Abstract Body (Do not enter title and authors here): Introduction: Fibrin formation begins with the thrombin-catalyzed cleavage of fibrinopeptide A from fibrinogen followed by exposure of the Gly-Pro-Arg(GPR) sequence called knob 'A'. This motif interacts with complementary holes ‘a’ in other fibrin molecules, driving fibrin polymerization. The GPRP peptide mimics knob 'A' and blocks competitively the knob-hole interactions, inhibiting fibrin assembly. Interruption of fibrin formation represents a novel strategy for anticoagulation to prevent or attenuate thrombosis.
Hypothesis: The synthesized GPRP-dextran conjugate possesses anticoagulant activity in vitro and in vivo.
Methods: GPRP was attached covalently to dextran to prolong its circulation time. The anticoagulant activity of the conjugate was assessed via aPTT and ROTEM in human PFP. Fibrin polymerization was evaluated by dynamic turbidimetry and clottable fibrinogen levels. Fibrin clot structure was analyzed using scanning electron microscopy (SEM) and fluorescent confocal microscopy. In vivo effects were studied in mice after intravenous injection of the conjugate, followed by bleeding time analysis and ex vivo blood clotting assays.
Results: GPRP–dextran significantly prolonged aPTT and changed the ROTEM parameters, including prolonged clotting time, reduced clot strengthening rate and clot firmness. The GPRP-dextran conjugate suppressed fibrin polymerization in a dose-dependent manner with an IC50 of ~ 40μM and reduced the clottable fibrinogen level, indicating that the conjugate can be used to push the equilibrium toward dissociation, without entirely abrogating clotting. SEM revealed that fibrin formed in the presence of GPRP–dextran was structurally abnormal with shortened and thickened fibers with barbed ends and clusters. Confocal microscopy confirmed these observations, showing disrupted fibrin networks and decreased fiber branching density in the GPRP-dextran-containing samples. After intravenous injection in mice and 15 minutes of circulation, ex vivo testing demonstrated a notable reduction in clottable fibrinogen levels. Importantly, the treatment did not prolong significantly tail bleeding time, indicating that the anticoagulant effect did not compromise hemostatic safety.
Conclusion: GPRP–dextran exhibits anti-fibrin polymerization effects in vitro and in vivo, highlighting its potential as a novel anticoagulant and antithrombotic agent with minimal bleeding risk.
This work was supported by American Heart Association grant#25POST1357254.