P83: Circulating Platelet-Derived Microparticles Modulate Platelet Adhesion Under Flow: Modeling Primary Thrombus Formation in a Microfluidic System

Background: Elevated levels of circulating platelet-derived microparticles (PDMPs) are associated with a range of pathologies, thought to increase thrombotic risk. We hypothesized that addition of PDMPs to whole blood would alter platelet adhesion and thrombogenicity, with its effect further modulated by shear stress magnitude. We tested the effect of addition of PDMPs to whole blood on platelet adhesion and primary thrombus formation on collagen under a range of shear flows using a microfluidic system. Methods: PDMPs were generated via sonication of gel-filtered human platelets and separated via differential centrifugation. Hirudin-anticoagulated human blood was stained with 3,3′-dihexyloxacarbocyanine iodide and circulated through a collagen-coated microchannel with and without PDMPs at defined shear stress magnitudes (13.5, 49.5, or 72 dyne/cm2) for 4 min. Platelet adhesion was visualized by fluorescence microscopy; images were analyzed using MATLAB software. Platelet adhesion and thrombogenicity was assessed via four parameters: 1. surface coverage (SC) – indicative of overall surface occupied by platelet thrombi; 2. number of thrombi (NT) – indicative of the number of sites of platelet adhesion on a collagen-covered surface; 3. mean thrombus area (MTA) and 4. mean fluorescence intensity (MFI) – both characterizing thrombus size. Results: Whole blood platelet exposure to increasing shear stress facilitated platelet adhesion to collagen, with increased SC, MTA and MFI (Fig. 1, WB group). Interestingly, the number of thrombi decreased (846±148 vs. 256±81 at 13.5 and 72 dyne/cm2, respectively) with increasing shear suggesting that increased platelet adhesion was due to increased thrombus size, rather than the number of sites of platelet adhesion. Circulating PDMPs significantly inhibited platelet adhesion to collagen, with a decrease of SC, MTI and MFI (but not NT) at all shear stress magnitudes (Fig. 1, WB+PDMPs vs. WB group, ANOVA: p<0.05). At high shear, PDMP-mediated inhibition of platelet adhesion was pronounced with a more than a 2-fold decrease of thrombus size, as indicated by MTA and MFI. Notably, adding PDMPs to recirculated whole blood increased NT adhered to collagen only at lower shear magnitudes. Conclusion: Shear stress of increasing magnitude promotes increased platelet adhesion to collagen, platelet thrombus size and surface coverage; while the number of sites of platelet adhesion decreases. Circulating PDMPs inhibit platelet adhesion under flow, resulting in a significant decrease in platelet thrombi size. Adding PDMPs to circulating blood increases the number of thrombi formed on collagen, but only at lower shear magnitudes. We speculate that increased levels of circulating PDMPs facilitate platelet adhesion and primary thrombus formation, contributing to an overall prothrombotic phenotype, yet with associated microthrombosis with embolic potential, imposing increased risk of distal ischemic and occlusive events.Figure 1. Circulating platelet-derived microparticles (PDMPs) inhibit platelet adhesion to collagen under shear stress: WB – whole blood, circulated without PDMPs; WB+PDMPs - whole blood, circulated without PDMPs (9:1). Mean ± SD, n = 7, ANOVA: * - p < 0.05, ** - p < 0.01.