Endogenous Nitric Oxide-Releasing Microgel Coating Prevents Clot Formation on Oxygenator Fibers Exposed to In Vitro Blood Flow

Background: Clot formation on foreign surfaces of extracorporeal membrane oxygenation systems is a frequent event. Herein, we show an approach that mimics the enzymatic process of endogenous nitric oxide (NO) release on the oxygenator membrane via a biomimetic, non-fouling microgel coating to spatiotemporally inhibit the platelet (PLT) activation and improve antithrombotic properties. This study aims to evaluate the potential of this biomimetic coating towards NO-mediated PLT inhibition and thereby the reduction of clot formation under flow conditions. Methods: Microgel-coated (NOrel) or bare (Control) poly(4-methyl pentene) (PMP) fibers were inserted into a test channel and exposed to a short-term continuous flow of human blood. The analysis included high-resolution PLT count, pooled PLT activation via β-Thromboglobulin (β-TG) and the visualization of remnants and clots on the fibers using scanning electron microscopy (SEM). Results: In the Control group, PLT count was significantly decreased, and β-TG concentration was significantly elevated in comparison to the NOrel group. Macroscopic and microscopic visualization showed dense layers of stable clots on the bare PMP fibers, in contrast to minimal deposition of fibrin networks on the coated fibers. Conclusion: Endogenously NO-releasing microgel coating inhibits the PLT activation and reduces the clot formation on PMP fibers under dynamic flow.

Temporal Roles of Platelet and Coagulation Pathways in Collagen- and Tissue Factor-Induced Thrombus Formation

In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.

SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet pro-coagulant activity

Background: Thrombosis of the lung micro-vasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting this activity. As TMEM16F plays an important role in the stimulation of the pro-coagulant activity of platelets, and considering that platelet abnormalities are common in COVID-19 patients, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. Methods: We produced SARS-CoV-2 Spike or VSV-G protein-pseudotyped virions, or generated cells expressing Spike on their plasma membrane, and tested their effects on platelet adhesion (fluorescence), aggregation (absorbance), exposure of phosphatidylserine (flow cytometry for annexin V binding), calcium flux (flow cytometry for fluo-4 AM), and clot formation and retraction. These experiments were also conducted in the presence of the TMEM16F activity inhibitors Niclosamide and Clofazimine. Results: Here we show that exposure to SARS-CoV-2 Spike promotes platelet activation, adhesion and spreading, both when present on the envelope of virions or upon expression on the plasma membrane of cells. Spike was effective both as a sole agonist or by enhancing the effect of known platelet activators, such as collagen and collagen-related peptide. In particular, Spike exerted a noticeable effect on the procoagulant phenotype of platelets, by enhancing calcium flux, phosphatidylserine externalisation, and thrombin generation. Eventually, this resulted in a striking increase in thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine almost abolished this Spike-induced pro-coagulant response. Conclusions: Together, these findings provide a pathogenic mechanism to explain thrombosis associated to COVID-19 lung disease, by which Spike present in SARS-CoV-2 virions or exposed on the surface of infected cells, leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing drugs targeting this protein, such as Niclosamide, for COVID-19 therapy.

Repeated Social Defeat Exaggerates Fibrin-Rich Clot Formation by Enhancing Neutrophil Extracellular Trap Formation via Platelet–Neutrophil Interactions

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl3-induced carotid arterial injury and were analyzed after 3 h. Although the volume of thrombi was comparable between the two groups, fibrin(ogen)-positive areas were significantly increased in defeated mice, in which Ly-6G-positive cells were appreciably co-localized with Cit-H3-positive staining. Treatment with DNase I completely diminished exaggerated fibrin-rich clot formation in defeated mice. Flow cytometric analysis showed that neutrophil CD11b expression before FeCl3 application was significantly higher in defeated mice than in control mice. In vitro NET formation induced by activated platelets was significantly augmented in defeated mice, which was substantially inhibited by anti-CD11b antibody treatment. Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NET formation, suggesting that NET can be a new therapeutic target in depression-related CVD.

A Review on Host-Leptospira Interactions: What We Know and Future Expectations

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is considered a neglected infectious disease of human and veterinary concern. Our group has been investigating proteins annotated as hypothetical, predicted to be located on the leptospiral surface. Because of their location, these proteins may have the ability to interact with various host components, which could allow establishment of the infection. These proteins act as adherence factors by binding to host receptor molecules, such as the extracellular matrix (ECM) components laminin and glycosaminoglycans to help bacterial colonization. Leptospira also interacts with the host fibrinolytic system, which has been demonstrated to be a powerful tool for invasion mechanisms. The interaction with fibrinogen and thrombin has been shown to reduce fibrin clot formation. Additionally, the degradation of coagulation cascade components by secreted proteases or by acquired surface plasmin could also play a role in reducing clot formation, hence facilitating dissemination during infection. Interaction with host complement system regulators also plays a role in helping bacteria to evade the immune system, facilitating invasion. Interaction of Leptospira to cell receptors, such as cadherins, can contribute to investigate molecules that participate in virulence. To achieve a better understanding of the host-pathogen interaction, leptospiral mutagenesis tools have been developed and explored. This work presents several proteins that mediate binding to components of the ECM, plasma, components of the complement system and cells, to gather research achievements that can be helpful in better understanding the mechanisms of leptospiral-host interactions and discuss genetic manipulation for Leptospira spp. aimed at protein function validation.

Synthesis of novel 1,3,4-oxadiazole derivatives of nicotinic acid and its in-vitro antithrombotic activity

The pharmacological characteristics of azole are diverse. Only a few medications can reduce the risk of clot formation, and they all have serious side effects. At present time there has to be more focus on to cure this type of complication beacause due to environmental, emotional, physical, other biological incidents are much related to this condition. Resistance has developed to existing medications, prompting the implementation of novel medications with a greater activity profile. The synthesis of a 1,3,4-oxadiazole derivative from nicotinic acid as well as its antithrombotic activity is described in this paper.

Bacteria-engineered porous sponge for hemostasis and vascularization

Background Hemostasis and repair are two essential processes in wound healing, yet early hemostasis and following vascularization are challenging to address in an integrated manner. Results In this study, we constructed a hemostatic sponge OBNC-DFO by fermentation of Acetobacter xylogluconate combined with TEMPO oxidation to obtain oxidized bacterial nanocellulose (OBNC). Then angiogenetic drug desferrioxamine (DFO) was grafted through an amide bond, and it promoted clot formation and activated coagulation reaction by rapid blood absorption. The further release of DFO stimulated the secretion of HIF-1α and the reconstruction of blood flow, thus achieving rapid hemostasis and vascularization in damaged tissue. This new hemostatic sponge can absorb approximately 38 times its weight in 28 seconds, rapidly enhancing clot formation in the early stage of hemostasis. In vitro and in vivo coagulation experiments (in rat tail amputation model and liver trauma model) demonstrated superior pro-coagulation effects of OBNC and OBNC-DFO to clinically used collagen hemostatic sponges (COL). They promoted aggregation and activation of red blood cells and platelets with shorter whole blood clotting time, more robust activation of endogenous coagulation pathways and less blood loss. In vitro cellular assays showed that OBNC-DFO prevailed over OBNC by promoting the proliferation of human umbilical vein endothelial cells (HUVECs). In addition, the release of DFO enhanced the secretion of HIF-1α, further strengthening vascularization in damaged skin. In the rat skin injury model, 28 days after being treated with OBNC-DFO, skin appendages (e.g., hair follicles) became more intact, indicating the achievement of structural and functional regeneration of the skin. Conclusion This hemostatic and vascularization-promoting oxidized bacterial nanocellulose hemostatic sponge, which rapidly activates coagulation pathways and enables skin regeneration, is a highly promising hemostatic and pro-regenerative repair biomaterial.

Approach to the Diagnosis of Bleeding Disorders in Children

Hemostasis is the cessation of bleeding in the intravascular compartment. This occurs by the formation of clot formation at the site of injury. The intricately related system also regulates the size of the clots by the activation of fibrinolysis. The disorders that lead to the bleeding outside the intravascular space is bleeding disorder. Bleeding occurs as a result of defective quality or quantitative deficiencies of platelets, coagulation problems in the extrinsic or intrinsic or the common pathways and abnormalities in the vascular walls to contain hemorrhage. Although the values of different coagulation and fibrinlytic factors are pretty much like that of the adults yet, neonates have considerable differences in these values than their senior counterparts even in pediatric age groups. Considering all these facts the history, physical examination and laboratory investigations always provide the clinicians some important clues to reach the diagnosis. Because of the complicated relationship among the different pathways of coagulations and fibrinolytic systems the whole procedure might look complex yet methodical approach paves the way of an intelligent clinician to arrive at the diagnosis with precision and clinical perfection. CBMJ 2020 July: Vol. 09 No. 02 P: 45-53

Small-Volume Noncontact Assessment of Blood Coagulation Via Acoustic Tweezing Coagulometry

Introduction: Blood coagulation analysis is routinely performed to assess bleeding and thrombotic risks in surgical and critical care patients as well as in patients with diseases that cause coagulation abnormalities (e.g., hemophilia, thrombophilia and sickle cell disease). Majority of coagulation assays are based on photo-optical measurement of coagulation onset in blood plasma such as prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT) and viscoelastic measurement of coagulating whole blood, often referred to as "global coagulation analysis", mostly done by thromboelastography (TEG, ROTEM) but they require large sample volume (> 0.5ml) requiring venipuncture, have poor standardization, and are unreliable tools to predict bleeding/thrombotic risk. Acoustic tweezing coagulometry (ATC) is an innovative noncontact drop-of-blood coagulation analysis technique that can perform both photo-optical and viscoelastic coagulation analysis with a sample volume as low as 4 μl to provide a comprehensive set of clinically relevant coagulation parameters such as blood viscosity, elasticity, reaction time, clotting rate, maximum clot stiffness, fibrin formation rate and cross-linking kinetics helpful for diagnosis and prediction of bleeding and thrombotic risks. ATC is particularly valuable for the pediatric patients as it enables safe and reliable point of care coagulation assessment with minimal sample volume. Materials and Methods: In this project, we demonstrate the feasibility of ATC for coagulation analysis by validation and standardization of the technique using whole blood collected from healthy adult volunteers and commercially purchased blood plasma. Further, we present the ability of ATC to assess bleeding risk in commercial blood plasma with coagulation FVIII deficiency with and without inhibitors, as well as whole blood collected from pediatric Hemophilia A patients without inhibitors. The time dependent changes in elasticity (elastic tweezograph, Figure 1A) and viscosity (viscous tweezograph, Figure 1B) of coagulating blood plasma or whole blood sample are used to extract the following coagulation parameters: clot initiation time (CIT), clotting rate (CR), clotting time (CT), time to firm clot formation (TFCF), and maximum clot stiffness (MCS) from elastic tweezograph; reaction time (RT), fibrin formation rate (FFR), and maximum fibrin level (MFL) from viscous tweezograph. Results and Discussion: Figure 1C shows the elastic tweezograph and figure 1D shows the viscous tweezograph of the healthy plasma, plasma with coagulation FVIII deficieny and plasma with inhibitors for coagulation FVIII activated via the intrinsic pathway of coagulation. The tweezographs suggest that the clot initiation is faster in healthy plasma compared to the FVIII deficient plasma and FVIII inhibitor plasma. The clotting rate is highest for healthy plasma followed by the FVIII deficient plasma and is the lowest for the plasma with FVIII inhibitors suggesting a delayed clot formation in the deficient and inhibitor groups. They all reach a similar final clot stiffness, but the time to firm clot formation is least in healthy plasma as expected and increases in the FVIII deficient group and further increases in the FVIII inhibitor group. Conclusions: Acoustic tweezing coagulometry can successfully measure the viscosity, elasticity and coagulation of whole blood and blood plasma with only a drop of the sample. This technique can successfully assess the bleeding risks in pediatric and adult patients with Hemophilia. Acknowledgements: This study has been supported by American Heart Association pre doctoral fellowship 20PRE35210991, U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559, PCT/US2018/014879 and PCT/US21/15336. Figure 1 Figure 1. Disclosures Kasireddy: Levisonics Inc.: Current Employment. Rafique: Pfizer Inc.: Consultancy; CSL Behring: Consultancy; HEMA Biologics: Consultancy. Khismatullin: Levisonics Inc.: Current equity holder in publicly-traded company; Levisonics Inc.: Patents & Royalties: PCT/US14/55559 (pending); Levisonics Inc.: Patents & Royalties: PCT/US2018/014879 (issued) ; Levisonics Inc.: Patents & Royalties: PCT/US21/15336 (pending)..

Ex Vivo and In Vivo Evaluation of Fibrinogen Concentrate to Mitigate Post-Surgical Bleeding in Neonates

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<0.0001). Functional microfluidic analysis of clot degradation demonstrated significantly faster degradation times among post-bypass samples when compared to baseline samples (baseline degradation rate = 11.061 ± 6.087, post-bypass degradation rate = 25.906 ± 9.990 microns/hour, p=0.04). The addition of 0.5 mg/mL HFC resulted in a slower degradation rate from the original post-CPB degradation rate, but did not reach statistical significance (0.5 mg/mL HFC=14.091 ± 2.241, p=0.14). However, the addition of 0.9 mg/mL HFC resulted in a significantly slower degradation rate (0.9 mg/mL HFC=8.594 ± 6.087, p=0.01). Studies comparing in vivo transfusion of cryoprecipitate and HFC demonstrated no significant difference between treatment groups in clot density or degradation rate for any sample time point. 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.