OC 36.4 - A Tethered Artificial Protease Reveals the Location of Key Regulatory Motifs in Factor V

Monday, July 11, 2022

11:30 AM – 11:45 AM

Location: ExCel Center, ICC Capital Suite Room 10&11

Presentation Format: In-Person

Presenting Author(s)

Francis Ayombil, PhD

Post-doctoral Research Fellow
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania, United States

Co-Author(s)

RT

Raffaella Toso

Research Fellow
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
RC

Rodney M. Camire

Professor of Pediatrics
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia. Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Philadelphia, Pennsylvania, United States

Background: Conserved acidic (AR2, 1493-1537) and basic (BR, 963-1008) regions within the FV B-domain play a key role in regulating cofactor function. Elimination of BR through splicing (e.g., FV-short) or proteolytic removal of BR and AR2 produce an active cofactor. Biochemical evidence suggests AR2, and BR form a tight interaction. The role of AR1 (656-697) at the C-terminus of the heavy chain (HC) is not defined.

Aims: To investigate the spatial relationships between AR1, AR2 and BR.

Methods: FV-short or FV-810 were incubated with BR fragments (FV-BR or TFPIα-BR) tethered to an artificial protease, FeBABE (Fe-p-bromoacetamidobenzyl-EDTA). Once activated, FeBABE cuts proteins if near ( < 12 Å) the contact site of the target protein. FV cleavage products were identified by immunoblotting and N-terminal sequencing.

Results: BR-FeBABE bound FV-short or FV-810 with high affinity. When activated, BR-FeBABE cut FV-short and FV-810 to yield distinct proteolytic fragments (~95 kDa and ~190 kDa) derived from the C-terminus of the A2- and B-domains. Using truncated HC derivatives of FVa, we found that cleavage within the A2 domain occurs near residues 675-685 which is part of AR1. Additional blotting and N-terminal sequencing revealed that cleavage yielding the ~190 kDa band is within AR2. These data suggest that BR engages both AR1 and AR2 and defines a key epitope for this functional unit. In control experiments, acetylated BR-FeBABE, free hydroxy radicals, or titration with unlabeled BR fragments, revealed that cleavage of FV-short and FV-810 was specific. Further, in competitive studies, FXaS195A, APC-DEGR, and TFPIα all inhibited cleavage of FV-short and FV-810 by BR-FeBABE consistent with their high affinity binding to cofactor-like species.

Conclusion(s): These studies document a physical interaction between BR and AR1/AR2, and help define how these key functional landmarks regulate the expression of cofactor activity when FV is converted to FVa or when FV-short is bound to TFPIα.