(G-278) Hemostatic fibrin knob triggered microgel to promote clotting in neonates with lowered thrombotic risks

Introduction:: Development of effective hemostatic therapies is critical for addressing life-threatening bleeding in neonates, particularly those undergoing complex surgeries [1]. The hemostatic system in neonates is immature and exhibits significant differences in coagulation parameters compared to adults. This immaturity increases the risk of both bleeding and thrombosis, with neonates exhibiting a higher incidence of thrombosis after cardiac surgery compared to other age groups [2, 3]. Current treatments, including the use of adult blood products such as platelets and cryoprecipitate, may exacerbate these risks, highlighting the need for alternative approaches. The usual treatment for neonatal bleeding is transfusion of adult blood products (ABP). However, ABP use in neonates is linked to an increased risk of thrombotic complications [4, 5]. Here, we develop hemostatic therapies that leverage age-dependent differences in clotting mechanisms to address life-threatening post-operative bleeding in neonates. In adults, clot formation is mainly driven by A:a knob hole binding, but neonatal clot formation occurs via B:b binding. We hypothesize that targeting B:b interactions with Bknob conjugated platelet like particles (BK-PLP) could crosslink neonatal fibrinogen, catalyze neonatal clot formation, and enhance clot strength in neonates with less thrombotic risk than ABP. This approach could help treat neonatal bleeding without the associated thrombotic complications of ABP. To this end, we synthesized pNIPAm microgels and conjugated them to B-knob (AHRPYAAK), A-knob (GPRPFPAK) mimetic peptides, and non-binding control peptides (GPSPFPAK) to prepare BK-PLP, AK-PLP and NB-PLP, respectively.


Materials and Methods:: Fibrin knob triggered microgel preparation: Ultra-low crosslinked (ULC) microgels were synthesized by mixing N-isopropylacrylamide and acrylic acid in ultrapure water at a 90:10 molar ratio. The microgels were then conjugated with B-knob mimicking peptide (AHRPYAAK), A-knob (GPRPFPAK), or a non-targeting control peptide (GPSPFPAK) using EDC-Sulfo-NHS chemistry. The peptide-conjugated microgels were purified by dialysis against ultrapure water. Particle deformation was evaluated using atomic force microscopy (AFM) dry imaging.

Determination of fibrin clot polymerization and stability: Fibrin clot polymerization and stability were assessed using an endogenous fibrinolysis assay. Clots of 100 μL volume were formed with 1 mg/mL of PLPs, 0.29 μg/mL human tissue plasminogen activator and 10.8 μg/mL human glu-plasminogen. The clots were formed in 96 well plates and clot absorbance was measured at 350 nm every 30 seconds for 3 hours. Area under the curve was calculated for all samples.

Clot mechanics: Clot stiffness was measured by generating 50 μL clots on a glass slide with 0.5 U/mL human thrombin and allowing them to polymerize for 1 hour prior to force measurements using AFM (Asylum MFP3D-Bio, Asylum Research, Morrisville, NC, USA). We used standard silicon nitride cantilevers from Nanoandmore (Watsonville, CA, USA) with a particle diameter of 1.98 µm.


Results, Conclusions, and Discussions:: We prepared three different types of peptide conjugated particles named BK-PLP, Ak-PLP, and Nonbinding-PLP (Fig. 1A). Their ability to alter fibrin clot morphology, stability, and stiffness was evaluated. Particle size was measured via AFM dry imaging (Fig. 1B) and diameters were 1.84±0.22, 1.33±0.05, and 1.30±0.04 µm for NB-PLP, AK-PLP, and BK-PLP, respectively (Fig. 1C).

An absorbance-based assay was utilized to assess the degradation rates of clots formed using either 0, 0.5, 1, 1.5, or 2 mg/mL microgels. Results show that the optimal particle concentration is 1 mg/mL, and BK-PLPs show significantly greater stability than AK-PLP, Nonbinding-PLP, or fibrin-only clots, as measured by the integrated area of the polymerization/degradation curve of each clot type, indicating that incorporation of BK-PLP into clots increases clot stability (Fig. 2A and B).

Clot microstructure was evaluated by CryoSEM (Fig. 3A). Quantification of pore size from images of different types of peptide-conjugated PLP-incorporated clots did not show significant differences. To determine how the structure of neonatal fibrin networks is affected by the addition of fibrin knob triggered microgels, we investigated the mechanical properties of clots formed from these same groups using AFM nano-indentation (Fig. 3A-B). There was a decrease in clot stiffness following the addition of AKnob and Nonbinding-PLPs when compared to clots without particles (P < 0.05). The possible explanation for this effect is that the nanoparticles may disrupt the clot network structure and increase the porosity of the clot, which could lead to a decrease in its stiffness.

Conclusion

Our study investigated the effects of three different types of peptide conjugate particles (BK-PLP, Ak-PLP, and Nonbinding-PLP) on fibrin clot morphology, density, stability, and stiffness. We found that the incorporation of BK-PLP into clots increased their stability compared to AK-PLP, Nonbinding-PLPs, or fibrin-only clots. Additionally, we observed a decrease in clot stiffness following the addition of AK-PLPs and Nonbinding-PLPs, indicating that these nanoparticles may disrupt the clot network structure and increase porosity. These results provide a promising foundation for further development of BK-PLP as a potential hemostatic therapy for neonates undergoing complex surgeries.


Acknowledgements (Optional): :

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