Decreased Platelet Adhesion Research Articles

Biomimetic modification of silicone tubes using sodium nitrite-collagen immobilization accelerates endothelialization.

Biomimetic coatings to increase endothelialization of blood-contacting materials in biomedical devices are promising to improve the biocompatibility of these devices. Although a stable extracellular matrix protein coating on a biomaterial's surface is a prerequisite for endothelial cell attachment, it also stimulates platelet adhesion. Therefore, antithrombotic additives, such as nitric oxide donors, to a stable protein coating might lead to successful endothelialization of a material's surface. We aimed to test whether immobilized bioactive nitrite and acidified nitrite-generating sodium nitrite-collagen conjugate on silicone tubes enhances endothelialization by increasing the number of endothelial cells as well as growth hormone production and by decreasing platelet adhesion. Stable collagen immobilization on acrylic acid-grafted silicone tubes decreased the water contact angle from 102° to 56°. Initial 25 µM sodium nitrite in conjugate resulted in maximal growth hormone production (2.5-fold increase) and endothelial cell number (1.8-fold increase) after 2 days. A 95% confluent endothelial cell monolayer on sodium nitrite-collagen conjugate coating was obtained after 6 days. Maximum (2.7-fold) inhibition of platelet adhesion was reached with initial 500 µM sodium nitrite in conjugate. Our data showing that sodium nitrite-collagen conjugate coating with 25-50 µM sodium nitrite on silicone tubes increases the number of endothelial cells attached and inhibits platelet adhesion suggest that this coating is highly promising for use in blood-contacting parts of biomedical devices. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1311-1321, 2016.

Analytical characterization of the role of phospholipids in platelet adhesion and secretion.

The cellular phospholipid membrane plays an important role in cell function and cell–cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography–tandem electrospray ionization mass spectrometry (UPLC–MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA). The relative enrichments of phosphatidylserine (PS) and phosphatidylethanolamine (PE) were characterized with UPLC–MS/MS, and the effects of the enrichment of these two phospholipids on both platelet secretory behavior and adhesion were examined. Results show that, in fact, both PS and PE influence platelet adhesion and secretion. PS was enriched dramatically and decreased platelet adhesion as well as secretion from δ-, α-, and lysosomal granules. PE enrichment was moderate and increased secretion from platelet lysosomes. These insights illuminate the critical connection between membrane phospholipid character and platelet behavior, and both the methods and results presented herein are likely translatable to other mammalian cell systems.

Development of Decellularized Aortic Valvular Conduit Coated by Heparin–SDF-1α Multilayer

Decellularization can reduce the immune response to aortic valve allograft tissue, but the thrombogenicity and in vivo remolding of these grafts remain controversial. The aim of the present study was to modify the surface of decellularized valvular conduits with heparin-stromal cell-derived factor-1α (SDF-1α) polyelectrolyte multilayer film and to test the thrombogenicity and biocompatibility in vitro and recellularization in vivo. The donor aortic valvular conduits were decellularized with a combination of different detergents and were coated with heparin and SDF-1α alternately to form a polyelectrolyte multilayer. Platelet adhesion and lactate dehydrogenase assay were used to evaluate the antiplatelet property. The adhesion, growth, and migration of bone marrow stem cells (BMSCs) to the scaffolds were assessed. For in vivo studies, the grafts were anastomosed to the infrarenal aorta, without or with heparin and SDF-1α multilayer. Functional assessment was performed by Doppler echography and micro-computed tomography at 2-week and 4-week time points after implantation. Explanted grafts were examined histologically and by immunohistochemistry. In vitro studies demonstrated that the heparin-SDF-1α multilayer film improved hemocompatibility with respect to a substantial reduction of platelet adhesion. BMSCs also achieved better adhesion, proliferation, and migration on the modified graft. For in vivo studies, the grafts in both groups remained patent after 4 weeks, but it was demonstrated that the modified decellularized grafts had better self-endothelialization and recruitment of endothelial progenitor cells. These results indicate that heparin-SDF-1α multilayer film can be used to cover the decellularized aortic valvular graft to decrease platelet adhesion while precipitating regeneration of the decellularized aortic valve allograft in vivo.

Recombinant VWF Promotes Platelet Adhesion Under Shear Stress Dependent on Its Multimer Size

Baxter has developed BAX 111, a recombinant von Willebrand factor (rVWF) currently being evaluated for the treatment of von Willebrand disease based on the results of a phase 3 clinical trial. BAX 111 contains ultra-large molecular weight multimers (ULMWM), which are the most hemostatically active type of multimer. ULMWM demonstrate higher functional activity than the multimers present in commercially available plasma-derived VWF (pdVWF) products in terms of VWF:RCo and VWF:CB activity. To investigate the contribution of VWF multimer size on BAX 111 hemostatic activity, fractions containing distinct portions of VWF multimers were generated and analyzed for their ability to mediate platelet adhesion under shear stress in vitro.rVWF fractions of varying VWF multimer size were purified by size-exclusion chromatography; size distribution was determined using low and high resolution agarose gel electrophoresis. A parallel-plate perfusion chamber system was used to investigate the rVWF-mediated adhesion of platelets from human blood to fibrillar collagen type I under shear stress. For this purpose, a VWF sample of 1.0 IU/mL VWF:Ag was added to healthy donor blood and the time course of surface coverage was determined by microscopy using fluorescent labeled platelets. Multiple pictures were taken every 5 seconds. Perfusion was performed for 3 minutes at +37°C and at a defined wall shear rate of 1500 s-1. Different batches of BAX 111, rVWF fractions of distinct multimer size, and three commercially available pdVWF products with low and high FVIII content were investigated.The endogenous VWF present in human whole blood mediated initial platelet adhesion with approximately 5% surface coverage after 120 seconds of perfusion. Additionally spiking human blood with 1.0 IU/mL of BAX 111 or rVWF fractions of different multimeric size resulted in increased, time-dependent platelet binding. The amount of platelets that became attached to collagen was clearly dependent on the size of rVWF. While highly multimerized BAX 111 effectively promoted platelet binding, lower-sized rVWF fractions containing VWF multimers of lower molecular weight showed decreased platelet adhesion properties. pdVWF products lacking ultra-large molecular weight multimers were less effective in mediating platelet adhesion to collagen, as observed by delayed platelet binding kinetics. After 60 seconds of perfusion, approximately twice the surface coverage was measured for BAX 111 compared to pdVWF products. The amount of FVIII contained in the pdVWF products had no influence on platelet adhesion.These results demonstrate that the improved hemostatic activity of BAX 111 over pdVWF is due to the presence of ULMWM and confirms their importance in mediating intact platelet binding. DisclosuresSchrenk:Baxter Innovations GmbH, Vienna, Austria: Employment. Schreiner:Baxter Innovations GmbH, Vienna, Austria: Employment. Gritsch:Baxter Innovations GmbH, Vienna, Austria: Employment. Turecek:Baxter Innovations GmbH, Vienna, Austria: Employment. Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

Improved in vitro blood compatibility of polycaprolactone nanowire surfaces.

There are a multitude of polymeric materials currently utilized to prepare a variety of blood-contacting implantable medical devices. These devices include tissue grafts, coronary artery and vascular stents, and orthopedic implants. The thrombogenic nature of such materials can cause serious complications in patients, and ultimately lead to functional failure. To date, there is no truly hemocompatible biomaterial surface. Nanostructured surfaces improve cellular interactions but there is a limited amount of information regarding their blood compatibility. In this study, the in vitro blood compatibility of four different surfaces (control, PCL; nanowire, NW; collagen immobilized control, cPCL; collagen immobilized nanowire, cNW) were investigated for their use as interfaces for blood-contacting implants. The results presented here indicate enhanced in vitro blood compatibility of nanowire surfaces compared control surfaces. Although there were no significant differences in leukocyte adhesion, there was a decrease in platelet adhesion on NW surfaces. Scanning electron microscopy images showed a decrease in platelet/leukocyte complexes on cNW surfaces and no apparent complexes were formed on NW surfaces compared to PCL and cPCL surfaces. The increase in these complexes likely contributed to a higher expression of specific markers for platelet and leukocyte activation on PCL and cPCL surfaces. No significant differences were found in contact and complement activation on any surface. Further, thrombin antithrombin complexes were significantly reduced on NW surfaces. A significant increase in hemolysis and fibrinogen adsorption was identified on PCL surfaces likely caused by its hydrophobic surface. This work shows the improved blood-compatibility of nanostructured surfaces, identifying this specific nanoarchitecture as a potential interface for promoting the long-term success of blood-contacting biomaterials.

Modulation of platelet activation and thrombus formation using a pan-PI3K inhibitor S14161.

The phosphatidylinositol 3–kinase (PI3K) signaling pathway is critical in modulating platelet functions. In the present study, we evaluated the effect of S14161, a recently identified pan-class I PI3K inhibitor, on platelet activation and thrombus formation. Results showed that S14161 inhibited human platelet aggregation induced by collagen, thrombin, U46619, and ADP in a dose-dependent manner. Flow cytometric studies showed that S14161 inhibited convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that S14161 decreased platelet adhesion on collagen-coated surface by about 80%. Western blot showed that S14161 inhibited phosphorylation of Akt at both Ser473 and Thr308 sites, and GSK3β at Ser9 in response to collagen, thrombin, or U46619. Comparable studies showed that S14161 has a higher potential bioavailability than LY294002, a prototypical inhibitor of pan-class I PI3K. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2 mg/kg) to male C57BL/6 mice significantly extended the first occlusion time (5.05±0.99 min, n = 9) compared to the vehicle controls (3.72±0.95 min, n = 8) (P<0.05), but did not prolong the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation without significant bleeding tendency and toxicity, and considering its potential higher bioavailability, it may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.

Chondroitin sulfate coatings display low platelet but high endothelial cell adhesive properties favorable for vascular implants.

This study highlights the advantages of chondroitin sulfate (CS) as a sublayer combining selective low-fouling properties, low-platelet adhesion and pro-adhesive properties on endothelial cells, making CS promising for vascular graft applications. These properties were evaluated by comparing CS with well-known low-fouling coatings such as poly(ethylene glycol) (PEG) and carboxymethylated dextran (CMD), which were covalently grafted on primary amine-rich plasma polymerized (LP) films. Protein adsorption studies by quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence measurements showed that CS is as effective as PEG in reducing fibrinogen adsorption (~90% reduction). CS also largely reduced adsorption of bovine serum albumin (BSA) as well as fetal bovine serum (FBS) but to a lower extent than PEG and CMD surfaces (72% vs 85% for BSA and 66% vs 89% for FBS). Whole blood perfusion assays indicated that, while LP surfaces were highly reactive with platelets, PEG, CMD, and CS grafted surfaces drastically decreased platelet adhesion and activation to levels significantly lower than polyethylene terephthalate (PET) surfaces. Finally, while human umbilical vein endothelial cell (HUVEC) adhesion and growth were found to be very limited on PEG and CMD, they were significantly increased on CS compared to that on bare PET and reached similar values as those for tissue culture polystyrene positive controls. Interestingly, HUVEC retention during perfusion with blood was found to be excellent on CS but poor on PET. Overall, our results suggest that the CS surface has the advantage of promoting HUVEC growth and resistance to flow-induced shear stress while preventing fibrinogen and platelet attachment. Such a nonthrombogenic but endothelial-cell adhesive surface is thus promising to limit vascular graft occlusion.

Real time visualization and characterization of platelet deposition under flow onto clinically relevant opaque surfaces

Although the thrombogenic nature of the surfaces of cardiovascular devices is an important aspect of blood biocompatibility, few studies have examined platelet deposition onto opaque materials used for these devices in real time. This is particularly true for the metallic surfaces used in current ventricular assist devices (VADs). Using hemoglobin depleted red blood cells (RBC ghosts) and long working distance optics to visualize platelet deposition, we sought to perform such an evaluation. Fluorescently labeled platelets mixed with human RBC ghosts were perfused across six opaque materials (a titanium alloy (Ti6Al4V), silicon carbide (SiC), alumina (Al2O3, 2-methacryloyloxyethyl phosphorylcholine polymer coated Ti6Al4V (MPC-Ti6Al4V), yttria partially stabilized zirconia (YZTP), and zirconia toughened alumina (ZTA)) for 5 min at wall shear rates of 400 and 1000 s(-1). Ti6Al4V had significantly increased platelet deposition relative to MPC-Ti6Al4V, Al2 O3 , YZTP, and ZTA at both wall shear rates (p < 0.01). For all test surfaces, increasing the wall shear rate produced a trend of decreased platelet adhesion. The described system can be a utilized as a tool for comparative analysis of candidate blood-contacting materials with acute blood contact.

Abstract 56: S14161 Inhibits Platelet Activation and Thrombus Formation via PI3K Signaling Pathway

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is critical in modulating platelet functions. In the present study, we evaluated the effect of S14161, a recently identified pan-class I PI3K inhibitor, on platelet activation and thrombus formation. Results showed that S14161 inhibited human platelet aggregation induced by collagen, thrombin, U46619, and ADP in a dose-dependent manner. For example, the maximal aggregation induced by collagen were decreased by 32.7% (P&lt;0.05), 59.4% (P&lt;0.001) and 89.2% (P&lt;0.001) (IC50=3.79±1.17 μM) when preincubated with 2.5, 5 and 10 μM of S14161, respectively. Interestingly, platelet aggregates were disaggregated after the peak of aggregation induced by ADP when platelets were pretreated with S14161 (10 μM). S14161 also inhibited platelet aggregation induced by GPVI selective ligand convulxin and CRP, PAR1 agonist SFLLRN, and calcium ionophore A23187. Flow cytometric studies showed that S14161 inhibited convulxin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that S14161 decreased platelet adhesion on collagen-coated surface by about 80%. Western blot showed that S14161 inhibited phosphorylation of Akt at both Ser473 and Thr308 sites, and GSK3β at Ser9 in response to collagen, thrombin, or U46619. Additionally, S14161 inhibited ERK1/2 activation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2 mg/kg) to male C57BL/6 mice significantly extended the first occlusion time (5.05±0.99 min, n=9) compared to the vehicle controls (3.72±0.95 min, n=8) (P&lt;0.05), but did not prolong the bleeding time (P&gt;0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.

Biocompatibility of Ir/Ti-oxide coatings: Interaction with platelets, endothelial and smooth muscle cells

Applying surface coatings on a biomedical implant is a promising modification technique which can enhance the implant's biocompatibility via controlling blood constituents- or/and cell-surface interaction. In this study, the influence of composition of IrxTi1−x-oxide coatings (x=0, 0.2, 0.4, 0.6, 0.8, 1) formed on a titanium (Ti) substrate on the responses of platelets, endothelial cells (ECs) and smooth muscle cells (SMCs) was investigated. The results showed that a significant decrease in platelet adhesion and activation was obtained on Ir0.2Ti0.8-oxide and Ir0.4Ti0.6-oxide coatings, rendering the surfaces more blood compatible, in comparison to the control (316L stainless steel, 316L-SS) and other coating compositions. Further, a substantial increase in the EC/SMC surface count ratio after 4h of cell attachment to the Ir0.2Ti0.8-oxide and Ir0.4Ti0.6-oxide coatings, relative to the 316L-SS control and the other coating compositions, indicated high potential of these coatings for the enhancement of surface endothelialization. This indicates the capability of the corresponding coating compositions to promote EC proliferation on the surface, while inhibiting that of SMCs, which is important in cardiovascular stents applications.

Ligand capture and activation of human platelets at monolayer modified gold surfaces.

Blood platelet adhesion is crucial in dictating haemocompatibility of medical implants and in platelet capture in diagnostics. Understanding the role of platelet activation in dictating platelet adhesion at chemically modified interfaces is important but relatively unexplored. Using scanning electron microscopy and confocal fluorescence microscopy a quantitative assessment of capture of blood platelets at self-assembled monolayers and mixed monolayers (SAMs) on gold as a function of the activation status of the platelets was conducted. Single and mixed monolayers were prepared using thiol-functionalized arginine-glycine-aspartic acid (RGD), C-Ahx-GRGDS (Ahx = aminohexanoic acid linker), thiolated poly(ethylene)glycol (PEG-COOH) and 1-octanethiol. When incubated with suspensions of resting platelets, RGD promoted platelet adhesion compared to bare or alkanethiol modified gold. Increasing the alkanethiol ratio in the deposition solution decreased the extent of platelet adhesion. Platelet adhesion increased approximately 3 fold at PEG-COO- modified surfaces compared to RGD-alone. Platelets adhered to RGD or mixed RGD : alkane SAM surfaces were found to be captured in their resting state. In contrast, platelets captured at PEG-COO- SAM surfaces were activated by these substrates. The effect of treating platelets with the chemical activators, Mn2+ or DTT or the physiological activator, thrombin, on the capture efficiency and activation at RGD modified surfaces was also investigated. Mn2+ treated platelets presented similar adhesion to untreated platelets, while surprisingly DTT yielded a very significant decrease in platelet adhesion. And, any platelets that were captured, were in a resting state. Thrombin activated platelets were captured with similar efficiencies as untreated platelets. However, the platelets captured were fully activated. The distinction between capture of chemically and physiologically activated platelet is interesting and likely to originate from differences in the conformation of the integrin induced by each process. Finally, platelet adhesion to each surface could be reversed by incubation with a solution of linear or cyclical RGD or PEG-COO- for the RGD and PEGCOO- surfaces respectively. The specificity of platelet removal confirmed that platelet adhesion at RGD surfaces is occurring through integrin-RGD interactions.

Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures:No relevant conflicts of interest to declare.

Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis

Derived from the root of Panax ginseng C.A.Mey, Panax notoginsenosides (PNS) is a widely used herbal medicine to treat atherothrombotic diseases in Asian medicine. Ginsenoside Rg1 is one of the main compounds responsible for the pharmaceutical actions of PNS. As platelets play pivotal roles in atherothrombogenesis, we therefore studied the effect of Rg1 on platelet activation and its underlying mechanisms. Human platelets are obtained from healthy subjects. Platelet activation and the inhibition of Rg1 were assessed by Born aggregometer, flow cytmetry, flow chamber and western blot. The in vivo thrombosis model was induced by 10% FeCl3 on mesenteric arterioles of wild type B57/b6 mice. Rg1 significantly inhibited platelet aggregation induced by thrombin, ADP, collagen and U46619, e.g., aggregation rate stimulated by 0.1UmL(-1) thrombin was decreased 46% by Rg1. Rg1 also reduced thrombin (0.1UmL(-1))-enhanced fibrinogen binding and P-selectin expression of single platelet by 81% and 66%, respectively. Rg1 affected αIIbβ3-mediated outside-in signaling as demonstrated by diminished platelet spreading on immobilized fibrinogen. Rg1 also decreased the rate of clot retraction in platelet rich plasma. Furthermore, Rg1 decreased platelet adhesion on collagen surface under a shear rate correlated to the arterial flow (1000s(-1)) by approximately 70%. Western blot showed that Rg1 potently inhibited ERK phosphrylation. The in vitro findings were further evaluated in the mouse model of in vivo arterial thrombosis, and Rg1 was found to prolong the mesenteric arterial occlusion time (34.9±4.1min without and 64.3±4.9min with Rg1; p<0.01). Rg1 inhibits platelet activation via the inhibition of ERK pathway, and attenuates arterial thrombus formation in vivo.

An anti-CD34 antibody-functionalized clinical-grade POSS-PCU nanocomposite polymer for cardiovascular stent coating applications: a preliminary assessment of endothelial progenitor cell capture and hemocompatibility.

In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs) from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs) were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU (5.0±1.0 vs. 1.7±0.6, p>0.05). However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05), as well as decreased platelet adhesion (76.8±7.8 vs. 8.4±0.7, p<0.05) and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34+ cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization.

Measurement of the effect of platelet activators and heparin on adhesion and spreading of human platelets using impedimetry

Purpose: Thrombogenesis has been recently focused in cardiology. Numerous methods exist for the assessment of platelet aggregation, but there is no well standardized and generally accepted technique for the isolated measurement of platelet adhesion. Impedimetry is a real-time technique for the dynamic measurement of cell adhesion, proliferation, viability and cytotoxicity. In this work we propose this novel method as a candidate for the measurement of platelet adhesion. Methods: The effect of activators (epinephrine, ADP and collagen) and unfractionated heparin on human platelets isolated from healthy individuals under no current drug therapy was investigated. For these experiments the xCELLigence SP system was used, where the platelets adhere to golden electrodes placed at the bottom of the wells. The change of electric impedance on the electrodes is in direct correlation with the number and the level of spreading of the adhering platelets. For the measurement of adhesion, platelets were introduced into wells that the drug had already been added to. For the measurement of spreading, platelets were seeded onto the electrodes and the drug was administered afterwards. Results: Both epinephrine and ADP increased platelet adhesion, 10 μM epinephrine caused 5.42 fold (p=0.002), 5 μM epinephrine caused 5.34 fold (p<0,001), 10 μM ADP caused 11.9 fold (p<0.001) and 5 μM ADP caused 9.89 fold (p<0.001) increase in slope; collagen showed no significant effect on platelet adhesion. Unfractionated heparin decreased platelet adhesion on pharmacological concentration, 250 IU/mL unfractionated heparin caused 0.48 fold (p=0.001) decrease in slope, on lower concentrations there was no significant effect. Platelet spreading was increased by ADP: 10 μM ADP caused 16.12 fold (p=0.011) and 5 μM ADP caused 10.89 fold (p=0.007) increase in slope. Epinephrine and collagen had inhibitory effects on the spreading of platelets. Conclusions: Platelet adhesion proved to be qualitatively and quantitatively well detectable by impedimetry. Characteristic slopes of data measured have a good correlation with the effect of substances used as laboratory reference and drugs of medical therapy. The novel method is offered as a useful technique for the more detailed evaluation of platelet function and the following of antithrombotic drug therapies.

The Effect of Heparin-VEGF Multilayer on the Biocompatibility of Decellularized Aortic Valve with Platelet and Endothelial Progenitor Cells

The application of polyelectrolyte multilayer films is a new, versatile approach to surface modification of decellularized tissue, which has the potential to greatly enhance the functionality of engineered tissue constructs derived from decellularized organs. In the present study, we test the hypothesis that Heparin- vascular endothelial growth factor (VEGF) multilayer film can not only act as an antithrombotic coating reagent, but also induce proliferation of endothelial progenitor cells (EPCs) on the decellularized aortic heart valve. SEM demonstrated the adhesion and geometric deformation of platelets. The quantitative assay of platelet activation was determined by measuring the production of soluble P-selectin. Binding and subsequent release of heparin and VEGF from valve leaflets were assessed qualitatively by laser confocal scanning microscopy and quantitatively by ELISA methods. Human blood derived EPCs were cultured and the adhesion and growth of EPCs on the surface modified valvular scaffolds were assessed. The results showed that Heparin-VEGF multilayer film improved decellularized valve haemocompatibility with respect to a substantial reduction of platelet adhesion. Release of VEGF from the decellularized heart valve leaflets at physiological conditions was sustained over 5 days. In vitro biological tests demonstrated that EPCs achieved better adhesion, proliferation and migration on the coatings with Heparin-VEGF multilayer film. Combined, these results indicate that Heparin-VEGF multilayer film could be used to cover the decellularized porcine aortic valve to decrease platelet adhesion while exhibiting excellent EPCs biocompatibility.

Evaluation of Platelet Adhesion Capacity and Its Hematological and Coagulation Correlates in Patients with Liver Cirrhosis

Purpose: Thrombocytopenia is a common hematological abnormality in patients with liver cirrhosis. Although thrombocytopenia may theoretically be associated with increased bleeding risk, it is often counterbalanced by elevated von Willebrand factor (vWF) levels and high factor VIII/protein C (FVIII/PC) ratios. However, cirrhotic patients may also display abnormal platelet function and platelet adhesion capacity, and this may increase their bleeding diathesis. Methods: In this study, our aim was to evaluate the platelet adhesion capacity and its hematological and coagulation correlates in patients with liver cirrhosis. We assessed platelet adhesion capacity by means of Platelet Function Analyzer-100 (PFA-100TM) in a series of 31 non-azotemic patients with cirrhosis. In these patients, we also assessed alanine aminotransferase (ALT), platelet count, hematocrit level, FVIII, vWF, PC, antithrombin III (ATIII) and correlated their results to those of PFA-100TM. Results were expressed as closure time (CT, in seconds) for the collagen-epinephrin cartridge (normal values, 85-165 sec), and PFA-100TM CT values >300 sec were considered non-closure. Data are shown as median values; comparisons between groups were carried out by means of Mann-Whitney U-test or Fisher's exact test. Correlation between variables was estimated using correlation coefficient. Results: PFA-100TM CT results were abnormal (i.e., CT >165 sec.) in 22 patients (71.0%), and non-closure (i.e., CT >300 sec.) was observed in 5 patients (16.1%). Patients with abnormal CT had significantly lower platelet counts (91x109/L versus 61x109/L; P=0.048), lower hematocrit levels (42.2% versus 36.8%; P=0.045), and higher ALT levels (83U/L versus 32U/L; P=0.007) as compared to patients with normal PFA-100TM results. Among coagulation factors, vWF were above the upper limit of normal in all patients (>150%), while FVIII, PC, and ATIII were not associated with abnormal PFA-100TM results. Serum albumin, bilirubin, and creatinine were not significantly different between patients with normal and abnormal PFA-100TM results. PFA-100TM CT showed a weak, inverse correlation with platelet count (r=-0.3671, P=0.042), although there was no difference in the proportion of patients with abnormal PFA-100TM CT between patients with platelet counts above or below 100x109/L. Conclusion: In patients with liver cirrhosis, there is evidence of a decreased platelet adhesion capacity. Decreased platelet adhesion capacity is associated with decreased platelet count and hematocrit level, and elevated ALT levels.

Fabrication of mesenchymal stem cells-integrated vascular constructs mimicking multiple properties of the native blood vessels

Mesenchymal stem cells (MSCs)-populated small diameter (6 mm) vascular constructs were fabricated. The constructs mimicked the native vessels in multiple levels, i.e. having similar structure and morphology to that of the extracellular matrix in the native blood vessels; recapitulating mechanical properties such as compliance and burst pressure of the native blood vessels; simulating the highly cellularized nature of the native blood vessels; and having an antithrombogenic lumen. The constructs were fabricated by simultaneously assembling poly(ester carbonate urethane) urea nanofibers and MSCs in an electrical field. The nanofibers had a diameter similar to that of the collagen and elastin fibers in the native blood vessels. MSCs were distributed evenly in the constructs. The constructs were highly cellularized when the cell loading density was exceeded 6 million/ml. The vascular constructs were strong and flexible with breaking strains of 144–202%, tensile strengths of 0.80–1.29 MPa, compliances of 13.23–21.96 × 10−4 mmHg−1, stiffness indexes of 7.3–9.8, and burst pressures greater than 1700 mmHg. These mechanical properties were similar to those of the native blood vessels. In vitro platelet deposition experiments showed that platelet adhesion was remarkably decreased in the MSCs-populated constructs compared to that in the construct without MSCs. An increase in MSC density in the constructs further decreased platelet adhesion. When cultured in a spinner flask, MSCs maintained their mitochondria viability and cell number during a two-week culture period, as confirmed by MTT and dsDNA assays. These vascular constructs may hold the potential to regenerate functional small diameter vessels for cardiovascular tissue repair.

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.