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 (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔT_m = 9.3 °C, similar to the ΔT_m when fibrinogen was converted to fibrin monomer (ΔT_m = 8.8 °C) or to fibrin (ΔT_m = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) T_m values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction.

Conclusions

TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin.

Significance

The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.

SUBMITTER: Crossen J 

PROVIDER: S-EPMC7752828 | biostudies-literature |

REPOSITORIES: biostudies-literature