A Procoagulant Peptide Analog of the SARS-CoV-2 Nucleocapsid C-terminal Domain

Background: Uncontrolled bleeding complicates trauma, surgery and many medical conditions. While currently available procoagulant therapies (e.g., plasma-derived factors, recombinant proteins, antifibrinolytics) have crucial limitations. Methods: N389 (CQQTVTLLPAADLDDFSC) was synthesized by Fmoc solid-phase chemistry, characterized by HPLC and LC-MS, then tested in normal human pooled plasma in microplate mechanical clot-formation assays using incubated and immediate addition formats. Kinetic parameters (plasma recalcification, PRT; maximum absorbance, Amax) were obtained from absorbance curves fit to four-parameter logistic models. Mixing studies with modified (i.e., aged, adsorbed) plasma probed factor dependence. Results: In plasma coagulation assays activated with 25 mM CaCl2, baseline clotting showed a PRT of 23.74 +/- 0.27 min and Amax of 0.1813 +/- 0.0043 (n = 3), whereas N389 significantly reduced PRT to 8.442 +/- 6.0395 min without incubation (p = 0.0012), further decreased PRT after incubation (p < 0.0001), increased Amax to 0.2523, and retained comparable activity across normal, adsorbed, and aged plasma, in contrast to S1255 which showed a faster but incubation-labile effect with PRT 2.353 +/- 1.3685 min (p = 0.0007) and marked attenuation in factor-depleted and aged plasma. Mixing studies showed N389 activity persisted across normal, aged and adsorbed plasma, consistent with a mechanism that does not require intact plasma coagulation factor profiles (specifically factors II, V, VIII, VII, IX, X). Discussion: Collectively with prior evidence on anionic surfaces, Ca2+-binding Gla domains, and peptide-modulated fibrin polymerization, these results support a model in which N389 functions as a stable, charge-based scaffold that coordinates divalent cations and/or directly nucleates fibrin(ogen), while highlighting limitations of bulk clotting assays and the need for targeted thrombin generation, binding, aggregation, and contact-activation studies. Conclusions: The aspartate-rich peptide N389 is a sustained, factor-independent procoagulant at least in vitro. N389 thus merits further mechanistic and translational evaluation as a synthetic hemostatic agent.