SummaryIn a continuing effort to create an agent which has both thrombolytic and antithrombotic properties, streptokinase (SK) was covalently bound to the potent antithrombin agent recombinant hirudin (rHir). Linkage of SK to 125I-rHir was accomplished via heterobifunctional crosslinkers in an average molar ratio of 1:1. The 125I-rHir-SK complex was purified from starting components by anion exchange and gel filtration chromatography. The major band containing covalently bound 125I-rHir had a molecular weight of 53 kDa as determined by SDS-PAGE and autoradiography. Biologic activity of each component was then assayed utilizing the chromogenic substrate for each compound. Complex bound 125I-rHir exhibited a 1.2 fold decrease in thrombin inhibition when compared to concentrations of, 25I-rHir greater than 3.13 nM. Complex bound i25I-SK, replacing the 125I label on rHir, displayed a 7.9-fold loss in plasminogen activation when compared to l25I-SK. These chromogenic assay results were not adversely altered in the presence of the converse compound’s substrate. The 125I-SK-rHir complex (examined at various concentrations) also demonstrated a 0. 17- to 17-fold greater affinity for thrombin immobilized onto Sepha- rose beads as compared to 125I-SK. These findings indicate the rHir-SK complex maintained both thrombolytic and antithrombin properties while also obtaining affinity for immobilized thrombin.
The purpose of this study was to examine the effect of the in vivo maturing ePTFE graft surface on platelet activation. Ten canines were randomized to receive either a carotid to infrarenal aorta ePTFE graft or sham operation. Animals were sampled at specific time points up to 3 months postoperatively. Whole blood platelet aggregometry (arachidonic acid, ADP, and collagen agonists) and ATP secretion (in response to arachidonic acid, ADP, collagen, and thrombin) were measured. Additionally, complete hematologic analysis and histology were performed. With time, graft animals showed significantly more decrease in platelet aggregation in response to ADP compared to sham animals (P = .023). The total amount of ATP per platelet was not different, as demonstrated by equivalent ATP release per platelet in response to thrombin. Over the first week, grafted dogs developed a decrease in systemic platelet count of 50% (P < .001) that persisted over the 3-month follow-up period. With time, overall regression model slopes of graft and sham platelet count data were not statistically different (P = .29). Histologically, the grafts demonstrated limited cellular ingrowth at both anastomoses, with fibrin matrix along the remainder of the blood-biomaterial interface. These data suggest that, similar to Dacron, exposure to an ePTFE surface results in significant changes in platelet biology, and these platelet-ePTFE interactions persist even after the graft has formed a mature pseudointima. The pseudointima appears to be the primary determinant of the blood-biomaterial interaction.
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