Near-Patient Thrombin Generation in Patients Undergoing Elective Cardiac Surgery.

BACKGROUND: Measuring thrombin generation (TG) in plasma increasingly gained attention as a diagnostic tool in the field of thrombosis and hemostasis. To include the contribution of all blood cells, recently, the whole blood TG method was developed. METHODS: We changed the calculation method of the standard calibrated automated thrombography (CAT) to a method only taking into account the data until the peak of TG, thereby considerably reducing the time from blood draw to result. By redesigning the method, the blood volume per test was reduced to 15 µL. RESULTS: For all TG parameters, the interassay variation proved to be below 15%. The interindividual variation of all parameters was comparable to the CAT method. Thirty-three patients undergoing cardiothoracic surgery were included to investigate whether our assay correlates with postoperative blood loss. On dividing patients into severe and mild bleeders, significant differences between both groups were found for the peak endogenous thrombin potential (peakETP) and peak values determined by our near-patient device. Importantly, patients with a peakETP below the median experienced significantly more blood loss compared to those with a peakETP above the median. A similar division based on the peak as well as the body mass index of the patient yielded similar significant differences. A combination of the peakETP, the body mass index, and the lag time even resulted in a better predictor of blood loss compared to each parameter separately. CONCLUSIONS: Our adapted whole blood TG assay can be used near patients and is indicative for the amount of blood loss post cardiothoracic surgery.

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network.

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.

Electrokinetic lab-on-a-biochip for multi-ligand/multi-analyte biosensing.

We present a simple electrokinetic lab-on-a-biochip (EKLB) with four microchannels integrated with a surface plasmon resonance imaging (iSPR) label-free biosensor that is operated using a single electrical voltage for the simultaneous transport of reagents in all microchannels without conventional fluidic plumbing. We demonstrate the utility of the simple approach with various biosensing experiments, including single injection kinetics (multiple varied ligand densities and single analyte concentration), one shot kinetics (single ligand densities and multiple varied analyte concentrations), and multi-ligand/multianalyte detection. In all cases, the binding kinetics and affinity were extracted using a conventional 1:1 interaction model. Since the reagent transport is done with a single electrical voltage source, scaling up to hundreds to thousands of simultaneous experiments is straightforward.

Electrokinetic label-free screening chip: a marriage of multiplexing and high throughput analysis using surface plasmon resonance imaging.

We present an electrokinetic label-free biomolecular screening chip (Glass/PDMS) to screen up to 10 samples simultaneously using surface plasmon resonance imaging (iSPR). This approach reduces the duration of an experiment when compared to conventional experimental methods. This new device offers a high degree of parallelization not only for analyte samples, but also for multiplex analyte interactions where up to 90 ligands are immobilized on the sensing surface. The proof of concept has been demonstrated with well-known biomolecular interactant pairs. The new chip can be used for high throughput screening applications and kinetics parameter extraction, simultaneously, of interactant-protein complex formation.

Integrated electrokinetic sample focusing and surface plasmon resonance imaging system for measuring biomolecular interactions.

Label-free biomolecular binding measurement methods, such as surface plasmon resonance (SPR), are becoming increasingly more important for the estimation of real-time binding kinetics. Recent advances in surface plasmon resonance imaging (iSPR) are emerging for label-free microarray-based assay applications, where multiple biomolecular interactions can be measured simultaneously. However, conventional iSPR microarray systems rely on protein printing techniques for ligand immobilization to the gold imaging surface and external pumps for analyte transport. In this article, we present an integrated microfluidics and iSPR platform that uses only electrokinetic transport and guiding of ligands and analytes and, therefore, requires only electrical inputs for sample transport. An important advantage of this new approach, compared to conventional systems, is the ability to direct a single analyte to a specific ligand location in the microarray, which can facilitate analysis parallelization. Additionally, this simple approach does not require complicated microfluidic channel arrangements, external pumps, or valves. As a demonstration, kinetics and affinity have been extracted from measured binding responses of human IgG and goat antihuman IgG using a simple 1:1 model and compared to responses measured with conventional pressure driven analyte transport. The measured results indicate similar binding kinetics and affinity between the electrokinetic and pressure-driven sample manipulation methods and no cross contamination to adjacent measurement locations has been observed.

Single injection microarray-based biosensor kinetics.

Binding affinity of biomolecular interactions can be directly extracted from measured surface plasmon resonance biosensor sensorgrams by fitting the data to the appropriate model equations. The conventional method for affinity estimation uses a series of analytes and buffers that are injected serially to a single immobilized ligand on the sensing surface, including a regeneration step between each injection, to generate information about the binding behavior. We present an alternative method to estimate the affinity using a single analyte concentration injected to multiple ligand densities in a microarray format. This parameter estimation method eliminates the need for multiple analyte injections and surface regeneration steps, which can be important for applications where there is limited analyte serum, fragile ligand-surface attachment, or the detection of multiple biomolecule interactions. The single analyte injection approach for binding affinity estimation has been demonstrated for two different interactant pairs, ß2 microglobulin/anti-ß2 microglobulin (ß2M) and human IgG/Fab fragments of anti-human IgG (hIgG), where the ligands are printed in a microarray format. Quantitative comparisons between the estimated binding affinities measured with the conventional method are ß2M: KD = 1.48 ± 0.28 nM and hIgG: KD = 12.6 ± 0.2 nM and for the single injection method are ß2M: KD = 1.52 ± 0.22 nM and hIgG: KD = 12.5 ± 0.6 nM, which are in good agreement in both cases.