Hemostatic B-knob–triggered microgels (BK-TriGs) to address bleeding in neonates

Abstract

Hemostatic immaturity in neonates presents critical challenges, especially during surgery, where bleeding and thrombosis risks are elevated. Current treatments rely on transfusing adult blood products, which may cause complications resulting from structural and functional differences between neonatal and adult fibrinogen. To address this, we developed hemostatic B-knob–triggered microgels (BK-TriGs) that target and bridge fibrinogen hole b sites. Functionalized with a fibrin hole b–specific peptide, BK-TriGs enhance clot density and resistance to degradation. In vitro studies using neonatal platelet-poor plasma (PPP) showed that at an optimal concentration, BK-TriGs increased clot density by more than 100% and improved stability by reducing fibrinolysis. Under flow conditions, BK-TriGs promoted robust clot formation compared to plasma-only controls. In an in vivo fib-null (Fga−/−) mouse model transfused with neonatal fibrinogen, BK-TriGs reduced blood loss by 50 to 60% and enhanced fibrin deposition at wound sites. This targeted approach offers a safer, more effective hemostatic solution tailored to neonatal clotting needs.

Targeted microgels enhance neonatal clotting, reducing blood loss and improving hemostasis without adult blood product risks.

INTRODUCTION

Uncontrolled bleeding presents a critical health care challenge, especially for neonates undergoing surgical interventions or experiencing trauma. This population faces heightened risks of bleeding and thrombosis because of an underdeveloped hemostatic system (1). Clinicians typically manage neonatal bleeding by transfusing adult blood products, such as platelets, packed red blood cells, fresh frozen plasma, and cryoprecipitate, which contain adult fibrinogen. However, these transfusions pose serious safety concerns by increasing morbidity, prolonging intensive care unit stays, and elevating posttransfusion thrombosis risks in neonates (2, 3). Specifically, adult fibrinogen transfusion may contribute to thrombosis as a result of structural and functional differences between neonatal and adult fibrin clots. Neonatal clots are softer, exhibit less fiber branching, and degrade more rapidly than adult fibrinogen–based clots. When neonates receive adult fibrinogen, their clots become stiffer and more resistant to fibrinolysis, increasing the risk of thrombotic complications (4).

To address these limitations, researchers have explored alternative therapies for neonatal bleeding, including clotting factor administration via prothrombin complex concentrate, fibrinogen concentrate, and recombinant factor VIIa (5–7). However, these approaches still carry risks of off-target thrombosis. Recent advances in biomaterial-based hemostatic solutions offer promising alternatives, particularly artificial platelet-mimetic materials (8). Such therapies could be used to enhance neonatal coagulation mechanisms without directly transfusing adult clotting factors. These materials directly interact with the body’s native coagulation mechanisms, efficiently treating both internal and external bleeding while reducing costs and minimizing risks of immunogenicity and infection. Among them, synthetic hemostatic particles—particularly highly deformable microgels known as platelet-like particles (PLPs)—demonstrate potential by mimicking key platelet functions (9). These microgels bind fibrin and exert mechanical force to stabilize clots, reduce clot size, and create a robust framework for wound healing (9). Their design highlights the importance of nanoparticle deformability and morphology in replicating platelet functionality and improving hemostasis (10). In addition, they exhibit excellent biocompatibility and clearance in vivo (9).

Neonates present unique challenges because of their immature coagulation system (11). Researchers have identified key differences in the coagulation cascade between neonates and adults, including neonatal platelets’ hyporeactivity and variations in clotting factor concentrations and activities (12). Neonatal fibrinogen exhibits lower activity and longer thrombin clotting times than adult fibrinogen (11–13). Our recent studies revealed age-dependent differences in fibrin knob:hole dynamics during clot polymerization. Following injury, thrombin cleaves fibrinopeptides A and B from the N-terminal ends of the Aα and Bβ chains of fibrinogen, exposing knobs A and B. These knobs bind complementary fibrin holes a and b on adjacent fibrin(ogen) molecules. In adults, fibrinopeptide A undergoes cleavage faster than fibrinopeptide B, and A:a interactions drive early fibrin monomer assembly into protofibrils. While researchers have yet to fully define the role of B:b interactions in fibrin polymerization, studies suggest that these interactions promote lateral aggregation of protofibrils to support fibrin fiber formation (14). In addition, fibrinopeptide B release appears to facilitate αC-αC interactions (15).

Competition assays using high concentrations of fibrin knob A and B mimetic peptides showed that knob A mimetics partially inhibited clot polymerization in adult clots but had a minimal effect on neonatal clots. Conversely, neonatal clot polymerization was greatly diminished in the presence of high concentrations of knob B mimetics but had a minimal effect on adult clots, indicating that neonatal fibrin polymerization is more strongly governed by B-knob:hole b interactions than by A-knob engagement (16). While these studies were performed with high concentrations of knob mimetics, preengagement of hole b using subsaturating concentrations of knob B peptides can enhance clot structure, likely through conformational changes that promote B:b interactions and tighter molecular packing (17).

Our prior studies also demonstrated that neonatal fibrinogen releases fibrinopeptide A at a reduced rate compared to adult fibrinogen, while fibrinopeptide B release increases. However, the relative timescale of fibrinopeptide B release remains similar between neonates and adults. Considering these findings, we hypothesized that B-knob–triggered microgels (BK-TriGs), created by conjugating fibrin knob B mimetic peptides to highly deformable microgels previously used for platelet-like particles, could capitalize on these interactions to enhance clotting and improve clot structure. We propose that BK-TriGs target and engage fibrinogen hole b before substantial thrombin-mediated B-knob release. This early binding may facilitate conformational changes or enhanced B:b interactions that promote lateral aggregation of protofibrils, strengthen the clot structure, and improve mechanical stability. Similar polymerization-modulating effects have been observed in prior studies using synthetic knob B peptides (17–19). Like first-generation platelet-like particles, BK-TriGs use highly deformable microgel particles, enabling them to induce mechanical reinforcement of the clot structure through a clot retraction–like mechanism. This action further stabilizes clots and improves long-term healing outcomes (9). BK-TriGs provide advantages over simple peptide delivery by mediating fibrin bridging and clot retraction.

In this study, we conducted in vitro experiments using neonatal platelet-poor plasma (PPP) to assess clot structure, density, and mechanical properties. We used confocal microscopy, intrinsic fibrinolysis assays, atomic force microscopy (AFM), and cryo–scanning electron microscopy (cryo-SEM) to visualize clot formation and quantify key parameters, including pore size, fiber intersection density, and clot stiffness. We also compared BK-TriGs to other microgel variants, including A-knob–targeting microgels [AK-ultralow cross-linked microgels (ULCs)] and nonbinding control particles (NB-ULCs).

To validate the clinical relevance of our findings, we conducted in vivo testing in a fib-null (Fga−/− FIB-KO) mouse model transfused with neonatal fibrinogen. We monitored blood loss in a traumatic liver laceration model to assess BK-TriGs’ ability to enhance clot stability and reduce bleeding. Our findings support BK-TriGs as a promising approach for improving hemostasis in neonates, offering a tailored, effective solution for this vulnerable patient population.

RESULTS

Micro–computed tomography analysis of age-dependent clot properties and synthesis and characterization of peptide-conjugated microgels

In this study, we evaluated the hemostatic efficacy of BK-TriGs by examining their effects on clot properties. We first characterized age-dependent structural differences in plasma clots by using micro–computed tomography (micro-CT) to quantify clot porosity. Clots formed from neonatal plasma exhibited a significantly higher porosity (95.9%) compared to those in adult plasma (54.1%). These findings highlight the age-dependent structural disparities of the clots investigated herein, which may influence clot stability and mechanical properties (Fig. 1, A to D). This comparison emphasizes the need for tailored hemostatic strategies that account for the unique properties of neonatal plasma.

Fig. 1. Micro-CT analysis of clots formed in neonatal and adult plasma and synthesis and characterization of peptide-conjugated microgel.

Micro-CT analysis of clots formed in adult plasma: (A) 3D reconstruction and (B) 2D cross-sectional view of the fibrin network structure. Corresponding 3D (C) and 2D (D) views for clots formed in neonatal plasma. The porosity analysis reveals differences in porosity between clots formed from neonatal (~95%) and adult (~54%) plasma, regardless of particle presence. (E) Schematic illustration of the synthesis process of ULCs, showing the polymerization of NIPAm and AAc to form pNIPAm-co-AAc microgels, followed by functionalization with EDC and sulfo-NHS to conjugate A-knob, B-knob, or NB peptides, creating NB-ULCs, AK-ULCs, and BK-TriGs. (F) AFM images of NB-ULCs, AK-ULCs, and BK-TriGs, showing the distinct morphology and surface characteristics of each particle type. Scale bars, 1 μm. (G) Quantitative analysis of particle size, with a comparison of the diameters of NB-ULCs, AK-ULCs, and BK-TriGs. Data are presented as the means ± SD, with significant differences indicated (****P < 0.0001 and **P < 0.01).

Our previous research on poly(N-isopropylacrylamide) (pNIPAm)–based ultrasoft microgels (ULCs) demonstrated that particle deformability plays a key role in inducing mechanical stabilization of clot structure in a manner reminiscent of clot retraction. Upon binding to fibrin, these particles undergo large shape changes, promoting effective clot stabilization (10, 20). We synthesized pNIPAm-co-acrylic acid (pNIPAm-co-AAc) ULCs via precipitation polymerization, producing microgels capable of substantial deformation post–fibrin binding, similar to the behavior of activated platelets (10).

Peptide conjugation is critical for enhancing the hemostatic function of these microgels, as it dictates their interaction with fibrin and overall clotting efficiency. To assess this effect, we compared BK-TriGs, conjugated to the B-knob peptide (AHRPYAAK) targeting fibrin hole b, with two additional microgel groups: AK-ULCs, conjugated to an A-knob peptide (GPRPFPAK), and NB-ULCs, conjugated to a nonbinding peptide (GPSPFPAK) as a control, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-sulfo-N-hydroxysuccinimide (NHS) chemistry (Fig. 1E). AFM images of the microgels are shown in Fig. 1F, with quantitative measurements of particle diameter presented in Fig. 1G. The measured diameters for the different microgels were 1846 ± 224 nm (NB-ULCs), 1330 ± 51 nm (AK-ULCs), and 1073 ± 176 nm (BK-TriGs). The decrease in diameter after peptide conjugation likely results from cross-linking, which slightly compresses the particle structure (9).

BK-TriGs increase clot density in neonatal plasma

We assessed how BK-TriGs, AK-ULCs, and NB-ULCs altered fibrin clot architecture by quantifying clot density in neonatal and adult PPP. Figure 2A illustrates the clot formation process with BK-TriG incorporation. Using confocal microscopy, we assessed the effect of BK-TriGs on clot density in neonatal and adult plasma (Fig. 2, B to D). In neonatal plasma, BK-TriGs at 1 mg/ml significantly increased the fiber density from 0.16 ± 0.07 (fibrin-only controls) to 1.32 ± 0.2 (P < 0.0001), supporting their role in clot stabilization. However, at higher concentrations (>1 mg/ml), the fiber density decreased. At 2 mg/ml, the fiber density dropped to 0.235 ± 0.1 (P ≤ 0.0001), suggesting a potential inhibitory effect on polymerization (Fig. 2, B and C, and fig. S1). This biphasic response may result from competition between synthetic knob B-hole b interactions and native fibrin polymerization at higher concentrations (19). We next compared this effect in adult plasma using the same microgel concentrations. In adult plasma, AK-ULCs were the most effective in enhancing clot density (Fig. 2, B and D). At 1, 1.5, and 2 mg/ml, AK-ULCs significantly increased clot density compared to NB-ULCs and BK-TriGs. The 2 mg/ml AK-ULC group exhibited the highest clot density (1.86 ± 0.41), outperforming all other groups. The AK-ULCs showed a dose-dependent response, with 1 mg/ml (1.61 ± 0.65) and 1.5 mg/ml (1.31 ± 0.52) concentrations also displaying substantially higher clot densities than NB-ULCs and BK-TriGs. All BK-TriG concentrations resulted in lower clot densities than their corresponding AK-ULC concentrations. Across all tested concentrations (0.5, 1, 1.5, and 2 mg/ml), NB-ULCs showed no statistically significant differences (P > 0.99) compared to BK-TriGs and controls, indicating minimal fibrin interaction. To additionally benchmark clot density changes when adult fibrinogen is added directly to clots, mimicking transfusion, we supplemented neonatal and adult plasma with endogenous adult fibrinogen (1 or 2.5 mg/ml) (fig. S2). Supplementation with adult fibrinogen (1 mg/ml) did not significantly alter clot density in either neonatal or adult plasma, whereas addition of adult fibrinogen (2.5 mg/ml) [representing the upper end of physiologic and therapeutic levels (21)] resulted in a marked increase in density. Using BK-TriGs to enhance neonatal-only fibrin clot formation circumvents the need to directly use adult fibrinogen, which creates more thrombotic, mixed fibrinogen clots.

Fig. 2. Impact of microgel incorporation on clot network density in neonatal and adult plasma.

(A) Illustration of the fibrin polymerization mechanism triggered by B-knob interactions. (B) Representative images of neonatal and adult plasma clot networks including NB-ULCs (1 mg/ml), AK-ULCs (1 mg/ml), and BK-TriGs (1 mg/ml), showing differences in clot density and network formation. (C) Quantitative analysis of clot density in neonatal plasma. Data are shown as the means ± SD with statistical significance indicated (**P < 0.01 and ****P < 0.0001). (D) Quantitative analysis of clot density in adult plasma. Data are presented as the means ± SD with significance levels indicated (****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05). ns, not significant.

To further examine clot formation, we used time-lapse confocal microscopy, incorporating fluorescently labeled BK-TriGs into the neonatal fibrin network (Fig. 3A) and AK-ULCs in adult plasma (fig. S4). Imaging at 0, 30, and 60 min post–clot formation confirmed that particles remained evenly distributed within the fibrin matrix without visible aggregation. Compared to neonatal plasma–only clots (Fig. 3B), BK-TriG–incorporated clots exhibited higher branching and density and particle distribution within the fibrin network. Compared to neonatal plasma–only clots (Fig. 3B), BK-TriG–incorporated clots exhibited higher branching and a denser fibrin network. Furthermore, time-lapse quantification showed increasing BK-TriG clustering over time, consistent with particle accumulation within forming fibrin-dense regions, as reflected by the rising number of clusters and total particle-associated area (fig. S3). Cryo-SEM imaging with false coloring further confirmed BK-TriG incorporation within the pore walls of fibrin clots, reinforcing the clot structure (Fig. 3C). Although high-magnification images reveal local particle clustering, lower-magnification confocal imaging demonstrates broad dispersion throughout the clot, consistent with the clot retraction model in which synthetic platelets contract fibrin fibers and pull incorporated materials, including particles, into denser regions during clot maturation (10). Findings demonstrate that BK-TriGs significantly enhance clot density in neonatal plasma at optimal concentrations. However, in adult plasma, AK-ULCs outperform BK-TriGs, particularly at higher concentrations. This study underscores the need to tailor hemostatic therapies to neonatal and adult patients to optimize clot formation and stability in clinical settings.

Fig. 3. Visualization of fibrin network formation and BK-TriG integration into clots.

(A) Confocal microscopy images of neonatal plasma networks formed over time in the absence of BK-TriGs, illustrating the evolution of the fibrin matrix at 0, 30, and 60 min. (B) Confocal images depicting the interaction and integration of BK-TriGs (red) within the fibrin network (green) over a time course of 0, 30, and 60 min, demonstrating enhanced fibrin network formation facilitated by BK-TriGs. (C) Scanning electron microscopy image highlighting the structural integration of BK-TriGs (false coloring) within the fibrin matrix, emphasizing the close association and distribution of the microgels within the fibrin architecture.

BK-TriGs improve fibrin clot stability

To assess fibrin clot resistance to enzymatic degradation, we quantified clot lysis profiles in the presence of microgels using a fibrinolysis assay. We hypothesized that the increased clot density observed with BK-TriGs in neonatal plasma would also translate into enhanced clot stability by reducing fibrinolysis. To test this, we performed an absorbance-based fibrinolysis assay, comparing clot stability across different BK-TriG concentrations (0.5, 1, 1.5, and 2 mg/ml) with control clots (plasma-only, NB-ULCs, and AK-ULCs) (representative curves shown in figs. S5 and S6). At the optimal concentration (1 mg/ml), BK-TriGs significantly increased clot stability, as demonstrated by a larger integrated area under the polymerization/degradation curve (1046 ± 290) compared to plasma-only clots (368 ± 171, P = 0.0001) and other control groups [AK-ULCs (1 mg/ml): 622 ± 71; NB-ULCs (1 mg/ml): 523 ± 121] (Fig. 4A). The polymerization and degradation rate, half-maximum polymerization time, maximum turbidity, and half-maximum degradation were measured to evaluate clot formation and stability (Fig. 4, B to E, and fig. S7A). BK-TriG clots exhibited lower degradation rates and higher maximum turbidity, further supporting their enhanced stability. These findings suggest that BK-TriGs mimic platelet-mediated stabilization by forming multipoint interactions with fibrin, thereby reinforcing the neonatal clot structure. Despite the similar size, AK-ULCs did not enhance the clot structure like BK-TriGs, suggesting that the effect is due to specific B-knob interactions rather than particle size.

Fig. 4. Intrinsic fibrinolysis assay demonstrating clot formation and degradation in neonatal and adult plasma treated with various microgels.

(A to D) Quantitative analysis of fibrinolysis demonstrating clot formation and degradation in neonatal plasma treated with various microgels. (A) Rate of fibrin degradation (min⁻¹) indicating the effect of microgels on fibrinolysis. (B) Maximum turbidity [absorbance at 350 nm (A₃₅₀)], representing fibrin clot density and stability in neonatal and adult plasma. (C) Rate of fibrin polymerization (min⁻¹), reflecting the efficiency of fibrin network formation. (D) Half-maximum polymerization time (min) showing the kinetics of clot formation. NB-ULCs, AK-ULCs, and BK-TriGs were tested at concentrations of 0.5, 1, 1.5, and 2 mg/ml. (E) Quantitative area under the curve (AUC) analysis for neonatal plasma. (F to I) Quantitative analysis of fibrinolysis demonstrating clot formation and degradation in adult plasma treated with various microgels. (F) Rate of fibrin degradation (min⁻¹), indicating the effect of microgels on fibrinolysis. (G) Maximum turbidity (A₃₅₀), representing fibrin clot density and stability in neonatal and adult plasma. (H) Rate of fibrin polymerization (min⁻¹), reflecting the efficiency of fibrin network formation. (I) Half-maximum polymerization time (min), showing the kinetics of clot formation. NB-ULCs, AK-ULCs, and BK-TriGs were tested at concentrations of 0.5, 1, 1.5, and 2 mg/ml. (J) Quantitative area under the curve analysis for neonatal plasma. Data are presented as the means ± SD with significance levels indicated (****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05).

To determine whether BK-TriG clot stabilization was age-dependent, we next examined adult plasma clots. In adult plasma, BK-TriGs and AK-ULCs both improved clot stability at higher concentrations (2 mg/ml) (Fig. 4F). However, unlike in neonatal plasma, BK-TriGs did not show preferential targeting. Specifically, AK-ULCs at 2 mg/ml significantly improved clot stability, but BK-TriGs at the same concentration exhibited a similar effect. Furthermore, in adult plasma, both AK-ULCs (1.5 mg/ml) and BK-TriGs (1.5 to 2 mg/ml) reduced degradation compared to other groups, with no significant differences in polymerization rate or maximum turbidity. Only high-dose BK-TriGs increased half-maximum turbidity, suggesting no clear preference for AK-ULCs or BK-TriGs in adults, unlike the neonatal setting (Fig. 4, G to J). Half-maximum degradation time analysis (fig. S7B) indicated prolonged clot persistence with both BK-TriGs and AK-ULCs compared to plasma-only controls, supporting their stabilizing effects in adult plasma. These results indicate that adult plasma does not exhibit the same selective response to BK-TriGs as neonatal plasma. Instead, A-knob interactions in AK-ULCs appear equally effective at higher concentrations, suggesting that adult fibrin polymerization mechanisms differ from neonatal clot formation. To further validate these findings under more physiologically relevant conditions, we next evaluated clot formation and degradation using a custom microfluidic device.

BK-TriGs enhance clot formation under flow

Using a custom T-junction microfluidic device, we observed clotting dynamics under flow conditions (Fig. 5 and fig. S8). A stationary fibrin clot was formed using plasma (neonatal and adult), thrombin (0.5 U/ml), and Alexa Fluor 488–labeled fibrinogen (10 μg/ml) for visualization. After polymerization, a solution containing Alexa Fluor 647–labeled BK-TriGs (1 mg/ml) in neonatal plasma, thrombin (0.1 U/ml), and Alexa Fluor 488–labeled fibrinogen (10 μg/ml) in Hepes buffer was flowed over the stationary clot for 20 min at a wall shear rate of 10 s⁻¹. Figure 5 illustrates the impact of BK-TriGs on clot formation and stability under flow conditions using the microfluidic device to assess clot growth and degradation. Figure 5A presents a schematic of the experimental setup, showing how the microfluidic channel enables the infusion of fibrinogen, thrombin, and microparticles to form a stationary clot at the interface. This setup allowed for real-time monitoring of clot growth and degradation (in subsequent experiments) over time. Figure 5B displays a confocal microscopy image of the clot reservoir within the microfluidic device, highlighting the fibrin clot structure with incorporated BK-TriGs. Figure 5C provides a magnified view of the clot/solution interface, showing BK-TriG incorporation (red) into the fibrin network (green). Figure 5 (D and E) presents time-lapse confocal images, demonstrating clot formation over 20 min. Movie S1 shows neonatal plasma alone under flow, and movie S2 shows BK-TriGs that promote clot growth under flow conditions compared to plasma only. BK-TriGs substantially promoted clot growth in neonatal plasma, whereas AK-ULCs were more effective in adult plasma (Fig. 5, D and E). Quantitative analysis of total clot growth area is shown in Fig. 5 (F and G) for neonatal and adult plasma, respectively. In neonatal plasma, BK-TriGs significantly increased clot growth compared to both NB-ULCs and AK-ULCs (P < 0.01), indicating specific B-knob interactions in neonatal plasma. However, in adult plasma, clot growth was comparable between BK-TriGs and AK-ULCs (P > 0.05).

Fig. 5. Evaluation of clot formation and degradation using a custom microfluidic device.

(A) Schematic representation of the microfluidic setup for assessing clot dynamics. (B) Wide-field image showing fibrin and microparticle distribution at the clot-solution interface. (C) Confocal images highlighting clot growth and degradation over time. Representative images of clot growth in neonatal (D and F) and adult plasma (E and G) treated with various microgels, demonstrating enhanced clot stability with BK-TriGs. (H to K) Clot degradation analysis for neonatal (H and J) and adult plasma (I and K), with quantitative assessments revealing significantly reduced degradation rates for BK-TriG–treated clots. h, hours. Dashed lines in time-lapse images indicate the region of interest used for quantifying clot growth or degradation over time. Data are expressed as the means ± SD, with significant differences indicated (*P < 0.05 and **P < 0.01).

Clot degradation rates using a custom microfluidic device

Clot degradation rates were assessed using a custom microfluidic device to compare the effects of different microgels in neonatal and adult plasma clots (Fig. 5, H and I). In neonatal plasma, the degradation rate was significantly higher in the control group (plasma only), measured at 49.2 ± 12.1 μm² s⁻¹, compared to the microgel-treated groups. The incorporation of AK-ULCs and BK-TriGs significantly reduced clot degradation rates. Notably, BK-TriGs demonstrated the most substantial reduction, with a degradation rate of 12.3 ± 5.5 μm² s⁻¹ compared to the control (P = 0.0092). AK-ULCs also showed a significant reduction, with a degradation rate of 14.8 ± 7.0 μm² s⁻¹ compared to the control (P = 0.0184). However, the difference between NB-ULCs and BK-TriGs was not statistically significant (P = 0.599), indicating that NB-ULCs were less effective in reducing clot degradation (Fig. 5J). In adult plasma, degradation rates were also significantly reduced with microgel incorporation, particularly with AK-ULCs and BK-TriGs. The control group exhibited the highest degradation rate at 43.7 ± 11.5 μm² s⁻¹, while AK-ULCs and BK-TriGs significantly decreased degradation rates to 23.5 ± 9.4 μm² s⁻¹ (P = 0.0272) and 22.8 ± 8.1 μm² s⁻¹ (P = 0.0396), respectively. However, the difference between AK-ULCs and BK-TriGs was not statistically significant (P > 0.9999), suggesting that both particles effectively stabilized the clot structure in adult plasma (Fig. 5K). These results indicate that both AK-ULCs and BK-TriGs significantly enhance clot stability by reducing degradation rates. However, BK-TriGs exhibited the most pronounced effect in neonatal plasma, supporting their targeted design for neonatal clot stabilization.

BK-TriGs increase clot stiffness in neonatal plasma

We used AFM to measure fibrin clot stiffness in neonatal and adult plasma with and without particle incorporation. These measurements evaluated clot stiffness in neonatal and adult plasma. AFM measurements for neonatal clot stiffness are shown in Fig. 6 (A and B). Force maps in Fig. 6A display the elastic modulus distribution for each group, with histograms illustrating the frequency distribution of stiffness values. The data demonstrated that BK-TriG clots were significantly stiffer than plasma-only controls (plasma only: 1030 ± 430 Pa; BK-TriGs: 2098 ± 1002 Pa; AK-ULCs: 1041 ± 390 Pa). The plasma-only (control) group exhibited a relatively uniform stiffness distribution, with a mean elastic modulus of ~1 kPa. Statistical analysis (Fig. 6B) confirmed significant differences in clot stiffness across groups. BK-TriGs at 1 mg/ml significantly increased clot stiffness compared to the control, indicating enhanced clot stability.

Fig. 6. Elastic modulus of neonatal and adult plasma clots with microgel treatments.

(A) AFM images and modulus histograms of neonatal plasma clots treated with NB-ULCs, AK-ULCs, and BK-TriGs compared to controls. (B) Quantified stiffness of neonatal clots, showing significant increases with BK-TriGs (****P < 0.0001, **P < 0.01, and *P < 0.05). (C) AFM images and histograms for adult clots. (D) Stiffness of adult clots, with AK-ULCs and BK-TriGs significantly stiffer than controls (****P < 0.0001). Error bars: means ± SD.

AFM measurements for adult plasma clots are presented in Fig. 6 (C and D). Similar to neonatal clots, force maps and histograms provide detailed stiffness distributions. However, in adult plasma, the addition of particles generally decreased the elastic modulus, suggesting that these particles soften the clot structure. In the NB-ULC group, concentrations above 0.5 mg/ml significantly reduce the elastic modulus, demonstrating a pronounced effect on clot stiffness. Similarly, in the AK-ULC group, concentrations greater than 1 mg/ml led to a significant decrease in the elastic modulus, with the 2 mg/ml concentration showing the most substantial reduction. The BK-TriG group also shows a reduction in elastic modulus compared to the control, suggesting that these particles contribute to a softer clot structure. Overall, NB-ULCs, AK-ULCs, and BK-TriGs each have a noticeable impact on reducing clot stiffness, with the effect varying on the basis of the concentration and type of particle, underscoring their potential role in modulating clot mechanical properties.

Clot microstructure in neonatal and adult plasma clots

To complement our initial fibrin density estimates from binarized fluorescence images, we performed cryo-SEM and quantified the pore size, percent porosity, and fiber intersection density, confirming that microgel incorporation alters the clot microstructure in both neonatal and adult plasma. Although both BK-TriGs and AK-ULCs showed similar overall dispersion in confocal images (Fig. 7), cryo-SEM, particularly in neonatal clots, revealed that BK-TriGs were more frequently embedded within the fibrin network (Fig. 3C).

Fig. 7. Cryo-SEM analysis and quantification of the clot microstructure in neonatal and adult plasma with and without particles.

(A) Representative cryo-SEM images of fibrin clots formed in neonatal plasma (top row) and adult plasma (bottom row) under different conditions: plasma only, AK-ULCs, and BK-TriGs. Yellow arrows highlight the less compact, porous fibrin network with thinner fibers and a more open pore structure observed in the neonatal plasma clot. (B) Quantification of fiber intersections per 100 μm², showing structural differences in clot density across conditions. (C) Percent porosity of the fibrin clots, highlighting significant differences between neonatal and adult plasma. (D) Pore size (μm²) distribution, demonstrating variations in clot architecture across different formulations. Statistical significance is indicated with *P < 0.05.

In neonatal plasma clots, the pore size measurements were 22.15 ± 9.0 μm² for plasma-only clots, 15.47 ± 3.7 μm² for AK-ULCs, and 14.65 ± 4.1 μm² for BK-TriGs. These results indicate that AK-ULCs and BK-TriGs reduced pore size compared to plasma-only clots (Fig. 7B). Percent porosity values were 61.69 ± 8.6% for plasma-only clots, 50.05 ± 11.5% for AK-ULCs, and 49.24 ± 8.2% for BK-TriGs, confirming a decrease in porosity with particle incorporation (Fig. 7C). Fiber intersection densities per 100 μm² were 3.61 ± 1.1 for plasma-only clots, 7.37 ± 1.7 for AK-ULCs, and 6.95 ± 1.8 for BK-TriGs, with BK-TriGs showing a significantly higher fiber intersection density than plasma-only clots (Fig. 7D). Cryo-SEM imaging (fig. S9A) revealed that neonatal plasma–only clots contained more frequent free-ended fibers, whereas BK-TriG–treated clots exhibited fewer free ends and a denser, more interconnected fibrin network. We note that some free ends may be attributable to sample preparation artifacts during cryofixation. However, it should also be noted that the appearance of the cryo-SEM and confocal images varies slightly, likely due to the differences in imaging modalities. Cryo-SEM reveals a fractured frozen surface at a high resolution, whereas confocal imaging captures the intact hydrated three-dimensional (3D) network at a lower resolution. Confocal microscopy (fig. S9B) supported these results, showing a sparser fibrin architecture in neonatal plasma–only clots compared to the compact, highly branched networks formed with BK-TriGs. Our quantitative analysis showed that BK-TriGs significantly increased the branch number, junction number, and branch length relative to plasma only, consistent with the formation of a more continuous and interconnected fibrin architecture (fig. S9, C to E).

In adult clots, the pore size measurements were 70.78 ± 67.4 μm² for plasma-only clots, 25.52 ± 6.6 μm² for AK-ULCs, and 46.59 ± 10.6 μm² for BK-TriGs. The significant reduction in pore size for all particle-incorporated groups compared to plasma-only clots suggests that the particles effectively compact the clot architecture. Percent porosity values in adult plasma clots were 51.3 ± 9.2% for plasma-only clots, 46.63 ± 0.9% for AK-ULCs, and 57.39 ± 5.5% for BK-TriGs. The AK-ULC group exhibited a significantly lower porosity than plasma-only clots, reflecting a denser clot structure. Fiber intersection densities per 100 μm² were 2.19 ± 1.5 for plasma-only clots, 8.31 ± 5.2 for AK-ULCs, and 3.85 ± 0.6 for BK-TriGs, with AK-ULCs demonstrating a significantly greater fiber intersection density than plasma-only clots.

The neonatal plasma clot structure appeared less compact and more porous, with a thin fibrin network, an open pore structure, and a distributed arrangement, as indicated by yellow arrows in Fig. 7A. This may be attributed to the distinct fibrin polymerization characteristics in neonatal plasma, where clot stability and density are generally lower than in adult plasma. In contrast, the adult plasma clot exhibited a denser and more compact fibrin network with thicker fibrin fibers (Fig. 7A). This structural tightness is characteristic of adult plasma clots, where increased fibrin cross-linking results in stronger, more resilient clot formation.

These cryo-SEM results are consistent with the findings from confocal imaging and AFM, reinforcing the observation that particle incorporation generally increases clot density and alters the microstructure in a manner that enhances clot stability. The significant reductions in pore size, variations in porosity, and fiber intersection across different groups highlight the impact of these particles on clot architecture.

BK-TriGs decrease blood loss and increase fibrin incorporation at wound sites in a liver laceration FIB-KO model of injury

To evaluate the hemostatic efficacy of BK-TriGs in vivo, we conducted a liver laceration injury experiment in fib-null (Fga−/−) mice transfused with neonatal fibrinogen (Fig. 8A). Given the difficulty of performing bleeding studies in neonatal mice, we selected this model to assess the influence of neonatal fibrinogen on BK-TriG efficacy before transitioning to future larger animal studies. Initially, we determined the optimal BK-TriG concentration by testing 10, 15, and 20 mg/kg doses. Doses of 10, 15, and 20 mg/kg were selected on the basis of prior studies using polymeric particles for hemostasis (19) and are consistent with standard preclinical dosing ranges (22); 15 mg/kg was chosen as the optimal dose on the basis of its efficacy and safety profile (9, 23, 24). The 15 mg/kg dose was the most effective, resulting in significantly lower blood loss (0.0096 ± 0.003 g blood/g animal) compared to the 10 mg/kg dose (0.017 ± 0.003 g blood/g animal, P = 0.0007). However, increasing the dose to 20 mg/kg led to significantly greater blood loss (0.033 ± 0.002 g blood/g animal) compared to both 10 mg/kg (P = 0.002) and 15 mg/kg (P = 0.0007) (Fig. 8, B and C, and fig. S10). This suggests that higher BK-TriG concentrations may inhibit clot formation, possibly due to competition with native fibrin knob interactions or steric hindrance within the clot network. These findings align with our previous studies in adult mice. On the basis of these results, we selected 15 mg/kg as the optimal dose for further experiments. This concentration was applied to all treatment groups, including BK-TriGs, other microgels, and saline. Each treatment was administered intravenously 5 min before inducing the liver laceration, and blood loss was monitored over time. The results demonstrated a trend of reduced bleeding in animals treated with BK-TriGs (15 mg/kg) compared to saline, NB-ULCs, and AK-ULCs at the same dose. Quantification of total blood loss over the 10-min injury period showed a significant reduction in blood loss for BK-TriG–treated animals (0.0096 ± 0.003 g blood/g animal) compared to saline-treated animals (0.017 ± 0.002 g blood/g animal, P = 0.006). Comparison with previously published platelet-mimetic particles using antibody-based or single-domain fragment (sdFv)–based targeting motifs showed that BK-TriGs achieved a comparable 50 to 60% reduction in blood loss, even in this fib-null animal model. Minimal differences were observed between saline and NB-ULC treatment groups (0.0181 ± 0.0003 g blood/g animal, P = 0.774), indicating that NB-ULCs were less effective in reducing blood loss (Fig. 8, D and E). BK-TriGs also significantly reduced blood loss relative to NB-ULCs (P = 0.025).

Fig. 8. In vivo assessment of hemostatic efficacy using a liver laceration model in FibKO mice.

(A) Schematic representation of the experimental timeline: neonatal fibrinogen injection, followed by microparticle injection, liver laceration, and subsequent blood loss monitoring over 10 min. (B) Time course of blood loss (grams of blood per gram of animal weight) in mice treated with saline or varying doses of BK-TriGs (10, 15, and 20 mg/kg), demonstrating dose-dependent hemostatic effects. (C) Total blood loss analysis showing significant reduction with BK-TriGs at 15 mg/kg compared to saline (P < 0.001) and highlighting increased blood loss at 20 mg/kg (P < 0.0001). (D) Comparative blood loss over time for mice treated with NB-ULCs, AK-ULCs, and BK-TriGs at 15 mg/kg, showing superior performance of BK-TriGs. (E) Total blood loss for the different particle treatments, indicating the significant efficacy of BK-TriGs (P < 0.05) compared to other groups. Data are presented as the means ± SD.

Immunohistochemical (IHC) analysis of fibrin deposition at the wound site revealed significantly higher fibrin deposition in animals treated with BK-TriGs (15 mg/kg) (60,066 ± 21437 μm²) compared to saline-treated (7207 ± 3185 μm², P = 0.0135) and NB-ULC–treated (840 ± 748 μm², P = 0.0099) animals (Fig. 9, A and B). This increased fibrin deposition was evident in both total fibrin counts and fibrin area at the wound site, supporting the enhanced hemostatic potential of BK-TriGs at the optimal dose. Hematoxylin and eosin staining of liver tissue indicated preserved general cellular architecture across all treatment groups, suggesting no adverse effects on the tissue structure (Fig. 9C). Martius Scarlet Blue–stained sections highlighted pronounced fibrin deposition (red) in BK-TriG–treated groups, particularly at the wound edges. This increased fibrin presence suggests effective clot formation and stabilization, supporting more efficient tissue repair (Fig. 9D). The weight of the liver tissue removed during injury showed no significant differences between experimental groups, supporting the notion that wound severity was consistent across treatments (fig. S11).

Fig. 9. IHC and histological analyses of fibrin deposition in liver tissue.

(A) Representative images of fibrin deposition in liver tissue following different treatments, visualized by immunofluorescence staining for fibrin (red) and cell nuclei [DAPI (4′,6-diamidino-2-phenylindole), blue]. (B) Quantitative analysis of fibrin deposition area in liver sections, revealing significantly greater fibrin deposition in the BK-TriG group (**P < 0.01) compared to saline and NB-ULC treatment (*P < 0.5). (C) Hematoxylin and eosin–stained liver tissue sections showing general tissue architecture across all groups. Scale bar, 100 µm. (D) Martius Scarlet Blue staining indicating fibrin distribution within the liver sections.

BK-TriGs exhibit targeted hemostatic effects without off-target fibrin deposition

To evaluate off-target fibrin deposition, we performed IHC analysis on healthy liver tissue loops to determine whether BK-TriG treatment induced unintended fibrin accumulation (fig. S12). The results confirmed that BK-TriGs did not cause fibrin accumulation in noninjured areas. In addition, we assessed peripheral organ tissues (heart, kidney, spleen, and liver) for fibrin deposition in animals treated with BK-TriGs (15 mg/kg), other microgels, or saline. IHC staining revealed no significant fibrin accumulation in peripheral organs compared to saline controls. In lung tissue, however, fibrin deposition was significantly higher in the saline group than in BK-TriG– and AK-ULC–treated groups (figs. S12 and S13). Notably, total fibrin deposition in peripheral tissues was significantly lower than at the injured liver site (Fig. 9), further emphasizing the targeted effect of BK-TriGs in promoting clot formation only at the wound site.

DISCUSSION

The present study aimed to evaluate the hemostatic efficacy of a BK-TriG synthetic platelet-like particle, which is a highly deformable microgel functionalized with a fibrin hole b–specific peptide. This investigation builds upon our previous work, where we demonstrated that microgel-based platelet-like particles could effectively stem bleeding and induce clot retraction by binding to fibrin fibers, spreading between them, and collapsing inward to facilitate clot consolidation (9, 10, 19, 25). These prior studies highlighted the importance of fibrin-binding affinity and microgel deformability as critical design features for achieving wound-targeted hemostasis and particle-mediated clot retraction. However, earlier designs relied on either full-length antibodies or sdFvs targeting fibrin’s E domain or polymerized fibrin, which, while effective, present challenges in terms of antibody biomanufacturing and scalability (26, 27).

In this study, we explored the potential of fibrin-targeting peptides, such as those used in BK-TriGs, as an alternative that not only retains strong hemostatic efficacy but also enhances the translational potential of these platelet-mimetic particles because of the more straightforward and cost-effective manufacturing processes associated with peptide synthesis (25, 28). We focused on the application of these particles in neonates, a population with unique hemostatic challenges because of their immature clotting systems. This study highlights the potential of BK-TriGs, designed for neonatal-specific clotting mechanisms, to address the heightened bleeding and thrombosis risks in neonates, who face 4.4 times higher postsurgery mortality (2–4, 12, 29).

Here, we used a fib-null (Fga^−/−) mouse model transfused with neonatal fibrinogen, representing a novel approach to mimic the neonatal hemostatic environment in vivo. This innovative model enabled the evaluation of BK-TriGs in a setting that replicates key aspects of neonatal fibrinogen polymerization and fibrinolytic sensitivity, providing preliminary insights into their potential clinical utility. However, it should be noted that this model does not fully replicate human neonatal physiology. Our research highlights key differences between neonatal and adult fibrin clots—neonatal clots are softer, degrade faster (around 60 times) (figs. S2, A and B, and S3), and exhibit minimal fiber branching (Figs. 2, B and C, and 3A).

Moreover, neonatal fibrin polymerization relies more on B interactions, unlike the A interactions predominant in adults (18, 30). These findings underscore the need for neonatal-specific hemostatic therapies like BK-TriGs, designed to target these unique clotting mechanisms and better address the hemostatic challenges in neonates. Our findings revealed a significant increase in clot density in vitro and a marked reduction in bleeding in the rodent trauma model (Figs. 2 and 8). These results suggest that BK-TriGs effectively mimic the fibrin-binding and clot-stabilizing abilities of previous designs while offering a more cost-effective and translational approach for hemostatic therapy.

BK-TriGs enhance fibrin network density through Brownian motion–induced deformation and intensified fibrin-particle interactions (19), mimicking platelet-mediated clot retraction, with an optimal concentration supporting localized fibrin-particle interactions (Fig. 2). In neonatal plasma, a biphasic response was observed: At 1 mg/ml, the fiber density increased significantly, while at 2 mg/ml, aggregation and competitive inhibition reduced the density. This highlights the importance of optimizing particle concentration to balance enhancement and avoid disruption of fibrin polymerization. In adult plasma, the AK-ULCs emerged as the most effective in promoting clot density across all tested concentrations. The AK-ULCs at 2 mg/ml achieved the highest clot density, outperforming both the NB-ULCs and BK-TriGs at corresponding concentrations. The dose-dependent increase in clot density observed with AK-ULCs suggests that these particles are more effective in adult plasma because of their ability to sustain and promote fibrin polymerization without the inhibitory effects seen with BK-TriGs at higher concentrations. The results indicate that while BK-TriGs are highly effective in neonatal plasma, AK-ULCs may be better suited for enhancing clot density in adult plasma, particularly at concentrations above 0.5 mg/ml (Fig. 2, B to D). We acknowledge that image binarization–based density analysis has limitations, including potential sensitivity to thresholding and oversimplification of intensity gradients. These measurements are more appropriately interpreted as relative comparisons between groups. Our conclusions are supported by complementary analyses of clot structure, including cryo-SEM, which together provide a more comprehensive assessment of clot architecture (Fig. 7). The observed effects are compatible with preferential particle localization to fibrin-dense regions, local increases in apparent fibrin concentration, and microgel-fibrin interactions that promote regional network organization (17, 19). On the basis of these observations, we describe BK-TriGs as supporting fibrin network formation rather than acting through a single, defined mechanistic interaction.

The comparison between BK-TriGs and previous iterations of platelet-mimetic particles using sdFvs highlights the effectiveness of the knob B mimetic peptide as a fibrin-binding motif (7). The enhanced clotting observed with BK-TriGs is consistent with the results seen in prior designs, suggesting that this peptide can serve as a viable alternative to antibody ligands for promoting clot formation. In neonatal samples, the incorporation of BK-TriGs led to an improvement in clot stability, as demonstrated by a substantial increase in the integrated area under the polymerization/degradation curve and higher maximum turbidity (Fig. 4A). These findings suggest that BK-TriGs effectively mimic the stabilizing functions of platelets by associating with fibrin at multiple points and supporting network organization. This effect was similarly observed in adult plasma, albeit requiring a higher concentration of 2 mg/ml to achieve comparable improvements in clot stability (Fig. 4B).

The observed increase in stiffness in BK-TriG clots for neonatal plasma, particularly at an optimal concentration, may be attributed to the interaction between the fibrin network and the highly deformable particles. These particles can increase the surface area of interaction with fibrin fibers, promoting a denser fibrin matrix structure. Confocal imaging data support this, showing significantly increased fiber density in BK-TriG clots compared to control. However, despite these interactions, the deformability of the particles means that they do not necessarily contribute to the overall stiffness in a linear manner. While BK-TriG clots in neonatal plasma are stiffer than plasma-only clots, increasing the particle concentration beyond a certain threshold may lead to a softening effect, as the deformable particles could disrupt the fibrin network without reinforcing it structurally. This explains the marked decrease in stiffness observed at higher concentrations in adult plasma, where the structural integrity of the clot is compromised by an excess of particles. In neonatal plasma, there is a concentration at which the incorporation of BK-TriGs leads to a stiffness significantly higher than that of plasma-only clots. The neonatal fibrin network, being looser and less developed than that in adult plasma, may benefit from the increased surface interaction and the reinforcing effect of particles up to an optimal point. Beyond this concentration, the benefit diminishes, and stiffness decreases because of particle-induced disruption of the fibrin structure.

In adult plasma, where the fibrin network is already more robust, the incorporation of particles like AK-ULCs tends to decrease clot stiffness as the concentration increases. This is likely because they interfere more robustly with fibrin polymerization in the denser fibrin network of adult clots, resulting in a softer clot. This effect is particularly pronounced at higher concentrations of particles, as seen in the significant decrease in elastic modulus in the AK-ULC and BK-TriG groups at 1 and 2 mg/ml concentrations. While deformable particles like BK-TriGs are beneficial in enhancing clot stiffness and density in neonatal plasma at optimal concentrations, they may lead to undesired softening effects at higher concentrations, especially in adult plasma. Moreover, BK-TriGs significantly enhance clot growth under dynamic flow, replicating physiological conditions, while stabilizing clots in static environments. This capability is vital for ensuring effective hemostasis in clinical scenarios where blood flow and shear stress are variable.

Building on the in vitro results, we proceeded to evaluate the hemostatic efficacy of BK-TriGs in vivo using a traumatic liver laceration model in fib-null (Fga^−/−) mice transfused with neonatal fibrinogen. This model was specifically chosen to closely mimic the neonatal environment. Performing bleeding studies in neonatal mice would be particularly challenging because of their small size; however, performing initial studies in rodents is a common screening strategy before moving forward with evaluation of lead candidates in larger animal models. This model allowed us to specifically evaluate the effects of BK-TriGs in a neonatal-like setting with a compromised fibrinogen system. The goal was to determine whether the enhanced clot stability and fiber density observed with BK-TriGs in vitro would effectively translate into reducing bleeding in a complex in vivo scenario.

The in vivo results reaffirmed the biphasic response observed earlier in vitro, where increasing the dose of BK-TriGs led to a significant reduction in blood loss up to an optimal concentration, beyond which the efficacy diminished. At a dose of 15 mg/kg, BK-TriGs significantly reduced total blood loss compared to saline-treated controls, demonstrating their superior hemostatic potential. This reduction in blood loss aligns with the performance of previous platelet-mimetic particles that used antibody-based or sdFv-based targeting motifs (10). The ability of BK-TriGs to achieve a ~50 to 60% reduction in blood loss, even in the fib-null model, highlights the use of a knob B–based peptide for fibrin targeting in stabilizing clots and preventing bleeding in vivo.

The results also showed that AK-ULCs, which were previously effective in enhancing clot density in adult plasma in vitro, did not perform as well as BK-TriGs in this neonatal-like model. Minimal differences in total blood loss were observed between saline treatment and NB-ULCs, indicating that NB-ULCs were less effective in reducing blood loss in this context.

However, it is critical to note that increasing the BK-TriG dose to 20 mg/kg resulted in significantly greater blood loss, similar to that observed in control animals. This outcome aligns with the in vitro observation that particle concentration must be optimized to avoid inhibiting clot formation. The likely mechanism involves competition with native fibrin knob interactions or steric hindrance within the clot network, disrupting the delicate balance required for effective clot stabilization. IHC analysis further supported these findings, revealing significantly higher fibrin incorporation at the wound site in animals treated with BK-TriGs. pNIPAm microgels evaluated by our group have been well tolerated in vivo at doses up to 200 mg/kg. Nonetheless, neonatal physiology differs from adult models; therefore, formal dose-escalation studies in neonatal systems will be required to validate the safety margin in the intended population (31).

A limitation of this study is the use of cord blood–derived fibrinogen as a substitute for neonatal plasma. While previous studies have demonstrated that cord blood–derived fibrinogen properties are highly similar to those of neonatal plasma, subtle differences may exist that were not captured in our experiments. These differences could potentially influence clot formation and stability in neonatal plasma. Future studies will address this limitation by validating our findings using direct neonatal plasma and larger animal models, enabling a more comprehensive assessment of the efficacy and safety of BK-TriGs in a broader context.

These findings highlight the potential of BK-TriGs as a promising synthetic platelet-mimetic approach for enhancing clot density and stability, particularly in neonatal plasma where traditional blood products may pose risks. The biphasic response observed in vitro and in vivo underscores the importance of optimizing BK-TriG concentration to balance enhancement and avoid inhibitory effects. Future studies should further validate these findings using neonatal plasma directly and larger animal models to ensure clinical translatability. The ability of BK-TriGs to achieve significant clot stabilization while maintaining biocompatibility suggests a potential role in tailored hemostatic interventions for neonates. Expanding this research to include different clinical bleeding scenarios will be essential to advancing these materials toward therapeutic applications. This is particularly relevant in neonates, where the most severe bleeding complications often arise in critical sites such as the gastrointestinal tract and the brain. In neonates, especially preterm infants, serious bleeding events frequently involve the gastrointestinal tract and the brain. Gastrointestinal hemorrhage is commonly linked to necrotizing enterocolitis or underlying coagulopathies, while intraventricular hemorrhage remains a major contributor to morbidity and mortality in very-low-birth-weight infants. These conditions are often associated with impaired fibrin formation and altered clot structure because of developmental fibrinogen deficiencies. A fibrin-targeted approach like BK-TriGs, which enhances clot formation without introducing systemic thrombotic risk, may offer a safer alternative to adult fibrinogen transfusions (32, 33).

MATERIALS AND METHODS

Experimental design

This study aimed to evaluate the hemostatic efficacy of BK-TriGs, a fibrin hole b–targeting microgel, in comparison to AK-ULCs and NB-ULCs in neonatal and adult plasma models. We assessed clot formation, stability, and mechanical properties using in vitro and in vivo models, including confocal microscopy, AFM, cryo-SEM, and a murine liver laceration model. Microfluidic assays and polymerization studies were used to determine clotting dynamics under flow conditions. Statistical analyses were performed to evaluate the significance of clot density, degradation rates, and hemostatic efficacy in animal models.

ULC synthesis and characterization

ULCs were synthesized via seeded precipitation polymerization previously described. NIPAm and AAc were dissolved in ultrapure water (90:10 molar ratio) at pH 5.0, with NIPAm recrystallized in hexanes. AAc was added 10 min before initiating polymerization with ammonium persulfate (APS). The reaction, carried out at 70°C under nitrogen for 6 hours, was confirmed visually by the solution turning opaque. After cooling overnight, the microgel solution was filtered, dialyzed for 3 days, lyophilized, and stored until further use.

Construction of particles by peptide conjugation to ULCs

Microgels were conjugated with either a knob B–mimicking peptide (AHRPYAAK), a knob A–mimicking peptide (GPRPFPAK), or a nontargeting control peptide (GPSPFPAK, GenScript) using EDC-sulfo-NHS chemistry. Each reaction contained 5 mM sulfo-NHS, 50 mM EDC, and a peptide-to-AAc molar ratio of 1:1, prepared in Hepes buffer at pH 7.4. Initially, microgels were incubated with EDC and sulfo-NHS for 30 min with gentle agitation, followed by the addition of the peptide and further incubation for 4 hours. After conjugation, BK TriGs (AHRPYAAK-microgels), AK-ULCs (GPRPFPAK-microgels), and control NB-ULCs (GPSPFPAK-microgels) were purified through dialysis against ultrapure water for 3 days. The purified microgels were then lyophilized and stored at 4°C until resuspension in ultrapure water for experiments.

Evaluation of the fibrin clot structure

Confocal microscopy was performed to investigate the structure of fibrin clots with different microgel concentrations and formulations. Clots were prepared using two types of pooled PPP: Human Platelet-Depleted Plasma and Human Cord Blood Recovered Plasma (Zen-Bio, Research Triangle Park, NC). Both plasma sources were used across all experiments. Cord blood plasma was used as a representative source of neonatal fibrinogen. In each 50-μl reaction, 45.25 μl of plasma was mixed with 1 μl of Alexa Fluor 488–labeled human fibrinogen (10 μg/ml final; Thermo Fisher Scientific), 1.25 μl of CaCl₂ (200 mM stock; final 5 mM), and 2.5 μl of human thrombin (10 U/ml; Enzyme Research Laboratories, US) to initiate polymerization [thrombin (final 0.5 U/ml)]. Clots were formed between a glass slide and coverslip and then allowed to polymerize for at least 2 hours before imaging. A Zeiss Laser Scanning Microscope (LSM 710, Zeiss Inc., White Plains, NY) was used to capture 5-μm z-stacks at ×63 magnification, acquiring at least three random z-stacks per clot. The z-stacks were converted into 8-bit 3D projections using ImageJ software, and the clot fiber density was assessed by calculating the ratio of black (fiber) to white (background) pixels in binary images. To assess the impact of particle concentration on the clot structure, clots were prepared with microgel concentrations of 0.5, 1, 1.5, and 2 mg/ml. In all experiments, microgels were incorporated before thrombin-induced clot polymerization. The optimal microgel concentration and formulation for promoting clot retraction were identified as those that most significantly enhanced clot density. The distribution of microgels within clots was assessed by labeling the particles with NHS ester Alexa Fluor 647 dye (Thermo Fisher Scientific). To achieve this, 5 mg of microgels was reacted with 0.2 mg of the dye in a 0.1 M sodium bicarbonate buffer (pH 8.4) for 1 hour. The labeled particles were then purified by dialysis using 1000-kDa molecular weight cutoff tubing (Spectrum Labs). These particles were subsequently incorporated into clots and imaged using a confocal microscope, as described earlier.

Determination of fibrin clot polymerization and stability

The rates of fibrin clot polymerization and degradation were measured using an established intrinsic fibrinolysis assay, which allows simultaneous tracking of both processes. Neonatal fibrinogen was purified from plasma samples using an ethanol precipitation method. Briefly, precooled 70% ethanol (4°C) was added to plasma maintained at 4°C in a 4:1 plasma-to-ethanol ratio. The mixture was incubated on ice for 20 min and then centrifuged at 1000g for 15 min at 4°C. After carefully removing the supernatant, the resulting fibrinogen pellet was resuspended by heating in a 37°C water bath, with the gradual addition of 20 mM sodium citrate buffer until it was fully dissolved (final volume: 0.25 to 0.5 ml). The fibrinogen concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). For experiments with adult fibrinogen, human fibrinogen (Fib 3) was purchased from Enzyme Research Laboratories. Clots (100 μl) were prepared as described for confocal microscopy, containing either 0.5, 1, 1.5, or 2 mg/ml of peptide-conjugated microgels, control microgels, or plasma alone. To assess both clot formation and breakdown, human tPA (tissue plasminogen activator; 0.29 μg/ml; Millipore Sigma) and human glu-plasminogen (10.8 μg/ml; Thermo Fisher Scientific, Waltham, MA) were added to all samples. Clots were formed in 96-well plates, and absorbance at 350 nm was repeated every 30 s over 3 hours using a plate reader. For each concentration, the area under the curve was calculated, along with the time to half-maximum turbidity, time to half-maximum degradation, maximum turbidity, polymerization rate, and degradation rate from the polymerization and degradation curves.

Analysis of mechanical clot properties via AFM

Clot stiffness was measured using AFM. Fifty microliters of each clot composition was prepared and allowed to polymerize on a charged glass slide for 2 hours. AFM imaging (Asylum MFP3D-Bio; Asylum Research) was conducted in force contact mode using cantilevers partially coated with chromium/gold on the detector side and equipped with a 1.98-μm particle tip (NanoAndMore). The cantilevers had a force constant of 0.01 N/m for enhanced sensitivity to soft biological samples. Three 20 by 20–μm force maps were captured per clot, focusing on the center to minimize edge effects. Each map contained a 16-by-16 grid of contact points, fitted to a Hertz model to calculate the elastic modulus, with the average value from all 256 points representing the clot’s stiffness.

Clot growth and degradation assay using a T-junction microfluidic device

A customized microfluidics-based microscopy assay, modified from Brown et al. (4), was used to evaluate clot growth and degradation. The assay used a polydimethylsiloxane (PDMS) device with a main channel and a perpendicular clot reservoir for fibrinolysis analysis (4, 14). Devices were fabricated by casting PDMS (Sylgard 184, Dow Corning, Midland, MI) in a 9:1 weight ratio of elastomer base to cross-linking agent into an acrylic mold. After curing for 24 hours, PDMS was plasma treated and bonded to a glass slide to form a sealed channel.

Clot growth under flow

To assess the impact of BK-TriGs on clot propagation under shear conditions, clot growth experiments were performed using the T-junction microfluidic device described above. A stationary fibrin clot was first formed at the junction by introducing a mixture of either neonatal or adult plasma with thrombin (0.5 U/ml) and Alexa Fluor 488–labeled fibrinogen (10 μg/ml). Following polymerization, a solution containing Alexa Fluor 647–labeled microgels (1 mg/ml; BK-TriGs, AK-ULC, or NB-ULC) suspended in the corresponding plasma type (neonatal or adult) or plasma alone supplemented with thrombin (0.1 U/ml) and Alexa Fluor 488–labeled fibrinogen (10 μg/ml) in Hepes buffer was perfused through the device for 20 min at a wall shear rate of 10 s⁻¹.

Time-lapse confocal imaging captured real-time clot growth and particle incorporation at the clot-solution interface. The clot growth area was quantified by measuring the expansion of the fibrin-rich region along the interface of the T-junction over time. The region of interest was defined using a fixed rectangular window extending from the junction point along the channel, as marked by dashed lines in the representative images.

Clot degradation in the T-junction device

Clot degradation was evaluated using the same T-junction microfluidic device under static conditions to compare the effects of different microgels in neonatal and adult plasma. Fibrin clots were first formed at the junction using plasma and thrombin, as described above. Following polymerization, fresh neonatal or adult plasma, with or without microgels, was introduced into the channel including human tPA (0.29 μg/ml) and human glu-plasminogen (10.8 μg/ml), and the system was incubated without flow to monitor spontaneous clot degradation over time. Clot degradation was assessed by measuring the reduction in the fluorescent clot area over time within a defined region of interest surrounding the initial clot location, as indicated by dashed lines in the representative images.

Characterization of the clot matrix structure via cryo-SEM

For cryo-SEM imaging, 150 μl of clots was quickly plunge-frozen in liquid nitrogen and then etched for 10 min before being sputter coated with gold. To ensure rapid and uniform freezing, 150 μl of clots was cast in thin layers and plunge-frozen in liquid nitrogen. Imaging was conducted on a JEOL scanning electron microscope (JEOL JSM-7600F, JEOL USA) at magnifications of ×1000, ×1500, ×2000, and ×2500. Two clots per group were imaged, with three random areas examined within each clot. ImageJ software was used to segment the images and analyze parameters such as pore size, percent porosity, fiber length, and the number of fiber intersections.

Fib-null murine liver laceration model of traumatic injury

A murine liver laceration model using fib-null mice was used to assess the hemostatic efficacy of BK-TriGs, AK-ULCs, and NB-ULCs. All procedures were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill (protocol number: 22-164). Eight-week-old (fib−/−) C57BL/6 mice were anesthetized with isoflurane (5% in oxygen) administered via a nose cone throughout the experiment. To mimic the neonatal coagulation environment, purified fibrinogen from pooled human cord plasma PPP (Zen-Bio, Research Triangle Park, NC) was prepared using the same ethanol precipitation protocol described in the intrinsic fibrinolysis assay. Neonatal fibrinogen was injected and allowed to transfuse for 5 min in fib-null mice to evaluate its role in clot formation and hemostasis. Treatments (BK-TriGs, AK-ULCs, NB-ULCs, and saline) were injected through the jugular vein (100 μl) and allowed to circulate for 5 min before inducing injury. To evaluate the dose-response, BK-TriGs were tested at concentrations of 10, 15, and 20 mg/kg, while AK-ULCs and NB-ULCs were administered at 15 mg/kg. A midline incision was made to expose the liver, followed by a 10-mm scalpel cut through the left lobe, creating a complete laceration. Blood loss was measured over a 10-min period by collecting blood on gauze placed adjacent to the wound. Gauze was replaced at 10-s intervals for the first 30 s postinjury, then every 30 s from 30 s to 3 min, and lastly, at 1-min intervals until 10 min. The gauze was immediately weighed after removal to prevent evaporation effects. Blood loss at each time point was calculated by subtracting the initial weight of the gauze from its final weight. Blood loss over time and total blood loss (normalized to animal weight) over 10 min were quantified. After blood collection, liver wound sections were harvested, fixed in 10% formalin for 48 hours, embedded in paraffin, and sectioned at 5 μm for histological analysis.

Statistical analysis

Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). All results are presented as the means ± standard deviation (SD), unless otherwise stated. Statistical significance was determined using one-way or two-way analysis of variance (ANOVA) for multiple comparisons. P < 0.05 was considered statistically significant. All experiments were performed at least in triplicate to ensure reproducibility.

Supplementary Materials

The PDF file includes:

Figs. S1 to S13

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Other Supplementary Material for this manuscript includes the following:

Movies S1 and S2

Data file S1