Prevention or treatment of bleeding episodes in patients with hemophilia A 

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Life cycle of FVIII, catabolic receptors, in vivo distribution.

Once released in the circulation, FVIII rapidly associates with VWF (Vlot et al., 1995; Dimitrov et al., 2012). The VWF-FVIII interaction increases FVIII half-life from about few minutes, as seen in patients with von Willebrand disease who lack FVIII-binding VWF, to 10- 12 hours. VWF stabilizes the heterodimeric structure of FVIII, protects FVIII from proteolytic degradation, avoids binding to phospholipids and association to FIXa and prevents the cellular uptake of FVIII (Lenting et al., 2007). After having played its role within the tenase complex, FVIII is inactivated through two distinct mechanisms: proteolytic degradation or spontaneous dissociation of the A2 domain. The proteolytic degradation of activated FVIII is mediated by activated FIX, activated FX and activated protein C (APC) and involves a cleavage in the acidic region a1 of the heavy chain at position 336 (Koedam et al., 1988; Rick et al., 1990). This proteolytic cleavage increases the dissociation rate of the A2 domain, thus impairing FVIII ability to bind and activate FX (Regan et al., 1996). APC also cleaves the A2 domain at position 562 thus disrupting the binding site of FVIII for FIXa (Fay et al., 1991). The spontaneous dissociation of FVIII is believe to be the consequence of the low affinity of the A2 domain for the metal ion-linked A1/A3-C1-C2 dimer (Persson et al., 1995).
Clearance studies have revealed that after injection to hemophilia A patients, the removal of FVIII from the circulation follows a biphasic model characterized by a fast phase and a slow phase (Saenko et al., 1999). Several catabolic receptors have been identified that mediate FVIII clearance.

Catabolic receptors for FVIII

The catabolism of FVIII implicates its uptake by specific receptors expressed on immune or non-immune cells. One of the first receptor identified as a catabolic receptor for FVIII uptake was the low density lipoprotein receptor-related protein (LRP, CD91) (Saenko et al., 1999). LRP is a major endocytic receptor which is expressed in the liver but is also present in the placenta, the brain and the lungs (Strickland et al., 1995). In vitro and in vivo studies have confirmed that LRP binds FVIII through the terminal part of the C2 domain in the light chain (Lenting et al., 1999) and the A2 domain of the heavy chain (Saenko et al., 1999) and leads to FVIII endocytosis and degradation. These observations have been confirmed by the use of a specific LRP inhibitor, the receptor-associated protein (RAP). Thus, the treatment of cells expressing LRP with RAP inhibited the uptake and the subsequent degradation of FVIII in vitro. Subsequently, the intravenous administration of RAP into mice completely inhibited the fast phase of FVIII clearance (Saenko et al., 1999).
Few years later, the membrane-bound heparan sulphate proteoglycans (HSPG) receptor was found to participate in the LRP-mediated FVIII uptake. The uptake of FVIII by HSPG was proposed to be mediated through the heparin-binding domain of FVIII located in the A2 domain. The identification of HSPG as a catabolic receptor for FVIII led to the description of two distinct pathway for FVIII elimination: an LRP-dependent pathway and an LRP-independent pathway (Sarafanov et al., 2001). However, the fact that blockade of both LRP and HSPG receptors does not completely prevent FVIII endocytosis in vitro suggests the implication of other receptors (Dasgupta et al., 2008). In this respect, the mannose-ending sugars present on FVIII have been implicated in FVIII endocytosis through the „macrophage mannose receptor‟ or CD206 (Dasgupta et al., 2007). Other sugar residues, particularly within the B domain of FVIII have been found to mediate FVIII uptake through the asialoglycoprotein receptor, an endocytic receptor for lectins, following exogenous removal of capping sialic acids using neuraminidase (Bovenschen et al., 2005). Recently, the sialic acid capping moiety on N-linked carbohydrate has been found to mediate FVIII endocytosis through the sialic-acid binding immunoglobulin-like lectin 5 (Siglec-5) (Pegon et al., 2012). To date, the nature of the receptors involved in FVIII uptake is still under investigation. We can speculate that structural changes in FVIII may lead to the generation of non-native molecular patterns which could participate in FVIII endocytosis through other receptors. In particular, as discussed later, one of our hypothesis is that oxidation of FVIII at the site of bleeding, where reactive oxygen species are released by the activated platelets and neutrophils may alter FVIII structure and immunogenicity.

In vivo distribution of FVIII

Following its infusion in the patient, FVIII circulate within the bloodstream. In vivo experiment in FVIII-deficient mice have demonstrated that the infused FVIII rapidly concentrates in the spleen and in the liver (Navarrete et al., 2009). In the spleen, FVIII co-localizes with the metallophilic macrophages in the marginal zone.

Prevention or treatment of bleeding episodes in patients with hemophilia A

Two different kinds of products are now available on the market. The first type of product that was used is called plasma-derived FVIII (pdFVIII). This product is obtained by purifying FVIII from the plasma of thousands of healthy donors. In a recent analysis, plasma-derived products were found to contain at least 124 other protein, among which some albumin, traces of fibronectin and VWF, which are co-purified during the manufacturing process (Basilico et al., 2010). The second type of product is called “recombinant” FVIII (rFVIII). Recombinant FVIII products are synthetized in vitro in stably transfected cell lines. Depending on the manufacturer, the rFVIII is produced in Chinese Hamster Ovary cells (CHO) or in Baby Hamster Kidney cells (BHK). Despite the fact that rFVIII is produced in vitro, the final formulation was found to contain at least 41 other proteins (Basilico et al., 2010). More recently, another type of rFVIII was developed in which the B domain, that has no known pro-coagulant function, has been deleted; this product is referred to as B-domain-deleted FVIII (BDD-FVIII).

Infusion regiment

FVIII can be administered to the patients following two distinct regimens, either on-demand or on a prophylactic basis. For on-demand treatments, the quantity of infused FVIII varies with the severity of the bleeding. Treatment of slight bleeds, such as epistaxis or moderate hematuria, can be achieved by restoring 30 to 50% of FVIII level. In cases of more severe bleeds, such as acute joint bleeds or large hematomas, FVIII is infused in the patients until bleeding stops. In cases of life-threatening bleeds, the continuous infusion of FVIII was found to reduce the consumption of FVIII (Batorova and Martinowitz, 2000). In the latter case, FVIII infusion may be prolonged for a period of up to 10 days, to prevent the recurrence of hemorrhages. In contrast with on-demand treatments, prophylactic treatments consist in the bi- or tri-weekly administration of FVIII and aim at maintaining a FVIII concentration between 3 and 5% of the normal levels. By preventing bleedings, this infusion regiment increases the patients‟ quality of life. Moreover, setting an early primary prophylaxis regiment has been shown to prevent the development of arthropathies, thus delaying or reducing the need for early joint replacement. To date, the development of easy-handling FVIII preparations has considerably improved the quality of life of the patients by allowing them to self-administrate FVIII.

Complications of the treatment

Until the 90‟s, the most serious complication of pdFVIII infusion was the transmission of infectious agents such as HIV or hepatitis B and C (Mauser-Bunschoten et al., 2009). Since then, progress in virus inactivation procedures along with the development of recombinant FVIII molecules have reduce the incidence of viral infection. Nowadays, the major complication consecutive to FVIII administration is the development of antibodies raised against therapeutic FVIII. These antibodies are capable of inhibiting FVIII activity and are thus called “inhibitors”. The development of an immune response towards FVIII abrogates the efficiency of the treatment and increases patient‟s mortality. This complication occurs in 25 to 30% of patients with severe hemophilia A, and 5% of patients with moderate/minor hemophilia A (Oldenburg and Pavlova, 2006).

Treatment of patients with FVIII inhibitors

The treatment of hemophilia A patients with FVIII-inhibitors can be achieved through several approaches. Classically, if a FVIII inhibitor is discovered in a hemophilia A patient, the first approach is the initiation of an immune tolerance induction (ITI) protocol. Indeed, almost 40 years ago, Brackmann and Gormsen reported that the repetitive injections of large amounts of FVIII was eradicating the FVIII inhibitors (Brackmann and Gormsen, 1977). Further studies have demonstrated that this protocol was efficient in up to 80% of the cases of inhibitor-positive patients (DiMichele and Kroner, 1999; Lenk, 1999). From a mechanistic point of view, the injection of high dose FVIII (currently ranging from 50 to 250 IU/kg/day) was found to eradicate FVIII-memory B cells in mice (Hausl et al., 2005) as well as in patients (Gilles et al., 1996; Sakurai et al., 2004; van Helden et al., 2010). However, it should be noted that all patients are not eligible for ITI. Moreover, many factors have been found to influence the success of ITI, such as the inhibitory titre at the time of ITI initiation (Mariani and Kroner, 1999), the delay between inhibitor detection and ITI initiation (Kreuz et al., 1995), as well as the age of the patient and the inhibitor peak titre after the start of ITI (Mariani et al., 1994; Kreuz et al., 1995). When all these parameters are fulfilled, then ITI may be initiated.

Polymorphisms in immune-related genes

The analysis of single nucleotide polymorphisms (SNPs) in different immune-related genes resulted in the identifications of variants in cytokine genes that were associated with the development of FVIII inhibitors. Thus, SNPs in the promoter of the genes encoding IL-10, TNF-α and CTLA-4 were associated with a higher incidence of FVIII inhibitor in patients with hemophilia A (Astermark et al., 2006a; Gouw and van den Berg, 2009; Pavlova et al., 2009). Indeed, the presence of a 134-bp-long variant of a CA repeat microsatellite in the promoter region of the IL-10 gene, previously associated with high levels of autoantibodies in several auto-immune diseases, was also associated with the presence of FVIII inhibitors in patients with severe hemophilia A (Astermark et al., 2006b). Similarly, within the TNF-α promoter, four SNPs were identified (-308 G>A, -827 C>T -238 G>A and -670 A>G) that affect the transcription of the TNF-α encoding gene. Subsequently, patients with severe hemophilia A possessing the combination of alleles -308AA -827CC -238GG and -670AA were found to be more susceptible to develop an inhibitor (Astermark et al., 2006a). Similarly, carriage of the G allele at position +49 of the CTLA-4 gene is associated with a decreased expression of CTLA-4, leading to an increased proliferation of activated T-cell, was associated with a higher incidence of inhibitor in patients with severe hemophilia A (Astermark et al., 2007). Recently, the Fc gamma receptor IIa (FcγRIIa or CD32) R131H polymorphism, that increases the binding affinity of the FcγRIIa to IgG1 and IgG2 isotypes was associated with inhibitor development in patients with severe hemophilia A (Eckhardt et al., 2014). Moreover, a genome-wide association study (GWAS) was conducted using a combined cohort involving 833 patients with severe hemophilia A from three independent cohorts (HIGS, MIBS and HGDS). This GWAS analysis evaluated the association of 13,331 SNPs from 1,081 genes with the presence of FVIII inhibitor in patients with severe hemophilia A. The authors identified 53 SNPs as significant predictors of inhibitor status. More precisely, within the 13 SNPs that were concordant in the three cohorts analyzed, 5 were associated with an increased risk for the development of FVIII-inhibitor (MAPK9, DOCK2, CD44, IQGAP2 and CSF1R) and 8 were found to be protective (PDGFRB, PCGF2, HSP90B1, F13A1, IGSF2, ALOX5AP, MAP2K4 and PTPRN2) (Astermark et al., 2013).

Table of contents :

I. INTRODUCTION
I.1. Hemophilia A
I.1.1. Generalities
I.1.1.1. Historical aspects
I.1.1.2. Phenotypic aspects
I.1.1.3. Genetic aspects
I.1.1.4 Bleeding complications in Hemophilia A
I.1.2 The coagulation cascade
I.1.2.1 Primary haemostasis
I.1.2.2 Secondary haemostasis
I.1.2.2.1 The extrinsic pathway
I.1.2.2.2 The intrinsic pathway
I.2.2.3 Pro-coagulant factor VIII
I.2.2.3.1 Synthesis, structure and post-traductional modifications
I.2.2.3.2 Activation of FVIII
I.2.2.3.3 Life cycle of FVIII, catabolic receptors, in vivo distribution, site of elimination
I.2.2.3.4 Catabolic receptors for FVIII
I.2.2.3.5 In vivo distribution of FVIII
I.1.3 Prevention or treatment of bleeding episodes in patients with hemophilia A
I.1.3.1 Factor VIII products
I.1.3.2 Infusion regiment
I.1.3.3 Complications of the treatment
I.1.3.4 Treatment of patients with FVIII inhibitors
I.2 The anti-factor VIII immune response
I.2.1 FVIII as seen by the immune system in patients with hemophilia A
I.2.2 Antigen-presenting cells
I.2.2.1 Dendritic cells
I.2.2.2 Macrophages
I.2.2.3 B cells
I.2.3 The spleen
I.2.4 The anti-FVIII immune response in hemophilia A patients
I.2.4.1 T-cell activation
I.2.4.2 B-cell activation
I.3 Risk factors
I.3.1 The danger signal theory
I.3.1.1 Exogenous danger signals
I.3.1.2 Endogenous danger signals
I.3.2 Genetic risk factors
I.3.2.1 FVIII mutations
I.3.2.2 HLA haplotype
I.3.2.3 Polymorphisms in immune-related genes
I.3.3 Non-genetic risk factors
I.3.3.1 Age at the start of treatment
I.3.3.2 Mode and intensity of FVIII administration
I.3.3.3 State of the immune system at the time of FVIII infusion
I.4 Purpose of the PhD work
II. RESULTS
II.1. Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphism in the HMOX1 promoter .
II.2. Hemarthrosis and arthropathies do not favor the development of factor VIII inhibitors in hemophilia A mice
II.3. Restoration of the oxidative balance in factor VIII-deficient mice reduces the immunogenicity of therapeutic factor VIII
II.4. Oxidation of therapeutic factor VIII aggravates its immunogenicity in factor-VIII deficient mice
III. DISCUSSION
IV. REFERENCES
V. ANNEXES 

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