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PubMed1983

Effect of phospholipid on factor VIII coagulant activity and coagulant antigen.

Incubation of purified factor VIII/von Willebrand factor complex or plasma with certain phospholipid preparation induced a two to threefold increase in apparent factor VIII activity, while a slight decrease of factor VIII coagulant antigen was observed. Between six or eightfold activity increases occurred when 10-3 units/ml of thrombin was present during the incubation. When the phospholipid was added first and the thrombin added 30 minutes later a twofold increase in apparent factor VIII activity occurred during the first part and this activity was then increased threefold further after the addition of thrombin. In separate experiments using gel filtration on Sepharose 2B, association of factor VIII activity to phospholipid vesicles could be demonstrated. This phospholipid bound factor VIII could be activated by thrombin. The relationship between these observations and the formation of factor X activating complex is discussed.

Antigens↗
PubMed1990

Validation of a procedure for potency assessing of a high purity factor VIII concentrate--comparison of different factor VIII coagulant assays and effect of prediluent.

The assessment of factor VIII coagulant activity (FVIII:C) in recently available highly purified and concentrated FVIII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVIII:C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVIII:C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVIII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVII-deficient plasma used as prediluents. In order to validate our "in vitro" data we performed 6 "in vivo" analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.

Blood Coagulation Tests↗
PubMed2004

Elevated coagulation factor VIII and the risk for recurrent early pregnancy loss.

Inherited and acquired thrombophilia are associated with recurrent pregnancy loss. Recently, an increased risk for thromboembolic disease was described for patients with elevated coagulation factor VIII, but it is unknown whether there is also an association to early pregnancy loss. We therefore evaluated the relation between recurrent early pregnancy loss and levels of coagulation factor VIII. We enrolled 49 unrelated Caucasian women with a history of 2-6 early pregnancy losses and 48 healthy controls, who had delivered at least one term infant and had never experienced pregnancy loss. We determined factor V Leiden-, G20210A prothrombin-, MTHFR C677T- and A1298C-gene mutations, levels of antithrombin, protein C, protein S, factor VIII, C-reactive protein and antiphospholipid antibodies. There was a significantly higher rate of pregnancy losses in women with Antiphospholipid Syndrome (p = 0.043). Furthermore, plasma levels of coagulation factor VIII were significantly higher in cases than in controls (130.5 IU/dl +/- 25.4 vs 119.5 IU/dl +/- 24.1; p = 0.032) and appeared independent of C-reactive protein (R = 0.146, p = 0.323 in cases; R = -0.028, p = 0.850 in controls). The relative risk for recurrent pregnancy loss in women with factor VIII levels above 151 IU/dl (90(th) percentile of controls) was 2.5 (0.7 - 8.9, 95 percent confidence interval), for levels above 156 IU/dl (95(th) percentile of controls) 3.9 (0.8 - 20.0, 95 percent confidence interval). Elevated maternal plasma levels of coagulation factor VIII tend to be associated with an increased risk for recurrent early pregnancy loss.

Abortion, Habitual↗
PubMed1987

Spectrophotometric method for the assay of human blood coagulation factor VIII.

A spectrophotometric method for the assay of human blood coagulation factor VIII in plasma is presented. The chromogenic assay for factor VIII:C in plasma is performed in 3 steps: activation of factor VIII by thrombin; activation of factor X in a mixture of factor X, factor IXa, phospholipids/Ca2+ and plasma containing activated factor VIII, and determination of the rate of factor Xa formation with the chromogenic substrate S2337. Within-assay variation was between 5 and 6.9% for factor VIII:C activities between 20 and 150%. Clotting and chromogenic factor VIII:C activities were compared in plasma of 50 normal healthy donors (coefficient of correlation r = 0.83).

Blood Coagulation Tests↗
PubMed1983

Development of an inhibitor specific to factor VIII: coagulant activity in a patient with platelet-type von Willebrand's disease.

Platelet-type von Willebrand's disease is a recently described autosomal dominant bleeding disorder characterized by decreased ristocetin cofactor activity, lack of the higher molecular weight von Willebrand Factor (vWF) multimers on SDS agarose gel electrophoresis, increased platelet aggregation with low concentrations of ristocetin, and increased ristocetin-induced binding of normal vWF to patient platelets. In this report the authors describe a 17-month-old male with Platelet-type von Willebrand's disease, inherited from the paternal side of his family, who developed an inhibitor specific to Factor VIII:C. The patient's plasma inhibited normal plasma VIII:C and partially purified VIII:C; it did not appear directed against normal VIIIR:Ag or ristocetin cofactor. This antibody is therefore similar to inhibitors that develop in some transfused hemophilia A patients. Since low VIII:C, VIII:CAg, and VIII:C/VIIIR:Ag ratio were encountered in his mother, it is likely that this patient has inherited hemophilia A in addition to Platelet-type von Willebrand's disease.

Adult↗
PubMed1980

Increase in coagulation factor VIII activity in man following acute hypoglycaemia: mediation via an adrenergic mechanism.

Coagulation factor VIII activity has been assayed in nine normal volunteers before and after acute hypoglycaemia. In all subjects there was a rise in activity with a mean increment of 96%. Control studies using saline failed to demonstrate any change. In four subjects with pre-ganglionic sympathetctomy no increase in activity was observed. In the normal subjects the increase was blocked by propranolol but not by metoprolol, indicating mediation via beta2 receptors.

Acute Disease↗
PubMed1986

Disseminated intravascular coagulation following Echis carinatus venom in dogs: effects of a synthetic thrombin inhibitor.

Disseminated intravascular coagulation (DIC) was produced by an infusion of a prothrombin activator (Echis carinatus venom; 30 minutes; 0.5 NIH thrombin equivalent U/kg) in mongrel dogs (Echis group, n = 7). Fibrinogen declined to below measurable levels (less than 25 mg/dl), and fibrin-fibrinogen degradation products appeared (53 +/- 8 micrograms/ml) at end venom infusion in the Echis group. These alterations were not seen when an irreversible thrombin inhibitor, D-phenylalanyl-L-prolyl-L-arginine-L-chloromethyl ketone (PPACK) (57 nmol/kg/min for 120 minutes), was given alone (PPACK group, n = 5) or in association with venom (Echis + PPACK group, n = 5). Factor II activity (1% +/- 1%) in the Echis and Echis + PPACK groups was significantly below the PPACK (55% +/- 9%) and the control (79% +/- 2%) levels at 120 minutes. In contrast, factor VIII coagulant (factor VIII:C) activity in the Echis group (1% +/- 1%) remained significantly below that in the Echis + PPACK (68% +/- 8%), PPACK (78% +/- 10%), and control (91% +/- 9%) groups at this interval. No change in factors X (91% +/- 7% to 81% +/- 7%, P not significant) and VII (64% +/- 10% to 48% +/- 11%, P not significant) activities were observed. Hemolysis was observed only in the Echis group, whereas thrombocytopenia and leukopenia were noted in both the Echis and the Echis + PPACK groups. These data show that large amounts of E. carinatus venom produce rapid DIC in vivo, because of the activation of prothrombin. In contrast, the decline in factor VIII:C activity appeared to be the result of the liberated thrombin. PPACK antagonized all of the venom-released thrombin without any major deleterious clotting abnormalities. This inhibitor appears to prevent thrombin-mediated DIC in vivo. In contrast, heparin was found to be an unreliable antagonist of the venom-released thrombin in vitro. PPACK also inhibited the marked hemolysis usually observed after venom. In addition, we found that the esterolytic (N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide HCL) activity of E. carinatus venom degrades fibrinogen in vitro.

Amino Acid Chloromethyl Ketones↗
PubMed2004

[Non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line].

To investigate the non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line, a recombinant plasmid vector, pRC/RSV-hFVIIIBDcDNA, was constructed by cloning B-domain-deleted (Delta760aa-1639aa) human factor VIII cDNA (hFVIIIBDcDNA) into plasmid vector, pRC/RSV. The plasmid RC/RSV-hFVIIIBDcDNA was then transfected by means of SuperFect Transfection Reagent into mouse 32D cell line. After screening with G418, the procoagulant activity (hFVIII:C) and antigen (hFVIII:Ag) of human factor VIII in the culture medium were detected using one-stage method and ELISA, respectively. Furthermore, RT-PCR was performed to observe the transcription of hFVIIIBDcDNA. The results showed that human coagulation factor VIII protein existed in culture medium with hFVIII:C up to 2.01 U/(10(6) cell x 24 hours) and hFVIII:Ag to 450.08 ng/(10(6) cell x 24 hours). RT-PCR displayed mRNA of hFVIIIBDcDNA in 32D cells. It is concluded that the recombinant plasmid RC/RSV-hFVIIIBDcDNA can successfully express human FVIII in mouse 32D cell line, and hFVIII expressed in vitro presents the similar coagulant activity to the native hFVIII existing in normal human plasma.

Animals↗
PubMed2006

Expression of human coagulation factor VIII in a human hybrid cell line, HKB11.

Loss of coagulation factor VIII (FVIII) function results in a bleeding disorder, hemophilia A, which requires FVIII replacement therapy. Owing to its large size and complexity, the expression level of recombinant FVIII is two to three orders of magnitude lower than other recombinant proteins produced in mammalian cell lines. To understand cellular factors limiting FVIII expression, we studied the expression of FVIII in a human cell line, HKB11 (a hybrid cell line of HEK293 and a human B cell line). In comparison with other cell lines, such as HEK293 and BHK-21, HKB11 showed increased FVIII expression levels. With unamplified, pooled stable cells, FVIII expression in HKB11 cells was 8- to 30-fold higher than the other cell lines tested. In this study, HKB11 clones expressing varying levels of FVIII were analyzed and FVIII secreted from these clones had similar specific activity. Characterization of these clones by immunofluorescence staining, Western blotting analysis, and flow cytometry showed that high-producing cells not only secreted more active FVIII but also accumulated more FVIII protein intracellularly. FVIII expression appears to be controlled by the rates of transcription, translation, and secretion, but transcription and translation may play more important roles than secretion in determining expression level in HKB11 cells. FACS analysis of live cells showed that the high-producing clones also had more FVIII on the HKB11 cell surface than low-producing cells, thus opening the possibility of using FACS to select high-producing cell lines. Expression levels of the chaperone protein Hsp70 and antiapoptotic proteins such as Bcl-2 and Bcl-xL were similar among HKB11 clones with different FVIII productivity. In conclusion, HKB11 is an efficient host cell line for expression of FVIII and possibly other recombinant proteins. Systematic approaches, such as gene expression profiling by DNA microarray, will be necessary to understand the global changes in the cells producing recombinant proteins.

Biotechnology↗
PubMed2001

Quantification of plasma-derived blood coagulation factor VIII by real-time biosensor measurements.

Plasma-derived blood coagulation factor VIII was analyzed in real time using biosensor technology. Monoclonal antibodies directed against the heavy and against the light chain of factor VIII were immobilized on different carboxymethyl dextran surfaces. Different factor VIII concentrations were injected over the antibody surfaces in parallel and response levels were determined from the dissociation phase at a fixed time after sample injection. Serial dilutions of plasma-derived factor VIII with known concentrations determined by a commercial FVIIIC:Ag ELISA were used as standards. A quantification limit of 0.9 I.U./ml with antibody 530p and 1.5 I.U./ml with antibody 531p was calculated. Intra-assay precision expressed as percent coefficient of variation was below 10% for concentrations above 0.6 I.U./ml. Inter-assay precision for antibody 530p was below 20% for concentrations higher than 0.6 I.U./ml. For 531p, inter-assay precision was below 10% for concentrations higher than 2 I.U./ml. A sensor chip lifetime in respect to regeneration of at least 100 cycles for both antibodies was found. The small sample requirement of 35 microl allows fast analysis of different FVIII products and the use of two monoclonal antibodies directed against two different FVIII domains provides additional information about the integrity of the FVIII molecule.

Biosensing Techniques↗
PubMed2000

ATP-dependent dissociation of non-disulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion.

Deficiency in coagulation factor VIII leads to the bleeding disorder hemophilia A. Previous studies demonstrated that factor VIII secretion is limited due to an ATP-requiring step early in the secretory pathway. In this report, we identified that this ATP-dependent rate-limiting step involves the dissociation of non-disulfide-linked aggregates within the endoplasmic reticulum (ER). In contrast to the numerous examples of interchain disulfide-linked aggregates, factor VIII is the first protein characterized to form non-disulfide-linked high molecular weight aggregates within the ER. Approximately a third of newly synthesized factor VIII was detected in high molecular weight aggregates. These aggregates disappeared over time as functional factor VIII appeared in the medium. The aggregated complexes did not require proteasomal degradation for clearance. Aggregate formation was enhanced by ATP depletion, and upon restoration of metabolic energy, these aggregates were dissociated and secreted. With the coexpression of von Willebrand factor (vWF), a small portion of vWF coaggregated with factor VIII. However, vWF dissociated from the aggregates more rapidly than factor VIII, supporting that these aggregates are dynamic. An increase in the factor VIII expression level elicited a corresponding increase in the fraction of factor VIII that was aggregated. In addition, a 110 amino acid sequence containing a hydrophobic beta-sheet within factor VIII was identified that may predispose factor VIII to aggregation. These data show that formation and ATP-dependent dissolution of nondisulfide-linked factor VIII aggregates is a dynamic, rate-limiting step during the folding process in the early secretory pathway. In summary, we have identified an unprecedented requirement for protein transport out of the ER that involves an ATP-dependent dissociation of non-disulfide-linked aggregates within the ER.

Acetylcysteine↗
PubMed2003

Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion.

Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.

Animals↗
PubMed1995

Molecular genetics of coagulation factor VIII gene and hemophilia A.

This review summarizes the structure of human coagulation factor VIII gene and its deduced protein sequence and the molecular etiology of hemophilia A in man. The gross DNA rearrangements including the common inversions of factor VIII (which account for about 45% of severe hemophilia A patients), and the point mutations are discussed. The functional consequences of certain missense mutations are illustrated.

Animals↗
PubMed1982

Simplified immunoradiometric assay for factor VIII coagulant antigen.

A simplified, non-competitive, solid phase immunoradiometric assay has been developed for the quantitation of factor VIII coagulant antigen (VIII:CAg)--the antigenic counterpart of FVIII coagulant activity (VIII:C). Both homologous and heterologous antibodies to human factor VIII (FVIII) were used in this assay. Initially, FVIII in a test sample was attached to immobilized, human IgG obtained from a polytransfused haemophilia A patient with a high titre antibody to VIII:C. The bound FVIII was then detected using rabbit 125I-IgG specific for human FVIII. The concentration of VIII:CAg correlated well with VIII:C levels in the plasma from normal donors (r = 0.84, n - 15). Homozygote von Willebrand's disease patients had undetectable levels of VIII:CAg in their plasma. Patients with severe haemophilia A (VIII:C less than 0.01 u/ml) could be divided into groups on the basis of the VIII:CAg levels, i.e. those having undetectable VIII:CAg and other with measurable VIII:CAg. VIII:CAg detected in normal serum was less than 0.002 u/ml. In this assay the use of human antibody to FVIII is considerably decreased compared to other methods for VIII:CAg, and the time-consuming steps to immunopurify human anti-FVIII antibody are eliminated.

Antigens↗
PubMed1992

The association of human coagulation factors VIII, IXa and X with phospholipid vesicles involves both electrostatic and hydrophobic interactions.

Blood coagulation factor X (FX) is converted to its active form (FXa) by a membrane bound multi-protein enzyme complex, comprised of factor VIII (FVIII), factor IXa (FIXa) and FX. Characterization of the molecular forces involved in the association of these proteins with phospholipids is crucial to understanding how these proteins bind to the lipid milieux of physiological membranes. In this report, the molecular forces involved in the association of FVIII, FIXa or FX with phospholipid vesicles (PLV) were characterized by ligand affinity chromatographic analyses. Treating FVIII-affinity columns with agents that disrupt electrostatic interactions caused elution of 15.2% of the total bound PLV, while agents that disrupt hydrophobic interactions caused elution of 84.8% of the total bound PLV. These results demonstrate that the association of PLV with FVIII is primarily hydrophobic. In contrast, the association of PLV with FIXa or FX is largely the result of electrostatic forces. This was established by observing that 71.3% and 78.9% of the total bound PLV was eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt electrostatic interactions. Of the total bound PLV, 28.7% and 21.2% were eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt hydrophobic interactions. These data demonstrate that hydrophobic forces play a heretofore unrecognized role in the association of PLV with FIXa or FX.

Blood Coagulation Factors↗
PubMedDate not supplied

Changes in factor VIII: coagulant activity and von Willebrand factor antigen concentration after subcutaneous injection of desmopressin in dogs with mild hemophilia A.

Four German Shepherd dogs with mild hemophilia A were given Desmopressin (Minirin intranasal solution, Ferring, Malmo, Sweden) subcutaneously at a range of doses in a controlled blind study. No substantial change in plasma FVIII activity was observed 0.5, 1, 2, 4, or 6 hours after administration of Desmopressin. Plasma von Willebrand factor antigen concentrations increased rapidly after doses of 0.4, 1.0, 2.0, and 5.0 micrograms/kg to mean values of 140%, 127%, 120%, and 140% of baseline, respectively. The duration of this increase was dependent on the dose of Desmopressin injected.

Animals↗