OC 76.3 - Molecular Basis for the Conformational Selectivity of Thrombomodulin for TAFI or Protein C

Wednesday, July 13, 2022

10:45 AM – 11:00 AM

Location: ExCel Center, ICC Capital Suite Room 2-4

Presentation Format: In-Person

Presenting Author(s)

Michael B. Boffa, PhD

Associate Professor
Western University, Robarts Research Institute
London, Ontario, Canada

Co-Author(s)

JA

John Ackersviller

Undergraduate student
Department of Biochemistry, University of Western Ontario
London, Ontario, Canada
HM

Haley Marier

MSc student
Department of Biochemistry, University of Western Ontario
London, Ontario, Canada
RM

Rayane Moric

Undergraduate student
Department of Biochemistry, University of Western Ontario
London, Ontario, Canada
HK

Hammaan Khan

Undergraduate student
Department of Biochemistry, University of Western Ontario
London, Ontario, Canada

Background: Thrombomodulin (TM) is an endothelial cell cofactor for the activation of protein C (PC) and thrombin-activatable fibrinolysis inhibitor (TAFI). EGF5 and 6 of TM bind thrombin, and EGF4 binds to PC in the ternary complex with thrombin. Met388 in the linker between EGF4 and EGF5 clamps these two domains in a specific orientation required for PC cofactor activity. TAFI cofactor activity additionally requires EGF3, which presumably binds to TAFI. However, the nature of the TM-thrombin-TAFI catalytic complex and how TM might discriminate between PC and TAFI remains unclear.

Aims: Our goal was to use recombinant variants of TM to reveal key structural features that mediate selectivity for PC or TAFI.

Methods: We expressed a series of soluble TM variants encompassing EGF3-6 in HEK293 cells. These included (i) systematic substitutions of Met388 for all other residues; and (ii) EGF3 and 4 domain swap variants consisting of EGF3-3-5-6, EGF4-3-5-6, and EGF4-4-5-6. We measured thrombin-mediated PC and TAFI activation in the presence of the TM variants.

Results: Most Met388 substitutions had minimal impact on TAFI cofactor activity, whereas PC cofactor activity was only retained when Met388 was conservatively substituted (Leu/Tyr/Phe/Ile). Oxidation of EGF3-6 with chloramine T virtually eliminated PC cofactor activity while only mildly decreasing TAFI cofactor activity. Of the domain swap variants, only EGF3-3-5-6 was able to support TAFI activation, and only EGF4-4-5-6 was able to support PC activation.

Conclusion(s): The specific orientation of EGF4 and 5 mediated by Met388 is dispensable for TAFI cofactor activity. In the context of TAFI activation, EGF4 acts as a flexible spacer to position TAFI-binding EGF3. These features provide a mechanism for TM to discriminate between PC and TAFI as substrates. Specifically, during inflammatory responses where Met388 might be oxidized by reactive oxygen species, TM would retain TAFI cofactor activity to maintain clot stability while losing its unwanted anticoagulant functions.