Enzymatic Activity Of Thrombin Research Articles

Flavonol and A-type procyanidin-rich extracts of Prunus spinosa L. flower exhibit anticoagulant activity through direct thrombin inhibition, but do not affect platelet aggregation in vitro

Background: Blackthorn flower (Prunus spinosa L.) is a traditional herbal remedy recommended for treating cardiovascular diseases (CVDs).Aim: This in vitro study investigates the effects of flavonol and A-type procyanidin-rich blackthorn flower extracts on the hemostatic system, including the blood plasma coagulation cascade and platelet aggregation.Methods: Six distinct extracts, characterized through various techniques, including LC-MS/MS, were assessed at in vivo-relevant levels (1–50 μg/mL) for their antithrombotic activity. The thrombin, prothrombin, and activated partial thromboplastin times were measured. Additionally, the thrombin enzymatic activity was tested using the chromogenic substrate S-2238 and fibrinogen as the physiological substrate of the enzyme. To gain insights into the mechanism of action, the interactions between the primary extracts’ constituents, their potential metabolites, and thrombin were examined in silico. The computational analyses were complemented by in vitro experiments and circular dichroism spectroscopy. The platelet aggregation in human platelet-rich plasma was assessed after ADP or collagen stimulation. Furthermore, the extracts’ biocompatibility was tested on human peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs).Results: The extracts slightly prolonged the prothrombin and thrombin times and effectively inhibited the thrombin’s enzymatic activity, reducing its amidolytic and proteolytic functions at 50 μg/mL by 91.2% and 74.8%, respectively. In silico molecular docking demonstrated a strong binding affinity of the examined polyphenols and their metabolites to thrombin. Most analytes bound exclusively within the enzyme active site; however, afzelin, kaempferitrin, and procyanidin A2 revealed the affinity to additional binding sites, including exosite I. The structure-activity relationship of flavonols as thrombin inhibitors was studied in vitro. Circular dichroism spectroscopy confirmed that the interactions between thrombin and the compounds (even at 1 μg/mL) induce alterations within the α-helices’ secondary structure, resulting in noticeable changes in the enzyme’s CD spectrum. On the other hand, the extracts did not influence platelet aggregation. Eventually, their cellular biocompatibility with PBMCs and RBCs was confirmed.Conclusion: The extracts directly inhibit thrombin, a critical serine protease in hemostasis and a prime anticoagulant drug target, and do not exhibit antiplatelet effects. This study enhances the knowledge of the biological activity of blackthorn flowers and supports their traditional use in CVDs.

Thrombin‐Loaded Magnetic Microbubbles for the Treatment of Pseudoaneurysms

AbstractThe drive toward minimally invasive surgery has yielded multiple benefits for patients but has also increased the incidence of pseudoaneurysms (PSA). The current standard of care is ultrasound‐guided thrombin injection to coagulate blood in the PSA sac and seal the ruptured vessel. There is, however, a risk of downstream thrombosis if thrombin escapes into the communicating vessel and this limits patient eligibility for thrombin injection. In this study, the feasibility of using magnetic targeting to reduce the risk of distal thrombosis is investigated. Thrombin‐loaded magnetic microbubbles are formulated and injected into tissue‐mimicking phantoms of PSAs with different geometries using either saline or whole (equine) blood. Ultrasound imaging is used to quantify the concentration of bubbles remaining in the sac with and without application of a custom‐built magnetic array. An absorbance‐based assay is also used to quantify the concentration of thrombin escaping from the sac. Magnetic targeting enables a significant increase in thrombin retention in all femoral artery PSA models except one, with up to 97% ± 2.5% of the injected thrombin being retained. It is also confirmed that the enzymatic activity of thrombin is maintained, and that clot formation can be successfully achieved in whole blood.

The Effects of Sorbus aucuparia L. Fruit Extracts on Oxidative/Nitrative Modifications of Human Fibrinogen, Impact on Enzymatic Properties of Thrombin, and Hyaluronidase Activity In Vitro.

Sorbus aucuparia L. fruits (rowanberries) are food products with acknowledged nutritional value, high phenolic content, and traditional application in diabetes. In this study, the effects of rowanberry extracts (phytochemically standardised, i.a., by LC-MS/MS) on some aspects of plasma haemostasis and vascular conditions were evaluated in vitro as possible mechanisms connected with cardiovascular complications of diabetes. The analyses of structural modifications of human fibrinogen under oxidative stress conditions (C-ELISA, SDS-PAGE and Western blot) revealed that the extracts (at a concentration of 1–5 µg/mL) considerably reduced the nitration of tyrosine residues and formation of high-molecular-weight aggregates. Moreover, they inhibited the enzymatic activity of thrombin (both amidolytic and proteolytic). Additionally, some promising outcomes might be expected regarding endothelial functions from the extracts ability to inhibit hyaluronidase. Parallel experiments on model polyphenols and correlation studies formed the basis for determining the contribution of different compounds, including hydroxycinnamic acid derivatives, flavonols, and low- or high-molecular-weight flavan-3-ols derivatives (proanthocyanidins), to the observed effects. The possible synergistic activity of individual constituents was also noticed. These results broaden the knowledge on the biological activity of rowanberries, partly confirming their health-promoting properties, and indicating that their functional applications might be promising.

A competitive thrombin-linked aptamer assay for small molecule: aflatoxin B1.

We described a competitive thrombin-linked aptamer assay for small molecule, using aflatoxin B1 (AFB1) as a model, taking advantage of aptamer affinity binding and enzymatic activity of thrombin. We designed a dual functional DNA probe that contained the aptamer sequence for thrombin and the aptamer sequence for AFB1. Thrombin was labeled on the DNA probe by affinity binding between thrombin and anti-thrombin aptamer. This thrombin-labeled DNA probe was attached on AFB1-bovine serum albumin conjugate (BSA-AFB1) coated on a microplate through the affinity interaction between AFB1 and anti-AFB1 aptamer. The thrombin attached on the microplate catalyzed the cleavage of peptide substrate into detectable product, generating signal for detection. In the presence of AFB1, free AFB1 competed with BSA-AFB1 in the binding to the limited amount of DNA probe, leading to signal decrease. Detection of AFB1 was achieved by measuring the signal change. Under optimized conditions, AFB1 was successfully detected in the range from 0.5nM to 1μM when fluorogenic peptide substrate of thrombin and fluorescence analysis were applied. The use of chromogenic peptide substrate in the assay allowed the detection of AFB1 ranging from 0.5 to 125nM by simple absorbance analysis. The thrombin-linked aptamer assay showed good selectivity towards AFB1, and enabled the detection of AFB1 spiked in diluted beer and corn flour. This TLAA strategy extends the analytical application of thrombin and aptamers in detection of small molecules. Graphical abstract.

To clot, or not to clot – Antithrombotic therapy is the question

Haemostasis and thrombosis rely on three components namely the vascular endothelial wall, blood platelets and the coagulation cascade. Non-physiologic excessive thrombosis occurs when haemostatic processes are dysfunctional, causing undue clot formation or reduced clot lysis. Antithrombotic agents including antiplatelet, anticoagulation and fibrinolytic agents are essential for the prophylaxis and pharmacological management of venous thromboembolism and arterial thrombosis. Anticoagulation treatment options have expanded steadily over the past few decades, providing a greater number of agents. Anticoagulants that directly target the enzymatic activity of thrombin and factor Xa have recently been developed to address the inadequacies of traditional vitamin K antagonists. Appropriate use of these agents requires knowledge of their individual characteristics, risks, and benefits.

PDI Inhibition Blocks Thrombin Generation in Humans By Interfering with Platelet Factor V Activation

Protein disulfide isomerase (PDI) is a ubiquitously expressed oxidoreductase that serves an essential role in protein folding in the endoplasmic reticulum by reshuffling disulfide bonds within nascent proteins. PDI can be released from vascular cells, including platelets, and inhibition or platelet-specific deletion of PDI blocks thrombus formation in vivo. However, the specific function of PDI in thrombus formation is poorly understood. Unlike the role of proteases in blood coagulation, which have been studied in depth, little is known about PDI substrates in the vasculature. Several platelet and endothelial integrins have been identified as putative substrates for PDI, but whether coagulation factors are directly targeted by extracellular PDI has not been established. We now identify platelet factor V as a principal coagulation substrate of extracellular PDI. We developed an unbiased strategy to identify novel substrates of PDI in washed platelets using PDI variants capable of trapping substrates: FLAG-tagged PDI mutants modified by a substitution of arginine or proline for histidine (CGHC → CGRC; CGHC → CGPC) in the catalytic motif of both the a and a’ domains. Whereas the AGHA-PDI variant which has no catalytic activity serves as a control. The CGRC-PDI variant co-precipitated with platelet factor V in a redox-sensitive manner while there was no platelet factor V detected with the AGHA-PDI variant, thus confirming that binding of PDI to platelet-derived factor V occurs through disulfide bond exchange. Platelet factor V associates with multimerin-1 through a disulfide bond. Trapping PDI mutants also bind to multimerin-1 in a reaction requiring disulfide bond exchange. To evaluate the effect of PDI inhibition on the activation of platelet factor V, washed platelets from healthy donors were stimulated with 0.1 U/mL of thrombin in the presence of varying concentrations of isoquercetin (0 to 50 µM), which has previously been shown to inhibit PDI function. We observed a dose-dependent reduction of factor Va following platelet activation despite the fact that isoquercetin did not inhibit platelet release of PF4 or block Xa or thrombin enzymatic activity directly. We next performed a clinical study designed to determine whether oral isoquercetin inhibits thrombin generation in human subjects via its ability to inhibit platelet Va generation. Plasma samples collected from healthy participants before and 4 hours after ingestion of 1000 mg of isoquercetin (N=17). In plasma samples, post-isoquercetin platelet-dependent thrombin generation decreased by 51% compared with pre-ingestion controls (P=0.0004). Furthermore, we observed an overall 26% reduction in FVa in non-FV depleted plasma (P<0.001), which corresponded with a 53% decrease in FVa generated from platelets (P<0.001). These data confirm a significant effect of PDI inhibition on the generation of FVa following platelet activation. Considering that isoquercetin reduces platelet FVa generation and similarly inhibits platelet-dependent thrombin generation in a PDI-dependent manner, we investigated whether the addition of FVa in vitro restored platelet-dependent thrombin generation. The pre-incubation of 7 µg/mL FVa prior to stimulation with low dose thrombin restored platelet-dependent thrombin generation to within 80% baseline of pre-treatment levels. We conclude that platelet factor V is an essential substrate in mediating PDI-dependent thrombin generation on platelets and propose that PDI cleaves a disulfide bond that links platelet factor V to multimerin-1, thereby releasing platelet factor V for activation and subsequent thrombin generation. DisclosuresZwicker:Quercegen Pharma: Research Funding.

Novel protease from the leaves of edible medicinal plantAster koraiensisNakai with antithrombotic activity: Purification and partial characterization

Nakaiase, a novel protease with antithrombotic and anticoagulant properties was purified from the leaves of Aster koraiensis Nakai, an edible medicinal plant. It had a molecular weight of 23 kDa, as confirmed by SDS-PAGE analysis. The protease was active at 30 °C and pH 7.0. The amidolytic activity of nakaiase was inhibited by EDTA, EGTA, and several metal ions (Ca, Ni, and Zn). Nakaiase inhibited fibrin clot formation and degraded blood clot. It also inhibit activated factor X and thrombin enzymatic activity. In addition, nakaiase prolonged activated partial thromboplastin time and prothrombin time. Further, the protease exerted significant protective effects against thrombin-induced pulmonary thromboembolism in mice. Nakaiase at a dose of 20 mg/kg was devoid of hemorrhagic activity. These results suggest that nakaiase has similar property to metalloprotease-like protease, and has potential as a therapeutic agent for thrombosis owing to its antithrombotic properties and lack of hemorrhagic activity. Practical applications Aster koraiensis Nakai, an edible medicinal plant, has long been used as a traditional medicine and healthy food source in Korea because of its pharmacological efficacy. This study provides beneficial information regarding the antithrombotic potential of A. koraiensis Nakai and bioactive enzymes. Our findings clarify the antithrombotic activities of a new purified protease, which is useful in the development of potential natural pharmacological resources and agents for functional food and clinical application.

Thrombin Induces Protease Activated Receptor (PAR)-3/-4 Interaction in Human Podocytes

INTRODUCTIONNephrotic Syndrome, one of the most common forms of glomerular disease, is characterized by massive proteinuria with structural and functional injury of specialized glomerular cells called podocytes.We and others have shown that thrombin generation is enhanced in nephrotic syndrome. Recent in vitro studies have demonstrated that exposure to high concentrations of thrombin can injure podocytes, suggesting that thrombin may exacerbate glomerular injury. However, the molecular mechanisms by which thrombin induces podocyte injury are not yet known. Thrombin activates platelets, leukocytes, and other cells via the protease activated receptor (PAR) system. Thus, we hypothesized that thrombin exacerbates glomerular injury by enhancing podocyte apoptosis in a PAR-dependent manner.METHODSExperiments were performed with differentiated, conditionally immortalized human podocytes. After 36 hours of thrombin (20nM) exposure podocyte apoptosis was determined by TUNEL assay. Specific PAR antibodies and activating peptides were utilized to determine which PARs mediate thrombin-induced podocyte apoptosis. Specific PAR antibodies (ab) included hPAR1ab (ATAP2), hPAR2 ab (SAM11), hPAR3 ab (8E8), hPAR4 ab (H-120). Activation peptides (AP) included PAR1 AP (TFFLRNPNDKNH2), PAR2 AP (SLIGRLNH2); PAR3 AP (TFRGAPOH); PAR4 AP (AYPGKFNH2) and a control peptide (FSLLRNNH2). Phalloidin assays were used to evaluate structural changes in the actin-cytoskeleton as a marker of podocyte stress. Proximity ligation assays (PLAs) allow detection of protein-protein interactions at the molecular level. PLAs were performed on podocytes grown in an 8-well chamber slide system. PLA was carried out using the DUOLinkTM kit (Sigma-Aldrich) according to the manufacturer's instructions and as described previously. Oligonucleotide-conjugated secondary antibodies against mouse and rabbit primary antibodies were used and protein-protein associations were detected by microscopy as bright red dots. One-Way ANOVA and t-tests were used to determine statistical significance (SigmaPlotTM).RESULTSThrombin exposure induced a significant increase in apoptosis of human podocytes from 1.8% to 42.87% (p <0.05). Blockade of PAR-3 or PAR-4 resulted in a significant decrease in apoptosis [9.2% with hPAR-3 ab and 11.7% with hPAR-4ab] (p <0.05). Inhibition of thrombin enzymatic activity with hirudin, a direct thrombin inhibitor, also resulted in a decrease in apoptosis to 2.1% (p <0.05). In comparison to a control peptide, PAR-4 activation peptide significantly increased apoptosis from 1.7% to 40.1% (p <0.05), while PAR-3 activation peptide did not. Analogous results were seen with the phalloidin assay. Thrombin caused actin cytoskeletal changes, while PAR-3 and PAR-4 blockade ameliorated these changes. In addition, only activation with PAR-4 activating peptide resulted in loss of actin stress fibers. These findings suggest that, in human podocytes, thrombin signaling is mediated via PAR-4, in a PAR-3-dependent manner.Thus to directly assess this hypothesis, we performed proximity ligation assays with PAR-3/PAR-4 antibodies in the presence and absence of thrombin which revealed the presence of PAR-3/PAR-4 interactions in thrombin exposed podocytes, but not in control podocytes. The quantification of the in situ PLA signals per cell was performed with a BZ900 microscope and software (Keyence, Osaka, Japan) equipped with automated 'dynamic cell count" feature (p =0.03).CONCLUSIONSThrombin-induced injury is mediated in a PAR-dependent fashion in human podocytes. Specifically, in this in vitro model, thrombin-induced apoptosis appears to be mediated in a PAR-3/-4 dependent manner. Furthermore, these data suggest that thrombin induced podocyte injury may be mediated in a manner dependent on interactions between PAR-3 and -4. This is a novel finding, as PAR-3/-4 interactions are known to mediate mouse platelet signaling, but have not been previously described in human cells. Collectively, our findings suggest that interrupting thrombin-mediated podocyte injury may offer a novel therapeutic approach to reduce podocyte injury during nephrotic syndrome. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thrombin Induces Apoptosis in Human and Rat Podocytes in a Protease Activated Receptor (PAR)-Dependent Manner

▪INTRODUCTIONNephrotic Syndrome, one of the most common forms of glomerular disease, is characterized by massive proteinuria with structural and functional injury of specialized glomerular cells called podocytes. There is evidence to indicate that thrombin generation is enhanced in nephrotic syndrome. The massive protein loss in nephrotic syndrome includes loss of key coagulation regulators, leading to a complex coagulopathy, enhanced thrombin activating capacity and, consequently, increased risk for devastating thromboembolic complications. Recent in vitro studies have demonstrated that exposure to high concentrations of thrombin can injure podocytes, suggesting that thrombin may exacerbate glomerular injury. However, the molecular mechanisms by which thrombin induces podocyte injury are not yet known. Thrombin activates platelets, leukocytes, and other cells via the protease activated receptor (PAR) system. Thus, we hypothesized that thrombin exacerbates glomerular injury by enhancing podocyte apoptosis in a PAR-dependent manner.METHODSExperiments were performed with differentiated, conditionally immortalized human and rat podocytes. After 36 hours of thrombin (20nM) exposure podocyte apoptosis was determined by TUNEL assay. In human podocytes, specific PAR antibodies and activating peptides were utilized to determine which PARs mediate thrombin-induced podocyte apoptosis. Specific PAR antibodies (ab) included hPAR1ab (ATAP2), hPAR2 ab (SAM11), hPAR3 ab (8E8), hPAR4 ab (H-120). Activation peptides (AP) included PAR1 AP (TFFLRNPNDKNH2), PAR2 AP (SLIGRLNH2); PAR3 AP (TFRGAPOH); PAR4 AP (AYPGKFNH2) and a control peptide (FSLLRNNH2). Phalloidin assays were used to evaluate structural changes in the actin-cytoskeleton as a marker of podocyte stress. We have established that human thrombin is hemostatically active and regulated in rat plasma. Thus, rat podocytes were exposed to human thrombin, to determine toxicity, and specific PAR antibodies to determine if blockade of PARs could ameliorate thrombin mediated toxicity. One-Way ANOVA and t-tests were used to determine statistical significance (SigmaPlotTM).RESULTSThrombin exposure induced a significant increase in apoptosis of human podocytes from 1.8% to 42.87% (p<0.05). Blockade of PAR-3 or PAR-4 resulted in a significant decrease in apoptosis [9.2% with hPAR-3 ab and 11.7% with hPAR-4ab] (p<0.05). Inhibition of thrombin enzymatic activity with hirudin, a direct thrombin inhibitor, also resulted in a decrease in apoptosis to 2.1% (p<0.05). In comparison to a control peptide, PAR-4 activation peptide significantly increased apoptosis from 1.7% to 40.1% (p<0.05), while PAR-3 activation peptide did not. Analogous results were seen with the phalloidin assay. Thrombin caused actin cytoskeletal changes, while PAR-3 and PAR-4 blockade ameliorated these changes. In addition, only activation with PAR-4 activating peptide resulted in loss of actin stress fibers. [Display omitted] [Display omitted] In rat podocytes human thrombin had a similar effect with increased apoptosis from 1% to 33.6% (p<0.05). We also demonstrated that this may be a PAR-mediated mechanism as blockade of PAR-1 and PAR-4 with specific antibodies ameliorated thrombin induced apoptosis [1.94% with PAR-1 ab and 3.5% with PAR-4ab] while blockade of PAR-2 and PAR-3 did not have a similar effect (p<0.05). [Display omitted] CONCLUSIONSThrombin-induced injury is mediated in a PAR-dependent fashion in both humans and rats. Specifically, in this in vitro model, thrombin induced apoptosis appears to be mediated in a PAR-3/-4 dependent manner in human podocytes but in a PAR-1/-4 manner in rat podocytes. Furthermore, these data suggest that thrombin induced podocyte injury may be mediated in a manner dependent on PAR heterodimerization. Our findings collectively suggest that interrupting thrombin-mediated podocyte injury may offer a novel future therapeutic approach to reduce podocyte injury in nephrotic syndrome. DisclosuresNo relevant conflicts of interest to declare.

Selective albumin-binding surfaces modified with a thrombin-inhibiting peptide

Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices.

Thrombin Activity Propagates in Space During Blood Coagulation as an Excitation Wave

Injury-induced bleeding is stopped by a hemostatic plug formation that is controlled by a complex nonlinear and spatially heterogeneous biochemical network of proteolytic enzymes called blood coagulation. We studied spatial dynamics of thrombin, the central enzyme of this network, by developing a fluorogenic substrate-based method for time- and space-resolved imaging of thrombin enzymatic activity. Clotting stimulation by immobilized tissue factor induced localized thrombin activity impulse that propagated in space and possessed all characteristic traits of a traveling excitation wave: constant spatial velocity, constant amplitude, and insensitivity to the initial stimulation once it exceeded activation threshold. The parameters of this traveling wave were controlled by the availability of phospholipids or platelets, and the wave did not form in plasmas from hemophilia A or C patients who lack factors VIII and XI, which are mediators of the two principal positive feedbacks of coagulation. Stimulation of the negative feedback of the protein C pathway with thrombomodulin produced nonstationary patterns of wave formation followed by deceleration and annihilation. This indicates that blood can function as an excitable medium that conducts traveling waves of coagulation.

Effects of heparinoids from algae on hemostasis and their action on the cycle cell

Heparin was the first component used as an anticoagulant and antithrombotic agent; therefore, its use presents limitations due to side effects, limited source of material and occurrence of contamination. Marine sulfated polysaccharides provide a low level of contamination by prions and viruses as they are extracted exclusively from a marine source. Heparin-like compounds, sulfated galactans, extracted from red marine algae Hypnea musciformis and fractionated with 1 volume of acetone (F1.0), were evaluated for their anticoagulant activity by activated partial thromboplastin time (aPTT) and prothrombin time (PT) tests by examining their action on enzymatic activity of thrombin and FXa, and their stimulatory action on antithrombotic heparan sulfate synthesis using [35S]-sodium sulfate, and determining how they promote the inhibition of cell proliferation. The aPTT test displayed a low anticoagulant activity at 350μg of F1.0 (∼90s) P<0.001. F1.0 caused an inhibition of around 50% on the enzymatic activity of the coagulation factor thrombin. This compound also exhibited a strong stimulatory action on antithrombotic heparan sulfate synthesis (4.6 times) by rabbit aortic endothelial cells (RAEC). The analysis showed that F1.0 causes inhibition of proliferation of RAEC cells due to blocking the cycle cell progression once the cells accumulate at the S phase of the cycle; nevertheless, F1.0 is not cytotoxic for this cell lineage because it does not cause apoptosis or necrosis in RAEC cells. Thus, this polymer has potential against the occurrence of thrombosis once it provides antithrombotic action.

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue. A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach.

Protease-resistant peptide ligands from a knottin scaffold library.

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 10(9) variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution and slow dissociation rates and were able to inhibit thrombin's enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a 2 h incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high-affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications.

Poly(methyl methacrylate) microchip affinity capillary gel electrophoresis of aptamer–protein complexes for the analysis of thrombin in plasma

Thrombin generation in blood serves as an important marker for various hemostasis-related diseases and conditions. Analytical techniques currently utilized for determining the thrombin potential of patients rely primarily on the enzymatic activity of thrombin. Microfluidic-based ACE using fluorescently labeled aptamers as affinity probes could provide a simple and efficient technique for the real-time analysis of thrombin levels in plasma. In this study, aptamers were used for the analysis of thrombin by affinity microchip CGE. The CGE used a poly(methyl methacrylate) (PMMA) microfluidic device for the sorting of the affinity complexes with a linear polyacrylamide (LPA) serving as the sieving matrix. Due to the fact that the assay was run under nonequilibrium electrophoresis conditions, the presence of the sieving gel was found to stabilize the affinity complex, providing improved electrophoretic performance compared to free-solution electrophoresis. Two fluorescently labeled aptamer affinity probes, HD1 and HD22, which bind to exosites I and II, respectively, of thrombin were investigated. With an electric field strength of 300 V/cm, two well-resolved peaks corresponding to free aptamer and the thrombin-aptamer complex were obtained in less than 1 min of separation time with a run-to-run and chip-to-chip reproducibility (RSD) of migration times <10% using both aptamers. HD22 affinity assays of thrombin produced baseline-resolved peaks with favorable efficiency due to its higher binding affinity, whereas HD1 assays showed poorer resolution of the free aptamer and complex peaks. HD22 was used in determining the level of thrombin in human plasma. Assays were performed directly on plasma that was diluted to 10% v/v. Thrombin was successfully analyzed by microchip CGE at a concentration level of 543.5 nM for the human plasma sample.

Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing

We selected DNA aptamers against insulin and developed an aptameric enzyme subunit (AES) for insulin sensing. The insulin-binding aptamers were identified from a single-strand DNA library which was expected to form various kinds of G-quartet structures. In vitro selection was carried out by means of aptamer blotting, which visualizes the oligonucleotides binding to the target protein at each round. After the 6th round of selection, insulin-binding aptamers were identified. These identified insulin-binding aptamers had a higher binding ability than the insulin-linked polymorphic region (ILPR) oligonucleotide, which can be called a “natural” insulin-binding DNA aptamer. The circular-dichroism (CD) spectrum measurement of the identified insulin-binding DNA aptamers indicated that the aptamers would fold into a G-quartet structure. We also developed an AES by connecting the best identified insulin-binding aptamer with the thrombin-inhibiting aptamer. Using this AES, we were able to detect insulin by measuring the thrombin enzymatic activity without bound/free separation.

Aptameric enzyme subunit for homogeneous protein sensing

We have developed an aptameric enzyme subunit (AES) for protein sensing. AES is composed of an enzyme-inhibiting aptamer and a target molecule-binding aptamer and it can allosterically control the corresponding enzyme activity by binding target molecules. Here, we applied AES to immunoglobulin E (IgE) sensing. We split the thrombin-inhibiting aptamer into two oligonucleotides and then connected IgE-binding aptamer and its complementary strand to the split thrombin-inhibiting aptamer. The hybrid of these two oligonucleotides inhibited thrombin enzymatic activity and it was decreased in the presence of IgE. We were able to detect IgE by using the AES in homogeneous assay and detection limit was 250 nM.

A comparative thermodynamic study for both natural and artificial RNA/DNA–protein binary complexes

In recent years, Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique has been developed into a fast growing field. In contrast to traditional recognition elements, like antibody, our interests focus on novel molecular recognition elements based on nucleic acids, which are of value both for the therapy and biosensors. A comparative study of thermodynamic for both natural and artificial RNA/DNA–protein complexes would establish bases for a specificity of complex formation. In particular, we have shown that aptamers could be used for a direct measuring of thrombin enzymatic activity in a solution.

The stabilization and release of hirudin from liposomes or lipid-assemblies coated with hydrophobically modified dextran.

Hirudin is a 65-amino acid peptide and the most potent and specific known inhibitor of thrombin (K(i) = 0.2 pM). The short elimination half-life of hirudin from the body (1 hour) necessitates the use of a sustained and controlled delivery system. A proliposome method was used to entrap hirudin in liposomes coated with palmitoyl dextran-coated liposomes and lipid-assemblies. In vitro release studies of hirudin were performed using the lipid systems enclosed in dialysis membranes or deposited in the pores of a vascular graft. The activity of hirudin and released hirudin was measured using a thrombin chromogenic substrate assay. Entrapment efficiencies of hirudin in lipid-assemblies approached 100%, however, the release of hirudin from these systems was rapid with 90% released in 17 hours. Entrapment efficiencies of hirudin in coated-liposomes ranged from 5% to 55% and were dependent on several variables. Palmitoyl dextran- coated-liposomes showed a burst of 30% hirudin released in 5 hours with an additional 10% to 35% released over the next 600 hours. In all samples, 30-40% of the hirudin remained associated with the lipid-systems even after 600 hours. The released hirudin retained only 33% of its ability to inhibit thrombin when released from uncoated liposomes. However, hirudin retained 95% of its thrombin inhibitory activity when released from palmitoyl dextran-coated liposomes. Coated liposomes were found to stabilize hirudin and result in greater retention of hirudin's ability to inhibit thrombin's enzymatic activity, although the mechanism is not yet understood.

Thrombin increases clusterin mRNA in glomerular epithelial and mesangial cells.

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.