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:3.4.21.6 (thromboplastin)
13,278 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

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

Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.
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PMID:Some human inhibitor antibodies interfere with factor VIII binding to factor IX. 963 9

FVII deficiency is a rather rare inherited hemocoagulation disorder that predisposes to hemorrhagic events, especially from mucous membranes, that are not predictable and severe as in hemophilia A. This defect produces prolonged prothrombin time (PT), reduced activity of FVII and normal activated partial thromboplastin time (aPTT). We report the case of a 43-year-old obese woman with severe deficiency of factor VII (FVII), probably genetic in nature, and meno-metrorrhagia associated with multiple fibromas of uterus. Our patient had no history of bleeding in infancy and young age, and in the past, before the disease was diagnosed, underwent major surgery operations (thyroidectomy and caesarian section) without hemorrhage. Patient's relatives with mild heterozygous deficiency of FVII (the father, a brother, a sister, a sister's daughter and the patient's son) did not show any bleeding tendency. This case report is discussed in the light of literature data ((source: Medline from 1964 to 1996). The different forms of congenital (isolated or combined with other clotting disorders) and acquired FVII deficiency, with the appropriate therapies, are reviewed. The clinician must consider FVII deficiency in cases of recurrent bleeding, and this disease, even if rather rare, should not be underestimated in clinical practice because it is potentially fatal.
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PMID:Genetic deficiency of factor VII and hemorrhagic diathesis. A case report and literature review. 978 23

A case of acquired hemophilia A in a 65-year-old woman is presented. The patient had been subjected to cholecystectomy 2 months before the bleeding tendency appeared. On admission, she had easy bruising and prolonged activated partial thromboplastin time, but during hospitalization she had severe hemorrhage into the right gluteal and femoral muscles. An inhibitor of the factor VIII coagulant protein (FVIII:C) of high Bethesda titer was found in her serum. The patient was successfully treated with activated recombinant human factor VII (rhFVIIa) and immunosuppression. We conclude that rhFVIIa is a safe, effective, and fast-acting preparation for the treatment of severe hemorrhage in patients with acquired hemophilia A, and that the simultaneous administration of azathioprine and corticosteroids may suppress production of the inhibitor.
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PMID:Severe acquired hemophilia A successfully treated with activated recombinant human factor VII. 979 81

Two patients who presented with active bleeding and were diagnosed with acquired hemophilia A (AHA) are reported herein. One was a 27-year-old woman who experienced spontaneous oozing from an episiotomy wound six days after her second normal delivery. Bleeding became progressively worse, despite treatment with primary sutures and curettage of the uterus at a local hospital. She underwent emergency exploratory laparotomy because of intra-abdominal bleeding, during which perforations of the uterus were discovered. Unremitting bleeding from the surgical wound occurred after surgery. The patient was finally diagnosed with AHA when Factor VIII (FVIII) inhibitor (titer, 19 Bethesda units (BU)/ml) was detected in her plasma. She died of refractory hemorrhaging, despite intensive treatment with Factor IX concentrate infusion and cyclophosphamide therapy. The second patient was a 22-year-old man who sustained spontaneous and recurrent intramuscular hemorrhage in the right thigh for one month. Laboratory studies including complete blood count, biochemical evaluation, coagulation screening and immunologic assays were all within normal limits, except for a prolonged activated partial thromboplastin time. Idiopathic AHA was diagnosed after the detection of plasma FVIII inhibitor with a concentration of 5.9 BU/ml. The patient's coagulopathy was successfully managed with plasma exchange and subsequent treatment with oral prednisolone and cyclophosphamide.
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PMID:Acquired hemophilia A: report of two cases. 979 3

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

Prothrombin time (PT) and activated partial thromboplastin time (APTT), popularized as a routine assay for screening blood coagulation disorders and monitoring anticoagulant therapy, still involve some issues regarding standardization. In this lecture, we present propositions to resolve these problems in respective laboratory. Although international normalized ratio (INR) calculated by international sensitivity index (ISI) of PT reagent seems to improve discrepancy of sensitivity between reagents, local calibration of sensitivity of PT reagent in respective laboratories (local SI) is reasonable to make INR/ISI system more useful. However, local calibration of reagent is not easy by WHO recommended method in a small size laboratory. By using AK calibrant (IMMUNO AG), one of calibration plasma for INR, we investigated its possibility to calibrate local SI in four different reagents, compared with the recommended methodology. The results led the following process to determine reagent and calibrate local SI for practical use of INR/ISI system. (a) Use PT reagent of which ISI is close to 1.0 if possible, and utilize manufacture's ISI as is for INR. (b) Select PT reagent labeled specific ISI for an instrument as the same as used in the lab., and use the manufacture's ISI as is, if impossible to choose small ISI reagent. (c) If use a reagent of which ISI is close to 2.0 and shown no specific ISI for used detector, adjustment of local SI by commercial calibration plasma is recommended when unavailable warfarinized patient plasma. In APTT, we attempted to evaluate sensitivity between five different APTT reagents with a patient model by hemophilia A plasma contained various FVIII: C. This model reflected difference of sensitivity between reagents in results. Because standardization of APTT is not improved in this point, certification of APTT pattern in each laboratories with patient models is required for not only monitoring of heparinization, but also screening of typical coagulation disorders such as hemophilia and von Willebrand disease.
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PMID:[Suggestions and propositions to resolve some issues for standardization of prothrombin time and activated partial thromboplastin time]. 1037 64

By virtue of a severely prolonged aPTT with a normal thromboplastin time (prothrombin time) and a normal thrombin time, severe FXII deficiency has been diagnosed in a woman without a bleeding diathesis or a history of thromboembolic complications. A deficiency of a factor of the contact activation system (FXII, prekallikrein, high molecular weight kininogen) is usually diagnosed during routine coagulation tests demonstrating a prolonged aPTT. The severe and partial deficiency of FXII, of prekallikrein or high molecular weight kininogen is not associated with a bleeding tendency. In contrast, severely factor XI deficient subjects may suffer from a mild hemorrhagic diathesis, whereas FVIII deficiency (hemophilia A, autoimmune "hemophilia", von Willebrand disease) and FIX deficiency (hemophilia B) are associated with a bleeding tendency of varying severity, depending on the clotting activity of FVIII or FIX, respectively. An isolated prolongation of the aPTT due to a lupus anticoagulant, however, is frequently associated with arterial and/or venous thrombosis. Therefore, in case of a prolongation of the aPTT, its cause has to be determined.
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PMID:[A patient with isolated prolongation of aPTT without hemorrhagic diathesis anamnesis: severe, hereditary factor XII deficiency]. 1051 21

Activated partial thromboplastin time may be prolonged as the result of either of two different autoimmune complications of chronic lymphocytic leukemia: the development of antiphospholipid antibodies, such as lupus anticoagulant or anticardiolipin antibodies, or anti-factor VIII inhibitors, such as acquired hemophilia A. In the rare simultaneous occurrence of both inhibitors, differential diagnosis of a prolonged activated partial thromboplastin time poses a number of problems during laboratory work-up, due to mutual interference of the commonly performed tests. Only careful clinical follow-up can disclose the significance of the laboratory findings. We report the case of concurrent antiphospholipid antibodies (lupus anticoagulant positivity, anticardiolipin antibodies; IgM 3880 MPL/mL and IgG 265 GPL/mL) and anti-factor VIII antibodies (46.8 Bethesda Units) in a patient with chronic B-cell lymphocytic leukemia who had prolonged activated partial thromboplastin time (78.8 s). The relationship between lymphoproliferative and antiphospholipid syndrome, laboratory work-up in the case of the association of antiphospholipid and anti-factor VIII antibodies, and related problems that occur during clinical management of the patient are also discussed.
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PMID:[The antiphospholipid syndrome during chronic lymphatic leukemia. An association with anti-factor VIII antibodies]. 1052 24


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