1. Investigating thrombin-loaded fibrin in whole blood clot microfluidic assay via fluorogenic peptide
Jennifer Crossen, Kaushik N Shankar, Scott L Diamond Biophys J. 2023 Feb 21;122(4):697-712. doi: 10.1016/j.bpj.2023.01.008. Epub 2023 Jan 12.
During clotting under flow, thrombin rapidly generates fibrin, whereas fibrin potently sequesters thrombin. This co-regulation was studied using microfluidic whole blood clotting on collagen/tissue factor, followed by buffer wash, and a start/stop cycling flow assay using the thrombin fluorogenic substrate, Boc-Val-Pro-Arg-AMC. After 3 min of clotting (100 s-1) and 5 min of buffer wash, non-elutable thrombin activity was easily detected during cycles of flow cessation. Non-elutable thrombin was similarly detected in plasma clots or arterial whole blood clots (1000 s-1). This thrombin activity was ablated by Phe-Pro-Arg-chloromethylketone (PPACK), apixaban, or Gly-Pro-Arg-Pro to inhibit fibrin. Reaction-diffusion simulations predicted 108 nM thrombin within the clot. Heparin addition to the start/stop assay had little effect on fibrin-bound thrombin, whereas addition of heparin-antithrombin (AT) required over 6 min to inhibit the thrombin, indicating a substantial diffusion limitation. In contrast, heparin-AT rapidly inhibited thrombin within microfluidic plasma clots, indicating marked differences in fibrin structure and functionality between plasma clots and whole blood clots. Addition of GPVI-Fab to blood before venous or arterial clotting (200 or 1000 s-1) markedly reduced fibrin-bound thrombin, whereas GPVI-Fab addition after 90 s of clotting had no effect. Perfusion of AF647-fibrinogen over washed fluorescein isothiocyanate (FITC)-fibrin clots resulted in an intense red layer around, but not within, the original FITC-fibrin. Similarly, introduction of plasma/AF647-fibrinogen generated substantial red fibrin masses that did not penetrate the original green clots, demonstrating that fibrin cannot be re-clotted with fibrinogen. Overall, thrombin within fibrin is non-elutable, easily accessed by peptides, slowly accessed by average-sized proteins (heparin/AT), and not accessible to fresh fibrinogen.
2. Internally quenched fluorogenic substrate for furin
H Angliker, U Neumann, S S Molloy, G Thomas Anal Biochem. 1995 Jan 1;224(1):409-12. doi: 10.1006/abio.1995.1058.
A new substrate for furin, Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO2)-Asp-OH, has been synthesized and characterized. The peptide is an internally quenched fluorogenic substrate. The kinetic parameters are Km = 3.8 microM, kcat = 29.3 s-1, and kcat/KM = 7,710,000 M-1 s-1. The substrate is efficiently cleaved by furin; its kcat/KM value is over 2000-fold higher than that of the commonly used substrate Boc-Arg-Val-Arg-Arg-AMC.
3. The behaviour of urokinase and porcine kidney cell plasminogen activator towards some synthetic peptides
B Walker, D T Elmore Thromb Res. 1984 Apr 15;34(2):103-7. doi: 10.1016/0049-3848(84)90066-5.
The behaviour of human urokinase and porcine kidney cell plasminogen activator towards some synthetic substrates has been investigated. Although N- benzyloxycarbonylglycylglycyl -L-arginine 4-methyl-7- coumarylamide (Z-Gly-Gly-Arg-Amc) (I), glutaryl-Gly-Arg-Amc (II) and Z-Gly-Gly-Arg-Val-OMe (III) were substrates, Boc-Gly-Gly-Arg-Val-Val-Gly-Gly-OEt (IV) and Z-Ala-Pro-Gly-Arg-Val-Val-Gly-Gly-OEt (V) were neither substrates nor inhibitors. Steady-state kinetic parameters for the hydrolysis of (II) and (III) by urokinase and porcine kidney cell plasminogen activator were similar.
