{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Crossen J"],"funding":["NHLBI NIH HHS","National Institutes of Health","NIH"],"pagination":["697-712"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC9989883"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["122(4)"],"pubmed_abstract":["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<sup>-1</sup>) 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<sup>-1</sup>). 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<sup>-1</sup>) 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."],"journal":["Biophysical journal"],"pubmed_title":["Investigating thrombin-loaded fibrin in whole blood clot microfluidic assay via fluorogenic peptide."],"pmcid":["PMC9989883"],"funding_grant_id":["U01-HL-131053","R01 HL103419","U01 HL131053","T32-HL-007971","R01-HL-103419","T32 HL007971"],"pubmed_authors":["Crossen J","Shankar KN","Diamond SL"],"additional_accession":[]},"is_claimable":false,"name":"Investigating thrombin-loaded fibrin in whole blood clot microfluidic assay via fluorogenic peptide.","description":"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<sup>-1</sup>) 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<sup>-1</sup>). 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<sup>-1</sup>) 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.","dates":{"release":"2023-01-01T00:00:00Z","publication":"2023 Feb","modification":"2026-06-17T06:34:11.631Z","creation":"2025-04-05T23:10:57.062Z"},"accession":"S-EPMC9989883","cross_references":{"pubmed":["36635963"],"doi":["10.1016/j.bpj.2023.01.008"]}}