A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A

Muczynski, Vincent; Verhenne, Sebastien; Casari, Caterina; Cherel, Ghislaine; Panicot‐Dubois, Laurence; Guéguen, Paul; Trossaërt, Marc; Dubois, Christophe; Lenting, Peter J.; Denis, Cécile V.; Olivier, Christophe

doi:10.1055/s-0039-1697662

Cited by 6 publications

(5 citation statements)

References 41 publications

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“…Examples include protease-activatable drugs (pro-drugs), antibodies (pro-antibodies), probes for imaging, and conjugates for photodynamic therapy. Notably, thrombin-cleavable drugs are under development to target the activity of the drug at sites where thrombin presents, such as the sites of clot formation, injury, and inflammation [ 44 , 45 , 46 , 47 ]. An example of a thrombin-activatable pharmaceutical closely related to our study (currently in clinical trials) is an EHL FVIII representing a recombinant FVIII genetically fused to the FVIII-binding domain of VWF [ 48 ].…”

Section: Discussionmentioning

confidence: 99%

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

Shestopal

Parunov

Olivares

et al. 2022

IJMS

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Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (VL and VH) are connected via an artificial linker. One particular scFv, iKM33, directed against blood coagulation factor VIII (FVIII) was shown to inhibit major FVIII functions and is useful in FVIII research. We aimed to investigate the properties of iKM33 enabled with protease-dependent disintegration. Three variants of iKM33 bearing thrombin cleavage sites within the linker were expressed using a baculovirus system and purified by two-step chromatography. All proteins retained strong binding to FVIII by surface plasmon resonance, and upon thrombin cleavage, dissociated into VL and VH as shown by size-exclusion chromatography. However, in FVIII activity and low-density lipoprotein receptor-related protein 1 binding assays, the thrombin-cleaved iKM33 variants were still inhibitory. In a pull-down assay using an FVIII-affinity sorbent, the isolated VH, a mixture of VL and VH, and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated VL and VH assembled into scFv-like heterodimer on FVIII, and the isolated VH alone also bound FVIII. We discuss the potential use of both protease-cleavable scFvs and isolated Fv subunits retaining high affinity to the antigens in various practical applications such as therapeutics, diagnostics, and research.

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Section: Discussionmentioning

confidence: 99%

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

Shestopal

Parunov

Olivares

et al. 2022

IJMS

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“…Some recent studies have reported that thrombin-activatable FX mutants enhanced hemostatic activity in in vitro and in vivo in hemophilia models [26,28,29], and the data suggested that FX-containing concentrates could offer the possibility of improved hemostatic treatment in the hemophilia human individuals. Also, previous Japanese studies highlighted the potential efficacy of pd-FVIIa/FX , consisting of a mixture of FVIIa and FX (w:w=1:10), in congenital hemophilia patients with inhibitors and in acquired HA [6][7][8][9].…”

Section: Discussionmentioning

confidence: 99%

“…Several studies utilizing in vitro and in vivo model systems have suggested that mutated FX or FXa might be useful for treatment in PwHA [26][27][28][29]. Analyses of binding affinity of emicizumab and FX determined by surface plasmon resonance-based assays have demonstrated, however, that the K d of emicizumab binding to FX was 1.85 μM [30],…”

Section: Accepted Manuscriptmentioning

confidence: 99%

“…Several studies utilizing in vitro and in vivo model systems have suggested that mutated FX or FXa might be useful for treatment in PwHA. [26][27][28][29] Analyses of binding affinity of emicizumab and FX determined by surface plasmon resonance-based assays have demonstrated, however, that the K d of emicizumab binding to FX was 1.85 μM, 30 indicating that the physiological level of FX in plasma ($130 nM 31 ) may be too low to influence the hemostatic potential of emicizumab. A recent study demonstrated that the more FX increased, the more FIX/FIXa-emicizumab-FX/FXa complex could generate.…”

Section: Introductionmentioning

confidence: 99%

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Additional Factor X Enhances Emicizumab-Driven Coagulation Function in Patients with Hemophilia A and Hemophilia A Mice

Shimizu,

Nakajima,

Takami

et al. 2024

Thromb Haemost

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Background: Bypassing agents are used for breakthrough bleedings in patients with hemophilia A with inhibitor (PwHAwI) receiving emicizumab prophylaxis. Previous study demonstrated a weak binding affinity between emicizumab and factor (F)X (Kd; 1.85 μM), and that this value was much greater than the plasma FX concentration (~130 nM). We speculated that increased FX levels could enhance coagulation potential in emicizumab-treated PwHA. Aims: To investigate the relationship between FX concentrations and emicizumab-driven coagulation. Methods: Plasma FX (up to 1,040 nM) and emicizumab (50 µg/mL) were added to FVIII-deficient plasmas, and plasma-derived FX (520 nM) or recombinant (r)FVIIa (2.2 µg/mL) was added to plasmas from three emicizumab-treated PwHAwI. The adjusted-maximum coagulation velocity (Ad|min1|) by clot waveform analysis and peak thrombin (PeakTh) by thrombin generation assay in them were evaluated. Emicizumab (3.0 mg/kg), human (h)FIX (100 IU/kg), and various doses of hFX (100-500 IU/kg) were intravenously administered to HA mice. Clotting time/clot formation time (CT/CFT) were assessed using rotational thromboelastometry, and blood loss was estimated by a tail-clip assay. Results: The addition of FX to FVIII-deficient plasma with emicizumab increased Ad|min1| and PeakTh. The coagulation parameters in emicizumab-treated PwHAwI spiked with additional FX remained within the normal range as well as the additional rFVIIa. In animal models, hFX injection shortened the CT and CT+CFT. The shorter CT and CT+CFT, and the lower blood loss were evident after 200 or 500 IU/kg hFX administration, and those indices were comparable to those in wild-type mice. Conclusion: Supplementation with FX may improve emicizumab-driven hemostasis in PwHA.

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“…FX variants (1 µM) and plasma-derived prothrombin (5.6-20 µM) were pretreated with APMSF (100 µM) and incubated for 60 minutes at 37°C in order to allow for the decomposition of unbound inhibitor, thereby preventing interference in the assay. 19 Steady-state initial velocities of macromolecular substrate cleavage were determined discontinuously at 25°C in assay buffer as described. 12 Briefly, unless otherwise stated, progress curves of prothrombin were obtained by incubating PCPS (50 μM), DAPA (10 µM), prothrombin (1.4 µM), and FV810 or v-ptFV (300 nM), and the reaction was initiated with 250 nM of FX, upon which the rate of prothrombin conversion was measured.…”

Section: Prothrombin Activationmentioning

confidence: 99%

Evolutionary Adaptations in Pseudonaja Textilis Venom Factor X Induce Zymogen Activity and Resistance to the Intrinsic Tenase Complex

et al. 2020

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The venom of the Australian snake Pseudonaja textilis comprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that of P. textilis, comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa–factor IXa tenase complex. Furthermore, our findings indicate that the human FX activation peptide comprises an essential binding site for the intrinsic tenase complex. Conversely, incorporation of FX into the extrinsic tissue factor–factor VIIa tenase complex is completely dependent on exosite-mediated interactions. Remarkably, the shortened activation peptide allows for factor V-dependent prothrombin conversion while in the zymogen state. This indicates that the active site of FX molecules comprising the v-ptFX activation peptide partially matures upon assembly into a premature prothrombinase complex. Taken together, the shortened activation peptide is one of the remarkable characteristics of v-ptFX that has been modified from its original form, thereby transforming FX into a powerful procoagulant protein. Moreover, these results shed new light on the structural requirements for serine protease activation and indicate that catalytic activity can be obtained without formation of the characteristic Ile16–Asp194 salt bridge via modification of the activation peptide.

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A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A

Cited by 6 publications

References 41 publications

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

Additional Factor X Enhances Emicizumab-Driven Coagulation Function in Patients with Hemophilia A and Hemophilia A Mice

Evolutionary Adaptations in Pseudonaja Textilis Venom Factor X Induce Zymogen Activity and Resistance to the Intrinsic Tenase Complex

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