A Microfluidic Approach For Evaluating Novel Antithrombotic Targets

Microfluidic systems allow precise control of the anticoagulation/pharmacology protocols, defined reactive surfaces, hemodynamic flow and optical imaging routines, and thus are ideal for studies of platelet function and coagulation response. This thesis describes the use of a microfluidic approach to investigate the role of the contact pathway factors XII and XI, platelet-derived polyphosphate, and thiol isomerases in thrombus growth and to evaluate their potential as safer antithrombotic drug targets. The use of low level of corn trypsin inhibitor allowed the study of the contact pathway on collagen/kaolin surfaces with minimally disturbed whole blood sample and we demonstrated the sensitivity of this assay to antithrombotic drugs. On collagen/tissue factor surfaces, we found the relative contributions of the extrinsic pathway, the contact pathway, and the thrombin feedback pathway vary with tissue factor surface concentration. Platelet-derived polyphosphate potentiated the thrombin feedback pathway at low tissue factor level but enhanced fibrin fiber structure regardless of tissue factor level. At locations with low tissue factor level, thrombosis may be druggable by contact pathway and polyphosphate inhibition, although thrombolytic susceptibility may benefit from polyphosphate antagonism regardless of tissue factor level. We developed a peptide-based platelet-targeting thiol reduction sensor to visualize thrombus-incorporated thiol reductase activity. Although distribution of thiol reductase activity was shown to be correlated with the level of platelet activation, protein disulfide isomerase inhibition showed a limited effect on platelet aggregation in microfluidic thrombosis assay. We also used the microfluidic system to explore the injury patch size limit for triggering clotting. We observed a full clotting response of platelet deposition, thrombin generation and fibrin polymerization on one of the smallest biological units of a single collagen fiber presenting tissue factor and von Willebrand factor suggesting the lack of physiological injury patch size limit. Finally, we made the first estimation of thrombin flux from growing thrombus under flow using the microfluidic thrombosis assay in combination with enzyme-linked immunosorbent measurement of thrombin-antithrombin complex. We found thrombin is robustly generated within clots by the extrinsic pathway, followed by late-stage factor XIa contributions, with fibrin localizing thrombin via its antithrombin activity as a self-limiting hemostatic mechanism