SummaryThe eradication of inhibitory antibodies in patients with haemophilia A can be accomplished by frequent administration of high or intermediate doses of factor VIII (FVIII), so-called immune tolerance induction (ITI). This study monitored the distribution of IgG subclasses of anti-FVIII antibodies during ITI. FVIII-specific antibodies of subclass IgG1 were detected in all inhibitor patients tested, anti-FVIII IgG4 in 16, IgG2 in 10 and IgG3 in one of 20 patients analysed. Levels of anti-FVIII IgG1 and IgG4 correlated well with inhibitor titres as measured by Bethesda assay. In low-titre inhibitor patients, anti-FVIII antibodies consisted primarily of subclass IgG1 whereas, anti-FVIII antibodies of subclass IgG4 were more prominent in patients with high titre inhibitors who needed prolonged treatment or who failed ITI. Longitudinal analysis of 14 patients undergoing ITI revealed that the relative contribution of IgG subclasses was constant for most of the patients analysed. In two patients, the relative contribution of IgG4 increased during ITI. Overall, our findings document the distribution and dynamics of anti-FVIII IgG subclasses during ITI. Future studies will need to address whether monitoring the relative contribution of anti-FVIII subclasses IgG1 and IgG4 may be useful for the identification of patients who are at risk of failing ITI.

Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (V H ) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the V H 1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from V H gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10-, DP-14-, and DP-88-encoded antibodies. In contrast, binding of the DP-5-encoded antibodies required residues in both the amino-and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5-encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from V H gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VIII. IntroductionHemophilia A is an X-linked bleeding disorder that is characterized by the absence or dysfunction of blood coagulation factor VIII. Current treatment of hemophilia A consists of infusion of therapeutic amounts of factor VIII that can evoke an immune response in some patients. The presence of neutralizing antibodies to factor VIII, commonly termed factor VIII inhibitors, presents a serious complication of hemophilia care. 1 The biochemical properties of factor VIII inhibitors have been extensively studied with emphasis on the epitope specificity and mode of action of these antibodies. Binding sites for inhibitors have been identified within the A2, A3, and C2 domains of factor VIII. [2][3][4][5][6] In general, heterogeneous mixtures of anti-factor VIII antibodies are present in plasma from patients with factor VIII inhibitors, and in more than 80% of inhibitor plasmas antibodies directed against the C2 domain are present. 7 Epitope mapping of anti-C2 domain antibodies revealed the presence of an inhibitor binding site comprising amino acid residues Val2248-Ser2312 of the C2 domain. 3 A second inhibitor epitope in the C2 domain has been attributed to region Glu2181-Val2243. 6 Antibodies reactive with the C2 domain prevent factor VIII from binding to phospholipid surfaces and von Willebrand factor. 8,9 Both findings are in agreement with the presence of binding sites for phospholipids and von Willebrand factor in the C2 domain. 10,11 An add...

Background: It is unclear how the LDL receptor family binds large protein ligands. Results: HDX and lysine scanning identified factor (F)VIII regions and specific lysine residues binding low-density lipoprotein receptor-related protein 1 (LRP1). Conclusion: FVIII-LRP1 interaction involves multiple "hot-spot" lysine residues in the A3C1 domains. Significance: Our study sheds light on interactions of complex ligands with the LDL receptor family.

BACKGROUND: Uncontrollable bleeding is the leading cause of death in traumatically injured patients. The extent to which direct factor Xa inhibitors interfere with the applied resuscitation measures is presently unknown. STUDY DESIGN AND METHODS:In this study, we investigated the effect of the resuscitation fluids saline, albumin, fresh frozen plasma (FFP) and solvent/ detergent (S/D)-treated plasma, fibrinogen concentrate, prothrombin complex concentrate (PCC), and combinations thereof on the hemostatic profile of rivaroxaban-anticoagulated whole blood and plasma. We used rivaroxaban-spiked whole blood and plasma from healthy donors, as well as plasma from patients on rivaroxaban, and mimicked a resuscitation approach in a 50% plasma dilution setting. Thromboelastography, thrombin generation, and fibrin generation clot lysis test were assessed using tissue factor to initiate coagulation and tissue plasminogen activator to induce clot lysis.ABBREVIATIONS: CAT = calibrated automated thrombography; CLT = clot lysis time; FGCLT = fibrin generation clot lysis test; FXa-I = factor Xa inhibitor; MA = maximal amplitude; PCC = prothrombin complex concentrate; TEG = thromboelastography; TF = tissue factor; tPA = tissue plasminogen activator; TXA = tranexamic acid.From the

Background In the initial absence of specific reversal agents for factor Xa inhibitors (FXa‐Is), prothrombin complex concentrate (PCC) as a hemostatic agent has been recommended by guidelines. Since 2017, idarucizumab has been registered for dabigatran reversal. Still, data on the clinical outcome of direct oral anticoagulant (DOAC)‐related emergencies (major bleeding or urgent interventions) is scarce. In addition, it is unknown to what extent PCC restores thrombin generation in FXa‐I–related emergencies. Our aim was to describe management and clinical outcomes of DOAC‐related emergencies and to assess the laboratory effect of PCC in patients with FXa‐I emergencies. Methods In this prospective cohort study in 5 Dutch hospitals, patients presenting with DOAC‐related emergencies were eligible. The primary outcome was effective hemostasis according to the ISTH definition. Safety outcomes were 30‐day mortality and thromboembolic rate. In patients treated with PCC, additional blood samples were taken to assess the effect on thrombin generation. Results We included 101 patients with major bleeding (FXa‐I, 76; dabigatran, 25) and 21 patients requiring an urgent intervention (FXa‐I, 16; dabigatran, 5). Of patients with major bleeding, 67% were treated with PCC or idarucizumab. Effective hemostasis, 30‐day mortality, and thromboembolism rate were 67%, 22%, and 1%, respectively. In a subset of bleeding patients on FXa‐I managed with PCC, thrombin generation increased, with 96% immediately after PCC administration. In patients requiring an urgent intervention, effective hemostasis, 30‐day mortality, and thromboembolic rate were 95%, 14%, and 5%. Conclusions Effective hemostasis was achieved in the majority of patients presenting with DOAC‐related emergencies;, thromboembolic complications were rare, and mortality was quite high.