This proposal focuses on the integrative and high throughput functional phenotyping of human blood, matched by Systems Biology and Bioengineering approaches for patient-specific training of computer models to identify and quantify responses to clotting triggers or pharmacological agents. High throughput phenotyping of individual blood samples will be used to train bottom-up and top-down models of blood clotting under static, venous, and arterial hemodynamic conditions.
Specific Aims are:
Aim 1 : Use high throughput intracellular calcium measurements to train neural network models to predict patient-specific response to combinatorial and sequential stimulation, thus testing the milieu that platelets actually experience during thrombosis. Furthermore, high throughput measures of inside-out signaling will be implemented for the development of large scale computational simulation of platelet metabolic pathways.
Aim 2 : Along with platelet phenotyping, we will use validated high throughput blood thrombin phenotyping to identify pathways and synergisms that are defective in patients with existing but undefined defects. These approaches then allow the development of a full platelet-plasma computer simulation of coagulation.
Aim 3 : Using validated tissue factor microarray-flow chambers and microfluidic chambers, we will functionally phenotype thrombus production and clot stability for normal donors and patients under hemodynamic conditions and pharmacological modulation.
Aim 4 : In vivo studies using a mouse laser injury model to follow evolving intrathrombic spatial gradients. The flow studies are supported by advanced multiscale Lattice Kinetic Monte Carlo (LKMC) simulation of clotting under flow using data from all three specific aims. These approaches represent the first full integration of platelet signaling models with realistic and hierarchical hemodynamic/mass transport simulations that regulate adhesive bond function and plasma protease networks. Better elucidation and quantitative measurement of blood reactions and platelet signaling pathways under hemodynamic conditions are directed at clinical needs in thrombosis risk assessment, anti-coagulation therapy during surgery, platelet targeted therapies, and stroke research.
Blood is ideal for Systems Biology research since it is easily obtained from donors or patients, amenable to high throughput liquid handling experiments, and clinically relevant. Clotting and bleeding diseases of aging are seldom due to acquired mutations and this drives the need for advanced functional phenotyping in concert with Systems Biology and other sequencing/genomic approaches.
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