Platelet Adhesion Measurements Research Articles

Facile method for covalent-bonding coating of crosslinked silicone layer onto poly(ester-urethane) surface to improve tensile properties and hemocompatibility

In this paper, a facile method was provided to prepare covalent-bonding coating of crosslinked silicone (CLS) layer onto the poly(ester-urethane) (PEU) surface for improving its properties. The PEU containing well-ordered hard segments was prepared by the direct chain extension of poly(ε-caprolactone) with diurethane diisocyanate (HBH), and the PEU films were acquired via solvent evaporation. The CLS layer was covalently coated onto the film surface by a three-step chemical treatment: the -NCO groups were first introduced onto the PEU film surface by chemical treatment of the film with hexamethylene diisocyanate; the free NCO groups were subsequently coupled with the NH2 groups of γ-aminopropyltriethoxysilane to fix the active alkoxysilane groups on the surface; finally, the alkoxysilane groups self-crosslinked to produce a CLS coating on the PEU surface (PEU-Si). FT-IR, XPS and SEM were used to characterize the modified surface. The studies on physicochemical properties of the films manifested that the PEU-Si film exhibited higher thermal stability, excellent tensile properties (broken elongation: 1389 %; ultimate strength: 41.6 MPa), and slower in vitro hydrolytic degradation (only 16.0 % weight loss after 18 months of degradation) than PEU film. The hemocompatibility evaluation of film surface was performed by protein adsorption and platelet adhesion measurements, and the results showed that the CLS-coated surface strongly inhibited protein adsorption (2.92 μg/cm2) and platelet adhesion (2,129 platelets/mm2). The PEU-Si exhibited excellent tensile properties, slow degradability, and superior surface hemocompatibility, indicating promise as a long-term implant.

Differentiation of Patients with Symptomatic Low von Willebrand Factor from Those with Asymptomatic Low von Willebrand Factor.

Accurate diagnosis of symptomatic low von Willebrand factor (VWF) remains a major challenge in von Willebrand disease (VWD). However, present tests do not adequately take into account flow forces that, at very high shear rates, reveal a weakness in the VWF-platelet glycoprotein glycoprotein Ib bond in normal subjects. The degree of this weakness is greater in symptomatic, but not asymptomatic, low VWF. The aim of this study is to distinguish patients with symptomatic low VWF (levels in the 30-50 IU/dL range) from those with asymptomatic low VWF and normal subjects. We measured platelet adhesion (PA)/aggregation in our novel microfluidic flow system that permits real-time assessment of PA (surface coverage) and PA/aggregation (V, aggregate volume) using epifluorescence digital videomicroscopy in flowing noncitrated whole blood at 4,000 second-1. Blood samples from 24 low VWF patients and 15 normal subjects were collected into plastic tubes containing 4 U/mL enoxaparin. MetaMorph software was used to quantify rates of PA and V increase. Rates of PA increase showed a bimodal distribution, with values for 16/24 patients (Group I) all below the 2.5th percentile of normal, and values for 8/24 patients (Group II) similar to controls. Bleeding scores (mean ± standard error) were 5.50 ± 0.45 versus 2.75 ± 0.45 (p = 0.00077), and 10 clinically significant bleeding events were observed in seven versus zero (p = 0.0295) Group I and Group II subjects, respectively. The present approach may offer a definitive means to distinguish symptomatic low VWF from either asymptomatic low VWF or normal controls.

Time-resolved MIET measurements of blood platelet spreading and adhesion

Human blood platelets are non-nucleated fragments of megakaryocytes and of high importance for early hemostasis. To form a blood clot, platelets adhere to the blood vessel wall, spread and attract other platelets. Despite the importance for biomedicine, the exact mechanism of platelet spreading and adhesion to surfaces remains elusive. Here, we employ metal-induced energy transfer (MIET) imaging with a leaflet-specific fluorescent membrane probe to quantitatively determine, with nanometer resolution and in a time-resolved manner, the height profile of the basal and the apical platelet membrane above a rigid substrate during platelet spreading. We observe areas, where the platelet membrane approaches the substrate particularly closely and these areas are stable on a time scale of minutes. Time-resolved MIET measurements reveal distinct behaviors of the outermost rim and the central part of the platelets, respectively. Our findings quantify platelet adhesion and spreading and improve our understanding of early steps in blood clotting. Furthermore, the results of this study demonstrate the potential of MIET for simultaneous imaging of two close-by membranes and thus three-dimensional reconstruction of the cell shape.

Design of comb-like poly(2-methyl-2-oxazoline) and its rapid co-deposition with dopamine for the study of antifouling properties

In this work, the comb-like poly(2-methyl-2-oxazoline) copolymer, poly(2-aminoethyl methacrylate-random-poly(2-methyl-2-oxazoline) (PMOXA-r-AEMA, PMA) is synthesized, and the CuSO4/H2O2-triggered dopamine/PMA co-deposition process is investigated. Ellipsometry, water contact angle (WCA), and X-ray photoelectron spectroscopy (XPS) are used to characterize the thickness, hydrophilicity, and surface composition of PMA-based coatings. PMA is facilely anchored to substrates within 60 min with the assistance of dopamine/polydopamine triggered by CuSO4/H2O2, and the coating thickness can achieve about 13 nm. Anti-protein adsorption and anti-blood platelet adhesion measurements are also studied to verify their antifouling properties. The adsorption capacity of FITC-BSA can be reduced to 5% of the original surface, and PMA-based coatings present excellent protein-resistant properties (∼95% reduction relative to gold surface) according to surface plasmon resonance (SPR) measurement, and it can resist adhesion of almost all blood platelets. Moreover, it is applied to a capillary inner wall for surface modification. An alkaline protein mixture and egg white proteins are successfully separated, and present excellent stability. The relative standard deviation (RSD) of migration time is lower than 0.71%. The practicability of this promising PMA-based coatings with this preparation strategy can be used for further proteomics analysis.

P721Shear stress induced unfolding of von willebrand factor may be involved in the premature development of myocardial infarction

Abstract Background Normally, von Willebrand factor (vWF) becomes highly reactive with platelets upon unfolding into a fibrillar conformation at critical shear rate (more than 5000 s–1), that may occur in stenotic arteries. At shear rates below critical value (1200–1300 s–1), which occur in intact coronary arteries, normally there is no conformational rearrangement of vWF. Pathologic unfolding of vWF at shear rates below critical value may increase a risk of the development of coronary thrombosis. There is little information on the role of shear stress induced conformational rearrangement of vWF in the development of myocardial infarction in young individuals. Purpose To investigate vWF-dependent platelet adhesion of patients with premature myocardial infarction at shear rates below critical value (1200–1300 s–1). Methods Using a microfluidic system, we measured platelet adhesion to a fibrinogen-coated optical surface at shear rates of 1200–1300 s–1 during 10 minutes. We assessed platelet-rich plasma of 8 male persons 40–52 years old, who had previous myocardial infarction at the age of 34–39. The control group comprised 6 healthy male volunteers 30–55 years old. We compared the intensity of scattered laser light measured in volts (V) at 10th minute. To study vWF-dependent platelet adhesion, we blocked GPIb receptor with monoclonal antibody to inhibit platelet interaction with vWF. To compare the intensity of vWF-dependent platelet adhesion with normally occurring adhesion to fibrinogen, we blocked GPIIb/IIIa receptor with monoclonal antibody. Results The inhibition of GPIb vWF-receptor decreased platelet adhesion to fibrinogen surface at shear rates of 1200–1300 s–1 by 17.8±4.7% in healthy volunteers and by 92±2.8% in persons with premature myocardial infarction (p<0.05). Inhibition of GPIIb/IIIa receptor decreased platelet adhesion by 91.5±3.8% in healthy volunteers and by 97.3±3.2% in persons with premature myocardial infarction. Conclusion Pathologic unfolding of vWF at shear rates below critical value may be involved in the development of premature myocardial infarction. Acknowledgement/Funding This work was supported by the grant of the Russian Science Foundation (project #16-15-10098)

Physicochemical evaluation and in vitro hemocompatibility study on nanoporous hydroxyapatite.

Hydroxyapatite is an ideal biomaterial for bone tissue engineering due to its biocompatibility and hemocompatibility which have been widely studied by many researchers. The incorporation of nanoporosity into hydroxyapatite could transform the biomaterial into an effective adsorbent for uremic toxins removal especially in artificial kidney system. However, the effect of nanoporosity incorporation on the hemocompatibility of hydroxyapatite has yet to be answered. In this study, nanoporous hydroxyapatite was synthesized using hydrothermal technique and its hemocompatibility was determined. Non-ionic surfactants were used as soft templates to create porosity in the hydroxyapatite. The presence of pure hydroxyapatite phase in the synthesized samples is validated by X-ray diffraction analysis and Fourier transform infrared spectroscopy. The TEM images show that the hydroxyapatite formed rod-like particles with the length of 21-90 nm and diameter of 11-70 nm. The hydroxyapatite samples exhibit BET surface area of 33-45 m2 g-1 and pore volume of 0.35-0.44 cm3 g-1. The hemocompatibility of the hydroxyapatite was determined via hemolysis test, platelet adhesion, platelet activation and blood clotting time measurement. The nanoporous hydroxyapatite shows less than 5% hemolysis, suggesting that the sample is highly hemocompatible. There is no activation and morphological change observed on the platelets adhered onto the hydroxyapatite. The blood clotting time demonstrates that the blood incubated with the hydroxyapatite did not coagulate. This study summarizes that the synthesized nanoporous hydroxyapatite is a highly hemocompatible biomaterial and could potentially be utilized in biomedical applications.

In Vitro Measurement and Modeling of Platelet Adhesion on VWF-Coated Surfaces in Channel Flow

The process of platelet adhesion is initiated by glycoprotein (GP)Ib and GPIIbIIIa receptors on the platelet surface binding with von Willebrand factor on the vascular walls. This initial adhesion and detachment of a single platelet is a complex process that involves multiple bonds forming and breaking and is strongly influenced by the surrounding blood-flow environment. In addition to bond-level kinetics, external factors such as shear rate, hematocrit, and GPIb and GPIIbIIIa receptor densities have also been identified as influencing the platelet-level rate constants in separate studies, but this still leaves a gap in understanding between these two length scales. In this study, we investigate the fundamental relationship of the dynamics of platelet adhesion, including these interrelating factors, using a coherent strategy. We build a, to our knowledge, novel and computationally efficient multiscale model accounting for multibond kinetics and hydrodynamic effects due to the flow of a cellular suspension. The model predictions of platelet-level kinetics are verified by our microfluidic experiments, which systematically investigate the role of each external factor on platelet adhesion in an in vitro setting. We derive quantitative formulas describing how the rates of platelet adhesion, translocation, and detachment are defined by the molecular-level kinetic constants, the local platelet concentration near the reactive surface determined by red-blood-cell migration, the platelet effective reactive area due to its tumbling motion, and the platelet surface receptor density. Furthermore, if any of these aspects involved have abnormalities, e.g., in a disease condition, our findings also have clinical relevance in predicting the resulting change in the adhesion dynamics, which is essential to hemostasis and thrombosis.

Platelet impedance adhesiometry: A novel technique for the measurement of platelet adhesion and spreading.

Thrombogenesis plays an important role in today's morbidity and mortality. Antithrombotics are among the most frequently prescribed drugs. Thorough knowledge of platelet function is needed for optimal clinical care. Platelet adhesion is a separate subprocess of platelet thrombus formation; still, no well-standardized technique for the isolated measurement of platelet adhesion exists. Impedimetry is one of the most reliable, state-of-art techniques to analyze cell adhesion, proliferation, viability, and cytotoxicity. We propose impedimetry as a feasible novel method for the isolated measurement of 2 significant platelet functions: adhesion and spreading. Laboratory reference platelet agonists (epinephrine, ADP, and collagen) were applied to characterize platelet functions by impedimetry using the xCELLigence SP system. Platelet samples were obtained from 20 healthy patients under no drug therapy. Standard laboratory parameters and clinical patient history were also analyzed. Epinephrine and ADP increased platelet adhesion in a concentration-dependent manner, while collagen tended to have a negative effect. Serum sodium and calcium levels and age had a negative correlation with platelet adhesion induced by epinephrine and ADP, while increased immunoreactivity connected with allergic diseases was associated with increased platelet adhesion induced by epinephrine and ADP. ADP increased platelet spreading in a concentration-dependent manner. Impedimetry proved to be a useful and sensitive method for the qualitative and quantitated measurement of platelet adhesion, even differentiating between subgroups of a healthy population. This novel technique is offered as an important method in the further investigation of platelet function.

In vitro measurement of platelet adhesion to intact endothelial cells under low shear conditions.

Prediction of thrombus formation at intact arterial walls under low shear flow conditions is clinically important particularly for better prognoses of embolisation in cerebral aneurysms. Although a new mathematical model for this purpose is necessary, little quantitative information has been known about platelet adhesion to intact endothelial cells. The objective of this study is to measure the number of platelets adhering to intact endothelial cells with a focus upon the influence of the shear rate. Endothelial cells disseminated in μ-slides were exposed to swine whole blood at different shear rates. Adenosine diphosphate (ADP) was used as an agonist. Adherent platelets were counted by means of scanning electron microscopy. At an ADP concentration of 1µM, 20.8 ± 3.1 platelets per 900µm2 were observed after 30-minute perfusion at a shear rate of 0.8s-1 whereas only 3.0 ± 1.4 per 900µm2 at 16.8s-1. The number of adherent platelets is determined by a balance between the shear and the degree of stimulation by the agonist. At an ADP concentration of 1µM, a limit to the shear rate at which platelets can adhere to intact endothelial cells is considered to be slightly higher than 16.8s-1.

Preparation and platelet adhesion of chitosan/PVA nanofibrous membrane

In this study, chitosan (CTS) dissolved in acetic acid was electrospun together with aqueous poly(vinyl alcohol) solution (PVA) to prepare membranes (CTS/PVA) with nanofibers. The morphology of the membranes varied with the electrospinning conditions. High polymer concentration, high distance or high voltage was facilitative in obtaining a membrane with nanofibers. A CTS/PVA membrane with evenly distributed nanofibers (50 nm dia.) was obtained under the following conditions: 4·5 g CTS in 55 ml 27% acetic acid, 0·4 g PVA in 5 ml water (H2O), 0·1 g sodium chloride (NaCl), electrospinning voltage of 40 kV and distance of 25 cm from the injector to the collector. The water contact angle of CTS/PVA membranes increased after cross-linking with glutaraldehyde steam. Platelet adhesion measurement expressed that CTS/PVA with only nanofibers had no platelet adhesion, indicating good blood compatibility. Cross-linked CTS/PVA mats were stable while in contact with phosphate-buffered saline solution.

Clovamide and clovamide-rich extracts of three Trifolium species as antioxidants and moderate antiplatelet agents in vitro

This in vitro study provides a new insight into the phytochemical, antioxidant, antiplatelet and cytotoxic profiles of commercial trans-clovamide and clovamide-rich extracts, isolated from aerial parts of three Trifolium species (i.e. T. clypeatum L., T. obscurum Savi and T. squarrosum L.).Under inorganic experimental conditions, the EC50 for the investigated substances varied between 4.87 (clovamide) and 7.91 μg/ml (T. clypeatum) according to the DPPH• assay. The most effective 1 mM ONOO− scavenger was clovamide (IC50 = 19.29 μg/ml), and the weakest was the T. obsucurum extract (IC50 = 41.79 μg/ml). The antioxidant action of the examined substances (1–50 μg/ml) was also evaluated in blood plasma (under 100 μM ONOO−-induced oxidative stress) using 3-nitrotyrosine, protein thiol groups, lipid hydroperoxides and thiobarbituric acid-reactive substances as biomarkers. All examined substances were more effective in limiting the oxidative damage to blood plasma components than the nitrative damage. Their anti-nitrative action was statistically significant only at the highest concentration (50 μg/ml). Measurements of platelet adhesion and aggregation found the compounds to have moderate antiplatelet properties of up to 20% inhibition of platelet adhesion at concentrations of 1–5 μg/ml. Additionally, clovamide and Trifolium extracts demonstrated no cytotoxicity towards blood platelets or peripheral blood mononuclear cells.

Hemocompatibility Evaluation of Polyurethane Film with Surface-Grafted Sugar- Based Amphipathic Compounds

Sugar-based amphipathic compounds (BA-CnAG) were successfully prepared. Polyurethane (PU) was grafted with glycidyl methacrylate (GMA) by the means of UV irradiation, and further modified with the BA-CnAG based on the ring opening of the epoxy groups. The surface graft polymerization was confirmed by ATR-FTIR and XPS. Water contact angle, protein adsorption, and platelet adhesion measurements were used to evaluate the hydrophilicity and hemocompatibility of the films. The results demonstrated that amphiphilic surfactant-containing polymer surfaces presented protein-resistant behavior and anti-platelet adhesion after functionalization with BA-CnAG. Besides, the hemocompatibility of the modified surface deteriorated as the length of hydrophobic chain of BA-CnAG increased.

Development of a platelet adhesion transport equation for a computational thrombosis model

Thrombosis is a significant issue for cardiovascular device development and use. While thrombosis models are available, very few are device-related and none have been thoroughly validated experimentally. Here, we introduce a surface adherent platelet transport equation into a continuum model to account for the biomaterial interface/blood interaction. Using a rotating disc system and polyurethane-urea material, we characterize steady and pulsatile flow fields using laser Doppler velocimetry. In vitro measurements of platelet adhesion are used in combination with the LDV data to provide further experimental validation. The rotating disc system is computationally studied using the device-induced thrombosis model with the surface platelet adherent transport equation. The results indicate that the flow field is in excellent agreement to the experimental LDV data and that the platelet adhesion simulations are in good agreement with the in vitro platelet data. These results provide good evidence that this transport equation can be used to express the relationship between blood and a biomaterial if the correct platelet adhesion characteristics are known for the biomaterial. Further validation is necessary with other materials.

Zwitterionic modification of polyurethane membranes for enhancing the anti-fouling property

Polyurethane (PU) is a biopolymer that has been commonly used for biomedical applications. However, the biofouling phenomenon on the hydrophobic PU surface is one of the crucial issues that embarrassing its applications. Here, we report a facile & efficient approach to improve the anti-biofouling ability of the PU substrates. Active residues were firstly generated on the PU surface by using the low temperature air-plasma treatment, promoting the immobilization of the atom transfer radical polymerization (ATRP) initiators on the surface. Then, three types of zwitterionic polymer brushes, as well as PEG brushes, have been fabricated on the PU substrates through surface-initiated ATRP (SI-ATRP). Robust surface characterizations that capable of revealing the surface chemistry (including X-ray photoelectron spectroscopy (XPS) and wettability tests), and antifouling evaluations of the PU substrates (protein adsorption, platelet adhesion, and cell adhesion measurements) were performed. Results showed that three types of zwitterionic brushes have been successful grafted on the PU surface, respectively. And the three types of zwitterionic brushes, in general, significantly inhibited the protein adsorption, the platelet adhesion, and the cell adhesion on the PU surface, endowing a significantly improved anti-fouling ability to the PU substrates. Furthermore, we found that this facial zwitterionic surface modification did not compromise the mechanical property of the PU substrates. This strategy could be easily exploited to PU-based biomaterials to improve their performance in many applications.

Preparation and hemocompatibility of electrospun O‐carboxymethyl chitosan/PVA nanofibers

ABSTRACTChitosan was deacetylated and carboxymethylated to prepare O‐carboxymethyl chitosan (CMC) for further electrospinning. CMC was characterized using FTIR, NMR, and chemical titration, indicating a degree of carboxymethylation of 51.4%. CMC was electrospun together with poly(vinyl alcohol) (PVA) to prepare membranes composed of nanofibers. The electrospinning conditions were optimized. The CMC/PVA membrane obtained at the conditions of 15.2 g/mL CMC 50 mL, 8 g/mL PVA 5 mL, 25 kV, and a distance of 23 cm, had nanofibers without beads, with diameters of 70–200 nm. The mats were crosslinked by glutaraldehyde before platelet adhesion measurement. The nanofibrous structure remained after crosslinking while the wettability decreased. CMC/PVA mats with higher CMC amount and fewer beads, had fewer adherent platelets and less platelets aggregation showing better hemocompatibility. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43565.

Versatile and Rapid Postfunctionalization from Cyclodextrin Modified Host Polymeric Membrane Substrate.

Surface modification has long been of great interest to impart desired functionalities to the bioimplants. However, due to the limitations of recent technologies in surface modification, it is highly desirable to explore novel protocols, which can advantageously and efficiently endow the inert material surfaces with versatile biofunctionalities. Herein, to achieve versatile and rapid postfunctionalization of polymeric membrane, we demonstrate a new strategy for the fabrication of β-cyclodextrin (β-CD) modified host membrane substrate that can recognize a series of well-designed guest macromolecules. The surface assembly procedure was driven by the host-guest interaction between adamantane (Ad) and β-CD. β-CD immobilized host membrane was fabricated via two steps: (1) epoxy groups enriched poly(ether sulfone) (PES) membrane was first prepared via in situ cross-linking polymerization and subsequently phase separation; (2) mono-6-deoxy-6-ethylenediamine-β-CD (EDA-β-CD) was then anchored onto the surface of the epoxy functionalized PES membrane to obtain PES-CD. Subsequently, three types of Ad-terminated polymers, including Ad-poly(styrenesulfonate-co-sodium acrylate) (Ad-PSA), Ad-methoxypoly(ethylene glycol) (Ad-PEG), and Ad-poly(methyl chloride-quaternized 2-(dimethylamino)ethyl methacrylate (Ad-PMT), were separately assembled onto the β-CD immobilized surfaces to endow the membranes with anticoagulant, antifouling, and antibacterial capability, respectively. Activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) measurements were carried out to explore the anticoagulant activity. The antifouling capability was evaluated via protein adsorption and platelet adhesion measurements. Moreover, Staphyllococcous aureus (S. aureus) was selected as model bacteria to evaluate the antibacterial ability of the functionalized membranes. The results indicated that well-regulated blood compatibility, antifouling capability, and bactericidal activity could be achieved by the proposed rapid postfunctionalization on polymeric membranes. This approach of versatile and rapid postfunctionalization is promising for the preparation of multifunctional polymeric membrane materials to meet with various demands for the further applications.

Synthesis of newly fluorinated thermoplastic polyurethane elastomers and their blood compatibility

In this paper, newly polyether polyol containing fluorine in the side chain, 6,9-polyoctafluorine hexanoic ether tetradecane glycol (PFEG) was successfully synthesized via cationic ring-opening polymerization method. By means of one-step bulk polymerization method, its mixture with polytetramethylene ether glycol (PTMEG) was used as soft segment to fabricate fluorinated thermoplastic polyurethane (FTPU) elastomers, which were further characterized by SEM, 1HNMR, FTIR, XRD, contact angle, tensile and thermal stability measurements. Hemocompatibility in vitro was evaluated through protein adsorption and platelet adhesion measurements, while cytocompatibility was assessed by MTT assay and cell adhesion. Results showed that the as-synthesized FTPU elastomers have satisfactory blood compatibility, cytocompatibility, mechanical and thermal stability properties.

Surface modification of polyurethane via covalent immobilization of sugar-based trisiloxane surfactants

Sugar-based trisiloxane surfactants (N-siloxanegluconamides and N-siloxanelactobionamides, denoted as Si(3)NGA and Si(3)NLA) with the amphiphilic structures were successfully prepared. Polyurethane (PU) was grafted with 2-carboxyethyl acrylate (CEA) by the means of UV-induced graft polymerization, and further introduction of the Si(3)NGA and Si(3)NLA by conjugating the amino groups and acid groups. The surface graft polymerization was confirmed by attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. Water contact angle, protein adsorption, and platelet adhesion measurements were used to evaluate the hydrophilicity and hemocompatibility of the films. It was found that the Si(3)NGA-modified substrates obviously suppressed protein adsorption and platelet adhesion, while Si(3)NLA-modified substrates exhibited a higher protein adsorption and a coagulating performance of blood.

Platelet GpIba binding to von Willebrand Factor under fluid shear:contributions of the D′D3-domain, A1-domain flanking peptide and O-linked glycans.

BackgroundVon Willebrand Factor (VWF) A1‐domain binding to platelet receptor GpIbα is an important fluid‐shear dependent interaction that regulates both soluble VWF binding to platelets, and platelet tethering onto immobilized VWF. We evaluated the roles of different structural elements at the N‐terminus of the A1‐domain in regulating shear dependent platelet binding. Specifically, the focus was on the VWF D′D3‐domain, A1‐domain N‐terminal flanking peptide (NFP), and O‐glycans on this peptide.Methods and ResultsFull‐length dimeric VWF (ΔPro‐VWF), dimeric VWF lacking the D′D3 domain (ΔD′D3‐VWF), and ΔD′D3‐VWF variants lacking either the NFP (ΔD′D3NFP─‐VWF) or just O‐glycans on this peptide (ΔD′D3OG─‐VWF) were expressed. Monomeric VWF‐A1 and D′D3‐A1 were also produced. In ELISA, the apparent dissociation constant (KD) of soluble ΔPro‐VWF binding to immobilized GpIbα (KD≈100 nmol/L) was 50‐ to 100‐fold higher than other proteins lacking the D′D3 domain (KD~0.7 to 2.5 nmol/L). Additionally, in surface plasmon resonance studies, the on‐rate of D′D3‐A1 binding to immobilized GpIbα (kon=1.8±0.4×104 (mol/L)−1·s−1; KD=1.7 μmol/L) was reduced compared with the single VWF‐A1 domain (kon=5.1±0.4×104 (mol/L)−1·s−1; KD=1.2 μmol/L). Thus, VWF‐D′D3 primarily controls soluble VWF binding to GpIbα. In contrast, upon VWF immobilization, all molecular features regulated A1‐GpIbα binding. Here, in ELISA, the number of apparent A1‐domain sites available for binding GpIbα on ΔPro‐VWF was ≈50% that of the ΔD′D3‐VWF variants. In microfluidics based platelet adhesion measurements on immobilized VWF and thrombus formation assays on collagen, human platelet recruitment varied as ΔPro‐VWF<ΔD′D3‐VWF<ΔD′D3NFP─‐VWF<ΔD′D3OG─‐VWF.ConclusionsWhereas VWF‐D′D3 is the major regulator of soluble VWF binding to platelet GpIbα, both the D′D3‐domain and N‐terminal peptide regulate platelet translocation and thrombus formation.

A combined microfluidic-microstencil method for patterning biomolecules and cells.

Despite the myriad of soft lithography based micropatterning methods available to researchers, it is still challenging to define small features (10-100 μm) that are spaced far apart (1-10 mm). In this report, we describe a combined microfluidic-microstencil patterning method that can produce multifunctional substrates of small features, O(10 μm), with a large pitch, O(1 mm). In that, we fabricate microstencils using an UV curable polyurethane (Norland Optical Adhesive 81) with dense arrays of 10-100 μm holes. Overlaying arrays of microfluidic channels over these microstencils allow for the control of the spacing between features and the ability to pattern multiple substrates. We show that this method is capable of patterning soluble proteins, fibrillar insoluble collagen, liposomes, cells, and nanoparticles. We demonstrate the utility of the method by measuring platelet adhesion under flow to three adhesive proteins (insoluble fibrillar collagen, laminin, and reconstituted acid solubilized collagen fibers) in a single assay.