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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1865; no. 2; p. 129805
• Main Authors: Crossen, J., Diamond, S.L.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2021
• Subjects: active sites; Amino Acid Chloromethyl Ketones - chemistry; Bleeding; calcium; Fibrin; Fibrin - chemistry; Fibrin Fibrinogen Degradation Products - chemistry; Fibrinogen; Fibrinogen - chemistry; Fibrinogens, Abnormal - chemistry; Humans; mutants; Oligopeptides - chemistry; polymerization; Protein Stability; Thrombin; Thrombin - chemistry; Thrombosis; Transition Temperature; Fibrin; Thrombin; Thrombosis; Bleeding; Fibrinogen
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129805

Thrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 μM) and the splice variant γ’-domain (Kd ~ 0.1 μM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability. A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin. Large increases in Tm 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 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 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ’-domain interaction. 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 low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants. [Display omitted] •Ligand bonding to a protein can impact its thermal stability.•Thermal denaturation of fibrinogen, fibrin monomer, thrombin, and fibrin complexes was studied.•Binding of Gly-Pro-Arg-Pro to fibrinogen induces a fibrin monomer like state.•Thrombin binding to fibrin did not alter fibrin stability.