Factor XI promotes hemostasis in factor IX‐deficient mice

Essentials

• Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model.
• FIX‐deficient mice displayed a hemostatic defect and FXI‐deficient mice were similar to wild type mice.
• Infusion of FXI or over‐expression of FXI in FIX‐deficient mice improved hemostasis.
• FXI may affect the phenotype of FIX‐deficiency (hemophilia B).

Summary

Background

In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI‐deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI‐deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model.

Objectives

To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B).

Methods

Wild‐type mice and mice lacking either FIX (F9^?) or FXI (F11^?/?) were tested in the SVB model. The plasma levels of FXI in F11^?/? mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection.

Results

F9^? mice showed a significant defect in the SVB model, whereas F11^?/? mice and wild‐type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9^? mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX‐binding site also improved hemostasis in F9^? mice.

Conclusions

Although we were unable to demonstrate a hemostatic defect in F11^?/? mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9^? mice through FIX‐independent pathways.

     

Recombinant factor XIII A‐subunit in a patient with factor XIII deficiency and recurrent pregnancy loss

Essentials

• Inherited factor XIII deficiency is a very rare bleeding disorder.
• We used recombinant factor XIII‐A in a pregnant patient with factor XIII‐A subunit deficiency.
• The patient had a successful pregnancy outcome with no pregnancy related complications.
• The dose of recombinant factor XIII‐A was minimized by using frequent trough level monitoring.

Summary

Inherited factor XIII deficiency is a very rare bleeding disorder, and is one of the causes of recurrent pregnancy loss. The use of plasma‐derived FXIII to improve pregnancy outcomes has been reported. We report a 26‐year‐old woman with FXIII A‐subunit (FXIII‐A) deficiency who was treated with recombinant FXIII‐A and had a successful pregnancy outcome with no pregnancy‐related complications. Our case illustrates that the dose of recombinant FXIII‐A can be minimized and adjusted on the basis of frequent trough level monitoring.

     

Immobilized transition metal ions stimulate contact activation and drive factor XII‐mediated coagulation

Summary. Background: Upon contact with an appropriate surface, factor XII (FXII) undergoes autoactivation or cleavage by kallikrein. Zn²⁺ is known to facilitate binding of FXII and the cofactor, high molecular weight kininogen (HK), to anionic surfaces. Objectives: To investigate whether transition metal ions immobilized on liposome surfaces can initiate coagulation via the contact pathway. Methods and results: Liposomes containing a metal ion‐chelating lipid, 1,2‐dioleoyl‐sn‐glycero‐3‐{(N[5‐amino‐1‐carboxypentyl]iminodiacetic acid)succinyl} ammonium salt (DOGS‐NTA), were prepared by membrane extrusion (20% DOGS‐NTA, 40% phosphatidylcholine, 10% phosphatidylserine, and 30% phosphatidylethanolamine). Ni²⁺ immobilized on such liposomes accelerated clotting in normal plasma, but not factor XI (FXI)‐deficient or FXII‐deficient plasma. The results were similar to those obtained with a commercial activated partial thromboplastin time reagent. Charging such liposomes with other transition metal ions revealed differences in their procoagulant capacity, with Ni²⁺ > Cu²⁺ > Co²⁺ and Zn²⁺. Plasma could be depleted of FXI, FXII and HK by adsorption with Ni²⁺‐containing beads, resulting in longer clot times. Consistent with this, FXI, FXII and HK bound to immobilized Ni²⁺ or Cu²⁺ with high affinity as determined by surface plasmon resonance. In the presence of Ni²⁺‐bearing liposomes, K_m and k_cat values derived for autoactivation of FXII and prekallikrein, as well as for activation of FXII by kallikrein or prekallikrein by FXIIa, were similar to literature values obtained in the presence of dextran sulfate. Conclusions: Immobilized Ni²⁺ and Cu²⁺ bind FXII, FXI and HK with high affinity and stimulate activation of the contact pathway, driving FXII‐mediated coagulation. Activation of the contact system by immobilized transition metal ions may have implications during pathogenic infection or in individuals exposed to high levels of pollution.     

Characterization of duplication breakpoints in the factor VIII gene

See also Goodeve AC. Another step towards understanding hemophilia A molecular pathogenesis. This issue, pp 2693–5. Summary. Background: Hemophilia A is caused by a wide spectrum of different mutations in the factor (F)VIII gene (F8), leading to deficiencies in coagulation FVIII activity and thus resulting in an inefficient blood clotting cascade. Large duplications comprising whole exons of F8 have been published for only a few cases so far. Results: In the current study, we characterized the exact breakpoints for a total of 10 exon‐spanning duplications of F8, including six novel duplications in seven unrelated patients. Seven breakpoints were located within long interspersed nuclear elements (LINEs), whereas short interspersed nuclear elements (SINEs) of the Alu‐repeat type were observed at both breakpoint sites in four of the 10 duplications. At three breakpoints, microhomologies of 2 bp and 3 bp each could be identified. Conclusions: Duplication breakpoints in F8 were shown to be located in repetitive elements, especially SINEs or LINEs, but also in unique sequences. In addition, microhomologies, particular genomic features or sequence motifs, contribute to the duplication formation mechanisms.     

Prothrombin complex concentrate and recombinant prothrombin alone or in combination with recombinant factor X and FVIIa in dilutional coagulopathy: a porcine model

Summary. Background: This study was conducted to assess whether newly developed recombinant clotting factor concentrates enable the reversal of dilutional coagulopathy. Methods: In 50 anesthetized pigs, ～ 60% of the blood volume was withdrawn and replaced with hydroxyethyl starch. Pigs were randomized to receive either 200 mg kg^?1 fibrinogen (n = 10), fibrinogen and 35 IU kg^?1 prothrombin complex concentrate (PCC) (n = 10), fibrinogen and 4 mg kg^?1 recombinant human factor II (rhFII) concentrate (n = 10), fibrinogen and a three‐factor combination (3F) of 4 mg kg^?1 rhFII, 0.006 mg kg^?1 recombinant human FVIIa and 0.32 mg kg^?1 recombinant human FX (n = 10), or saline (n = 10). Thereafter, a standardized liver laceration was performed to induce uncontrolled hemorrhage. Survival time and blood loss were determined, and standard coagulation tests and thrombelastometry were performed. Results: Fibrinogen combined with rhFII or PCC improved survival. Blood loss was significantly decreased in all groups as compared with the animals receiving saline. Clotting time was significantly shortened in the animals treated with fibrinogen and PCC, as well as in those treated with fibrinogen and 3F. One animal died after administration of fibrinogen and PCC. Conclusion: Following hemodilution, a combination of fibrinogen and PCC, rhFII or 3F enhances coagulation and final clot strength. Mortality was reduced statistically significantly only in the animals treated with fibrinogen and rhFII or PCC, whereas administration of the combination of fibrinogen and PCC caused a fatal thromboembolic complication. The combination of fibrinogen and rhFII might be effective in reversing dilutional coagulopathy and may reduce blood loss in cases of dilutional coagulopathy.     

Bortezomib delays the onset of factor VIII inhibitors in experimental hemophilia A,but fails to eliminate established anti‐factor VIII IgG‐producing cells

Summary. Background: Replacement therapy with exogenous factor VIII to treat hemorrhages induces inhibitory anti‐FVIII antibodies in up to 30% of patients with hemophilia A. Current approaches to eradicate FVIII inhibitors using high‐dose FVIII injection protocols (immune tolerance induction) or anti‐CD20 depleting antibodies (Rituximab) demonstrate limited efficacy; they are extremely expensive and/or require stringent compliance from the patients. Objectives: To investigate whether the proteasome inhibitor bortezomib, which depletes plasmocytes, modulates the anti‐FVIII immune response in FVIII‐deficient mice. Methods and results: Preventive 4‐week treatment of naïve mice with bortezomib at the time of FVIII administration delayed the development of inhibitory anti‐FVIII IgG, and depleted plasma cells as well as different lymphoid cell subsets. Conversely, curative treatment of inhibitor‐positive mice for 10 weeks, along with FVIII administration, failed to eradicate FVIII inhibitors to extents that would be clinically relevant if achieved in patients. Accordingly, bortezomib did not eradicate anti‐FVIII IgG‐secreting plasmocytes that had homed to survival niches in the bone marrow, despite significant elimination of total plasma cells. Conclusions: The data suggest that strategies for the efficient reduction of anti‐FVIII IgG titers in patients with hemophilia A should rely on competition for survival niches for plasmocytes in the bone marrow rather than the mere use of proteasome inhibitors.     

Dangerous liaisons: how the immune system deals with factor VIII

Summary. Only a fraction of patients with hemophilia A develop a neutralizing antibody (inhibitor) response to therapeutic infusions of factor VIII. Our present understanding of the underlying causes of the immunogenicity of this protein is limited. In the past few years, insights into the uptake and processing of FVIII by antigen‐presenting cells (APCs) have expanded significantly. Although the mechanism of endocytosis remains unclear, current data indicate that FVIII enters APCs via its C1 domain. Its subsequent processing within endolysosomes allows for presentation of a heterogeneous collection of FVIII‐derived peptides on major histocompatibility complex (MHC) class II, and this peptide–MHC class II complex may then be recognized by cognate effector CD4⁺ T cells, leading to anti‐FVIII antibody production. Here we aim to summarize recent knowledge gained about FVIII processing and presentation by APCs, as well as the diversity of the FVIII‐specific T‐cell repertoire in mice and humans. Moreover, we discuss possible factors that can drive FVIII immunogenicity. We believe that increasing understanding of the immune recognition of FVIII and the cellular mechanisms of anti‐FVIII antibody production will lead to novel therapeutic approaches to prevent inhibitor formation in patients with hemophilia A.     

Clinical guidelines and off‐license recombinant activated factor VII: content,use, and association with patient outcomes

Summary. Background: Recombinant activated factor VII (rFVIIa) is increasingly being used off‐license for treating critical bleeding. Guidelines may therefore be useful for improving processes and outcomes. Little is known regarding guidelines for off‐license rFVIIa or their association with patient outcomes. Objectives: To investigate the availability of hospital guidelines for off‐license rFVIIa use and the association between these guidelines and mortality. Methods: Data were extracted from the Haemostasis Registry, which collects all cases of off‐license rFVIIa use in participating institutions in Australia and New Zealand. Contributing hospitals were requested to supply a copy of the institutional guideline relating to off‐license rFVIIa administration. The characteristics of patients treated in accordance with all elements of the guidelines were compared with those of patients for who one or more guideline elements had been violated. The relationship between guideline‐directed treatment and 28‐day mortality was investigated using stepwise logistic regression. Results: Two thousand five hundred and fifty‐one patients in 75 hospitals were available for analysis. Of these hospitals, 58 provided a guideline for analysis. Patients complying with all guideline elements (n = 530) did not differ from patients receiving care that violated guidelines (n = 1035) regarding age, size of dose, or gender. Guideline‐directed treatment was not found to have an association with 28‐day mortality following logistic regression. Conclusions: Few patients are treated in accordance with the criteria of rFVIIa guidelines, despite their availability in the majority of hospitals. Moreover, 28‐day mortality does not appear to be associated with the use of guidelines in this patient group. Refinement of guidelines relating to the off‐license use of rFVIIa is therefore required.     

A phase 1 ascending dose study of a subcutaneously administered factor IXa inhibitor and its active control agent

Summary. Background: The REG2 anticoagulation system consists of pegnivacogin, a subcutaneously administered aptamer factor IXa inhibitor, and its intravenous control agent, anivamersen. Objectives: To assess the safety, tolerability and pharmacokinetic and pharmacodynamic responses of REG2. Patients/Methods: In this phase 1a study, 36 healthy volunteers were enrolled into five cohorts and given one dose of pegnivacogin. Cohorts 1 (n = 6) and 1A (n = 4) received 0.5 mg kg^?1; cohort 2 (n = 6) received 1.0 mg kg^?1; cohort 3 (n = 6) received 3.0 mg kg^?1; and cohort 4 (n = 8) received 2.0 mg kg^?1. In cohorts 1–3, two subjects were randomized to placebo. Cohort 4 subjects were subsequently randomized to single‐dose (n = 4) or multidose (n = 4) anivamersen. Results: The mean maximum observed concentrations of pegnivacogin in cohorts 1, 1A, 2 and 3 at median time were 5.16 μg mL^?1 at 84 h, 5.19 μg mL^?1 at 72 h, 9.32 μg mL^?1 at 90 h, and 32.5 μg mL^?1 at 84 h, respectively. The maximum relative activated partial thromboplastin time and time needed to achieve this were 1.18 at 2 days, 1.16 at 2 days, 1.27 at 3 days, and 1.85 at 2 days, respectively. The calculated mean half‐life and mean residence times of pegnivacogin were 6.12 days and 9.6 days, respectively. There was rapid reversal with intravenous anivamersen, although subsequent reaccumulation of pegnivacogin was observed. Conclusions: In our first‐in‐human study, REG2 was well tolerated and provided dose‐proportional anticoagulation for several days after a single subcutaneous dose, with complete, although transient, reversal by its control agent. This study demonstrates the first application of a subcutaneously administered aptamer, and represents a potential advance in aptamer therapeutics.     

Tissue‐regenerating functions of coagulation factor XIII

The protransglutaminase factor XIII (FXIII) has recently attracted attention within the field of tissue regeneration, as it has been found that FXIII significantly influences wound healing by exerting a multitude of functions. It supports hemostasis by enhancing platelet adhesion to damaged endothelium, and by its cross‐linking activity it stabilizes the formed fibrin clot. Furthermore, FXIII limits bacterial dissemination from the wound and incorporates macromolecules of importance for cellular infiltration, supporting cell migration and survival. FXIII‐mediated complex formation of the vascular endothelial growth factor receptor 2 and α_Vβ₃ integrin is important for angiogenesis, supporting the formation of granulation tissue. Chronic inflammatory conditions involving bleeding and activation of the coagulation cascade have been shown to lead to reduced FXIII levels in plasma. Of particular importance for this review is the fact that patients suffering from inflammatory bowel disease (IBD) have reduced FXIII antigen levels and activity. Furthermore, these patients show impaired mucosal healing, which supports the inflammatory state of the disease. This review summarizes the role of FXIII in the healing of wounds, and briefly summarizes the previous use of FXIII in clinical settings. Moreover, it addresses the potential role for FXIII as a therapeutic agent in the healing of persistent wounds during chronic conditions, with an emphasis on IBD.