fibrin and thrombin-aptamer

We found for the first time that a thrombin-binding DNA aptamer (TBA) is selectively entrapped in fibrin gels during the gel growth reaction catalyzed by thrombin. Furthermore, using this phenomenon, we successfully demonstrated multiple incorporation of amphiphilic aliphatic groups into fibrin gels via chemically modified TBA.

Topics: Aptamers, Nucleotide; Base Sequence; Cell Proliferation; Fibrin; Fibrinogen; Gels; HeLa Cells; Humans; Microscopy, Fluorescence; Thrombin

Simple 2'-OMe-chemical modification in the loop region of the 15mer G-rich DNA sequence GGTTGGTGTGGTTGG is reported. The G-quadruplex structure of this thrombin-binding aptamer (TBA), is stabilized by single modifications (T → 2'-OMe-U), depending on the position of the modification. The structural stability also renders significantly increased inhibition of thrombin-induced fibrin polymerization, a process closely associated with blood-clotting.

Topics: Aptamers, Nucleotide; Binding Sites; Blood Coagulation; Dose-Response Relationship, Drug; Fibrin; G-Quadruplexes; Molecular Structure; Polymerization; Structure-Activity Relationship; Thrombin

Molecular intervention during transient stages of various metastatic pathways may lead to development of promising therapeutic technologies. One of such involves soluble fibrin (sFn) that has been implicated as a cross-linker between circulating blood or tumor cells and endothelial cell receptors, promoting cell arrest on the endothelium during circulation. sFn generation is a result of thrombin-mediated fibrinogen (Fg) cleavage due to either vascular injuries or a tumor microenvironment. For cancer therapy, thrombin-mediated conversions of Fg to sFn thus serve as potential intervention points to decrease circulating tumor cell adhesion to the endothelium and subsequent metastatic events. The purpose of this work was to investigate the function of an anti-thrombin oligonucleotide aptamer in reducing tumor cell arrest. Both molecular and cellular interactions were examined to demonstrate the binding and inhibitory effects of anti-thrombin aptamer. The results show that the aptamer is capable of inhibiting thrombin-mediated Fg conversion, thereby reducing sFn-mediated tumor cell adhesion in a concentration-dependent manner. Notably, the aptamer is able to bind thrombin under dynamic flow conditions and reduce tumor cell adhesive events at various physiological shear rates. This study further indicates that oligonucleotide aptamers hold great promise as therapeutic regulators of tumor cell adhesion, and consequently, metastatic activity.

Topics: Aptamers, Nucleotide; Cell Adhesion; Electrophoresis, Polyacrylamide Gel; Endothelium; Fibrin; Humans; Neoplasms; Surface Plasmon Resonance; Thrombin; Tumor Cells, Cultured

Topics: Allosteric Regulation; Amino Acid Chloromethyl Ketones; Aptamers, Nucleotide; Binding, Competitive; Dimerization; Fibrin; Protein Binding; Thrombin; Time Factors

We investigated the in vitro effects of the site-directed thrombin inhibitor-a single-stranded oligonucleotide aptamer (GGTTGGTGTGGTTGG)-on thrombin proteolytic activity towards its two natural substrates: fibrinogen and platelet thrombin receptor (PAR-1). The thrombin aptamer was shown to strongly affect thrombin clotting activity at nanomolar concentrations and thrombin-dependent degradation of proteolytically activatable receptor, PAR-1, exposed on platelet surface membrane at micromolar concentrations. The incubation of PPP with thrombin in the presence of 100-1000 nM aptamer resulted in the significant concentration-dependent prolongation of thrombin time (up to fourfold, P<.0001). Aptamer significantly reduced the thrombin-induced platelet degranulation (46+/-20% inhibition at 0.15 U/ml thrombin, P<.001), as well as thrombin-mediated platelet aggregation in PRP (7+/-10% inhibition at 1 U/ml thrombin, P<.05). Furthermore, aptamer inhibited the thrombin-catalysed cleavage of PAR-1 in a dose-dependent manner, i.e., by 17%, 27% and 70%, respectively, for the concentrations of 100, 500 and 1000 nM (P<.025 by randomised block analysis; P(regression slope)<.0001). We conclude that aptamer is able to considerably attenuate thrombin proteolytic activity regardless of the molecular size of thrombin substrates. Our observations directly proved that aptamer may be successfully used for the inhibition of thrombin activity towards various physiological targets: one related to fibrin generation in the final stage of coagulation cascade, and another concerning the interaction of thrombin with its surface membrane receptor, PAR-1, in blood platelets.

Topics: Adult; Aptamers, Nucleotide; Blood Coagulation Tests; Blood Platelets; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Fibrin; Flow Cytometry; Humans; Male; Middle Aged; Oligonucleotides; Platelet Aggregation; Receptor, PAR-1; Receptors, Thrombin; Thrombin