PROJECT SUMMARY The purpose of the Bold New Bioengineering Research for Heart, Lung, Blood and Sleep Disorder and Diseases is to support early phases of groundbreaking bioengineering projects that have the potential to revolutionize diagnostics and treatment. In response to NOSI (NOT-HL-21-024), we propose a bioengineering research idea that challenges the status quo in platelet function testing, which has been hampered by low throughput, high variance, and unreliable methods that fail to capture the full complexity of platelet physiology. The long-term goal of this line of research is to engineer technologies that can diagnose the spectrum of bleeding disorders at increased specificity, sensitivity, and throughput than is currently available. The overall objective of this application is to design elastic hydrogel microparticles as a platform to measure platelet adhesion, activation, aggregation, and contraction. The rationale for this project focuses on inherited platelet disorders that are challenging to detect using conventional clinical assays, or lack a specific diagnosis as in the case of many acquired bleeding disorders associated with acute or chronic inflammation. We will meet our overall objective with two specific aims: 1) Immobilized particle assay to measure platelet function kinetics; and 2) Suspended particle assay to measure platelet function equilibrium. We will synthesize peptide-functionalized polyethylene glycol (PEG) particles that present ligands in a combinatorial manner using microfluidic single and double emulsion approaches. In Aim 1, the kinetics or speed of platelet adhesion, activation, aggregation, and contraction will be measured by optical microscopy on immobilized beads in a channel. The equilibrium or strength of these same platelet subfunctions will be measured in suspension in a well-defined shear flow using a rotational rheometer. This approach simulates force-dependent platelet function that is regulated by blood flow in vivo, but with the throughput of a flow cytometer. By creating a platform where each particle serves as an individual test site and each assay includes thousands of test sites, we aim to improve the reliability and reproducibility of platelet function testing, as well as identify new patterns of underlying bleeding disorders. The proposed research is innovative, in our opinion, because it represents the creation of a diagnostic approach that incorporates four key elements of platelet function—adhesion, aggregation, activation, and contraction—within a high throughput format via a lab-on-a-particle platform. This contribution will be significant because it has the potential to improve the lives of people with bleeding disorders by standardizing diagnostic criteria which is currently undefined.