Decreased Platelet Adhesion Research Articles

In vitro effects of serotonin and noradrenaline reuptake inhibitors on human platelet adhesion and coagulation

BackgroundAlthough several studies show that there is an increased risk of bleeding events during antidepressant treatment with selective serotonin reuptake inhibitors (SSRIs), few studies show direct effects in vitro of SSRIs on hemostasis. MethodsThis study was undertaken to investigate the effects on platelet adhesion and plasma coagulation (APTT and PT) of two common SSRIs, citalopram and sertraline, the selective noradrenaline reuptake inhibitor reboxetine, and the serotonin and noradrenaline reuptake inhibitor venlafaxine. ResultsNone of the compounds affected plasma coagulation significantly but all compounds except for venlafaxine inhibited platelet adhesion by approximately 50% or more at the highest concentration (100μg/l, p<0.01). The potency of respective compound to inhibit platelet adhesion to both collagen and fibrinogen surfaces was in the following order; citalopram > sertraline > reboxetine. In contrast, venlafaxine caused a weak but statistically significant increased platelet adhesion to fibrinogen. ConclusionThis study showed that sertraline, citalopram and reboxetine direct and acutely decrease platelet adhesion to both collagen and fibrinogen in vitro. These results also indicate that increased risk for bleeding complications in antidepressant users may not only be explained by depletion of serotonin in platelets.

Decreased Platelet Adhesion and Enhanced Endothelial Cell Functions on Nano and Submicron-Rough Titanium Stents

Endothelialization of a vascular stent is a critical step to prevent late stent thrombosis. In this study, electron beam deposition was utilized to create different scales of roughness on titanium stents, including flat features (F-Ti), a mixture of nanometer and submicron features (S-Ti), and nanometer features (N-Ti). The role of stent surface roughness on initial protein adsorption, platelet adhesion, rat aortic endothelial cell adhesion, migration, and nitric acid/endothelin-1 secretion was investigated in vitro. Results revealed the highest endothelial cell attachment on S-Ti after 4 h. Moreover, under flow conditions, the endothelial cell layer remained the most intact on S-Ti. These results were positively correlated with improved vitronectin adsorption on S-Ti. Endothelial cells also showed the fastest migration on S-Ti of all the samples over a 96 h time period. Endothelial cells on S-Ti exhibited the highest nitric acid/endothelin-1 ratio of all the samples, indicating potentially the best antithrombic endothelial cellular phenotype. This study also revealed the lowest platelet adhesion on S-Ti of all the samples. In summary, without using pharmaceutical agents, significantly less platelet and greater endothelial responses on nanometer to submicron rough titanium were observed in this study compared to flat titanium and, thus, nanometer to submicron surface features on titanium should be further studied for vascular stent applications.

Beta-90Sr irradiation treatment of silicon wafer coated with extracellular matrix protein to mimic the beta-32P radiation application in intravascular brachytherapy

Wu YP et al. in the International Journal of Cardiology reported that beta-radiation reduced the reactivity of extra cellular matrix proteins in intravascular brachytherapy (IVBT) which resulted in decreased platelet adhesion [1]. Beta-radiation treatment decreases the thrombogenicity of the vessel wall extracellular matrix proteins, in particular, vWF, fibrinogen, fibronectin and collagen, resulting in decreased platelet adhesion and aggregation. The vessel wall becomes lowly prone to thrombosis and might help reduce the onset of acute coronary events and of acute coronary occlusion after intravascular interventions.

Depression and heart failure

[1] Wu YP, Stella PR, Chen SF, et al. Beta-radiation reduces the reactivity of extra cellular matrix proteins in intravascular brachytherapy (IVBT), resulting in decreased platelet adhesion. Int J Cardiol 2012;156:283–8. [2] Xu JY, Jin B, Zhao Y, Wang K, Xia XH. In situ monitoring of the DNA hybridization by attenuated total reflection surface-enhanced infrared absorption spectroscopy. Chem Commun (Camb) 2012;48:3052–4. [3] Raizner AE, Kaluza GL, Ali NM. Clinical experience with a spiral balloon centering catheter for the delivery of intracoronary radiation therapy. Cardiovasc Radiat Med 1999;1:214–9. [4] Goodfellow M, Hayes A, Murphy S, et al. A retrospective study of (90)Strontium plesiotherapy for feline squamous cell carcinoma of the nasal planum. J FelineMed Surg 2006;8:169–76. [5] Hammond GM, Gordon IK, Theon AP, et al. Evaluation of strontium Sr 90 for the treatment of superficial squamous cell carcinoma of the nasal planum in cats: 49 cases (1990–2006). J Am Vet Med Assoc 2007;231:736–41. [6] Belbachir Karima, Noreen Razia, Gouspillou Gilles, Petibois Cyril. Collagen types analysis and differentiation by FTIR spectroscopy. Anal Bioanal Chem 2009;395:829–37.

A silver nanocomposite biomaterial for blood‐contacting implants

Cardiovascular implants must resist infection and thrombosis. A nanocomposite polymeric material [polyhedral-oligomeric-silsesquioxane-poly(carbonate-urea)urethane; POSS-PCU] demonstrates ideal properties for cardiovascular applications. Silver nanoparticles or nanosilver (NS) are recognized for efficient antibacterial properties. This study aims to determine the influence of NS integrated POSS-PCU on thrombogenicity. Silver nitrate was reduced with dimethylformamide and stabilized by the inclusion of fumed silica nanoparticles to prevent aggregation of NS and were incorporated into POSS-PCU to form a range of POSS-PCU-NS concentrations (by weight); 0.20% (NS16), 0.40% (NS32), 0.75% (NS64), and 1.50% (NS128). Surface wettability was determined with sessile-drop water contact angles. Platelets were introduced onto test samples and Alamar Blue (AB), mitochondrial-activity assay, quantified the degree of platelet adhesion whilst platelet-factor-4 (PF4) ELISA quantified the degree of platelet activation. Thromboelastography (TEG) determined the profiles of whole blood kinetics while hemolysis assay demonstrated the degree of blood compatibility. Increasing levels of NS induced greater hydrophilicity. A concentration dependant decrease in platelet adhesion and activation was observed with AB and PF4 readings, respectively. TEG demonstrated that the antithrombogenic properties of POSS-PCU were retained with POSS-PCU-NS16, and enhanced with POSS-PCU-NS32, but was reduced with POSS-PCU-NS64 and POSS-PCU-NS128. POSS-PCU-NS64 and POSS-PCU-NS128 demonstrated a hemolytic tendency, but no hemolysis was observed with POSS-PCU-NS16 and POSS-PCU-NS32. Overall, POSS-PCU-NS32 rendered potent antithrombogenic properties.

ARC15105 Is a Potent Antagonist of Von Willebrand Factor Mediated Platelet Activation and Adhesion

We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. Platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time with the platelet function analyzer-100 and platelet aggregation by multiple electrode aggregometry. Platelet adhesion was measured on denuded porcine aortas and in a flow chamber. Aptamer stability was assessed by incubation in nuclease rich human, monkey, and rat serum for up to 72 hours. Pharmacokinetic and pharmacodynamic profiles were tested in cynomolgus monkeys after IV and SC administration. The median IC(100) and IC(50) to prolong collagen/adenosine diphosphate-induced closure timewere 27 nmol/L and 12 nmol/L, respectively. ARC15105 (1.3 μmol/L) completely inhibited ristocetin-induced platelet aggregation in whole blood (P<0.001), but also diminished collagen, ADP, arachidonic acid, and thrombin receptor activating peptide-induced platelet aggregation to some extent (P<0.05). ARC15105 (40 nmol/L) decreased platelet adhesion by >90% on denuded porcine aortas (P<0.001), which was comparable to the degree of inhibition obtained with abciximab. ARC15105 (100 nmol/L) also inhibited platelet adhesion to collagen under arterial shear in a flow chamber by >90% (P<0.001). The IV and SC terminal half-lives in cynomolgus monkeys were 67 and 65 hours, respectively, and the SC bioavailability was ≈98%. Allometric scaling estimates the human T(1/2) would be ≈217 hours. Pharmacodynamic analysis confirms that ARC15105 inhibits von Willeband factor activity >90% in blood samples taken 300 hours after a 20 mg/kg IV or SC dose in monkeys. The potency, pharmacokinetic profile, and SC bioavailability of ARC15105 support its clinical investigation for chronic inhibition of von Willeband factor -mediated platelet activation.

Construction of heparinylated multilayer films on Ti[sbnd]O via streptavidin/biotin interaction

Construction of heparinylated multilayer films on TiO via streptavidin/biotin interaction was conducted in the present study. An organic layer of 3-aminopropylphosphonic acid (APP) was first introduced on TiO by self-assembling, and then biotin was immobilized by photochemical methods. So streptavidin and biotinylated heparin were assembling through biorecognition, and a desired 6-layer heparinylated multilayer was obtained through layer-by-layer driven by streptavidin/biotin interaction. The in vitro platelet adhesion and activation were investigated by a static platelet adhesion test. The clotting time was examined by activated partial thromboplastin time (APTT). Results show that the heparinylated multilayer coated TiO can significantly decrease platelet adhesion and activation, and prolong clotting time of APTT compared to untreated TiO, which indicates the heparinylated multilayer coated TiO displays more excellent anticoagulation performance than that of the bare TiO.

In vitro blood compatibility of polyethylene terephthalate with covalently bounded hirudin on surface

Polyethylene terephthalate (PET, Dacron) was modified by surface immobilization of hirudin with glutaraldehyde(GA) as coupling reagent to improve the blood compatibility. Hirudin-immobilized PETs were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The blood compatibility of the PETs was evaluated by platelet adhesion evaluation and fibrinogen conformational change measurements in vitro. The results showed the decrease of platelet adhesion and activation on hirudin-immobilized PET with increasing of glutaraldehyde concentration. Fibrinogen experiment showed that fibrinogen adherence and conformational changes of PET-HRD were less than those of untreated PET, which made the materials difficult to form thrombus. The proper reason of blood compatibility improvement was low interface tension between hirudin-immobilized PETs and blood, as well as blood proteins, and low ratio of dispersive/polar component of the surface energy(γsd/γsp) and high hydrophilicity.

Anticoagulant, anti-aggregation and antithrombotic effects of a novel hexapeptide

Hexapeptide is a novel synthetic oligopeptide with a structure similar to that of eptifibatide. This study was designed to investigate the anticoagulant, anti-aggregation, disaggregation and anti-thrombogenesis effects of hexapeptide. The effects of antiplatelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin, and the effect of disaggregation of platelet aggregation induced by ADP were determined. The anticoagulation indexes were determined by different kits. Hexapeptide 1 × 10(-5) -1 × 10(-4) m could significantly prolong rabbit blood clotting time, thrombin time, prothrombin time and activated partial thromboplastin enzyme time, and reduce the length, wet weight, dry weight and the index of thrombus in a concentration-dependent manner. Hexapeptide 1 × 10(-4) m decreased platelet adhesion rate by 40.2%. The platelet aggregation inhibition of hexapeptide in dogs and humans was more obvious than in rabbits and rats. The aggregation inhibition rate of 1 × 10(-5) m hexapeptide in dogs, rabbits, rats and humans induced by ADP was 93.9 ± 1.3%, 66.2 ± 1.4%, 76.1 ± 3.2% and 99.8 ± 0.2%, respectively; the 50% inhibitory concentration (IC50) of hexapeptide was 7.24 × 10(-8), 3.24 × 10(-6), 6.61 × 10(-6) and 8.91 × 10(-8) m, respectively. For the aggregation inhibition rate of hexapeptide in dogs, rabbits and humans induced by AA, the IC50 was 1.29 × 10(-9), 1.32 × 10(-6) and 9.33 × 10(-8) m, respectively; the IC50 of aggregation inhibition rates induced by thrombin was 2.88 × 10(-6), >1 × 10(-5) and 4.17 × 10(-6) m, respectively. The disaggregation rate of 1 × 10(-4) m hexapeptide in dog induced by ADP was 68.8 ± 7.4%. Hexapeptide has anticoagulant, antiplatelet aggregation, disaggregation and antithrombotic effects in vitro.

Platelet and leukocyte adhesion to albumin binding self-assembled monolayers

This study reports the use of tetraethylene glycol-terminated self-assembled monolayers (EG(4) SAMs) as a background non-fouling surface to study the effect of an 18 carbon ligand (C18) on albumin selective and reversible adsorption and subsequent platelet and leukocyte adhesion. Surface characterization techniques revealed an efficient immobilization of different levels of C18 ligand on EG(4) SAMs and an increase of surface thickness and hydrophobicity with the increase of C18 ligands. Albumin adsorption increased as the percentage of C18 ligands on the surface increased, but only 2.5%C18 SAMs adsorbed albumin in a selective and reversible way. Adherent platelets also increased with the amount of immobilized C18. Pre-immersion of samples in albumin before contact with platelets demonstrated an 80% decrease in platelet adhesion. Pre-immersion in plasma was only relevant for 2.5%C18 SAMs since this was the only surface to have less platelet adhesion compared to buffer pre-immersion. EG(4) SAMs adhered negligible amounts of leukocytes, but surfaces with C18 ligands have some adherent leukocytes. Except for 10%C18 SAMs, which increased leukocyte adhesion after albumin pre-adhesion, protein pre-immersion did not influence leukocyte adhesion. It has been shown that a surface with a specific surface concentration of albumin-binding ligands (2.5%C18 SAMs) can recruit albumin selectively and reversibly and minimize the adhesion of platelets, despite still adhering some leukocytes.

Shear Stress Responses of Adult Blood Outgrowth Endothelial Cells Seeded on Bioartificial Tissue

Human blood outgrowth endothelial cells (HBOECs) are expanded from circulating endothelial progenitor cells in peripheral blood and thus could provide a source of autologous endothelial cells for tissue-engineered vascular grafts. To examine the suitability of adult HBOECs for use in vascular tissue engineering, the shear stress responsiveness of these cells was examined on bioartificial tissue formed from dermal fibroblasts entrapped in tubular fibrin gels. HBOECs adhered to this surface, deposited collagen IV and laminin, and remained adherent when exposed to 15 dyn/cm(2) shear stress for 24 h. The shear stress responses of HBOECs were compared to human umbilical vein endothelial cells (HUVECs). As with HUVECs, HBOECs upregulated vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 when exposed to tumor necrosis factor (TNF)-α and shear stress decreased the expression of these adhesion molecules on TNF-α-activated monolayers. Nitric oxide production was elevated by shear stress, but did not vary between cell types. Both cell types decreased platelet adhesion to the bioartificial tissue, whereas pre-exposing the cells to flow decreased platelet adhesion further. These results illustrate the potential utility for HBOECs in vascular tissue engineering, as not only do the cells adhere to bioartificial tissue and remain adherent under physiological shear stress, they are also responsive to shear stress signaling.

A Diet Rich in Whole Grain Flaxseeds has Antithrombotic Effects Without Increasing Oxidative Stress in Experimental Atherosclerosis

We hypothesized that high doses of flaxseed used for antithrombotic effects could increase oxidative stress and vitamin E was added to prevent the lipid oxidation. Therefore, we investigated platelet functions and oxidative stress in ovariectomized hamsters fed on high-fat diet supplemented with flaxseeds and/or vitamin E. Eighty six months-old Golden Syrian hamsters were either sham-operated (Sham) or ova-riectomized (Ovx) and received one of the following diets: 1. high-fat diet (40 % energy as fat); 2. high-fat diet supplemented with flaxseed (15 g Linum usitatissimum /100 g of food) (Linum); 3. high-fat diet supplemented with vitamin E (40 mg alpha-tocopherol/100 g of food) (E); 4. high-fat diet supplemented with flaxseed and vitamin E (Linum+E) for 8 weeks. The ovariectomized groups fed on Linum/Vitamin E enriched diet showed a less advanced stage of atherosclerosis lesions and decreased aortic cholesterol content as compared to the high-fat diet group. The addition of flaxseed led to a significant decreased platelet adhesion in Ovx group. In Ovx hamsters, serum and liver TBARS were significantly decreased and liver GSH was significantly increased by supplementation of diet with flaxseed and combined diet augmented the antioxidative effect of flaxseed. The combined diet (Linum + vitamin E) did not seem to bring more benefits than flaxseed alone. Our results demonstrated that cardio-protective effect of high doses of ground flaxseed could be due to its antiplatelet and antioxidative effects and the addition of supplementary antioxidant is not required.

In vivo performance of a phospholipid‐coated bioerodable elastomeric graft for small‐diameter vascular applications

There remains a great need for vascular substitutes for small-diameter applications. The use of an elastomeric biodegradable material, enabling acute antithrombogenicity and long-term in vivo remodeling, could be beneficial for this purpose. Conduits (1.3 mm internal diameter) were obtained by electrospinning biodegradable poly(ester urethane)urea (PEUU), and by luminally immobilizing a non-thrombogenic, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer. Platelet adhesion was characterized in vitro after contact with ovine blood. The conduits were implanted as aortic interposition grafts in the rat for 4, 8, 12, and 24 weeks. Surface treatment resulted in a 10-fold decrease in platelet adhesion compared to untreated material. Patency at 8 weeks was 92% for the coated grafts compared to 40% for the non-coated grafts. Histology at 8 and 12 weeks demonstrated formation of cellularized neotissue consisting of aligned collagen and elastin. The lumen of the grafts was confluent with cells qualitatively aligned in the direction of blood flow. Immunohistochemistry suggested the presence of smooth muscle cells in the medial layer of the neotissue and endothelial cells lining the lumen. Mechanically, the grafts were less compliant than rat aortas prior to implantation (4.5 ± 2.0 × 10(-4) mmHg(-1) vs. 14.2 ± 1.1 × 10(-4) mmHg(-1) , respectively), then after 4 weeks in vivo they approximated native values, but subsequently became stiffer again at later time points. The novel coated grafts exhibited promising antithrombogenic and mechanical properties for small-diameter arterial revascularization. Further evaluation in vivo will be required to demonstrate complete remodeling of the graft into a native-like artery.

Beta-radiation reduces the reactivity of extra cellular matrix proteins in intravascular brachytherapy (IVBT), resulting in decreased platelet adhesion

BackgroundIntravascular Brachytherapy as a tool to reduce restenosis is thought to alter vascular wall biology and vessel wall protein function. Platelet accumulation is also indeed important in the genesis of restenosis.We examine the in vitro effects of beta-radiation on the certain vessel wall extra cellular matrix proteins. We hypothesized that vessel wall (proteins) had become less prone to thrombosis. MethodsWe examined platelet adhesion to 20-Gy beta radiation treated extra cellular matrix proteins under flow conditions. Platelet flow adhesion was evaluated or quantified by image analysis, aggregation size analysis using the Watershed program and real-time fluorescence images of thrombus formation. The effect of beta radiation on vWF was further showing by measuring the binding of domain-specific antibodies to radiation treated vWF. Results20-Gy beta radiation significantly decreased platelet adhesion to extra cellular matrix protein; vWF and collagen Type III and had no effect on the adhesion upon fibrinogen and fibronectin. The beta-radiation affected mostly the AI, A2 and A3 domains of the vWF molecule on the surface, whereas the D′–D3 and B–C1 domains on the surface remain unaffected and suggesting a significant decrease in vWF binding capacity to the GPIb, heparin and collagen ligands. ConclusionBeta radiation treatment can alter the reactivity of the certain vessel wall extra cellular matrix proteins, in particular vWF and collagen. The vessel wall may become less prone to platelet adhesion, which results in decrease thrombus formation. It might help to reduce the onset of acute coronary occlusion after the intervention.

Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3-kinase.

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms. SAA dose-dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP-enhanced platelet P-selectin expression and fibrinogen binding, which consequently hampered ADP-induced platelet-leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside-in signaling. Under an arterial shear rate of 1000 s(-1), SAA decreased platelet adhesion on collagen surfaces by approximately 40%. Western blot analysis showed that SAA, like the phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and TGX-221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild-type mice (35 + or - 2 min without SAA and 56 + or - 4 min with SAA; P < 0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in Ldlr(tm1Her) mutant mice (21 + or - 2 min without SAA and 45 + or - 4 min with SAA; P < 0.01). SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.

Modulation of hemocompatibility of polysulfone by polyelectrolyte multilayer films

Polyelectrolyte multilayer (PEM) films have been recently applied to surface modification of biomaterials. Cellular interactions with PEM films consisted of weak polyelectrolytes are greatly affected by the conditions of polyelectrolyte deposition, such as pH of polyelectrolyte solution. Previous studies indicated that the adhesion of several types of mammalian cells to PAH/PAA multilayer films was hindered by low pH and high layer numbers. The objective of this study is to evaluate whether the hemocompatibility of polysulfone can be modulated by deposition of poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) multilayer films. PAH/PAA multilayer films with different layer numbers were assembled onto polysulfone at either pH 2.0 or pH 6.5. The number of platelet adhesion and the morphology of adherent platelets were determined to evaluate hemocompatibility of modified substrates. Compared to non-treat polysulfone, the PEM films developed at pH 2.0 decreased platelet adhesion, while those built at pH 6.5 enhanced platelet deposition. Platelet adhesion was found positively correlated to polyclonal antibodies binding to surface-bound fibrinogen. The extent of platelet spreading was increased with layer numbers of PEM films, suggesting that the adherent platelets on thick PEM films were prone to activation. In conclusion, PAH/PAA films with few layers developed at pH 2.0 possessed better hemocompatibility compared to other substrates.

Inhibitors of Platelet Adhesion

Platelet aggregation, in fundamental terms, is considered a biological end point that contributes to the occurrence of clinical events among patients with advanced atherosclerotic coronary artery disease. Acute coronary syndrome, including non–ST elevation myocardial infarction (NSTEMI) and ST elevation myocardial infarction (STEMI), accounts for upward of 733 000 hospital admissions yearly in the United States.1 The primary pathophysiological mechanism responsible for the majority of acute coronary syndromes is endothelial plaque disruption with and subsequent platelet adhesion, activation, and thrombus formation.2 The end result is formation of thrombus within a coronary artery, leading to subtotal vessel occlusion with NSTEMI and complete occlusion of the artery with STEMI. In this review, we provide a contemporary view of platelet adhesion as a highly coordinated and teleologically conserved process achieved by surface receptors, protein ligands, and matrix proteins operating at the platelet–subendothelium interface. We also discuss drugs in development, including monoclonal antibodies, inhibitory peptides, and oligonucleotides; preclinical data; and, where available, clinical trial results, highlighting the potential translation of fundamental constructs in platelet biology to patient care. Platelets are derived from a hematopoietic bone marrow stem cell precursor.3 Through a highly conserved program of cellular differentiation, this stem cell precursor becomes a megakaryocyte at a rate of approximately 100 000 000 megakaryocytes produced per day.3 Subsequently, each individual megakaryocyte will give rise to approximately 500 platelets via additional developmental steps within the bone marrow that involve a proplatelet intermediate stage.3 Once released into circulation, a mature platelet has an expected life span of 7 to 10 days.3 Platelet maturation and development involve the expression of receptors on the platelet cell surface. These receptors facilitate platelet adhesion and activation, and they promote thrombus development through receptor–ligand interactions, with several ligands expressed on the surface of endothelial cells, within the …

Toward Engineering a Human Neoendothelium with Circulating Progenitor Cells

Tissue-engineered vascular grafts may one day provide a solution to many of the limitations associated with using synthetic vascular grafts. However, identifying a suitable cell source and polymer scaffold to recreate the properties of a native blood vessel remains a challenge. In this work, we assess the feasibility of using endothelial progenitor cells (EPCs) found in circulating blood to generate a functional endothelium on poly(1,8-octanediol-co-citrate) (POC), a biodegradable elastomeric polyester. EPCs were isolated from human blood and biochemically differentiated into endothelial-like cells (HE-like) in vitro. The differentiated cell phenotype and function was confirmed by the appearance of the characteristic endothelial cell (EC) cobblestone morphology and positive staining for EC markers, von Willebrand factor, vascular endothelial cadherin, flk-1, and CD31. In addition, HE-like cells cultured on POC express endothelial nitric oxide synthase at levels comparable to aortic ECs. Furthermore, as with mature endothelial cells, HE-like cell populations show negligible expression of tissue factor. Similarly, HE-like cells produce and secrete prostacyclin and tissue plasminogen activator at levels comparable to venous and aortic ECs. When compared to fibroblast cells, HE-like cells cultured on POC show a decrease in the rate of plasma and whole-blood clot formation as well as a decrease in platelet adhesion. Finally, the data show that HE-like cells can withstand physiological shear stress of 10 dynes/cm(2) when cultured on POC-modified expanded poly(tetrafluoroethylene) vascular grafts. Collectively, these data are the foundation for future clinical studies in the creation of an autologous endothelial cell-seeded vascular graft.

Similar inhibition of platelet adhesion, P-selectin expression and plasma coagulation by ioversol, iodixanol and ioxaglate

Contrast media (CM) are reported to possess both prothrombotic and anticoagulant properties. The mechanisms are not clearly understood, and early reports are contradictory. To study the effects of CM on haemostasis, we analysed the ex vivo effects of ioversol and iodixanol on platelet adhesion and P-selectin expression, and the in vitro effects of ioversol, iodixanol and ioxaglate on platelet adhesion, P-selectin expression and plasma coagulation. A novel enzymatic assay was used to measure platelet adhesion to protein surfaces, and an enzyme-linked immunosorbent assay was used to measure platelet P-selectin surface expression. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were used to measure plasma coagulation. The ex vivo study consisted of blood from 27 outpatients administered ioversol and 9 patients administered iodixanol intravenously. Samples were collected before and 5 min after CM administration. Healthy donors were used for the in vitro studies on the effects of CM. The ex vivo study showed significantly (p<0.05) decreased platelet adhesion and P-selectin expression after administration of ioversol and iodixanol. Adhesion was more affected than P-selectin expression. The in vitro study showed that ioversol, iodixanol and ioxaglate significantly (p<0.05) and dose-dependently (beginning at 3 mg ml(-1)) decreased platelet adhesion and P-selectin expression. APTT and PT were significantly (p<0.01) prolonged at concentrations of 10 mg ml(-1) and 30 mg ml(-1), respectively. In conclusion, ioversol, iodixanol and ioxaglate inhibit platelet adhesion and P-selectin expression, as well as plasma coagulation. Platelets are more sensitive in relation to the inhibiting effect on plasma coagulation.

Serotonin stimulates platelet receptor shedding by tumor necrosis factor-alpha-converting enzyme (ADAM17).

Peripheral serotonin (5-hydroxytryptamine, 5-HT) is transported by platelets and released upon stimulation. In the platelet cytoplasm, 5-HT is transamidated to small GTPases, promoting alpha-granule release and primary hemostasis. We hypothesized that 5-HT could also stimulate platelet receptor shedding after binding to the membrane 5-HT receptor (5-HT2AR). Western blot and flow cytometry were used to determine levels of the adhesion receptor glycoprotein (GP)Ibalpha on platelets or its shed fragment glycocalicin in plasma and serum from wild-type mice, Tph1(-/-) mice lacking peripheral 5-HT, and mice lacking functional tumor necrosis factor-alpha-converting enzyme (TACE, ADAM17). Flow chamber experiments and intravital microscopy were used to examine the adhesive properties of platelets after stimulation of 5-HT2AR. Glycocalicin was significantly reduced in Tph1(-/-) plasma and serum. In isolated platelets, 5-HT induced shedding of GPIbalpha, which was increased to 60% when 5-HT uptake was inhibited by the selective serotonin reuptake inhibitor fluoxetine. Specific 5-HT2AR agonism and antagonism suggested activation of this receptor. The shedding could not be induced in TACE(DeltaZn/DeltaZn) platelets, suggesting that activated TACE mediated the shedding of GPIbalpha. Intracellular signaling involved phosphorylation of p38 mitogen-activated protein kinase rather than G-protein signaling. 5-HT2AR stimulation decreased platelet adhesion to collagen-bound von Willebrand factor under arterial shear (1500 s(-1)) and incorporation into FeCl3-induced thrombi in mesenteric arterioles. Stimulation of 5-HT2AR on platelets induces TACE-mediated shedding of GPIbalpha, the key adhesion molecule under high shear conditions. Our observations demonstrate a new pathway through which 5-HT could modulate cardiovascular disease.