Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrin-dependent plasmin generation on thrombin-activated platelets.

BACKGROUND: Thrombin-activated platelets can promote fibrinolysis by binding plasminogen in a fibrinogen-dependent manner and enhancing its activation by tissue-type plasminogen activator (t-PA). Whether t-PA also binds to activated platelets and the mechanism for regulation of platelet-dependent fibrinolysis remain unknown. OBJECTIVES: Determine the mechanism of plasminogen and t-PA binding on thrombin-activated platelets and its regulation by activated thrombin-activatable fibrinolysis inhibitor (TAFIa). METHODS: Plasminogen and t-PA binding with or without TAFIa treatment was quantified using flow cytometry. Plasmin generation on platelets was quantified using a plasmin-specific substrate. Mass spectrometry analyses identified fibrinogen as a potential target of TAFIa. Thrombus formation was studied in mice lacking fibrinogen (Fg-/- ) using intravital microscopy. RESULTS: Plasminogen and t-PA bind to platelets activated by thrombin but not by other agonists, including protease-activated receptor agonists (PAR-AP). Plasminogen binds via its kringle domains because ε-aminocaproic acid eliminates binding, whereas t-PA binds via its finger and kringle domains. Plasminogen binding is fibrinogen-dependent because it is abolished on (a) Fg-/- platelets, and (b) thrombi in Fg-/- mice. Binding requires thrombin-mediated fibrinogen modification because addition of batroxobin to PAR-AP activated platelets has no effect on plasminogen binding but induces t-PA binding. TAFIa reduces plasminogen and t-PA binding to thrombin-activated platelets and attenuates plasmin generation in a concentration-dependent manner. Mass spectrometry identified K556 on the fibrinogen alpha-chain as a potential thrombin cleavage site that generates a TAFIa sensitive C-terminal lysine residue. CONCLUSION: These findings provide novel mechanistic insights into how platelets activated by thrombin at sites of vascular injury can influence fibrinolysis.

A proof of concept application of aptachain: ligand-induced self-assembly of a DNA aptamer.

A challenge for the use of aptamers as biosensors is how to signal the occurrence of their ligand binding event into a signal that can be exploited in a detection scheme. Here, we present the concept of "aptachain" formation, where an aptamer is split into two overlapping or staggered strands and assembles into an extended oligomer upon ligand binding. This assembly of aptamers can then be used as a way to detect ligand binding by the aptamer. As an example of this concept, we employed the cocaine-binding aptamer as a model system, used its ability to tightly bind quinine and demonstrated its capability in a gold nanoparticle-based biosensing application. We used isothermal titration calorimetry to demonstrate that, when split into two overlapping DNA strands, the aptamer remains functional. Size-exclusion chromatography showed that the quinine-bound oligos form a larger assembly of aptamer units than in the absence of ligand. Finally, we used the oligomer forming ability of the aptachain oligos in a biosensor application for quinine that brings gold nanoparticles closer together resulting in a shift in their plasmonic resonance to a longer wavelength and an observed colour shift. We propose that splitting aptamers into overlapping strands that form oligomers in the presence of a ligand, aptachain formation, will be generally applicable to aptamers and prove useful in a variety of biotechnology applications.

Apolipoprotein A-IV binds αIIbß3 integrin and inhibits thrombosis.

Platelet αIIbß3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet ß3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbß3 integrin, and the direct apoA-IV-αIIbß3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbß3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbß3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases.

Optimizing Stem Length To Improve Ligand Selectivity in a Structure-Switching Cocaine-Binding Aptamer.

Understanding how aptamer structure and function are related is crucial in the design and development of aptamer-based biosensors. We have analyzed a series of cocaine-binding aptamers with different lengths of their stem 1 in order to understand the role that this stem plays in the ligand-induced structure-switching binding mechanism utilized in many of the sensor applications of this aptamer. In the cocaine-binding aptamer, the length of stem 1 controls whether the structure-switching binding mechanism for this aptamer occurs or not. We varied the length of stem 1 from being one to seven base pairs long and found that the structural transition from unfolded to folded in the unbound aptamer is when the aptamer elongates from 3 to 4 base pairs in stem 1. We then used this knowledge to achieve new binding selectivity of this aptamer for quinine over cocaine by using an aptamer with a stem 1 two base pairs long. This selectivity is achieved by means of the greater affinity quinine has for the aptamer compared with cocaine. Quinine provides enough free energy to both fold and bind the 2-base pair-long aptamer while cocaine does not. This tuning of binding selectivity of an aptamer by reducing its stability is likely a general mechanism that could be used to tune aptamer specificity for tighter binding ligands.

Comparison of the free and ligand-bound imino hydrogen exchange rates for the cocaine-binding aptamer.

Using NMR magnetization transfer experiments, the hydrogen exchange rate constants (k ex ) of the DNA imino protons in the cocaine-binding aptamer have been determined for the free, cocaine-bound, and quinine-bound states. The secondary structure of the cocaine-binding aptamer is composed of three stems built around a three-way junction. In the free aptamer the slowest exchanging imino protons are located in the middle of the stems. The highest k ex values were found for a nucleotide in the GAA loop of stem 3 and for nucleotides at the end of the stems that form the three-way junction structure and in the tandem GA mismatch. Upon ligand binding, the k ex values of nucleotides at the ligand binding site are reduced, indicating that these base pairs become more stable or less solvent accessible in the bound state. The imino proton k ex values of nucleotides located away from the binding site are only minimally affected by ligand binding.

Salt-mediated two-site ligand binding by the cocaine-binding aptamer.

Multisite ligand binding by proteins is commonly utilized in the regulation of biological systems and exploited in a range of biochemical technologies. Aptamers, although widely utilized in many rationally designed biochemical systems, are rarely capable of multisite ligand binding. The cocaine-binding aptamer is often used for studying and developing sensor and aptamer-based technologies. Here, we use isothermal titration calorimetry (ITC) and NMR spectroscopy to demonstrate that the cocaine-binding aptamer switches from one-site to two-site ligand binding, dependent on NaCl concentration. The high-affinity site functions at all buffer conditions studied, the low-affinity site only at low NaCl concentrations. ITC experiments show the two ligand-binding sites operate independently of one another with different affinities and enthalpies. NMR spectroscopy shows the second binding site is located in stem 2 near the three-way junction. This ability to control ligand binding at the second site by adjusting the concentration of NaCl is rare among aptamers and may prove a useful in biotechnology applications. This work also demonstrates that in vitro selected biomolecules can have functions as complex as those found in nature.

Utilizing a Key Aptamer Structure-Switching Mechanism for the Ultrahigh Frequency Detection of Cocaine.

Aptasensing of small molecules remains a challenge as detection often requires the use of labels or signal amplification methodologies, resulting in both difficult-to-prepare sensor platforms and multistep, complex assays. Furthermore, many aptasensors rely on the binding mechanism or structural changes associated with target capture by the aptameric probe, resulting in a detection scheme customized to each aptamer. It is in this context that we report herein a sensitive cocaine aptasensor that offers both real-time and label-free measurement capabilities. Detection relies on the electromagnetic piezoelectric acoustic sensor (EMPAS) platform. The sensing interface consists of a S-(11-trichlorosilyl-undecanyl)benzenethiosulfonate (BTS) adlayer-coated quartz disc onto which a structure-switching cocaine aptamer (MN6) is immobilized, completing the preparation of the MN6 cocaine aptasensor (M6CA). The EMPAS system has recently been employed as the foundation of a cocaine aptasensor based on a structurally rigid cocaine aptamer variant (MN4), an aptasensor referred to by analogy as M4CA. M6CA represents a significant increase in terms of analytical performance, compared to not only M4CA but also other cocaine aptamer-based sensors that do not rely on signal amplification, producing an apparent K(d) of 27 ± 6 µM and a 0.3 µM detection limit. Remarkably, the latter is in the range of that achieved by cocaine aptasensors relying on signal amplification. Furthermore, M6CA proved to be capable not only of regaining its cocaine-binding ability via simple buffer flow over the sensing interface (i.e., without the necessity to implement an additional regeneration step, such as in the case of M4CA), but also of detecting cocaine in a multicomponent matrix possessing potentially assay-interfering species. Finally, through observation of the distinct shape of its response profiles to cocaine injection, demonstration was made that the EMPAS system in practice offers the possibility to distinguish between the binding mechanisms of structure-switching (MN6) vs rigid (MN4) aptameric probes, an ability that could allow the EMPAS to provide a more universal aptasensing platform than what is ordinarily observed in the literature.

Ultra-high frequency piezoelectric aptasensor for the label-free detection of cocaine.

This paper describes a label-free and real-time piezoelectric aptasensor for the detection of cocaine. The acoustic wave sensing platform is a quartz substrate functionalized with an adlayer of S-(11-trichlorosilyl-undecanyl)-benzenethiosulfonate (BTS) cross-linker onto which the anti-cocaine MN4 DNA aptamer is next immobilized. Preparation of the sensor surface was monitored using X-ray photoelectron spectroscopy (XPS), while the binding of cocaine to surface-attached MN4 was evaluated using the electromagnetic piezoelectric acoustic sensor (EMPAS). The MN4 aptamer, unlike other cocaine aptamer variants, has its secondary structure preformed in the unbound state with only tertiary structure changes occurring during target binding. It is postulated that the highly sensitive EMPAS detected the binding of cocaine through target mass loading coupled to aptamer tertiary structure folding. The sensor achieved an apparent Kd of 45 ± 12 µM, and a limit of detection of 0.9 µM. Repeated regenerability of the sensor platform was also demonstrated. This work constitutes the first application of EMPAS technology in the field of aptasensors. Furthermore, it is so far one of the very few examples of a bulk acoustic wave aptasensor that is able to directly detect the binding interaction between an aptamer and a small molecule in a facile one-step protocol without the use of a complex assay or signal amplification step.

Adlayer-mediated antibody immobilization to stainless steel for potential application to endothelial progenitor cell capture.

This work describes the straightforward surface modification of 316L stainless steel with BTS, S-(11-trichlorosilylundecanyl)-benzenethiosulfonate, a thiol-reactive trichlorosilane cross-linker molecule designed to form intermediary coatings with subsequent biofunctionalization capability. The strategy is more specifically exemplified with the immobilization of intact antibodies and their Fab' fragments. Both surface derivatization steps are thoroughly characterized by means of X-ray photoelectron spectroscopy. The antigen binding capability of both types of biofunctionalized surfaces is subsequently assessed by fluorescence microscopy. It was determined that BTS adlayers achieve robust immobilization of both intact and fragmented antibodies, while preserving antigen binding activity. Another key finding was the observation that the Fab' fragment immobilization strategy would constitute a preferential option over that involving intact antibodies in the context of in vivo capture of endothelial progenitor cells in stent applications.

Engineering a structure switching mechanism into a steroid-binding aptamer and hydrodynamic analysis of the ligand binding mechanism.

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.

Defining the secondary structural requirements of a cocaine-binding aptamer by a thermodynamic and mutation study.

Isothermal titration calorimetry (ITC) was used to measure the binding affinity and thermodynamics of a cocaine-binding aptamer as a function of pH and NaCl concentration. Tightest binding was achieved at a pH value of 7.4 and under conditions of no added NaCl. These data indicate that ionic interactions occur in the ligand binding mechanism. ITC was also used to measure the binding thermodynamics of a variety of sequence variants of the cocaine-binding aptamer that analyzed which regions and nucleotides of the aptamer are important for maintaining high-affinity binding. Individually, each of the three stems can be shortened, resulting in a reduced binding affinity. If all three stems are shortened, no binding occurs. If all three of the stems in the aptamer are lengthened by five base pairs ligand affinity increases. Changes in nucleotide identity at the three-way junction all decrease the affinity of the aptamer to cocaine. The greatest decrease in affinity results from changes that disrupt the GA base pairs and the identity of T19.

Defining a stem length-dependent binding mechanism for the cocaine-binding aptamer. A combined NMR and calorimetry study.

We have used a combined approach of NMR spectroscopy and isothermal titration calorimetry (ITC) to determine the ligand-binding mechanism employed by a cocaine-binding aptamer. We found that the length of the stem containing the 3' and 5' termini determines the nature of the binding mechanism. When this stem is six base pairs long, the secondary structure of the aptamer is fully folded in the free form and only putative tertiary interactions form with ligand binding. If this stem is shortened by three base pairs, the free form of the aptamer contains little secondary structure, and ligand binding triggers secondary structure formation and folding. This binding mechanism is supported by both NMR spectral changes and the ITC measured heat capacity of binding (ΔC(p)°). For the aptamer with the long stem the ΔC(p)° value is -557 ± 29 cal mol(-1) K(-1) and for the aptamer with the short stem the ΔC(p)° value is -922 ± 51 cal mol(-1) K(-1). Chemical shift perturbation data and the observation of intermolecular NOEs indicate that the three-way junction is the site of ligand binding.