Amidolytic Activity Of Thrombin Research Articles

Thrombin and Factor Xa Hydrolysis of Chromogenic Substrates in the Presence of Sulfated Derivatives of Galactomannan and Galactoglucomannan Natural Gels.

Polysaccharides are important structural components of all plant species. Gel-like polysaccharides have found wide application in various fields, including medicine, construction, and the food industry. In the present work, galactomannan and galactoglucomannan gel-like polysaccharides were modified with sulfate groups and their anticoagulant activity was studied. Sulfation with chlorosulfonic acid in pyridine and with sulfamic acid in pyridine and a sulfamic acid-urea deep eutectic solvent were used as synthesis routes. The resulting gel-like polysaccharide sulfates were studied by elemental analysis, Fourier-transform infrared spectroscopy, and gel permeation chromatography. It was established that the anticoagulant effect of sulfated galactoglucomannan (SGGM) and galactomannan (SGM-1 and SGM-2) is related to an independent antithrombin-independent decrease in the amidolytic activity of thrombin and factor Xa. It is shown that the inhibitory activity of SGGM and SGM-2 against the collagen-induced platelet aggregation can be an additional factor in selecting compounds that are most promising for modifying polymer surfaces to ensure resistance to blood clotting.

Screening of the Promising Direct Thrombin Inhibitors from Haematophagous Organisms. Part I: Recombinant Analogues and Their Antithrombotic Activity In Vitro.

The success in treatment of venous thromboembolism and acute coronary syndromes using direct thrombin inhibitors has stimulated research aimed at finding a new anticoagulant from haematophagous organisms. This study deals with the comparison between hirudin-1 from Hirudomedicinalis(desirudin), being the first-known and most well-studied natural anticoagulant, along with recombinant analogs of haemadin from the leech Haemadipsa sylvestris, variegin from the tick Amblyomma variegatum, and anophelin from Anopheles albimanus. These polypeptides were chosen due to their high specificity and affinity for thrombin, as well as their distinctive inhibitory mechanisms. We have developed a universal scheme for the biotechnological production of these recombinant peptides as pharmaceutical substances. The anticoagulant activities of these peptides were compared using the thrombin amidolytic activity assay and prolongation of coagulation time (thrombin time, prothrombin time, and activated partial thromboplastin time) in mouse and human plasma. The preliminary results obtained suggest haemadin as the closest analog of recombinant hirudin-1, the active substance of the medicinal product Iprivask (Aventis Pharmaceuticals, USA) for the prevention of deep venous thrombosis in patients undergoing elective hip or knee replacement surgery. In contrast, variegin can be regarded as a natural analog of bivalirudin (Angiomax, The Medicines Company), a synthetic hirudin-1 derivative certified for the treatment of patients undergoing percutaneous coronary intervention and of patients with unstable angina pectoris after percutaneous transluminal coronary angioplasty.

Evaluation of the Biocompatibility of Water-Soluble Pristine С60 Fullerenes in Rabbit

C60 fullerenes have proved their therapeutic effects and efficacy by the results of countless experiments. For further usage of these nanoparticles, the systematic toxicological investigations are required. Blood compatibility should be studied for C60 fullerenes due to the potential blood contact. Currently, available data is not systematic and has not provided insights into possible side effects of C60 fullerenes on blood components. In this study, water-soluble pristine C60 fullerenes were tested in vitro to assess their biocompatibility in rabbit. The blood compatibility has been evaluated looking at the impact of C60 fullerenes on erythrocyte integrity, platelet aggregation, and some blood factors involved in coagulation. Our results revealed that C60 fullerenes cannot elicit hemolysis at studied concentrations and did not show any effect on coagulation process. C60 fullerenes in concentration-dependent manner increased ADP-dependent platelet aggregation and changed the key kinetic parameters of these processes. C60 fullerenes inhibited thrombin amidolytic activity but did not affect the activities of other studied coagulation factors. The prothrombotic property of C60 fullerenes could be the potential risk factor that leads to enhancement of vascular thrombosis. The ability of fullerene to inhibit thrombin activity is important for the pharmacological use of these carbon nanoparticles as anticoagulant agents.

Phenolic fractions from nine Trifolium species modulate the coagulant properties of blood plasma in vitro without cytotoxicity towards blood cells.

The study covers an evaluation of the influence of extracts (1-50 μg/ml), isolated from aerial parts of nine Trifolium L. species (i.e. T. alexandrinum, T. fragiferum, T. hybridum, T. incarnatum, T. pallidum, T. pratense, T. resupinatum var. majus, T. resupinatum var. resupinatum and T. scabrum) on haemostatic properties of blood plasma. The clot formation and fibrinolysis assay (CFF), blood clotting times, the extrinsic and intrinsic coagulation pathway-dependent polymerization of plasma fibrin were measured. The effects of plant extracts on amidolytic activity of thrombin were also evaluated and compared with argatroban, an antithrombotic drug. Cytotoxicity was assessed in a model of blood platelets and as the viability of peripheral blood mononuclear cells. While no changes in blood clotting times or fibrinolytic properties of blood plasma were found, some fractions impaired the blood plasma coagulation induced by the intrinsic coagulation pathway. Reduction in the maximal velocity of fibrin polymerization was also observed in the clot formation and fibrinolysis assay. No cytotoxicity of Trifolium extracts towards the investigated cells was recorded. The most efficient anticoagulant activity in plasma was found for T. fragiferum and T. incarnatum extracts, while the T. alexandrinum fraction was the most effective inhibitor of thrombin amidolytic activity.

Effect of IgG from multiple sclerosis patients on amidolytic activity of coagulation and anticoagulation factors of hemostasis

Background: Immunoglobulin G (IgG) is a major immunoglobulin (Ig) in blood that accumulates to a greater extent in the bloodstream of patients impacted by neuroimmunological disorders such as multiple sclerosis (MS). The aim of this study was to determine the effect of IgG obtained from MS patients on the amidolytic activity of coagulation and on anticoagulation factors, and to compare those effects to the effects of IgG from healthy donors.
 Methods: Spectrophotometric hydrolysis of specific chromogenic substrate by key haemostasis factors was examined.
 Results: Our study shows that unlike healthy individuals, patients suffering from MS express IgG which enhances the amidolytic activity of thrombin and protein C, but inhibits the activity of factor Xa.
 Conclusion: Our study shows that IgG and coagulation factors, indeed, interact with each other. IgG may be key mediators of neuroinflammation and, therefore, may serve as a potential target for therapeutic strategies for MS and other neuroimmunological diseases.

Stability of erythrocyte membrane and overall hemostasis potential - A biocompatibility study of mebrofenin and other iminodiacetic acid derivatives.

Intravenous injection seems to be the most convenient way of administering drugs and contrast agents, which makes components of the blood the first and usually unwanted target of their action. Binding of intravenously administered compounds to erythrocytes, blood platelets and vascular wall may have serious clinical implications. The aim of this study was to examine the influence of four iminodiacetic acid derivatives, potential ligands for gadolinium complexation, on the process of coagulation and fibrinolysis, activity of thrombin and hemolysis. Kinetic parameters of coagulation and fibrinolysis process were determined during an optical CL-test based on measurement of transmittance alterations. Thrombin (0.5 IU/mL) and t-PA (240 ng/mL) were used to obtain a clotting and lysis curve. The activity of thrombin was determined with a chromogenic substrate S-2238. Hemolysis was examined spectrophotometrically and expressed as a percentage of released hemoglobin. Exposure to iminodiacetic acid derivatives resulted in a significant increase in the overall potential of clotting and lysis (CLAUC), as well as with the significant changes in the key parameters of these processes (thrombin time, initial plasma clotting velocity, clot stabilization time). Furthermore, iminodiacetic acid derivatives caused a significant decrease in the amidolytic activity of thrombin and enhanced hemolysis in a concentration-dependent manner. Despite their influence on the process of coagulation and fibrinolysis, amidolytic activity of thrombin and hemolysis, iminodiacetic acid derivatives should be generally considered safe as the significant effects were observed mostly at 4 μmol/mL, which is about 10-fold higher than the theoretical plasma concentration of these compounds.

PP.35.07

Objective: The clinical studies indicate that co-administration of aldosterone receptor blocker with standard therapy reduces mortality in patients with heart failure. We confirmed previously a strong prothrombotic effect of aldosterone and angiotensin II in the experimental models of thrombosis. Therefore, we suggest that the additional benefits of dual renin-angiotensin-aldosterone system (RAAS) blockade may be related to the effects of drugs on haemostasis. The aim of the present study was to investigate the effect of combined spironolactone (SPIRO) and quinapril (QUIN) administration on haemostasis in hypertensive rats. Design and method: Wistar 2K1C-hypertensive rats were used (n = 10–12). SPIRO (20 mg/kg), QUIN (3 mg/kg), or VEH (gummi arabici, 5% aq.sol.) were administered p.o. for 10 days. Systolic blood pressure was measured. The venous thrombosis was induced by the vena cava ligation on the 11th day. The formed thrombus was removed and weighted. The effect of drugs on platelets adhesion to collagen (ex vivo and in vitro), bleeding time (BT) and amidolytic activity of thrombin was evaluated. Tissue plasminogen activator (t-PA), plasminogen activator inhibitor type-1 (PAI-1), tissue factor (TF) and thrombin-activatable fibrinolysis inhibitor (TAFI) plasma levels were measured by ELISA. The plasma levels of some oxidative stress markers (H2O2, MDA) were measured. Results: SPIRO and QUIN reduced TF, PAI-1, TAFI plasma level (p < 0.001 vs VEH), while effects of QUIN were stronger (p < 0.001 vs SPIRO) and the most pronounced changes were observed after drugs co-administration. SPIRO/QUIN markedly reduced H2O2 and MDA plasma levels, but the strongest effect was observed after combined treatment. SPIRO/QUIN (ex vivo) or their metabolites (in vitro) reduced platelets adhesion to collagen. Only dual RAAS blockade significantly reduced thrombus weight and diminished thrombosis incidences. Conclusions: The most pronounced changes in haemostasis, leading to marked reduction of venous thrombosis, were obtained after combined SPIRO and QUIN administration. The mechanism was related to inhibition of coagulation, fibrinolysis augmentation, platelets adhesion reduction and oxidative stress impairment. This study indicates that additional benefits of dual RAAS blockade observed clinically may be also a result of drugs effect on haemostatic balance restoration.

PAMAM Dendrimers as Potential Carriers of Gadolinium Complexes of Iminodiacetic Acid Derivatives for Magnetic Resonance Imaging

This is the first study describing the utilization of PAMAM dendrimers as delivery vehicles of novel magnetic resonance imaging (MRI) contrast agents. The purpose of this paper was to establish the potential of G4 PAMAM dendrimers as carriers of gadolinium complexes of iminodiacetic acid derivatives and determine imaging properties of synthesized compounds inin vivostudies. Furthermore, we examined the influence of four synthesized complexes on the process of clot formation, stabilization, and lysis and on amidolytic activity of thrombin. Biodistribution studies have shown that the compounds composed of PAMAM G4 dendrimers and gadolinium complexes of iminodiacetic acid derivatives increase signal intensity preferably in liver in range of 59–116% in MRI studies which corresponds with the greatest accumulation of gadolinium after administration of the compounds. Synthesized compounds affect kinetic parameters of the proces of clot formation, its stabilization, and lysis. However, only one synthesized compound at concentration 10-fold higher than potential plasma concentrations contributed to the increase of general parameters such as the overall potential of clot formation and lysis (↑CLAUC) and total time of the process (↑T). Results of described studies provide additional insight into delivery properties of PAMAM dendrimers but simultaneously underscore the necessity for further research.

Total Synthesis of Homogeneous Variants of Hirudin P6: A Post‐Translationally Modified Anti‐Thrombotic Leech‐Derived Protein

AbstractHirudin P6 is a leech‐derived anti‐thrombotic protein which possesses two post‐translational modifications, O‐glycosylation and tyrosine sulfation. In this study we report the ligation‐based synthesis of a library of hirudin P6 proteins possessing homogeneous glycosylation and sulfation modifications. The nature of the modifications incorporated was shown to have a drastic effect on inhibition against both the fibrinogenolytic and amidolytic activities of thrombin and thus highlights a potential means for attenuating the biological activity of the protein.

Total Synthesis of Homogeneous Variants of Hirudin P6: A Post‐Translationally Modified Anti‐Thrombotic Leech‐Derived Protein

Hirudin P6 is a leech-derived anti-thrombotic protein which possesses two post-translational modifications, O-glycosylation and tyrosine sulfation. In this study we report the ligation-based synthesis of a library of hirudin P6 proteins possessing homogeneous glycosylation and sulfation modifications. The nature of the modifications incorporated was shown to have a drastic effect on inhibition against both the fibrinogenolytic and amidolytic activities of thrombin and thus highlights a potential means for attenuating the biological activity of the protein.

Thrombin inhibitory activity of some polyphenolic compounds

Thrombin, also known as an active plasma coagulation factor II, belongs to the family of serine proteases and plays a crucial role in blood coagulation process. The process of thrombin generation is the central event of the hemostatic process and regulates blood coagulant activity. For this reason, thrombin inhibition is key to successful novel antithrombotic pharmacotherapy. The aim of our present study was to examine the effects of the well-known polyphenolic compounds on the activity of thrombin, by characterization of its interaction with selected polyphenols using different biochemical methods and biosensor BIAcore analyses. Only six compounds, cyanidin, quercetin, silybin, cyanin, (+)-catechin and (−)-epicatechin, of all examined in this study polyphenols caused the inhibition of thrombin amidolytic activity. But only three of the six compounds (cyanidin, quercetin and silybin) changed thrombin proteolytic activity. BIAcore analyses demonstrated that cyanidin and quercetin caused a strong response in the interaction with immobilized thrombin, while cyanin and (−)-epicatechin induced a low response. Lineweaver–Burk curves show that used polyphenol aglycones act as competitive thrombin inhibitors. Our results suggest that polyphenolic compounds might be potential structural bases and source to find and project nature-based, safe, orally bioavailable direct thrombin inhibitors.

Coagulation and fibrynolitic parameters in women and the effects of hormone therapy; comparison of transdermal and oral administration

It is established that hormone therapy (HT) is related with significant increased prothrombotic risk factor. The aim of our study was to assess the effects of oral hormone therapy (o-HT) and transdermal hormone therapy (t-HT) on hemostasis parameters: fibrinogen (Fg) concentration, the maximum velocity of polymerization of clot formation, fibrin half-time lysis, plasma level of thrombin inhibitor of fibrinolysis (TAFI) and activity of generated thrombin and plasmin amidolytic activity. We observed that values of initial velocity of polymerization in o-HT group were increased (94.64 mOD/min vs. 131.50 mOD/min, p < 0.001) compared to control group. Fibrin lysis half-time increased in both groups with HT (controls - 18.26 min vs. 32.43 min (o-HT); 23.34 min transdermal hormone therapy (t-HT) p < 0.001) compared to controls. The activity of thrombin was statistically higher in plasma of women after o-HT (72.6 ± 8.5 mOD/min) than in patients with t-HT (53.7 ± 10.1 mOD/min) and controls (51.2 ± 10 mOD/min. Plasmin activity was the highest in controls (84.5 ± 10.2 mOD/min). The highest level of TAFI we observed in patients after oral hormones (80.38 ± 8.23%); women on transdermal HT had 61.58 ± 9.81% and the lowest concentration of TAFI we noted in controls 44.70 ± 10.16). The results of our study show that HT may partly explain the increase in venous thrombosis (VTE) and cardiovascular events reported after the use of it, especially the oral form of treatment.

Antithrombin Effect of Polyphenol‐Rich Extracts from Black Chokeberry and Grape Seeds

Thrombin is a serine protease that cleaves the peptide bonds in proteins located on the carboxyl side of arginine. Thrombin plays a central role in thromboembolic diseases, which are the major cause of mortality. The aim of the study was to estimate the effects of plant extracts on proteolytic properties of thrombin. Thrombin was incubated with polyphenol-rich extracts from berries of Aronia melanocarpa or seeds of Vitis vinifera (0.5, 5, 50 µg/mL) and with polyphenols ((+)-catechin, (-)-epicatechin, gallic acid, chlorogenic acid, procyanidin B1, cyanidin, cyanidin 3-glucoside, quercetin). The in vitro experiments showed that both extracts in all used concentrations inhibited proteolytic activity of thrombin observed as inhibition of thrombin-induced fibrinogen polymerization, stabilized fibrin formation, and platelet aggregation. Moreover, thrombin amidolytic activity was inhibited by polyphenols belonging to the flavonoid class. Results presented in this study indicate that polyphenol-rich extracts from berries of A. melanocarpa and seeds of V. vinifera may become promising dietary supplements in the prevention of thrombotic states.

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI ( A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant ( K i) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito’s development.

Pomegranate fruit components modulate human thrombin

Pomegranate (Punica granatum) is an important source of polyphenols with assessed antioxidant properties. The aims of this study were: (i) the characterization of the monomeric phenolic variability on each isolated fruit component (endocarp, mesocarp, aril); (ii) the study on the effect of pomegranate fruit components on human thrombin amidolytic activity. Collectively, our data show that pomegranate components contain bioactive metabolites (mainly ellagic acid) and suggest a potential role for the pomegranate extract in the regulation of a number of physio-pathological processes involving thrombin (or thrombin-like proteinase).

Anticoagulant activity of original synthetic peptide derivatives

Original synthetic peptide derivatives exhibit anticoagulant activity in vitro and in vivo. They delayed fibrin clot formation from human blood plasma in tests for the intrinsic coagulation pathway (activated partial thromboplastin time) and final stage of plasma coagulation (thrombin time) and inhibited amidolytic activity of thrombin. We determined the minimum effective dose of the most active compound providing a 2-fold lengthening of blood clotting time (activated partial thromboplastin time test and thrombin time test), which persisted for 2-3 h.

Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.

A thrombin inhibitor from the gut of Boophilus microplus ticks

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin-Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules.

Arginine Inhibits Serpins

Serine protease inactivators (serpins) are important regulators in biochemistry. Often it is necessary to block the serpin action, that is, to stabilize the sample. The guanidine group of arginine is the ligand for the active center pocket of many serine proteases. Arginine or guanidine inhibits serine proteases, and arginine belongs to the reactive P1-P1' center of many serpins. The plasmatic antithrombin, antiplasmin, or anti-C1-esterase activity was determined: A total of 20 microL of pooled normal plasma or 7% human albumin was added to 100 microL of 0-2.67 M arginine, pH 8.6, 10 microL of 26 mIU/mL thrombin in 7% human albumin, and 30 microL of 1.7 mM CHG-Ala-Arg-pNA (37 degrees C). DeltaA at 405 nm was determined, by using a microtiter plate reader. Thrombin was substituted by plasmin or C1-esterase, and the chromogenic peptide substrates <Glu-Phe-Lys-pNA or MeOC-Lys(eCBO)-Gly-Arg-pNA, respectively, were used. The IC(50) of arginine against plasmatic antithrombin activity is 580 mM; the IC( 25) is 440 mM. The IC(25) of arginine against plasmatic alpha( 2)-antiplasmin or C1-inactivator is 1650 mM. The amidolytic activity of thrombin, plasmin, and C1-esterase is inhibited similarly by arginine: the IC(50) for arginine against the amidolytic activity of these proteases is about 400 mM. Arginine at very high concentrations inhibits serpins. This is important, if stabilization of a biological fluid is a prerequisite for valid activities of serine proteases. In addition, these high concentrations of arginine might be a new gentle principle to inhibit pathogens that need serpins for their pathophysiology.

Flavonoids inhibit the amidolytic activity of human thrombin

The effect of a group of natural flavonoids on human thrombin amidolytic activity was investigated using a spectrophotometric inhibition assay while information on the kinetics and thermodynamics was obtained using optical biosensor techniques. All the flavonoids tested acted as reversible inhibitors, and the quercetin–thrombin complex was found to be most stable at pH=7.5. Docking analysis indicated that quercetin's inhibitory behavior could be related to its planar structure and low steric hindrance, and to its ability to form a critical H-bond with thrombin His57.