Peptides Derived from a Plant Protease Inhibitor of the Coagulation Contact System Decrease Arterial Thrombus Formation in a Murine Model, without Impairing Hemostatic Parameters

**Background:** Atherothrombotic diseases account for over 25% of deaths worldwide. Current antithrombotic therapies (anticoagulants and antiplatelet agents) are limited by increased major bleeding risk. The Delonix regia trypsin inhibitor (DrTI), a Kunitz-type plant protease inhibitor, inhibits trypsin (Ki_app = 2.2 × 10⁻⁸ M), plasma kallikrein (Ki_app = 5.25 × 10⁻⁹ M), factor XIIa (Ki_app = 3.1 × 10⁻⁷ M), and factor XIa (Ki_app = 1.3 × 10⁻⁶ M), and prolongs arterial occlusion time without increasing bleeding time. However, proteins face limitations including cost, stability, and multifaceted mechanisms. This study evaluated two synthetic peptides derived from DrTI's primary sequence as potential antithrombotic agents.

**Methods:** Two peptides (pDrTI and its enantiomer pDrTI-RI, synthesized with D-amino acids) were tested. Inhibitory activity against bovine trypsin and plasma kallikrein (PKa) was measured using chromogenic substrates. In vitro coagulation assays (aPTT) were performed on pooled human plasma. Platelet aggregation was assessed in human PRP using ADP (2 mM), arachidonic acid (1 mM), and collagen I (1 µg/mL) as agonists. Arterial thrombosis was induced in C57BL/6J mice by photochemical injury (Rose Bengal dye + 542 nm laser) after IV peptide administration (0.5 mg/kg). Platelet behavior was monitored by intravital microscopy following FeCl₃-induced injury. Bleeding time was measured by tail transection. Statistical analysis used Kruskal-Wallis with Dunn's multiple comparisons (p < 0.05).

**Key Results:** Neither peptide inhibited bovine trypsin at concentrations up to 680 µM. At 25 µM, neither inhibited PKa, but at high concentrations pDrTI inhibited PKa while pDrTI-RI did not, indicating stereospecificity. In vitro, pDrTI prolonged aPTT 1.5-fold at 0.55 mM; pDrTI-RI showed dose-dependent prolongation up to 8-fold at the highest concentration, with 0.27 µM producing approximately 2-fold prolongation. Ex vivo aPTT showed no changes at analyzed concentrations. Both peptides inhibited ADP- and arachidonic acid-induced platelet aggregation at low concentrations (complete inhibition at 200 µM for arachidonic acid) but did not inhibit collagen-induced aggregation. In the arterial thrombosis model, pDrTI achieved occlusion at 88.6 ± 9 min and pDrTI-RI at 97.8 ± 22 min, versus 53 ± 7 min for control and 68.5 ± 14 min for heparin (p < 0.05 for both peptides vs. control). Intravital microscopy showed both peptides reduced platelet adhesion/aggregation, with fluorescence peaking at 9 min and decreasing by 12 min, similar to native DrTI. Bleeding time was not significantly altered by either peptide, while heparin rendered mice uncoagulable.

**Clinical Implications:** These DrTI-derived peptides represent promising candidates for antithrombotic therapy that dissociate antithrombotic efficacy from bleeding risk—the major limitation of current therapies. Their antiplatelet action without prolonging bleeding time, combined with the enhanced stability conferred by D-amino acids in pDrTI-RI, addresses key translational barriers. The peptides' structural similarity to bradykinin suggests potential interaction with bradykinin B2 receptors, which could explain their effects on platelet adhesion and nitric oxide-mediated vasodilation. Further studies are needed to characterize the mechanism of action, toxicity, and pre-formulation before clinical development.