Extracorporeal medical machines such as the pump-oxygenator and artificial kidney rely on systemic heparinization to improve blood compatibility. However, heparin can lead to serious complications such as bleeding. With the prospect of even longer perfusion times with machines such as the membrane oxygenator, the problems due to heparinization become more severe. Although many approaches have been explored to solve this problem, such as the use of neutralizing compounds or heparin bonded surfaces, there still remains no real alternative to systemic heparinization. We propose a new method to control heparin levels using a blood filter containing immobilized heparinase. Such a filter might be used in situations where it is desired to heparinize the extracorporeal circuit without simultaneous heparinization of the patient. Alternatively, it could eliminate the use of neutralizing compounds such as protamine. Because the amount of data on heparinase has, until now, been limited ad the methods of producing it are inadequate for large scale use, research has focused not only on the development and testing of the filter, but on enzyme production and purification. Thus far we have been able (1) to define critical requirements for nutrients for fermentation which greatly reduced the cost of these procedures by eliminating the need for heparin as the inducer; (2) to completely purify heparinase to homogeneity and to characterize its entire amino acid content; (3) to demonstrate that the heparin degradation products do not build up in the body and are cleared much more rapidly than the parent compound, heparin, as demonstrated by both radio-labeled and radioautographic studies; (4) to develop approaches for immobilizing heparinase and develop models that can predict the behavior of the immobilized enzyme; (5) to test this reactor in a sheep model using both the reactor by itself and the reactor in conjunction with a dialyzer at flow rates as high as 200 cc/min. in as many as 16 separate perfusions in a given animal, and (6) to demonstrate that heparinase, in contrast to protamine, can neutralize a variety of low molecular weight heparins. Having made these findings, our specific aims are (1) improving purification and production procedures using genetic engineering and other procedures, (2) developing improved reactors and modeling them in vivo and (3) testing these reactors in hemodialysis and cardiovascular applications in vivo.
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