Switch to pdVWF:pdFVIII concentrate for prophylaxis in a paediatric patient with Type 3 von Willebrand disease: a case report.

OBJECTIVES: To report the long-term prophylaxis management of a child with type 3 von Willebrand disease by switching to Wilate (Octapharma AG), a plasma-derived, double virus-inactivated concentrate of freeze-dried of a 1 to 1 ratio of active Von Willebrand Factor and Factor VIII (pdVWF:pdFVIII) recently marketed as Eqwilate in France. METHODS: This is a case report of 12.6-year-old boy with congenital Type 3 VWD who had a history of frequent bleeds. Prophylaxis started at the age of 38 months with FVIII-poor pdVWF concentrate (Wilfactin, LFB) and FVIII (Wilstart, LFB). Pharmacokinetics and thrombin generation assay were performed. Annualized bleeding rate was derived from the bleeding episodes documented in the medical record during a 24-month period before and after starting pdVWF:pdFVIII concentrate. RESULTS: Both product injections promptly raised the endogenous thrombin potential (ETP). However, the maximal concentration of formed thrombin was higher following pdVWF:pdFVIII injection. Due to a high bleeds frequency and better results regarding FVIII levels and thrombin generation, the prophylaxis regimen was changed to the same dose and frequency of pdVWF:pdFVIII concentrate (42 IU/kg per day, three times a week). During the last 24 months, annualized total, trauma, and spontaneous bleeding rates were 7.5, 4.5, and 3, respectively. These rates decreased to 2, 1.5, and 0.5 respectively during the next two years. The mother reported a marked improvement in the quality of life of his son and hers. CONCLUSION: Switch to pdVWF:pdFVIII concentrate for long-term prophylaxis in a young type 3 VWD patient was safe and effective in reducing bleeds.

Preparation of magnetic poly(ethylene glycol dimethacrylate-N-Methacryloyl-(L)-glutamic acid) particles for thrombin purification.

In this study, magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-glutamic acid) (mPEGDMA-MAGA) particles were prepared by the dispersion polymerization in order to purify thrombin effectively. mPEGDMA-MAGA particles were synthesized by adding different ratios of magnetite (Fe3O4) to the medium in addition to the monomer phases EGDMA and MAGA. The characterization studies of mPEGDMA-MAGA particles were used by fourier transform infrared spectroscopy, zeta size measurement, scanning electron microscopy and electron spin resonance. mPEGDMA-MAGA particles were used in thrombin adsorption studies from aqueous thrombin solutions in both batch and magnetically stabilized fluidized bed (MSFB) system. Maximum adsorption capacity in pH 7.4 phosphate buffer solution is 964 IU/g polymer and 134 IU/g polymer in MSFB system and batch system, respectively. The developed magnetic affinity particles enabled the separation of thrombin from different patient serum samples in one step. It has also been observed that magnetic particles can be used repeatedly without significant reduction in adsorption capacity.

Towards understanding the effect of fibrinogen interactions on fibrin gel structure.

Fibrin gelation involves the enzymatic conversion of the plasma protein fibrinogen to fibrin monomers which then polymerize to form the gel that is a major structural component of a blood clot. Because fibrinogen provides the material from which fibrin is made, it is generally regarded as promoting the gelation process. However, fibrinogen can bind to a site on a fibrin oligomer, preventing another fibrin oligomer from binding there, thus slowing the polymerization process. "Soluble fibrin oligomers," which are mixtures of fibrin and fibrinogen, are found in the blood plasma and serve as biomarkers for various clotting disorders, so understanding the interplay between fibrin and fibrinogen during fibrin polymerization may have medical importance. We present a kinetic gelation model of fibrin polymerization which accounts for the dual and antagonistic roles of fibrinogen. It builds on our earlier model of fibrin polymerization that proposed a novel mechanism for branch formation, which is a necessary component of gelation. This previous model captured salient experimental observations regarding the determinants of the structure of the gel, but did not include fibrinogen binding. Here, we add to that model reactions between fibrinogen and fibrin, so oligomers are now mixtures of fibrin and fibrinogen, and characterizing their dynamics leads to equations of substantially greater complexity than previously. Using a moment generating function approach, we derive a closed system of moment equations and we track their dynamics until the finite time blow-up of specific second moments indicates that a gel has formed. In simulations begun with an initial mixture of fibrin and fibrinogen monomers, a sufficiently high relative concentration of fibrinogen prevents gelation; the critical concentration increases with the branch formation rate. In simulations begun with only fibrinogen monomers that are converted to fibrin at a specified rate, the rates of conversion, fibrinogen binding to oligomers, and branch formation together determine whether a gel forms, how long it takes to form, and the structural properties of the gel that results.

Protein labeling and crosslinking by covalent aptamers.

In this chapter, a new approach to the selective modification of native proteins is discussed, using electrophilic covalent aptamers. These biochemical tools are generated through the site-specific incorporation of a label-transferring or crosslinking electrophile into a DNA aptamer. Covalent aptamers provide the ability to transfer a variety of functional handles to a protein of interest or to irreversibly crosslink to the target. Methods for the aptamer-mediated labeling and crosslinking of thrombin are described. Thrombin labeling is fast and selective, in both simple buffer and in human plasma and outcompetes nuclease-mediated degradation. This approach provides facile, sensitive detection of labeled protein by western blot, SDS-PAGE, and mass spectrometry.

Spinal epidural fibrosis following hemostatic agent employment.

OBJECTIVE: Failed Back Surgery Syndrome (FBSS) refers to a subset of patients who have new or persistent pain after spinal surgery for back or leg pain. Epidural fibrosis (EF) is a common cause of FBSS. Many agents aiming to prevent EF have been tested. However, hemostatic agents are readily available at hospitals, easy to reach and frequently used. For these reasons, oxidized regenerated cellulose, polysaccharide hemostat, hemostatic thrombin-gelatin matrix and chitosan linear polymer were evaluated for their effects on epidural fibrosis on rats after laminectomy. METHODS: 40 Sprague-Dawley rats were randomly divided into 5 equal groups including the control group where only the laminectomy was performed. The other 4 groups received hemostatic agents after laminectomy. The rats were euthanized 45 days later and were assessed by a blinded observer to grade the fibrosis level. RESULTS: The study revealed that oxidized regenerated cellulose, polysaccharide hemostat and hemostatic thrombin-gelatin matrix lowered the epidural fibrosis grade which was statistically significant (p < 0.001). Although chitosan linear polymer created fibrosis similar to the control group it was not proven to be statistically significant (p = 0.8999). However, when compared with other hemostatic agents it resulted in a higher fibrosis grade (p < 0.001). CONCLUSION: The results obtained from this experimental study revealed that Pahacel, Sealfoam and Surgiflo, were effective in reducing epidural fibrosis after laminectomy in rats.

Diabetes type 2: relationships between lysosomal exocytosis of circulating normal-sized platelets and in vitro ɑ-thrombin-evoked platelet responses.

BACKGROUND/OBJECTIVE: Type 2 diabetes is a major risk factor for atherosclerotic disease. It is well agreed that the reactivity of diabetic platelets is increased but how platelet reactivity regulates is unknown. In our laboratory, density separated platelets have been investigated extensively and high- and low-density platelets circulate in an activated state. The density distribution of circulating platelets is altered in diabetes type 2 as well. We hypothesize that such platelets modify whole blood (WB) in vitro α-thrombin-evoked (10 µM/mL) activity in type 2 diabetes. Thus, the study aims to identify features of circulating normal-sized density subpopulations affecting whole blood (WB) platelet reactivity in type 2 diabetes. PATIENTS AND METHODS: Patients with type 2 diabetes (n = 16) were enrolled. Their normal-sized platelets were divided into density subfractions (n = 16) using continuous polyvinylpyrrolidone-coated silica (Percoll™) gradients (density span, 1.090-1.040 kg/L) containing prostaglandin E1. The proportions (%) of such density-separated platelets expressing lysosomal-associated membrane protein 1 (LAMP-1) were analyzed using a flow cytometer. Further, determinations of WB ɑ-thrombin-evoked (10 U/mL) surface LAMP-1 (an assessment of lysosomal release), the fibrinogen (αIIbß3) receptor activity, annexin V (binds to exposed membrane phosphatidylserine), and mitochondrial transmembrane potentials (an estimate of organelle integrity) were performed. Surface LAMP-1 expressions of individual normal-sized platelet density subpopulations were stratified into equal-sized groups (n = 2) depending on reactivity, as judged from the ɑ-thrombin-induced WB activity markers. RESULTS: With some exceptions, the proportion of normal-sized circulating platelets showing spontaneous LAMP-1 was strongly associated with WB ɑ-thrombin-evoked (10 U/mL) surface LAMP-1 and αIIbß3 receptor activity. LAMP-1-expressing normal-sized platelets also displayed inverse associations with WB ɑ-thrombin-induced surface annexin V and mitochondrial damage, which are features of procoagulant platelets. CONCLUSIONS: From the current descriptive work only involving type 2 diabetes, it is impossible to judge whether the findings are features of the disease or if they occur in healthy individuals as well. However, the study describes LAMP-1 expressing subpopulations of circulating normal-sized platelets that associate with WB α-thrombin (10 U/mL) responses in vitro. Increased proportions of such platelets induced lysosomal release and αIIbß3 receptor activity, whereas lower proportions promoted WB agonist-induced procoagulant platelet creation. It is to hypothesize that the new described regulatory mechanism could in the future offer a possibility to influence platelet behavior in type 2 diabetes.Key messagesLysosomal exocytosis of circulating platelets influences reactivity, as determined by agonist-induced platelet reactions in vitroThus, the low release of lysosomes by normal-sized platelets in vivo increases agonist-evoked procoagulant platelet production.Higher lysosomal exocytosis of circulating normal-sized platelets promotes platelet aggregation and secretion.

Novel therapeutic perspectives for crescentic glomerulonephritis through targeting parietal epithelial cell activation and proliferation.

INTRODUCTION: Kidney injury is clinically classified as crescentic glomerulonephritis (CrGN) when ≥50% of the glomeruli in a biopsy sample contain crescentic lesions. However, current strategies, such as systemic immunosuppressive therapy and plasmapheresis for CrGN, are partially effective, and these drugs have considerable systemic side effects. Hence, targeted therapy to prevent glomerular crescent formation and expansion remains an unmet clinical need. AREAS COVERED: Hyperproliferative parietal epithelial cells (PECs) are the main constituent cells of the glomerular crescent with cell-tracing evidence. Crescents obstruct the flow of primary urine, pressure the capillaries, and degenerate the affected nephrons. We reviewed the markers of PEC activation and proliferation, potential therapeutic effects of thrombin and thrombin receptor inhibitors, and how podocytes cross-talk with PECs. These experiments may help identify potential early specific targets for the prevention and treatment of glomerular crescentic injury. EXPERT OPINION: Inhibiting PEC activation and proliferation in CrGN can alleviate glomerular crescent progression, which has been supported by preclinical studies with evidence of genetic deletion. Clarifying the outcome of PEC transformation to the podocyte phenotype and suppressing thrombin, thrombin receptors, and PEC hyperproliferation in early therapeutic strategies will be the research goals in the next ten years.

It is clinically classified as crescentic glomerulonephritis (CrGN) when more than 50% of the glomeruli of the kidney in a biopsy sample contain crescentic lesions (crescent shaped injuries). However, current strategies, such as immunosuppressive therapy and plasmapheresis (the removal, treatment and returning of blood) for CrGN, are partially effective, and these drugs have considerable side effects. In order to seek targeted therapy for CrGN, we reviewed the current research evidences. First, the hyperproliferative parietal epithelial cells (PECs) are the main cells within the glomerular crescent seen with cell-tracing evidence. The activated PECs can express specific markers and altered biological characteristics, such as cell growth and multiplication, migration, and extracellular matrix production. CD44, CD74, CD9, and pERK-1/2 are specific markers for PEC activation, and also as the potential therapeutic targets with evidence of gene knockout and inhibitor. Second, during the formation of glomerular crescents, PECs grow and multiply also through cross-talking with podocyte cells by the AngII/SDF-1/CXCR4/ERK1/2, HB-EGF/EGFR/JAK/STAT3, and PDGF/PDGFR signaling pathways, suggesting that the intervention of key molecules in these disease processes may be promising therapeutic targets for CrGN. Third, thrombin and protease-activated receptors (PARs) participate in the excessive proliferation of PEC through activation of the coagulation cascade reaction, PAR-1 and PAR-2. Therefore, anticoagulation therapy, especially inhibition of PAR-1 and PAR-2, is expected to be an effective strategy for the early prevention and treatment of CrGN. The drug vorapaxar selectively antagonizes PAR-1 and is the most promising candidate. These findings will not only improve the outlook for CrGN treatment, but will also help in the treatment of other glomerular diseases with crescentic lesions. [Figure: see text].

Tin(II) and Tin(IV) Complexes Incorporating the Oxygen Tripodal Ligands [(η5-C5R5)Co{P(OEt)2O}3]-, (R = H, Me; Et = -C2H5) as Potent Inflammatory Mediator Inhibitors: Cytotoxic Properties and Biological Activities against the Platelet-Activating Factor (PAF) and Thrombin.

Metal complexes displaying antiplatelet properties is a promising research area. In our methodology, Platelet-Activating Factor (PAF), the most potent lipid pro-inflammatory mediator, serves as a biological probe. The antiplatelet activity is exerted by the inhibition of the PAF-induced aggregation in washed rabbit platelets (WRPs) and in rabbit plasma rich in platelets (rPRPs). Herein, the synthesis and biological investigation of a series of organometallic tin(II) and tin(IV) complexes, featuring the oxygen tripodal Kläui ligands [(η5-C5R5)Co{P(OEt)2O}3]-, {R = H, (LOEt-); Me (L*OEt-)}, are reported. Reaction of NaLOEt (1a) and NaL*OEt (1b) with SnCl2, yielded the rare four-coordinate LOEtSnCl (2a) and L*OEtSnCl (2b) complexes. Accordingly, LOEtSnPh3 (3a) and L*OEtSnPh3 (3b) were prepared, starting from Ph3SnCl. Characterization includes spectroscopy and X-ray diffraction studies for 2a, 2b and 3b. The antiplatelet activity of the lead complexes 2b and 3a (IC50 = 0.5 µΜ) is superior compared to that of 1a and 1b, while both complexes display a pronounced inhibitory activity against thrombin (IC50 = 1.8 µM and 0.6 µM). The in vitro cytotoxic activities of 3a and 2b on human Jurkat T lymphoblastic tumor cell line is higher than that of cisplatin.

Comparative Analysis of Roots from Vicatia thibetica de Boiss and Angelica sinensis Based on Chemical Composition, Antioxidant, Nitrite-Scavenging and Enzyme Inhibition Activities.

Radix Vicatia thibetica de Boiss (RVT) is locally known as "Xigui" or "Dujiao-danggui" in Tibetan medicine and is often used as a substitute for Radix Angelica sinensis (RAS) in daily nourishing diets and clinical applications. In this study, we determined and compared the contents of polysaccharides, total coumarins, ferulic acid, total phenols, total flavonoids, chlorogenic acid, protein, and amino acids, and the composition of volatile oil in RVT and RAS. Biological activities, including antioxidants, scavenging of nitrite, inhibition of tyrosinase, thrombin, and coagulation FXa, were comparatively evaluated. Results showed that RVT contains more polysaccharides, phenols, flavonoids, proteins, glutamic acid, and lysine as compared to RAS. Among volatile compounds, 14 species are similar, and 20 species are different in RVT and RAS. Overall, among volatile compounds, the content of 3-N-Butylphthalide was higher, whereas the content of ligustilide was lower in RVT volatile oil. A significant difference was reported in the bioactivity of RVT and RAS. The biological activity of RVT had higher antioxidant, nitrite scavenging, and tyrosinase inhibitory activities, whereas it showed much lower thrombin and FXa inhibitory activities. Correlation analysis showed that the antioxidant, nitrite scavenging, and tyrosinase inhibitory activities were related to the phenol and flavonoid content, whereas the thrombin and FXa inhibitory activities were related to ferulic acid and volatile oil content. This study presents a comparative analysis of RAS and RVT's chemical compositions of antioxidant, nitrite-scavenging, inhibition of tyrosinase, thrombin, and coagulation FXa activities. It was found that both RVT and RAS have their unique advantages, and RVT has the potential to be utilized as functional foods, cosmetics, and medical products.

Allogeneic Solid Platelet-Rich Plasma for Persistent Epithelial Neurotrophic Defects: A Protocol and Pilot Study.

PURPOSE: Here, we present the procedure to obtain allogeneic solid platelet-rich plasma (PRP) and its use in a pilot study of patients with persistent neurotrophic epithelial defects. METHODS: We included 4 eyes of 4 patients with persistent neurotrophic epithelial defects unresponsive to other therapies from a single institution. PRP and thrombin were produced by the Department of Transfusion Medicine from healthy blood donors. PRP was activated in its solid form in the operating room with addition of thrombin and calcium gluconate 10% and applied on the cornea with fibrin glue and soft contact lens. Corneal healing time, corneal esthesiometry, visual acuity, Oxford staining score, Ocular Surface Disease Index questionnaire, and Schirmer I test were recorded. Anterior segment optical coherence tomography and in vivo confocal microscopy were also evaluated over the 4-month follow-up period. RESULTS: The persistent epithelial defect healed in all patients in the first 10 days. During the follow-up, there was an absence of recurrences. For all patients, there was a reduction in Ocular Surface Disease Index questionnaire score (case 1: -55 points, -73.3%; case 2: -26.3 points, -58.4%; case 3: -56 points, -69.1%; case 4: -20 points, -26.6%; mean reduction: 39.3 points, 56.85%) and Oxford staining score (case 1, 2, and 3: 3 points decrease; case 4: 2 points decrease; mean reduction: -2.75 points). CONCLUSIONS: Allogeneic solid PRP in combination with fibrin glue may facilitate wound healing in neurotrophic persistent epithelial defects. Further prospective studies are needed to quantify its efficacy.

Anticoagulant Property of a Sulfated Polysaccharide with Unique Structural Characteristics from the Green Alga Chaetomorpha aerea.

Sulfated polysaccharides from marine algae have attracted a great amount of attentions for the development of marine drugs due to their unique structural features, and they are great potential sources of naturally occurring anticoagulant agents. The genus Chaetomorpha is one of the largest genera in green algae and has a worldwide distribution. In the present study, a homogeneous polysaccharide from Chaetomorpha aerea, designated as PCA, was obtained by alkali extraction, anion-exchange and size-exclusion chromatography. Based on the results of chemical and spectroscopic analyses, PCA was a sulfated galactoarabinan which was mainly constituted of a backbone of →4)-ß-l-Arap-(1→ unit, partially sulfated at C-3 of →4)-ß-l-Arap-(1→ and C-4 of →6)-α-d-Galp-(1→. The side chains consisting of →6)-α-d-Galp-(1→ and →5)-α-l-Araf-(1→ residues were in C-2 of →4)-ß-l-Arap-(1→ unit. PCA had a strong anticoagulant activity in vitro as evaluated by the assays of activated partial thromboplastin time, thrombin time and fibrinogen level. The obvious anticoagulant activity in vivo of PCA was also found. PCA significantly inhibited the activities of the intrinsic coagulation factors XII, XI, IX and VIII, and exhibited weak inhibition effects on the common coagulation factors II and X. The anticoagulant mechanism of PCA was attributed to strong thrombin inhibition potentiated by heparin cofactor II or antithrombin III, and it also possessed an apparent inhibition effect on coagulation factor Xa mediated by antithrombin III. The investigation demonstrated that PCA could be a promising anticoagulant agent for health promotion and the treatment of thrombotic diseases.

Investigation of cofactor activities of endothelial microparticle-thrombomodulin with liposomal surrogate.

Thrombomodulin (TM) is a type I transmembrane glycoprotein mainly expressed on the endothelial cells, where it binds thrombin to form the thrombin-TM complex that can activate protein C and thrombin-activable fibrinolysis inhibitor (TAFI) and induce anticoagulant and anti-fibrinolytic reactions, respectively. Cell activation and injury often sheds microparticles that contain membrane TM, which circulate in biofluids like blood. However, the biological function of circulating microparticle-TM is still unknown even though it has been recognized as a biomarker of endothelial cell injury and damage. In comparison with cell membrane, different phospholipids are exposed on the microparticle surface due to cell membrane ''flip-flop'' upon cell activation and injury. Liposomes can be used as a microparticle mimetics. In this report, we prepared TM-containing liposomes with different phospholipids as surrogates of endothelial microparticle-TM and investigated their cofactor activities. We found that liposomal TM with phosphatidylethanolamine (PtEtn) showed increased protein C activation but decreased TAFI activation in comparison to liposomal TM with phosphatidylcholine (PtCho). In addition, we investigated whether protein C and TAFI compete for the thrombin/TM complex on the liposomes. We found that protein C and TAFI did not compete for the thrombin/TM complex on the liposomes with PtCho alone and with low concentration (5%) of PtEtn and phosphatidylserine (PtSer), but competed each other on the liposomes with higher concentration (10%) of PtEtn and PtSer. These results indicate that membrane lipids affect protein C and TAFI activation and microparticle-TM may have different cofactor activities in comparison to cell membrane TM.

Increased In Vivo Thrombin Generation in Patients with Localized Non-Small Cell Lung Cancer Unfit for Surgery.

Patients with lung cancer face a substantially increased risk of thromboembolic disease. Patients with localized non-small cell lung cancer (NSCLC) who are unfit for surgery due to age or comorbidity have additional thrombotic risk factors. Thus, we aimed to investigate markers of primary and secondary hemostasis, since this could assist in treatment decisions. We included 105 patients with localized NSCLC. Ex vivo thrombin generation was determined by calibrated automated thrombogram and in vivo thrombin generation was determined by measurement of thrombin-antithrombin complex (TAT) levels and prothrombin fragment F1 + 2 concentrations (F1 + 2). Platelet aggregation was investigated by impedance aggregometry. Healthy controls were used for comparison. TAT and F1 + 2 concentrations were significantly higher in NSCLC patients than in healthy controls (P < .001). The levels of ex vivo thrombin generation and platelet aggregation were not increased in the NSCLC patients. Patients with localized NSCLC considered unfit for surgery had significantly increased in vivo thrombin generation. This finding should be further investigated as it could be relevant for the choice of thromboprophylaxis in these patients.

Target-Synergized Biologically Mediated RAFT Polymerization for Electrochemical Aptasensing of Femtomolar Thrombin.

The assay of thrombin levels is integral to the assessment of coagulation function and clinical screening of coagulation disorder-related diseases. In this work, we illustrate the ingenious use of the target-synergized biologically mediated reversible addition-fragmentation chain transfer (RAFT) polymerization (tsBMRP) as a novel amplification strategy for the electrochemical aptamer-based biosensing of thrombin at the femtomolar levels. Briefly, the tsBMRP-based strategy relies on the boronate affinity-mediated decoration of the glycan chain(s) of the target itself with RAFT agents and the subsequent recruitment of signal labels via BMRP, mediated by the direct reduction of RAFT agents by NADH into initiating/propagating radicals. Obviously, the tsBMRP-based strategy is biologically friendly, low-cost, and simple in operation. As thrombin is a glycoconjugate, its electrochemical aptasensing involves the use of the thrombin-binding aptamer (TBA) as the recognition receptor, the site-specific decoration of RAFT agents to the glycan chain of thrombin via boronate affinity, and further the recruitment of ferrocene signal labels via the BMRP of ferrocenylmethyl methacrylate (FcMMA). As boronate affinity results in the decoration of each glycan chain with tens of RAFT agents while BMRP recruits hundreds of signal labels to each RAFT agent-decorated site, the tsBMRP-based strategy allows us to detect thrombin at a concentration of 35.3 fM. This electrochemical aptasensor is highly selective, and its applicability to thrombin detection in serum samples has been further demonstrated. The merits of high sensitivity and selectivity, low cost, good anti-interference capability, and simple operation make the tsBMRP-based electrochemical thrombin aptasensor great promise in biomedical and clinical applications.

Construction and application of thrombin-activated fluorescence-SERS dual-mode optical nanoprobes.

Thrombin (TB) plays a key role in the pathological and physiological coagulation of diseases. In this work, a TB-activated fluorescence-surface-enhanced Raman spectroscopy (SERS) dual-mode optical nanoprobe (MRAu) was constructed by linking rhodamine B (RB)-modified magnetic fluorescent nanospheres with AuNPs through TB-specific recognition peptides. In the presence of TB, the polypeptide substrate could specifically be cleaved by TB, resulting in the weakening of SERS hotspot effect and the reduction of Raman signal. Meanwhile, the fluorescence resonance energy transfer (FRET) system was destroyed, and the RB fluorescence signal originally quenched by AuNPs was recovered. Using MRAu, SERS and fluorescence methods were combined to extend the TB detection range to 1-150 pM, and the detection limit was as low as 0.35 pM. In addition, the ability to detect TB in human serum also verified the effectiveness and practicality of the nanoprobe. The probe was also successfully employed to evaluate the inhibitory effect against TB of active components in Panax notoginseng. This study provides a new technical means for the diagnosis and drug development of abnormal TB-related diseases.

Thrombin generation in real life bleeding patients on oral anticoagulants reversed (or not) with (activated) prothrombin complex concentrate.

BACKGROUND: Bleeding during oral anticoagulant therapy is currently codified by expert guidelines. Monitoring of coagulation during bleeding events is challenging. Our study sought to assess thrombin generation assay (TGA) in direct oral anticoagulant-treated patients without bleeding (WB), bleeding without reversal therapy (BR-), and bleeding with reversal therapy (BR+). METHODS: We conducted a prospective, monocentric study from June 2015 to June 2018. For all bleeding groups, TGA was evaluated using platelet-poor plasma collected upon arrival at emergency (T0), and 30 min (T1), 6 h (T2) and 24 h (T3) after reversal therapy (if indicated) following activation by tissue factor 5 pM and phospholipids. RESULTS: Overall, 292 patients participated, including 91 BR+, 94 BR-, and 107 WB patients. At T0, vitamin K antagonist reversed (VKA-BR+) patients experienced a significant decrease in TGA parameters (ETP and peak) compared with VKA without bleeding (VKA-WB). Compared with healthy controls, VKA-BR+ patients reversed by four-factor prothrombin complex concentrate (4F-PCC) displayed comparable TGA 's ETP and peak at T1, T2, and T3, whereas direct anti-Xa BR+ patients reversed by 4F-PCC or activated prothrombin complex concentrate (aPCC) reached thrombin generation parameters that exceeded normal range at T2 and T3. CONCLUSIONS: In VKA-treated patients reversed by 4F-PCC, TGA parameters were normalized, whereas in rivaroxaban or apixaban-treated patients reversed by 4F-PCC or aPCC, TGA parameters exceeded normal range. Further studies are needed to compare the efficacy and safety of a different dose of reversal therapy and the impact on coagulation parameters.

PARIN5, a Novel Thrombin Receptor Antagonist Modulates a Streptozotocin Mice Model for Diabetic Encephalopathy.

Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research.

Comparison of the International Normalized Ratio Between a Point-of-Care Test and a Conventional Laboratory Test: the Latter Performs Better in Assessing Warfarin-induced Changes in Coagulation Factors.

Background: Point-of-care testing (POCT) coagulometers are increasingly used for monitoring warfarin therapy. However, in high international normalized ratio (INR) ranges, significant discrepancy in the INR between POCT and conventional laboratory tests occurs. We compared the INR of POCT (CoaguChek XS Plus; Roche Diagnostics, Mannheim, Germany) with that of a conventional laboratory test (ACL TOP 750; Instrumentation Laboratory SpA, Milan, Italy) and explored possible reasons for discrepancy. Methods: Paired POCT and conventional laboratory test INRs were analyzed in 400 samples from 126 patients undergoing warfarin therapy after cardiac surgery. Coagulation factor and thrombin generation tests were compared using the Mann-Whitney U test. Correlations between coagulation factors and INRs were determined using Pearson correlation coefficients. Results: The mean difference in the INR between the tests increased at high INR ranges. Endogenous thrombin potential levels were decreased at INR <2.0 for CoaguChek XS Plus and 2.0< INR <3.0 for ACL TOP 750 compared with those at INR <2.0 for both tests, indicating a better performance of ACL TOP 750 in assessing thrombin changes. The correlation coefficients of coagulation factors were stronger for ACL TOP 750 INR than for CoaguChek XS Plus INR. Vitamin K-dependent coagulation factors were found to contribute to the INR discrepancy. Conclusions: Decreases in vitamin K-dependent coagulation and anticoagulation factors can explain the significant discrepancy between the two tests in high INR ranges. Since conventional laboratory test INR values are more reliable than POCT INR values, a confirmatory conventional laboratory test is required for high INR ranges.

Enhanced Thrombin Formation in Patients With Ventricular Assist Devices Experiencing Bleeding: Insights From the Multicenter PREVENT Study.

The aim of this investigation was to characterize the hemostatic status of heart failure patients with implanted left ventricular assist devices (LVADs) to propose a mechanism associated with bleeding. Patients (n = 300) from 23 US hospitals were enrolled in the PREVENtion of HeartMate II Pump Thrombosis through Clinical Management (PREVENT) study. A biobank was established with serum and plasma samples prospectively collected from a cohort of 175 patients preimplant baseline (BL) and 3 months (3M) postimplant. Outcomes were collected for 6 months. Thrombin (prothrombin fragment 1.2 [F1.2], functional thrombin generation [TG]) and fibrinolytic activity (D-dimer, plasminogen activator inhibitor-1 [PAI-1]), but not contact activation (complement C5a), were elevated in heart failure patients at BL. F1.2, TG, and PAI-1 levels decreased 3M after LVAD implantation ( p < 0.01) but did not revert to normal in all patients; conversely, D-dimer increased BL to 3M ( p < 0.01). Compared with patients without events, thrombin activity (F1.2) was increased in patients with late bleeding (3-4 months postimplant) ( p = 0.06) and in those with late gastrointestinal (GI) bleeding ( p = 0.01). Patients with 3M F1.2 levels above the cohort mean had a higher incidence of bleeding ( p < 0.001) and GI bleeding ( p < 0.001) compared with those with below mean F1.2. Patients experiencing multiple bleeding events were more likely to have 3M F1.2 greater than the cohort mean. Despite anticoagulation with aspirin and warfarin, LVAD implanted patients exhibit hemostatic activation. Excess thrombin formation, particularly shown by increased F1.2, was demonstrated in association with bleeding in LVAD implanted patients.