Thermal shift assay to probe melting of thrombin, fibrinogen, fibrin monomer, and fibrin: Gly-Pro-Arg-Pro induces a fibrin monomer-like state in fibrinogen.

ABSTRACT:

Background

Thrombin activates fibrinogen and binds the fibrin E-domain (K_d ~ 2.8 μM) and the splice variant γ'-domain (K_d ~ 0.1 μM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability.

Methods

A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (T_m) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin.

Results

Large increases in T_m indicated that calcium led to protein stabilization (0 vs. 2 mM Ca²⁺) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the T_m of thrombin (58.

SUBMITTER: Crossen J 

PROVIDER: S-EPMC7752828 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature