Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies were carried out on the effects of different doses of hydroxyethyl starch 200/0.5 (HES) on plasma clotting factors in dogs, as an animal model for the human clotting system. In 8 German shepherd dogs 15% of the total blood was isovolemically substituted either by Ringer's solution with lactate alone (controls) or with 0.6, 1.3, 1.9, 2.5 g HES/kg b.w. Immediately after the infusion, the HES concentration in the recipients' plasma amounted to 8 mg/ml up to 38 mg/ml. In the following 6 h, the HES decreased to 25% in each case. It was found that the higher the plasma HES content was, the lower the haematocrit. Neither the thrombin-nor the batroxobin-time showed any significant change, irrespective of the plasma HES concentration. The prothrombin-time was decreased directly after the infusion in parallel to the haematocrit. The single clotting factors FI, FII, FV, FVII, FVIII, FX, and FXII behaved approximately in the same way: their activities directly after infusion, but also 6 h later, were lowered in proportion to the amount of HES infused. The loss of factor activity correlated with the volume-expanding effect of HES shortly after the infusion, but not 6 h later. It is concluded that there are two different modes of HES action on clotting factors: the dilution by plasma volume expansion and a non-dilutional action. Cautious handling might be required in patients with clotting disturbances as well as in long-term treatment.
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PMID:Action of hydroxyethyl starch (HES) on the activity of plasmatic clotting factors. 969 31

In 18 sportsmen the platelet count, thrombin time, prothrombin time, activated partial thromboplastin time (APTT), cloting factors (F):I, VII, VIII and fibrin/fibrinogen degradation products (FDP) were measured before, immediately after progressive incremental exercise--and 30 min later. All post-exercise changes of the values were corrected for the plasma volume changes, which were calculated from hematocrit values. Platelet count, thrombin time, prothrombin time FI and FVII did not change significantly after exercise. Immediately post-exercise APTT was significantly shortened, but FVIII and FDP were significantly elevated. 30 min later FDP level normalized but FVIII and APTT changes persisted. The obtained data show that the applied physical exercise caused shift of the existing balance between coagulation and fibrinolysis towards hypercoagulability. They also confirm the necessity of taking into account post-exercise hemostasis changes in cardiological rehabilitation and endurance training in adults.
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PMID:[The effect of progressive incremental exercise on some parameters of hemostasis]. 973 91

We have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the Al subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1(336)/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.
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PMID:Interaction of the A1 subunit of factor VIIIa and the serine protease domain of factor X identified by zero-length cross-linking. 975 21

The addition of a pasteurisation step to a solvent/detergent (SD) treated FVIII concentrate has recently resulted in enhanced inhibitor incidence in patients in Germany and Belgium. We have investigated the effect of virus inactivation procedures on FVIII function by preparing experimental concentrates from the same starting cryoprecipitate with the following procedures: none (N); dry heat (DH); pasteurisation (P); solvent/detergent (SD); solvent detergent + dry heat (SDDH); solvent detergent + pasteurisation (SDP). In addition, several clinical SD concentrates with and without pasteurisation were studied. There were no significant differences in fibrinogen and vWF content and in the ratio of one-stage/chromogenic FVIII activity among any of the samples studied. In thrombin proteolysis and FXa generation experiments, there were no differences in results on samples N, DH, P, and SDDH from those on sample SD. However sample SDP gave markedly different results from sample SD in the following respects: slower thrombin proteolysis (t(1/2) = 12.0 min vs 1.9 min); more rapid FXa generation (rate 2.5 times that of SD); enhanced phospholipid binding (K(D) = 3.89 x 10(-11) M vs 5.53 x 10(-10) M). Similar differences between SDP and SD were seen in the clinical samples. The observed changes in the FVIII activity occurred in combination with SD and pasteurisation, but not with either treatment alone. These results suggest that SDP treatment may enhance exposure of the phospholipid binding site in the C2 domain of FVIII, and since inhibitors to the SDP product are predominantly against C2, these findings could be relevant to the enhanced immunogenicity of the SDP product.
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PMID:Modification of factor VIII in therapeutic concentrates after virus inactivation by solvent-detergent and pasteurisation. 979 82

Three model systems have been used to study the dynamics of the blood clotting process initiated by tissue factor (TF): synthetic plasma mixtures prepared with purified coagulation proteins and inhibitors; mathematical models based on the reaction constants, stoichiometries and thermodynamics of individual catalyst and inhibitor reactions; and contact suppressed whole blood induced to clot in vitro by the addition of exogenous TF. In the three models, the generation of thrombin can be described in terms of an initiation phase in which pmol/l concentrations of the coagulation serine proteases are generated and the cofactor proteins factor V (FV) and FVIII are activated. Subsequently, explosive thrombin generation occurs during a propagation phase. The complementary inhibitory pathways extinguish the generation of thrombin. Tissue factor pathway inhibitor (TFPI), present in low concentrations, primarily influences the duration of the initiation phase and has little influence on the propagation phase. Antithrombin III (ATIII), present in higher concentrations, has little influence during the initiation phase, but decreases the rate of thrombin generation during the propagation phase. The protein C pathway cannot act in the absence of thrombin and therefore only influences the duration of the propagation phase by inactivating activated FV. Thus combinations of TFPI plus ATIII and TFPI plus protein C pathway components contribute to the synergistic inhibitory processes. As a consequence of the roles of pro, and anti-coagulants, the generation of thrombin by the TF pathway becomes a threshold limited process.
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PMID:The role of the tissue factor pathway in initiation of coagulation. 981 22

We have used a cell-based model system to examine some aspects of coagulation. Unactivated platelets and tissue factor (TF)-bearing cells were mixed with plasma levels of zymogen factors IX (FIX), FVIII, FX, FV, and prothrombin, as well as coagulation inhibitors antithrombin III and TF pathway inhibitor. Reactions were initiated with plasma levels (0.2 nmol/l) of activated factor VII (FVIIa). We were able to measure platelet activation and subsequent thrombin generation in this system and have established parameters for the normal amount of thrombin generation and the range of values seen with different individuals. If FIX or FVIII were not added to this system, platelet activation but not thrombin generation was seen. We have used this system to examine the mechanism of action of high-dose FVIIa. If platelets were activated with the thrombin receptor agonist peptide SFLLRN and incubated with inhibitors and zymogen factors X, V, and prothrombin, no thrombin generation was observed. Addition of increasing amounts of FVIIa gave increasing amounts of thrombin generation. At the FVIIa concentrations present in the plasma of patients given 60 microg/kg recombinant FVIIa (NovoSeven, Novo Nordisk, Bagsvaerd, Denmark), 10-40 nmol/l, thrombin generation in the model system approached the normal amount seen in the TF-initiated model system. When FIX and FVIII were included in the above reaction, FVIIa could initiate thrombin generation at levels three to four times the amount seen in the TF-initiated model system. We speculate that this platelet-localized thrombin generation may, in part, account for the clinical efficacy of high-dose FVIIa.
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PMID:A possible mechanism of action of activated factor VII independent of tissue factor. 981 24

Numerous recent publications point to significant improvements in haemostasis in the bleeding patient suffering from haemophilia with inhibitors when a recombinant activated factor VII (rFVIIa) molecule is administered in high doses. In theory, activated factor VII (FVIIa) is believed to initiate haemostasis through its physiological interaction with tissue factor at sites of cellular injury, whereby factor X (FX) activation and, in consequence, thrombin formation is amplified. There has been speculation, however, whether high circulating FVII procoagulant (FVII:C) levels may induce systemic coagulation activation. The present retrospective investigation was undertaken to study, ex vivo, the influence of treatment with rFVIIa as assessed by the sensitive marker of prothrombin conversion, prothrombin fragment F1+2, in plasma samples. Study subjects consisted of: seven people suffering from thrombocytopenia participating in a study of the influence of rFVIIa on the bleeding time, in whom serial plasma samples had been collected before and subsequently at 10, 60 and 180 min after infusion of rFVIIa; four haemophilia A patients with inhibitors to FVIII undergoing surgery; two haemophilia A patients with inhibitors treated with rFVIIa for minor bleedings on 16 occasions in whom plasma samples had been collected before and 10-15 min after rFVIIa infusion; and two FVII-deficient patients undergoing treatment with rFVIIa. A group of seven haemophilia A patients with no signs of inhibitors subjected to a pharmacokinetic study of a plasma-derived FVIII concentrate served as controls. In the group of thrombocytopenic patients our results showed a mean increase in F1+2 following doses of 50 microg/kg body weight and 100 microg/kg body weight of rFVIIa of 1.1 and 1.4 nmol/l, respectively, with a gradual increase over time, but there was no significant correlation between FVII:C and the corresponding values of F1+2. During and after haemophilic inhibitor surgery, a mean increase in F1+2 of 1.44 nmol/l (range 0.6-3.2 nmol/l) was found, whereas 16 matched samples collected during treatment for minor bleedings showed a mean increase in F1+2 of 0.10 nmol/l (range -0.12 to 0.20 nmol/l). In FVII-deficient individuals, the mean rise in F1+2 was <0.10 nmol/l. In the control group, the mean elevation of F1+2 was 0.13 nmol/l (range -0.5 to 0.7 nmol/l). Hence, our results show that only discrete changes in F1+2 follow administration of rFVIIa.
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PMID:Levels of prothrombin activation peptide F1+2 in patients with a bleeding tendency. 981 44

Patients with diabetes have an increased prevalence of premature atherosclerotic vascular disease, and alterations in plasma coagulation proteins have been incriminated as a possible cause. The roles of hyperglycemia and hyperinsulinemia in the pathogenesis of these changes are unknown. To examine the effects of prolonged hyperglycemia and of selective hyperinsulinemia on the tissue factor pathway of blood coagulation, nine healthy young men were infused with glucose to maintain levels at 11.1 mmol/l (approximately 200 mg/dl) for 18-72 h (hyperglycemia-hyperinsulinemia group). Five normal men were infused with regular insulin to maintain levels comparable to that in the previous group (900 pmol/l, approximately 150 microU/ml) and with glucose to maintain levels at 5.6 mmol/l (approximately 100 mg/dl) (euglycemia-hyperinsulinemia group). Measured were plasma activated factor VII activity (FVIIa), FVII coagulant (FVIIC) activity, FVIII coagulant (FVIIIC) activity, tissue factor pathway inhibitor (TFPI) antigen, and thrombin markers; and serum glucose, insulin, and electrolytes. Plasma FVIIa, FVIIC, FVIIIC, and TFPI rose during hyperglycemic-hyperinsulinemia but not during euglycemic-hyperinsulinemia. Markers of thrombin generation rose transiently and inconsistently during hyperglycemia-hyperinsulinemia. We concluded that in normal subjects, hyperglycemia-hyperinsulinemia induced activation of the tissue factor pathway, reflected by increases in plasma FVIIa, FVIIC, and TFPI. This activation was independent of hyperinsulinemia, hypertriglyceridemia, and hyperosmolality. The elevations in plasma coagulation factors during hyperglycemia-hyperinsulinemia, characteristic of type 2 diabetes, may constitute a potential for enhanced thrombin generation and thrombosis when triggered by exposure of tissue factor, such as during arterial plaque rupture.
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PMID:Activation of the tissue factor pathway of blood coagulation during prolonged hyperglycemia in young healthy men. 1033 23

Granulocyte colony-stimulating factor (G-CSF) is used in healthy donors of peripheral blood stem cells (PBSC) for allogeneic transplantation. However, some data have recently suggested that G-CSF may induce a hypercoagulable state, prompting us to study prospectively 22 PBSC donors before and after G-CSF 5 microg/kg twice daily. We sought evidence for changes in the following parameters: platelet count, von Willebrand factor antigen (vWF:Ag) and activity (vWF activity), beta-thromboglobulin (beta-TG), platelet factor 4 (PF-4), platelet activation markers (GMP-140 and PAC-1), activated partial thromboplastin time (aPTT), prothrombin time (PT), coagulant factor VIII (FVIII:C), thrombin-antithrombin complex (TAT), prothrombin fragment 1+2 (F1+2), thrombomodulin (TM) and tissue plasminogen activator antigen (tPA:Ag) prior to G-CSF and immediately before leukapheresis. ADP-induced platelet aggregation studies were also performed. G-CSF administration produced only mild discomfort. We found a significant increase in vWF:Ag (from 0.99 +/- 0.32 U/ml to 1.83 +/- 0.69 U/ml; P < 0.001), in vWF activity (from 1.04 +/- 0.34 U/ml to 1.78 +/- 0.50 U/ml; P < 0.001) and in FVIII:C (from 1.12 +/- 0.37 U/ml to 1.73 +/- 0.57 U/ml; P < 0.001) after G-CSF. Of note, four donors with low baseline vWF had a two- to three-fold increase after receiving G-CSF. G-CSF had no impact on the platelet count, beta-TG, PF-4, GMP-140 or PAC-1. The final% of platelet aggregation decreased from 73 +/- 22% to 37 +/- 26% after G-CSF (P < 0.001). We found a significant decrease in aPTT after G-CSF (29.9 +/- 3.1 s to 28.3 +/- 3.3 s; P = 0.004), but the PT was unaffected. In addition, we also observed a significant increase in TAT, F1+2 and TM, but not in tPA:Ag. Our data suggest that G-CSF may possibly induce a hypercoagulable state by increasing levels of FVIII:C and thrombin generation. In contrast to this information, we found reduced platelet aggregation after G-CSF administration. The clinical implications of these findings remain unclear and larger studies are definitely required.
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PMID:A prospective study of G-CSF effects on hemostasis in allogeneic blood stem cell donors. 1037 63

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.
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PMID:Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII. 1041 Mar 9


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