Thrombin generation test based on a 96-channel pipettor for evaluation of FXIa procoagulant activity in pharmaceuticals.

Thrombin generation (TG) assays are used widely to investigate both diseases and drugs that impact thrombosis and bleeding. TG assays were also instrumental in the identification of thrombogenic impurities in immune globulin products, which were associated with thrombotic adverse events in patients. TG assays are therefore now used by quality control laboratories of plasma derivative drug manufacturers and regulatory agencies responsible for the safety testing and release of immune globulin products. In this protocol, we describe a robust and sensitive version of the TG assay for quantitative measurement of thrombogenic activity in immune globulin products. Compared with the version of the assay commonly used in clinical laboratories that compares individual patient plasma samples with normal donor samples, our TG assay is suitable for quick (170-260 min) semiautomated analysis of multiple drug samples against the World Health Organization international standard for factor XIa. Commercially available reagents can be used for the assay, and it does not require specialized equipment. The protocol can be easily adapted for the measurement of the procoagulant activity of other biopharmaceuticals, e.g., coagulation factors.

Stimulus-responsive self-assembly of protein-based fractals by computational design.

Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns in fractal-shaped branched objects, such as trees, lungs and sponges, results in a high surface area to volume ratio, which provides key functional advantages including molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies could provide access to functional biomaterials. Here we describe a computational design approach for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Guided by atomic-resolution models, we develop fusions of Src homology 2 (SH2) domain or a phosphorylatable SH2-binding peptide, respectively, to two symmetric, homo-oligomeric proteins. Mixing the two designed components resulted in a variety of dendritic, hyperbranched and sponge-like topologies that are phosphorylation-dependent and self-similar over three decades (~10 nm-10 µm) of length scale, in agreement with models from multiscale computational simulations. Designed assemblies perform efficient phosphorylation-dependent capture and release of cargo proteins.