Thrombin Binding Site Research Articles

High-density aptamer synthesis based on primer exchange reaction for label-free electrochemical biosensing

We report an electrochemical aptamer-based (E-AB) sensor for label-free quantitative detection of thrombin. In-situ synthesis of high-density aptamer replicas on the electrode is accomplished based on primer exchange reaction (PER), by which a DNA primer is continuously extended with the presence of a catalytic hairpin template and DNA polymerase. The PER enables the synthesis of long single-stranded DNA with replicated aptamer sequence for thrombin binding. The PER products on the surface of a gold electrode increase the density of aptamers on a limited electrode area, which can provide more effective binding sites for thrombin and therefore improve the performance of the sensor based on electrochemical impedance spectroscopy. Compared with the conventional E-AB sensor based on a 15-mer aptamer immobilized on the electrode, our method has a lower detection limit of 0.91 pM, and a wider linear range of 1.0 pM to 50 nM. Therefore, we believe our method is promising for label-free electrochemical detection of biomarkers.

Diversity of Plasmatic Thrombin Pools Regulate Thrombin Generation and Blood Clotting: Interference By a Novel Nanobody

Background: Thrombin has a multitude of roles in the initiation and propagation of (anti)coagulation, in platelet activation and fibrin clot formation. The assessment of thrombin generation (TG) and thrombin-dynamics, combined with multi-omics data, has provided in-depth understanding of the plasma factors contributing to the (in)activation of thrombin in triggered plasma and whole-blood. By default, the generated thrombin is considered to act as a single pool. Here, we present evidence for at least two major proteolytically active thrombin pools with non-overlapping functions in blood clotting. Methods: A novel Syn-Nb-AF106 anti-fibrin nanobody (Nb106) was characterized, targeting a species-conserved thrombin-binding site exposed by fibrinopeptide A cleavage from fibrinogen (Sun et al, this meeting). Thrombin generation was assessed by calibrated automated thrombography using various triggers with control (multi-donor) and (reconstituted) coagulation factor-deficient plasmas, platelet-rich plasma or whole blood. Fibrin clot formation was evaluated by scanning electron microscopy (SEM). Clot retraction was macroscopically assessed. Results: In tissue factor-triggered thrombin generation,we discovered that Nb106 dose-dependently reduced the thrombin peak level and endogenous thrombin potential (ETP) to 54-62%, without affecting kinetic thrombin generation parameters. We observed a similar reduction in platelet-free plasma, platelet-rich plasma and whole blood. Biochemically, Nb106 displaced thrombin from a binding site on cleaved fibrinogen (Sun et al., abstract). Nb106 inhibited tissue factor-induced thrombin generation, using plasma deficient in factor IX, XI or XII, but not using plasmas deficient in fibrinogen or antithrombin. Reconstitution experiments showed that the inhibition depended on the fibrinogen concentration. A-specific control nanobodies were without effect. Together, the fibrin(ogen)-dependent and thrombin-inhibiting effect indicated that Nb106 operates by releasing proteolytically-active thrombin from fibrin to allow inactivation by antithrombin. Time-dependent, late spiking experiments revealed that Nb106 reduced the generation of thrombin only when added at 0-15 minutes after trigger, but no longer after 30 minutes, suggesting that the inhibitory capacity was due to a shortened thrombin binding to fibrin rather than to a continued protection towards antithrombin. In plasmas from a cohort of 64 healthy subjects, application of Nb106 showed a consistent reducing effect on the parameters peak and ETP, with a moderate positive correlation with the fibrinogen level. Nb106 suppressed the thrombin generation amplitude, but not the kinetics in the presence of a panel of direct oral anticoagulants (DOAC). Strikingly, brightfield microscopy and SEM indicated that Nb106, but not control nanobodies, in triggered plasma, platelet-rich plasma and whole-blood, fully abrogated the formation of elongating fibrin fibers. In agreement with this, it strongly suppressed whole-blood clot retraction. Conclusion: Collectivity, these data point to the existence of two major pools of proteolytically active thrombin, both of which contributing to thrombin generation, that are formed upon plasma and blood coagulation. One pool of soluble thrombin steers the proteolytic coagulation cascade, while another pool is bound to growing fibrin fibers. The latter pool still cleaves the conventional AMC thrombin substrate, is temporarily protected against antithrombin, and is required for fibrin fiber extension and blood clotting and clot retraction. The Nb106, replacing thrombin from its binding site on fibrin, selectively annuls the second pool, thereby blocking the clotting process. The replacement ensures thrombin inactivation by antithrombin. Perspective: The novel anti-fibrin nanobody Syn-Nb-AF106 or a derivative has the potential to act as an alternative anticoagulant in thrombotic diseases with pathological fibrin formation.

Hypochlorite-induced oxidative modification of fibrinogen

Fibrinogen (FG) is a protein that plays a key role in the hemostasis system and is most susceptible to oxidative modification compared to other plasma proteins. FG undergoes post-translational modifications that can potentially disrupt its structure and function.For the first time, using high resolution mass spectrometry (HPLC/MS/MS), the consequences of hypochlorite (HOCl)- induced FG oxidation were studied, and a list of FG amino acid residue oxidatively modifications. Samples of nonoxidized and treated with 50 μM HOCL fibrinogen were analyzed by mass spectrometry and it was found that many amino acid residues localized in all three polypeptide chains and the main structural elements of fibrinogen, with the exception of the E region, are involved in oxidation.Thus, the amino acid residues localized in the E region, which are involved in thrombin binding, were not subject to oxidative modification, which indicates the preservation of the thrombin-binding sites of the fibrinogen molecule during oxidation. The αС region contains the largest number of oxidizing sites, which confirms the hypothesis that this region can serve as a trap for reactive oxygen species (ROS) molecules.

Sensitivity analysis of a reduced model of thrombosis under flow: Roles of Factor IX, Factor XI, and γ'-Fibrin.

A highly reduced extrinsic pathway coagulation model (8 ODEs) under flow considered a thin 15-micron platelet layer where transport limitations were largely negligible (except for fibrinogen) and where cofactors (FVIIa, FV, FVIII) were not rate-limiting. By including thrombin feedback activation of FXI and the antithrombin-I activities of fibrin, the model accurately simulated measured fibrin formation and thrombin fluxes. Using this reduced model, we conducted 10,000 Monte Carlo (MC) simulations for ±50% variation of 5 plasma zymogens and 2 fibrin binding sites for thrombin. A sensitivity analysis of zymogen concentrations indicated that FIX activity most influenced thrombin generation, a result expected from hemophilia A and B. Averaging all MC simulations confirmed both the mean and standard deviation of measured fibrin generation on 1 tissue factor (TF) molecule per μm2. Across all simulations, free thrombin in the layer ranged from 20 to 300 nM (mean: 50 nM). The top 2% of simulations that produced maximal fibrin were dominated by conditions with low antithrombin-I activity (decreased weak and strong sites) and high FIX concentration. In contrast, the bottom 2% of simulations that produced minimal fibrin were dominated by low FIX and FX. The percent reduction of fibrin by an ideal FXIa inhibitor (FXI = 0) ranged from 71% fibrin reduction in the top 2% of MC simulations to only 34% fibrin reduction in the bottom 2% of MC simulations. Thus, the antithrombotic potency of FXIa inhibitors may vary depending on normal ranges of zymogen concentrations. This reduced model allowed efficient multivariable sensitivity analysis.

Abstract 9308: Extreme Gamma Prime Fibrinogen Levels in COVID-19 Patients

Introduction: COVID-19 disease progression can be accompanied by a "cytokine storm" that leads to secondary sequelae such as thrombosis and acute respiratory distress syndrome. Several inflammatory cytokines have been associated with COVID-19 disease progression, but have far too much daily intra-individual variability to be useful in tracking the course of the disease. In contrast, we have shown that the inflammatory biomarker γ' fibrinogen (γ' Fbg) has a 6-fold lower coefficient of variability compared to other inflammatory markers such as hs-CRP. The aims of the study were to measure γ' Fbg in serial blood samples from COVID-19 patients at a tertiary care medical center in order to investigate its association with clinical measures of disease progression. Hypothesis: Our hypothesis was that γ' Fbg levels would be elevated in COVID-19 patients compared to historical controls, and that the degree of elevation would be associated with disease severity. Methods: COVID-19 patients at a tertiary care medical center were retrospectively enrolled between 3/16/2020 and 8/1/2020. γ' Fbg was measured using the GammaCoeur ELISA (Gamma Diagnostics, Patent Pending). Results: Our results showed that ten out of the eighteen patients with COVID-19 had the highest levels of γ' Fbg ever recorded. The previous highest γ' Fbg level of 80.3 mg/dL was found in a study of 10,601 participants in the ARIC study. γ' Fbg levels were significantly associated with the need for ECMO and mortality. Conclusions: We found that COVID-19 patients can develop extraordinarily high levels of γ' Fbg. This has several important clinical implications. γ' Fbg contains a high affinity binding site for thrombin that binds to anion-binding exosite II on thrombin and protects it from inactivation by heparin. High levels of γ' Fbg therefore provide a reservoir of heparin-resistant clot-bound thrombin when the γ' Fbg is clotted. These findings have potential clinical implications regarding prophylactic anticoagulation of COVID-19 patients and suggest that heparin prophylaxis may be less effective than using other anticoagulants, particularly direct thrombin inhibitors.

Structural and Binding Effects of Chemical Modifications on Thrombin Binding Aptamer (TBA)

The thrombin binding aptamer (TBA) is a promising nucleic acid-based anticoagulant. We studied the effects of chemical modifications, such as dendrimer Trebler and NHS carboxy group, on TBA with respect to its structures and thrombin binding affinity. The two dendrimer modifications were incorporated into the TBA at the 5′ end and the NHS carboxy group was added into the thymine residues in the thrombin binding site of the TBA G-quadruplex (at T4, T13 and both T4/T13) using solid phase oligonucleotide synthesis. Circular dichroism (CD) spectroscopy confirmed that all of these modified TBA variants fold into a stable G-quadruplex. The binding affinity of TBA variants with thrombin was measured by surface plasmon resonance (SPR). The binding patterns and equilibrium dissociation constants (KD) of the modified TBAs are very similar to that of the native TBA. Molecular dynamics simulations studies indicate that the additional interactions or stability enhancement introduced by the modifications are minimized either by the disruption of TBA–thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental data. Overall, this study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference, which could be useful in the design and development of more functional TBA analogues.

Role for PARS and Thrombin-Gpibα Interaction for Thrombin Induced Procoagulant Platelet Formation

Procoagulant platelets, a subpopulation of platelets which support thrombin generation, play a role in regulating pathological thrombosis. Using a novel flow cytometry assay (Hua et at, Blood 2015) to identify procoagulant platelets based upon the cell death marker GSAO and activation marker P-selectin, we found that patients with angiogram proven coronary artery disease had heightened thrombin procoagulant platelet formation compared to healthy controls, (25.2% vs 12.2%, p<0.001). Inhibition of this is an attractive target for atherothrombosis prevention. However, the mechanism of thrombin induced procoagulant platelet formation remains controversial with discrepant results regarding requirement for the thrombin binding domain of N terminal GPIbα (Dorman et al, Blood 2000 and Ravanat et al, Blood 2010) and recent reports implicating PAR4 (French et al JTH, 2016). Here we use a recently described novel assay to investigate the roles of PARs and GPIbα measuring procoagulant platelets by FACS (GSAO+/P-selectin+) in whole blood, plasma and washed platelets after treatment with heparin, PAR and GPIbα inhibitors prior to thrombin or PAR agonist stimulation.First, using active site inhibited PPACK-thrombin we demonstrated that catalytically active thrombin is required for procoagulant platelet formation in our in vitro system. PAR1 and PAR4 are implicated in procoagulant platelet formation, however, preincubation with combination of PAR1 and PAR 4 inhibitors (vorapaxar and a PAR4 polyclonal inhibitory antibody), resulted in only partial inhibition of thrombin induced procoagulant platelets (reduction 40%±5.7%, p<0.01) suggesting a role for an additional non-PAR pathway. Thrombin-GPIbα interaction was implied when competition studies with recombinant soluble GPIbα, glycocalicin showed a 49%±7.7% reduction. Involvement of the N-terminus of GPIbα was confirmed by a 39.5%±2.9% reduction after cleavage of GPIbα proximal to the thrombin binding site by NK protease. Pretreatment with PAR1 inhibitor after NK protease cleavage of GPIbα reduced thrombin stimulated procoagulant platelet formation to unstimulated levels implying both PAR cleavage and GPIbα-thrombin interaction is required for thrombin induced procoagulant platelet formation.Consistent with a role of thrombin-GPIbα interaction, we noted a reduction in thrombin induced procoagulant platelets after heparin bolus in patients undergoing coronary angiogram (16.3±10.5% vs 6.1%±2.4%, p<0.001). Heparin is known to known to displace thrombin from GPIbα in addition to anti-thrombin effects via enhancement of antithrombin (AT). Using platelets resuspended in AT-deficient plasma, we demonstrated high dose heparin suppressed procoagulant platelet formation via an AT-independent mechanism, this was confirmed by heparin suppression of thrombin induced procoagulant platelets in washed platelets. In similar experiments, the heparinoid danaparoid, which has no anti-thrombin effect, also inhibited thrombin induced procoagulant platelet formation. Heparin inhibition was not due to PAR inhibition as heparin had no effect on direct PAR activation by PAR1 agonist TRAP or PAR4 agonist AYPGKF. These data suggest heparin has a direct effect on platelets in suppression of thrombin induced procoagulant platelet formation, most likely relating to displacement of thrombin from GPIbα.Together this data indicates that a thrombin-GPIbα interaction via the N terminal fragment is required in addition to thrombin mediated PAR activation. This mechanism is a potential target for modifying atherothrombotic risk.Figure 1: Whole blood was treated with PAR1 and PAR4 inhibitors and washed platelets were treated with glycocalicin, NK protease or NK and PAR1 inhibitor prior to thrombin stimulation (2 U/mL). Data are expressed as fold change from vehicle control ((*p<0.05; **p<0.01, statistics are calculated for n≥3). Figure 2: Plasma, washed platelets or whole blood were treated or not with heparin (12.8 U/ml) prior to thrombin stimulation (2 U/mL), PAR1 (20 µM) or PAR4 (300 µM) agonists. Data are expressed as the ratio of heparin-treated sample to untreated (***p<0.001, statistics are calculated for n≥3). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Deletion of the Thrombin Proteolytic Site in Neurofascin 155 Causes Disruption of Nodal and Paranodal Organization.

In the central nervous system, myelin is attached to the axon in the paranodal region by a trimolecular complex of Neurofascin155 (NF155) in the myelin membrane, interacting with Caspr1 and Contactin1 on the axolemma. Alternative splicing of a single Neurofascin transcript generates several different Neurofascins expressed by several cell types, but NF155, which is expressed by oligodendrocytes, contains a domain in the third fibronectinIII-like region of the molecule that is unique. The immunoglobulin 5–6 domain of NF155 is essential for binding to Contactin1, but less is known about the functions of the NF155-unique third fibronectinIII-like domain. Mutations and autoantibodies to this region are associated with several neurodevelopmental and demyelinating nervous system disorders. Here we used Crispr-Cas9 gene editing to delete a 9 bp sequence of NF155 in this unique domain, which has recently been identified as a thrombin binding site and implicated in plasticity of the myelin sheath. This small deletion results in dysmyelination, eversion of paranodal loops of myelin, substantial enlargement of the nodal gap, a complete loss of paranodal septate junctions, and mislocalization of Caspr1 and nodal sodium channels. The animals exhibit tremor and ataxia, and biochemical and mass spectrometric analysis indicates that while NF155 is transcribed and spliced normally, the NF155 protein is subsequently degraded, resulting in loss of the full length 155 kDa native protein. These findings reveal that this 9 bp region of NF155 in its unique third fibronectinIII-like domain is essential for stability of the protein.

Genetic Variants in the FGB and FGG Genes Mapping in the Beta and Gamma Nodules of the Fibrinogen Molecule in Congenital Quantitative Fibrinogen Disorders Associated with a Thrombotic Phenotype

Fibrinogen is a hexameric plasmatic glycoprotein composed of pairs of three chains (Aα, Bβ, and γ), which play an essential role in hemostasis. Conversion of fibrinogen to insoluble polymer fibrin gives structural stability, strength, and adhesive surfaces for growing blood clots. Equally important, the exposure of its non-substrate thrombin-binding sites after fibrin clot formation promotes antithrombotic properties. Fibrinogen and fibrin have a major role in multiple biological processes in addition to hemostasis and thrombosis, i.e., fibrinolysis (during which the fibrin clot is broken down), matrix physiology (by interacting with factor XIII, plasminogen, vitronectin, and fibronectin), wound healing, inflammation, infection, cell interaction, angiogenesis, tumour growth, and metastasis. Congenital fibrinogen deficiencies are rare bleeding disorders, characterized by extensive genetic heterogeneity in all the three genes: FGA, FGB, and FGG (enconding the Aα, Bβ, and γ chain, respectively). Depending on the type and site of mutations, congenital defects of fibrinogen can result in variable clinical manifestations, which range from asymptomatic conditions to the life-threatening bleeds or even thromboembolic events. In this manuscript, we will briefly review the main pathogenic mechanisms and risk factors leading to thrombosis, and we will specifically focus on molecular mechanisms associated with mutations in the C-terminal end of the beta and gamma chains, which are often responsible for cases of congenital afibrinogenemia and hypofibrinogenemia associated with thrombotic manifestations.

Thrombin contributes to cancer immune evasion via proteolysis of platelet-bound GARP for LTGF-β activation

Abstract Cancer-associated thrombocytosis and high concentration of circulating TGFβ1 are hallmarks of progressive tumor disease in cancer patients. Using genetic and pharmacological approaches, we show a direct link between thrombin catalytic activity and maturation of LTGFβ bound to platelet GARP. We found that GARP protein has two conserved thrombin binding sites through which thrombin cleaves platelet-bound GARP to release active TGFβ from GARP/LTGFβ complex. Crystal structure models docked thrombin onto the concave surface of GARP supporting the notion that thrombin is able to bind and cleave GARP/LTGFβ complex. We observed that inhibition of thrombin obliterates TGFβ maturation in platelet releasate and in serum of WT mice, which translates in better tumor control alone and in combination with checkpoint blockade therapy, in two tumor preclinical models. Of note, the reduction of serum active TGFβ and the anti-tumor properties of thrombin were not observed in absence of platelet GARP. In the tumor microenvironment we found that blocking thrombin reduced active TGFβ signaling and increased the infiltrating CD8+ T cells as well as the innate NK cells and neutrophils. Also, we demonstrated that thrombin cleavage releases soluble GARP/LTGFβ complex and that high concentration of GARP in plasma, alone or in complex with LTGFβ, is a biomarker of poor clinical outcome in prostate cancer patients. We conclude that thrombin-GARP interaction and consequent GARP cleavage induce the activation of TGFβ derived from platelets. Considering that platelets are the major TGFβ reservoir, these observations demonstrated that blocking GARP cleavage opens a new therapeutic window to overcome TGFβ-mediated resistance to immune check point inhibitors.

Residues 1680-1684 in the A3 Domain of Factor VIII Contain a Novel Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689

Thrombin-catalyzed activation of factor (F)VIII by proteolytic cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies FX activation as a cofactor of FIXa. We previously reported that thrombin interacts with FVIII through the A2 domain (residues 392-394 and 484-509) and C2 domain, and these interactions governed the cleavages at Arg740, Arg372 and Arg1689, respectively (Nogami, JBC 2000, 2005, BJH 2008). A previous report suggested, however, that FVIII lacking the C2 domain retained &gt;50% cofactor activity (Wakabayashi, JBC 2010), supportive of the presence of other thrombin-binding site responsible for cleavage at Arg1689 within the A3-C1 domain. Recently, we focused on similar sequence in A3 acidic region to hirugen residues 54-65, and demonstrated that two region of residues 1659-1669 and 1675-1685 might contained the thrombin-binding site(s) responsible for cleavage at Arg1689 by functional and binding experiments using synthetic peptide (Minami. ASH 2015). In this study, to identify the crucial residues, seven acidic clustered residues and two sulfated Tyr residues in these regions as a series of rFVIII mutants were converted to Ala, and 9 single mutants for D1663A, Y1664A, D1665A, D1666A, Y1680A, D1681A, E1682A, D1683A, E1684A and 2 double mutants for D1665A/D1666A, D1683A/E1684A, were prepared by using a BHK system. Specific activities of FVIII in all of 5 mutants (D1663A, Y1664A, D1665A, D1666A, D1665/D1664) in the former region and 4 mutants (Y1680A, D1683A, E1684A, D1683A/E1684A) in the latter region, assessed by a one-stage clotting assay were 40-70% and 50-70% of wild type (WT), respectively. These mutants exhibited the assay discrepancy for FVIII activity between one-stage clotting assay and FVIIIa-dependent FXa generation assay (one-stage &lt; FXa generation assay), guessing possible association with these mutated residues for thrombin reaction. Next, FVIII mutants (10 nM) were examined for activation by thrombin (0.4 nM) in a one-stage clotting assay. Regards FVIII mutants in the 1663-1666 region, FVIII activation by thrombin in all mutants were not significant difference from that in WT. We further examined thrombin-catalyzed cleavage at Arg1689 of these mutants by using SDS-PAGE and Western blot using an anti-FVIII monoclonal antibody recognizing the A3 acidic region for detection. The initial velocity of thrombin cleavage at Arg1689 in these mutants showed no significant difference from that in WT. In addition, the rate of thrombin cleavage at Arg372 in them was also almost similar to that in WT. These results indicated that the 1663-1666 region appeared unlikely to participate in the functional association between FVIII and thrombin. On the other hand, regards the 1680-1684 region, peak activity in FVIII activation by thrombin for D1683A, E1684A and D1683A/E1684A mutants were modestly reduced with an ~60% level of WT, and that for Y1680A mutant was significantly diminished with peak activity of ~30% of WT. FVIII activation by thrombin in D1681A and E1682A mutants was not significant difference from WT, however. Evaluated by SDS-PAGE, the initial velocities of cleavage at Arg1689 of D1681A and E1682A were comparable to WT, whilst those of D1683A, E1684A and D1683A/E1684A mutants were ~60% level of WT. It was note that Y1680A mutant showed a ~10% of velocity rates of WT on the cleavage at Arg1689, supportive of the results obtained from thrombin-catalyzed activation of FVIII. However, the rate of thrombin cleavage at Arg372 in these mutants was almost similar to that in WT. These results suggested that three residues were involved with the cleavage at Arg1689. In conclusion, we for the first time identified that that 1680-1684 residues in A3 acidic region, in particular sulfated Tyr1680, played a key role in thrombin-interactive sites responsible for cleavage at Arg1689. Disclosures No relevant conflicts of interest to declare.

Modulation of Thrombin-Induced Platelet Activation By Defibrotide

Defibrotide is a mixture of single-stranded phosphodiester oligonucleotides 9-80 bases in length derived from mammalian tissue and it is the only medication that is FDA approved for the prevention and treatment of post-transplant veno-occlusive disease, also known as sinusoidal obstructive syndrome (VOD/SOS). The mechanism of action of defibrotide is not well understood. A better understanding of the molecular mechanism by which defibrotide prevents and reverses the microvascular occlusion seen in VOD/SOS will be critical in the development of novel therapeutics for VOD/SOS and other related disorders. Our central hypothesis is that single stranded phosphodiester oligonucleotide mixtures such as defibrotide alter thrombin-induced platelet and endothelial cell activation primarily via altered signaling through protease activated receptors (PARs). To determine the effect of defibrotide on thrombin-induced platelet activation we performed flow cytometry on washed platelets from humans and mice. Washed platelets were incubated with agonist (1 nM thrombin, 100 μM synthetic PAR1 agonist TRAP6, or 100 μM synthetic PAR4 agonist PAR4AP) in the presence or absence of 1 mg/mL of defibrotide. Fluorescent antibodies against P-selectin (anti-CD62P antibody) and against the high affinity conformation of the αIIβ3 receptor (PAC1 antibody for human samples and JON/A antibody for murine samples) were added to assess for inhibition of platelet activation by defibrotide. Fluorescent anti-PAR1 antibodies SPAN12 (which spans the canonical thrombin cleavage site) and WEDE15 (which binds at the hirudin-like domain) were also added to separate samples to assess whether defibrotide inhibits cleavage of the PAR1 N-terminal domain by thrombin. After twenty minutes of incubation at room temperature the samples were analyzed by flow cytometry. We calculated the geometric mean of the fluorescence intensity (MFI) for three replicate samples and data are shown as the fold change compared to the washed platelet control without addition of agonist or defibrotide. For thrombin-stimulated human platelets, both anti-CD62P binding (MFI fold over control 5.7 vs. 1.2) and PAC1 antibody binding (MFI fold over control 7.1 vs. 1.7) are significantly reduced in the presence of defibrotide (See Figure 1a). For thrombin-stimulated murine platelets there is a small decrease in anti-CD62P antibody binding (MFI fold over control 8.4 vs. 6.6), but no significant change in JON/A antibody binding (See Figure 1b). Binding of both anti-CD62P and PAC1 antibodies to human platelets stimulated with synthetic PAR agonists was unaffected by the presence of defibrotide (See Figure 1c). There is no significant difference in SPAN12 or WEDE15 binding in thrombin-stimulated platelets in the absence or presence of defibrotide. However, defibrotide alone does appear to interfere with WEDE15 binding in unstimulated platelets (See Figure 1d). In conclusion, we showed that defibrotide inhibits thrombin-induced human platelet activation at low concentrations of thrombin, as shown by a decrease in surface platelet activation markers measured by flow cytometry. This effect is much less pronounced under the same conditions if mouse platelets are used, supporting the hypothesis that defibrotide inhibits platelet activation by alteration of PAR1 receptor signaling, since PAR1 receptors are not present on mouse platelets. Defibrotide does not interfere with TRAP6-induced platelet activation, evidence against interference with the intracellular portion of the signaling pathway, since TRAP6 is a synthetic PAR1 agonist that binds directly into the ligand-binding pocket of the receptor, eliminating the need for cleavage of the PAR1 extracellular domain. The decrease in WEDE15 antibody binding to washed platelets seen with defibrotide alone suggests that the mechanism may involve alterations in the hirudin-like domain of the PAR1 receptor which is crucial for thrombin binding and cleavage site specificity. Ongoing studies including investigating the effect of defibrotide on peptide mimics of the extracellular N-terminal domain of the PAR1 receptor and the effect of defibrotide on the activation of endothelial cells will likely shed light on the exact mechanism of platelet response modulation by defibrotide and may provide new targets for treatment of the microvascular occlusion that characterizes VOD/SOS. Disclosures No relevant conflicts of interest to declare.

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).

Highly sensitive fluorometric determination of thrombin by on-chip signal amplification initiated by terminal deoxynucleotidyl transferase.

The article describes an on-chip amplification scheme initiated by a terminal deoxynucleotidyl transferase (TdT) for highly sensitive fluorometric determination of protein. Two thrombin-binding aptamers were designed to capture thrombin as they can form a sandwich structure for improved specificity. An amino-modified aptamer (TBA29) was first immobilized on a silicon chip. After captureof thrombin, a secondaptamer (TBA15) was conjugated to the second binding site of thrombin. The 3'-terminal of aptamer TBA15 isexposed on the chip surface, and then fluorescein-labeled 12-dATP associates to the 3'-terminal with the help of TdT. This results in signal amplification, and eventually leads to highly sensitive detection. Under optimal conditions, fluorescence intensity is linearly related to the logarithm of thrombin concentration in the range of 100 fM - 0.1 μM, andthe detection limit is as low as 2.0 fM. The assay is sensitive and selective even over potentially interfering proteins and in the presence of human serum. Graphical abstract Schematic strategy for thrombin detection. Two thrombin-binding aptamers were designed to capture thrombin to form a sandwich structure for improved specificity. The protein detection is based on TdT initiated on-chip fluorescent amplification.

OP-613 - Idarucizumab for Urgent Dabigatran Reversal in a Patient with Hemothorax and Perforation of Right Ventricular Free Wall by Pacemaker Lead

Background: Idarucizumab is a humanized monoclonal antibody fragment that binds the thrombin-binding site of dabigatran with high affinity, effectively neutralizing its anticoagulant effects. Herein, we present our experience with use of idarucizumab for urgent dabigatran reversal in a patient with hemothorax and perforation of right ventricular free wall by pacemaker lead.

Probing the Dynamics of Clot-Bound Thrombin at Venous Shear Rates

In closed system models of fibrin formation, exosite-mediated thrombin binding to fibrin contributes to clot stability and is resistant to inhibition by antithrombin/heparin while still susceptible to small, active-site inhibitors. Each molecule of fibrin can bind ∼1.6 thrombin molecules at low-affinity binding sites (Kd = 2.8 μM) and ∼0.3 molecules of thrombin at high-affinity binding sites (Kd = 0.15 μM). The goal of this study is to assess the stability of fibrin-bound thrombin under venous flow conditions and to determine both its accessibility and susceptibility to inhibition. A parallel-plate flow chamber (7 × 50 × 0.25 mm) for studying the stability of thrombin (0–1400 nM) adhered to a fibrin matrix (0.1–0.4 mg/mL fibrinogen, 10 nM thrombin) under a variety of venous flow conditions was developed using the thrombin-specific, fluorogenic substrate SN-59 (100 μM). The flow within this system is laminar (Re < 1) and reaction rates are driven by enzyme kinetics (Pe = 100, Da = 7000). A subpopulation of active thrombin remains stably adhered to a fibrin matrix over a range of venous shear rates (46–184 s−1) for upwards of 30 min, and this population is saturable at loads >500 nM and sensitive to the initial fibrinogen concentration. These observations were also supported by a mathematical model of thrombin adhesion to fibrin, which demonstrates that thrombin initially binds to the low-affinity thrombin binding sites before preferentially equilibrating to higher affinity sites. Antithrombin (2.6 μM) plus heparin (4 U/mL) inhibits 72% of the active clot-bound thrombin after ∼10 min at 92 s−1, while no inhibition is observed in the absence of heparin. Dabigatran (20 and 200 nM) inhibits (50 and 93%) clot-bound thrombin reversibly (87 and 66% recovery). This model illustrates that clot-bound thrombin stability is the result of a constant rearrangement of thrombin molecules within a dense matrix of binding sites.

Measurable Amount of Active Thrombin Is Bound to Circulating D-Dimers. Is There Any Impact on Diagnosis and Pathophysiology of Thrombosis?

IntroductionThrombosis is a common pathology underlying ischemic heart disease, stroke, and venous thromboembolism. D-dimer is a global indicator of blood coagulation activation and fibrinolysis and, therefore, an indirect marker of thrombotic activity. D-dimers half-life is 48 hours. D-dimer determination is the standard procedure in the treatment of thrombosis. Serine protease thrombin plays pivotal roles in thrombosis and hemostasis, blood coagulation and platelet activation. Thrombin has a short half-life and naturally occurring inhibitors, such as antithrombin, rapidly bind thrombin, which makes the direct measurements of in vivo active thrombin difficult. Thrombin binds to fibrin where it is quite efficiently protected from inhibition by heparin-antithrombin but susceptible to inactivation by different antithrombin-independent inhibitors. There are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrinogen E region, and one high affinity thrombin non-substrate binding site on fibrinogen gamma' chains inside the D region (Meh DA, Siebenlist KR, Mosesson MW, J Biol Chem. 1996, 23121-5). We have shown that thrombin bound to fibrin promotes further fibrin growth in the presence of fibrinogen and absence of free thrombin in solution (Riedel T, Brynda E, Dyr JE, Houska, M, J. Biomed. Mater. Res. Part A 2009, 437-447). The aim of this project was to look for any thrombin activity on isolated D-dimers using several commercial D-dimer kits in groups of patients with elevated D-dimers.MethodsThree D-dimers kits were used (ELISA kits CEA506Hu, USCN and D-Dimer (D2D), BioAssay™; and immunoturbidimetric assay D-Dimer KAI-090, K-ASSAY). To detect thrombin activity on captured D-dimers one chromogenic (S-2238, Chromogenix) and two fluorogenic (SN-59, Haematologic Technologies, Inc.; (p-tosyl-Gly-Pro-Arg)2-R110, Thermo Fisher Scientific, Inc.) specific substrates and specific inhibitors (hirudine and PPACK) were used. Independently, bound proteins were removed from immobilized antibodies in 8 M urea and after treatment with trypsin analyzed by mass spectrometry (TripleTOF 5600, Sciex). 34 patients with high, moderate, and low levels of D-dimers and with different diagnosis were analyzed.ResultsOut of 18 patients with main diagnosis linked with thrombosis 61 % exhibited active thrombin on D-dimers. In these patients we found active thrombin bound to D-dimers captured by antibodies in all applied D-dimer kits. Using mass spectrometry thrombin bound to isolated D-dimers was detected. Specificity of thrombin activity related to D-dimers was determined by combination of several specific thrombin substrates and inhibitors. The activity of bound thrombin was remarkably stable over the period of more than 24 hours. It showed that precise measurement of even very low activity of thrombin was possible. In the group of 16 non-thrombotic patients with elevated D-dimers only 19 % exhibited thrombin activity. Interestingly, differences in values of thrombin activity were up to five orders of magnitude and differences in the activities related to the value of captured D-dimers were even greater.ConclusionThis, to our knowledge, is the first report showing the presence of active thrombin bound to circulating D-dimers. Although the group of patients we were so far able to evaluate is too small to being statistically tested, the results are highly encouraging. The amount of bound active thrombin on fibrin degradation products reflects the way of thrombus formation and its degradation (times, durations and rates of relevant reactions and especially the rate of thrombin generation). The thrombin concentration present at the time of fibrin gelation plays an important role in formation of fibrin clot structure, including its mechanical and fibrinolytic stability. It remains to be seen in further studies with much larger cohorts of patients if the presence of active thrombin has any impact on pathophysiology of thrombosis and if its determination may be of importance for the improvement of diagnosis of thrombotic events.Supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.1.16/3.1.00/28007. DisclosuresNo relevant conflicts of interest to declare.

Oral thrombin inhibitor aggravates platelet adhesion and aggregation during arterial thrombosis.

In patients with atrial fibrillation, oral anticoagulation with oral thrombin inhibitors (OTIs), in contrast to vitamin K antagonists (VKAs), associates with a modest increase in acute coronary syndromes (ACSs). Whether this observation is causatively linked to OTI treatment and, if so, whether OTI action is the result of a lower antithrombotic efficacy of OTI compared to VKA or reflects a yet undefined prothrombotic activity of OTI remain unclear. We analyzed platelet function in patients receiving OTI or dose-adapted VKA under static and flow conditions. In vivo, we studied arterial thrombosis in OTI-, VKA-, and vehicle-treated mice using carotid ligation and wire injury models. Further, we examined thrombus formation on human atherosclerotic plaque homogenates under arterial shear to address the relevance to human pathology. Under static conditions, aggregation in the presence of ristocetin was increased in OTI-treated blood, whereas platelet reactivity and aggregation to other agonists were only marginally affected. Under flow conditions, firm platelet adhesion and thrombus formation on von Willebrand factor, collagen, and human atherosclerotic plaque were increased in the presence of OTI in comparison to VKA. OTI treatment was associated with increased thrombus formation in injured carotid arteries of mice. Inhibition or ablation of GPIbα-thrombin interactions abolished the effect of OTI on thrombus formation, suggesting a mechanistic role of the platelet receptor GPIbα and its thrombin-binding site. The effect of OTI was also abrogated in the presence of aspirin. In summary, OTI treatment has prothrombotic activity that might contribute to the increase in ACS observed clinically in patients.

An allosteric disulfide bond is involved in enhanced activation of factor XI by protein disulfide isomerase

Background Reduction of factor (F) XI by protein disulfide isomerase (PDI) has been shown to enhance the ability of FXI to cleave its chromogenic substrate. Three disulfide bonds in FXI (Cys118-Cys147, Cys362-Cys482, and Cys321-Cys321) are involved in this augmented activation. Objectives To characterize the mechanisms by which PDI enhances FXI activity. Methods FXI activity was measured following PDI reduction. Thiols that were exposed in FXI after PDI reduction were labeled with 3-(N-maleimidopropionyl)-biocytin (MPB) and detected with avidin. The rate of conversion of FXI to activated FXI (FXIa) following thrombin activation was assessed with western blotting. FXI molecules harboring mutations that disrupt the three disulfide bonds (C147S, C321S, and C482S) were expressed in cells. The antigenicity of secreted FXI was measured with ELISA, and its activity was assessed by the use of a chromogenic substrate. The effect of disulfide bond reduction was analyzed by the use of molecular dynamics. Results Reduction of FXI by PDI enhanced cleavage of both its chromogenic substrate, S2366, and its physiologic substrate, FIX, and resulted in opening of the Cys362-Cys482 bond. The rate of conversion of FXI to FXIa was increased following its reduction by PDI. C482S-FXI showed enhanced activity as compared with both wild-type FXI and C321S-FXI. MD showed that disruption of the Cys362-Cys482 bond leads to a broader thrombin-binding site in FXI. Conclusions Reduction of FXI by PDI enhances its ability to cleave FIX, probably by causing faster conversion of FXI to FXIa. The Cys362-Cys482 disulfide bond is involved in enhancing FXI activation following its reduction, possibly by increasing thrombin accessibility to FXI.

The Acidic Region of Factor VIII Light Chain Contains the Thrombin-Binding Site(s) Responsible for the Cleavage at Arg1689

Thrombin-catalyzed activation of factor (F)VIII by cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies the activation of FX as a cofactor of FIX. We had already demonstrated that thrombin interacts with FVIII through the residues 392-394 and 484-509 in the A2 domain and the C2 domain, and each association regulates cleavage at Arg740, Arg372, and Arg1689, respectively (Nogami K, JBC 2000, 2005; BJH 2008), and recently reported that the A1 acidic clustered region 340-350 involving the sulfated tyrosine regulate the cleavage of Arg372 (Minami et al. 55th ASH). On the other hand, Fay and colleague suggested that recombinant FVIII lacking the C2 domain retains greater than 50% cofactor activity (JBC 2010), supporting the presence of other thrombin-binding region responsible for cleavage at Arg1689 of the light chain. In this study, we attempted to identify this thrombin-binding site(s). We focused on the acidic residues 1659-1669 and 1675-1685 within the light chain, which had similar sequence to the A1 residues 340-350 in terms of involving the clustered acidic residues and sulfated tyrosine as well as hirugen residues 54-65. We prepared four of synthetic peptides corresponding to the residues 1659-1669 and 1675-1685 with sulfated tyrosine, P(1659-69s) and P(1675-85s), and with non-sulfated tyrosine, P(1659-69) and P(1675-85). The inhibitory effect on the thrombin-catalyzed FVIII activation by each peptide was evaluated in a one-stage clotting assay. Each peptide showed a dose-dependent inhibition on thrombin-catalyzed activation. These inhibitory effects were greater in order of P1675-85s, P1659-69s, P1675-85, P1659-69, and the IC50 were 25, 67, 71 and 225 µM, respectively. The peptides with sulfated tyrosine had approximately 3-fold greater inhibition of the FVIII activation by thrombin than with non-sulfated tyrosine. The IC50 in the presence of mixture of P1675-85s and P1659-69s was 30.4 µM, suggesting that these peptides had no an additive effect. The impacts of P1659-69s and P1675-85s on the thrombin-catalyzed cleavage at Arg1689 were examined by SDS-PAGE/western blotting. These peptides blocked the cleavage at Arg1689 in dose-dependent fashions. In timed-course assay, the presence of P1659-69s and P1675-85s decreased the cleavage rate of Arg1689 by 61.3 % and 81.8 %, respectively compared to its absence. The direct binding of P1659-69s and P1675-85s to thrombin was examined by surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based assay using a Biacore T200TM, thrombin bound to immobilized P1659-69s and P1675-85s directly with high affinity. The Kd values adjusted to 1:1 binding model of global fitting were 203 nM and 94 nM, respectively. EDC cross-linking in fluid-phase assay revealed that formation of EDC cross-linking products between biotinylated P1659-69s or P1675-85s and thrombin were observed in dose-dependent fashions. The products between the biotinylated peptides (800 nM) and thrombin were competitively reduced by the addition of non-biotinylated peptides. Moreover, N-terminal sequence analysis of cross-linking products between both peptides-thrombin indicated that thrombin bound to the residues 1664-1669 and 1683-1684. Taken together, we demonstrated that the A3 residues 1659-1669 (QEEIDYDDTIS) and residues 1675-1685 (EDFDIYDEDEN) contained the thrombin binding-sites responsible for proteolytic cleavage at Arg1689 of the A3 domain. DisclosuresNogami:Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding. Shima:Chugai Pharmaceutical Co., Ltd. and F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.