Thrombin In Samples Research Articles

Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

IntroductionHematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment.We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. MethodsThis is a single-centre, case-control, non-interventionist, prospective study.During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed:Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway.Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software.TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA).The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. ResultsThose patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests.Table 1 shows the results obtained.Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI.Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI.Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. ConclusionsUBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient.Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes.TFPI inhibition might emerge as a good therapy for some of these patients.Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field.This work was supported by Novo Nordisk Pharma S.A.Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LTlagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. [Display omitted] DisclosuresAlvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Thrombin Bionsensors: A Rapid and Ultrasensitive Thrombin Biosensor Based on a Rationally Designed Trifunctional Protein (Adv. Healthcare Mater. 12/2020)

In article number 2000364 by Yanyan Wang, Zefang Wang, and co-workers, an ultrasensitive and rapid thrombin biosensor is developed based on rationally designed trifunctional protein HTs, comprising three functional units, including a far-red fluorescent protein smURFP, hydrophobin HGFI, and a thrombin cleavage site (TCS). smURFP is used as a detection signal to eliminate any interference from the autofluorescence of sample matrix to increase detection sensitivity. HGFI serves as an adhesive unit to allow rapid immobilization of HTs on a multiwall plate. The TCS linking HGFI and smURFP functions as a sensing element to recognize and detect thrombin. As a result, the intensity of the fluorescent signal on the surface of multiwall plates changes depending on the concentration of thrombin. The sensing mechanism of this biosensor relies on the ability of active thrombin in samples to specifically recognize and hydrolyze its cleavage site in the fusion protein, resulting in a subsequent decrease in the fluorescence intensity of the sample.

A Rapid and Ultrasensitive Thrombin Biosensor Based on a Rationally Designed Trifunctional Protein.

Rapid and sensitive detection of thrombin is imperative for the early diagnosis, prevention, and treatment of thrombin-related diseases. Here, an ultrasensitive and rapid thrombin biosensor is developed based on rationally designed trifunctional protein HTs, comprising three functional units, including a far-red fluorescent protein smURFP, hydrophobin HGFI, and a thrombin cleavage site (TCS). smURFP is used as a detection signal to eliminate any interference from the autofluorescence of sample matrix to increase detection sensitivity. HGFI serve as an adhesive unit to allow rapid immobilization of HTs on a multiwall plate. The TCS linking HGFI and smURFP function as a sensing element to recognize and detect thrombin. HTs immobilization is symmetrically optimized and characterized. Thrombin assay reveals the specific recognition of active thrombin in samples and the hydrolysis of the immobilized HTs, resulting in a decrease in the fluorescence intensity of the sample in a thrombin concentration-dependent manner. The limit of detection (LOD) is as low as 0.2 am in the serum. To the authors' knowledge, this is the lowest LOD ever reported for any thrombin biosensor. This study sheds light on the engineering of multifunctional proteins for biosensing.

Rapid and reagentless detection of thrombin in clinic samples via microfluidic aptasensors with multiple target-binding sites

A reusable and straightforward aptasensor with the implementation of open-ended porous silicon (OEPSi) membranes was introduced for thrombin detection. When passing through the nanochannels of OEPSi integrated in a microfluidic cell, thrombin in sample solution could be captured by thrombin-binding aptamers (TBA) immobilized along the inner walls. The formation of thrombin-aptamer complex causes refractive index changes which can be measured by reflective interferometric Fourier transform spectroscopy (RIFTS). And this flow-through configuration with OEPSi has proven more efficient in capturing thrombin than the flow-over configuration with closed-ended PSi. For higher sensitivity, we investigated how the pore size, ionic strength, pH and aptamers affected the thrombin-aptamer interaction in nanopores. Under optimized conditions, the limits of detection (LOD) for thrombin detection in the buffer and serum were ∼6.70 nM and ∼8.21 nM respectively and a wide linear detection range (10–1000 nM) was observed. More importantly, this work reveals the sensitivity of the label-free biosensor can be significantly improved by attaching newly designed aptamers with two thrombin-binding sites. This phenomenon also indicates the potential of aptamer probes in adjusting effective pore size and enhancing the interaction between aptamers and targets through meticulous sequence design. Furthermore, the proposed strategy has been applied in thrombin detection in clinic samples successfully, which was verified by Enzyme-Linked Immunosorbent Assays (ELISA).

Bi-cell surface plasmon resonance detection of aptamer mediated thrombin capture in serum

The serine protease coagulation factor thrombin functions primarily in hemostasis, but is also involved in atherosclerosis, thromboembolic disease, cancer and inflammatory disease. Direct measurement of coagulation proteins including thrombin in plasma samples poses a significant challenge because of lack of specific probes and low thrombin concentrations. In addition, high plasma protein concentrations in samples can result in high backgrounds. These challenges were overcome using a bi-cell surface plasmon resonance (SPR) spectrometer with an immobilized thrombin aptamer to measure thrombin in samples passed through a low volume flow cell. For thrombin in Tris–EDTA buffer, the limit of detection (LOD) was 25 nM. Coefficient of variation (CV) for detection of 50 nM was 12.2% and 12.4% for intra and inter-day measurements respectively. This detection was specific for both thrombin aptamer and for thrombin. Using serum samples spiked with thrombin, the LOD was 50 nM with a linear range of detection from 50 nM to 200 nM. However use of serum samples was associated with consistent, low-level background drift. The contributions of nonspecific protein absorption onto the sensor surface and sample flow speed were assessed, and strategies to reduce this background drift were explored. We conclude that the bi-cell SPR platform with an aptamer capture probe can be employed as a highly sensitive real-time, label-free biosensor for the detection of coagulation factors in plasma samples.

First Experiences with the Measurement of Thrombin Generation (TGA) on the Ceveron® Alpha in the Routine Lab

Aims: Aim of the study was to show the suitability of the new coagulation analyzer Ceveron® alpha for determination of thrombin generation (TGA) in routine laboratories for discrimination of normal plasma samples from haemophilic and thrombophilic patient samples, from patients with a Lupus syndrome and patients with bleeding disorders.Methods: TGA was determined by a thrombin generation assay based on monitoring the formation of thrombin by means of a fluorigenic substrate upon activation of the coagulation cascade by tissue factor and phospholipids using 2 reagents with different phospholipid concentrations (RClow and RChigh) on Ceveron® alpha. Thrombin generation is measured with a specially adapted TGA fluorimetric module which is placed over the cuvette rotor. Readout parameters were lag time (tLag), peak thrombin (Peak), time to peak(tPeak), velocity index (VI) and area under the curve (AUC). In case of patients with Lupus Anticoagulants a ratio of peak thrombin values triggered with RClow and RChigh was formed. Four laboratory groups participated in the study. All patient samples used for thrombin generation measurement were characterized by routine parameters.Results: Group 1 analyzed samples with bleeding disorders. Peak thombin values showed the best discrimination between Hemophilia B patients (41.9, SD12.7nM), Hemophilia B patient after treatment (188.9nM) and normal controls (208.5nM, SD= 74.0). In Hemophilia A patients tLag was the best parameter to discriminate severe Hemophilia A (8.5min) from acquired hemophilia A and VWD (4.6 min, SD=2.1).Normal ranges for TGA established in group 1 (n=16) were tLag 2.7min (SD=0.8), tPeak 7.8min (SD=1.9), peak thrombin 208.5nM (SD= 74), AUC 1769.4 (SD=327.9).Group 2 analyzed samples of thrombophilic patients with pregnancy (n=30). Patient samples showed peak thrombin values (310.9nM, SD= 65.7) above normals (174.2nM, SD= 22.5). Even patients with heparin treatment had high peak thrombin values (340.5nM, SD=67.2). In heparin treated patients D-dimer values were pathologic as well.Group 3 determined thrombin generation in patients with Lupus Anticoagulants determined by dRVVT, APTT-LA, Lupus-Ceveron and anti-cardiolipin antibodies (ACA). Patients only with high ACA values showed normal peak thrombin ratios of RClow/RChigh 0.6 (SD= 0.07) whereas the ratio in patients with high lupus anticoagulants as measured in the coagulation tests was 0.98 (SD= 0.31) for high antibody titer and 0.83 (SD 0.01) for low antibody titer. Inter-assay CVs in the normal control determined by this laboratory were 2.67% for peak thrombin and 4.83% for AUC and in thrombophilic plasma 3.0% for peak thrombin and 1.41% for AUC.Group 4 determined TGA in orally anticoagulated patients. Normal range for peak thrombin established in this laboratory is 220nM (SD=52, n=12). Oral anticoagulant treatment lowered the AUC (from 1713.3nM, SD= 435.4 thrombin in samples with INR in the range 1 – 1.5 to 894.4nM, SD= 229.2 thrombin in samples with INR in the range 2 – 2.5) and peak thrombin formation (from 204.1nM, SD= 73.5 peak thrombin in samples with INR in the range 1 –1.5 to 79.0nM, SD= 35.2 peak thrombin in samples with INR in the range 2 – 2.5) and prolonged the tLag (from 5.5min, SD= 1.7 in samples with INR in the range 1 –1.5 to 7.6min, SD= 2.4 in samples with INR in the range 2 – 2.5.Conclusions: TGA can be measured reproducibly with low variation under routine laboratory conditions. Peak thrombin is the best parameter for discrimination of patient samples from normals in haemophiliacs, in thrombophilia, patients with a Lupus syndrome, pregnancy and bleeding disorders. With automated, standardised thrombin generation measurement on the Ceveron® alpha large prospective studies can be conducted to confirm these results.