We propose an experimental and theoretical investigation of blood clot dissolving reactions under realistic hemodynamic conditions. We seek to define the quantitative relationship between the pharmacodynamics of a given thrombolytic therapy, the composition and location of a thrombus, and the consequent reperfusion time and reperfusion flow rate. Particular attention is placed on the penetration rates of plasma constituents into thrombi (driven by hemodynamic pressures) and the consequent dissolution dynamics. We seek to identify rate-controlling steps and criteria for optimal thrombolysis. The first area of study is an investigation of fundamental adsorption and heterogeneous kinetic rate processes of fibrinolytic mediators interacting with fibrin, partially-degraded crosslinked fibrin, and lysis-resistant fibrins. We will measure binding, reaction, and penetration of fibrinolytic mediators to clots subjected to venous and arterial pressures. These experiments will utilize epifluorescence video microscopy and various binding assays to quantify the penetration and binding rates of fluorescently labeled and radiolabeled proteins within fibrin gels and in vitro clots. We will evaluate local instantaneous concentrations of reactants at the plasma- clot interface during lysis. This interface is the critical 100 microM zone that can dictate the outcome of thrombolytic therapy. In particular we seek to evaluate tPA levels in degrading clots under arterial conditions and elucidate the mechanisms by which clots are slow to lyse at high tPA dosages. This is particularly important in light of our recent discovery that recombinant single-chain tPA can interfere with plasmin activity on fibrin, i.e., clots can be overdosed with tPA. The second area of research is to determine the causes of clot cannulation whereby highly thrombogenic material remains on the vessel walls after reperfusion takes place. Rheological, structural, and cellular determinants of nonuniform lysis will be characterized. Thirdly, we will also seek to optimize dissolution regimes for these nonoccluding residual thrombi under defined shear conditions. The fourth area is the advancement of computer simulation of the clot dissolving reactions using biphasic, multicomponent convection-dispersion-reaction equations for erodible fibrin structures with heterogeneous adsorption and reaction. Coupled with pharmacodynamic modeling of the systemic circulation, these computer simulations will help predict rates of clot reperfusion, causes of cannulation, as well as, help evaluate therapeutic approaches for annular clots remaining after cannulation. Overall, the understanding of spatial dynamics in dissolving thrombi will help facilitate the rational design and optimization of lytic therapy, particularly those approaches involving catheters or combined therapeutic agents.
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