Analysis of morphology of platelet aggregates formed on collagen under laminar blood flow

In a focal injury model, platelets adhere and activate under flow on a collagen-coated surface, creating a field of individual platelet aggregates. These aggregates exhibit distinct structural characteristics that are linked to the local flow conditions. By combining image analysis techniques and epifluorescence microscopy, we developed a robust strategy for quantifying the characteristic instantaneous width and length of a growing platelet deposit. We have confirmed the technique using model images consisting of ellipsoid objects and quantified the shear rate-dependent nature of aggregate morphology. Venous wall shear rate conditions (100 s⁻¹) generated small, circular platelet deposits, whereas elevated arterial shear rates (500 and 1000 s⁻¹) generated platelet masses elongated twofold in the direction of flow. At 2000 s⁻¹, an important regime for von Willebrand Factor (vWF)-mediated recruitment, we observed sporadic platelet capture events on collagen that led to rapidly growing deposits. Furthermore, inter-donor differences were investigated with respect to aggregate growth rate. After perfusion at elevated shear rates (1000 s⁻¹) for 5 min, we identified a twofold increase in aggregate size (81.5 ± 24.6 μm; p < 0.1) and a threefold increase in growth rate parallel to the flow (0.40 ± 0.09 μm/s; p < 0.01) for an individual donor. Suspecting a role for vWF, we found that this donor had a twofold increase in soluble vWF relative to the other donors and pooled plasma. Microfluidic devices in combination with automated morphology analysis offer new tools for characterizing clot development under flow.     

Rapid platelet accumulation leading to thrombotic occlusion

Platelet thrombosis under arterial conditions remains a large clinical problem. Previous in vitro experiments have concentrated on early adherence without thrombotic occlusion. We have developed a controllable hemodynamic system that creates intravascular thrombosis to occlusion. Lightly heparinized (3.5 USP units/mL), whole, porcine blood is perfused through a 1.5 mm inner diameter, tubular, collagen-coated stenosis. The microscopic growth of thrombus is optically recorded using a high resolution CCD camera. Occlusive thrombus is examined using microcomputed tomography and histology. Thrombus consistently formed in the throat of the stenosis where wall shear rates were greatest. Rapid platelet accumulation (RPA) reached rates as high as 13.7 μm³ μm⁻² min⁻¹. Total occlusion of flow occurred after 17 ± 2.6 min (n = 6). The average thrombus volume accumulation of 7.8 ± 3.5 μm³ μm² min⁻¹ occurred under very high wall shear rates exceeding 100,000 s⁻¹. Significant volumes of thrombus did not form until 7.6 ± 3.6 min after the onset of flow, a delay consistent with activation of adherent mural platelets. Platelets did not accumulate with large volume for normal wall shear rates <2000 s⁻¹. Very high wall shear rates stimulate the capture of millions of circulating platelets with exposure times <2 ms in an arterial stenosis.     

Microfluidic device to study arterial shear-mediated platelet-surface interactions in whole blood: reduced sample volumes and well-characterised protein surfaces

We report a novel device to analyze cell-surface interactions under controlled fluid-shear conditions on well-characterised protein surfaces. Its performance is demonstrated by studying platelets interacting with immobilised von Willebrand Factor at arterial vascular shear rates using just 200 μL of whole human blood per assay. The device’s parallel-plate flow chamber, with 0.1 mm² cross sectional area and height-to-width ratio of 1:40, provides uniform, well-defined shear rates along the chip surface with negligible vertical wall effects on the fluid flow profile while minimizing sample volumetric flow. A coating process was demonstrated by ellipsometry, atomic force microscopy, and fluorescent immunostaining to provide reproducible, homogeneous, uniform protein layers over the 0.7 cm² cell-surface interaction area. Customized image processing quantifies dynamic cellular surface coverage vs. time throughout the whole-blood-flow assay for a given drug treatment or disease state. This device can track the dose response of anti-platelet drugs, is suitable for point-of-care diagnostics, and is designed for adaptation to mass manufacture.     

Breast adenocarcinoma cell adhesion to the vascular subendothelium in whole blood and under flow conditions: effects of alphavbeta3 and alphaIIbbeta3 antagonists

Tumour cell adhesion to vascular extracellular matrix (ECM), an important step of metastatic progression, is promoted by platelets. The aim of our study was to investigate, in whole blood under venous and arterial shear conditions, the respective role of tumour cell α_vβ₃ and platelet α_IIbβ₃integrins in MDA-MB-231 breast adenocarcinoma cell adhesion to human umbilical vein endothelial cell ECM. For that purpose, blood containing MDA-MB-231 cells was incubated with non-peptide antagonists specific for platelet α_IIbβ₃ (lamifiban) or tumour cell α_vβ₃ (SB-273005). At 300 s⁻¹, each antagonist used alone did not modify tumour cell adhesion, whereas, at 1500 s⁻¹, tumour cell adhesion was decreased by 25% in presence of lamifiban indicating a role of platelet α_IIbβ₃ at higher shear rate. However, a combination of SB-273005 and lamifiban, or c7E3 Fab (a potent inhibitor of both α_IIbβ₃ and α_vβ₃) inhibited tumour cell adhesion by 40–45%, at either shear rate applied, indicating a cooperation between these two integrins in MDA-MB-231 cell adhesion to ECM, as well as the participation of other adhesive receptors on tumour cells and/or platelets. Thus, efficient anti-metastatic therapy should target multiple receptors on tumour cells and platelets. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microfluidic array for three-dimensional perfusion culture of human mammary epithelial cells

The ability to culture cells in three dimensional extracellular matrix (3D ECM) has proven to be an important tool for laboratory biology. Here, we demonstrate a microfluidic perfusion array on a 96-well plate format capable of long term 3D ECM culture within biomimetic microchambers. The array consists of 32 independent flow units, each with a 4 μl open-top culture chamber, and 350 μl inlet and outlet wells. Perfusion is generated using gravity and surface tension forces, allowing the array to be operated without any external pumps. MCF-10A mammary epithelial cells cultured in Matrigel in the microfluidic array exhibit acinus morphology over 9 days consistent with previous literature. We further demonstrated the application of the microfluidic array for in vitro anti-cancer drug screening.     

The role of fibrinogen spacing and patch size on platelet adhesion under flow

Platelet adhesion to the vessel wall during vascular injury is mediated by platelet glycoproteins binding to their respective ligands on the vascular wall. In this study we investigated the roles that ligand patch spacing and size play in regulating platelet interactions with fibrinogen under hemodynamic flow conditions. To regulate the size and distance between patches of fibrinogen we developed a photolithography-based technique to fabricate patterns of proteins surrounded by a protein-repellant layer of poly(ethylene glycol). We demonstrate that when mepacrine labeled whole blood is perfused at a shear rate of 100 s^?1 over substrates patterned with micron-sized wide lines of fibrinogen, platelets selectively adhere to the areas of patterned fibrinogen. Using fluorescent and scanning electron microscopy we demonstrate that the degree of platelet coverage (3–35%) and the ability of platelet aggregates to grow laterally are dependent upon the distance (6–30 μm) between parallel lines of fibrinogen. We also report on the effects of fibrinogen patch size on platelet adhesion by varying the size of the protein patch (2–20 μm) available for adhesion, demonstrating that the downstream length of the ligand patch is a critical parameter in platelet adhesion under flow. We expect that these results and protein patterning surfaces will be useful in understanding the spatial and temporal dynamics of platelet adhesion under physiologic flow, and in the development of novel platelet adhesion assays.     

Design of a Novel Apparatus to Study Nitric Oxide (NO) Inhibition of Platelet Adhesion

Nitric oxide (NO) is a simple biological molecule which inhibits adhesion and aggregation of platelets. A novel NO delivery device has been developed to quantitatively study the effects of NO concentration and flux on the adhesion of platelets to a surface. The slit-flow device is lined with a protein-coated membrane through which NO gas permeates into a perfusing platelet suspension. A model predicting spatial NO concentrations and fluxes within the flow slit was validated. At a wall shear rate of 250s^-1, platelet adhesion was inhibited 87% relative to controls for exposures as low as 0.1 ppm NO. Corresponding model predictions of the aqueous NO concentration and fluxes at the surface were 0.15 nM, and between 0.5 and 1.1 nanomoles cm^-2 s_-1, respectively. Endo-thelial cells, which release NO to inhibit platelet adhesion in vivo, generate NO at an estimated flux similar to the above values. At a NO exposure of 0.02 ppm, platelet inhibition was only 10%. The delivery device is useful for other studies in which a knowledge of the spatial NO fluxes or concentrations is desired. Knowledge of these fluxes or concentrations is beneficial towards the design of biomaterials incorporating NO to inhibit platelet adhesion. © 1998 Biomedical Engineering Society. PAC98: 8745Hw, 8722Fy, 8780+s, 8710+e, 8380Lz     

The targeting of endothelial progenitor cells to a specific location within a microfluidic channel using magnetic nanoparticles

A common problem with the in vivo therapeutic applications of cells is that cells can rapidly disappear into the circulatory system after an injection. Magnetic nanoparticles can be used to solve this problem. Bacterial magnetic nanoparticles were used in this study for targeting stem cells at a specific location within a microfluidic channel. Magnetic nanoparticles were isolated from Magnetospirillum sp. AMB-1 and delivered to endothelial progenitor cells (EPCs). Cellular uptake of magnetic nanoparticles and their functional feasibility was characterized in vitro. The environment of a human blood vessel was simulated using a microfluidic channel. Magnetic nanoparticle-incorporated EPCs were injected into a microchannel and the flow rate of cells was uniformly controlled by use of a syringe pump. EPCs were effectively targeted to a specific location within the microchannel by an external magnetic field (about 400 mT). About 40% of EPCs were efficiently targeted with a flow rate of 5 μl min⁻¹ when 10 μg of magnetic nanoparticles were used per 10⁴ cells. This microfluidic system provides a useful tool towards a better understanding of the behavior of magnetic nanoparticle-incorporated cells within the human circulatory system for clinical use.     

Surface and bulk effects on platelet adhesion and aggregation during simple (laminar) shear flow of whole blood

This study attempts to clarify the role of the artificial surface and the fluid bulk on platelet adhesion and aggregation events during simple shear flow of whole blood. The experimental approach involved the shearing of fresh whole blood samples over the shear rate range of 720-5680 s^-1, which corresponded to a shear stress maximum of about 150 dyn cm^-2. Results on platelet adhesion, measured as surface coverage by platelets, and platelet aggregation, measured in terms of reduction in platelet count and adenosine diphosphate (ADP) release, were determined as a function of the surface to volume ratio (S/V); and artificial surface used. Both shear-induced platelet adhesion and platelet count reduction showed significant variation over the range of S/V employed. The ratios between the three different S/V values used in this system (10:6:4) were about the same as the ratio of the shear rate-averaged results obtained. Also, for shear-induced hemolysis, an increase in the release of hemoglobin from red blood cells was found as S/ V was increased, again with ratios between the shear rate-averaged values similar to the ratio of S/V values employed. The shear-induced release of ADP, presumably from platelets and from red blood cells indicated a different dependence of ADP release on S/ V than was observed for the other parameters reported. Irreversible platelet aggregation was expected to occur because the amount of ADP that was released as a result of the shear was substantial. Models proposed to explain the experimental results were found to support a surface-controlled mechanism.     

High Content Evaluation of Shear Dependent Platelet Function in a Microfluidic Flow Assay

The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 μL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50–920 s^?1). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 μm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater than 20 μm was necessary to support platelet adhesion under flow, suggesting a threshold injury size is necessary for stable platelet adhesion. Integrating 50 μm CTF microspots into a multishear microfluidic device yielded a high content assay from which we extracted platelet accumulation metrics (lag time, growth rate, total accumulation) on the spots using Hoffman modulation contrast microscopy. This method has potential broad application in identifying platelet function defects and screening, monitoring, and dosing antiplatelet agents.     

Alternating current electrophoresis of human red blood cells

An a.c. electrophoretic technique in the frequency range between 2–10 Hz, using a rectangular chamber design, has been developed for measuring the surface electric charge density of human red blood cells. With the a.c. approach, mobility can be determined rapidly, electrode polarization is minimized and the construction of the chamber simplified. For frequencies under 10 Hz and a chamber thickness of 150 μm, a theoretical analysis of the electroosmotic flow profile shows that it is almost identical to the d.c. case. With the a.c. method and using a 0.145 N NaCl solution buffered with NaHCO₃, the mobility of red cells measured at the lower stationary level is found to be −1.07±0.02 (S.D.) μmsec⁻¹ V⁻¹ cm. The zeta potential and charge density calculated from the mobility are −14.03±0.26 (S.D.) mV and −3603±69 (S.D.) esu cm⁻², respectively.     

Micropatterned surfaces for controlling cell adhesion and rolling under flow

Cell adhesion and rolling on the vascular wall is critical to both inflammation and thrombosis. In this study we demonstrate the feasibility of using microfluidic patterning for controlling cell adhesion and rolling under physiological flow conditions. By controlling the width of the lines (50–1000 μm) and the spacing between them (50–100 μm) we were able to fabricate surfaces with well-defined patterns of adhesion molecules. We demonstrate the versatility of this technique by patterning surfaces with 3 different adhesion molecules (P-selectin, E-selectin, and von Willebrand Factor) and controlling the adhesion and rolling of three different cell types (neutrophils, Chinese Hamster Ovary cells, and platelets). By varying the concentration of the incubating solution we could control the surface ligand density and hence the cell rolling velocity. Finally by patterning surfaces with both P-selectin and von Willebrand Factor we could control the rolling of both leukocytes and platelets simultaneously. The technique described in this paper provides and effective and inexpensive way to fabricate patterned surfaces for use in cell rolling assays under physiologic flow conditions.     

New strategy of platelet substitutes for enhancing platelet aggregation at high shear rates: cooperative effects of a mixed system of fibrinogen γ-chain dodecapeptide- or glycoprotein Ibα-conjugated latex beads under flow conditions

To construct platelet substitutes that have hemostatic properties over a wide range of shear rates, we used fibrinogen γ-chain carboxy-terminal sequence HHLGGAKQAGDV (H12), which recognizes activated platelets at low shear rates, and a recombinant water-soluble moiety of the platelet glycoprotein (rGPIbα), which recognizes von Willebrand factor at high shear rates. Three kinds of samples were prepared for this purpose: H12-conjugated latex beads (H12-latex beads), rGPIbα-latex beads, and H12/rGPIbα-latex beads. These samples were evaluated in thrombocytopenia-imitation blood at various flow conditions. Based on ADP-induced platelet aggregation studies, the H12-latex beads significantly enhanced platelet aggregation via H12 binding with GPIIb/IIIa activated on the surface of activated platelets, whereas the rGPIbα-latex beads did not support platelet aggregation. In the case of the H12/rGPIbα-latex beads, the function of H12 was suppressed by steric hindrance from the larger rGPIbα bound to the latex bead. A mixture of the H12-latex beads and the rGPIbα-latex beads adhered to a collagen surface over a wide range of shear rates. In particular, at high shear rates, a cooperative effect was observed in the enhancement of platelet thrombus formation compared with H12-latex beads or rGPIbα-latex beads alone. We propose that a mixed system of H12- and rGPIbα-conjugated nanoparticles is a more effective platelet substitute than each of the beads used alone and has enhanced platelet aggregation properties.     

Haemodynamic forces on in vitro thrombi: a numerical analysis

The flow of blood past an axisymmetric thrombus analogue, within an in vitro geometry, is computed via solution of the discrete three-dimensional (3D) Navier–Stokes equations. Particle tracking is used to model the behaviour of thrombocytes (platelets) moving throughout the domain and to investigate behaviour with respect to the platelets. The system is explored using shear rate to quantify the effects an idealised thrombus has with respect to an undisturbed in vitro geometry over ‘Poiseuille flow’ shear rate conditions applicable to in vivo and in vitro experiments (1,200–10,000 s⁻¹). Local shear rate variations show peaks in shear rate greater than double that of Poiseuille flow conditions. These local shear rate variations are observed to be non-linear, despite the low Reynolds number (5.2–43.4) within the system. Topological transitions of shear rate are observed, limiting the height of peak shear rate within the system, suggesting a thrombus growth limiting behaviour. Temporal gradients of shear rate, measured with respect to individual platelets, were calculated. Multiple regions of peak shear rate gradient were observed throughout the flow, suggesting that platelet–platelet interaction may not be limited to regions near to the surface of the thrombus.     

Platelet interactions with liposomes carrying recombinant platelet membrane glycoproteins or fibrinogen: approach to platelet substitutes.

Liposomes carrying both recombinant platelet membrane glycoproteins GPIa/IIa (rGPIa/IIa) and GPIb alpha (rGPIb alpha) (rGPIa/IIa-Ib alpha-liposomes), or fibrinogen (Fbg-liposomes) were prepared. Their interactions with platelets on a collagen surface under flow conditions were evaluated using a recirculating flow chamber, mounted on an epifluorescence microscope, which allows for real-time visualization of fluorescence-labeled liposomes or platelets interacting with the surface. Adhesion of platelets to the collagen surface increased with increasing the shear rate from 600 to 2400 s(-1). Also, the percentages of surface coverage of rGPIa/IIa-Ib alpha-liposomes or Fbg-liposomes increased with increasing platelet adhesion. These phenomena were attenuated by a peptide containing arginine-glycine-aspartic acid (RGD-peptide), or prostaglandin E1 (PGE), but not by a peptide containing arginine-glycine-glutamic acid (RGE-peptide). In a homogeneous solution, rGPIa/IIa-Ib alpha-liposomes and Fbg-liposomes enhanced platelet aggregation in a dose-dependent manner, as evaluated using an aggregometer. These findings suggest that rGPIa/IIa-Ib alpha-liposomes and Fbg-liposomes form aggregates at the site of injury in blood vessels, resulting in stationary adhesion together with activated platelets.     

A micro circulating PCR chip using a suction-type membrane for fluidic transport

A new micromachined circulating polymerase chain reaction (PCR) chip is reported in this study. A novel liquid transportation mechanism utilizing a suction-type membrane and three microvalves were used to create a new microfluidic control module to rapidly transport the DNA samples and PCR reagents around three bio-reactors operating at three different temperatures. When operating at a membrane actuation frequency of 14.29 Hz and a pressure of 5 psi, the sample flow rate in the microfluidic control module can be as high as 18 μL/s. In addition, an array-type microheater was adopted to improve the temperature uniformity in the reaction chambers. Open-type reaction chambers were designed to facilitate temperature calibration. Experimental data from infrared images showed that the percentage of area inside the reaction chamber with a thermal variation of less than 1°C was over 90% for a denaturing temperature of 94°C. Three array-type heaters and temperature sensors were integrated into this new circulating PCR chip to modulate three specific operating temperatures for the denaturing, annealing, and extension steps of a PCR process. With this approach, the cycle numbers and reaction times of the three separate reaction steps can be individually adjusted. To verify the performance of this circulating PCR chip, a PCR process to amplify a detection gene (150 base pairs) associated with the hepatitis C virus was performed. Experimental results showed that DNA samples with concentrations ranging from 10⁵ to 10²copies/μL can be successfully amplified. Therefore, this new circulating PCR chip may provide a useful platform for genetic identification and molecular diagnosis.     

Shear Stress Induced Release of Von Willebrand Factor and Thrombospondin-1 in Uvec Extracellular Matrix Enhances Breast Tumour Cell Adhesion

Endothelial cells in vivo are exposed to blood shear forces and flow perturbations induce their activation. Such modifications of hemodynamic can be observed in patients with cancer. We have submitted endothelial cells (HUVEC) to shear stress (13 dynes/cm²) and isolated their extracellular matrix (ECM) prior perfusion with breast adenocarcinoma cells (MDA-MB-231) in whole blood at a shear rate of 1500 s⁻¹. Exposure of HUVEC to 13 dynes/cm² (τ₁₃) for 2 h enhanced the secretion of von Willebrand factor (vWF) and thrombospondin-1 (TSP-1) in the ECM. Moreover, MDA-MB-231 cell adhesion was enhanced to such treated-ECM. This over-adhesion was inhibited by pre-incubating the ECM with anti-vWF or anti-TSP-1 antibodies, or by blocking tumour cell α_vβ₃ integrin. Although blood platelets were involved in tumour cell adhesion to ECM, blockade of platelet GPIb or α_IIbβ₃ receptors did not specifically inhibit the enhanced tumour cell adhesion observed on τ₁₃. ECM. These findings indicate that shear stress can modulate the expression of adhesive proteins in ECM, which favours direct tumour cell adhesion via α_vβ₃ and other receptors.     

Can blood flow in separate small tubes be quantitatively assessed by high-resolution laser Doppler imaging?

A method is suggested for quantitative flow assessment of whole-blood perfusing tubes with diameters in the range from 500 μm to 1.5 mm, for velocities below 9 mm s⁻¹. The algorithm is based both on the Doppler broadening of backscattered laser light and the magnitude of the diameter of the perfused tube. A bandwidth-modified high-resolution laser Doppler perfusion imaging system is used to record the Doppler broadening. A flow model, consisting of a linearly narrowing tube (inner diameter 620–1330 μm), is connected to a precision infusion pump and perfused by human whole blood of volume flows ranging from 0 to 6.6 mm³ s⁻¹. empirical data are fitted into a regression model, and the parameters of the algorithm can be determined, resulting in a correlation coefficient of 0.975 between the predicted and true volume flows. Using this algorithm, volume flows in tubes of inner diameters of 500 μm, 750 μm and 1.4 mm are predicted, with accuracies corresponding to correlation coefficients of 0.994, 0.993 and 0.996.     

A physiologically realistic in vitro model of microvascular networks

Existing microfluidic devices, e.g. parallel plate flow chambers, do not accurately depict the geometry of microvascular networks in vivo. We have developed a synthetic microvascular network (SMN) on a polydimethalsiloxane (PDMS) chip that can serve as an in vitro model of the bifurcations, tortuosities, and cross-sectional changes found in microvascular networks in vivo. Microvascular networks from a cremaster muscle were mapped using a modified Geographical Information System, and then used to manufacture the SMNs on a PDMS chip. The networks were cultured with bovine aortic endothelial cells (BAEC), which reached confluency 3–4 days after seeding. Propidium iodide staining indicated viable and healthy cells showing normal behavior in these networks. Anti-ICAM-1 conjugated 2-μm microspheres adhered to BAEC cells activated with TNF-α in significantly larger numbers compared to control IgG conjugated microspheres. This preferential adhesion suggests that cultured cells retain an intact cytokine response in the SMN. This microfluidic system can provide novel insight into characterization of drug delivery particles and dynamic flow conditions in microvascular networks.     

Rac1 is essential for phospholipase C-γ2 activation in platelets

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cβ or PLCγ2. Active PLCs trigger Ca²⁺ mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCβ isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCγ2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP₃) and intracellular calcium mobilization suggesting inappropriate activation of PLCγ2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ^−/− platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA₂ analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ^−/− mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCγ2 activation in platelets and that this is critical for thrombus formation in vivo. Irina Pleines, Margitta Elvers, Amrei Strehl: these authors contributed equally to this work.