HemoDens: A global hemostasis analyzer for kinetic characterization of clot formation, retraction, and lysis

During clot retraction (CR), platelets use their actomyosin apparatus to contract the cross-linked fibrin-platelet meshwork of a nascent thrombus. As CR depends on thrombin generation, fibrin polymerization/cross-linking, platelet activation and fibrinolysis, measurements of CR can provide valuable information about global hemostatic function. However, a lack of standardized methods for simple and reproducible assessment of CR remains a major obstacle to a more widespread adoption of CR assays for clinical testing. Herein, we developed a whole-blood hemostasis analyzer (HemoDens) to enable rapid and reproducible kinetic characterization of clot formation (CF), CR, and exogenous clot lysis (eCL) from minute quantities of citrated whole blood. In our assay, double-end-anchored clots were formed inside hemitoroidal fluoropolymer sample chambers. This approach provided spatiotemporal standardization by focusing macroscale contractile forces on a geometrically defined region-of-interest (ROI) from which clots retracted uniformly. Fluorescence densitometry was used to monitor the redistribution of RBCs in this ROI during experiments. A set of analytical parameters provided a detailed kinetic characterization of CF, CR and eCL from a single experiment. The specific effects of CR on our assay were verified experimentally by inhibiting platelet fibrin(ogen) binding, actin polymerization, and non-muscle myosin II. We verified the sensitivity of our assay to variations in blood counts, and to the effects of unfractionated heparin (UFH), low molecular weight heparin (LMWH), dabigatran, and recombinant tissue plasminogen activator (rtPA). We also show that our assay could detect the reversal of heparins and rtPA with protamine and tranexamic acid.