Antithrombotic Potential Research Articles

Commentary on Antithrombotic Effect of Protopanaxatriol Saponins from Panax notoginseng Using Zebrafish Model.

Thrombosis continues to pose a significant challenge in cardiovascular and cerebrovascular diseases, contributing to severe health complications such as myocardial infarction, acute ischemic stroke, and venous thromboembolism. Despite the wide array of anti-thrombotic drugs available, these treatments frequently carry substantial risks, notably including bleeding complications. In this paper, we comment the findings reported by Liu et al. about the anti-thrombotic potential of protopanaxatriol saponins from panax notoginseng.

PROTACs in platelets: emerging antithrombotic strategies and future perspectives.

Proteolysis-targeted chimeras (PROTACs) are heterobifunctional compounds that selectively target proteins for degradation and are an emerging therapeutic modality to treat diseases such as cancer and neurodegenerative disorders. This review will widen the area of application by highlighting the ability of PROTACs to remove proteins from the anucleate platelets and evaluate their antithrombotic potential. Proteomic and biochemical studies demonstrated that human platelets possess the Ubiquitin Proteasomal System as well as the E3 ligase cereblon (CRBN) and therefore may be susceptible to PROTAC-mediated protein degradation. Recent findings confirmed that CRBN ligand-based PROTACs targeting generic tyrosine kinases, Btk and/or Fak lead to efficacious and selective protein degradation in human platelets. Downregulation of Btk, a key player involved in signalling to thrombosis, but not haemostasis, resulted in impaired in-vitro thrombus formation. Platelets are susceptible to targeted protein degradation by CRBN ligand-based PROTACs and have limited ability to resynthesise proteins, ensuring long-term downregulation of target proteins. Therefore, PROTACs serve as an additional research tool to study platelet function and offer new therapeutic potential to prevent thrombosis. Future studies should focus on enhancing cell specificity to avoid on-target side effects on other blood cells.

Antipolyphosphate monoclonal antibodies derived from autoimmune mice

BackgroundInorganic polyphosphates (polyPs) are linear chains of phosphates that accelerate blood clotting. Targeting polyP in vivo has been shown to reduce thrombosis. ObjectivesTo identify and characterize anti-polyP monoclonal antibodies that could be used as analytical tools and as antithrombotic agents. MethodsHybridomas were prepared from spleen cells from autoimmune NZBWF1/J female mice and screened for anti-polyP antibodies. Antibodies that bound polyP using enzyme-linked immunosorbent assay and pull-down assays were further characterized with plate binding, surface plasmon resonance, and plasma-based clotting assays. Antithrombotic potential was evaluated in a murine ferric chloride–induced carotid artery thrombosis model. ResultsOf 4 antibodies that bound polyP in our pull-down assay, 2 (PP2069 and PP2099) were available for further characterization. While analyzing these anti-polyP antibodies, we found secretory leukocyte peptidase inhibitor (SLPI) to be a common contaminant of these antibodies and that SLPI binds polyP. We removed SLPI quantitatively from our purified immunoglobulin G. Both PP2069 and PP2099 immunoglobulin G displayed high affinity for polyP but also bound to other polyanions such as DNA, heparin, and certain other glycosaminoglycans, indicating limited specificity. Both antibodies inhibited polyP-initiated plasma clotting in vitro. When tested in vivo in a mouse thrombosis model, however, neither PP2069 nor PP2099 exhibited a significant antithrombotic effect. ConclusionAutoimmune mice spontaneously produce antibodies against polyP. The 2 examples of anti-polyP monoclonal antibodies studied here not only bound to polyP with high affinity but also cross-reacted with DNA and heparin. Neither antibody protected against thrombosis in a mouse model, but they might have some utility for in vitro studies of polyP.

Anti-Inflammatory and Antithrombotic Potential of Metal-Based Complexes and Porphyrins

Inflammation and thrombosis are implicated in several chronic disorders. Recent studies have outlined the way in which several compounds can offer protection against inflammation. Within this comprehensive review the so-far reported anti-inflammatory health-promoting effects of several metal-based complexes, both in vitro and in vivo, are thoroughly presented. These metal-based compounds usually interfere with various biochemical processes associated with the inflammatory response and thrombus formation and become capable of inhibiting these biochemical pathways with proposed health benefits. Emphasis is given to the multifaceted actions of metal-based complexes that have exhibited potent anti-inflammatory and antithrombotic activities against the inflammatory mediator, platelet-activating factor (PAF), and its thrombo-inflammatory signaling, as well as on their anti-platelet and antitumor health promoting properties. Furthermore, the enhancement of the anti-inflammatory potency of well-established bioactive compounds by their incorporation as ligands in several metal-based complexes is discussed. Metal-based complexes bearing natural anti-inflammatory bioactives are also outlined. Characteristic examples of both free and metal-based porphyrins are explored. These compounds are recognized to have anti-inflammatory and antithrombotic assets, in addition to other pleiotropic advantages including antibacterial or anticancer actions. Additionally, applications of metal complexes in various models of inflammatory and thrombotic complications are demonstrated. The combined results of this study show that further research is required towards the preparation of several metal-based complexes with improved pharmacological profiles. Finally, restrictions on the application of these metal-based compounds are also covered, along with their prospects for the future and the need for additional study in order to improve their efficacy and safety.

Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

Protease activated receptor-4: ready to be part of the antithrombosis spectrum.

Cardiovascular disease is a major cause of death worldwide. Platelets play a key role in this pathological process. The serine protease thrombin is a critical regulator of platelet reactivity through protease activated receptors-1 (PAR1) and PAR4. Since targeting PAR4 comes with a low chance for bleeding, strategies blocking PAR4 function have great antithrombotic potential. Here, we reviewed the literature on platelet PAR4 with a particular focus on its role in thromboinflammation. Functional PAR4 variants are associated with reduced venous thrombosis risk (rs2227376) and increased risk for ischemic stroke (rs773902). Recent advances have allowed for the creation of humanized mouse lines in which human PAR4 is express instead of murine PAR4. This has led to a better understanding of the discrepancies between human and murine PAR4. It also made it possible to introduce single nucleotide polymorphisms (SNPs) in mice allowing to directly test the in vivo functional effects of a specific SNP and to develop in vivo models to study mechanistic and pharmacologic alterations induced by a SNP. PAR4 plays an important role in cardiovascular diseases including stroke, myocardial infarction and atherosclerosis. Targeting PAR4 hold great potential as a safe antithrombotic strategy.

Hydroxychloroquine inhibits hemolysis-induced arterial thrombosis ex vivo and improves lung perfusion in hemin-treated mice

BackgroundFree labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis. ObjectivesTo investigate the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced platelet activation and arterial thrombosis. MethodsThe effect of hydroxychloroquine on hemin-induced platelet activation and hemolysis-induced platelet recruitment and aggregation was measured in washed platelets and hemolyzed blood, respectively. Its effect on ferric-chloride (FeCl3)-induced arterial thrombosis and lung perfusion following hemin injection was assessed in wild-type mice. ResultsErythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited ferric-chloride–induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi. ConclusionHydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion.

Variability of the antithrombotic effect of acetylsalicylic acid with the administration of different dosages: reality or myth?

Abstract Introduction We rely on the antithrombotic effect of acetylsalicylic acid (ASA) in a number of pathologies, although other beneficial effects are known, such as its anti-inflammatory properties, when administered in higher doses (500mg, 1000mg per os). However, whether the anti-inflammatory effect decreases the antithrombotic potency of ASA is not known. This gap in evidence may lead to an unnecessary use of two drugs (one antithrombotic and one anti-inflammatory) that could be replaced by ASA alone. This scenario presents frequently: post-infarct pericarditis or when in doubt between non-ST segment elevation myocardial infarction vs myopericarditis. In such cases, the use of ASA could assure both antithrombotic and anti-inflammatory effects. Since the antithrombotic effect of ASA is not scientifically proved for higher doses, currently we use ASA 100mg as an antithrombotic agent and ibuprofen as an anti-inflammatory. This experimental study intends to assess whether ASA maintains its antithrombotic effect when administered in an anti-inflammatory dose. Methods Twenty healthy volunteers were recruited. They all had their platelet function assessed in a qualitative manner, using PFA-200 Innovance technology. The volunteers were randomized into four groups, with 5 participants each. The participants of groups 1, 2, 3 and 4 ingested ASA 100mg, 300mg, 500mg and 1000mg respectively, in a blind experiment. One hour after ingestion (peak of action), the volunteers’ platelet function was reassessed. PFA-200 evaluates platelet function through platelet occlusion time (OT), which is measured in milliseconds (ms). Normal platelet function translates into OT of 82-150ms. If the OT is higher than 150ms, the patient is anti-aggregated. Results Prior to ASA administration, 19/20 volunteers had OT within the reference range. Afterwards, in all four groups, volunteers reached OT > 150ms, regardless of the ASA dose administered. Mean OT values for each group were 188ms [SD-23] , 205ms [SD-63], 262ms[SD-44] and 232ms [SD-47], respectively. Overall SD was 32ms. Conclusion ASA maintains its antithrombotic effect when administered in an anti-inflammatory dose. There is no clear correlation between the potency of antithrombotic effect and the ASA dose administered. This was a pilot study that supports the maintenance of the antithrombotic effect of ASA in higher doses, but further and larger studies are required to corroborate these results.

PB0422 The Piperazinyl-Purine Analogue MK177 is a Bifunctional Drug with Promising Anti-Ischemic and Anti-Thrombotic Potential

PB0422 The Piperazinyl-Purine Analogue MK177 is a Bifunctional Drug with Promising Anti-Ischemic and Anti-Thrombotic Potential

Antioxidant and antithrombotic properties of fruit, leaf, and seed extracts of the Halhalı olive (Olea europaea L.) native to the Hatay region in Turkey

The olive (Olea europaea L.) is one of the most important plants grown in many Mediterranean countries that has a high economic value. Olives, which are specific to each region, have different bioactive components. In this study, we investigated the phenolic/flavonoid contents, as well as antioxidant, antimicrobial, and antithrombotic activities of the fruit, leaf, and seed extracts obtained from the Halhalı olive grown in Arsuz district of Hatay, Turkey.
 Antioxidant activities of the phenolic compounds found in the olive fruit, seed, and leaf extracts were determined by employing established in vitro systems. Total phenolics were determined as gallic acid equivalents, while total flavonoids were determined as quercetin equivalents. Also, we evaluated a possible interaction between oleuropein and aggregation-related glycoproteins of the platelet surface via docking studies.
 The extracts showed effective antioxidant activity. The seed extract had the highest phenolic content of 317.24 μg GAE, while the fruit extract had the highest flavonoid content of 4.43 μg. The highest potential for metal chelating activity was found in the leaf extract, with an IC50 value of 13.33 mg/mL. Also, the leaf extract showed higher levels of antioxidant, antithrombotic, and antimicrobial activity, compared to the fruit and seed extracts. The docking scores of oleuropein against the target molecules GPVI, α2β1, and GPIbα were calculated as –3.798, –4.315, and –6.464 kcal/mol, respectively.
 The olive fruit, leaf, and seed extracts used as experimental material in our study have remarkable antioxidant, antimicrobial, and antithrombotic potential.

Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type.

In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2β1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2β1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs. Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2β1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28. In this first comparison of four inhibitors of platelet-collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2β1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2β1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.

Synthesis of pyrrolo[3,2-d]pyrimidineone derivatives as novel FXa inhibitors

A series of pyrrolo[3,2-d]pyrimidineone compounds have been designed and synthesized as novel FXa inhibitors. Bioassay of the tested compounds showed moderate to excellent anticoagulant potency in vitro. Further FXa inhibitory and bioactivity evaluation in rats, the FeCl3-induced venous thrombosis model, showed that the compound 17a has good FXa inhibitory activity (IC50 = 1.57 nM) and in vivo antithrombotic potency. The anticoagulant effects of compound 17a were dose dependent whether in vitro or in vivo. The results further confirmed our hypothesis that the large conjugated structure is an ideal skeleton binding FXa.

In vivo antithrombotic potential of protease from Bacillus thuringiensis HSFI-12

Background/Aim: Cardiovascular diseases (CVDs) are the primary noncommunicable disease at the global level due to abnormal platelet aggregation by fibrin forming clots in blood vessels called thrombus. The search for thrombolytic drugs is largely carried out to treat thrombosis. Crude extract and dialysate protease of Bacillus thuringiensis HSFI-12 is known to have thrombolytic activity in vitro. The in vivo thrombolytic activity evaluation of concentrated protease of the bacterium is yet to be done. This study aimed to evaluate in vivo thrombolytic activity of concentrated protease produced by ultrafiltration of crude B thuringiensis HSFI-12 protease using Rattus norvegicus as animal model. Methods: Carrageenan was used as thrombosis induction agent in rats. Intravenous injection of B thuringiensis HSFI-12 concentrated protease doses of 75, 150, 300, 600 µg/kg body weight (BW) was administered to rats, then induction of carrageenan was given intravenously to the rats' tails 30 min after injection of B thuringiensis HSFI-12 protease concentrate. The average length of the infarct area in the tail of the rat was shorter in the rats that were given various doses of B thuringiensis HSFI-12 protease concentrate compared to the negative control (rats induced by carrageenan 20 mg/kg BW). Results: The PT examination results showed a prolonged PT time at 300 µg/kg BW dose, while there was at risk of bleeding at 600 µg/kg BW dose. The activated partial thromboplastin time (aPTT) examination results showed that time elongation beyond the normal range did not occur in rats after treatment. The amount leukocytes (WBC) and erythrocytes (RBC) after treatment were within the normal range indicating that they did not affect the haemostasis mechanism, while the platelet count (PLT) assay showed decrease in the number of platelets (thrombocytopenia). However, after treatment the number of platelets (PLT) showed a positive response as seen from an increase in values close to normal range. As conclusion, induction of carrageenan conducted had successfully caused thrombosis in R norvegicus' tail used as the thrombosis model. Conclusion: Concentrated protease of B thuringiensis HSFI-12 showed in vivo antithrombotic potential with an effective dose of based on PT, aPTT and blood count evaluation at 150 µg/kg BW.

GPIIb/IIIa Receptor Targeted Rutin Loaded Liposomes for Site-Specific Antithrombotic Effect.

Rutin (RUT) is a flavonoid obtained from a natural source and is reported for antithrombotic potential, but its delivery remains challenging because of its poor solubility and bioavailability. In this research, we have fabricated novel rutin loaded liposomes (RUT-LIPO, nontargeted), liposomes conjugated with RGD peptide (RGD-RUT-LIPO, targeted), and abciximab (ABX-RUT-LIPO, targeted) by ethanol injection method. The particle size, ζ potential, and morphology of prepared liposomes were analyzed by using DLS, SEM, and TEM techniques. The conjugation of targeting moiety on the surface of targeted liposomes was confirmed by XPS analysis and Bradford assay. In vitro assessment such as blood clot assay, aPTT assay, PT assay, and platelet aggregation analysis was performed using human blood which showed the superior antithrombotic potential of ABX-RUT-LIPO and RGD-RUT-LIPO liposomes. The clot targeting efficiency was evaluated by in vitro imaging and confocal laser scanning microscopy. A significant (P < 0.05) rise in the affinity of targeted liposomes toward activated platelets was demonstrated that revealed their remarkable potential in inhibiting thrombus formation. Furthermore, an in vivo study executed on Sprague Dawley rats (FeCl3 model) demonstrated improved antithrombotic activity of RGD-RUT-LIPO and ABX-RUT-LIPO compared with pure drug. The pharmacokinetic study performed on rats demonstrates the increase in bioavailability when administered as liposomal formulation as compared to RUT. Moreover, the tail bleeding assay and clotting time study (Swiss Albino mice) indicated a better antithrombotic efficacy of targeted liposomes than control preparations. Additionally, biocompatibility of liposomal formulations was determined by an in vitro hemolysis study and cytotoxicity assay, which showed that they were hemocompatible and safe for human use. A histopathology study on rats suggested no severe toxicity of prepared liposomal formulations. Thus, RUT encapsulated nontargeted and targeted liposomes exhibited superior antithrombotic potential over RUT and could be used as a promising carrier for future use.

Sodium ferulate-functionalized silver nanopyramides with synergistic antithrombotic activity for thromboprophylaxis

Thrombosis is one of the feared complications contributing to death in patients with cardiovascular diseases. Ferulic acid (FA), a bioactive compound extracted from traditional Chinese medicines, has drawn tremendous attention in prevention and treatment of thrombosis because of its notable antithrombotic potency. However, the poor aqueous solubility and pharmacokinetic profile of FA limit its clinical use. In this study, sodium ferulate-functionalized silver nanopyramides (SF-pAgNPs) with narrow size distribution were synthesized to overcome these obstacles and enhance the suppression effect of platelet activation and thrombosis formation. The cytotoxicity and hemolysis assays demonstrated that the prepared nanopyramides have a favorable biocompatibility pattern. In vitro studies revealed that SF-pAgNPs could effectively suppress platelet activation, aggregation and adhesion through the synergetic antithrombotic potential of SF and nano silver. Furthermore, SF-pAgNPs exhibited potent antithrombotic activity and prolonged inhibitory effect, much better than PEG-Ag and free SF in mouse model. With enhanced antithrombotic effect and acceptable biocompatibility, we believe the sodium ferulate-functionalized silver nanopyramides might hold the potential to be a promising strategy for the prevention and treatment of thrombosis.

Antithrombotic potential of Lippia alba: A mechanistic approach

Ethnopharmacological relevanceLippia alba (Mill.) N.E.Br. ex Britton & P. Wilson is traditionally used in Brazil as an adjunct in the relief of mild anxiety, as an antispasmodic, and as an antidyspeptic. This medicinal species was included in the Phytotherapeutic Form of the Brazilian Pharmacopeia 2nd edition (2021) and has already been described as the most used medicinal plant in a study with patients from an Anticoagulation Clinic in Brazil. Meanwhile, no studies were found that support the safety of the use of L. alba in patients using anticoagulants, a drug with several safety limitations. Aim of the studyProvide scientific evidence to ensure the safety of the concomitant use of L. alba and warfarin and support the management of these patients by evaluating its in vitro anticoagulant effect and chemical composition. And, as a timely complementation, evaluate the potential of this medicinal species in the development of new antithrombotics. MethodsThe chemical profile of L. alba derivatives was analyzed by chromatographic methods such as Ultra-Performance Liquid Chromatography (UPLC) coupled with electrospray ionization mass spectrometry (ESI-MS), qualitative UPLC using Diode-Array Detection, and Thin Layer Chromatography. The anticoagulant activity was evaluated by the innovative Thrombin Generation Assay by Calibrated Automated Thrombogram method and using traditional coagulometric tests: prothrombin time, activated partial thromboplastin time, and plasma fibrinogen measurement. ResultsExtracts and fractions prolonged the coagulation time in all the tests and reduced thrombin formation in thrombin generation assay. Coagulation times with the addition of ethanloic extract (2.26 mg/mL) was 17.78s, 46.43s and 14.25s respectively in prothrombin time, activated partial thromboplastin time and fibrinogren plasma measurement. In thrombin generation test, this same extract showed ETP as 323 nM/min compared to control (815 nM/min) with high tissue factor and 582 nM/min compared to control (1147 nM/min) using low tissue factor. Presence of flavonoids, phenylpropanoids, and triterpenes were confirmed by chromatographic methods and 13 compounds were identified by UPLC-ESI-MS. Based on these results and on the scientific literature, it is possible to propose that phenylpropanoids and flavonoids are related to the anticoagulant activity observed. ConclusionThe results demonstrate the in vitro anticoagulant activity of L. alba, probably due to the activation of intrinsic and extrinsic pathways. It is concluded, then, that there is a potential for interaction, which needs to be further studied, between L. alba and warfarin. Also, this medicinal species shows a great potential for use in the development of new antithrombotics.

Design and efficacy of all-in-one sandwich-like multifunctional platform for drug delivery

Through electrostatic and H-bonding interaction, polyelectrolyte could be self-assembled onto the medical devices via layer-by-layer (LBL) method. However, the built-in instability of LBL coating has seriously limited its further application. The polyphenols were incorporated into the polyelectrolyte coating, which provided various synergistic forces, including electrostatic, H-bonding, π-π stacking, and weak chemical cross-linking. The surface engineering of polyphenol-based sandwich-like films with polyethyleneimine and heparin via LBL self-assembly. This design improved the loading capacity and long-term release of drugs, such as heparin, rapamycin, and gentamicin sulfate. These experiments suggested enhanced durability of antithrombotic potential (over 90 days), good anti-inflammatory and anti-bacteria performances, and promotion of vascular healing. Therefore, all-in-one sandwich-like coatings might be a straightforward, versatile, and durable surface modification method for implantable medical devices.

Antithrombotic potential of a single‐domain antibody enhancing the activated protein C‐cofactor activity of protein S

BackgroundProtein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factors V (FVa) and VIII (FVIIIa), but also for tissue factor pathway inhibitor α (TFPIα) in the inhibition of activated factor X (FXa). ObjectiveFor therapeutic purposes, we aimed at generating single‐domain antibodies (sdAbs) that could specifically modulate the APC‐cofactor activity of PS in vivo. MethodsA llama‐derived immune library of sdAbs was generated and screened on recombinant human PS by phage display. PS binders were tested in a global activated partial thromboplastin time (APTT)‐based APC‐cofactor activity assay. ResultsA PS‐specific sdAb (PS003) was found to enhance the APC‐cofactor activity of PS in our APTT‐based assay, and this enhancing effect was greater for a bivalent form of PS003 (PS003biv). Further characterization of PS003biv demonstrated that PS003biv also enhanced the APC‐cofactor activity of PS in a tissue factor (TF)‐induced thrombin generation assay and stimulated APC in the inactivation of FVa, but not FVIIIa, in plasma‐based assays. Furthermore, PS003biv was directed against the sex hormone‐binding globulin (SHBG)‐like domain but did not inhibit the binding of PS to C4b‐binding protein (C4BP) and did not interfere with the TFPIα‐cofactor activity of PS. In mice, PS003biv exerted an antithrombotic effect in a FeCl3‐induced thrombosis model, while not affecting physiological hemostasis in a tail‐clip bleeding model. DiscussionAltogether, these results showed that pharmacological enhancement of the APC‐cofactor activity of PS through an original anti‐PS sdAb might constitute a promising and safe antithrombotic strategy.

The Antithrombotic Potential of Bioactive Peptides Induced by Buffalo Milk Probiotic Cheddar Cheese

Cheddar cheese undergoes significant changes resulting in numerous microbiological and biochemical processes called glycolysis, lipolysis, and proteolysis, accountable for a unique texture, aroma, appearance, and taste. Specific bioactive peptides developed during these biochemical reactions impart health benefits. Addition of probiotics boots the development of bioactive peptides in foods. Objective: The current research investigated the therapeutic potential of water-soluble peptides (WSPs) extracts from buffalo milk probiotic Cheddar cheese regarding anti-thrombic facets. Methods: The appropriateness of Buffalo milk for Cheddar manufacturing was assessed by analysing its pH, acidity, fat, protein, and total solids content. Two batches of Cheddar cheese were produced, one having a mixture of Probiotic microorganisms and commercially available mesophilic cheese starter and the second containing only commercially available cheese starters. Both of the cheese batches were analysed for their physicochemical properties. Water-soluble extract of Cheddar cheese samples was analysed for anti-thrombic effects after two-month intervals during ripening. Results: Three concentrations of WSE of buffalo milk cheddar cheese were used to assess the antithrombotic effect during 60, 120, and 180 days of ripening at 4°C. Antithrombotic activity increased with the ripening period for both control and probiotic cheddar cheese samples Conclusion: A significantly increased effect of antithrombotic activity was observed by Probiotic adjunct on control cheddar cheese.

The Anti-Inflammatory and Antithrombotic Properties of Bioactives from Orange, Sanguine and Clementine Juices and from Their Remaining By-Products

The anti-oxidant properties of vitamin C and of phenolic compounds of citrus fruits are well established. However, the evaluation of the anti-inflammatory and antithrombotic potential of both vitamin C and of the more amphiphilic and lipophilic components of citrus fruits needs further attention. In this study, the anti-inflammatory and antithrombotic properties of vitamin C and of freshly squeezed juices and their lipid bioactives from the Navalina and Sanguine orange varieties and the Clementine variety of mandarins, as well as from their remaining by-products, were evaluated against the inflammatory and thrombotic pathways of the platelet-activating factor (PAF) and thrombin in platelets, as well as against PAF-biosynthesis in leukocytes. The non-oxidized juices of these citrus fruits and a vitamin C supplement showed stronger anti-PAF and antithrombin effects than their oxidized versions through their general anti-oxidant effect in platelets. The total lipids (TLs) and the HPLC-derived fractions of phenolic compounds and of polar lipid bioactives from both juices and their peels’ by-products showed a more specific stronger inhibitory effect against the inflammatory and thrombotic pathways of PAF and thrombin in platelets, while these bioactives strongly inhibited also the specific enzyme activities of the main biosynthetic enzymes of PAF in leukocytes. The stronger bioactivity of the dietary bioactives found in the juices of these citrus fruits against specific biochemical pathways of inflammation and thrombosis seems to act with synergy with the anti-oxidant potential of their vitamin C content, which further supports the notion that these juices are functional foods with anti-inflammatory protective health benefits. In addition, the presence of these dietary bioactive phenolic compounds and polar lipid bioactives in the remaining peels’ wastes further enhance the valorization of such food industry by-products as potential sources of anti-inflammatory bioactives to be used as ingredients for novel functional products.