A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab′)2 possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab′)2s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab′)2, as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab′)2. Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab′)2 in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.

The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na ؉ binds to the substrate bound FXa with K d ϳ39 mM in the absence and ϳ9.5 mM in the presence of Ca 2؉ . Sodium-bound FXa (sodium-Xa) has ϳ18-fold increased catalytic efficiency (ϳ4.5-fold decrease in K m and ϳ4-fold increase in k cat ) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca 2؉ further increases this k cat ϳ1.4-fold. Ca 2؉binds to the protease domain of substrate bound FXa with K d ϳ705 M in the absence and ϳ175 M in the presence of Na ؉ . Ca 2؉ binding to the protease domain of FXa (Xa-calcium) has no effect on the K m but increases the k cat ϳ4-fold in hydrolyzing S-2222, and Na ؉ further increases this k cat ϳ1.4-fold. In agreement with the K m data, sodium-Xa has ϳ5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and ϳ4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca 2؉ (؎Na ؉ ) has no effect on these interactions. Antithrombin binds to Xa-calcium with a ϳ4-fold faster rate, to sodium-Xa with a ϳ24-fold faster rate and to sodium-Xa-calcium with a ϳ28-fold faster rate. Thus, Ca 2؉ and Na ؉ together increase the catalytic efficiency of FXa ϳ28-fold. Na ؉ enhances Ca 2؉ binding, and Ca 2؉ enhances Na ؉ binding. Further, Na ؉ enhances S1 site occupancy, and S1 site occupancy enhances Na ؉ binding. Therefore, Na ؉ site is thermodynamically linked to the S1 site as well as to the protease domain Ca 2؉ site, whereas Ca 2؉ site is only linked to the Na ؉ site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.Factor X is a vitamin K-dependent plasma glycoprotein that plays a crucial role in blood coagulation. The human protein circulates as a zymogen with a molecular weight of ϳ59,000 and consists of a light chain (amino acids 1-139) and a heavy chain (amino acids 143-448) held together by a single disulfide bond between Cys-132 and Cys-302 (1). Upon activation by factor VIIa, Ca 2ϩ , and tissue factor or by factor IXa, Ca 2ϩ phospholipid, and factor VIIIa, a single peptide bond in factor X between residues Arg-194(c15) 1 and Ile-195(c16) is cleaved with resultant formation of a serine protease, factor Xa (FXa), 2 and release of a 52-residue activation peptide (1, 2). FXa is converted to its ␤-form where a ϳ4-kDa peptide is cleaved off from the COOH terminus of the heavy chain; this, however, does not result in a loss of coagulant activity (3). FXa converts prothrombin to thrombin (IIa) in a reaction requiring Ca 2ϩ , phospholipid membrane, and factor Va (FVa) (2, 4). Through its active site Ser-397(c195), FXa also binds to the serpin antithrombin (AT) (5) and to the second Kunitz domain of tissue factor pathway inhibitor (TFPI) (6).The NH 2 termi...

No abstract

Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.