Inhibit Blood Coagulation Research Articles

Innovative approaches to assessing risk factors, diagnostics and treatment of neonatal thrombosis

Compared to children of other ages, neonates especially seriously ill and premature subjects comprise a high thrombotic risk group. A decline in the incidence of neonatal thrombosis may be accounted for by improved treatment of severe conditions in newborns and increased survival of premature infants. Neonatal and adult hemostasis exhibit distinct physiological features: difference in concentration, synthesis rate of blood coagulation factors, metabolic rate, thrombin and plasmin levels. At the same time, neonatal threshold values for natural blood coagulation inhibitors (protein C, protein S, antithrombin, heparin cofactor II) and vitamin K-dependent coagulation factors (FII, FVII, FIX, FX) are quite low, whereas that of FVIII and von Willebrand factor exceeds those found in adults. Thus, newborns have lower plasma fibrinolytic activity. The main risk factors for developing thrombotic complications are as follows: central venous catheters, altered body fluid volume, liver disease, as well as sepsis and inflammatory processes particularly COVID-19. The significance of congenital and acquired maternal and neonatal thrombophilia may pose an additional risk factor for thrombotic complications. Low-molecular weight heparins are the first-choice drugs in treatment and prevention of neonatal thrombosis.

Platelets provide robustness of spatial blood coagulation to the variation of initial conditions

Activated platelets provide phospholipid surface and secrete coagulation factors, enhancing blood clotting. We investigated the role of platelets in the regulation of blood coagulation spatial dynamics. We activated blood clotting with tissue factor-bearing (TF) surface in platelet-rich plasma (PRP) or platelet-free plasma (PFP). When blood coagulation was initiated by high TF density, clot growth rate (V) in PRP (2 × 105/μL platelets) was only 15 % greater than in PFP. Spatial distribution of thrombin in PRP had a peak-like shape in the area of the fibrin clot edge, while in PFP thrombin was distributed in the shape of descending plateau. Platelet inhibition with prostaglandin E1 or cytochalasin D made spatial thrombin distribution look like in the case of PFP. Inhibition of blood coagulation by natural endogenous inhibitor heparin was diminished in PRP, while the effect of the exogenous or artificial inhibitors (rivaroxaban, nitrophorin, hirudin) remained undisturbed in the presence of platelets. Ten times decrease of the TF surface density greatly depressed blood coagulation in PFP. In PRP only clotting initiation phase was, while the propagation phase remained intact. Coagulation factor deficiency greatly reduced amount of thrombin and decreased V in PFP rather than in PPR. Thus, platelets were redundant for clotting in normal plasma under physiological conditions but provided robustness of the coagulation system to the changes in initial conditions.

Efficacy and Mechanism Study of 6S-5-Methyltetrahydrofolate-Calcium Against Telencephalon Infarction Injury in Zebrafish Model of Ischemic Stroke.

Ischemic stroke is a heterogeneous brain injury with complex pathophysiology and it is also a time sensitive neurological injury disease. At present, the treatment options for ischemic stroke are still limited. 6S-5-methyltetrahydrofolate-calcium (MTHF-Ca) is the calcium salt of the predominant form of dietary folate in circulation. MTHF-Ca has potential neuroprotective effect on neurocytes, but whether it can be used for ischemic stroke treatment remains unknown. We established zebrafish ischemic stroke model through photothrombotic method to evaluate the protective effect of MTHF-Ca on the ischemic brain injury of zebrafish. We demonstrated that MTHF-Ca reduced the brain damage by reducing motor dysfunction and neurobehavioral defects of zebrafish with telencephalon infarction injury. MTHF-Ca counteracted oxidative damages after Tel injury by increasing the activities of GSH-Px and SOD and decreasing the content of MDA. RNA-seq and RT-qPCR results showed that MTHF-Ca played a neuroprotective role by alleviating neuroinflammation, inhibiting blood coagulation, and neuronal apoptosis processes. Overall, we have demonstrated that MTHF-Ca has neuroprotective effect in ischemic stroke and can be used as a potential treatment for ischemic stroke.

Enhancement of apixaban's solubility and dissolution rate by inclusion complex (β-cyclodextrin and hydroxypropyl β-cyclodextrin) and computational calculation of their inclusion complexes.

Apixaban (AP) is a factor X inhibitor, an orally active drug that inhibits blood coagulation for better prevention of venous thromboembolism. It has poor solubility, dissolution rate and low bioavailability. The aim of this study was to improve the aqueous solubility and dissolution rate of oral AP as a step to enhance its bioavailability by preparing it as an inclusion complex with beta- and hydroxy propyl beta-cyclodextrin. A simple, rapid method of analysis of AP was developed using ultraviolet spectrophotometry (UV) and partially validated in terms of linearity, precision and accuracy, recovery, and robustness. AP was prepared as a complex with beta cyclodextrin (βCD) and hydroxy propyl beta cyclodextrin (HPβCD) in weight ratios 1:1, 1:2, and 1:3 by kneading, solvent evaporation and spray drying methods and characterized by Fourier transfer infra-red (FTIR), differential scanning calorimetry (DSC), and percent drug content in each of the prepared complex. Using the computer simulation, the interactions of AP with βCD and HPβCD were investigated. The phase solubility study showed that the solubility of AP was greatly enhanced from 54×10-3 mmol /L to 66 mmol/L using HPβCD with acceptable stability constant. Computer docking supports the formation of a stable 1:1 complex between AP and CD's. The dissolution test results showed that the complex gave a significantly higher percentage of drug release (95%) over one hour compared to the free AP (60%) (p<0.05). AP- HPβCD complex in the ratio of 1:2 (w/w) can significantly improve the solubility and in vitro dissolution rate of AP.

Decellularized Scaffold-Based Artificial Vascular Patch for Porcine Vascular Repair.

Vascular transplantation is an effective strategy against cardiovascular diseases (CVD), and artificial vascular patches are of urgent need across the world. In this work, we designed a multifunctional decellularized scaffolds (DCS)-based vascular patch for porcine vascular repair. Ammonium phosphate zwitter-ion (APZI) and poly(vinyl alcohol) (PVA) hydrogel were coated on the surface of DCS to improve the mechanical properties and biocompatibility of an artificial vascular patch. Then a heparin (Hep)-loaded metal-organic framework (MOF) further decorated the artificial vascular patches to inhibit blood coagulation and promote vascular endothelialization. The obtained artificial vascular patch showed suitable mechanical properties, good biocompatibility, and blood compatibility. In addition, the proliferation and adhesion of endothelial progenitor cells (EPCs) on the surface of artificial vascular patch improved a lot when compared with unmodified PVA/DCS. According to the results of B-ultrasound and CT images, the artificial vascular patch could maintain the patency of the implant site after implanting into the pig carotid artery. The current results solidly support that a MOF-Hep/APZI-PVA/DCS vascular patch would be an excellent vascular replacement material.

Go West: Hirudins and Decorsin/Ornatin-like Platelet Aggregation Inhibitors in Two Representatives of American Hematophagous Leeches

Hematophagous leeches express a broad variety of secretory factors in their salivary glands; among them are hirudins, inhibitors of blood coagulation, and decorsins/ornatins, inhibitors of platelet aggregation. Here, we describe the identification and molecular and functional characterization of putative hirudins and decorsins/ornatins in two leech species of American origin, Limnobdella mexicana and Haementeria vizottoi. The leech species represent two orders of leeches, the proboscis-bearing Rhynchobdellida and the non-proboscis-bearing Arhynchobdellida. Members of the hirudin superfamily, such as hirudins or decorsins/ornatins, are described for the first time in the genus Haementeria. Both species expressed very potent inhibitors of platelet aggregation, but only the putative hirudins of L. mexicana displayed high thrombin-inhibitory potency, whereas the putative hirudin of H. vizottoi turned out to be a hirudin-like factor. The results of our study provide new insights into the evolutionary background of the blood-sucking lifestyle in leeches.

Particularity of environmental biotic factors impact on mammal’s organism

This article explores ecological significance of animal venom as a stressful biotic factor and its role in disruption of homeostatic processes in mammal’s organism. The variety of venomous species of animals and their widely distribution in the world forces other species of animals to adapt to the coexistence with them. Thus, venom toxins have an important ecological significance, because they act as stressors and appreciably impact on the biotic interaction between organisms. According to scientists’ estimates, 220.000 species of animals, or 15% of the world’s biodiversity, are venomous, and there are 3.709 species of venomous snakes. The increase of snakebites cases is not only related to geographical distribution of snakes, but also to climate changes (high humidity and an increase of average annual temperature), which contributes to the reproduction of snakes and a shift of their natural habitats. Every year, around 5.4 million people are bitten by snakes worldwide. This leads to heavy social and economic costs in many countries of the world and it is the reason for constant attention to this problem. Among the variety of venomous animals, snakes have attracted the greatest attention of scientists due to peculiarities of the composition of their venom, its biochemical properties and the possibility of use in pharmacology and medicine. However, despite annual large-scale studies of the composition of snake toxins, the specifics of their effects on animals and human is not fully indentified. It has been established that among venomous snakes only vipers are common in Ukraine. Among them there are two subspecies of the common adder (Vipera berus Linnaeus, 1758): Vipera b. berus, which can be found in Polissia, Forest Steppe and the Carpathians, and Nikolsky’s viper (Vipera berus nikolskii Vedmederja, Grubant et Rudaeva, 1986), which occurs mainly in the Left Forest Steppe. Their bites can be very painful, but are rarely fatal for adults. Vipera berus venom has hemolytic, proteolytic and cytotoxic properties due to its composition: phospholipase A2, serine proteases, metallo proteases, amino acid L-oxidase, hemorrhagic factors and blood coagulation inhibitors, vasoactive peptides, cysteine-rich secretory protein. The composition of Vipera b. berus and Vipera b. nikolskii venom and mechanism of its action in victim’s organism were analyzed too. It is known that among the components of Nikolsky viper’s venom (Vipera b. nikolskii) the most part belongs to various enzymes. Of all enzymes, phospholipase A2 reached about 65% of the dry weight, serine proteases — 19%. Experiments on mammals (mice) showed that Nikol­sky’s viper (Vipera b. nikolskii) venom cause both local symptoms — swelling, pain, lymphangitis, and systemic symptoms — hypotension, mild neurotoxic effect. In addition, there are reports of damage to the cranial nerves and progressive paralysis of the limbs in mice. The components of Vipera b. berus viper venom have mainly a hemotoxic effect due to variety of enzymes from the metalloproteinase family, serine proteases, L-amino acid oxidases and C-type lectinlike proteins and cause enhanced blood coagulation. It is known that L-amino acid oxidases block the neuromuscular transmission of impulses and cause the destruction of cell membranes. The venom of this viper also contains several proteins that have a neurotoxic effect. A comprehensive and thorough analysis of experimental research data testify that the composition of toxins of various animal species, including vipers, is extremely diverse. Plenty of studies state about the hemovasotoxic and neurotoxic effects of these venoms. There are also reports of damage to the cardiovascular, urinary, respiratory, and digestive systems. It is noted that not all targets of viper toxins in mammal’s organism have been identified yet, and not all mechanisms of its action are understood, so further research is relevant.

Thrombosis prevention and anti-thrombus formation by zinc (Ⅱ) ions against COVID-19 infection

Zinc induced COVID-19 thrombosis prevention and anti-thrombus formation have been established by that zinc promotes COVID-19 thrombosis prevention and anti-thrombus formation and zinc ions-induced activated anti-thrombus activity is proceeded to prevent acute ischemic stroke among COVID-19 patients Zinc can reduce neurological outcomes in COVID-19 patients that Zn2+ promotes inflammatory cytokine as a neurodegenerative disorder and the coronaviruses can affect the nervous system through blood circulation, causing neuro-inflammation. Zinc supplementation affects bronchial mucosal epithelial integrity, both under normal and zinc deficient conditions. The other, zinc ions inhibit COVID-19 lung inflammation that zinc ions promote platelet activation function that inhibits pulmonary thromboembo-lism, in which platelets could respond to changes in extracellular and intracellular Zn2+ concentration. Zn2+ plays a major role in the regulation of coagulation that zinc inhibit blood coagulation against COVID-19 infection, in which Zn2+ can modulate platelet and coagulation activation pathways, including fibrin formation that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation. Zinc-induced platelet aggregation, low concentrations of ZnSO4 and zinc chelation involve platelet activation and potentiated platelet aggregation. Persistent zinc intake for severe aggravation of COVID-19 has been suggested to be 8–11 mg/day for adults (tolerable upper intake level 40 mg/day) and suggesting that a zinc intake of 30–70 mg/day might aid in the RNA viruses control. Thus, zinc ions can inhibit inflammation, platelet behaviour function, blood coagulation, and neurological thrombus formation during ROS production and excessive oxidative stress against COVID-19 infection. Zn2+ ions-binding with many proteins-molecular mechanism has been clarified that Zn2+ ions may be bound with COVID-19 inflammatory, platelet, coagulation, thrombus proteins by Zn2+ ions-centered tetrahedrally binding protein molecular coordination pattern.

The Endocannabinoid System: A Potential Therapeutic Target for Coagulopathies.

Abnormal blood coagulation or coagulopathy is a common manifestation of many pathological conditions. It occurs when there is an imbalance between the activities of the coagulation system and the fibrinolytic system, leading to excessive or impaired intravascular blood clot formation, which can disturb blood flow causing ischemia or hemorrhage in the affected tissues. A growing body of evidence has demonstrated blood coagulation abnormalities in association with cannabinoid use, suggesting the involvement of the endogenous cannabinoid system (ECS) in modulating blood coagulation. However, the evidence in the literature has been controversial on whether cannabinoids promote or inhibit blood coagulation. The ECS has been extensively studied in recent years for its potential as a therapeutic target for many diseases. This review provides a brief introduction to the ECS and discusses the reported anticoagulatory and procoagulatory effects of various cannabinoids, highlighting some possible mechanisms that might underlie the observed effects. Understanding the coagulatory effects of cannabinoids and the interaction between the coagulation system and the ECS is vital for developing novel therapeutics for coagulopathies.

Zinc (Ⅱ)-Induced Anti-Thrombus Formation against Severe COVID-19 Infection

Zinc(Ⅱ) induced neurological COVID-19 anti-thrombus formation has been established by that zinc induced inhibitive bronchial thrombosis and modulatory pulmonary thromboembolism are attained, causing COVID-19 activated anti-thrombus activity and leading to COVID-19 anti-thrombus formation. Zinc supplementation affected bronchial mucosal epithelial integrity, both under normal and zinc deficient conditions that there was an interaction between the individual zinc status. The other, zinc ions inhibit COVID-19 lung inflammation and promote platelet activation function that inhibits pulmonary thromboembolism, in which platelets could respond to changes in extracellular and intracellular Zn2+ concentration. Zinc-induced platelet aggregation, low concentrations of ZnSO4 and zinc chelation involve platelet activation and potentiated platelet aggregation, in which Zn2+ plays a major role in the regulation of coagulation that zinc inhibit blood coagulation against COVID-19 infection. Zn2+ can modulate platelet and coagulation activation pathways, including fibrin formation that the release of ionic Zn2+ store contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation. Further, zinc may reduce neurological resultings in COVID-19 patients that Zn2+ promotes inflammatory cytokine as a neurodegenerative disorder and the coronaviruses can affect the nervous system through blood circulation, causing neuro-inflammation. Zinc ions can inhibit inflammation, platelet behaviour function, and blood coagulation. Hence, zinc ions promote neurological anti-thrombosis formation during ROS production and excessive oxidative stress against COVID-19 infection. Persistent zinc intake for severe aggravation of COVID-19 has been suggested to be 8–11 mg/day for adults (upper intake level 40 mg/day) and suggesting that a zinc intake of 30–70 mg/day might aid in the RNA virus control. Accordingly, Zn2+ ions-binding molecular mechanism has been clarified that Zn2+ ions may be bound with COVID-19 inflammatory, platelet, coagulation, and thrombus various proteins by Zn2+ ions-centered tetrahedrally binding protein molecular coordination pattern.

The Therapeutic Potential of Anticoagulation in Organ Fibrosis.

Fibrosis, also known as organ scarring, describes a pathological stiffening of organs or tissues caused by increased synthesis of extracellular matrix (ECM) components. In the past decades, mounting evidence has accumulated showing that the coagulation cascade is directly associated with fibrotic development. Recent findings suggest that, under inflammatory conditions, various cell types (e.g., immune cells) participate in the coagulation process causing pathological outcomes, including fibrosis. These findings highlighted the potential of anticoagulation therapy as a strategy in organ fibrosis. Indeed, preclinical and clinical studies demonstrated that the inhibition of blood coagulation is a potential intervention for the treatment of fibrosis across all major organs (e.g., lung, liver, heart, and kidney). In this review, we aim to summarize our current knowledge on the impact of components of coagulation cascade on fibrosis of various organs and provide an update on the current development of anticoagulation therapy for fibrosis.

Functionalized Silicone Elastomer via Alkaline Solution to Coat Phosphorylcholine-Based Copolymer Containing Organosilane to Improve Hemocompatibility for Medical Devices

Surface modification of hemocompatible copolymers on silicone elastomers (SEs) is crucial for the long-term use of medical devices. Both physical adsorption and chemical conjugation are important for modification of SE. Oxygen plasma treatment is widely used to produce silanol groups on SE for silane coupling. However, the plasma reaction is difficult to apply to the surface modification of three-dimensional complex devices. This study demonstrated an appropriate and efficient method with alkaline solution for producing silanol groups on SE for modifying phosphorylcholine-based copolymer with organosilane (cross-MPC copolymer). A 2.5 wt% aqueous solution of potassium hydroxide (KOH) was effective in producing silanol groups and for coating the cross-MPC copolymer. Additionally, we successfully modified the cross-MPC copolymer on the inner surface of SE tubes after pretreatment with the 2.5 wt% KOH aqueous solution, and the copolymer film was coated homogeneously. The cross-MPC copolymer film on SE was stable for one month under fluidic condition with a shear stress of 3.2 Pa. The hollow fiber membrane with the polymer coating inhibited blood coagulation after one week implantation with extracorporeal circulation device using a goat. Therefore, pretreatment of SE using an alkaline solution is an appropriate method for producing silanol groups for coating the cross-MPC copolymer by silane-coupling reaction.

Antithrombin Effect of Jidabokuippo and Identification of Active Compounds

Static blood (Oketsu) is a blood disorder of Kampo medicine. Many natural medicines have been used to cure static blood, and these drugs are called blood stasis-resolving formula. Some of these were reported to inhibit blood coagulation. Jidabokuippo (JDI), one of the blood stasis-resolving formulas, has been used to treat bruises and sprains. In this study, we evaluated the antithrombin activity of JDI and its active compounds using thrombin and an artificial substrate. Bioassay-guided isolation was performed for JDI, and the active compounds were identified using spectral data (NMR spectroscopy and mass spectrometry) by comparison with reported data. JDI depressed thrombin activity in a dose-dependent manner, ranging from 0.125 to 1 mg/mL. Chrysophanol-1- O-β-D-glucoside (1) and chrysophanol-8- O-β-D-glucoside (2) were obtained from JDI and showed thrombin inhibitory activity. This study provided the first report of the antithrombin effect of JDI and the isolation from it of two active compounds (1 and 2). These findings might contribute to the understanding of the mechanism of static blood and blood stasis-resolving formula.

MOLECULAR MECHANISMS OF INTRAVASCULAR INHIBITION AND STIMULATION OF EXTRAVASCULAR THROMBOSIS

The hemostasis system is designed to maintain a balance between the processes of blood clotting, anticoagulation, as well as fibrinolysis, to ensure constant effective blood circulation in the body and rapid cessation of bleeding in the event of their occurrence. The procoagulant potential of the hemostasis system is based on molecular mechanisms that lead to the formation of fibrin in the bloodstream, which is the framework of the thrombus, and to the aggregation of platelets — the basis of the thrombus body. The anticoagulant potential of blood plasma is provided by mechanisms aimed at inhibiting blood coagulation processes. Thorough study and understanding of these mechanisms will open up numerous treatments for pathologies associated with both intravascular thrombosis and bleeding of various origins. The purpose of this review is to analyze ways to prevent intravascular thrombosis and stimulate extravascular thrombosis. The review describes and analyzes available and promising means of thrombosis prevention, in particular, direct and indirect anticoagulants and antiplatelets, as well as methods of effective stimulation of thrombosis, which is necessary in case of vascular damage. The result of this analysis is to determine the nodal points of the protein network of the hemostasis system, the action of which by specific molecular effectors will control the process of thrombosis.

Thiol Isomerase Activity of β3 Integrin Psi Domain and L33P Polymorphism: Implications in Blood Coagulation and Anti-Thrombotic Therapy

Background: Integrins are α and β subunit heterodimeric receptors expressed ubiquitously on metazoan cells that play key roles in cell adhesion, movement, and signaling. The αIIbβ3 integrin on platelets is essential for thrombosis and hemostasis through its binding of fibrinogen and other ligands to mediate platelet adhesion and aggregation. The plexin-semaphorin-integrin (PSI) domain is an approximately 54 amino acid sequence at the N-terminus of the β3 subunit located in close proximity to the “knee” region. Our lab recently discovered that two “CXXC” motifs within PSI domains in the integrin family exert endogenous thiol isomerase activity, and blockade of this activity in β3 integrin with our novel anti-PSI domain monoclonal antibodies (mAbs) significantly attenuated platelet adhesion and aggregation without impairing hemostasis (Blood, 2017). Interestingly, intravital microscopy studies demonstrate that our anti-PSI antibodies inhibited thrombosis in vivo 10-30 fold more potent than their effects in vitro under anti-coagulant conditions, suggesting a possible inhibitory effect on blood coagulation. Notably, A L33P polymorphism (HPA-1b) within the β3 integrin PSI domain is linked to an approximately two-fold increased risk for cardiovascular disease (CVD), however, the underlying mechanism for this remains unclear.Methods/Results: To investigate the role of PSI domain in blood coagulation, we first employed thromboelastography (TEG) to compare our anti-PSI domain mAbs with other anti-β3 mAbs that do not directly bind to β3 PSI domain. Results show that anti-PSI domain mAbs inhibited blood coagulation in human and murine whole blood or platelet-rich plasma (PRP) significantly more than anti-αIIbβ3 antibodies JAN-D1, M1, and Abciximab precursor 7E3. To address whether the L33P polymorphism affects PSI domains thiol isomerase activity, we generated L33P PSI domain via site-directed mutagenesis in E. coli. Using a scrambled RNase assay, we found that L33P polymorphism enhanced thiol-isomerase activity relative to WT PSI domain. We further corroborated these findings through an insulin β chain reduction assay, and a MPB (N-Maleimidopropionyl-biocytin) western blot assay, which quantifies thiol isomerase activity through MPB binding to free thiols that have not been oxidized into RNase. Interestingly, TEG results show that recombinant human PSI domain enhanced blood coagulation in platelet-microparticle (PMP) free plasma, which was generated through high-speed centrifugation (17,000 x g) of platelet poor plasma (PPP)for 15 minutes that removed residual platelets and microparticles .Conclusion: We have discovered a novel role of integrin β3 PSI domain in blood coagulation, which is enhanced by the L33P polymorphism (HPA-1b). These data highlight the β3 PSI domain as a suitable therapeutic target for its roles in both platelet adhesion/aggregation, and blood coagulation. Furthermore, these data may explain the increased risk of CVD such as myocardial infarction and deep vein thrombosis for individuals with the L33P polymorphism. DisclosuresNo relevant conflicts of interest to declare.

Prediction of the biological activity of a compound depending on its NH-acidity

Abstract: Acetamides are building blocks for the synthesis of compounds containing pharmacophores in their structure, manifesting a diverse range of biological activity. The drugs based on these substances possess antidiabetic effect and inhibit blood coagulation. Some of them act as chemosensitizers (i.e., cancer cell inhibitors). However, the full potential of these compounds remains to be fully accomplished. In a previous study, we synthesised acetamides with the RCONHCH (R´) CCl3 general formula (where R = CH3, CH2Cl; R´ = C6H5, C6H4CH3, C6H4OCH3, C6H4OH) and studied their acid-base behaviour. The NH-acidity of the studied acetamides is controlled by the polar effects of substituents. In this paper, the potential biological activity of the previously obtained acetamides is calculated, and the dependence of their biological potential on the NH-acidity values is elucidated. Prediction of biological activity was carried out using the PASS software. An analysis of the types of biological activity occurring in all compounds allowed us to determine a linear dependence between the probability of biological potential and the value of dissociation constant.

Laboratory Assays to Address the Problem of Heparin-Protamine Balance Following Neonatal Open Heart Surgery

Introduction: Neonates are especially susceptible to bleeding during and after cardiopulmonary bypass (CPB) surgery seemingly by virtue of a proportionately larger foreign surface exposure area for blood, significant hemodilution, and a coagulation system that differs from older patients. During CPB, heparin is administered to prevent thrombin formation. After CPB, it is reversed with protamine sulfate. Although administered to reverse anticoagulation, high levels of protamine sulfate paradoxically inhibit blood coagulation. This study evaluated the effect of heparin anticoagulation and protamine sulfate reversal on the accuracy of diagnostic coagulation tests and bleeding outcomes in a neonatal population undergoing CPB for congenital heart defects in the first 30 days of life.Methods: Blood samples were obtained at baseline (Draw 1), on CPB (Draw 2), following protamine reversal (Draw 3) and upon admission to the cardiac intensive care unit (Draw 4). Thrombin time (±protamine), FXa activity, activated partial thromboplastin time (APTT), Rotational Thromboelastometry (ROTEM) and Calibrated Automated Thrombography (CAT) were performed. Excessive bleeding was defined by meeting one or more of the following criteria: ≥ 84 mL/kg total chest tube output (CTO) in the first 24 hours, ≥ 7 mL/kg/hr CTO for ≥2 consecutive hours in the first 12 hours, or re-exploration in the first 24 hours for bleeding or tamponade.Results: This prospective observational study enrolled 44 neonates. Of these, 16 (36%) had excessive bleeding. The thrombin time assay was sensitive to heparin but was only minimally impacted by protamine sulfate, unless heparin was in the sample. Therefore, it was selected as the “gold-standard” assay for measurement of heparin. Relative heparin levels, determined by rate of fibrin formation, inversely correlated with thrombin generation measured using CAT, particularly in comparison to the thrombin generation rate in Draw 4 samples (exponential fit R2= 0.333). Higher plasma heparin concentration was present in 85% of the patients at Draw 4 than at Draw 3, indicating postoperative heparin rebound. This was important because, although the plasma heparin concentration in bleeders varied greatly, those patients with the two highest heparin concentrations at Draw 4 had excessive bleeding. The FXa activity assay was altered by protamine and by heparin. Therefore, samples with elevated FXa activity but normal thrombin time likely had high protamine. There were 4 such samples and 2 were bleeders. Additional coagulation assays were compared to the thrombin time assay to assess their performance for detection of heparin. ROTEM and APTT assays also correlated weakly (R2= 0.0531 with P value 0.1527 and R2 = 0.1549 with P value 0.0099, respectively) with the thrombin time assay at Draw 4. However, no correlation was present at Draw 3.Conclusions: Bleeding remains a common complication in neonates undergoing CPB for repair of heart defects. While there are many possible causes for the bleeding, excess heparin or protamine may contribute to its severity in a subset of cases. The simple to perform and inexpensive thrombin time test readily and accurately detects excess heparin. Increased use of this test may be beneficial, particularly for detection of heparin rebound that is observed in approximately 85% of patients. Excess protamine may also contribute to bleeding in these patients, and can be assessed using a combination of thrombin time and factor Xa activity assays. DisclosuresMast:Novo Nordisk: Research Funding.

The trypsin inhibitor-like domain is required for a serine protease inhibitor of Haemonchus contortus to inhibit host coagulation

Haemonchus contortus, a blood-feeding nematode, inhibits blood coagulation at the site of infection to facilitate blood-sucking and digesting for successful parasitism. However, the mechanism underlying anti-coagulation at the host-parasite interface is largely unknown. In the current study, Hc-spi-i8, which has two greatly different transcripts named Hc-spi-i8a and Hc-spi-i8b, respectively, was described. Hc-SPI-I8A was a serine protease inhibitor containing a trypsin inhibitor-like cysteine rich (TIL) domain, while Hc-SPI-I8B was not. Hc-SPI-I8A/B were primarily expressed in the hypodermis, intestines and gonads in the parasitic stages of H. contortus. Hc-SPI-I8A interacted with Ovis aries TSP1-containing protein (OaTSP1CP), which was determined by yeast two-hybrid, co-immunoprecipitation (Co-IP), pull down and co-localization experiments. The blood clotting time contributed by the TIL domain was prolonged by Hc-SPI-I8A. Hc-SPI-I8A is most likely interfering in the extrinsic coagulation cascade by interacting with OaTSP1CP through its TIL domain and intrinsic coagulation cascade by an unknown mechanism. These findings depict a crucial point in the host-parasite interaction during H. contortus colonization, which should contribute to drug discovery and vaccine development in fighting against this important parasite worldwide.

Controlled sulfation of mixed-linkage glucan by Response Surface Methodology for the development of biologically applicable polysaccharides

Endogenous and exogenous sulfated polysaccharides exhibit potent biological activities, including inhibiting blood coagulation and protein interactions. Controlled chemical sulfation of alternative polysaccharides holds promise to overcome limited availability and heterogeneity of naturally sulfated polysaccharides. Here, we established reaction parameters for the controlled sulfation of the abundant cereal polysaccharide, mixed-linkage β(1,3)/β(1,4)-glucan (MLG), using Box-Behnken Design of Experiments (BBD) and Response Surface Methodology (RSM). The optimization of the degree-of-substitution (DS) was externally validated through the production of sulfated MLGs (S-MLGs) with observed DS and Mw values deviating less than 20% and 30% from the targeted values, respectively. Simultaneous optimization of DS and Mw resulted in the same range of deviation from the targeted value. S-MLGs with DS > 1 demonstrated a modest anticoagulation effect versus heparin, and a greater P-selectin affinity than fucoidan. As such, this work provides a route to medically important polymers from an economical agricultural polysaccharide.

Modulation of Host Immune System by Staphylococcal Superantigen-like (SSL) Proteins

Staphylococcus aureus is a common pathogen causing a wide range of infectious diseases in humans and animals. This bacterium secretes a variety of exoproteins, including toxins known as superantigens, such as toxic shock syndrome toxin-1 (TSST-1) and enterotoxins. Staphylococcal superantigen-like (SSL) proteins are a family of exoproteins showing structural similarities with superantigens but no superantigenic activity. This family is composed of 14 members (SSL1-SSL14), and recent studies have revealed that these members exhibit various immunomodulatory activities: e.g., inhibition of antibody and complement functions, impairment of leukocyte trafficking, modulation of receptor functions, inappropriate activation of immunocytes, and inhibition of blood coagulation. These activities have been proposed to contribute to immune evasion of the bacteria. The interactions between SSL proteins and their target molecules in the host immune system and the pathophysiological roles of SSL proteins in the bacterial infections are reviewed in this article.