Abstract Number: PB0403
Meeting: ISTH 2021 Congress
Background: Thrombin activates fibrinogen and binds the fibrin E-domain (Kd~2.8 mM) and the splice variant γ’-domain (Kd~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 (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin.
|Sample||Average ΔTm (°C)|
|Fibrinogen||0.0 ± 0.4|
|Fibrinogen; 5 mM GPRP||9.3 ± 0.4|
|Fibrin; 5 mM GPRP||8.8 ± 0.4|
|Fibrin||10.4 ± 0.4|
|Fibrinogen; 300 nM PPACK-thrombin||-0.3 ± 0.4|
|Fibrin; 300 nM PPACK-thrombin||9.7 ± 0.2|
As indicated by large increases in Tm, 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 Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3°C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm= 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 molar ratio to fibrin (or fibrinogen) had little effect on fibrinogen or fibrin Tm 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.
To cite this abstract in AMA style:Crossen J, Diamond S. 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]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). 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/. Accessed November 28, 2023.
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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/