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PubMed1983

Comparison of immunochemical and biological properties of human anti thrombin during blood clotting and upon interaction with exogenous thrombin.

Modification of immunological and biological properties of human antithrombin were studied in plasma-serum pairs and in defibrinated plasma supplemented with human thrombin. Modified antithrombin obtained through whole-blood clotting or upon addition of exogenous thrombin appeared the same with regards to its electrophoretic or biological properties. However, amounts of thrombin higher than that physiologically available, had to be used to obtain a "serum-like" antithrombin in thrombin supplemented plasma suggesting different pathways for this transformation. This was in agreement with the observation in plasma of a modification of antithrombin antigenic properties upon thrombin addition whereas no difference was demonstrated when comparing serum to normal plasma. It may be concluded that the inactivation of antithrombin and the appearance of electrophoretically modified forms in normal serum is not mainly due to the formation of enzyme-inhibitor complexes and therefore that proteolytically modified, enzyme-free forms of antithrombin demonstrated in purified systems (Fish et al. 1979) could be of physiological relevance.

Antithrombins↗
PubMed1998

Highly selective mechanism-based thrombin inhibitors: structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes.

The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined and refined to R values 0.152-0. 170 at 1.8-2.1 A resolution. Since the selectivity of two of the inhibitors was >1600 with respect to trypsin, the structures of trypsin-inhibited complexes of these inhibitors were also determined (R = 0.142-0.157 at 1.9-2.1 A resolution). The selectivity appears to reside in the inability of a benzenesulfonamide group to bind at the equivalent of the D-enantiomorphic S3 site of thrombin, which may be related to the lack of a 60-insertion loop in trypsin. All the inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. Differences in the binding constants of these inhibitors are correlated with their interactions with thrombin and trypsin. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism. The structures of both thrombin and trypsin complexes show similar well-defined transition states in the S1 site and at the electrophilic carbon atom and Ser195OG. The trypsin structures, however, suggest that the first step in a two-step kinetic mechanism may involve formation of a weak transition-state complex, rather than binding dominated by the P2-P4 positions.

Aldehydes↗
PubMed1996

Thrombin-mediated activation of endogenous factor XI in plasma in the presence of physiological glycosaminoglycans occurs only with high concentrations of thrombin.

The variable bleeding tendency associated with a genetic deficiency of factor XI (FXI) and the lack of bleeding disorders in individuals with a genetic deficiency of factor XII (FXII) suggest an alternative mechanism for FXI activation in vivo. Recently, thrombin has been shown to activate FXI. However, in plasma this activation has been shown to occur only with exogenous FXI and a non-physiological cofactor (sulphatides), and the occurrence of this reaction in a plasma environment has been questioned. Using recently developed sensitive assays for FXIa-inhibitor complexes we found thrombin-mediated and FXII-dependent activation of endogenous FXI in plasma in the presence of heparan sulphate, heparin, dermatan sulphate or dextran sulphate. Using heparan sulphate, which is present in the human vascular system, activation of about 1-2% of plasma FXI was observed, however, only after addition of very high amounts (500 nmol/l) of human alpha-thrombin to FXII-deficient plasma (at a 1 to 4 final dilution). We conclude that endogenous FXI in plasma can be activated by thrombin in the presence of various glycosaminoglycans, including the physiological compounds heparan sulphate and dermatan sulphate, but only at very high concentrations of thrombin, corresponding to 100% prothrombin activation in undiluted plasma.

Blood Coagulation↗
PubMed2002

Thrombin potential and thrombin generation after exhaustive exercise.

Exhaustive exercise leads to an activation of blood coagulation, but the implications of this activation are still unclear. The aim of this study was to investigate if a hypercoagulant stage exists after exhaustive treadmill- or cycle exercise; intrinsic and extrinsic endogenous thrombin potential (ETP) were measured by using the method of Hemker et al. Thirteen healthy male subjects underwent an exhaustive treadmill (TR) or cycle (CY) ergometer test and a control-day in random order. Blood samples were taken, repeatedly, after a 30 min rest, immediately before and after, and 1 h after exercise for measuring intrinsic and extrinsic total thrombin potential (TTPin, TTPex) (including free and alpha 2 -macroglobulin-bound thrombin) and endogenous thrombin potential (ETPin, ETPex), aPTT, PT, F1 + 2 and TAT. In comparison to the pre-value taken immediately before the exercise, the intrinsic TTP was significantly (p < 0.05) increased directly after exercise (TR-TTPin, + 11.6 %; CY-TTPin, + 11.5 %). In contrast, ETPin remained unchanged after both exercises. Additionally for TTPex and ETPex, no changes after exercise were detectable. aPTT was significantly (p < 0.05) shorter after exercise (TR-aPTT, - 16.2 %; CY-aPTT - 17.5 %), F1 + 2-concentrations were higher (p < 0.05) (TR-F1 + 2, + 21.2 %; CY-F1 + 2, + 9.8 %), but TAT remained unchanged. Differences between TR or CY could not be determined. These results show the expected shortening of aPTT and the increase of F1 + 2 indicating an activation of the coagulation system during exercise. However, the unchanged intrinsic and extrinsic ETP lead to the conclusion that in healthy young male subjects the potential for thrombin generation is insignificant, is directly counterbalanced by alpha 2-macroglobulin and is independent of the type of exhaustive exercise done.

Adult↗
PubMed2001

Quantitative measurements of mouse brain thrombin-like and thrombin inhibition activities.

Thrombin-like enzymatic activity was measured in mouse brain homogenates and slices by cleavage of a peptide substrate, N-p-Tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin. The activity was localized mainly to white matter. However, it was not affected by specific thrombin inhibitors, and was found to represent the sum of at least two enzyme activities, a prolyl endopeptidase and an aminopeptidase. By specifically inhibiting this endogenous activity in combination with exogenously added thrombin, mouse brain tissue was shown to express a capacity of thrombin inhibitory activity equivalent to 0.2 mU thrombin/mg brain tissue. The present study offers a simple and reliable method for measuring total thrombin inhibitory activity in brain.

Aminopeptidases↗
PubMed1991

Characterization of the cellular binding domain and the effects of monoclonal antibodies and thrombin inhibitors on the binding and internalization of the antithrombin-III--thrombin complex by cultured cells.

Antithrombin III (AT) binds to cultured cells mainly as a complex with thrombin or other serine proteases rather than in its free form. This implies that, upon complex formation, a new determinant appears on the AT molecule which is recognized by the cells. Fragmentation of AT by cyanogen bromide exposes this determinant and an 8-kDa fragment is recognized by cultured cells. The binding of this fragment to cultured cells is inhibited by antithrombin-III-thrombin (AT-T) complex, but not by free AT. The putative cellular binding domain of AT-T is located over amino acid residues 253-314 of AT, in the large loop close to the carboxy-terminus of the molecule. The cell-associated AT-T is internalized and degraded, forming the thrombin-modified AT (ATM). This process is inhibited by lysosomal degradation inhibitors, such as chloroquine or benzamidine. Thus, the appearance of ATM in cells is not a result of its binding to the cell surface, but probably a result of lysosomal degradation of cell-associated AT-T. Another degradation product, with a molecular mass of 43 kDa, appears in cells along with the appearance of ATM. Hirudin, a specific inhibitor of thrombin, inhibits the cellular internalization of AT-T complexes, indicating a possible role for thrombin activity in the internalization process of complexes. Monoclonal antibodies (mAb) against AT, whose epitopes on the AT molecule have been located, affect the binding of AT-T to cultured cells. Two mAb, A10 whose epitope is found outside of the cellular binding site, and B108 whose epitope may overlap this sequence, totally inhibit the binding of the complex to cells. Inhibition of binding results either from steric hindrance or induced changes in the binding site.

Animals↗
PubMed1991

In vitro non-thrombogenicity of a thrombin-substrate-immobilized polymer surface by the inhibition of thrombin activity.

Derivatives of thrombin substrate were synthesized and immobilized on a poly(acrylic acid)-grafted polyurethane film. The carboxyl terminal of the thrombin substrate peptide should be blocked for a higher inhibitory effect of the thrombin activity. Immobilization of the thrombin substrate peptide enhanced adsorption and inactivation of thrombin on the polymer film to prolong the time for fibrin network formation, and suppressed adhesion and deformation of platelets on the film. Consequently, in vitro thrombus formation on the polymer film was strongly suppressed.

Amino Acid Sequence↗
PubMedDate not supplied

Binding of biotinylated thrombin receptor peptide to cloned human thrombin receptor overexpressed in baby hamster kidney cells.

Baby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblast growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Analysis of the binding to live BHK cells yielded an equilibrium dissociation constant (Kd) of 3.0 +/- 0.3 mu mol/l and a maximal binding capacity (Bmax) of 31.0 +/- 0.5 nmol/mg of protein. In competitive binding experiments, the thrombin receptor agonist peptide (SFLLRN), which is a strong inducer of human platelet aggregation, was the most potent competitor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPNDKYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system using the biotinylated thrombin receptor peptide as a labeled ligand and the cloned thrombin receptor overexpressed in BHK cells may be useful for exploring specific antagonists of the receptor.

Amino Acid Sequence↗
PubMed2000

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity.

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

Adult↗
PubMed1988

An automated approach to characterize, in simple kinetic terms, the generation and inactivation of thrombin and the interaction of fibrinogen with thrombin.

Here, we used a fully automated, computer-directed centrifugal analyzer (which permitted simultaneous turbidimetry and calculation of results) and purified thrombin, fibrinogen, and various inhibitors to study clot formation. The Km and Vm for these reactions were useful in detecting and partly characterizing anticoagulants. We also explored the generation and inactivation of thrombin, using the two-stage prothrombin time and antithrombin activity tests. The amount of thrombin instantaneously generated and inactivated was monitored under artificially created pathological conditions. The pseudo-first-order rate constant for thrombin generation and inactivation and the instantaneous concentration of enzymatically active and inactive thrombin were used in the characterization of these conditions. We believe this approach is suitable for routine clinical use.

Algorithms↗
PubMed1977

Binding of thrombin to functionally defective platelets: a hypothesis on the nature of the thrombin receptor.

The initial step in the interaction of thrombin with human platelets is binding of thrombin to specific structures on the platelet surface. Data are presented which show that platelets from thrombasthenic patients bind thrombin similar to controls. In contrast, platelets from a patient with Bernard-Soulier syndrome had lower thrombin binding capacity. It is suggested that glycoprotein one (GP-I) on the platelet surface might be the receptor for thrombin.

Blood Platelet Disorders↗
PubMed1993

Immunization by bovine thrombin used with fibrin glue during cardiovascular operations. Development of thrombin and factor V inhibitors.

Brief case histories of three patients aged 58, 38, and 44 years are reported. All underwent cardiovascular operations. Subsequently hemostasis test abnormalities developed between the seventh and eighth postoperative days after exposure to bovine thrombin used with fibrin glue. These were characterized by an increased activated partial thromboplastin time (64 to 147 seconds), prothrombin time (19 to 24 seconds), bovine thrombin time (> 120 seconds) and a markedly reduced factor V level (< 10% in two patients and 16% in the third patient). A patient plasma dilution of 1 in 200 with a normal plasma pool was necessary to correct bovine thrombin time. No fast-acting or progressive inhibitor against factor V could be detected by coagulation tests, and fresh frozen plasma perfusion had no effect. Plasmapheresis was performed preventatively to avoid bleeding, and factor V levels stabilized at around 50% after two to four exchanges. Immunologic studies showed that the inhibitors were directed not only against bovine factors but also against human ones. Therefore factor V decrease could have been the result of rapid clearance from the circulation of complexes formed with a nonneutralizing inhibitor that is not detected by clotting tests. These antibodies were purified by standard methods and immunoaffinity. Fast immunization could be explained by a prior sensitization to bovine thrombin exposure during previous operations. It is suggested that bovine thrombin used with fibrin glue contains small amounts of factor V and may be responsible for these abnormalities. This is in agreement with previous literature reports. However, these described neutralizing factor V inhibitors, which were easily detected.

Adult↗
PubMed1995

Thrombin generation in plasma: its assessment via the endogenous thrombin potential.

Thrombin generation is a pivotal function of plasma in haemostasis and thrombosis. Its mechanism is essentially the classical cascade, the velocity of which is governed by the availability of factors Va and VIIIa and that is confined to the surface of the procoagulant membranes which appear at the site of injury. There is no routine test that quantitatively renders the thrombin forming capacity of a plasma sample. Clotting times (PT-prothrombin time, APTT-activated partial tromboplastin time) do not reflect the over all thrombin generation and are insensitive to hypercoagulative states. The endogenous thrombin potential (ETP), i.e. the area under the thrombin generation curve, better represents this function. We developed a method to assess the ETP in the routine laboratory. The first results suggest that it is a sensitive indicator for every form of anticoagulation. It is increased in hypercoagulable states thus far studied, both congenital and acquired and can be designed to indicate deficiencies in protein C and S and APC (activated protein C) resistance.

Animals↗
PubMed1992

Prophylactic antithrombin III administration during pregnancy immediately reduces the thrombin hyperactivity of congenital antithrombin III deficiency by forming thrombin-antithrombin III complexes.

We examined the changes of haemostatic molecular markers after antithrombin III (AT III) administration in a 22-year-old woman with congenital AT III deficiency in the third trimester of pregnancy who did not have thrombosis. Various markers including fibrinopeptide A (FPA), thrombin-antithrombin III complex (TAT), prothrombin fragment F1 + 2 (F1 + 2), plasmin-alpha 2antiplasmin, D-dimer, beta-thromboglobulin, and platelet factor 4 were measured before and just after 3,000 U of AT III concentrate, which was given three times per week from the 34 week of pregnancy until delivery. Just after AT III administration, F1 + 2 and FPA levels decreased on most occasions, while TAT sometimes increased. Plasma FPA levels were markedly decreased on all 8 occasions when the plasma FPA levels was above 2.0 ng/ml before AT III administration. Plasma FPA levels were always greater than or equal to 6.4 ng/ml before AT III administration on the 4 occasions when TAT increased to above 115%. The changes of plasma F1 + 2 levels were significantly correlated with the AT III level. These results suggest that prophylactic AT III administration in the third trimester immediately inactivates intravascular thrombin to form TAT and reduce the plasma FPA level. Thus, the transient TAT elevation following AT III administration may not only be due to extraction of thrombin from the fibrin clots of thrombi but also to intravascular thrombin which is not attached to thrombi. FPA is the best molecular marker for thrombin hyperactivity and it should be monitored in AT III-deficient pregnant women in the third trimester.

Adult↗
PubMed2004

[Thrombin markers in pregnant women: measurements of prothrombin fragments F 1+2 and thrombin-antithrombin III complexes (TAT) in the cord blood, the mother's blood and in amniotic fluid].

OBJECTIVES: Hypercoagulability is a well known feature of pregnancy, while hypocoagulability is attributed to newborns. The level of thrombin markers in the blood reflects the relations in coagulation system. We have measured two markers of thrombin generation, e.i prothrombin fragments F 1+2 and thrombin-antithrombin III complexes (TAT), in the cord blood and the mother's blood, as well as in amniotic fluid. MATERIAL AND METHODS: The study group consisted of 33 parturient women, 24.2 +/- 3.6 years old, 20 primiparas and 13 multiparas with normal course of pregnancy. The level of F 1+2 and the level of TAT were estimated by ELISA method. RESULTS: The highest level of F 1+2 and TAT was in amniotic fluid, e.i. 29.99 (9.15 - 50.75) nmol/l vs. 519.62 +/- 270.51 microg/l. In the blood cord the level of F 1+2 was 7.15 (5.05 - 22.05) nmol/l, and the level of TAT was 151.57 +/- 134.17 microg/l. In the mother's blood plasma the levels of F 1+2 and TAT were significantly lower than in cord blood (5.15, range 3.50 - 6.05 nmol/l vs. 36.30 +/- 18.65 microg/l respectively, p < 0.001). CONCLUSION: Increased generation of thrombin in foetal blood which is reflected in increased levels of F 1+2 and TAT, is one of the features of "foetal phenomenon" concerning foetal coagulation system. High concentration of F 1+2 and TAT in amniotic fluid can be consider to be a result of increased thrombin generation or the lowered metabolism of F 1+2 and TAT.

Adult↗
PubMed1996

Synergistic actions of a thrombin-derived synthetic peptide and a thrombin receptor-activating peptide in stimulating fibroblast mitogenesis.

We measured the ability of the thrombin receptor activating peptide, SFLLR-NH2 (P5A) to stimulate 3H-thymidine incorporation in hamster CCL-39 fibroblasts either alone or in combination with the thrombin-derived polypeptides, YPPWNKNFTENDLL (TDP-1) and AGYKPDEGKRGDACEGDSGGPFV (TDP-2). In the presence (but not absence) of the amino peptidase inhibitor amastatin (10 microM), P5A alone (7.5 to 100 microM) caused a 1.5- to 2-fold stimulation of thymidine incorporation above basal, even though this inhibitor did not abrogate the degradation of P5A by other peptidases present in the assay medium. Neither TDP-1 nor TDP-2 alone had any effect on thymidine incorporation. However, TDP-1 (30 to 90 microM) considerably augmented P5A-mediated thymidine incorporation at low P5A concentrations (7.5 to 30 microM), shifting the P5A concentration-effect curve to the left. TDP-2 was inactive in this regard. The EC50 for this potentiating action of TDP-1 was approximately 40 microM. Further, thrombin, rendered proteolytically inactive by a low-molecular-weight bifunctional inhibitor, hirutonin-6, also acted synergistically with P5A to stimulate CCL-39 cell thymidine incorporation. We hypothesize that thrombin can cause its cellular effects, such as thymidine incorporation, not only via the proteolytic activation of its G-protein-coupled receptor, but also via the concurrent and synergistic interaction of its TDP-1 peptide domain with a separate cell surface docking site.

Amino Acid Sequence↗
PubMed1990

Thrombin receptor antagonists. Structure-activity relationships for the platelet thrombin receptor and effects on prostacyclin synthesis by human umbilical vein endothelial cells.

Structure-activity studies on a series of analogues of N-(3-methyl-S-(1-pyrrolidinyl carbonyl) butyl)-D-alanine ethyl ester hydrochloride (SC42619) have defined the features of this dipeptide analogue required for observation of thrombin receptor antagonist activity on the human platelet. The affinity for SC42619, and for its structural analogue SC43583 is enhanced by pretreatment of the platelets with chymotrypsin. Endothelial cell prostacyclin (PGI2) synthesis induced by thrombin and trypsin is selectively inhibited by SC42619 provided that prolonged exposure to this antagonist is avoided. However inhibition of PGI2 synthesis by SC42619 is not overcome by increasing the thrombin concentration. The data provide further support for identification of SC42619 and certain of its analogues as selective antagonists at the platelet thrombin receptor but suggest that these compounds may have more complex, and possibly non-selective effects on the endothelial cell.

Antithrombins↗
PubMed1982

Further studies on the interaction between thrombin and GP Ib using crossed immunoelectrophoresis. Effect of thrombin inhibitors.

The platelet surface protein GP Ib (glycocalicin-related protein) has been shown to be retarded by thrombin-Sepharose 4B in a crossed immunoelectrophoresis system. The interaction between GP Ib and thrombin was abolished when thrombin was blocked either at the active serine site with tosyl-lysine-chloromethyl-ketone (TLCK) or phenylmethylsulfonylfluoride (PMSF) or at the fibrinogen binding site (macromolecular binding site) with N-bromosuccinimide (NBS) or heparin, indicating that both sites have to be freely accessible for the retention of the glycocalicin-related protein by thrombin.

Animals↗