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1.
The effect of thrombin on human factor VIII. Cleavage of the factor VIII procoagulant protein during activation 总被引:12,自引:0,他引:12
Human VIII: C has been partially purified by immunoabsorbent chromatography and agarose gel filtration in the presence of 2 mM DFP. The ratio of VIII:C to VIIIR:Ag in these preparations was greater than 1000:1, and the VIII:C procoagulant and immunologic (VIII:CAg) activities eluted together from Sephadex G-200 with a Kav of 0.05, a value consistent with a Stokes radius of 88 A. The molecular weight estimated from this measurement and sucrose density-gradient centrifugation studies (8.2S) is 285,000. VIII:C activation was detected when the purified procoagulant was incubated with 2 x 10(-5) to 10(-2) U/ml highly purified human alpha-thrombin. Although 2 mM DEP inhibits VIII:C activation by alpha-thrombin, DFP added after activation did not prevent subsequent loss of activity. When VIII:C was incubated for 4 hr with dilute alpha-thrombin, 2 x 10(-5) to 2 x 10(-3) U/ml, the activated procoagulant (VIII:C/VIII:CAg > 1) eluted from Sephadex G-200 with a Kav of about 0.2 This gel filtration pattern corresponds to a protein with a Stokes radius of 60 A and, taken together with a preliminary estimate of sedimentation properties (5S), suggests a molecular weight of about 116,000. Higher concentration of thrombin, 10(-3) to 10(-1) U/ml, inactivated VIII:C in these experiments, and the nonfunctional protein was identified by VIII:CAg immunoassay. The inactivated VIII:CAg eluted from Sephadex G-200 in a broad peak of Kav approximately 0.3. THese data suggest that alpha-thrombin activates and inactivates human VIII:C by proteolytic modification of VIII:C structure and that thrombin-activated VIII:C is smaller than the unactivated procoagulant. 相似文献
2.
Platelet receptor-mediated factor X activation by factor IXa. High-affinity factor IXa receptors induced by factor VIII are deficient on platelets in Scott syndrome. 总被引:4,自引:2,他引:2
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S S Ahmad R Rawala-Sheikh B Ashby P N Walsh 《The Journal of clinical investigation》1989,84(3):824-828
We have studied factor IXa binding and factor X activation with normal platelets and with platelets obtained from a patient with a bleeding disorder and an isolated deficiency of platelet procoagulant activity termed Scott syndrome. In the absence of factor VIIIa and factor X, normal, thrombin-treated platelets exposed 560 +/- 35 sites for factor IXa with a Kd of 2.75 +/- 0.27 mM, compared with 461 +/- 60 sites per patient platelet with Kd of 3.2 +/- 0.33 nM. The addition of factor VIIIa and factor X resulted in a decrease in the Kd for normal platelets to 0.68 nM but had no effect on the Kd for patient platelets. The concentrations of factor IXa required for half-maximal rates of factor X activation for normal (0.52 nM) and patient platelets (2.5 nM) were similar to those determined from equilibrium binding studies. Kinetic parameters for factor X activation by factor IXa showed that the Km and Kcat were identical for normal and patient platelets in the absence of factor VIIIa. In the presence of factor VIIIa, and kcat for patient platelets (163 min-1) was only 33% of that for normal platelets (491 min-1): This result can be explained by the difference in affinity for factor IXa between normal and patient platelets in the presence of factor VIIIa, suggesting impaired factor VIIIa binding to Scott syndrome platelets. 相似文献
3.
Interaction of factor VIII antigen in hemophilic plasmas with human antibodies to factor VIII. 总被引:2,自引:1,他引:1
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T S Zimmerman L de la Pointe T S Edgington 《The Journal of clinical investigation》1977,59(5):984-989
By utilizing a simple modification of previous immunological assays, we have demonstrated that most, if not all, hemophilic plasmas contain antigen reactive with human antibodies directed against Factor VIII procoagulant activity (VIIIc). Antibodies developing in a nonhemophiliac patient and in a hemophiliac patient gave similar results. The VIIIc antigen so identified was removed from hemophilic plasmas with immobilized rabbit antibody which reacted with normal VIIIc and von Willebrand's disease antigen. These data suggest that there are greater antigenic similarities between normal and hemophilic Factor VIII than previously thought. 相似文献
4.
Heterogeneity of human factor VIII. I. Characterization of factor VIII present in the supernatant of cryoprecipitate 总被引:6,自引:0,他引:6
J Over B N Bouma J A van Mourik J J Sixma R Vlooswijk I Bakker-Woudenberg 《The Journal of laboratory and clinical medicine》1978,91(1):32-46
Recent observations suggest that plasma F VIII consists of a series of molecules with different molecular weights. The data described in this paper suggest that sup F VIII represents the molecules with relatively low molecular weights whereas the molecules with the highest molecular weights appear in cryo F VIII. Sup F VIII was associated with VIII:C and VIIIR:Ag, but ristocetin cofactor activity was lacking. Although the immunoprecipitation characteristics of sup F VIII with rabbit antifactor VIII were different from those of cryo F VIII, immunological identity was observed in immunodiffusion and crossed immunoelectrophoresis. In 0.8M NaCl sup F VIII dissociated into VIIIR:Ag of relatively high molecular weight and VIII:C of low molecular weight. No indications were obtained that the presence of sup F VIII was the result of proteolytic degradation of factor VIII. VIII:C of sup F VIII was more labile in vitro than VIII:C in plasma. It could be activated by traces of thrombin in a way similar to plasma F VIII. In patients with classic von Willebrand's disease relatively more VIII:C remained in the supernatant after cryoprecipitation of plasma. 相似文献
5.
T. SOEDA K. NOGAMI T. MATSUMOTO K. OGIWARA M. SHIMA 《Journal of thrombosis and haemostasis》2010,8(11):2494-2503
Summary. Background: Factor (F)VIIa, complexed with tissue factor (TF), is a primary trigger of blood coagulation, and has extremely restricted substrate specificity. The complex catalyzes limited proteolysis of FVIII, but these mechanisms are poorly understood. Objectives: In the present study, we investigated the precise mechanisms of FVIIa/TF‐catalyzed FVIII activation. Results: FVIII activity increased ~4‐fold within 30 s in the presence of FVIIa/TF, and then decreased to initial levels within 20 min. FVIIa (0.1 nm ), at concentrations present physiologically in plasma, activated FVIII in the presence of TF, and this activation was more rapid than that induced by thrombin. The heavy chain (HCh) of FVIII was proteolyzed at Arg740 and Arg372 more rapidly by FVIIa/TF than by thrombin, consistent with the enhanced activation of FVIII. Cleavage at Arg336 was evident at ~1 min, whilst little cleavage of the light chain (LCh) was observed. Cleavage of the HCh by FVIIa/TF was governed by the presence of the LCh. FVIII bound to Glu‐Gly‐Arg‐active‐site‐modified FVIIa (Kd, ~0.8 nm ) with a higher affinity for the HCh than for the LCh (Kd, 5.9 and 18.9 nm ). Binding to the A2 domain was particularly evident. Von Willebrand factor (VWF) modestly inhibited FVIIa/TF‐catalyzed FVIII activation, in keeping with the concept that VWF could moderate FVIIa/TF‐mediated reactions. Conclusions: The results demonstrated that this activation mechanism was distinct from those mediated by thrombin, and indicated that FVIIa/TF functions through a ‘priming’ mechanism for the activation of FVIII in the initiation phase of coagulation. 相似文献
6.
7.
Treatment of human platelets with purified bovine Factor VIII caused three types of aggregation: (a) primary agglutination; (b) secondary aggregation involving the platelet release reaction; and (c) super-aggregation, in which the platelets were gathered into only a few large clumps. Removal of calcium ions or treatment with p-hydroxymercuiriphenyl sulfonate blocked the release reaction, but not primary agglutination or super-aggregation. Platelets treated with formalin were not aggregated by ADP, thrombin, or collagen, but were agglutinated by bovine Factor VIII, although they did not show super-aggregation. For malin-treated platelets were agglutinated by phytohemagglutinin P less extensively and less rapidly than by bovine Factor VIII. Treatment of platelets and Factor VIII with neuraminidase released 60 and 53%, respectively, of the sialic acid residues without affecting the agglutination reaction or the procoagulant activity of the Factor VIII. Agglutination was inhibited by high salt concentrations, dextran sulfate, and heparin. During agglutination, both the procoagulant and platelet-agglutinating activities of Factor VIII became bound to the platelet surface. 相似文献
8.
Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid. 总被引:4,自引:2,他引:4
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Most antibodies to factor VIII have recently been shown to react with discrete regions of the factor VIII light chain (within the C2 domain) and/or the factor VIII heavy chain (within the amino-terminal segment of the A2 domain). The mechanism by which these antibodies, usually designated "factor VIII inhibitors," interfere with factor VIII function has been examined by determining their effect on factor VIII binding to a phospholipid. Factor VIII-phosphatidylserine binding was prevented by all seven factor VIII inhibitors that had strong factor VIII light chain reactivity and reduced by two inhibitors with weak anti-light chain reactivity. None of four inhibitors with heavy chain reactivity prevented factor VIII-phosphatidylserine interaction, though a partial reduction (less than 50%) was noted for the intact IgG preparations. However, when Fab' fragments were substituted, no detectable reduction in factor VIII-phosphatidylserine binding was noted for the anti-heavy chain inhibitors and complete inhibition was retained by the anti-light chain inhibitors. These data suggest that a subset of factor VIII inhibitors, those that bind to light chain determinants, inactivate factor VIII by preventing its effective interaction with phospholipid. 相似文献
9.
Autoantibodies to factor VIII (FVIII)(spontaneous inhibitors) often inactivate FVIII in a complex fashion (type II inhibitors) as compared with alloantibodies (hemophilic inhibitors), which usually demonstrate second-order reaction kinetics (type I inhibitors). The infusion of porcine FVIII in patients with spontaneous inhibitors may give rise to anti-porcine FVIII antibodies. The purpose of this study was to determine whether these were type I or type II inhibitors. Plasma from 8 patients with spontaneous inhibitors and 6 patients with hemophilia with inhibitors were studied. Equal volumes of patient plasma and either pooled normal human plasma or porcine FVIII (Hyate-C) were incubated at 37 degrees C for 90 minutes. Aliquots were removed immediately after mixing and at 30-minute intervals and assayed for FVIII by using a two-stage method. The values for residual FVIII were log-transformed and plotted against the time of incubation, and the resultant curves were analyzed for goodness of fit (coefficient of determination, r2) by using linear and exponential equations. The values were examined by paired t tests; P values were two-tailed. Values are expressed as mean +/- SD. The titers of spontaneous inhibitors against human FVIII ranged from 2.6 to 416 Bethesda Units (BU), and those against porcine FVIII ranged from 0.7 to 47 BU. Samples were diluted so that the FVIII levels in the mixtures before incubation were similar: human, 0.44 U/ml; porcine, 0.47 U/ml; P = not significant. Four of the 8 patients with spontaneous inhibitors inactivated human FVIII in a fashion consistent with complex kinetics; their r2 values with the linear equation were less than 0.90. All r2 values improved when the exponential equation was used (linear, 0.90 +/- 0.08; exponential, 0.92 +/- 0.06; P = .007). In contrast, r2 values with porcine FVIII were the same (0.94) with either the linear or the exponential equation (P = not significant). r2 for the 6 hemophilic inhibitors showed no significant difference between the linear and exponential equations; the values were 0.99 with human FVIII and 0.95 with porcine FVIII. In non-hemophilic patients, antibodies developing to porcine FVIII have kinetics of inhibition that are second order (type I), even though antibodies to human FVIII in these same patients may have complex (type II) kinetics. 相似文献
10.
Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder haemophilia B. FIX contains a Gla module, two epidermal growth factor-like (EGF) modules, and a serine protease region. I characterized a monoclonal antibody and found that it recognizes an epitope around residues 72 and 80 in the C-terminal part of EGF1 in human FIX. The antibody exhibited 10-fold greater affinity for activated FIX (FIXa) than for the zymogen FIX, indicating the existence of intra-molecular communication between the serine protease region and EGF1. Binding of the antibody did not affect the amidolytic activity of FIXa, hence I could use the antibody during activation of FIX to show that the C-terminal part of EGF1 is of importance for the interaction with FXIa but not with FVIIa/TF. Considering activation of FX, it is a matter of debate whether EGF1 or FIXa interacts directly with FVIIIa. I activated FX in the presence and absence of the antibody and/or FVIIIa. The addition of antibody caused only a minor decrease in k cat,app , and the major increase in k cat,app caused by the addition of FVIIIa occurred even in the presence of the antibody. This implies that EGF1 of FIXa is not directly involved in interaction with FVIIIa in the Xase complex. A model of the FIXa-FVIIIa complex, based on my findings and results from the literature, was constructed and indicated that EGF1 of FIXa does not interact directly with FVIIIa. 相似文献
11.
Summary. Activation of precursor proteins by specific and limited proteolysis is a hallmark of the hemostatic process. The homologous coagulation factors (F)V and FVIII circulate in an inactive, quiescent state in blood. In this so‐called procofactor state, these proteins have little, if any procoagulant activity and do not participate to any significant degree in their respective macromolecular enzymatic complexes. Thrombin is considered a key physiological activator, cleaving select peptide bonds in FV and FVIII which ultimately leads to appropriate structural changes that impart cofactor function. As the active cofactors (FVa and FVIIIa) have an enormous impact on thrombin and FXa generation, maintaining FV and FVIII as inactive procofactors undoubtedly plays an important regulatory role that has likely evolved to maintain normal hemostasis. Over the past three decades there has been widespread interest in studying the proteolytic events that lead to the activation of these proteins. While a great deal has been learned, mechanistic explanations as to how bond cleavage facilitates conversion to the active cofactor species remain incompletely understood. However, recent advances have been made detailing how thrombin recognizes FV and FVIII and also how the FV B‐domain plays a dominant role in maintaining the procofactor state. Here we review our current understanding of the molecular process of procofactor activation with a particular emphasis on FV. 相似文献
12.
Monoclonal antibodies to human procoagulant factor VIII 总被引:2,自引:0,他引:2
F Rotblat A H Goodall D P O'Brien E Rawlings S Middleton E G Tuddenham 《The Journal of laboratory and clinical medicine》1983,101(5):736-746
VIII:C was purified from intermediate-purity concentrate by adsorption on polyelectrolyte E5 and affinity chromatography on Sepharose/anti-VIIIR:Ag. The highly purified VIII:C preparation (sp. act. 1598 U/mg) was used to immunize Balb-C mice. Spleen cells from a mouse with a serum antibody titer of 963 U/ml were fused with P3 NSI mouse myeloma cells. Hybrid clones were screened by a coagulation inhibition assay and by a four-layer antibody adsorption procedure. Nine monoclonal antibodies specific to VIII:C were produced. Five of these antibodies have been cloned and grown in mouse ascitic fluid. Antibody titers from ascitic fluid ranged from 35 to 82,000 BU/ml. The antibodies, when radiolabeled, form a high-molecular-weight complex with antigens present in normal plasma and factor VIII concentrate, but not when incubated with CRM-negative hemophilic plasma. A two-site assay using a combination of monoclonal antibodies is able to detect VIII:CAg in normal plasma and in factor VIII concentrate. Sensitive two-site immunoradiometric assays using monoclonal antibodies as the solid phase have been set up. 相似文献
13.
J. L. PLANTIER V. ROLLI C. DUCASSE Y. DARGAUD N. ENJOLRAS H. BOUKERCHE C. NÉGRIER 《Journal of thrombosis and haemostasis》2010,8(2):286-293
Summary. Background: Factor VIII (FVIII) and its activated form (FVIIIa) are subject to proteolysis that dampens their cofactor function. Among the proteases that attack FVIII (activated factor X (FXa), activated protein C (APC) and plasmin), only APC cleaves within the FVIII A2 domain at R562 to fully abolish FVIII activity. Objectives: We investigated the possible involvement of the FXa cleavage at R562 within the A2 domain in the process of FVIII inactivation. Methods: An antibody (GMA012/R8B12) that recognizes the carboxy‐terminus extremity of the A2 domain (A2C) was used to evaluate FXa action. A molecule mutated at R562 was also generated to assess the functional role of this particular residue. Results and Conclusions: The appearance of the A2C domain as a function of time evidenced the identical cleavage within the A2 domain of FVIII and FVIIIa by FXa. This cleavage required phospholipids and occurred within minutes. In contrast, the isolated A2 domain was not cleaved by FXa. Von Willebrand factor and activated FIX inhibited the cleavage in a dose‐dependent manner. Mutation R562K increased both the FVIII specific activity and the generation of FXa due to an increase in FVIII catalytic efficiency. Moreover, A2C fragment could not be identified from FVIII‐R562K cleavage. In summary, this study defines a new mechanism for A2 domain‐mediated FVIII degradation by FXa and implicates the bisecting of the A2 domain at R562. 相似文献
14.
The activation of factor X and prothrombin by recombinant factor VIIa in vivo is mediated by tissue factor. 总被引:14,自引:1,他引:14
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H ten Cate K A Bauer M Levi T S Edgington R D Sublett S Barzegar B L Kass R D Rosenberg 《The Journal of clinical investigation》1993,92(3):1207-1212
The human coagulation system continuously generates very small quantities of Factor Xa and thrombin. Current evidence suggests that basal level activation of the hemostatic mechanism occurs via Factor VIIa-dependent activation of Factor X, but direct proof has not been available for the participation of tissue factor in this pathway. To examine this issue, we infused relatively high concentrations of recombinant Factor VIIa (approximately 50 micrograms/kg body wt) into normal chimpanzees and observed significant increases in the plasma levels of Factor IX activation peptide, Factor X activation peptide, and prothrombin activation fragment F1+2. Metabolic turnover studies with radiolabeled Factor IX activation peptide, Factor X activation peptide, and F1+2 indicate that elevated levels of the activation peptides are due to accelerated conversion of the three coagulation system zymogens into serine proteases. The administration of a potent monoclonal antibody to tissue factor, which immediately neutralizes function of the Factor VIIa-tissue factor complex in vitro, abolishes the activation of Factor X and prothrombin mediated by the infused recombinant protein, and also suppresses basal level activation of Factor IX and Factor X. The above results suggest that recombinant Factor VIIa functions as a prohemostatic agent by interacting with endogenous tissue factor sites, but definitive proof will require studies in hemophilic animals using relevant hemostatic endpoints. 相似文献
15.
Summary Plasma-exchange is one of many treatments available for the prevention and management of hemorrhage in patients with factor
VIII inhibitors. Details of exchange technique and the frequency of treatment are determined by the clinical situation, venous
access and the availability of equipment, staff and blood products. In view of the many difficulties associated with plasma-exchange,
other treatment options are likely to be preferred in most circumstances. The technique, remains attractive as a means for
reducing inhibitory activity before elective surgery and during life-threatening hemorrhage. 相似文献
16.
A Trimarchi D Minestrini G Palazzesi M Cassetti 《International Journal of Clinical & Laboratory Research》1992,22(2):115-118
We examined the effect of the lipoproteins on the activation of human factor X in an in vitro system containing purified human factor VII, low levels of tissue factor and calcium ions. In the absence of the lipoproteins the maximum activation was achieved within 10 min of the start of incubation; after this period the formation of factor Xa ceases. When very low-density lipoproteins, low-density lipoproteins or high-density lipoproteins were present at normal or subnormal plasma concentrations, the factor Xa generated was almost doubled after 10 min. This increase could be abolished by treating each lipoprotein subfraction with the phospholipase A2; hence, the treated lipoproteins lowered the factor Xa activity. We conclude that the phospholipids favor factor Xa formation and protect the tissue factor/factor VIIa/factor Xa complex from a potent inhibitor contained in the lipoprotein subfractions. 相似文献
17.
目的:探讨降脂药物非诺贝特激活法尼醇X受体FXR的机制。方法 :分离小鼠肝脏原代细胞,予非诺贝特处理,通过实时定量PCR检测FXR下游基因的表达情况,并通过荧光素酶双报告基因实验,分析非诺贝特对FXR下游基因调控的机制。结果:①非诺贝特处理可诱导FXR下游靶基因SHP和BSEP的表达,进而降低三酰甘油合成关键转录因子SREBP1c的表达。②非诺贝特能促进FXR上调SHP和BSEP启动子活性,而FXR配体结合区域及转录激活域缺失型则丧失该功能。结论:非诺贝特可能是潜在的FXR激动型配体,可通过激活FXR信号通路,降低三酰甘油合成基因的表达,从而抑制肝脏三酰甘油的沉积。 相似文献
18.
19.
Studies of the human factor VIII/von Willebrand factor protein. III. Qualitative defects in von Willebrand's disease. 总被引:2,自引:6,他引:2
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The Factor VIII/von Willebrand factor protein was characterized in two unrelated patients with von Willebrand's disease in whom procoagulant and Factor VIII/von Willebrand factor antigen levels were normal. In both patients evidence of an abnormal protein was observed on crossed antigen-antibody electrophoresis. In one patient the Factor VIII/von Willebrand factor protein eluted from Sepharose 4B in a position and distribution identical to normal with normal levels of procoagulant activity and antigen. However, the partially purified Factor VIII/von Willebrand factor protein had markedly reduced von Willebrand factor activity in a ristocetin assay. In the second patient the peak of Factor VIII/von Willebrand factor protein, antigen, and procoagulant activity eluted from a Sepharose 4B column with an estimated molecular weight of approximately half that of normal. This protein had no von Willebrand factor activity. In both patients the reduced Factor VIII/von Willebrand factor protein subunit was indistinguishable from normal on polyacrylamide gel electrophoresis. These studies indicate that in some patients with von Willebrand's disease there is a qualitative defect of the Factor VII/von Willebrand factor protein; the total amount of protein, antigen, and procoagulant activity are normal while the von Willebrand factor activity is deficient. 相似文献
20.
Subunit structure of factor VIII antigen synthesized by cultured human endothelial cells. 总被引:2,自引:0,他引:2
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Cultured human endothelial cells were labeled with (3H)leucine, and the radioactive Factor VIII antigen present in the postculture medium was isolated by double anitbody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction with dithiothreitol. The Factor VIII antigen synthesized by cultured endothelial cells was found to contain the same single polypeptide subunit (mol wt 225,000) present in plasma Factor VIII antigen. These results suggest that in vivo, the endothelial cell is a major site of synthesis of circulating Factor VIII antigen. 相似文献