A. V. Udovenko, Ye. M. Makogonenko, V. O. Chernyshenko

Palladin Institute of biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine;
*e-mail: ymakogonenko@gmail.com

Received: 07 May 2026; Revised: 27 June 2026;
Accepted: 27 July 2026; Available on-line: August 2026

Background. Thrombin is the key enzyme of blood coagulation that converts fibrinogen into fibrin and activates multiple coagulation factors. However, the temporal relationship between thrombin-mediated fibrin formation and the availability of thrombin for other physiological substrates during the initiation of coagulation remains insufficiently understood. Objective. To investigate the role of fibrinogen and fibrin in regulating thrombin activity during the initiation phase of thrombin generation in plasma. Methods. Thrombin generation was initiated in human plasma using activated partial thromboplastin time (APTT) reagent or Thromborel. Fibrin formation was monitored turbidimetrically at 405 nm. Thrombin amidolytic activity toward the chromogenic substrate S2238 was measured spectrophotometrically at 405 nm. Fibrin polymerization was inhibited with the synthetic peptide GPRP. The effects of plasma dilution and inhibition of fibrin polymerization on the temporal profiles of fibrin formation and thrombin activity were analyzed. Results. During initiation of thrombin generation with both APTT-reagent and Thromborel, the beginning of cleavage of S2238 by thrombin coincided with the end of fibrin clot formation, marked by turbidimetric curve. At 1:3 plasma dilution, fibrin formation lag time was 210 s, amidase activity lag phase was 410 s. At 1:10 dilution, the lag time for fibrin formation was 73 s, amidase activity was 200 s, indicating that dilution (and lower fibrinogen) accelerates thrombin generation. With 1 mM GPRP, fibrin and amidase activity lag times increased by an order of magnitude. Conclusions. Thrombin generated during initiation of thrombin generation acts primarily on fibrinogen, remaining bound to the molecule throughout its conversion into fibrin and clot formation. Its amidase activity emerges only after protofibrils undergo lateral association into fibrin clot. Once the binding sites in the fibrin clot are saturated, thrombin becomes available for other substrates (FV, FVIII, FXI) and physiological functions.

Keywords: fibrin, fibrinogen, thrombin, thrombin generation assay