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)

Factor VIII functions as an essential cofactor in the blood coagulation cascade for the factor IXa-mediated activation of factor X. Factor VIII contains 6 tyrosine residues at positions 346, 718, 719, 723, 1664, and 1680 that are modified by post-translational sulfation. This modification is required for full factor VIII procoagulant activity. We have employed site-directed mutagenesis to identify the individual sulfated tyrosines within factor VIII that influence activity. The molecules were expressed in COS-1 monkey cells by transient transfection, and the resultant proteins were characterized. Metabolic incorporation of [35S]sulfate demonstrated that all 6 tyrosine residues are sulfated in factor VIII. Sulfation at residues 346 and 1664 was required for full activity in a factor VIII clotting assay but did not affect factor VIII activity monitored by a factor Xa generation assay. The Tyr346-->Phe and Tyr1664-->Phe mutants displayed delayed thrombin activation that correlated with delayed cleavage at residues 372 and 1689, respectively. In contrast, these mutants were efficiently activated by factor Xa. A triple Tyr to Phe mutant at residues 718, 719, and 723 displayed both reduced factor VIII clotting activity and factor Xa generation activity. Finally, a Tyr1680-->Phe mutant factor VIII displayed a 5-fold reduced affinity for von Willebrand factor. The results demonstrate that 1) sulfation at tyrosine residues 346 and 1664 increases factor VIII activity by increasing the rate of thrombin activation and cleavage; 2) sulfation at tyrosine residues 718, 719, and 723 increases the intrinsic activity of factor VIIIa; and 3) sulfation at tyrosine residue 1680 increases the affinity for vWF. In addition, the results implicate that thrombin interacts with three distinct sites within factor VIII, two of which are required for proteolytic activation. The results demonstrate that the six sites of tyrosine sulfation modulate factor VIII activity through different mechanisms.
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PMID:Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage. 805 Oct 97

Factor VIII is an important cofactor in the intrinsic activation of factor X. To function effectively as a cofactor, factor VIII must be activated. In plasma, factor VIII circulates in a complex with von Willebrand factor, and although thrombin can activate complexed factor VIII, the activation by activated factor X is inhibited by von Willebrand factor. In this study, the effect of von Willebrand factor on the generation of factor Xa by the factor IXa-VIII complex was investigated. Purified human factors VIII, IXa, and X were incubated on human umbilical vein endothelial cells or phospholipid vesicles in the presence of calcium ions, and the generation of factor Xa was followed. In the presence of von Willebrand factor, a prolonged lag-phase and a dose-dependent inhibition of factor X activation was observed. These effects were not observed when von Willebrand factor was preincubated with a monoclonal antibody directed against von Willebrand factor that blocks factor VIII binding. When factor VIII was activated with thrombin before the incubation, neither the monoclonal antibody nor von Willebrand factor had an effect on the rate of factor X activation. Preincubation of endothelial cells with the monoclonal antibody resulted in a somewhat higher rate of factor X activation. When endothelial cells from a patient with von Willebrand's disease type I were used, preincubation of the monoclonal antibody had no effect on the rate of factor X activation. We conclude that von Willebrand factor on the surface of endothelial cells can modulate the intrinsic factor X activation. This effect is greatly enhanced, however, by the addition of exogenous von Willebrand factor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:von Willebrand factor as a regulator of intrinsic factor X activation. 814 7

Factor VIII and factor V function as cofactors in the blood coagulation cascade to accelerate the rate of activation of factor X and prothrombin, respectively. Both cofactors require proteolytic activation by either activated factor X or thrombin for functional activity. Human factor VIII and factor V expressed in mammalian cells are both modified by posttranslational sulfation of tyrosine residues. In the present study, the posttranslational addition of sulfate in factor V expressed in transfected Chinese hamster ovary (CHO) cells was demonstrated by [35S]sulfate incorporation into the thrombin-cleaved 94-kDa heavy chain and the 150-kDa activation peptide. The presence of tyrosine sulfate in recombinant factor V was confirmed by barium hydroxide hydrolysis and two-dimensional thin-layer electrophoresis. The importance of sulfation for factor V secretion and activity was evaluated by characterizing factor V expressed in Chinese hamster ovary cells grown in the presence of sodium chlorate, a potent inhibitor of posttranslational sulfation in intact cells. Increasing concentrations of sodium chlorate inhibited the incorporation of [35S]sulfate into factor V but did not inhibit the synthesis or secretion of factor V. However, the specific activity of factor V secreted in the presence of sodium chlorate was reduced 5-fold. The reduced activity was attributed to (1) slower cleavage and activation by thrombin and (2) a reduced intrinsic activity of factor Va. In contrast, sulfation of factor V did not affect the rate of activation mediated by factor Xa. These results show that sulfation of factor V is required for efficient thrombin activation but not for activation by factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Posttranslational sulfation of factor V is required for efficient thrombin cleavage and activation and for full procoagulant activity. 820 29

A pilot study was performed on the storage of plasma and cryosupernatant plasma at 4 degrees C for up to 28 days. Eight bags, four of CPD fresh-frozen plasma (FFP) and four of CPD cryosupernatant plasma (CSP, plasma without cryoprecipitate), were sampled during storage for assays of pH; factors V, VIII, IX, and XI; fibrinogen; prothrombin time; activated partial thromboplastin time (APTT); plasma protein electrophoresis; viscosity; and C1q binding. No changes were found in viscosity or the plasma protein electrophoretic pattern, and there was no detectable immune complex formation. The fibrinogen concentration remained constant, and the prothrombin time showed a gradual increase of 2.5 seconds for both groups of plasma. The labile coagulation factor V decreased gradually for FFP and CSP to 58 and 64 percent of its initial value, respectively (51 +/- 8% and 54 +/- 6% of the value of fresh pooled plasma). Factor VIII decreased to 36 percent of its initial value in FFP (48 +/- 14% of fresh pooled plasma). In CSP, factor VIII decreased after 28 days to 7 percent of its initial value (7 +/- 1% of fresh pooled plasma). The APTT increased for FFP from 28 to 35.8 +/- 1.1 seconds and for CSP from 36 to 49.5 +/- 4.9 seconds. The only chemical change observed for both plasmas was a rise in pH, from 7.27 to 7.56, after 28 days. The results of this pilot study indicate that FFP can be stored at 4 degrees C for 28 days with sufficient recovery of coagulation factors to maintain hemostasis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Coagulation parameters of CPD fresh-frozen plasma and CPD cryoprecipitate-poor plasma after storage at 4 degrees C for 28 days. 821 20

An acquired factor VIII inhibitor was found in an 82-year-old woman who presented with numerous spontaneously appearing ecchymoses. Coagulation studies revealed the presence of a prolonged activated partial thromboplastin time that was not corrected by 1:1 mixture with normal fresh plasma after a 2-h incubation. Factor VIII:C was 4%, and the titer for factor VIII inhibitor was 9 Bethesda units. Three months later, after a retroperitoneal hemorrhage, a lymphocytosis was found in her peripheral blood with morphological and surface immunophenotype characteristics of B-cell chronic lymphocytic leukemia, later confirmed by bone marrow morphological and immunocytochemical examinations. To our knowledge, this is the first report of an autoimmune factor VIII inhibitor associated with chronic lymphocytic leukemia.
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PMID:Acquired factor VIII inhibitor preceding chronic lymphocytic leukemia. 828 Aug 15

We report here 2 cases of elderly patients who were admitted to our Department of Geriatrics for a hemorrhagic syndrome. The patients were found to be affected by a coagulopathy with prolonged activated partial thromboplastin time (aPTT), caused by the presence of an acquired inhibitor of Factor VIII. This syndrome, due to anti-Factor VIII antibodies, is surely an important clinical entity that is not easy to diagnosed, but in concomitance with a hemorrhagic syndrome and prolonged aPPT it should always be suspected. In fact, a quick diagnosis is very important in order to apply specific treatment as soon as possible.
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PMID:A hemorrhagic syndrome of the elderly patient caused by anti-factor VIII antibodies. 829 58

The emergence of an autoantibody directed against factor VIII may be responsible for severe, life-threatening haemorrhages. This rare disease is usually idiopathic, but it may be consecutive to an autoimmune disease or to the absorption of certain drugs such as penicillin. The diagnosis rests on the finding of a prolonged activated thromboplastin time with presence of a circulating anticoagulant and deep fall in factor VIII level. Two cases of severe haemorrhage successfully treated with porcine factor VIIIc are reported. The first case concerned an 80-year old woman presenting with a large haematoma of the thigh uncontrolled by injections of human factor VIIIc. The second case was that of a 24-year old woman in a state of shock due to a pleural blood effusion that occurred during heparin treatment of cerebral thrombophlebitis, combined with penicillin treatment of bronchial superinfection. In both cases the high-titer autoantibody to the human factor VIIIc did not, or little, cross with porcine factor VIIIc. Factor VIII rose after the first injection of the porcine factor, and the haemorrhage was rapidly controlled. In both cases, the autoantibody disappeared within a few months, either spontaneously or after treatment with immunosuppressants.
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PMID:[Acquired autoantibodies directed against human factor VIIIc. Treatment of 2 hemorrhagic accidents with porcine factor VIIIc]. 851 Oct 68

Factor VIII replacement therapy for patients with hemophilia A is conventionally monitored using a plasma-based factor VIII:C assay (a modified activated partial thromboplastin time [APTT] test). The plasma factor VIII assay requires the preparation of plasma from citrated whole blood and measurement of the clotting times of mixtures of patient plasma, factor VIII-deficient substrate, and APTT reagent. Results are not routinely available in less than 1.5 hr, reducing the clinical value of the laboratory data regarding the ability to immediately adjust patient therapy. Results from the whole blood factor VIII assay, performed on a portable coagulation analyzer and using test tubes prefilled with the necessary APTT and factor VIII-deficient reagents, are available within 5-7 min. This immediate determination of the factor VIII:C level from citrated whole blood provides the opportunity to greatly reduce turnaround time and improve the efficacy of factor VIII replacement therapy. Based on clotting time, factor VIII:C activity is read from a standard curve. A clinical evaluation of this whole blood test was performed in two hemophilia centers. A high degree of correlation was seen (r=0.813, n=220) between the whole blood values obtained and conventional laboratory results. This level of correlation was superior to that obtained when comparing two different plasma-based systems(r=0.753, n=23). Factor VIII:C activity levels measured using the whole blood assay system were similar,irrespective of the test operator (laboratory technologist, nurse clinician, or patient). This study indicates that the whole blood factor VIII assay provides results comparable to those of conventional plasma-based assays, but in a more rapid and efficient manner. It provides an opportunity to reduce unnecessary patient consumption of replacement preparations, hence reducing the cost of hemophilia A maintenance and prophylaxis regimens, and to reduce overall patient exposure to human blood products.
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PMID:Bedside measurement of factor VIII:C activity in individuals with hemophilia A. 861 97

Various coagulation defects have been associated with Gaucher's disease, including factor IX deficiency and acquired von Willebrand's disease (VWD). We performed repeated coagulation assays in 9 patients with Gaucher's disease over a period of 2 years. The prothrombin time (PT) and fibrinogen levels were normal in 8 of 9 patients, while the partial thromboplastin time (PTT) was abnormal in 5 of 9; all mixing PTT tests showed correction. Factor IX was normal repeatedly in the 7 of 7 patients tested. In contrast, factor XI was decreased in 3 of 9 patients assayed. Anticardiolipin (ACL) IgM was normal in all patients. ACL IgG was highly variable; levels were abnormal at least once in 6 of 8 patients, but were also normal at least once in 7 of 8 patients. Factor VIII was also quite variable: levels were decreased at least once in 4 of 9 patients, and normal at least once in 8 of 9 patients. Von Willebrand factor antigen (VWF Ag) studies were normal in 7 of 8 patients, but VWF activity was decreased at least once in 4 of 8 patients. In some patients, these problems could be overcome by specimen dilution. In ony 1 patient was VWF Ag decreased; this patient had a factor VIIIC level of 13% , and VWF activity of 18.7%. Coagulation assays performed before and after alglucerase administration failed to demonstrate any significant improvement in these assays, and neither was there a consistent improvement over the duration of therapy. We suggest that previously reported decreases in factor IX and VWF may be secondary to the interfering presence of increased cerebroside levels. Caution must be used in the interpretation of clotting assays in the patient with Gaucher's disease.
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PMID:Coagulation abnormalities in patients with Gaucher's disease: effect of therapy. 861 5

Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481-525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most potent inhibition was observed for peptide VP493 (residues 493-506), with 50% inhibition at 2.5 microM. VP493 also inhibited FXa in plasma in FXa-1-stage clotting assays by 50% at 3 microM. When the C-terminal carboxamide group of VP493 was replaced by a carboxyl group, most prothrombinase inhibitory activity was lost. VP493 preincubated with FXa inhibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Affinity-purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa-1-stage clotting times. VP493 also blocked the ability of protein S to inhibit prothrombinase independently of APC. Immobilized VP493 bound specifically with similar affinity to both FXa and protein S (Kd approximately 40 nM), but did not measurably bind prothrombin or APC. These studies suggest that FVa residues 493-506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa toward APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage causes 40% decrease in FVa activity and facilitates inactivation of FVa.
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PMID:Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va. 888 Sep 12


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