Effect of In Vivo Administration of Fibrinogen Concentrate Versus Cryoprecipitate on Ex Vivo Clot Degradation in Neonates Undergoing Cardiac Surgery.

BACKGROUND: Neonates undergoing cardiac surgery require fibrinogen replacement to restore hemostasis after cardiopulmonary bypass (CPB). Cryoprecipitate is often the first-line treatment, but recent studies demonstrate that fibrinogen concentrate (RiaSTAP; CSL Behring) may be acceptable in this population. This investigator-initiated, randomized trial compares cryoprecipitate to fibrinogen concentrate in neonates undergoing cardiac surgery (ClinicalTrials.gov NCT03932240). The primary end point was the percent change in ex vivo clot degradation from baseline at 24 hours after surgery between groups. Secondary outcomes included intraoperative blood transfusions, coagulation factor levels, and adverse events. METHODS: Neonates were randomized to receive cryoprecipitate (control group) or fibrinogen concentrate (study group) as part of a post-CPB transfusion algorithm. Blood samples were drawn at 4 time points: presurgery (T1), after treatment (T2), arrival to the intensive care unit (ICU) (T3), and 24 hours postsurgery (T4). Using the mixed-effect models, we analyzed the percent change in ex vivo clot degradation from a patient's presurgery baseline at each time point. Intraoperative blood product transfusions, coagulation factor levels, perioperative laboratory values, and adverse events were collected. RESULTS: Thirty-six neonates were enrolled (intent to treat [ITT]). Thirteen patients in the control group and seventeen patients in the study group completed the study per protocol (PP). After normalizing to the patient's own baseline (T1), no significant differences were observed in clot degradation at T2 or T3. At T4, patients in the study group had greater degradation when compared to those in the control group (826.5%, 95% confidence interval [CI], 291.1-1361.9 vs -545.9%, 95% CI, -1081.3 to -10.4; P < .001). Study group patients received significantly less median post-CPB transfusions than control group patients (ITT, 27.2 mL/kg [19.0-36.9] vs 41.6 [29.2-52.4]; P = .043; PP 26.7 mL/kg [18.8-32.2] vs 41.2 mL/kg [29.0-51.4]; P < .001). No differences were observed in bleeding or thrombotic events. CONCLUSIONS: Neonates who received fibrinogen concentrate, as compared to cryoprecipitate, have similar perioperative ex vivo clot degradation with faster degradation at 24 hours postsurgery, less post-CPB blood transfusions, and no increased bleeding or thrombotic complications. Our findings suggest that fibrinogen concentrate adequately restores hemostasis and reduces transfusions in neonates after CPB without increased bleeding or thrombosis risk.

Influence of Fibrinogen Concentrate on Neonatal Clot Structure When Administered Ex Vivo After Cardiopulmonary Bypass.

BACKGROUND: Bleeding is a serious complication of cardiopulmonary bypass (CPB) in neonates. Blood product transfusions are often needed to adequately restore hemostasis, but are associated with significant risks. Thus, neonates would benefit from other effective, and safe, hemostatic therapies. The use of fibrinogen concentrate (FC; RiaSTAP, CSL Behring, Marburg, Germany) is growing in popularity, but has not been adequately studied in neonates. Here, we characterize structural and degradation effects on the neonatal fibrin network when FC is added ex vivo to plasma obtained after CPB. METHODS: After approval by the institutional review board and parental consent, blood samples were collected from neonates undergoing cardiac surgery and centrifuged to yield platelet poor plasma. Clots were formed ex vivo from plasma obtained at several time points: (1) baseline, (2) immediately post-CPB, and (3) post-transfusion of cryoprecipitate. In addition, we utilized post-CPB plasma to construct the following conditions: (4) post-CPB +0.5 mg/mL FC, and (5) post-CPB +0.9 mg/mL FC. The resultant fibrin networks were imaged using confocal microscopy to analyze overall structure, fiber density, and alignment. Clots were also analyzed using a microfluidic degradation assay. Fibrinogen content was quantified for all plasma samples. RESULTS: The addition of 0.5 or 0.9 mg/mL FC to post-CPB samples significantly enhanced the median fiber density when compared to untreated post-CPB samples (post-CPB = 0.44 [interquartile range {IQR}: 0.36-0.52], post-CPB +0.5 mg/mL FC = 0.69 [0.56-0.77], post-CPB +0.9 mg/mL FC = 0.87 [0.59-0.96]; P = .01 and P = .006, respectively). The addition of 0.9 mg/mL FC to post-CPB samples resulted in a greater fiber density than that observed after the in vivo transfusion of cryoprecipitate (post-transfusion = 0.54 [0.45-0.77], post-CPB +0.9 mg/mL FC = 0.87 [0.59-0.96]; P = .002). Median fiber alignment did not differ significantly between post-CPB samples and samples treated with FC. Degradation rates were not statistically significant from baseline values with either 0.5 or 0.9 mg/mL FC. In addition, we found a significant correlation between the difference in the baseline and post-CPB fibrinogen concentration with patient age ( P = .033) after controlling for weight. CONCLUSIONS: Our results show that clots formed ex vivo with clinically relevant doses of FC (0.9 mg/mL) display similar structural and degradation characteristics compared to the in vivo transfusion of cryoprecipitate. These findings suggest that FC is effective in restoring structural fibrin clot properties after CPB. Future studies after the administration of FC in vivo are needed to validate this hypothesis.

Point-of-Care Cardiac Troponin I Assay Evaluation in New Zealand White Rabbits (Oryctolagus cuniculus).

Cardiac troponin I (cTnI) is a cardiac-specific biomarker, used for the detection of myocardial injury. While rabbits develop naturally occurring cardiovascular disease, they are also an animal model for human disease; thus, rapid detection of cTnI has implications for both veterinary and human medicine. The objective of this study was to validate and establish a reference interval for a point-of-care (POC) cTnI assay in New Zealand White rabbits. In the first portion of the study, rabbit cardiac and skeletal muscle tissues were used to create homogenates, serially diluted with saline or rabbit whole blood, and run by repeated analysis on the POC assay. In the second portion of the study, a reference interval of peripheral whole blood cTnI was determined by robust methods from 49 New Zealand White rabbits. The least diluted cardiac muscle homogenates produced detectable cTnI (mean 23.12 ± 3.557 ng/mL), while skeletal muscle homogenates produced low to undetectable cTnI. The CV ranged from 0.00% to 32.51% for cTnI of diluted cardiac muscle homogenates. Rabbit cardiac homogenate diluted in blood had a linear relationship to cTnI concentration (Y = 0.2254 × X + 0.5396, R² = 0.975). The reference interval for cTnI in this population was less than 0.04 ng/mL. This POC assay may be useful when rapid detection of cTnI is needed and differentiation between normal and elevated values is required. Given the high CV, this assay may not be appropriate for cases that require high sensitivity or detection of low concentrations of cTnI.

Clot-Targeted Nanogels for Dual-Delivery of AntithrombinIII and Tissue Plasminogen Activator to Mitigate Disseminated Intravascular Coagulation Complications.

Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo, single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.

Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly (N-isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

COVID-19 patient fibrinogen produces dense clots with altered polymerization kinetics, partially explained by increased sialic acid.

BACKGROUND: Thrombogenicity is a known complication of COVID-19, resulting from SARS-CoV-2 infection, with significant effects on morbidity and mortality. OBJECTIVE: We aimed to better understand the effects of COVID-19 on fibrinogen and the resulting effects on clot structure, formation, and degradation. METHODS: Fibrinogen isolated from COVID-19 patients and uninfected subjects was used to form uniformly concentrated clots (2 mg/ml), which were characterized using confocal microscopy, scanning electron microscopy, atomic force microscopy, and endogenous and exogenous fibrinolysis assays. Neuraminidase digestion and subsequent NANA assay were used to quantify sialic acid residue presence; clots made from the desialylated fibrinogen were then assayed similarly to the original fibrinogen clots. RESULTS: Clots made from purified fibrinogen from COVID-19 patients were shown to be significantly stiffer and denser than clots made using fibrinogen from noninfected subjects. Endogenous and exogenous fibrinolysis assays demonstrated that clot polymerization and degradation dynamics were different for purified fibrinogen from COVID-19 patients compared with fibrinogen from noninfected subjects. Quantification of sialic acid residues via the NANA assay demonstrated that SARS-CoV-2-positive fibrinogen samples contained significantly more sialic acid. Desialylation via neuraminidase digestion resolved differences in clot density. Desialylation did not normalize differences in polymerization, but did affect rate of exogenous fibrinolysis. DISCUSSION: These differences noted in purified SARS-CoV-2-positive clots demonstrate that structural differences in fibrinogen, and not just differences in gross fibrinogen concentration, contribute to clinical differences in thrombotic features associated with COVID-19. These structural differences are at least in part mediated by differential sialylation.

Fibrin-specific poly(N-isopropylacrylamide) nanogels for targeted delivery of tissue-type plasminogen activator to treat thrombotic complications are well tolerated in vivo.

Targeted drug delivery for maintaining blood fluidity can reduce the risks associated with systemic anticoagulants that can lead to off-target bleeding. Recently, there has been much interest in targeted delivery of tissue-type plasminogen activator (tPA) for treating thrombotic complications. The work presented here characterizes a fibrin-specific nanogel (FSN) design for targeted delivery of tPA to treat thrombotic complications. Fibrin binding and clot degradation were characterized in vitro, and animal models of thrombosis were used to examine nanogel effects on coagulation parameters. In vitro assays showed tPA-FSNs attach to fibrin in a dose-dependent manner independent of tPA loading. In animal models of thrombosis, including an electrolytic injury to monitor clot properties in real time, and a lipopolysaccharide-induced disseminated intravascular coagulation (DIC) animal model, tPA-FSNs modulated fibrin/fibrinogen and platelet incorporation into clots and at optimized dosing could recover consumptive coagulopathy in DIC. Distribution of unloaded and tPA-loaded FSNs showed potential clearance of tPA-FSNs after 24 h, although unloaded FSNs may be retained at sites of fibrin deposits. Maximum tolerated dose studies showed tPA-FSNs have minimal toxicity up to 20 times the optimized therapeutic dose. Overall, these studies demonstrate the therapeutic efficacy of targeted fibrinolysis for systemic microthrombi and begin to evaluate key translational parameters for tPA-FSN therapeutics, including optimal tPA-FSN dosage in a DIC rodent model and safety of intravenous tPA-FSN therapeutics.

Neonatal coagulopathies: A review of established and emerging treatments.

Despite the relative frequency of both bleeding and clotting disorders among patients treated in the neonatal intensive care unit, few clear guidelines exist for treatment of neonatal coagulopathies. The study and treatment of neonatal coagulopathies are complicated by the distinct hemostatic balance and clotting components present during this developmental stage as well as the relative scarcity of studies specific to this age group. This mini-review examines the current understanding of neonatal hemostatic balance and treatment of neonatal coagulopathies, with particular emphasis on emerging treatment methods and areas in need of further investigative efforts.