Inhibited Platelet Adhesion Research Articles

Depurative capacity toward medium molecules of the dialyzer Toray NV-U® Hydrolink™: A new hydrophilic membrane to perform online hemodiafiltration

Introduction: New dialysis membranes with new properties are being developed to improve efficacy and tolerance. The hemocompatibility of a polymeric biomaterial is influenced by the layer of water at the blood membrane interface. The new dialyzer TORAY NV-U® has a membrane Hydrolink™, designed to suppress platelet adhesion and to improve the hemocompatibility. Until now, there is no experience in online hemodiafiltration (OL-HDF). The objective of the present study is to evaluate the efficacy of this new membrane in OL-HDF therapy compared to another membrane commonly used. Other objectives are to evaluate the inflammatory response, hemodynamic tolerance, and the anticoagulation regimes. Methods: This is a prospective pilot study performed in five anuric patients receiving OL-HDF. For 1 month patients were kept with their usual dialyzer FX1000® (FMC). Subsequently, the dialyzer was changed to TORAY NV-U® (Hydrolink®) for 1 month. In the last dialysis session of each dialyzer, blood tests were performed to evaluate inflammation and depurative capacity. Results: We did not find differences in medium size removal molecules and convective volume: FX1000®: 31 ± 9 l per session and Hydrolink™ 30 ± 8 l; p = 0.7); β2microglobulin reduction ratio (RR) FX1000® FMC 83 ± 3%; Hydrolink™ 79 ± 4; p = 0.14; Myoglobin RR FX1000® FMC 72 ± 7%; Hydrolink™ 76 ± 4; p = 0.28. We did not find differences in inflammation parameters: serum IL6 with FX1000® 6.0 ± 4.2 pg/mL; Hydrolink™ 7.6 ± 5.0 pg/mL; p = 0.3. During all sessions with the two dialyzers there was adequate plasmatic filling, reaching 85 % filling. All patients had “good” dialyzer status in all dialysis sessions with TORAY NV-U®, while the dialyzer status with FX1000® was “good” in 20% of the sessions, “medium” in 30%, and “dirty” in the remaining 50% dialysis sessions. Conclusions: The new dialyzer Hydrolink™, TORAY NV-U® is not inferior to perform OL-HDF compared to dialyzers usually used for this therapy, and could allow decrease heparin doses. Further studies with a bigger sample size and longer follow-up will answer if Hydrolink improves inflammation and assess a better hemodynamic tolerance.

Rosuvastatin inhibit ox-LDL-induced platelet activation by the p38/MAPK pathway.

This study intends to explore the effects of Rosuvastatin on ox-LDL induced platelet activation and its molecular mechanism. Platelet aggregation rate was detected by aggregometer. ELISA kit was used to detect the levels of cAMP. Immunofluorescence staining was used to detect the platelet adhesion. The expression levels of platelet surface markers CD62p and PAC-1 were detected by flow cytometry. The protein levels of p-p38, p-IKKa and p-IKKB in platelets were detected by western blot. We found that rosuvastatin significantly inhibited platelet aggregation and increased the level of cAMP in a dose-dependent manner. Immunofluorescence staining results showed that rosuvastatin could inhibit platelet adhesion. Flow cytometry results showed that rosuvastatin could reduce the expression of platelet activation markers. Western blot results showed that rosuvastatin could down-regulate the expression levels of p-p38, p-IKKa and p-IKKb. Our study revealed the rosuvastatin could inhibit the aggregation, adhesion and activation of platelet induced by ox-LDL, its mechanism may be related to inhibition of p38/MAPK signal pathway.

Effects of Panax notoginseng saponins on alleviating low shear induced endothelial inflammation and thrombosis via Piezo1 signalling

Ethnopharmacological relevancePanax notoginseng saponins (PNS) are the major effective components of Panax notoginseng (burk) F.H.Chen which is one of the classic promoting blood circulation herbs in traditional Chinese medicine. PNS is widely used in China for the treatment of cerebral ischemic stroke. Pathological low shear stress is a causal factor in endothelial inflammation and thrombosis. However, the mechanism of PNS against low shear related endothelial inflammation is still unclear. Aim to the studyThis study aims to investigate the effects of PNS against endothelial inflammation induced by low shear stress and to explore the underlying mechanical and biological mechanisms. Materials and methodsMouse model of carotid partial ligation for inducing low endothelial shear stress was established, the pharmacodynamic effect and mechanism of PNS against endothelial inflammation induced by low shear stress through Piezo1 were explored. Yoda1-evoked Piezo1 activation and expression in human umbilical vein endothelial cells (HUVECs) were determined at static condition. Microfluidic channel systems were used to apply shear stress on HUVECs and Piezo1 siRNA HUVECs to determine PECAM-1, p-YAP and VCAM-1 expression. And platelet rich plasma (PRP) was introduced to low shear treated endothelial cells surface to observe the adhesion and activation by fluorescence imaging and flowcytometry. ResultsPNS attenuated endothelial inflammation and improved blood flow in a reasonable dose response pattern in carotid partial ligation mouse model by influencing Piezo1 and PECAM-1 expression, while suppressing yes-associated protein (YAP) nuclear translocation. We found Piezo1 sensed abnormal shear stress and transduced these mechanical signals by different pathways in HUVECs, and PNS relieved endothelial inflammation induced by low shear stress through Piezo1. We also found Piezo1 signalling has interaction with PECAM-1 under low shear stress, which were involved in platelets adhesion to endothelial cells. Low shear stress increased YAP nuclear translocation and increased VCAM-1 expression in HUVECs which might activate platelets. PNS inhibited low shear induced Piezo1 and PECAM-1 expression and YAP nuclear translocation in HUVECs, furthermore inhibited platelet adhesion and activation on dysfunctional endothelial cells induced by low shear stress. ConclusionPNS ameliorated endothelial inflammation and thrombosis induced by low shear stress through modulation of the Piezo1 channel, PECAM-1 expression, and YAP nuclear translocation. PNS might serve as a potential therapeutic candidate for ameliorating endothelial inflammation induced by abnormal blood shear stress.

Preparation of an anticoagulant polyethersulfone membrane by immobilizing FXa inhibitors with a polydopamine coating

Anticoagulation treatment for patients with high bleeding risk during hemodialysis is challenging. Contact between the dialysis membrane and the blood leads to protein adsorption and activation of the coagulation cascade reaction. Activated coagulation Factor X (FXa) plays a central role in thrombogenesis, but anticoagulant modification of the dialysis membrane is rarely targeted at FXa. In this study, we constructed an anticoagulant membrane using the polydopamine coating method to graft FXa inhibitors (apixaban and rivaroxaban) on the membrane surface. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used to characterize the membranes. The apixaban- and rivaroxaban-modified membranes showed lower water contact angles, decreased albumin protein adsorption, and suppressed platelet adhesion and activation compared to the unmodified PES membranes. Moreover, the modified membranes prolonged the blood clotting times in both the intrinsic and extrinsic coagulation pathways and inhibited FXa generation and complement activation, which suggested that the modified membrane enhanced biocompatibility and antithrombotic properties through the inhibition of FXa. Targeting FXa to design antithrombotic HD membranes or other blood contact materials might have great application potential.

Stability Enhancement by Hydrophobic Anchoring and a Cross-Linked Structure of a Phospholipid Copolymer Film for Medical Devices.

Surface modification using zwitterionic 2-methacryloyloxyethylphosphorylcholine (MPC) polymers is one of the most reasonable ways to prepare medical devices that can suppress undesired biological reactions such as blood coagulation. Usable MPC polymers are hydrophilic and water soluble, and their surface modification strategy involves exploiting the copolymer structures by adding physical or chemical bonding moieties. In this study, we developed copolymers composed of MPC, hydrophobic anchoring moiety, and chemical cross-linking unit to clarify the role of hydrophobic interactions in achieving biocompatible and long-term stable coatings. The four kinds of MPC copolymers with cross-linking units, such as 3-methacryloxypropyl trimethoxysilane (MPTMSi), and four different hydrophobic anchoring moieties, such as 3-(methacryloyloxy)propyltris(trimethylsiloxy)silane (MPTSSi) named as PMMMSi, n-butyl methacrylate (BMA) as PMBSi, 2-ethylhexyl methacrylate (EHMA) as PMESi, and lauryl methacrylate as PMLSi, were synthesized and coated on polydimethylsiloxane, polypropylene (PP), and polymethyl pentene. These copolymers were uniformly coated on the substrate materials PP and poly(methyl pentene) (PMP), to achieve hydrophilic and electrically neutral coatings. The results of the antibiofouling test showed that PMBSi repelled the adsorption of fluorescence-labeled bovine serum albumin the most, whereas PMLSi repelled it the least. Notably, all four copolymers suppressed platelet adhesion similarly. The variations in protein adsorption quantities among the four copolymer coatings were attributed to their distinct swelling behaviors in aqueous environments. Further investigations, including 3D scanning force microscopy and neutron reflectivity measurements, revealed that the PMLSi coating exhibited a higher water intake under aqueous conditions in comparison to the other coatings. Consequently, all copolymer coatings effectively prevented the invasion of platelets but the proteins penetrated the PMLSi network. Subsequently, the dynamic stability required to induce shear stress was evaluated using a circulation system. The results demonstrated that the PMMMSi and PMLSi coatings on PMP and PP exhibited exceptional platelet repellency and maintained high stability during circulation. This study highlights the potential of hydrophobic moieties to improve hemocompatibility and stability, offering potential applications in medical devices.

Intelligent Design of Antithrombotic Peptide Targeting Collagen.

Binding of blood components to collagen was proved to be a key step in thrombus formation. Intelligent Design of Protein Matcher (IDProMat), a neural network model, was then developed based on the principle of seq2seq to design an antithrombotic peptide targeting collagen. The encoding and decoding of peptide sequence data and the interaction patterns of peptide chains at the interface were studied, and then, IDProMat was applied to the design of peptides to cover collagen. The 99.3% decrease in seq2seq loss and 58.3% decrease in MLP loss demonstrated that IDProMat learned the interaction patterns between residues at the binding interface. An efficient peptide, LRWNSYY, was then designed using this model. Validations on its binding on collagen and its inhibition of platelet adhesion were obtained using docking, MD simulations, and experimental approaches.

Fast-polymerized lubricant and antibacterial hydrogel coatings for medical catheters

Medical catheters are highly susceptible to friction and infection when intervening or implanting in the body, and to thrombosis when in contact with blood. Coating medical catheters with lubricant and antibacterial hydrogel is an effective strategy but struggle to achieve. Here, hydrogel coating on the catheter surface with lubrication and antibacterial properties was constructed by the rapid polymerization of acrylamide (AAM) and sulfobetaine methacrylate (SBMA) in the presence of quaternary ammonium chitosan (QCS) based on the redox-based cross-linking method. First, the medical catheter surface was decorated with a polyethyleneimine (PEI) adhesive layer. The pre-gel solution was then applied to the catheter surface, followed by immersion in an aqueous FeSO4·7H2O solution, where a large amount of SO4-• was generated by a redox reaction between ammonium persulfate (APS) and Fe2+, which rapidly triggered the monomer to cross-link to form the coating. Polyelectrolyte PEI as the adhesive layer could bind the coating to the substrate through hydrogen bonding interactions and confer antimicrobial properties to the coating with the assistance of QCS (antibacterial rate of 95.88 % against E. coli and 97.11 % against B. subtilis). The hydrogel coatings exhibited outstanding lubricity with a low coefficient of friction (COF ≈ 0.053) and excellent antifouling properties (resists non-specific protein adsorption, inhibits platelet adhesion and prevents thrombosis (The clotting time was extended from 25.6 s to 74.7 s)) based on the integration of SBMA. This work shed new light on the design of hydrogel coatings to achieve modification of the substrate surface.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

Development of 3D printed electrospun vascular graft loaded with tetramethylpyrazine for reducing thrombosis and restraining aneurysmal dilatation.

Small-diameter vascular grafts have become the focus of attention in tissue engineering. Thrombosis and aneurysmal dilatation are the two major complications of the loss of vascular access after surgery. Therefore, we focused on fabricating 3D printed electrospun vascular grafts loaded with tetramethylpyrazine (TMP) to overcome these limitations. Based on electrospinning and 3D printing, 3D-printed electrospun vascular grafts loaded with TMP were fabricated. The inner layer of the graft was composed of electrospun poly(L-lactic-co-caprolactone) (PLCL) nanofibers and the outer layer consisted of 3D printed polycaprolactone (PCL) microfibers. The characterization and mechanical properties were tested. The blood compatibility and in vitro cytocompatibility of the grafts were also evaluated. Additionally, rat abdominal aortas were replaced with these 3D-printed electrospun grafts to evaluate their biosafety. Mechanical tests demonstrated that the addition of PCL microfibers could improve the mechanical properties. In vitro experimental data proved that the introduction of TMP effectively inhibited platelet adhesion. Afterwards, rat abdominal aorta was replaced with 3D-printed electrospun grafts. The 3D-printed electrospun graft loaded with TMP showed good biocompatibility and mechanical strength within 6months and maintained substantial patency without the occurrence of acute thrombosis. Moreover, no obvious aneurysmal dilatation was observed. The study demonstrated that 3D-printed electrospun vascular grafts loaded with TMP may have the potential for injured vascular healing.

Construction and study of blood purification membrane modified with PDE inhibitor: Investigation of antiplatelet activity and hemocompatibility

The recent “cell-based theory” of coagulation suggests that platelets serve as the site of coagulation factor reactions, making platelets an effective target for inhibiting membrane thrombosis. Unfortunately, there is limited research on how blood purification membranes affect platelet intracellular signaling. In this study, we modified polyethersulfone (PES) membranes with the platelet phosphodiesterase (PDE) inhibitor dipyridamole (DIP) and investigated the effects of the DIP/PES (DP) membranes on platelet adhesion, activation, aggregation, and secretion, as well as the role of the PDE-cyclic adenosine monophosphate (cAMP) intracellular signaling pathway. Additionally, we evaluated the hemocompatibility and preliminary in vivo safety of DP membranes. Our results demonstrate that the modified DP membranes effectively inhibited platelet adhesion, membrane CD62P expression, and plasma soluble P-selectin activation levels. Furthermore, we confirmed that DP membranes achieved platelet aggregation inhibition and reduced platelet factor 4 and β-thromoglobulin secretion levels by inhibiting platelet intracellular PDE-cAMP signaling. Moreover, the modified DP membranes exhibited good anticoagulant and red blood cell membrane stability and complement resistance and demonstrated preliminary biocompatibility in mouse experiments. Collectively, these findings highlight the potential application of DP dialysis membranes in blood purification for critically ill patients.

Qihuang Zhuyu formula alleviates coronary microthrombosis by inhibiting PI3K/Akt/αIIbβ3-mediated platelet activation

BackgroundCoronary microembolism (CME) is commonly seen in the peri-procedural period of Percutaneous Coronary Intervention (PCI), where local platelet activation and endothelial cell inflammation crosstalk may lead to micro thrombus erosion and rupture, with serious consequences. Qihuang Zhuyu Formula (QHZYF) is a Chinese herbal compound with high efficacy against coronary artery disease, but its antiplatelet mechanism is unclear. Hypothesis/PurposeThis study aimed to elucidate the effects and mechanisms of QHZYF on sodium laurate-induced CME using network pharmacology and in vitro and in vivo experiments. MethodsWe employed high-performance liquid chromatography mass spectrometry to identify the main components of QHZYF. Network pharmacology analysis, molecular docking and surface plasmon resonance (SPR) were utilized to predict the primary active components, potential therapeutic targets, and intervention pathways mediating the effects of QHZYF on platelet activation. Next, we pretreated a sodium laurate-induced minimally invasive CME rat model with QHZYF. In vivo experiments were performed to examine cardiac function in rats, to locate coronary arteries on heart sections to observe internal microthrombi, to extract rat Platelet-rich plasma (PRP) for adhesion assays and CD62p and PAC-1 (ITGB3/ITGA2B) flow assays, and to measure platelet-associated protein expression in PRP. In vitro clot retraction and Co-culture of HUVECs with PRP were performed and the gene pathway was validated through flow cytometry and immunofluorescence. ResultsCombining UPLC-Q-TOF/MS technology and database mining, 78 compounds were finally screened as the putative and representative compounds of QHZYF, with 75 crossover genes associated with CME. QHZYF prevents CME mainly by regulating key pathways of the inflammation and platelets, including Lipid and atherosclerosis, Fluid shear stress, platelet activation, and PI3K-Akt signaling pathways. Five molecules including Calyson, Oroxin A, Protosappanin A,Kaempferol and Geniposide were screened and subjected to molecular docking and SPR validation in combination with Lipinski rules (Rule of 5, Ro5). In vivo experiments showed that QHZYF not only improved myocardial injury but also inhibited formation of coronary microthrombi. QHZYF inhibited platelet activation by downregulating expression of CD62p receptor and platelet membrane protein αIIbβ3 and reduced the release of von Willebrand Factor (vWF), Ca2+ particles and inflammatory factor IL-6. Further analysis revealed that QHZYF inhibited the activation of integrin αIIbβ3, via modulating the PI3K/Akt pathways. In in vitro experiments, QHZYF independently inhibited platelet clot retraction. Upon LPS induction, the activation of platelet membrane protein ITGB3 was inhibited via the PI3K/Akt pathway, revealing an important mechanism for attenuating coronary microthrombosis. We performed mechanistic validation using PI3K inhibitor LY294002 and Akt inhibitor MK-2206 to show that QHZYF inhibited platelet membrane protein activation and inflammation to improved coronary microvessel embolism by regulating PI3K/Akt/αIIbβ3 pathways, mainly by inhibiting PI3K and Akt phosphorylation. ConclusionQHZYF interferes with coronary microthrombosis through inhibition of platelet adhesion, activation and inflammatory crosstalk, thus has potential in clinical anti-platelet applications. Calyson, Oroxin A, Protosappanin A, Kaempferol and Geniposide may be the major active ingredient groups of QHZYF that alleviate coronary microthrombosis.

Dealloyed nano-porous Ti[sbnd]Cu coatings with controlled copper release for cardiovascular devices

TiCu coatings with controlled copper release and nano-porous structures were fabricated as biocompatible, blood-contacting interfaces through a two-step process. Initially, coatings with 58 % Cu were created using HiPIMS/DC magnetron co-sputtering, followed by immersion in a dilute HF solution for varying durations to achieve dealloying. The presence of Ti elements in the as-deposited TiCu coatings facilitated their dissolution upon exposure to the dilute HF solution, resulting in the formation of nanopores and increased nano-roughness. Dealloying treatment time correlated with higher Cu/(Ti + Cu) values, nanopore size, and nano-roughness in the dealloyed samples. The dealloyed TiCu coatings with 87 % Cu exhibited a controlled release of copper ions and displayed nanopores (approximately 80 nm in length and 31.0 nm in width) and nano-roughness (Ra roughness: 82 nm). These coatings demonstrated inhibited platelet adhesion and suppressed smooth muscle cell behavior, while supporting favorable endothelial cell viability and proliferation, attributed to the controlled release of copper ions and the extent of nanostructures. In contrast, the as-deposited TiCu coatings with 85 % Cu showed high copper ion release, leading to decreased viability and proliferation of endothelial cells and smooth muscle cells, as well as suppressed platelet adhesion. The TiCu coatings met medical safety standards, exhibiting hemolysis rates of <5 %. The technology presented here paves the way for the simple, controllable, and cost-effective fabrication of TiCu coatings, opening new possibilities for surface modification of cardiovascular devices such as vascular stents and inferior vena cava filters.

Fine-Tuning the Nanostructured Titanium Oxide Surface for Selective Biological Response.

Cardiovascular diseases are a pre-eminent global cause of mortality in the modern world. Typically, surgical intervention with implantable medical devices such as cardiovascular stents is deployed to reinstate unobstructed blood flow. Unfortunately, existing stent materials frequently induce restenosis and thrombosis, necessitating the development of superior biomaterials. These biomaterials should inhibit platelet adhesion (mitigating stent-induced thrombosis) and smooth muscle cell proliferation (minimizing restenosis) while enhancing endothelial cell proliferation at the same time. To optimize the surface properties of Ti6Al4V medical implants, we investigated two surface treatment procedures: gaseous plasma treatment and hydrothermal treatment. We analyzed these modified surfaces through scanning electron microscopy (SEM), water contact angle analysis (WCA), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) analysis. Additionally, we assessed in vitro biological responses, including platelet adhesion and activation, as well as endothelial and smooth muscle cell proliferation. Herein, we report the influence of pre/post oxygen plasma treatment on titanium oxide layer formation via a hydrothermal technique. Our results indicate that alterations in the titanium oxide layer and surface nanotopography significantly influence cell interactions. This work offers promising insights into designing multifunctional biomaterial surfaces that selectively promote specific cell types' proliferation─which is a crucial advancement in next-generation vascular implants.

Antiplatelet Agents Inhibit Platelet Adhesion and Aggregation on Glass Surface Under Physiological Flow Conditions: Toward a Microfluidic Platelet Functional Assay Without Additional Adhesion Protein Modification.

As the pathogenesis of arterial thrombosis often includes platelet adhesion and aggregation, antiplatelet agents are commonly used to prevent thromboembolic events. Here, a new microfluidic method without additional adhesion protein modification was developed to quantify the inhibitory effect of antiplatelet drugs on the adhesion and aggregation behavior of platelets on glass surfaces under physiological flow conditions. Polydimethylsiloxane-glass microfluidic chips were fabricated by soft photolithography. Blood samples from healthy volunteers or patients before and after taking antiplatelet drugs flowed through the microchannels at wall shear rates of 300 and 1500 second -1 , respectively. The time to reach 2.5% platelet aggregation surface coverage (Ti), surface coverage (A 150s ), and mean fluorescence intensity (F 150s ) were used as quantitative indicators. Aspirin (80 μM) prolonged Ti and reduced F 150s . Alprostadil, ticagrelor, eptifibatide, and tirofiban prolonged Ti and reduced A 150s and F 150s in a concentration-dependent manner, whereas high concentrations of alprostadil did not completely inhibit platelet aggregation. Aspirin combined with ticagrelor synergistically inhibited platelet adhesion and aggregation; GPIb-IX-von Willebrand factor inhibitors partially inhibited platelet aggregation, and the inhibition was more pronounced at 1500 than at 300 second -1 . Patient administration of aspirin or (and) clopidogrel inhibited platelet adhesion and aggregation on the glass surface under flow conditions. This technology is capable of distinguishing the pharmacological effects of various antiplatelet drugs on inhibition of platelet adhesion aggregation on glass surface under physiological flow conditions, which providing a new way to develop microfluidic platelet function detection method without additional adhesive protein modification for determining the inhibitory effects of antiplatelet drugs in the clinical setting.

In vitro biological responses of plasma nanocoatings for coronary stent applications.

In-stent restenosis and thrombosis remain to be long-term challenges in coronary stenting procedures. The objective of this study was to evaluate the in vitro biological responses of trimethylsilane (TMS) plasma nanocoatings modified with NH3 /O2 (2:1 molar ratio) plasma post-treatment (TMS + NH3 /O2 nanocoatings) on cobalt chromium (CoCr) alloy L605 coupons, L605 stents, and 316L stainless steel (SS) stents. Surface properties of the plasma nanocoatings with up to 2-year aging time were characterized by wettability assessment and x-ray photoelectron spectroscopy (XPS). It was found that TMS + NH3 /O2 nanocoatings had a surface composition of 41.21 ± 1.06 at% oxygen, 31.90 ± 1.08 at% silicon, and 24.12 ± 1.7 at% carbon, and very small but essential amount of 2.77 ± 0.18 at% nitrogen. Surface chemical stability of the plasma coatings was noted with persistent O/Si atomic ratio of 1.292-1.413 and N/Si atomic ratio of ~0.087 through 2 years. The in vitro biological responses of plasma nanocoatings were studied by evaluating the cell proliferation and migration of porcine coronary artery endothelial cells (PCAECs) and smooth muscle cells (PCASMCs). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay results revealed that, after 7-day incubation, TMS + NH3 /O2 nanocoatings maintained a similar level of PCAEC proliferation while showing a decrease in the viability of PCASMCs by 73 ± 19% as compared with uncoated L605 surfaces. Cell co-culture of PCAECs and PCASMCs results showed that, the cell ratio of PCAEC/PCASMC on TMS + NH3 /O2 nanocoating surfaces was 1.5-fold higher than that on uncoated L605 surfaces, indicating enhanced selectivity for promoting PCAEC growth. Migration test showed comparable PCAEC migration distance for uncoated L605 and TMS + NH3 /O2 nanocoatings. In contrast, PCASMC migration distance was reduced nearly 8.5-fold on TMS + NH3 /O2 nanocoating surfaces as compared to the uncoated L605 surfaces. Platelet adhesion test using porcine whole blood showed lower adhered platelets distribution (by 70 ± 16%), reduced clotting attachment (by 54 ± 12%), and less platelet activation on TMS + NH3 /O2 nanocoating surfaces as compared with the uncoated L605 controls. It was further found that, under shear stress conditions of simulated blood flow, TMS + NH3 /O2 nanocoating significantly inhibited platelet adhesion compared to the uncoated 316L SS stents and TMS nanocoated 316L SS stents. These results indicate that TMS + NH3 /O2 nanocoatings are very promising in preventing both restenosis and thrombosis for coronary stent applications.

Continuous NO dual-generation by ZnO nanoparticle conjugated with α-lipoic acid for functional biodegradable vascular stent

Coronary artery diseases are treated with a biodegradable vascular scaffold (BVS) intervention. However, BVS need to be enhanced to avoid post-insertion side effects, which can be done by controlling the nitric oxide (NO) concentration in blood vessels. NO has excellent functions, such as re-endothelialization, inhibition of smooth muscle cells, and prevention of activation and aggregation of platelets, and is essential in maintaining a healthy heart and blood vessels. In this study, a novel nanoparticle with fortified NO-generating ability was synthesized by combining two mechanisms capable of long-term NO production: the decomposition of the NO donor by the redox reaction of zinc oxide and the thiol/disulfide exchange reaction between the NO donor and α-lipoic acid. Next, a poly(L-lactic acid) (PLLA) scaffold containing synthesized nanoparticles was fabricated, and the potent functional recovery ability of unhealthy vessels was demonstrated in vitro and in vivo. The NO generated from scaffolds inhibited platelet adhesion and activation, increased endothelial cell proliferation, and decreased smooth muscle cell proliferation. In addition, the scavenging ability of reactive oxygen species and their inhibition of toxic NO production were investigated. The scaffolds implanted subcutaneously in mice suppressed the inflammatory response and induced healthy blood vessel formation, offering a new tool for designing cardiovascular stents and various biological applications.

P83: Circulating Platelet-Derived Microparticles Modulate Platelet Adhesion Under Flow: Modeling Primary Thrombus Formation in a Microfluidic System

Background: Elevated levels of circulating platelet-derived microparticles (PDMPs) are associated with a range of pathologies, thought to increase thrombotic risk. We hypothesized that addition of PDMPs to whole blood would alter platelet adhesion and thrombogenicity, with its effect further modulated by shear stress magnitude. We tested the effect of addition of PDMPs to whole blood on platelet adhesion and primary thrombus formation on collagen under a range of shear flows using a microfluidic system. Methods: PDMPs were generated via sonication of gel-filtered human platelets and separated via differential centrifugation. Hirudin-anticoagulated human blood was stained with 3,3′-dihexyloxacarbocyanine iodide and circulated through a collagen-coated microchannel with and without PDMPs at defined shear stress magnitudes (13.5, 49.5, or 72 dyne/cm2) for 4 min. Platelet adhesion was visualized by fluorescence microscopy; images were analyzed using MATLAB software. Platelet adhesion and thrombogenicity was assessed via four parameters: 1. surface coverage (SC) – indicative of overall surface occupied by platelet thrombi; 2. number of thrombi (NT) – indicative of the number of sites of platelet adhesion on a collagen-covered surface; 3. mean thrombus area (MTA) and 4. mean fluorescence intensity (MFI) – both characterizing thrombus size. Results: Whole blood platelet exposure to increasing shear stress facilitated platelet adhesion to collagen, with increased SC, MTA and MFI (Fig. 1, WB group). Interestingly, the number of thrombi decreased (846±148 vs. 256±81 at 13.5 and 72 dyne/cm2, respectively) with increasing shear suggesting that increased platelet adhesion was due to increased thrombus size, rather than the number of sites of platelet adhesion. Circulating PDMPs significantly inhibited platelet adhesion to collagen, with a decrease of SC, MTI and MFI (but not NT) at all shear stress magnitudes (Fig. 1, WB+PDMPs vs. WB group, ANOVA: p<0.05). At high shear, PDMP-mediated inhibition of platelet adhesion was pronounced with a more than a 2-fold decrease of thrombus size, as indicated by MTA and MFI. Notably, adding PDMPs to recirculated whole blood increased NT adhered to collagen only at lower shear magnitudes. Conclusion: Shear stress of increasing magnitude promotes increased platelet adhesion to collagen, platelet thrombus size and surface coverage; while the number of sites of platelet adhesion decreases. Circulating PDMPs inhibit platelet adhesion under flow, resulting in a significant decrease in platelet thrombi size. Adding PDMPs to circulating blood increases the number of thrombi formed on collagen, but only at lower shear magnitudes. We speculate that increased levels of circulating PDMPs facilitate platelet adhesion and primary thrombus formation, contributing to an overall prothrombotic phenotype, yet with associated microthrombosis with embolic potential, imposing increased risk of distal ischemic and occlusive events.Figure 1. Circulating platelet-derived microparticles (PDMPs) inhibit platelet adhesion to collagen under shear stress: WB – whole blood, circulated without PDMPs; WB+PDMPs - whole blood, circulated without PDMPs (9:1). Mean ± SD, n = 7, ANOVA: * - p < 0.05, ** - p < 0.01.

NITRIC OXIDE IN BLOOD PLASMA OF WISTAR RATS RECEIVING VITAMIN D3

Recently, much data has been published on the correlation between vitamin D and endothelium. Which is known to regulate vascular homeostasis and hemodynamics. Nitric oxide is characterized by a strong vasodilating effect. It is also a powerful inhibitor of platelet aggregation and adhesion to the vascular wall. Vitamin D and its receptors can regulate the nitric oxide synthesis by changing the activity of endothelial NO synthase.&#x0D; The purpose of the study was to examine the effect of vitamin D3 (emulsion) consumption on the level of nitric oxide in the blood of Wistar rats.&#x0D; Materials and Methods. The study was carried out on mature male Wistar rats weighing 200–300 g (aged 2–3 months). The rats were randomly divided into two groups: 1) control (n=6), kept in vivarium during the whole experiment, 2) experimental (n=6), daily receiving vitamin D3 in emulsion for 30 days. The calculation of vitamin D3 dose was based on the recommended daily amount of vitamin D3 for an adult person (4000 IU). A biochemical blood test determined 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and the level of stable nitric oxide metabolites. Statistica software was used for statistical analysis.&#x0D; Results. The trial study showed that a 30-day intake of vitamin D3 in emulsion increased its concentration and the nitric oxide level in the blood of rats. It may indicate the intensification of the enzymatic nitric oxide synthesis and the improvement in endothelial function.&#x0D; Conclusion. Thus, vitamin D and nitric oxide are important functionally correlated compounds. Adequate vitamin D status can mitigate the development of cardiovascular disease.

Interaction between adenosine diphosphate receptors and protein-kinase C isoforms in platelet adhesion under flow condition

Adenosine diphosphate (ADP) receptors and protein-kinase C (PKC) isoforms play different role in platelet activity. In the present study, whole blood platelet adhesion at 200 - 1800 s-1 shear rates was investigated by Impact-R system, measuring percent of surface coverage (SC) by platelets. Gradual heightened shear rate par-alleled increase of platelet adhesion. At relatively low shear (200 and 1000 s-1) blockade of neither P2Y1 receptor nor P2Y12 receptor (by A2P5P and 2MeSAMP, respectively) affected SC. At high shear rate (1800 s-1) reduction of SC was observed by 2MeSAMP. Treatment of blood with PKCδ inhibitor (rottlerin) but not PKCα,β inhibitor (Gö6976) diminished platelet adhe-sion. Among all the agents, only combination of 2MeSAMP and rottlerin used at subthreshold concentrations was able to inhibit platelet adhesion under high shear condition. We suggest that platelet agonist-induced P2Y12 and PKCδ signaling essentially stimulates platelet adhesion under flow condition, the important initiating step of thrombin formation.

Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets

Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.