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)

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.
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PMID:Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. 934 26

The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor V(LEIDEN) mutation, which leads to the substitution of Arg506 --> Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII-deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor V(LEIDEN) mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa x tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (approximately 6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor V(LEIDEN) mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor V(LEIDEN) concentration. When the factor V(LEIDEN) concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor V(LEIDEN) level to understand the effect of factor V(LEIDEN) in hemophilia A patients. The presented data support the hypothesis that the factor V(LEIDEN) mutation can increase thrombin formation in severe hemophilia A.
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PMID:An in vitro analysis of the combination of hemophilia A and factor V(LEIDEN). 937 87

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 mumol/L) and the S558F (960 mumol/L) mutants compared with wild-type FVIII (> 2,000 mumol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 mumol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.
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PMID:The molecular basis for cross-reacting material-positive hemophilia A due to missense mutations within the A2-domain of factor VIII. 942 7

Factor VIII is a trace plasma glycoprotein involved as a cofactor in the activation of factor X by factor IXa. Inherited deficiency of factor VIII results in the X-linked bleeding disorder hemophilia A which has been documented in humans, horses, sheep and dogs. In this report, the putative proximal promoter, 5' untranslated region, complete coding sequence and 3' untranslated region of the canine factor VIII gene have been characterized. When compared to the human gene, the 5' flanking region shows conservation of transcription factor binding sites in the 5' untranslated region. Alignment of the amino acid sequence with that of the previously reported human, mouse and pig proteins demonstrates sequence identity of between 77 and 92% for the A1, A2, A3, C1 and C2 domains but an identity of only between 44 and 62% for the central B domain. The three thrombin cleavage sites are conserved in the canine sequence as are the protein C cleavage sites and the von Willebrand factor binding region. In addition, all six tyrosine residues that are known to undergo sulfation in the human protein are conserved in the dog. The 3' untranslated region of the canine gene extends 1.5 kilobases. The initial 700 basepairs of this sequence are highly GC rich and the sequence terminates with 2 alternative potential polyadenylation sites. The knowledge of this sequence, in combination with a well described canine model of hemophilia A, provides the necessary starting point for studies addressing the long-term evaluation of factor VIII gene therapy using a homologous transgene.
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PMID:The canine factor VIII cDNA and 5' flanking sequence. 949 83

We studied the effects of porcine factor VIII (P-FVIII; Hyate:C) and other coagulation products employed in the management of patients with hemophilia A, on platelet activation in vitro. Exposure of normal resting platelets to P-FVIII resulted in platelet activation, as manifested by increased expression of the platelet surface activation markers CD62, CD63, and activated-GPIIbIIIa, and by activation-induced modulation of expression of normal platelet membrane glycoproteins CD41, CD42, and CD36. In contrast, platelet activation was not observed after exposure of the platelets to human FVIII, FEIBA, recombinant FVIIa, or cryosupernatant plasma. As with thrombin, exposure of platelets to P-FVIII resulted in the generation of platelet microparticles, an effect not seen not with the other products. In contrast to the characteristic reduction in expression in the number of CD42 molecules detected on thrombin-activated platelets, P-FVIII-stimulated platelets showed a small increase in CD42 expression. In contrast to thrombin, P-FVIII did not cause platelet dense granule release. The results indicate that therapeutic P-FVIII activates platelets, likely in ways that are different from the platelet activation seen with thrombin. The observed platelet activation and microparticle generation may provide a "hypercoagulable" mechanism for hemostasis with P-FVIII therapy separate from, and additional to, that due to increased circulating FVIII levels.
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PMID:Platelet activation induced by porcine factor VIII (Hyate:C). 949 69

Current therapies for treatment of hemophilia A involve infusion of factor VIII, but are ineffective for patients who develop inhibitory antibodies. We have previously proposed that bypassing the intrinsic pathway (VIIIa/IXa) with reversibly acylated factor Xa offers an improvement on existing therapies as it provides a time-dependent release of procoagulant activity without the addition of factors VIII or IX. The present study was designed to determine the effect of substituted 4-amidinophenyl benzoates on the acylation of factor Xa, as well as the subsequent deacylation rates of the resulting acyl Xa. A subset of this series of acyl Xa's were incorporated into the prothrombinase complex and recovery of catalytic activity was measured by activation of prothrombin to thrombin. Similarly, some acyl Xa's were also evaluated for their capacity to enhance clotting times of human plasma. Our study indicates that by choosing the appropriate acyl Xa, the time course of factor Xa regeneration can be modulated extensively. Animal studies will be required to show that the use of acyl Xa as a procoagulant agent is feasible in an in vivo system.
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PMID:Reversible acylation of factor Xa as a potential therapy for hemophilia. 952 60

We further characterised the abnormal factor VIII molecule (factor VIII Leiden) of a Crm+, mild hemophilia A patient with a factor VIII activity of 0.18 IU/ml and a factor VIII antigen of 0.95 IU/ml. Mutation analysis of the coding region, promoter and 3' untranslated region of the factor VIII gene revealed the presence of a C to T substitution at codon 527. This nucleotide change predicts the replacement of an arginine to tryptophan in the A2 domain close to a suggested binding site for factor IXa. Since a previous study of this mutant factor VIII protein suggested that this protein had a reduced affinity for factor IXa, position 527 in the protein might be involved in the interaction with factor IXa. In this study we gathered evidence for our hypothesis that the Arg to Trp mutation at position 527 is the cause of the reduced activity of factor VIII Leiden. Replacement of the mutated A2 domain by wild type A2 domain partially corrected the defect. Factor VIII from normal and factor VIII Leiden plasma was concentrated by cryoprecipitation, activated with thrombin and incubated with excess wild type A2 domain. Competition with excess isolated human A2 domain resulted in a partial reconstitution of the factor VIIa activity of thrombin treated factor VIII Leiden. This supports the hypothesis that the mutation in the A2 domain is the cause of the reduced factor VIII activity.
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PMID:Partial reconstitution of factor VIII activity from a mild Crm+ hemophilia A patient by replacement of the defective A2 domain. 960 26

Tissue factor (TF)-induced coagulation was compared in contact pathway suppressed human blood from normal, factor VIII-deficient, and factor XI-deficient donors. The progress of the reaction was analyzed in quenched samples by immunoassay and immunoblotting for fibrinopeptide A (FPA), thrombin-antithrombin (TAT), factor V activation, and osteonectin. In hemophilia A blood (factor VIII:C <1%) treated with 25 pmol/L TF, clotting was significantly delayed versus normal, whereas replacement with recombinant factor VIII (1 U/mL) restored the clot time near normal values. Fibrinopeptide A release was slower over the course of the experiment than in normal blood or hemophilic blood with factor VIII replaced, but significant release was observed by the end of the experiment. Factor V activation was significantly impaired, with both the heavy and light chains presenting more slowly than in the normal or replacement cases. Differences in platelet activation (osteonectin release) between normal and factor VIII-deficient blood were small, with the midpoint of the profiles observed within 1 minute of each other. Thrombin generation during the propagation phase (subsequent to clotting) was greatly impaired in factor VIII deficiency, being depressed to less than 1/29 (<1.9 nmol TAT/L/min) the rate in normal blood (55 nmol TAT/L/min). Replacement with recombinant factor VIII normalized the rate of TAT generation. Thus, coagulation in hemophilia A blood at 25 pmol/L TF is impaired, with significantly slower thrombin generation than normal during the propagation phase; this reduced thrombin appears to affect FPA production and factor V activation more profoundly than platelet activation. At the same level of TF in factor XI-deficient blood (XI:C <2%), only minor differences in clotting or product formation (FPA, osteonectin, and factor Va) were observed. Using reduced levels of initiator (5 pmol/L TF), the reaction was more strongly influenced by factor XI deficiency. Clot formation was delayed from 11.1 to 15.7 minutes, which shortened to 9.7 minutes with factor XI replacement. The maximum thrombin generation rate observed ( approximately 37 nmol TAT/L/min) was approximately one third that for normal (110 nmol/L TAT/min) or with factor XI replacement (119 nmol TAT/L/min). FPA release, factor V activation, and release of platelet osteonectin were slower in factor XI-deficient blood than in normal blood. The data demonstrate that factor XI deficiency results in significantly delayed clot formation only at sufficiently low TF concentrations. However, even at these low TF concentrations, significant thrombin is generated in the propagation phase after formation of the initial clot in hemophilia C blood.
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PMID:Blood coagulation in hemophilia A and hemophilia C. 961 54

Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional factor VIII (FVIII) protein and are termed cross-reacting material (CRM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit procoagulant activity. Thrombin-activated FVIII is a heterotrimer with the A2 subunit (amino acid residues 373 to 740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIII. Recently, a phenotype of CRM-positive hemophilia A patients has been characterized whose plasma displays a discrepancy between their FVIII activities, where the one-stage clotting assay displays greater activity than the two-stage clotting assay. One example is a missense mutation where ARG531 has been substituted by HIS531. An FVIII cDNA construct was prepared containing the ARG531(HIS) mutation and the protein was expressed in COS-1 monkey cells by transient DNA transfection. Metabolic labeling with [35S]-methionine demonstrated that ARG531(HIS) was synthesized at an equal rate compared with FVIII wild-type (WT) but had slightly reduced antigen in the conditioned medium, suggesting a modest secretion defect. A time course of structural cleavage of ARG531(HIS) demonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes, ARG531(HIS) had discrepant activity as measured by a one-stage activated partial thromboplastin time (aPTT) clotting assay (36% +/- 9.6% of FVIII WT) and a variation of the two-stage assay using a chromogenic substrate (COAMATIC; 19% +/- 6.9% of FVIII WT). Partially purified FVIII WT and ARG531(HIS) proteins were subjected to functional activation by incubation with thrombin. ARG531(HIS) demonstrated significantly reduced peak activity and was completely inactivated after 30 seconds, whereas FVIII WT retained activity until 2.5 minutes after activation. Because the ARG531(HIS) missense mutation predicts a charge change to the A2 subunit, we hypothesized that the ARG531(HIS) A2 subunit could be subject to more rapid dissociation from the heterotrimer. The rate of A2 dissociation, using an optical biosensor, was determined to be fourfold faster for ARG531(HIS) compared with FVIII WT. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increased rate of inactivation and reduced specific activity.
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PMID:Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo. 986 59

To evaluate the potential of the ex vivo bone marrow stromal cell (BMSC) system as a gene therapy for hemophilia A, we studied the in vitro expression of human factor VIII (hFVIII) in canine BMSCs following transfection with plasmid vectors and transduction with retroviral vectors. Vectors were composed of B domain-deleted forms of hFVIII that either retain or delete the proteolytic site at amino acid 1648. On transfection of BMSCs, vectors supported expression and secretion of similar levels of up to 386 mU/10(6) cells/24 hr, even though only 3-9% of the cells expressed hFVIII while 42-48% of transfected cells harbored plasmid vector. Much higher percentages (approximately 70%) of cells expressing hFVIII were achieved when BMSCs were transduced by retroviral vectors, resulting in expression and secretion as high as 1000-4000 mU/10(6) cells/24 hr. Western analysis demonstrated that the B domain-deleted forms possessing the proteolytic site were secreted predominantly as heavy and light chain heterodimers that resemble native forms found in plasma. In contrast, the hFVIII lacking the proteolytic site was expressed mostly as unprocessed, single heavy-light chains. Both hFVIII forms were correctly cleaved and activated by thrombin. The proteolyzed hFVIII form possessed > or = 93% normal biological activity while the unproteolyzed form possessed consistently less than 55% normal biological activity and was therefore considered less suitable for therapeutic application. These results demonstrate that the BMSC system has potential utility in gene therapy for hemophilia A and stress the importance of selecting the appropriate hFVIII structure for prospective clinical use.
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PMID:Bone marrow stromal cell-mediated gene therapy for hemophilia A: in vitro expression of human factor VIII with high biological activity requires the inclusion of the proteolytic site at amino acid 1648. 1002 31


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