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)

Recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Denmark) induces hemostasis in life- and limb-threatening bleeds and in major surgery of hemophilia A and B patients, regardless of inhibitor titer. A total of more than 6,500 patients have been treated, and NovoSeven has been administered in more than 180,000 standard doses. Experience gained from these clinical situations suggests that NovoSeven should be administered as a 90- to 110-microg/kg bolus dose every second hour. Hemophilia patients with mild to moderate bleeding episodes require two to three doses to achieve complete hemostasis, whereas patients with severe bleeding episodes may require more doses. For major surgery and in cases of life-threatening bleeding, dosing every second hour for the first 24 hours may be required. Thereafter, the same dose, but with longer intervals between doses, is recommended. Recent in vitro experiments indicate that even higher doses of NovoSeven may be needed to achieve full thrombin generation in the absence of factor VIII (FVIII), factor IX (FIX), and factor XI (FXI).
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PMID:Recombinant factor VIIa (Novoseven) as a hemostatic agent. 1173 10

Recombinant factor VIIa (rFVIIa) is a novel prohemostatic drug for patients with hemophilia who have developed inhibitory antibodies. The postulation has been made that hemophilia is not only a disorder of coagulation, but that hyperfibrinolysis due to a defective activation of thrombin activatable fibrinolysis inhibitor (TAFI) might also play a role. In this in vitro study, the potential of rFVIIa to down-regulate fibrinolysis via activation of TAFI was investigated. rFVIIa was able to prolong clot lysis time in plasmas from 17 patients with severe hemophilia A. The prolongation of clot lysis time by rFVIIa was completely abolished by addition of an inhibitor of activated TAFI. The concentration of rFVIIa required for half maximal prolongation of clot lysis time (C(lys 1/2)-VIIa) varied widely between patients (median, 73.0 U/mL; range, 10.8-250 U/mL). The concentration of rFVIIa required for half maximal reduction of clotting time (C(clot 1/2)-VIIa) was approximately 10-fold lower than the C(lys 1/2)-VIIa value (median, 8.4 U/mL; range, 1.7-22.5 U/mL). Inhibition of TFPI with a polyclonal antibody significantly decreased C(lys 1/2)-VIIa values (median, 2.6 U/mL; range, 0-86.9 U/mL), whereas C(clot 1/2)-VIIa values did not change (median, 7.2 U/mL; range, 2.2-22.5 U/mL). On addition of 100 ng/mL recombinant full-length TFPI, a nonsignificant increase of C(lys 1/2)-VIIa values was observed (median, 119.2 U/mL; range, 12.3-375.0 U/mL), whereas C(clot 1/2)-VIIa values did not change (median, 8.8 U/mL; range, 2.6-34.6 U/mL). In conclusion, this study shows that rFVIIa both accelerates clot formation and inhibits fibrinolysis by activation of TAFI in factor VIII-deficient plasma. However, a large variability in antifibrinolytic potential of rFVIIa exists between patients.
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PMID:Inhibition of fibrinolysis by recombinant factor VIIa in plasma from patients with severe hemophilia A. 1175 68

The ability of factor VIIa to initiate thrombin generation and clot formation in blood from healthy donors, blood from patients with hemophilia A, and in anti-factor IX antibody-induced ("acquired") hemophilia B blood was investigated. In normal blood, both factor VIIa-tissue factor (TF) complex and factor VIIa alone initiated thrombin generation. The efficiency of factor VIIa was about 0.0001 that of the factor VIIa-TF complex. In congenital hemophilia A blood and "acquired" hemophilia B blood in vitro, addition of 10 to 50 nM factor VIIa (pharmacologic concentrations) corrected the clotting time at all TF concentrations tested (0-100 pM) but had little effect on thrombin generation. Fibrinopeptide release and insoluble clot formation were only marginally influenced by addition of factor VIIa. TF alone had a more pronounced effect on thrombin generation; an increase in TF from 0 to 100 pM increased the maximum thrombin level in "acquired" hemophilia B blood from 120 to 480 nM. Platelet activation was considerably enhanced by addition of factor VIIa to both hemophilia A blood and "acquired" hemophilia B blood. Thus, pharmacologic concentrations of factor VIIa cannot restore normal thrombin generation in hemophilia A and hemophilia B blood in vitro. The efficacy of factor VIIa (10-50 nM) in hemophilia blood is dependent on TF.
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PMID:Mechanism of factor VIIa-dependent coagulation in hemophilia blood. 1209 17

The process of tissue factor initiated blood coagulation is discussed. Reactions of the blood coagulation cascade are propagated by complex enzymes containing a vitamin K-dependent serine protease and an accessory cofactor protein that are assembled on a membrane surface in a calcium-dependent manner. These complexes are 105-109-fold more efficient in proteolyses of their natural substrates than enzymes alone. Based upon data acquired using several in vitro models of blood coagulation, tissue factor initiated thrombin generation can be divided into two phases: an initiation phase and a propagation phase. The initiation phase is characterized by the generation of nanomolar amounts of thrombin, femto- to picomolar amounts of factors VIIa, IXa, Xa, and XIa, partial activation of platelets, and almost quantitative activation of procofactors, factors V and VIII. The duration of this phase is primarily influenced by concentrations of tissue factor and TFPI. The characteristic features of the propagation phase are: almost quantitative prothrombin activation at a high rate, completion of platelet activation, and solid clot formation. This phase is primarily regulated by antithrombin III and the protein C system. Thrombin generation during the propagation phase is remarkably suppressed in the absence of factor VIII and IX (hemophilia A and B, respectively) and at platelet counts <5% of mean plasma concentration. The majority of data accumulated in in vitro models and discussed in this review are in good agreement with the results of in vivo observations.
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PMID:Blood coagulation. 1184 35

In an earlier study, a site directed mutant rFVIII (rFVIII(m), Arg(336) --> Gln(336)) expressed in baculovirus-insect cell (Sf9) system was found to sustain high level activity during incubation at 37 degrees celsius for 24 h while the cofactor activity of normal plasma was declined steadily. In this study, a mutant B-domain deleted rFVIII(m), Arg(336) --> Gln(336) expressed in baculovirus-insect cell (Sf9) system was characterized for its enzymatic and chemical properties. The expressed rFVIII(m) and plasma FVIII (pFVIII) were purified by immunoaffinity column chromatography and identified by Western blot analysis. The partially purified rFVIII(m) exhibited cofactor specific activity of 2.01 x 10(3)units/mg protein. The molecular weight of rFVIII(m) ranged between 40 to 150 kDa with a major band at 150 kDa. Treatment of both rFVIII(m) and pFVIII with thrombin increased their cofactor activity in a similar pattern. Treatment of both the activated rFVIII(m) and native FVIII with APC decreased their cofactor activities, however, the former exhibited a slower decrease than the latter, although no significant difference was present. rFVIII(m) formed a complex with vWF, resulting in a stabilized form, and the lag period of thrombin-mediated activating was extended by vWF association. These results implicated that rFVIII(m) expressed in baculovirus-insect cell system had a comparable capacity as FVIII cofactor activity and might be a good candidate for the FVIII replacement therapy for hemophilia A patients.
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PMID:Expression and characterization of a mutant recombinant blood coagulation factor VIII (rFVIII (m)). 1221 15

Hemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.
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PMID:Expression and characterization of recombinant murine factor VIII. 1235 75

Diffuse alveolar hemorrhage (DAH) is a potentially life-threatening pulmonary toxicity that occurs in 1-21% of patients following bone marrow transplantation. The syndrome is associated with a high mortality rate; and current treatment options are limited. Recombinant factor VIIa (rFVIIa, Novoseven) has recently been approved for the treatment of bleeding in patients with hemophilia A/B with inhibitors. A greater understanding of the mechanism by which rFVIIa restores hemostasis has recently become available; with in vitro evidence supporting that the thrombin burst achieved by rFVIIa is independent of the presence or binding to tissue factor. This insight has suggested a range of other potential clinical uses for the drug; including the setting of pulmonary hemorrhage. We review our experience with using rFVIIa for treatment of DAH in a patient with acute myelogenous leukemia following a matched unrelated donor bone marrow transplant. Boluses of 90 microg/kg rFVIIa were given every 3 h x 4 doses/day, concurrently with high-dose corticosteroids and maintenance of a platelet count >50 000/mm(3). Rapid clinical and radiological improvement was noted within several doses of rFVIIa, with discontinuation of the drug after eight doses. However, the patient's clinical condition began to rapidly deteriorate following cessation of rVIIa, resulting in reinstitution of therapy 24 h later. The patient again exhibited rapid clinical improvement; and rFVIIa was continued for an additional 16 doses with no further evidence of pulmonary hemorrhage noted. No toxicity or adverse events were observed with rFVIIa treatment. Our experience indicates that rFVIIa may be an effective treatment option for DAH post bone marrow transplant; although further clinical studies are needed before recommendations can be made regarding off label use of rFVIIa in this clinical setting.
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PMID:Treatment of diffuse alveolar hemorrhage after allogeneic bone marrow transplant with recombinant factor VIIa. 1247 94

Recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Denmark) induces hemostasis in life- and limb-threatening bleeds and in major surgery of hemophilia A and B patients, regardless of inhibitor titer. A total of more than 6,500 patients have been treated, and NovoSeven has been administered in more than 180,000 standard doses. Experience gained from these clinical situations suggests that NovoSeven should be administered as a 90- to 110-microg/kg bolus dose every second hour. Hemophilia patients with mild to moderate bleeding episodes require two to three doses to achieve complete hemostasis, whereas patients with severe bleeding episodes may require more doses. For major surgery and in cases of life-threatening bleeding, dosing every second hour for the first 24 hours may be required. Thereafter, the same dose, but with longer intervals between doses, is recommended. Recent in vitro experiments indicate that even higher doses of NovoSeven may be needed to achieve full thrombin generation in the absence of factor VIII (FVIII), factor IX (FIX), and factor XI (FXI).
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PMID:Recombinant factor VIIa (NovoSeven) as a hemostatic agent. 1252 27

Factor (F) VIII is a large gene located near the terminus of the long arm of the X chromosome. It contains 26 exons that code for a signal peptide and a 2332 amino acid polypeptide with three different types of domains, namely A1-A2-B-A3-C1-C2. The A domains are homologous with each other and those of ceruloplasmin; substitution into the known crystal structure of the copper binding protein produces molecular models. The large, central B domain is highly glycosylated but has a variable sequence, even among FVIIIs from different species. Most of B can be deleted and the resulting recombinant protein has essentially normal survival in circulation and corrects the bleeding tendency in hemophilia A patients. The C domains are similar to each other, and the crystal structure of a recombinant human C2 domain is known, allowing construction of a molecular model of C1. The FVIII protein is secreted as a heterodimer following at least two intracellular cleavages within the B domain. In circulation it is stabilized by binding to von Willebrand factor (vWF) with a plasma half-life of about 10 hours. After specific thrombin cleavages that remove the remainder of the B domain and one of the high-affinity von Willebrand factor binding sites, FVIII becomes heterotrimeric FVIIIa, capable of enhancing intrinsic FX activation by FIXa. Inactivation of FVIIIa occurs by A2 dissociation or by specific cleavages within A1 and A2 by activated protein C. Control of intrinsic FX activation is critical for hemostasis and thrombosis.
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PMID:Structure and function of the factor VIII gene and protein. 1264 May 60

High plasma levels of soluble P-selectin are associated with thrombotic disorders and may predict future cardiovascular events. Mice with high levels of soluble P-selectin have more microparticles in their plasma than do normal mice. Here we show that chimeras of P-selectin and immunoglobulin (P-sel-Ig) induced formation of procoagulant microparticles in human blood through P-selectin glycoprotein ligand-1 (PSGL-1; encoded by the Psgl1 gene, officially known as Selpl). In addition, Psgl1-/- mice produced fewer microparticles after P-sel-Ig infusion and did not spontaneously increase their microparticle count in old age as do wild-type mice. Injected microparticles specifically bound to thrombi and thus could be involved in thrombin generation at sites of injury. Infusion of P-sel-Ig into hemophilia A mice produced a 20-fold increase over control immunoglobulin in microparticles containing tissue factor. This significantly improved the kinetics of fibrin formation in the hemophilia A mice and normalized their tail-bleeding time. P-sel-Ig treatment could become a new approach to sustained control of bleeding in hemophilia.
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PMID:Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A. 1289 56


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