PB0752 - Competitive inhibition of TLT-1/fibrinogen molecular interaction disrupts platelet function in ex vivo platelet assays.

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

SB

Siobhan L. Branfield

PhD Candidate
Oakland University
Auburn hills, Michigan, United States

Co-Author(s)

XV

Xariana Vales

PhD Student
Stanford University
Stanford, California, United States
SL

Susan Lagrand

Graduate Student
Oakland University
Rochester Hills, Michigan, United States
CW

Colin Wu

Professor
Oakland University
Rochester Hills, Michigan, United States
BM

Barbara Manfredi

post doctoral fellow
Oakland University
Rochester, Michigan, United States
PA

Pearl Akamine

Research Scientist
University of Puerto Rico
San Juan, Puerto Rico, United States
YM

Yadira Marie Cantres

Researcher
Oakland University
San Juan, Puerto Rico, United States
JG

Jose González

Research Scientist
University of Puerto Rico
San Juan, Puerto Rico, United States
AB

Abel Baerga

Professor
University of Puerto Rico
San Juan, Puerto Rico, United States
LM

Loyda Melendez Aponte

Professor
University of Puerto Rico
San Juan, Puerto Rico, United States
VW

Valance Washington

Professor
Oakland University
Rochester, Michigan, United States

Background: Platelets facilitate thrombosis, hemostasis, and immunity through their interaction with fibrinogen. Fibrinogen is the ligand for the platelet receptors IIb3 and TREM-Like Transcript-1 (TLT-1), TLT-1 is an Ig-domain containing receptor released from platelet -granules upon platelet activation. Unlike IIb3, little is known about the TLT-1/fibrinogen interaction.

Aims: Delineate the TLT-1/fibrinogen molecular interaction and determine if, peptides representing this interaction can regulate platelet function and inflammatory hemostasis.

Methods: Octet Qke Bio-layer Interferometry and atomic force microscopy (AFM) were used to evaluate soluble TLT-1 binding to fibrinogen. Immunoprecipitation and a peptide array were used to determine which fibrinogen peptides bound TLT-1. Flow cytometry using FITC-labelled fibrinogen binding to activated human platelets and human platelet aggregometry in the presence of these peptides were used in competitive inhibition assays. Additionally, competitive ELISAs using peptides representing regions the TLT-1 extracellular domain incubated with soluble TLT-1 in a fibrinogen coated wells were used to identify the region(s) on TLT-1 interaction with fibrinogen.

Results: We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1/Fibrinogen interaction. AFM suggests that sTLT-1 mediates fibrinogen polymerization. Competitive inhibition assays suggest complementary determining loop-1 and LP-17 (a 17 amino acid region of TLT-1) mediate TLT-1’s interaction to fibrinogen. The 3D modeling of our identified peptides show that they form a binding complex on the B module of the D-domain rather than a single binding site. These peptides competitively reduce platelet aggregation in aggregation assays and decrease fibrinogen binding to platelets in flow cytometry assays compared to controls. Cell binding and clotting assays are underway and the current status of projected will be reported here.

Conclusion(s): TLT-1 uses LP17 and CDR-1 to interact with the newly identified TLT-1 binding complex on fibrinogen. This TLT-1 binding surface may represent a targetable intervention to regulate thrombosis and inflammation.