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PubMed2003

Thrombophilic families with inheritably associated high levels of coagulation factors VIII, IX and XI.

We describe six families in which associated high levels of coagulation factors (F) XI, FIX and FVIII (each with a plasma concentration higher than the 95th percentile found in a control group of 500 asymptomatic individuals: respectively, 135, 145 and 155 IU dL-1) were inherited as a dominant autosomic genetic traits. In these six families, this syndrome is associated with venous thromboembolic events (Odds ratio 41 [4.9-353], P = 0.0006). It seems to predispose to idiopathic events and, as age increases, is often associated with recurrence. First thrombotic episodes occur in young patients (50% of the carriers are symptomatic at the age of 32 years) and in women, can be unmasked by hormonal treatments, mainly oral contraceptives. The association of high levels of coagulation FXI, FIX and FVIII is thus a new rare high-risk inherited thrombophilia syndrome.

Adolescent↗
PubMed1986

The functional domains of coagulation factor VIII:C.

A lack of factor VIII:C, manifested as a bleeding disorder due to the absence of clot formation, is known as hemophilia A, an X chromosome-linked inherited disease afflicting 1-2 males/10,000. To determine the minimum functional domain(s) essential for factor VIII:C activity, we have expressed the amino-terminal (92-kDa) and carboxyl-terminal (80-kDa) proteolytic cleavage products as individual, secreted polypeptides in monkey cells without the 909-residue central region. We have found that neither terminal domain alone is able to promote coagulation in factor VIII:C-deficient plasma. However, when the 92- and 80-kDa peptides are co-expressed, clotting activity is readily detected. Thus, these two chains alone constitute an active or activatable complex. The central domain is required neither for activity nor for the assembly of an active complex from two chains expressed in trans. These results suggest that a truncated derivative of factor VIII:C may be useful in coagulation therapy.

Amino Acid Sequence↗
PubMed1977

Factor VIII-related antigen and factor VIII coagulant activity in normal and pre-eclamptic pregnancy.

The changes in the ratio between factor VIII-related antigen and factor VIII activity were compared in ten patients with normal pregnancies and in ten patients with severe pre-eclampsia. In the patients with pre-eclampsia, a highly significant increase in the ratio was observed during the third trimester. No difference in the ratio between the two groups was found on day 7 of the puerperium. In the pre-eclamptic patients, the highest ratios for factor VIII-related antigen to factor VIII activity were associated with either a perinatal death or with the delivery of a severely growth retarded infant. These findings are in keeping with an increased rate of thrombin production in women with pre-eclampsia, and suggest that the ratio of factor VIII-related antigen to factor VIII activity may reflect the severity of the effect of the disease process on the fetus.

Antigens↗
PubMed1995

Binding of blood coagulation factor VIII and its light chain to phosphatidylserine/phosphatidylcholine bilayers as measured by ellipsometry.

Factor VIII is a plasma protein which plays an essential role in the coagulation system. When assembled with the enzyme Factor IXa on a phospholipid membrane, it functions as a cofactor in the enzyme complex that cleaves the zymogen Factor X to Factor Xa. We studied the binding of both Factor VIII and the Factor VIII light chain to planar phospholipid bilayers consisting of 25% dioleoylphosphatidylserine and 75% dioleoylphosphatidylcholine (PSPC) by ellipsometry. Equilibrium-binding studies revealed that both Factor VIII and its light chain bind with high affinity to PSPC bilayers. The binding affinity of Factor VIII, with a dissociation constant Kd of 0.24 nM, was comparable with that of the Factor VIII light chain (Kd 0.49 nM). Maximal binding was 2.3 mmol of protein per mol of PSPC for Factor VIII and 7.1 mmol of protein per mol of PSPC for the Factor VIII light chain. Adsorption kinetics of both Factor VIII and its light chain conformed to the classical Langmuir adsorption model yielding dissociation constants calculated from the rates of adsorption that were similar to those obtained by equilibrium-binding studies. In contrast, measurements of rates of desorption revealed a deviation from those expected for a single class of binding sites. The desorption rate of Factor VIII increased with increasing residence time on the lipid membrane. This indicates transition of Factor VIII to a configuration with a lower binding affinity. As this time-dependent change in affinity could affect the validity of the measurement of binding parameters, in particular equilibrium-binding determinations carried out on a long timescale, binding affinity was also estimated from adsorption kinetics at half-maximal surface coverage, a relatively rapid procedure for the determination of the affinity. A Kd of 0.087 nM was obtained under these conditions. Measurement of equilibrium binding to small PSPC vesicles, a system in which equilibrium is rapidly attained, resulted in similar binding parameters (Kd = 0.13 nM and a maximal binding of 2.8 mmol of protein per mol of PSPC). These data confirm the results of equilibrium binding to planar bilayers. Taken together, our results indicate that Factor VIII, by means of its 80 kDa light chain, binds to PSPC bilayers with a dissociation constant below the concentration of Factor VIII in plasma and therefore may readily bind to exposed phospholipid membranes under physiological conditions.

Adsorption↗
PubMed1999

Proteolysis of blood coagulation factor VIII by the factor VIIa-tissue factor complex: generation of an inactive factor VIII cofactor.

Activation of factor VIII by thrombin occurs via limited proteolysis at R372, R740, and R1689. The resultant active factor VIIIa molecule consists of three noncovalently associated subunits: A1-a1, A2-a2, and A3-C1-C2 (50, 45, and 73 kDa respectively). Further proteolysis of factor VIIIa at R336 and R562 by activated protein C subsequently inactivates this cofactor. We now find that the factor VIIa-tissue factor complex (VIIa-TF/PL), the trigger of blood coagulation with restricted substrate specificity, can also catalyze limited proteolysis of factor VIII. Proteolysis of factor VIII was observed at 10 sites, producing 2 major fragments (47 and 45 kDa) recognized by an anti-factor VIII A2 domain antibody. Time courses indicated the slow conversion of the large fragment to 45 kDa, followed by further degradation into at least two smaller fragments. N-Terminal sequencing along with time courses of proteolysis indicated that VIIa-TF/PL cleaved factor VIII first at R740, followed by concomitant cleavage at R336 and R372. Although cleavage of the light chain at R1689 was observed, the majority remained uncleaved after 17 h. Consistent with this, only a transient 2-fold increase in factor VIII clotting activity was observed. Thus, heavy chain cleavage of factor VIII by VIIa-TF/PL produces an inactive factor VIII cofactor no longer capable of activation by thrombin. In addition, VIIa-TF/PL was found to inactivate thrombin-activated factor VIII. We hypothesize that these proteolyses may constitute an alternative pathway to regulate coagulation under certain conditions. In addition, the ability of VIIa-TF/PL to cleave factor VIII at 10 sites greatly expands the known protein substrate sequences recognized by this enzyme-cofactor complex.

Amino Acid Sequence↗
PubMed1989

Secretion of coagulant factor VIII activity and antigen by in vitro cultivated rat liver sinusoidal endothelial cells.

Different types of liver cells and a few extrahepatic cell types were analysed for the presence and production of factor VIII activity (VIII:C). Only freshly prepared suspensions of rat liver sinusoidal cells and pure monolayer cultures of rat liver endothelial cells (LEC) were found to contain and secrete detectable amounts of the coagulation factor. Secretion of VIII:C by cultured LEC was inhibited by cycloheximide and by monensin. Constant levels of VIII:C were produced for at least 48 h suggesting continuous synthesis rather than a burst release of stored material. VIII:C, as measured spectro-photometrically by conversion of X to Xa, was inhibited by anti-human VIII:C antiserum. Indirect immunocytochemistry using this antiserum gave positive staining only with LEC. Immunoprecipitation of metabolically labelled proteins in conditioned rat LEC medium with the anti human VIII:C antiserum revealed the presence of proteins of similar sizes to those reported for human VIII:C. These results indicate that rat LEC are an important site for production and secretion of procoagulant factor VIII and are not only a site for storage and release of the factor. The established conditions for synthesis of VIII:C in in vitro cultivated rat LEC should provide the means to study the regulation of VIII:C synthesis.

Animals↗
PubMed1998

Differential interaction of coagulation factor VIII and factor V with protein chaperones calnexin and calreticulin.

Factor VIII (FVIII) and factor V (FV) are homologous coagulation cofactors sharing a similar domain organization (A1-A2-B-A3-C1-C2) and are both extensively glycosylated within their B-domains. In mammalian cell expression systems, compared with FV, the FVIII primary translation product is inefficiently transported out of the endoplasmic reticulum. Here we show that FVIII is degraded within the cell by a lactacystin-inhibitable pathway, implicating the cytosolic 20 S proteasome machinery. Protein chaperones calnexin (CNX) and calreticulin (CRT) preferentially interact with glycoproteins containing monoglucosylated N-linked oligosaccharides and are proposed to traffic proteins through degradative and/or secretory pathways. Utilizing co-immunoprecipitation assays, intracellular FVIII was detected in association with CNX maximally within 30 min to 1 h following synthesis, whereas FV could not be detected in association with CNX. In contrast, both FVIII and FV displayed interaction with CRT during transit through the secretory pathway. B-domain deleted FVIII significantly reduced the CNX and CRT interaction, indicating the B-domain may represent a primary CNX and CRT interaction site. In the presence of inhibitors of glucose trimming, the interactions of FVIII with CNX, and of FVIII and FV with CRT, were significantly reduced whereas the secretion of FVIII, and not FV, was inhibited. In addition, transfection in a glucosidase I-deficient Chinese hamster ovary cell line (Lec23) demonstrated that both degradation and secretion of FVIII were inhibited, with little effect on the secretion of FV. These results support that CNX and CRT binding, mediated at least in part by the B-domain of FVIII, is required for efficient FVIII degradation and secretion. In contrast, FV does not require CNX interaction for efficient secretion. The results suggest a unique requirement for carbohydrate processing and molecular chaperone interactions that may limit the productive secretion of FVIII.

Animals↗
PubMed1986

[Surgery and the acquired inhibitor of factor VIII coagulant].

A case is reported of an 80 year old woman with old sarcoidosis and acquired factor VIIIc inhibitor undergoing orthopaedic surgery. This was successfully carried out after she had been given a total of 48,420 units of factor VIII concentrate: there were no haemorrhagic complications. The main pathological states in which acquired factor VIIIc inhibitor may be found are related: principal treatments available nowadays are discussed: immunosuppressors, human factor VIII, animal factor VIII, prothrombin complex concentrate, plasma exchange.

Aged↗
PubMed2002

Molecular defects in coagulation Factor VIII and their impact on Factor VIII function.

Molecular defects in Factor VIII (FVIII), such as haemophilia A-related mutations or denaturative conformational changes, may affect the stability of FVIII as well as its interactions with physiological activators, von Willebrand Factor, phospholipid, or conformationally sensitive antibodies. We summarize the contemporary assays which allow identification of impaired functional interactions of FVIII that cause a reduction or loss of its cofactor activity and/or increased immunogenicity. These assays can potentially be used for detection of molecular defects in FVIII and elucidation of the function impaired by these defects.

Blood Coagulation↗
PubMed2003

Blood coagulation factor VIII: An overview.

Factor VIII (FVIII) functions as a co-factor in the blood coagulation cascade for the proteolytic activation of factor X by factor IXa. Deficiency of FVIII causes hemophilia A, the most commonly inherited bleeding disorder. This review highlights current knowledge on selected aspects of FVIII in which both the scientist and the clinician should be interested.

Factor VIII↗
PubMed2002

High level expression of recombinant porcine coagulation factor VIII.

Recombinant human factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine factor VIII molecule. Recombinant B-domain-deleted porcine factor VIII expression levels are 10- to 14-fold greater than recombinant B-domain-deleted human factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units x 10(6) cells(-1) x 24 h(-1) was observed from a baby hamster kidney-derived cell line stably expressing recombinant porcine factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state factor VIII mRNA levels. The identification of sequence(s) in porcine factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit factor VIII expression.

Animals↗
PubMed1981

Immunoradiometric assay of factor VIII: coagulant antigen using four human antibodies. Study of 27 cases of haemophilia A.

Immunoradiometric assay (IRMA) of VIII:C antigen was performed using either IgG or monovalent Fab fragments from four antibodies arisen in polytransfused haemophilia A patients (titre between 100 and 1500 U/ml). Using IgG isolated by a solid or a liquid phase system, only the high titre (greater than or equal to 1000 U/ml) antibodies could be used for IRMA, with a sensitivity of 0.2% VIII:CAg. Using Fab fragments isolated by liquid phase, high and low (less than or equal to 150 U/ml) titre antibodies could be used and the IRMA was significantly improved with a 10-fold higher sensitivity. The affinity of the antibodies for VIII:CAg, studied by displacement curves using a modification of the IRMA, was found not to depend upon the titre of the antibody. Comparative levels of VIII:C and VIII:CAg in 27 cases of haemophilia A emphasize the heterogeneity of this disorder, two types of severe and three types of mild haemophilia being observed.

Antibody Affinity↗