Background: Thrombin activates fibrinogen and binds the fibrin E-domain (K_d~2.8 mM) and the splice variant γ’-domain (K_d~0.1 mM).  Previous studies have shown that thrombin becomes “trapped” within fibrin fibers and will remain in the clot for extended periods of time after buffer wash.

Aims: We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability. We also confirmed previous DCS analysis of fibrin(ogen) with thermal shift assay (TSA) and looked at thermal stability of fibrin monomers in comparison to GPRP-bound fibrinogen

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

Results: Representative fibrinogen and fibrin melt curve derivative with and without GPRP. 1 mg/mL fibrinogen, 5x SYPRO, 2 mM Ca2+ in each well. (A) Fibrinogen comparison. No GPRP (black) and 5 mM GPRP (red). (B) Fibrin (50 nM thrombin). No GPRP (black) and 5 mM GPRP (red).

Summary of shift in Tm measurements from reference fibrinogen on each well plate. N = 3 plates, n = 9 wells
As indicated by large increases in T_m, 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). As expected, fibrin was more stable than fibrinogen (+Ca²⁺). 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 molar ratio to fibrin (or fibrinogen) had little effect on fibrinogen or fibrin 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. The assay can be used in future experiments to determine fibrinogen interactions in which a small volume is required due to rarity of reagents.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/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/