with an ∼1.8-fold increased risk, whereas the O2 haplotype tends to be slightly protective (odds ratio, ∼0.80). In addition, although the A1 and B blood groups are associated with increased von Willebrand factor and factor VIII plasma levels, only the A1 blood group is associated with ICAM levels, but in an opposite direction, leaving additional avenues to be explored to fully understand the spectrum of biological effects mediated by ABO locus on cardiovascular traits.

Introduction: The pneumatic tube system (PTS) is widely used for sample delivery. We aimed to investigate the impacts of PTS on hemostasis assays., Methods: Triplicate samples from 30 healthy volunteers were delivered to the core laboratory manually by human courier or via the 500 m long-distance PTS or via the 1000 m long-distance PTS. Comparisons of 19 hemostasis tests were conducted., Results: Although PT, INR, APTT, FII, FV, FVII FIX, FX, FXII, DD, α2-PI, and PC had statistical significance (all P < .05), all had low average bias remaining within clinical acceptable limits. PTS transportation only resulted in a statistically significant and clinically relevant decrease in FVIII activity. In the 500 m-PTS group, 66.7% (20/30) of samples for FVIII testing had a bias greater than 8.3%. Moreover, in the 1000 m-PTS group, 96.7% (29/30) of samples had a bias of over 8.3%, and the maximal bias achieved 42.1%., Conclusions: Pneumatic tube system in our institution could be used to deliver blood samples for hemostasis tests evaluated in this study except FVIII activity assay., (© 2020 John Wiley & Sons Ltd.)

Myocardial infarction is a life-threatening emergency with a high mortality rate. A high plasma level of factor VIII is an established risk for both arterial and venous thrombotic events. In this mini-review, we report the case of a 41-year-old woman without cardiovascular risk factors or a previous history of thrombotic events, admitted for ST-elevation myocardial infarction, in whom coronary angiography showed a thrombotic occlusion in the left anterior descending artery. The patient underwent primary percutaneous coronary intervention (PCI), with GPIIB-IIIA antagonist, then, a pre-dilation with a semi-compliant balloon-catheter, followed by implantation of 2 stents. The etiological assessment revealed a high level of coagulation factor VIII (FVIII). She underwent anticoagulation therapy (with acenocoumarol) with well-controlled international normalised ratio (INR)., Competing Interests: The authors declare no competing interests., (Copyright: Falmata Laouan Brem et al.)

Introduction: Factor VIII activity (FVIII:C) is measured by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Significant differences in FVIII:C between OSA (FVIII:C 1st ) and CSA (FVIII:C Chr ) are described as assay discrepancy in nonsevere haemophilia A (HA). A large number of reagent combinations (APTT reagent and FVIII-deficient plasma) are used for OSA, but the impact of variations in reagent combinations on assay discrepancy has not been fully characterized., Aim: To clarify the variations in FVIII:C 1st /FVIII:C Chr ratios according to OSA reagent combination in HA subjects with/without assay discrepancy., Methods: Thirty-nine patients previously diagnosed with nonsevere HA were enrolled, and their FVIII genes were investigated and FVIII:C levels were assessed by a single CSA reagent and 11 OSA reagent combinations. Receiver operating characteristic (ROC) curve analysis was used to predict possible cut-off values of the FVIII:C 1st /FVIII:C Chr ratio to define FVIII assay discrepancy for each reagent combination., Results: Patients were categorized into nondiscrepant (n = 25), discrepant (n = 5) and unclassified (n = 9) groups according to their genotypes and information in the database. The FVIII:C 1st /FVIII:C Chr ratio in nondiscrepant HA varied widely, depending on the APTT reagents and FVIII-deficient plasma used. The ratio in discrepant HA patients differed with respect to their genotype and the reagent combination used. ROC curve analyses revealed that cut-off values to distinguish the assay discrepancy differed depending on the reagents used, but revealed two novel genotype variants, p.Cys573Gly and p.Gly582Arg, associated with FVIII assay discrepancy., Conclusion: Our findings showed that the FVIII:C 1st /FVIII:C Chr ratio is dependent on the reagent combination used for OSA., (© 2020 John Wiley & Sons Ltd.)

Introduction: Activated partial thromboplastin time (aPTT) and antifactor Xa (anti-Xa) activity are used to monitor unfractionated heparin therapy in children on extracorporeal membrane oxygenation (ECMO). Elevated C-reactive protein (CRP) can prolong aPTT and cause discrepancy between these two assays. We aimed to evaluate CRP effect on aPTT and anti-Xa assays in the presence of heparin and to determine whether elevated CRP affects laboratory monitoring in pediatric ECMO patients., Materials and Methods: Citrated normal specimens were spiked with CRP, heparin, and recombinant factor VIII (FVIII) and followed by measurement of aPTT and anti-Xa activity. Additionally, aPTT, anti-Xa activity, FVIII, fibrinogen, and CRP were measured in 18 ECMO specimens., Results: Elevated CRP prolonged aPTT in normal specimens with or without heparin, but did not affect anti-Xa assay. In contrast, ECMO specimens showed similar aPTT and anti-Xa values regardless of CRP level. Elevated CRP in specimens was accompanied by increased fibrinogen and FVIII activity. Additional in vitro experiments confirmed that FVIII spiked simultaneously with CRP attenuated CRP-induced aPTT prolongation in heparinized specimens., Conclusion: In vitro CRP-induced aPTT prolongation is not observed in pediatric ECMO samples due to concomitant FVIII increase. Discordant changes of CRP and FVIII in plasma could contribute to aPTT/anti-Xa discrepancies observed during heparin therapy in the pediatric population. The anti-Xa assay is preferable for heparin monitoring in pediatric ECMO settings., (© 2020 John Wiley & Sons Ltd.)

Acquired hemophilia A (AHA) is a severe bleeding disorder caused by inhibiting autoantibodies to coagulation factor VIII (FVIII). For hemostatic treatment, bypassing agents and human or porcine FVIII are currently standard of care. Emicizumab is a bispecific, FVIII-mimetic therapeutic antibody that reduced the annualized bleeding rates in congenital hemophiliacs. Here, we report on 6 male and 6 female patients with AHA treated with emicizumab (all data medians and interquartile range), age 74 (64-80) years, initial FVIII <1%; inhibitor titer 22.3 Bethesda units (BU)/mL (range, 3-2000). Eight patients had severe bleeding. Emicizumab was started, 3 mg/kg subcutaneously, weekly for 2 to 3 doses, followed by 1.5 mg/kg every 3 weeks to keep the lowest effective FVIII levels. For FVIII monitoring, chromogenic assays with human and bovine reagents were used. All patients received immunosuppression with steroids and/or rituximab. After the first dose of emicizumab, activated partial thromboplastin time normalized in 1 to 3 days, FVIII (human reagents) exceeded 10% after 11 (7.5-12) days. Hemostatic efficacy was obtained and bypassing therapy stopped after 1.5 (1-4) days. FVIII (bovine reagents) exceeded 50%, indicating complete remission after 115 (67-185) days, and emicizumab was stopped after 31 (15-79) days. A median of 5 injections (range, 3-9) were given. No patient died of bleeding or thromboembolism, and no breakthrough bleeding was observed after the first dose of emicizumab. In conclusion, emicizumab seems to be an effective hemostatic therapy for AHA, with the advantages of subcutaneous therapy, good hemostatic efficacy, early discharge, and reduction of immunosuppression and adverse events., (© 2021 by The American Society of Hematology.)

Bleeding frequency and severity within clinical categories of hemophilia A are highly variable and the origin of this variation is unknown. Solving this mystery in coagulation requires the generation and analysis of large data sets comprised of experimental outputs or patient samples, both of which are subject to limited availability. In this review, we describe how a computationally driven approach bypasses such limitations by generating large synthetic patient data sets. These data sets were created with a mechanistic mathematical model, by varying the model inputs, clotting factor, and inhibitor concentrations, within normal physiological ranges. Specific mathematical metrics were chosen from the model output, used as a surrogate measure for bleeding severity, and statistically analyzed for further exploration and hypothesis generation. We highlight results from our recent study that employed this computationally driven approach to identify FV (factor V) as a key modifier of thrombin generation in mild to moderate hemophilia A, which was confirmed with complementary experimental assays. The mathematical model was used further to propose a potential mechanism for these observations whereby thrombin generation is rescued in FVIII-deficient plasma due to reduced substrate competition between FV and FVIII for FXa (activated factor X).

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.

Von Willebrand factor (VWF) is a plasma glycoprotein that circulates noncovalently bound to blood coagulation factor VIII (fVIII). VWF is a population of multimers composed of a variable number of ∼280 kDa monomers that is activated in shear flow to bind collagen and platelet glycoprotein Ibα. Electron microscopy, atomic force microscopy, small-angle neutron scattering, and theoretical studies have produced a model in which the conformation of VWF under static conditions is a compact, globular "ball-of-yarn," implying strong, attractive forces between monomers. We performed sedimentation velocity (SV) analytical ultracentrifugation measurements on unfractionated VWF/fVIII complexes. There was a 20% per mg/ml decrease in the weight-average sedimentation coefficient, s w , in contrast to the ∼1% per mg/ml decrease observed for compact globular proteins. SV and dynamic light scattering measurements were performed on VWF/fVIII complexes fractionated by size-exclusion chromatography to obtain s w values and z-average diffusion coefficients, D z . Molecular weights estimated using these values in the Svedberg equation ranged from 1.7 to 4.1 MDa. Frictional ratios calculated from D z and molecular weights ranged from 2.9 to 3.4, in contrast to values of 1.1-1.3 observed for globular proteins. The Mark-Houwink-Kuhn-Sakurada scaling relationships between s w , D z and molecular weight, [Formula: see text] and [Formula: see text] , yielded estimates of 0.51 and -0.49 for a s and a D , respectively, consistent with a random coil, in contrast to the a s value of 0.65 observed for globular proteins. These results indicate that interactions between monomers are weak or nonexistent and that activation of VWF is intramonomeric., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Acquired hemophilia A (AHA) is a severe auto-immune bleeding disorder. Treatment of AHA is burdensome and optimal management is still unresolved. Therefore a retrospective nationwide multi-center cohort study (1992-2018) was performed to evaluate clinical presentation and treatment efficacy and safety of AHA in the Netherlands. Multivariate logistic and Cox regression analysis was used to study independent associations between patient characteristics and clinical outcomes. A total of 143 patients (median age 73 years; 52.4% male) were included with a median follow-up of 16.8 months (IQR 3.6-41.5 months). First-line immunosuppressive treatment was mostly steroid monotherapy (67.6%), steroids/cyclophosphamide (11.9%) and steroids/rituximab (11.9%), with success rates of 35.2%, 80.0% and 66.7% respectively, P < .05. Eventually 75% of patients achieved complete remission (CR). A high anti-FVIII antibody titer, severe bleeding and steroid monotherapy were associated with lower CR rates. Infections, the most important adverse event, occurred significantly more often with steroid combination therapy compared to steroids alone (38.7% vs 10.6%; P = .001). Overall mortality was 38.2%, mostly due to infections (19.2%) compared to 7.7% fatal bleeds. Advanced age, underlying malignancy and ICU admission were predictors for mortality. This study showed that AHA is characterized by significant disease-related and treatment-related morbidity and mortality. A high anti-FVIII titer, severe bleeding and steroid monotherapy were associated with a lower CR rate. The efficacy of steroid combination therapies however, was overshadowed by higher infection rates and infections represented the most important cause of death. The challenging and delicate balance between treatment effectivity and safety requires ongoing monitoring of AHA and further identification of prognostic markers., (© 2020 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Chronic liver disease is one of the leading causes of death in the United States. Coagulopathy is often a sequela of chronic liver disease, however, the role and regulation of coagulation components in chronic liver injury remain poorly understood. Clinical and experimental evidence indicate that misexpression of the procoagulant factor VIII (FVIII) is associated with chronic liver disease. Nevertheless, the molecular mechanism of FVIII-induced chronic liver injury progression remains unknown. This review provides evidence supporting a pathologic role for FVIII in the development of chronic liver disease using both experimental and clinical models., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The FVIII activity in patients treated with several extended half-life FVIII (EHL-FVIII) agents different when various activated partial thromboplastin time (APTT) reagents were used. The present study examined the difference in clot waveform analysis (CWA) findings and FVIII activity when various APTT reagents and CWA were used. The CWA including FVIII activity was measured using 12 APTT reagents, and the FIX activation based on a small amount of tissue factor assay (sTF/FIX) were examined in reference plasma (RP), EHL-FVIII (Jivi ® ) and Kovaltry ® . The 3 APTT reagents were associated with high variation in the peak time and height in the CWA when analyzing low concentrations of FVIII. The peak time and height could not be measured with one APTT reagent, and there were marked differences in the CWA findings between Jivi ® and Kovaltry ® among APTT reagents. Several APTT reagents showed a markedly lower FVIII activity with Jivi ® than with Kovaltry ® . In the FVIII assay, the peak time measured with sTF/FIX did not differ markedly between Jivi ® and Kovaltry ® ; however, the FVIII activity in Jivi ® (as measured by the peak height) tended to be higher than in Kovaltry ® . The CWA findings for monitoring Jivi ® varied for monitoring Jivi ® depending on the APTT reagents used, and sTF/FIX assay may be able to measure the EHL-FVIII.