Search

Thrombogenicity is a known complication of COVID-19, resulting from SARS-CoV-2 infection, with significant effects on morbidity and mortality.We aimed to better understand the effects of COVID-19 on fibrinogen and the resulting effects on clot structure, formation, and degradation.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.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.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.

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.

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.

Introduction: Bleeding is a serious complication among neonates undergoing cardiopulmonary bypass (CPB) and it is linked to significant morbidity and mortality. Current standard of care treatment for bleeding after CPB focuses on the transfusion of adult blood products, including platelets and cryoprecipitate. However, prior work by Nellenbach et al. has demonstrated structural differences between neonatal and adult clotting components. Importantly, neonatal and adult fibrin do not fully integrate during clot formation which may contribute to ineffective clot formation and/or increased thrombotic risk following transfusion of adult cryoprecipitate to neonates. There has been increased interest in using human fibrinogen concentrate (HFC) in treating bleeding in the post-CPB neonate; however, HFC has not been validated in this population through evidence-based means. This study analyzed structural and degradation properties of post-CPB clots +/- the ex vivo addition of HFC and compared structural and degradation properties of post-CPB clots after the in vivo transfusion of HFC versus cryoprecipitate. Methods: Human neonatal plasma samples were collected from patients undergoing CPB at the Children's Hospital of Atlanta. For ex vivo studies, samples were taken at baseline, post-bypass, and post-transfusion of cryoprecipitate (n = 18 patients). Clots were formed for analysis from samples alone as well as post-bypass samples with the addition of 0.5 or 0.9 mg/mL HFC (RiaSTAP, CSL Behring) and structure was examined through confocal microscopy. Clot degradation was assessed through a microfluidic fibrinolysis assay. For in vivo studies, samples were taken at baseline, post-transfusion of cryoprecipitate or HFC, upon ICU arrival, and at 24 hours post-surgery (n = 36 patients). Clots were formed from samples and structure was examined through confocal microscopy. Clot degradation was assessed through a plate-based fibrinolysis assay. Results: In ex vivo studies, clot structural analysis demonstrated no significant differences in fiber density between samples collected at different time points (baseline = 0.541 ± 0.105, post-bypass = 0.431 ± 0.111, post-transfusion = 0.594 ± 0.170). The addition of 0.5 mg/mL or 0.9 mg/mL HFC to post-bypass samples led to a significant increase in fiber density (0.5 mg/mL HFC=0.654 ± 0.158, p=0.02; 0.9 mg/mL HFC= 0.797 ± 0.193, p Conclusion: We identify patterns in structural properties of clots formed after the transfusion of HFC that are consistent with successful hemostasis. However, caution is warranted regarding potentially thrombotic risks and should be carefully analyzed in future studies. Figure: Effect of Ex Vivo HFC Addition on Clot Structure and Degradation. (A) Representative confocal imaging of clots formed from different samples and HFC dosages (scale = 50 um). (B) Effect of HFC Addition on Clot Fiber Density. Addition of both 0.5 and 0.9 mg/mL HFC dosages to post-bypass sample result in statistically significant increases in fiber density compared to post-bypass samples. (C) Effect of HFC Addition on Clot Degradation Profiles. Addition of 0.9 mg/mL HFC to post-bypass sample leads to statistically significant slower fibrinolysis. Figure 1 Figure 1. Disclosures Brown: Selsym Biotech, Inc.: Other: Co-Founder and CEO. OffLabel Disclosure: RiaSTAP (human fibrinogen concentrate) is FDA approved for the treatment of congenital hypofibrinogenemia.