One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates its EPCR‐dependent hemostatic activity in vivo

Pavani, Giulia; Zintner, Shannon M.; Ivanciu, Lacramioara; Small, Juliana C.; Stafford, Katherine A.; Szeto, Julia; Margaritis, Paris

doi:10.1111/jth.13607

Cited by 5 publications

(14 citation statements)

References 13 publications

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“…Although earlier studies suggest that FVIIa-EPCR interaction may contribute to the hemostatic effect of FVIIa in hemophilia therapy, 5;14;20 it is not entirely clear the mechanism by which EPCR-FVIIa interaction contributes to the hemostatic effect. EPCR-FVIIa interaction could contribute to the hemostatic effect of FVIIa by competing with protein C for the EPCR and thus downregulating EPCR-mediated APC generation, 1;5;19 by FVIIa-EPCR directly activating FX, or by EPCR tethering of FVIIa on the endothelium thus extending the locale of procoagulant reactions.…”

Section: Discussionmentioning

confidence: 99%

“…It is unlikely that the differences between human and murine FVIIa to interact with murine TF or EPCR alone were the reasons for this discrepancy since murine FVIIa variants that effectively interact with both TF and EPCR were also used at 3 mg/Kg dose to restore hemostasis in hemophilia B mice. 14;20 The restoration of hemostasis in FVIII Ab-induced hemophilia, where the bleeding phenotype is not as severe as in FVIII genetic deficient mice (FVIII −/− mice) because of residual FVIII activity in the Ab-induced hemophilia model, required 4 to 10 times less rFVIIa (0.25 to 1 mg/Kg) than necessary in FVIII −/− mice (1 to 10 mg/Kg).…”

Section: Discussionmentioning

confidence: 99%

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Factor VIIa interaction with EPCR modulates the hemostatic effect of rFVIIa in hemophilia therapy: mode of its action

et al. 2017

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Recent studies established that clotting factor VIIa (FVIIa) binds endothelial cell protein C receptor (EPCR). It has been speculated that FVIIa interaction with EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy. Active-site inhibited FVIIa, which is capable of binding to EPCR but has no ability to activate factor X, reduced the concentration of rFVIIa required to correct the bleeding following the saphenous vein injury in mouse hemophilia model systems. Higher doses of rFVIIa were required to restore hemostasis in EPCR overexpressing hemophilia mice compared to hemophilia mice expressing normal levels of EPCR. Administration of FVIII antibody induced only mild hemophilic bleeding in EPCR-deficient mice, which was corrected completely with a low dose of rFVIIa. Administration of therapeutic concentrations of rFVIIa increased plasma protein C levels in EPCR overexpressing mice, indicating the displacement of protein C from EPCR by rFVIIa. EPCR levels did not significantly alter the bioavailability of rFVIIa in plasma. Overall, our data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating hemophilia. Our present findings suggest that FVIIa displacement of anticoagulant protein C from EPCR that results in down-regulation of activated protein C generation and not the direct effect of EPCR-FVIIa on FX activation is the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Factor VIIa interaction with EPCR modulates the hemostatic effect of rFVIIa in hemophilia therapy: mode of its action

et al. 2017

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“…This analog exhibited a 3‐fold increase in hemostatic activity compared with wild‐type mFVIIa . More recently, the L4F substitution was found to be solely responsible for the EPCR‐dependent increase in hemostatic activity . They concluded that binding to EPCR on the endothelium may augment the procoagulant effect of FVIIa.…”

Section: Discussionmentioning

confidence: 99%

“…Pavani et al . suggested a direct role for EPCR in FVIIa‐mediated hemostasis . They engineered a murine FVIIa (mFVIIa) analog with three mutations (L4F/L8M/W9R) that allow it, unlike wild‐type mFVIIa, to bind murine EPCR.…”

Section: Discussionmentioning

confidence: 99%

Human platelets express endothelial protein C receptor, which can be utilized to enhance localization of factor VIIa activity

Fager

Machlus

Ezban

et al. 2018

Journal of Thrombosis and Haemostasis

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Background High-dose factor VIIa (FVIIa) is routinely used as an effective bypassing agent to treat hemophilia patients with inhibitory antibodies that compromise factor replacement. However, the mechanism by which FVIIa binds activated platelets to promote hemostasis is not fully understood. FVIIa-DVQ is an analog of FVIIa with enhanced tissue factor (TF)-independent activity and hemostatic efficacy relative to FVIIa. Our previous studies have shown that FVIIa-DVQ exhibits greater platelet binding, thereby suggesting that features in addition to lipid composition contribute to platelet-FVIIa interactions. Objectives Endothelial cell protein C receptor (EPCR) also functions as a receptor for FVIIa on endothelial cells. We therefore hypothesized that an interaction with EPCR might play a role in platelet-FVIIa binding. Methods/results In the present study, we used flow cytometric analyses to show that platelet binding of both FVIIa and FVIIa-DVQ is partially inhibited in the presence of excess protein C or an anti-EPCR antibody. This decreased binding results in a corresponding decrease in the activity of both molecules in FXa and thrombin generation assays. Enhanced binding to EPCR was sufficient to account for the increased platelet binding of FVIIa-DVQ compared with wild-type FVIIa. As EPCR protein expression has not previously been shown in platelets, we confirmed the presence of EPCR in platelets using immunofluorescence, flow cytometry, immunoprecipitation, and mass spectrometry. Conclusions This work represents the first demonstration that human platelets express EPCR and suggests that modulation of EPCR binding could be utilized to enhance the hemostatic efficacy of rationally designed FVIIa analogs.

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“…Recombinant rat FVIIa was purified exclusively in a 2-chain form (Figure 1B), similar to what we previously observed with human, mouse, and canine FVIIa engineered transgenes. 12,14,[17][18][19] Using a prothrombin time (PT) clotting assay, we found that rat FVIIa tissue factor-dependent activity was similar to rhFVIIa (Figure 1C; P . .05).…”

Section: Purification Of Rat Fviia and In Vitro Activitymentioning

confidence: 99%

Gene-based FVIIa prophylaxis modulates the spontaneous bleeding phenotype of hemophilia A rats

et al. 2019

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A sizable proportion of hemophilia inhibitor patients fails immune tolerance induction and requires bypass agents for long-term bleed management. Recombinant human-activated coagulation Factor VII (rhFVIIa) is an on-demand bypass hemostatic agent for bleeds in hemophilia inhibitor patients. Prophylactic use of rhFVIIa may enable sustained hemostatic management of inhibitor patients, but the critical relationship of rhFVIIa circulating levels and clinical outcome in that setting remains unclear. To address this in vivo, we used the rat hemophilia A (HA) model that exhibits spontaneous bleeds and allows longitudinal studies with sufficient statistical power. We simulated activated Factor VII (FVIIa) prophylaxis by adeno-associated virus (AAV) gene transfer of a rat FVIIa transgene. Compared with naive HA animals, rat FVIIa continuous expression affected the overall observed bleeds, which were resolved with on-demand administration of recombinant rat FVIIa. Specifically, although 91% of naive animals exhibited bleeds, this was reduced to 83% and 33% in animals expressing less than 708 ng/mL (<14 nM) and at least 708 ng/mL (≥14 nM) rat FVIIa, respectively. No bleeds occurred in animals expressing higher than 1250 ng/mL (>25 nM). Rat FVIIa expression of at least 708 ng/mL was also sufficient to normalize the blood loss after a tail vein injury. Continuous, AAV-mediated rat FVIIa transgene expression had no apparent adverse effects in the hemostatic system of HA rats. This work establishes for the first time a dose dependency and threshold of circulating FVIIa antigen levels for reduction or complete elimination of bleeds in a setting of FVIIa-based HA prophylaxis.

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One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates its EPCR‐dependent hemostatic activity in vivo

Cited by 5 publications

References 13 publications

Factor VIIa interaction with EPCR modulates the hemostatic effect of rFVIIa in hemophilia therapy: mode of its action

Factor VIIa interaction with EPCR modulates the hemostatic effect of rFVIIa in hemophilia therapy: mode of its action

Human platelets express endothelial protein C receptor, which can be utilized to enhance localization of factor VIIa activity

Gene-based FVIIa prophylaxis modulates the spontaneous bleeding phenotype of hemophilia A rats

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