Characterization and in vitro digestibility of soybean tofu: Influence of the different kinds of coagulant.

This study explored the effect of diverse coagulants (glucono-δ-lactone (GDL), gypsum (GYP), microbial transglutaminase (MTGase), and white vinegar (WVG)) on microstructure, quality, and digestion properties of tofu. The four kinds of tofu were significantly different in their structure, composition, and digestibility. Tofu coagulated with MTGase had the highest springiness and cohesiveness while GDL tofu had the highest enthalpy (6.54 J/g). However, the WVG and GYP groups outperformed others in terms of thermodynamic, and digestion properties. The WVG group exhibited the highest nitrogen release (84.3%), water content, denaturation temperature, and the highest free-SH content but the lowest S-S content. Compared to WVG, the GYP group had the highest ash content, hardness, and chewiness. Results demonstrated that the tofu prepared by WVG and GYP show high digestibility. Meanwhile, the former has better thermal properties and the latter has better texture properties.

Chitosan: A review of molecular structure, bioactivities and interactions with the human body and micro-organisms.

Chitosan has many desirable attributes e.g. antimicrobial properties and promoting wound healing, and is used in various applications. This article first discusses how degree of deacetylation (DD) and molecular weight (MW) impacts on what level of bioactivities chitosan manifests, then introduces the "molecular chain configuration" model to explain various possible mechanisms of antimicrobial interactions between chitosan with different MW and different types of bacteria. Similarly, the possible pathways of how chitosan reacts with cancer and the body's immune system to demonstrate immune and antitumor effects are also discussed by using this model. Moreover, the possible mechanisms of how chitosan enhances coagulation and wound healing are also discussed. With these beneficial bioactivities in mind, the application of chitosan in surgery, tissue engineering and oncology is outlined. This review concludes that as chitosan demonstrates many beneficial bioactivities via multiple mechanisms, it is an important polymer with a promising future in medicine.

Virus-Inspired Gold Nanorod-Mesoporous Silica Core-Shell Nanoparticles Integrated with tTF-EG3287 for Synergetic Tumor Photothermal Therapy and Selective Therapy for Vascular Thrombosis.

Synergetic therapy includes the combination of two or more conventional therapeutic approaches and can be used for tumor treatment by combining the advantages and avoiding the drawbacks of each type of treatment. In the present study, truncated tissue factor (tTF)-EG3287 fusion protein-encapsulated gold nanorod (GNR)-virus-inspired mesoporous silica core-shell nanoparticles (vinyl hybrid silica nanoparticles; VSNP) (GNR@VSNP-tTF-EG3287) were synthesized to achieve synergetic therapy by utilizing selective vascular thrombosis therapy (SVTT) and photothermal therapy (PTT). By integrating the targeted coagulation activity of tTF-EG3287 and the high tumor ablation effect of GNR@VSNP, local hyperthermia could induce a high percentage of apoptosis of vascular endothelial cells by using near-infrared light. This provided additional phospholipid sites for tTF-EG3287 and enhanced its procoagulant activity in vitro. In addition, the nanoparticles, which had unique topological viral structures, exhibited superior cellular uptake properties leading to significant antitumor efficacy. The in vivo antitumor results further demonstrated an interaction between SVTT and PTT, whereas the synergetic therapy (SVTT and PTT) achieved an enhanced effect, which was superior to the respective treatment efficacy of each modality or the additive effect of their individual efficacies. In summary, the synthesized GNR@VSNP-tTF-EG3287 exerted synergetic effects and enhanced the antitumor efficiency by avoiding multiple injections and suboptimal administration. These effects simultaneously affected both tumor blood supply and cancer cell proliferation. The data suggested that the integration of SVTT induced by tTF-EG3287 and PTT could provide potential strategies for synergetic tumor therapy.

Global sources of cryoprecipitate demonstrate variability in coagulant factor levels and functional hemostasis.

Cryoprecipitate (cryo) is a plasma-derived blood product utilized during trauma resuscitation, surgery, and other major bleeding. Although local quality control metrics exist, inherent donor variability, and processing may confer differences in hemostatic effect between sources. The purposes of this study were to quantify procoagulant content in three global sources of cryo and evaluate their functional hemostatic effect. In this Institutional Review Board exempt study, 24 units of group A cryo from three different sources, American Red Cross single donor and pooled donor, Australian Red Cross single donor, Southwestern United States single donor, and Southwest pooled donor, were evaluated. Procoagulant factors were quantified from each source using ELISA and automated clot-based assays. Functional hemostasis was evaluated using rotational Thromboelastometry (ROTEM). Microparticles isolated from cryo units were enumerated and evaluated for cellular origin by flow cytometry, as well as their capacity to support thrombin generation. Southwestern United States single donor units demonstrated highest levels of fibrinogen, fibronectin, factor VIII, and von Willebrand factor in the selected units. In the coagulopathy model, successive doses from all cryo units were significantly correlated to decreasing coagulation time (P = 0.0100), and increasing maximum clot firmness (P = 0.0002) and alpha angle (P = 0.0009). Southwest pooled donor demonstrated significantly shorter coagulation time at all three doses (P = 0.02) than other sources. Microparticles support prothrombinase activity and thrombin generation. In this study of global cryo sources, procoagulant activity and in-vitro clot formation varied by source. This could be explained by variance in production and storage protocols. Further study is warranted to assess functional variance in cryo to optimize and standardize the use of cryo products.

Generation of hyperfunctional recombinant human factor IX variants expressed in human cell line SK-Hep-1.

OBJECTIVE: To develop recombinant factor IX (FIX) variants with augmented clotting activity. RESULTS: We generated three new variants, FIX-YKALW, FIX-ALL and FIX-LLW, expressed in SK-Hep-1 cells and characterized in vitro and in vivo. FIX-YKALW showed the highest antigen expression level among the variants (2.17 µg-mL), followed by FIX-LLW (1.5 µg-mL) and FIX-ALL (0.9 µg-mL). The expression level of FIX variants was two-five fold lower than FIX-wild-type (FIX-WT) (4.37 µg-mL). However, the biological activities of FIX variants were 15-31 times greater than FIX-WT in the chromogenic assay. Moreover, the new variants FIX-YKALW, FIX-LLW and FIX-ALL also presented higher specific activity than FIX-WT (17, 20 and 29-fold higher, respectively). FIX variants demonstrated a better clotting time than FIX-WT. In hemophilia B mice, we observed that FIX-YKALW promoted hemostatic protection. CONCLUSION: We have developed three improved FIX proteins with potential for use in protein replacement therapy for hemophilia B.

Characterisation of recombinant factor IX before and after GlycoPEGylation.

The effect of the GlycoPEGylation process used for prolonging the half-life of recombinant factor IX (rFIX) has no impact on the primary and higher order structure of activated factor IX. Characterisation work performed on recombinant factor IX and on the GlycoPEGylated form of rFIX (N9-GP), confirm that the primary structure as well as the post translational modifications (PTMs) (disulphide bonds, γ-carboxylation, ß-hydroxylation, sulphation and O- and N-linked glycan structures) were comparable for rFIX and N9-GP. Three O-linked glycan sites were identified in the activation peptide (Thr159, Thr163 and Thr169), where Thr163 has not been reported previously. For N9-GP, the mono GlycoPEGylation is directed toward one of the two N-linked glycans present at Asn157 and Asn167 in the activation peptide in a one to one ratio. Spectroscopic techniques, such as far and near UV Circular Dichroism studies show comparable secondary and tertiary structures of rFIX and N9-GP. The thermally induced unfolding of rFIX and N9-GP shows that the unfolding temperature is approximately 1 °C higher for N9-GP than that of the rFIX. Furthermore, the pH dependent degradation was reduced due to the GlycoPEGylation of rFIX. GlycoPEGylated rFIX (N9-GP) is used for the manufacturing of Refixia® (nonacog beta pegol, Rebinyn®, Novo Nordisk A/S, Bagsvaerd, Denmark).

The Procoagulant Snake Venom Serine Protease Potentially Having a Dual, Blood Coagulation Factor V and X-Activating Activity.

A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell's viper venom in lupus anticoagulant testing, thus achieving greater reliability of the analysis. As a blood-coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of view for treating patients suffering from hemophilia.

Evaluation of the procoagulant properties of a newly developed platelet modified lysate product.

BACKGROUND: Platelet transfusion is associated with logistical problems with the national storage guidelines of platelets. This results in decreased function in vivo as a result of the platelet storage lesion, and complications such as allergic or hemolytic reactions and thrombosis. We evaluated a new, freshly prepared platelet modified lysate (PML) product designed to be more procoagulant than fresh and stored platelets. METHODS: Fresh platelets were concentrated, sonicated, and centrifuged to produce PML. Samples of both washed and unwashed PML were evaluated for particle size, concentration, and activity, and then tested for clot kinetics and thrombin generation. PML samples were also stored at various temperatures for durations up to 6 months and evaluated for clot kinetics and thrombin generation throughout. RESULTS: PML showed significantly higher concentration of platelet microparticles, increased procoagulant properties, and increased thrombin generation as compared to fresh and stored platelets. In addition, PML maintained its clot kinetics over a 6-month storage period with variable storage conditions. CONCLUSIONS: The newly proposed PML product is more procoagulant, stable, and has additional potential applications than currently available platelet products. Further studies will be performed to assess its functions in vivo and to assess thrombotic potential.

Enteromorpha prolifera polysaccharide based coagulant aid for humic acids removal and ultrafiltration membrane fouling control.

Polyacrylamide (PAM) has been used as a coagulant aid in water treatment process for past decades, but it has caused great damages to human nervous system. Developing new coagulant aid with high biological safety is urgently demanded. This study provides a natural biomacromolecule coagulant aid with good biosecurity-Enteromorpha prolifera polysaccharide (Ep). Its coagulant aid efficiency and mechanism were investigated in terms of organics removal, floc properties and membrane fouling degree. In addition, contrast experiments were conducted with PAM to evaluate its potential of industrial applications. Results showed that organics removal could be increased by 23% when 0.3 mg/L Ep was used, which exhibited comparable aid effects to PAM. Due to the bridging-sweep aid role of Ep, flocs sizes, growth rate and recovery factor reached 470 µm, 62.6 µm/min and 0.492, respectively, while only 170 µm, 14.0 µm/min and 0.326 were obtained by PAM. Additionally, flocs exhibited more porous and multi-branched structures when Ep was applied, which caused less ultrafiltration membrane fouling (eventual J/J0 value = 0.52). As a result, Ep could be considered as a potential substitute of PAM, since better biosecurity, higher organics removal and lower membrane fouling could be obtained simultaneously by Ep addition.

Aluminium Drinking Water Treatment Residuals and Their Toxic Impact on Human Health.

Aluminium exerts undeniable human health effects, so its concentration should be controlled in water treatment plants. The article presents and discusses the results of studies on the influence of selected properties of aluminium coagulants on the concentration of aluminium remaining in the purified water. The coagulants used were classical hydrolysing aluminium salts: aluminium sulphate (VI) and sodium aluminate as well as pre-hydrolysed polyaluminium chlorides: Flokor 105B and PAX XL10 that had different the alkalinity coefficient r = [OH-]/[Al3+]. The Al species distribution in the coagulants samples were analysed by the Ferron complexation timed spectrophotometry. On the basis of their reaction rates with ferron reagent, the aluminium species were divided into three categories: monomeric (Ala), medium polymerised (Alb) and colloidal (Alc). The usefulness of the tested aluminium coagulants due to the concentration of residual aluminium and dissolved aluminium, which is easily assimilated by the human body, was increased according to the following series: sodium aluminate (Ala = 100%, Alb = 0) < aluminium sulphate (VI) (Ala = 91%, Alb = 9%) < PAX XL 10 (Ala = 6%, Alb = 28%, r = 2.10) < Flokor 105B (Ala = 3%, Alb = 54%, r = 2.55).

Coagulant Effects and Mechanism of Schefflera heptaphylla (L.) Frodin.

Schefflera heptaphylla (L.) Frodin, are commonly used in anti-inflammatory, analgesic, traumatic bleeding and hemostasisas. In this paper, the coagulation effect of the ethanol extract (Set), ethyl acetate phase (Sea) and n-butanol phase (Sbu) was evaluated by prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen content (FIB) assays in vitro. Then, Three main lupanine triterpenes (compounds A-C) were isolated and identified from Sea and Sbu by a bioassay-guided method and their structure were identified as 3α-Hydroxy-lup-20(29)-ene-23, 28-dioic acid, betulinic acid 3-O-sulfate and 3α-Hydroxy-lup-20(29)-ene-23, 28-dioic acid 28-O-(α-l-rhamnopyranosyl(1→4)-O-ß-d-glucopyranosyl(1→6))-ß-d-glucopyranoside) by spectroscopic data analysis. Among of them, compound B was confirmed to have significant coagulant effect in vitro. Furthermore, the pro-coagulation mechanism of S. heptaphylla extracts and compound B were investigated by measuring whole blood viscosity (WBV), plasma viscosity (PV), erythrocyte sedimentetion rate (ESR), pack cell volume (PCV), APTT, PT, TT, and FIB in vivo. Meanwhile, the levels of thromboxane B2 (TXB2), 6-keto prostaglandin F1α (6-keto-PGF1α), endothelial nitric oxide synthase (eNOS) and (endothelin-1) ET-1 were detected. The bleeding time (BT) was tested by tail bleeding method, which proved the traumatic bleeding and hemostasis activities of S. heptaphylla. The pharmacology experiments showed that the Set, Sea, Sbu and compound B has significant pro-coagulation effect. In addition, compound B might be the main constituent of pro-coagulation in S. heptaphylla These results could support the fact that S. heptaphylla could be used traditionally to cure traumatic bleeding, and the pro-coagulation effects were associated with the regulation of vascular endothelium active substance and hemorheology parameters.

Characterization of L-amino Acid Oxidase Derived from Crotalus adamanteus Venom: Procoagulant and Anticoagulant Activities.

Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from Crotalus adamanteus venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.

Amphiphilic multi-charged cyclodextrins and vitamin K co-assembly as a synergistic coagulant.

Balancing and neutralizing heparin dosing after surgeries and hemodialysis treatment is of great importance in medical and clinical fields. In this study, a series of new amphiphilic multi-charged cyclodextrins (AMCD)s as anti-heparin coagulants were designed and synthesized. The AMCD assembly was capable of selective heparin binding through multivalent bonding and showed a better neutralizing effect towards both unfractionated heparin and low molecular weight heparin than protamine in plasma. Meanwhile, an AMCD and vitamin K (VK) co-assembly was prepared to realize heparin-responsive VK release and provide a novel VK deficiency treatment for hemodialysis patients. This AMCD-VK co-assembly for heparin neutralization & vitamin K supplementation synergistic coagulation represents a promising candidate as a clinical anti-heparin coagulant.

Comparative proteomes, immunoreactivities and neutralization of procoagulant activities of Calloselasma rhodostoma (Malayan pit viper) venoms from four regions in Southeast Asia.

The intraspecific geographical venom variations of Calloselasma rhodostoma from Malaysia (CR-M), Indonesia (CR-I), Thailand (CR-T) and Vietnam (CR-V) were investigated through 1D SDS-PAGE and nano-ESI-LCMS/MS. The venom antigenicity, procoagulant activities and neutralization using Thai C. rhodostoma Monovalent Antivenom (CRMAV) were also investigated. SDS-PAGE patterns of the venoms were relatively similar with minor variations. Proteomic analysis revealed that snake venom metalloproteinases (SVMPs, particularly P-I class), serine proteases (SVSPs) and snaclecs dominated the venom protein composition (68.96-81.80%), followed by L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2) (7.37-11.08% and 5.18-13.81%, respectively), corroborating C. rhodostoma envenoming effects (hemorrhage, consumptive coagulopathy, thrombocytopenia and local tissue necrosis). Other proteins of lower abundances (2.82-9.13%) identified include cysteine-rich secretory proteins (CRISP), phospholipase B, phosphodiesterase, nerve growth factor, 5'-nucleotidase, aminopeptidase and hyaluronidase. All four venoms exhibited strong procoagulant effects which were neutralized by CRMAV to different extents. CRMAV immunoreactivity was high toward venoms of CR-M, CR-I and CR-T but relatively low for CR-V venom. Among the venom samples from different locales, CR-V venom proteome has the smallest SVMP composition while SVSP, PLA2 and phosphodiesterase were more abundant in the venom. These variations in C. rhodostoma venom protein composition could partly explain the differences seen in immunoreactivity. (198 words).

Pharmacokinetic and safety considerations when switching from standard to extended half-life clotting factor concentrates in hemophilia.

Introduction: Plenty of new FVIII/IX concentrates have been developed and entered the market of hemophilia treatment. Others are going to end the long/demanding procedures for approval. Changes of the FVIII molecule (single chain), pegylation of B-domain deleted FVIII, and fusion with Fc succeeded to improve the FVIII half-life, about 4 hours. Pegylation and fusion with albumin or Fc of rFIX caused a substantial increase of half-life, approximately 3-4 times that of FIX standard concentrates. Area covered: Extended Half-life concentrates may allow a longer time interval between the prophylaxis bolus, a feature very well accepted by young patients. Also, adherence of adolescents can be improved by these new, less demanding, concentrates. The immunogenicity of these new molecules is so far under post-marketing evaluation. The incidence of neutralizing antibodies is very low in previously treated patients, but the data on previously untreated patients are not yet assessed. The cost of some Extended Half-Life concentrates is higher than that of standard ones, and some concerns have been raised about the cost for public or private health care institutions. Expert opinion: An accurate evaluation of patients' needs, individual pharmacokinetics, and cost/effectiveness might allow a more appropriate usage of these new and expensive concentrates.

De Novo Assessment and Review of Pan-American Pit Viper Anticoagulant and Procoagulant Venom Activities via Kinetomic Analyses.

Snakebite with hemotoxic venom continues to be a major source of morbidity and mortality worldwide. Our laboratory has characterized the coagulopathy that occurs in vitro in human plasma via specialized thrombelastographic methods to determine if venoms are predominantly anticoagulant or procoagulant in nature. Further, the exposure of venoms to carbon monoxide (CO) or O-phenylhydroxylamine (PHA) modulate putative heme groups attached to key enzymes has also provided mechanistic insight into the multiple different activities contained in one venom. The present investigation used these techniques to characterize fourteen different venoms obtained from snakes from North, Central, and South America. Further, we review and present previous thrombelastographic-based analyses of eighteen other species from the Americas. Venoms were found to be anticoagulant and procoagulant (thrombin-like activity, thrombin-generating activity). All prospectively assessed venom activities were determined to be heme-modulated except two, wherein both CO and its carrier molecule were found to inhibit activity, while PHA did not affect activity (Bothriechis schlegelii and Crotalus organus abyssus). When divided by continent, North and Central America contained venoms with mostly anticoagulant activities, several thrombin-like activities, with only two thrombin-generating activity containing venoms. In contrast, most venoms with thrombin-generating activity were located in South America, derived from Bothrops species. In conclusion, the kinetomic profiles of venoms obtained from thirty-two Pan-American Pit Viper species are presented. It is anticipated that this approach will be utilized to identify clinically relevant hemotoxic venom enzymatic activity and assess the efficacy of locally delivered CO or systemically administered antivenoms.

Treprostinil treatment decreases circulating platelet microvesicles and their procoagulant activity in pediatric pulmonary hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) results from pulmonary vascular disease and may eventually lead to right heart failure and death. Vasodilator therapy has greatly improved PAH prognosis. Circulating microvesicles are considered as surrogate markers of endothelial and hematopoietic cell activation. AIM: Thus, our purpose was to determine if MVs are upregulated in pediatric PAH such as reported in adult patients, and to analyze the impact of vasodilator therapies on MV count and function. PATIENTS: Population study consisted of 26 patients of median age 6.09 years, with Congenital Heart Disease (CHD) and elevated pulmonary vascular resistance (CHD-PAH) or idiopathic PAH (iPAH). RESULTS: Compared to healthy controls, all circulating MV subpopulations were found higher in untreated PAH patients. No significant differences of annexin-V+ total MV, endothelial, or leukocyte derived-MV counts were found between untreated patients and those receiving oral vasodilator therapies. Conversely, platelet MVs were significantly lower in the group treated with SC-treprostinil compared with both untreated PAH and oral therapy groups (P = 0.01), and exhibited a significant decrease of phospholipid procoagulant activity. Control samples treated in vitro with treprostinil at therapeutic concentrations showed as expected a significant decrease of platelet aggregation but also a reduced spontaneous MV generation. CONCLUSION: Our results suggest that treprostinil, besides vasodilation, might exert its beneficial effect through an inhibition of platelet activation, resulting in a decreased number and procoagulant activity of circulating MVs.

Application of enteromorpha polysaccharides as coagulant aid in the simultaneous removal of CuO nanoparticles and Cu2+: Effect of humic acid concentration.

Humic acid (HA) influences the aggregation and stability of nanoparticles (NPs), which determine the removal performance of NPs in coagulation. Consequently, in this study, the impact of HA concentration on the simultaneous removal of CuO NPs, Cu2+, and natural organic matter (NOM) was investigated. Enteromorpha polysaccharides (Ep), as the novel recycling coagulant aid, were integrated with polyaluminum chloride (PAC) to treat composite contaminants in this coagulation process. Removal performance, floc properties, zeta potential, scanning electron microscope (SEM) images, and Fourier Transform Infrared Spectra (FT-IR) were measured and analyzed. Results showed that PAC with Ep (PAC-Ep) was more beneficial for improving removal performances than PAC alone. Further, the coagulation performance became better with the increase in HA concentration. When the HA concentration was 10 mg/L (used PAC-Ep), the removal efficiencies of CuO NPs and Cu2+ were both more than 80%; and in particular, the highest removal efficiency of turbidity was 98%. However, excessive HA (more than 10 mg/L) reduced the removal efficiency of Cu by more than 31%. Smaller and denser flocs with better recoverability were formed as the HA concentration increased. Furthermore, because of affluent functional groups, HA was easily adsorbed on the surface of NPs and combined with dissociative Cu2+, thereby forming a composite contaminant. During the coagulation process, the colloidal system was destabilized preferentially by the charge neutralization between composite contaminants and PAC. Furthermore, a chelated reticular structure was formed by the conjunction of carboxyl and hydroxyl groups (from Ep) and Al (III) species (from PAC). Flocs were further enlarged and precipitated by bridging and sweeping of this structure.

Effects of Low- and High-Dose Chemotherapy Agents on Thrombogenic Properties of Extracellular Vesicles Derived from Breast Cancer Cell Lines.

BACKGROUND: The involvement of extracellular vesicles (EVs) in cancer-associated thrombosis (CT) is unclear. This study aimed to explore the properties of EVs derived from breast cancer (BC) cells following exposure to high- or low-dose chemotherapeutic agents and evaluate thrombogenic effects of these EVs on endothelial cells (ECs). METHODS: EVs were isolated from BC cell lines (non-metastatic MCF7, high-metastatic MDA-MB-231), pre-exposed to serum-free medium (control), with or without increasing doses of doxorubicin or paclitaxel. EV structure and size were studied using electron microscopy and Nano-sight. Antigen levels were measured by fluorescence-activated cell sorting (FACS). EV effects on EC thrombogenicity were assessed using FACS, factor Xa chromogenic assay and RT-PCR. RESULTS: Serum-free medium BC cell resulted in EV shedding that additionally increased when MDA-MB-231 cells were exposed to high doses of both agents. Tissue factor (TF) levels were similarly low (9-13%) in all EVs compared with the high expression on their parental MDA-MB-231 cells (76-83%). EVs derived from MDA-MB-231 cells stimulated with high-dose doxorubicin demonstrated significantly (fivefold; p < 0.001) elevated levels of negatively charged phospholipids, a 97% decrease in TF pathway inhibitor (TFPI) levels and a sixfold increase (p < 0.001) in procoagulant activity. These EVs also enhanced EC thrombogenicity. Effects of EVs originating from MCF7 cells were less pronounced. CONCLUSION: These findings suggest that thrombogenic properties of BC-derived EVs may depend on the type and dose of the applied chemotherapy agent and may also be affected by the cell metastatic nature.

Suppressive Role of Tissue Factor Pathway Inhibitor-α in Platelet-Dependent Fibrin Formation under Flow Is Restricted to Low Procoagulant Strength.

Tissue factor pathway inhibitor-alpha (TFPI-α) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI-α thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI-α regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-α in plasma is a function of the local procoagulant strength-defined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI-α enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 0 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-α antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-α is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.