The Ability of Thrombin Inhibitors to Reduce the Thrombin Activity Generated in Plasma on Extrinsic and Intrinsic Activation

1997 ◽  
Vol 77 (03) ◽  
pp. 498-503 ◽  
Author(s):  
D Prasa ◽  
L Svendsen ◽  
J Stürzebecher
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Anion Binding ◽  
Thrombin Inhibitors ◽  
Coagulation System ◽  
Thrombin Activity ◽  
Continuous Registration ◽  
High Concentrations ◽  
20 Nm ◽  
Generation Test

SummaryIn a thrombin generation test with continuous registration of thrombin activity in plasma we studied the ability of a variety of thrombin inhibitors of different type and mechanism of action to influence the activity of thrombin after activation of the coagulation system. Depending on the inhibitor, the peak of thrombin activity is delayed and/or reduced.By blocking the active site of generated thrombin inhibitors cause a concentration dependent reduction of the thrombin peak and inhibit feed-back reactions of thrombin resulting in a delay of thrombin generation. Highly potent synthetic active-site directed inhibitors (Ki ≤ 20 nM) reduce the thrombin activity formed in plasma after extrinsic or intrinsic activation with the same efficiency (IC50 0.1 - 0.6 μM) as hirudin. The delay and reduction of thrombin generation by inhibitors of the anion-binding exosite 1 of thrombin is only attributed to an inhibition of feed-back reactions of thrombin. For a 50% reduction of thrombin activity in plasma by this type of inhibitors relatively high concentrations were determined.

Inhibition of Thrombin Generation in Plasma by Inhibitors of Factor Xa

1997 ◽  
Vol 78 (04) ◽  
pp. 1215-1220 ◽  
Author(s):  
D Prasa ◽  
L Svendsen ◽  
J Stürzebecher
Keyword(s):  
Thrombin Generation ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Thrombin Generation Assay ◽  
Ic50 Values ◽  
Fxa Inhibitor ◽  
20 Nm ◽  
Tick Anticoagulant Peptide ◽  
Inhibition Of Thrombin

SummaryA series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations.The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

Natural inhibitors of thrombin

2014 ◽  
Vol 111 (04) ◽  
pp. 583-589 ◽  
Author(s):  
James Huntington
Keyword(s):  
Blood Flow ◽  
Crystal Structures ◽  
Active Site ◽  
Anion Binding ◽  
Thrombin Inhibitors ◽  
Multiple Functions ◽  
Endogenous Inhibitors ◽  
Active Site Cleft ◽  
Single Protein ◽  
Natural Inhibitors

SummaryThe serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

Acute Pancreatitis in Man and Blood Coagulation Disturbances

1979 ◽  
Author(s):  
H. Kõtering ◽  
M. Hasenbein ◽  
H. Artmann ◽  
U. Kasten ◽  
J. Kellermann
Keyword(s):  
Acute Pancreatitis ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
The Other ◽  
Phospholipase A ◽  
Coagulation System ◽  
Consumption Coagulopathy ◽  
Blood Coagulation System ◽  
Severe Pancreatitis ◽  
Generation Test

The pathogenesis of blood coagulation-disturbances in patients with acute pancreatitis in man is still unknown. Therefore we studied repeatedly the blood coagulation system of all patients with acute pancreatitis, who were admitted to our clinic or were transferred from other hospitals after complications occurred. 19 patients with a severe pancreatitia were studied. Most of them showed oliguria, pancreatic lungs, thrombosis or haemorrhage. Only 9 determinations (in 5 patients) resulted an enhancement of thrombin generation in the Thrombin-Generation-Test (TGT). All the other patients showed already hypocoagulsbility in the TGT and severe signs of DIC and consumption coagulopathy with a loss of platelets, fibrinogen and prothrombin complex. In 9 patients, who died, we found histomorphologicaliy fibrin deposites and hyaline thrombi. In comparison to 58 patients with elevated amylases but no severe pancreatitiS we found, that the initial alteration of blood coagulation system in pancreatitis is a hypercoagulaoility, possibly caused by trypsin, phospholipase A or elastase.

scholarly journals Thrombin generation assays and their clinical application

2014 ◽  
Vol 155 (22) ◽  
pp. 851-857 ◽  
Author(s):  
Anita Kern ◽  
Katalin Várnai ◽  
Barna Vásárhelyi
Keyword(s):  
Arterial Thrombosis ◽  
Thrombin Generation ◽  
Coagulation System ◽  
Anticoagulant Treatment ◽  
Oral Anticoagulant Treatment ◽  
Novel Oral Anticoagulant ◽  
Key Enzyme ◽  
Generation Test ◽  
Bleeding Severity

Thrombin is a key enzyme of the coagulation system, having both pro- and anticoagulant functions. Thus, the generation of thrombin is one of the most important steps in coagulation. Global haemostasis assay, the so-called thrombin generation test is appropriate for its assessment. Since thrombin generation is sensible for both pro- and anticoagulant processes it can be applied for the general characterisation of the risk of thrombosis and bleeding, too. Clinical studies confirmed augmented thrombin generation in patients with high risk of venous or arterial thrombosis. Anticoagulant therapy (also novel oral anticoagulant treatment) can be monitored by thrombin generation. In case of haemophilia thrombin generation assays reflect bleeding severity. It is applicable for monitoring of both conventional haemophilia treatment and inhibitor-bypassing therapy, which is needed when inhibitors develop in patients. Standardization of thrombin generation methods and determination of cut off values are required before its application in clinical practice. Orv. Hetil., 2014, 155(22), 851–857.

Evaluation of Hypercoagulability in Obese Children With Thrombin Generation Test and Microparticle Release: Effect of Metabolic Parameters

2011 ◽  
Vol 17 (6) ◽  
pp. 585-589 ◽  
Author(s):  
Zeynep Şıklar ◽  
Gönül Öçal ◽  
Merih Berberoğlu ◽  
Bülent Hacıhamdioğlu ◽  
Şenay Savas Erdeve ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Body Mass Index ◽  
Thrombin Generation ◽  
Standard Deviation Score ◽  
Metabolic Parameters ◽  
Coagulation System ◽  
Release Time ◽  
Healthy Children ◽  
Obese Children ◽  
Generation Test

Obesity is associated with a hypercoagulable state. Thrombin generation test (TGT) and microparticle levels were not studied in obese children extensively. It is aimed to determine whether any differences in the coagulation system between obese and normal weighed children exist with the use of TGT and microparticles release. A total of 120 obese and 38 healthy children were included to the study. An increase of thrombin generation and microparticles levels were found in obese children. Hyperinsulinism could not find a risk factor for hypercoagulability in our obese children. None of the parameters of TGT has been shown to be related to metabolic parameters and metabolic syndrome. Microparticles release time is found to correlate only to body mass index (BMI) Standard deviation score (SDS) in obese children. Hypercoagulability is associated with childhood obesity. Significant correlation between degree of obesity and microparticles release suggested that high adipokine levels secreted from adipose tissue can stimulate procoagulant status-independent metabolic dearrangements.

Endotoxins And Procqagulant Activities Of Ascitic Fluid

1981 ◽  
Author(s):  
H Murr ◽  
P Schüssler ◽  
G E Rindfleisch ◽  
J Eisenburg ◽  
E Hiller
Keyword(s):  
Ascitic Fluid ◽  
Thrombin Generation ◽  
Procoagulant Activity ◽  
Cirrhosis Of The Liver ◽  
Coagulation System ◽  
Factor X ◽  
Monocytes And Macrophages ◽  
Tissue Thromboplastin ◽  
Generation Test ◽  
Limulus Test

Procoagulant activities in ascitic fluid of patients with cirrhosis of the liver have been reported repeatedly. It has been suggested that they cause DIC after reinfusicn of ascitic fluid, however, the nature of these factors is unknown. Since endotoxins have been found in ascitic fluid and it is known that they can greatly augment the procoagulant activities of monocytes and macrophages, we assayed for the presence of endotoxin and compared the procoagulant activities in each of 15 specimens of ascitic fluid caused either by cirrhosis of the liver or by metastasizing tumors.Endotoxins were assayed by the limulus test. Procoagulant activities were determined by thrcmbelastography, thrombin generation test, the presence of soluble fibrin monomer complexes ( SFMC ) and the ability to activate prothrombin complex and factor X.Endotoxins were detected in 12 of 15 specimens of ascitic fluid caused by cirrhosis of the liver. In 11 of the endotoxin positive and in 2 of the 3 endotoxin negative samples procoagulant activity was present ( i.e. shortened reaction time, increased thrombin generation, activation of prothrombin complex ). In contrast, in only one out of the 15 samples of tumerous ascitic fluid the limulus test was positive. Nevertheless, in 9 of these 15 specimens procoagulant activity was present. In all specimens increased amounts of SFMC were found.The precise mechanism for the activation of the coagulation system following ascitic fluid reinfusion remains to be established. In addition to stimulation of macrophages by endotoxin other factors like tissue thromboplastin release from tumor cells or injured tissues may account for the procoagulant activity of ascitic fluid.

Differential Inhibition of Thrombin Activity and Thrombin Generation by a Synthetic Direct Thrombin Inhibitor (Napsagatran, Ro 46-6240) and Unfractionated Heparin in Patients with Deep Vein Thrombosis

1999 ◽  
Vol 81 (04) ◽  
pp. 498-501 ◽  
Author(s):  
Henri Bounameaux ◽  
Herbert Ehringer ◽  
Alain Gast ◽  
Johan Hulting ◽  
Herbert Rasche ◽  
...  
Keyword(s):  
Deep Vein Thrombosis ◽  
Unfractionated Heparin ◽  
Thrombin Generation ◽  
Thrombin Inhibitors ◽  
Fixed Dose ◽  
Thrombin Inhibitor ◽  
Direct Thrombin Inhibitors ◽  
Vein Thrombosis ◽  
Thrombin Activity ◽  
Deep Vein

Summary Background: Direct thrombin inhibitors belong to a new class of antithrombotic drugs whose effects on blood coagulation in vivo in patients suffering from acute thrombotic conditions have not yet been fully explored. Methods and Results: One hundred and five patients with acute proximal deep-vein thrombosis were randomized to receive a continuous intravenous infusion of napsagatran, a novel synthetic thrombin inhibitor, at a fixed dose of 5 mg/h (n = 36) or 9 mg/h (n = 25) for five days, or APTT-adjusted unfractionated heparin (UFH, n = 44) for the same time. In these patients, thrombin activity and thrombin generation could be assessed by measuring thrombin-antithrombin III complexes (TAT) and prothrombin fragment 1+2 (F1+2), respectively, on three occasions. At baseline, TAT and F1+2 did not differ among the three groups. On Day 2 (steady state), TAT significantly decreased in all groups, and the decrease was significantly more pronounced in the patients given higher-dose napsagatran. F1+2 decreased significantly only in UFH-treated patients. Two hours after cessation of the infusion, the TAT levels increased in the two napsagatran groups but not in the UFH group, whilst F1+2 went back to the baseline levels in the napsagatran-treated patients but remained low in the UFH-treated patients. There was no rebound effect. Conclusions: The data presented suggest that direct thrombin inhibition with napsagatran at 9 mg/h is more potent than UFH in attenuating thrombin activity, but is less potent than UFH in inhibiting thrombin generation. The real significance of these findings will have to be substantiated in further trials with clinically relevant endpoints.

scholarly journals Аspects of the methodology of laboratory studies of hemostasis in pediatric hematology-oncology and general approaches in the pathology of hemostasis in leukemia

2018 ◽  
Vol 5 (3) ◽  
pp. 74-88
Author(s):  
E. M. Koltsova ◽  
A. N. Balandina ◽  
E. A. Seregina ◽  
A. V. Poletaev ◽  
T. A. Vuymo ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Thrombin Generation ◽  
Clinical Manifestations ◽  
Underlying Disease ◽  
Screening Tests ◽  
Coagulation System ◽  
Central Vein ◽  
Standard Laboratory ◽  
Pediatric Hematology ◽  
Generation Test

Children with acute leukemia are faced with high risks of thrombotic and hemorrhagic complications. The pathogenesis of haemostasis disorders in hemoblastoses is complex because, in addition to the disease itself, the aggressiveness of the therapy and the need for numerous invasive manipulations also make a significant contribution. Patients with hemoblastoses are equally susceptible to thrombosis and hemorrhage, which makes it possible to speak of multidirectional shifts in the balance of the hemostatic system in each individual patient. Standard laboratory hemostasis tests (clotting times, marker tests) are designed to assess the concentrations of individual proteins and the functioning of individual components of the hemostasis, and in do not assess the balance between its procoagulant and anticoagulant components. Global hemostatic tests designed to assess the coagulation balance, such as thromboelastography, thrombin generation test, and thrombodynamics, can be the alternative for the standard coagulation assays. The review focuses on the mechanisms of various laboratory hemostasis tests, as well as an assessment of their informative value in frequent complications of the underlying disease (sepsis leading to the development of disseminated intravascular coagulation (DIC) syndrome, thrombocytopenia) and catheterization, which is present in the majority of patients with hemoblastosis. General screening tests of the blood coagulation system have little diagnostic value in the DIC syndrome in patients with acute leukemia, mainly due to their insensitivity to hypercoagulability. Standard markers (for example, D-dimers) are non-specific and only confirm the clinical manifestations of clotting disorder in sepsis and septic shock, but are unable to predict the dynamics of this process at earlier stages of the inflammatory response. In this case, the thrombin generation test and thrombodynamics make it possible to reveal the hypercoagulable phase of the DIC syndrome. Thrombocytopenia accompanies almost all protocols of chemotherapy. In this case, the degree of bleeding does not always depend only on the concentration of platelets, since chemotherapeutic drugs can affect not only the quantity, but also the functional characteristics of platelets, which are not determined by standard examination of patients. The catheterization that accompanies the treatment of hemoblastoses is the leading cause of thrombosis in children with acute leukemia. Thromboembolism of the pulmonary artery due to thrombosis in the central vein system occurs in 8–15 % of patients. The prediction of catheter-associated thromboses using standard laboratory methods for assessing the state of the hemostasis is not possible. Absence of sensitive tests in modern diagnostic schemes leads to the fact that the attending physician is forced to focus exclusively on the clinical picture of thrombosis or bleeding. The development of new functional methods of hemostasis allows one to think that today the existing standard panel of coagulation tests can be expanded and made much more informative in terms of the prediction of thrombohemorrhagic complications in pediatric hematology-oncology.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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