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Structure and Function of Trypsin-Loaded Fibrinolytic Liposomes.

ABSTRACT: Protease encapsulation and its targeted release in thrombi may contribute to the reduction of haemorrhagic complications of thrombolysis. We aimed to prepare sterically stabilized trypsin-loaded liposomes (SSL_T) and characterize their structure and fibrinolytic efficiency. Hydrogenated soybean phosphatidylcholine-based SSL_T were prepared and their structure was studied by transmission electron microscopy combined with freeze fracture (FF-TEM), Fourier transform infrared spectroscopy (FT-IR), and small-angle X-ray scattering (SAXS). Fibrinolytic activity was examined at 45, 37, or 24°C on fibrin or plasma clots with turbidimetric and permeation-driven lysis assays. Trypsin was shown to be attached to the inner surface of vesicles (SAXS and FF-TEM) close to the lipid hydrophilic/hydrophobic interface (FT-IR). The thermosensitivity of SSL_T was evidenced by enhanced fibrinolysis at 45°C: time to reduce the maximal turbidity to 20% decreased by 8.6% compared to 37°C and fibrin degradation product concentration in the permeation lysis assay was 2-fold to 5-fold higher than that at 24°C. SSL_T exerted its fibrinolytic action on fibrin clots under both static and dynamic conditions, whereas plasma clot dissolution was observed only in the permeation-driven assay. The improved fibrinolytic efficiency of SSL_T under dynamic conditions suggests that they may serve as a novel therapeutic candidate for dissolution of intravascular thrombi, which are typically exposed to permeation forces.

SUBMITTER: Tanka-Salamon A

PROVIDER: S-EPMC5512056 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Structure and Function of Trypsin-Loaded Fibrinolytic Liposomes.

Tanka-Salamon Anna A Bóta Attila A Wacha András A Mihály Judith J Lovas Miklós M Kolev Krasimir K

BioMed research international 20170703

Protease encapsulation and its targeted release in thrombi may contribute to the reduction of haemorrhagic complications of thrombolysis. We aimed to prepare sterically stabilized trypsin-loaded liposomes (SSL<sub>T</sub>) and characterize their structure and fibrinolytic efficiency. Hydrogenated soybean phosphatidylcholine-based SSL<sub>T</sub> were prepared and their structure was studied by transmission electron microscopy combined with freeze fracture (FF-TEM), Fourier transform infrared spe ...[more]

PMID: 28758116

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Project description:Microbial fibrinolytic proteases are therapeutic enzymes responsible to ameliorate thrombosis, a fatal cardiac-disorder which effectuates due to excessive fibrin accumulation in blood vessels. Inadequacies such as low fibrin specificity, lethal after-effects and short life-span of available fibrinolytic enzymes stimulates an intensive hunt for novel, efficient and safe substitutes. Therefore, we herewith suggest a novel and potent fibrinolytic enzyme RFEA1 from Bacillus cereus RSA1 (MK288105). Although, attributes such as in-vitro purification, characterization and thrombolytic potential of RFEA1 were successfully accomplished in our previous study. However, it is known that structure-function traits and mode of action significantly aid to commercialization of an enzyme. Also, predicting structural model of a protein from its amino acid sequence is challenging in computational biology owing to intricacy of energy functions and inspection of vast conformational space. Our present study thus reports In-silico structural-functional analysis of RFEA1. Sequence based modelling approaches such as-Iterative threading ASSEmbly Refinement (I-TASSER), SWISS-MODEL, RaptorX and Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) were employed to model three-dimensional structure of RFEA1 and the modelled RFEA1 was validated by structural analysis and verification server (SAVES v6.0). The modelled crystal structure revealed the presence of high affinity Ca1 binding site, associated with hydrogen bonds at Asp147, Leu181, Ile185 and Val187residues. RFEA1 is structurally analogous to Subtilisin E from Bacillus subtilis 168. Molecular docking analysis using PATCH DOCK and FIRE DOCK servers was performed to understand the interaction of RFEA1 with substrate fibrin. Strong RFEA1-fibrin interaction was observed with high binding affinity (-21.36 kcal/mol), indicating significant fibrinolytic activity and specificity of enzyme RFEA1. Overall, the computational research suggests that RFEA1 is a subtilisin-like serine endopeptidase with proteolytic potential, involved in thrombus hydrolysis.

Dataset Information

Structure and Function of Trypsin-Loaded Fibrinolytic Liposomes.

Publications

Structure and Function of Trypsin-Loaded Fibrinolytic Liposomes.

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