Construction and characterization of a t-PA mutant for use in ATTEMPTS: a drug delivery system for achieving targeted thrombolysis.
Construction and characterization of a t-PA mutant for use in ATTEMPTS: a drug delivery system for achieving targeted thrombolysis.
复制标题
用于 ATTEMPTS 的 t-PA 突变体的构建和表征：用于实现靶向溶栓的药物输送系统。
DOI：
10.1016/j.jconrel.2005.09.027
复制
发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Liang,JunF
中科院分区：
文献类型：
--
作者：
Yang,VictorC;Naik,SaritaS;Song,Hui;Dombkowski,AlanA;Crippen,Gorden;Liang,JunF
To resolve the bleeding risk associated with thrombolytic therapy, we have designed an approach, termed ATTEMPTS (Antibody Targeted Triggered Electrically Modified Prodrug Type Strategy), to deliver t-PA to the clot site in an inactive form and then trigger its conversion to the active form, so that it would selectively activate the clot bound plasminogen while alleviating the bleeding risk. This delivery system was composed of a large protein complex, consisting of two components: (i) a heparin-modified, negatively charged fibrin-targeting antibody; and (ii) a cationic peptide-modified, positively charged t-PA. Both in vitro and in vivo studies have confirmed the feasibility of this targeted drug delivery approach. A site-specific thrombolysis was observed in animals, without concomitant depletion of the coagulation factors — the phenomenon in conventional thrombolytic therapy that contributes to the bleeding risk. Despite promise, the chemical conjugation method employed previously in the preparation of the cationic peptide-modified t-PA also revealed several major shortcomings. The primary drawback was that the number of the cationic peptides and the location at which these peptides were attached to a t-PA molecule could not be regulated by using the chemical conjugation method. As a consequence, the resultant modified t-PA possessed a wide range of heparin-binding strength, rendering the inhibition of t-PA activity by heparin binding ineffective. In this paper, we present a new strategy in producing the desired modified t-PA, utilizing the genetic engineering approach. A computer simulation-guided rational design strategy was adopted to identify the most desirable site in t-PA (i.e. the 37-loop) for incorporation of the heparin-binding peptide sequence. By altering the amino acid composition via mutation at three locations, i.e. Ser300to Cys, Gly302to Arg, and Glu303to Arg, a highly cationic nanomer sequence consisting of297KHRRCPRRR304and possessing a well-demonstrated heparin-binding domain was established within the 37-loop. To ensure the binding of heparin to this specifically modified domain, a cysteine residue (i.e. Cys300) was created to allow for site-specific conjugation of an additional heparin-binding peptide (i.e. the LMWP peptide previously developed in our laboratory) to this domain via the chemical conjugation method. In vitro fibrinolysis assays showed that both the t-PA mutant and the LMWP-attached t-PA mutant exhibited a fibrinolytic potency similar to that of the wild type t-PA. Inhibition studies using small chromogenic substrates demonstrated that the activity of mutant tPA-LMWP could be significantly inhibited by heparin binding. In conclusion, using computer simulation and molecular biology approaches, a mutated t-PA that meets the needs of the ATTEMPTS system, in providing a safe thrombolytic therapy, could be readily prepared.
