Monoclonal antibody R11D10 to human cardiac myosin, which also cross-reacted with canine cardiac myosin, was used to demonstrate in vivo localization and visualization by gamma scintigraphy of experimental myocardial infarction. R11D10 Fab with a Ka of 5 X 10(8) M-1 was labeled with technetium-99m (99mTc) by the dithionite reduction method of technetium pertechnetate, via a bifunctional chelating agent, diethylene triamine pentaacetic acid (DTPA). Uptake of 99mTc R11D10 Fab in the infarct can be visualized as early as 2 h after intravenous administration. Comparison of R11D10 uptake to thallium-201, an analogue of potassium which is sequestered by normal myocardium, showed an inverse relation (r = -0.75, -0.87, -0.89), similar to that obtained with 125I labeled polyclonal antimyosin Fab. Ratios of R11D10 Fab in the infarct to normal myocardium were as high as 30:1 where access of antibody to antigen was not blood flow limited. However, with severe blood-flow restriction, the ratios were lower at about 10:1. Despite the theoretical limitation of a single epitope per myosin molecule available for binding by R11D10 Fab, the immense excess of myosin in the infarcted myocardium allowed adequate concentration of radiolabeled R11D10 for visualization of the infarct by external gamma scintiscanning.
Summaryrt-PA-K, a variant of recombinant tissue-type plasminogen activator (rt-PA) with substitution of amino acids 296 to 299 with alanine (KHRR296-299AAAA) has increased fibrin-specificity and reduced sensitivity to plasminogen activator inhibitor-1; rt-PA-T, with threonine 103 replaced by asparagine has an additional glycosylation site and a reduced clearance; and rt-PA-N, with asparagine 117 mutagen-ized to glutamine lacks the high mannose carbohydrate side chain. We have investigated whether combination of these properties in a single molecule might yield an improved thrombolytic agent.The thrombolytic potency and fibrin-specificity of the combination mutant rt-PA-TNK was compared with that of rt-PA in a combined venous whole blood clot model and platelet-rich arterial eversion graft thrombosis model in dogs given intravenous heparin and aspirin. Infusion of 0.125 to 1.0 mg/kg over 60 min in groups of 4 to 5 dogs produced dose-dependent fibrin-specific venous clot lysis. The thrombolytic potency (percent lysis per mg compound administered per kg body weight) of rt-PA-TNK was significantly higher than that of rt-PA as evidenced by a higher maximal rate of lysis of 480 ± 100% versus 140 ± 40% within the 2 h observation period per mg of compound administered per kg body weight (mean ± SEM, p = 0.004) and a significantly lower dose of 0.08 ± 0.01 versus 0.21 ± 0.04 mg/kg body weight at which the maximal rate of lysis was obtained (p = 0.004). This higher thrombolytic potency was the result of a significantly reduced clearance (240 ± 32 versus 540 ± 49 ml/min, p = 0.002) and a similar specific thrombolytic activity (percent lysis per |ig/ml plasma antigen level). Arterial reflow was obtained with 1 mg/kg rt-PA and with 0.5 mg/kg rt-PA-TNK, but with each agent recanalization was consistently associated with cyclic reocclusion and reflow. The frequency of arterial recanalization was somewhat higher with rt-PA-TNK (10/12) than with rt-PA (4/12) (p = 0.07) but the total patency times during a 2 h observation period were similar.
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