Quantification of free tissue-type plasminogen activator in plasma from patients undergoing coronary thrombolysis

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Background We have shown that the activity of tissue-type plasminogen activator (t-PA) in plasma is dependent on concentrations of free rather than total t-PA after administration of t-PA. The purpose of this study was to develop and validate a rapid immunoassay capable of quantifying free single- and two-chain t-PA in plasma from patients and experimental animals. Methods Monoclonal antibody specific for free t-PA was developed, purified, characterized, and used to develop a immunoassay for free t-PA antigen in plasma. Results Monoclonal antibody was demonstrated to attenuate t-PA activity and did not recognize complexes of t-PA with type-1 plasminogen activator inhibitor, C1 -esterase inhibitor and α2-macroglobulin. The interassay and intraassay coefficient of variations were approximately 10% and 5%, respectively. The lower limit of detection was 2 ng/mL. Quantification of the pharmacokinetics of free t-PA antigen in plasma samples from 18 patients with infarction given infusions of t-PA and experimental animals (n = 3) given intravenous injections of t-PA indicated that concentrations of t-PA activity in plasma correlated with concentrations of free t-PA antigen. Results obtained with the assay for free t-PA were not confounded by the presence of complexes of t-PA with inhibitors in contrast to results obtained with assay of total t-PA. Conclusions We have developed a rapid immunoassay capable of accurate and reproducible quantification of free t-PA in plasma. The availability of this assay should facilitate the evaluation of novel-dose regimens of t-PA and regimens involving the use of conjunctive agents.