Microfluidic flow-based platforms for induction and analysis of dynamic shear-mediated platelet activation—Initial validation versus the standardized hemodynamic shearing device

Abstract

A microfluidic flow-based platform (μFP), able to stimulate platelets via exposure of shear stress patterns pertinent to cardiovascular devices and prostheses, was compared to the Hemodynamic Shearing Device (HSD)-a state-of-the-art bench-top system for exposure of platelets to defined levels and patterns of shear. Platelets were exposed to time-varying shear stress patterns in the two systems; in detail, platelets were recirculated in the μFP or stimulated in the HSD to replicate comparable exposure time. Shear-mediated platelet activation was evaluated via (i) the platelet activity state assay, allowing the measurement of platelet-mediated thrombin generation and associated prothrombotic tendencies, (ii) scanning electron microscopy to evaluate morphological changes of sheared platelets, and (iii) flow cytometry for the determination of platelet phosphatidylserine exposure as a marker of shear activation. The results revealed good matching and comparability between the two systems, with similar trends of platelet activation, formation of microaggregates, and analogous trends of activation marker exposure for both the HSD and microfluidic-stimulated samples. These findings support future translation of the microfluidic platform as a Point-of-Care facsimile system for the diagnosis of thrombotic risk in patients implanted with cardiovascular devices.

Shear-Mediated Platelet Microparticles Demonstrate Phenotypic Heterogeneity as to Morphology, Receptor Distribution, and Hemostatic Function

“…Gel-filtered platelets (GFP) were isolated from PRP by gel chromatography through Sepharose-2B. 83 Platelet fractions were stored and handled at room temperature to minimize storage-associated platelet function decline. 84 Similarly, to limit 37°C-stimulated platelet apoptosis 85, 86 and associated alterations of the surface expression of platelet receptors 45 and microparticle generation, 87 most experiments were performed at room temperature if not otherwise specified.…”

DNA Origami–Platelet Adducts: Nanoconstruct Binding without Platelet Activation

“…Platelet-rich plasma was obtained by centrifugation of the blood at 400 g for 15 min at room temperature. Gel-filtered platelets (GFPs) were isolated from platelet-rich plasma by gel chromatography through Sepharose-2B, and the platelet count was measured on the Z1 particle counter (Beckman Coulter, Indianapolis, IN) . Gel filtration through Sepharose-2B allows for efficient platelet separation from blood plasma proteins and other components without cell damage or alterations of their biochemical and physiological properties .…”

“…Gel-filtered platelets (GFPs) were isolated from platelet-rich plasma by gel chromatography through Sepharose-2B, and the platelet count was measured on the Z1 particle counter (Beckman Coulter, Indianapolis, IN). 59 Gel filtration through Sepharose-2B allows for efficient platelet separation from blood plasma proteins and other components without cell damage or alterations of their biochemical and physiological properties. 60 Gel filtration was performed using a small column packed with Sepharose-2B gel beads and equilibrated with modified Tyrode's buffer, pH 7.4.…”

Platelet Activation via Shear Stress Exposure Induces a Differing Pattern of Biomarkers of Activation versus Biochemical Agonists

“…Gel-filtered platelets (GFP) were isolated from PRP via gel filtration as previously described. 27 Platelet fractions were stored and handled at room temperature if not otherwise indicated. Platelet count was quantified with Z1 Coulter Particle Counter (Beckman Coulter Inc., Indianapolis, Indiana, United States).…”