Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C–mediated proteolysis

Abstract

Factor VIII (FVIII) inhibitor antibodies are classified into 2 groups according to the kinetic pattern of FVIII inactivation. Type 2 antibodies are more commonly observed in patients with acquired hemophilia A and do not completely inhibit FVIII activity; in most cases, substantial levels of circulating FVIII are detected. Three type 2 autoantibodies from patients who had normal levels of FVIII antigen despite having low levels of FVIII activity were studied. The antibodies reacted exclusively with the light chain of FVIII but not with the C2 domain, and their epitopes were therefore ascribed to the regions in the A3-C1 domains. Heavy and light chains of FVIII were detected in plasmaderived immune complexes extracted by using protein G Sepharose. Direct binding assays using anhydro-activated protein C (anhydro-APC), a catalytically inactive derivative of activated protein C (APC) in which the active-site serine is converted to dehydroalanine, were used to examine the relation between immune complexes and APC. IntroductionFactor VIII (FVIII) inhibitors develop as alloantibodies in patients with hemophilia A who have received multiple transfusions or as autoantibodies in previously hemostatically normal individuals, particularly elderly people, patients with autoimmune diseases, pregnant women, and women in the postpartum period. 1 Appearance of the autoantibodies causes a reduction in FVIII activity (FVIII:C) and usually results in a severe bleeding tendency known as acquired hemophilia A.FVIII is a glycoprotein cofactor that accelerates the blood coagulation tenase complex. 2 Mature FVIII is synthesized as a single-chain polypeptide consisting of 2332 amino acid residues, 3,4 and it consists of 3 domains arranged in the following order: A1-A2-B-A3-C1-C2. 5 As a result of proteolytic processing, FVIII circulates as a divalent, cation-linked heterodimer formed by a heavy chain (HCh) composed of the A1, A2, and B domains and a light chain (LCh) composed of the A3, C1, and C2 domains. 4,5 FVIII is transformed into its active form, an A1/A2/A3-C1-C2 heterotrimer, by limited proteolysis at Arg372, Arg740, and Arg1689 by thrombin and factor Xa. 6 Additional proteolytic cleavage at Arg336 by activated protein C (APC) inactivates FVIII. 6 The binding site for APC was found within the residues His2009 to Val2018 in the LCh. 7 In plasma, FVIII is noncovalently bound to von Willebrand factor (vWF), and this complex formation protects FVIII from inactivation by APC. 8 Common epitopes of autoantibodies, as well as hemophilic alloantibodies, have been found in either the A2 domain, the C2 domain, or both. 9 Most autoantibodies, however, appear to be directed against a single domain rather than both domains, with anti-C2 antibodies being more common than anti-A2 antibodies. 9 In contrast, most hemophilic alloantibodies appear to recognize both domains. 10 Most inhibitor antibodies with a C2 epitope inhibit binding of FVIII to both vWF and phospholipid (PL). 11,12 However, a new dominant epitope in the A3-C1 domains has been id...

A novel mechanism of factor VIII protection by von Willebrand factor from activated protein C–catalyzed inactivation

“…The inhibitory effect of the IgG on binding of FVIII to anhydro-APC was comparable to that of anti-FVIII autoantibodies with A3-C1 epitopes. 21 Notably, all of these autoantibodies were found to bind to the same peptides, supporting the view that the APC binding site is located within the defined peptide region 2009 to 2018.The inhibitory effect of VWF on FVIII binding to anhydro-APC was confirmed in the absence of PL. The present findings indicated that VWF and APC binding sites are in close proximity to each other.…”

“…Analysis of FVIII cleavage by APC was assessed as previously reported. 21 Briefly, anti-FVIII MoAb JR8 (0.2 M) was immobilized on microtiter wells. After blocking the wells with 4% HSA, 10 nM FVIII, incubated with serial dilutions of antipeptide IgG for 1 hour at 37°C, was added to each well, followed by addition of 5 nM APC, 10 M PL vesicles, and 2.5 mM CaCl 2 .…”

“…Anhydro-APC, a catalytically inactive derivative of APC in which the active-site serine is replaced by dehydroalanine, was prepared as previously described. 21 The purified anhydro-APC did not inactivate FVIII activity. PL vesicles were prepared by sonication of a phosphatidylserine/phosphatidylcholine mixture (20:80 molar ratio) as previously described.…”

Omental implantation of BOECs in hemophilia dogs results in circulating FVIII antigen and a complex immune response

“…However, persistent circulating IgG immune complexes are not uncommon and are found in patients with different types of allergies, 44 and in some patients with acquired hemophilia A. 25 Consistent with our observations, the acquired hemophilia patients in Nogami et al 25 had disproportionally higher levels of FVIII antigen relative to activity, and antibodies that interacted with the APC cleavage site. Furthermore, dog #3 had antibodies that interacted with the LRP and membrane phospholipid binding sites which may inhibit FVIII clearance from the circulation and contribute to the elevated levels of circulating FVIII antigen.…”

“…The details outlining the purification of total IgG antibodies 25,26 and screening the peptide library are provided in supplemental Methods, available on the Blood Web site.…”