PB0893 - Characterization of the TLT-1 Fibrinogen ligand interaction.

Background: Platelets play a central role in thrombosis and hemostasis, additionally, they act as immune cells to regulate various vascular inflammatory conditions. Upon activation, platelets release TREM-Like Transcript-1 (TLT-1) from their -granules. Amino link columns and immunoblotting identifies fibrinogen as a ligand for TLT-1. Fibrinogen is a plasma protein that facilitates thrombosis and inflammation.
TLT-1 binding fibrinogen was surprising since αIIbβ3, the most abundant platelet receptor, binds fibrinogen, facilitating platelet aggregation. Although TLT-1 may assist in clot formation and hemostasis like αIIbβ3, TLT-1’s main association is with regulating inflammatory-derived bleeding. Little is known about the TLT-1-fibrinogen interaction.

Aims: Delineate the TLT-1-fibrinogen molecular interaction.

Methods: We used Octet Qke Bio-layer Interferometry (BLI) to determine binding affinity (Figure 1). Subsequently we digested fibrinogen, carried out an immunoprecipitation (IP) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to identify potential interacting peptides of TLT-1 and fibrinogen. Effects of these peptides on competitive ELISAs, cell binding assays, platelet aggregometry, and flow cytometry were used to determine which of the identified peptides have the greatest inhibitory effect. ELISAs adding TLT-1 to a fibrinogen coated plate were designed to determine the region on TLT-1 responsible for binding fibrinogen.

Results: We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1-fibrinogen interaction, demonstrating a high affinity interaction. Alpha chain peptides 272-287, 548-558 (GGSTSYGTGSETESPR, GSESGIFTNTK respectively) and beta chain peptides 256-264, 271-283 (QDGSVDFGR and QGFGNVATNTDGK respectively) that potentially bind TLT-1 were identified by mass spectometry. Using these identified peptides, TLT-1 extracellular domain loops and a 17-mer (LP17) in competitive platelet aggregometry, flowcytometric assays, and binding of HEK293 cells stably transfected with TLT-1, to fibrinogen, peptides 548-558 GSESGIFTNTK and 271-283 QGFGNVATNTDGK of fibrinogen, P17 and CDR3 loop of TLT-1 demonstrate the highest inhibition.

Conclusion(s): These results suggest potential regions of interaction between TLT-1-fibrinogen. Our poster will report the current state of these studies.