ADP and Thromboxane Inhibitors Both Reduce Global Contraction of Clot Length, While Thromboxane Inhibition Attenuates Internal Aggregate Contraction.

Platelet contractility drives clot contraction to enhance clot density and stability. Clot contraction is typically studied under static conditions, with fewer studies of wall-adherent platelet clots formed under flow. We tested the effect of inhibitors of ADP and/or thromboxane A2 (TXA2) signaling on clot contraction. Using an eight-channel microfluidic device, we perfused PPACK-treated whole blood (WB) ± acetylsalicylic acid (ASA), 2-methylthioAMP (2-MeSAMP), and/or MRS-2179 over collagen (100/s) for 7.5 min, then stopped flow to observe contraction for 7.5 minutes. Two automated imaging methods scored fluorescent platelet percent contraction over the no-flow observation period: (1) "global" measurement of clot length and (2) "local" changes in surface area coverage of the numerous platelet aggregates within the clot. Total platelet fluorescence intensity (FI) decreased with concomitant decrease in global aggregate contraction when ASA, 2-MeSAMP, and/or MRS-2179 were present. Total platelet FI and global aggregate contraction were highly correlated ( R 2 = 0.87). In contrast, local aggregate contraction was more pronounced than global aggregate contraction across all inhibition conditions. However, ASA significantly reduced local aggregate contraction relative to conditions without TXA2 inhibition. P-selectin display was significantly reduced by ADP and TXA2 inhibition, but there was limited detection of global or local aggregate contraction in P-selectin-positive platelets across all conditions, as expected for densely packed "core" platelets. Our results demonstrate that global aggregate contraction is inhibited by ASA, 2-MeSAMP, and MRS-2179, while ASA more potently inhibited local aggregate contraction. These results help resolve how different platelet antagonists affect global and local clot structure and function.

A Robust Fabrication Technique for Hydrogel Films Containing Micropatterned Opal Structures via Micromolding and an Integrated Evaporative Deposition-Photopolymerization Approach.

Opal-structured thin-film hydrogel materials with micropatterns hold great potential for utility in a wide range of sensing applications. Micropatterning offers key advantages such as ready addressability, high throughput assay, and multiplexing. However, controlled fabrication of such films in a rapid, inexpensive, and reliable manner remains a challenge. Existing techniques suffer from long opal deposition times and often involve complex and arduous steps. In this report, we examined a simple micromolding-based evaporation-polymerization method for the fabrication of poly(ethylene glycol)-based hydrogel films containing micropatterned opal structures. Specifically, intense and uniform opalescent colors were achieved by evaporative deposition of polystyrene bead solution in patterned micromolds. These opal micropatterns were then captured in hydrogel films by simple photopolymerization of a UV-curable PEG diacrylate solution. The as-prepared films show high tunability as well as responsiveness to various environmental cues readily manifested via shifts in color. Combined with UV-vis reflectance spectroscopy and scanning electron microscopy results, these findings illustrate the robust, simple, and reliable nature of our integrated deposition-polymerization approach for controlled fabrication of optically active and stimuli-responsive functional materials. We thus envision that the results and the facile approach reported here can be extended to many application areas including environmental monitoring, diagnostics, and biosensing applications.

Core and shell platelets of a thrombus: A new microfluidic assay to study mechanics and biochemistry.

BACKGROUND: Hemostatic clots have a P-selectin positive platelet core covered with a shell of P-selectin negative platelets. OBJECTIVE: To develop a new human blood microfluidic assay to interrogate core/shell mechanics. METHODS: A 2-stage assay perfused whole blood over collagen/± tissue factor (TF) for 180 seconds at 100 s-1 wall shear rate, followed by buffer perfusion at either 100 s-1 (venous) or 1000 s-1 (arterial). This microfluidic assay used an extended channel height (120 µm), allowing buffer perfusion well before occlusion. RESULTS: Clot growth on collagen stopped immediately with buffer exchange, revealing ~10% reduction in platelet fluorescence intensity (at 100 s-1) and ~30% (at 1000 s-1) by 1200 seconds. Thrombin generation (on collagen/TF) reduced erosion at either buffer flow rate. P-selectin-positive platelets were stable (no erosion) against 1000 s-1, in contrast to P-selectin negative platelets. Thrombin inhibition (with D-Phe-Pro-Arg-CMK) reduced the number of P-selectin-positive platelets and lowered thrombus stability through the reduction of P-selectin-positive platelets. Interestingly, fibrin inhibition (with H-Gly-Pro-Arg-Pro-OH acetate salt) increased the number of P-selectin-positive platelets but did not lower stability, suggesting that fibrin was only in the core region. Thromboxane inhibition reduced P-selectin-positive platelets and caused a nearly 60% reduction of the clot at arterial buffer flow. P2Y1 antagonism reduced clot size and the number of P-selectin-positive platelets and reduced the stability of P-selectin-negative platelets. CONCLUSION: The 2-stage assay (extended channel height plus buffer exchange) interrogated platelet stability using human blood. Under all conditions, P-selectin-positive platelets never left the clot.