In vitro effects of combining Mim8 with factor VIII, FVIIa, and activated prothrombin complex concentrates in thrombin generation assays.

BACKGROUND: Mim8 is a novel antifactor IXa/antifactor X bispecific antibody in clinical development for prophylactic treatment of hemophilia A with and without inhibitors. Patients treated with Mim8 may need supplementary bleed treatment under certain conditions such as surgery or major trauma. OBJECTIVES: This study aimed to better understand the response of Mim8 in thrombin generation assays (TGAs) alone or in combination with other hemostatic proteins. METHODS: We used TGAs with different activators (tissue factor (TF) and activated factor XI) to better understand the similarities and differences between the mode of action of Mim8 and factor VIII (FVIII). Following this, we investigated the effects of mixing Mim8 with the main bleed treatment options for persons with hemophilia A with or without inhibitors: FVIII, activated factor VII (FVIIa), and activated prothrombin complex concentrates (aPCC). RESULTS: The results indicated that for patients without inhibitors, Mim8 does not interfere with FVIII's mode of action. For patients with inhibitors, Mim8 mixed with aPCC results in a strong synergistic effect causing thrombin generation far exceeding the normal levels. Contrary to this, mixing Mim8 with FVIIa results in a more controlled additive effect, visible only when using TF as a trigger, which does not exceed the normal level of thrombin generation. CONCLUSION: These findings support the use of approved clinical doses of FVIIa for bleed treatment of patients with FVIII inhibitors treated with Mim8. Additionally, the findings suggest that concomitant use of FVIII and Mim8 is safe for managing breakthrough bleeds.

A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa.

Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two ß-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1-7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2-3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.

Site-specific functional roles of the Factor X activation peptide in the intrinsic tenase-mediated Factor X activation.

The conversion of zymogen Factor X (FX) to an active protease involves the removal of a 52-residue long activation peptide (AP). Through site-directed mutagenesis, we investigate the role of the AP and demonstrate that the high abundance of proline residues is important for efficient proteolysis of FX. Moreover, we identify an essential interaction site for Factor IXa (FIXa) between residues 22 and 30 (AP numbering) and find that the residues between 31 and 41 may provide an important interaction site for the intrinsic tenase complex, composed of Factor IXa (FIXa) and Factor VIIIa (FVIIIa). Finally, we suggest that the carbohydrate chain at Asn-39 restricts the activator specificity, as elimination of this glycosylation site increases the activation rate for activation by FIXa and FXa.

The functional role of the autolysis loop in the regulation of factor X upon hemostatic response.

BACKGROUND: The regulation of factor X (FX) is critical to maintain the balance between blood coagulation and fluidity. OBJECTIVES: To functionally characterize the role of the FX autolysis loop in the regulation of the zymogen and active form of FX. METHODS: We introduced novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. The activity and inhibition of recombinant FX variants was quantified in pure component assays. The in vitro thrombin generation potential of the FX variants was evaluated in FX-depleted plasma. RESULTS: The factor VIIa (FVIIa)-mediated activation and prothrombin activation was reduced, presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, whereas ATIII inhibition in the presence of the heparin-analog Fondaparinux was modestly increased by 2- to 15-fold compared with wild-type. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FXa variants. CONCLUSION: The autolysis loop participates in all aspects of FX regulation. In plasma-based assays, a modest decrease in FX activation rate appeared to knock down the procoagulant response even when down regulation of FXa activity by inhibitors was reduced.

A factor VIIIa-mimetic bispecific antibody, Mim8, ameliorates bleeding upon severe vascular challenge in hemophilia A mice.

Hemophilia A is a bleeding disorder resulting from deficient factor VIII (FVIII), which normally functions as a cofactor to activated factor IX (FIXa) that facilitates activation of factor X (FX). To mimic this property in a bispecific antibody format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by 4 orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation, with potencies 13 and 18 times higher than a sequence-identical analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.

Monoclonal antibodies targeting the disintegrin-like domain of ADAMDEC1 modulates the proteolytic activity and enables quantification of ADAMDEC1 protein in human plasma.

Decysin-1 (ADAMDEC1) is an orphan ADAM-like metalloprotease with unknown biological function and a short domain structure. ADAMDEC1 mRNA has previously been demonstrated primarily in macrophages and mature dendritic cells. Here, we generated monoclonal antibodies (mAbs) against the mature ADAMDEC1 protein, as well as mAbs specific for the ADAMDEC1 pro-form, enabling further investigations of the metalloprotease. The generated mAbs bind ADAMDEC1 with varying affinity and represent at least six different epitope bins. Binding of mAbs to one epitope bin in the C-terminal disintegrin-like domain efficiently reduces the proteolytic activity of ADAMDEC1. A unique mAb, also recognizing the disintegrin-like domain, stimulates the caseinolytic activity of ADAMDEC1 while having no significant effect on the proteolysis of carboxymethylated transferrin. Using two different mAbs binding the disintegrin-like domain, we developed a robust, quantitative sandwich ELISA and demonstrate secretion of mature ADAMDEC1 protein by primary human macrophages. Surprisingly, we also found ADAMDEC1 present in human plasma with an approximate concentration of 0.5 nM. The presence of ADAMDEC1 both in human plasma and in macrophage cell culture supernatant were biochemically validated using immunoprecipitation and Western blot analysis demonstrating that ADAMDEC1 is secreted in a mature form.

Evidence for restricted reactivity of ADAMDEC1 with protein substrates and endogenous inhibitors.

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1' position. Cys(392), present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys(392) for a Ser increased the reactivity with α2-macroglobulin but not with casein or Cm-Tf. Substitution of Asp(362) for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.

Heparin-binding mechanism of the IGF2/IGF-binding protein 2 complex.

IGF1 and IGF2 are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF-binding proteins (IGFBP1-IGFBP6) are primary regulators of IGF half-life and receptor availability. Generally, the binding of IGFBPs inhibits IGF receptor activation. However, it has been shown that IGFBP2 in complex with IGF2 (IGF2/IGFBP2) stimulates osteoblast function in vitro and increases skeletal mass in vivo. IGF2 binding to IGFBP2 greatly increases the affinity for 2- or 3-carbon O-sulfated glycosaminoglycans (GAGs), e.g. heparin and heparan sulfate, which is hypothesized to preferentially and specifically target the IGF2/IGFBP2 complex to the bone matrix. In order to obtain a more detailed understanding of the interactions between the IGF2/IGFBP2 complex and GAGs, we investigated heparin-binding properties of IGFBP2 and the IGF2/IGFBP2 complex in a quantitative manner. For this study, we mutated key positively charged residues within the two heparin-binding domains (HBDs) in IGFBP2 and in one potential HBD in IGF2. Using heparin affinity chromatography, we demonstrate that the two IGFBP2 HBDs contribute differentially to GAG binding in free IGFBP2 and the IGF2/IGFBP2 protein complex. Moreover, we identify a significant contribution from the HBD in IGF2 to the increased IGF2/IGFBP2 heparin affinity. Using molecular modeling, we present a novel model for the IGF2/IGFBP2 interaction with heparin where all three proposed HBDs constitute a positively charged and surface-exposed area that would serve to promote the increased heparin affinity of the complex compared with free intact IGFBP2.

ADAMDEC1 is a metzincin metalloprotease with dampened proteolytic activity.

ADAMDEC1 (Decysin-1) is a putative ADAM (a disintegrin and metalloprotease)-like metalloprotease with an unknown physiological role, selectively expressed in mature dendritic cells and macrophages. When compared with other members of the ADAM family, ADAMDEC1 displays some unusual features. It lacks the auxiliary cysteine-rich, EGF, and transmembrane domains, as well as the cytoplasmic tail. The active site of ADAMDEC1 is unique by being the only mammalian ADAM protease with a non-histidine zinc ligand, having an aspartic acid residue instead. Here we demonstrate that ADAMDEC1, despite these unique features, functions as an active metalloprotease. Thus, ADAMDEC1 is secreted as a mature, glycosylated, and proteolytically active metalloprotease, capable of cleaving macromolecular substrates. In the recombinant form, three of the four potential N-linked glycosylation sites are modified by carbohydrate attachment. Substitution of basic residues at the predicted proprotein convertase cleavage site blocks proprotein processing, revealing both specific ADAMDEC1-dependent and specific ADAMDEC1-independent cleavage of the prodomain. The pro-form of ADAMDEC1 does not have proteolytic activity, demonstrating that the prodomain of ADAMDEC1, like in other members of the ADAM family, confers catalytic latency. Interestingly, the proteolytic activity of mature ADAMDEC1 can be significantly enhanced when a canonical ADAM active site with three zinc-coordinating histidine residues is introduced.

Mutations in calmodulin cause ventricular tachycardia and sudden cardiac death.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe dominantly inherited form of CPVT-like arrhythmias, we mapped the disease locus to chromosome 14q31-32. Sequencing CALM1 encoding calmodulin revealed a heterozygous missense mutation (c.161A>T [p.Asn53Ile]) segregating with the disease. A second, de novo, missense mutation (c.293A>G [p.Asn97Ser]) was subsequently identified in an individual of Iraqi origin; this individual was diagnosed with CPVT from a screening of 61 arrhythmia samples with no identified RYR2 mutations. Both CALM1 substitutions demonstrated compromised calcium binding, and p.Asn97Ser displayed an aberrant interaction with the RYR2 calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac death.