Optimization of a dual enzyme system and its effect on collagenous surfaces
Lokapur, A. K
MetadataShow full item record
The main purpose of this thesis is to co-immobilize heparin and plasmin onto collagenous substrates in an effort to produce a biomaterial possessing both fibrinolytic and thromboresistant properties. The path followed in this research has been to immobilize heparin onto collagenous substrates such as collagen powder, human umbilical vein grafts (HUVG) and chymotrypsin treated goat carotid arteries (GCA), and optimize its activity and stability over a period of time. Several pretreatment procedures have been studied in the case of heparin, which include fibronectin. Endothelial cell growth factor (ECGF),* methanol and hydrazine, hydrogen peroxide, and ammonium hydroxide. The crosslink agent 1-ethy1-3(3-dimethy1-aminopropy1) carbodiimide (EDO has been used to activate the carboxyl groups of the heparin structure. Heparin and plasmin have been co-immobi1ized on collagen powder and the activity of each has been studied in co-bound state. Experiments have shown that heparin in excess of 0.0 1 mg/sq cm can be immobilized on chymotrypsin treated GCA. that would remain stable for more than 3 weeks. In vivo studies have been performed on goats and dogs, the results of which show a patency rate of 807. after 1 week and 707Â« after 2 weeks, for the 10 samples that have been tested. Of all the pretreatment procedures, ammonium hydroxide appears to produce the most active and stable heparin complex. Co-immobi1ization of heparin and plasmin has shown that bound plasmin activity is increased if collagen powder is preimmobi1ized with heparin. Heparin, however, loses its activity. On the other hand, heparinization of plasmin bound collagen has not been successful. Furthermore, originally bound plasmin loses part of its activity. Experiments have been performed to determine the constants V^g>^ and K^j^ assuming M i chael i s-Menten kinetics for plasmin. The activation energy Eg has also been estimated for plasmin in soluble as well as immobilized form.