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PubMed1996

Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V.

The proteolytic cleavage and subsequent inactivation of recombinant human factor VIII (rhFVIII) and human factor VIIIa (rhFVIIIa) by recombinant human activated protein C (rAPC) was analyzed in the presence and absence of human protein S and human factor V (FV). Membrane-bound rhFVIIIa spontaneously looses most of its initial cofactor activity after 15 minutes of incubation at pH 7.4. The remaining activity can be eliminated after incubation with rAPC. Complete inactivation of the membrane-bound rhFVIII and rhFVIIIa by APC correlates with cleavage at Arg336. The inactivation of rhFVIII and human plasma FV by rAPC were also compared. Under similar experimental conditions, complete inactivation of membrane-bound FVIII (60 nmol/L) by rAPC (10 nmol/L) requires 4 hours of incubation, in contrast to 5 minutes for FV (60 nmol/L). The presence of protein S (100 nmol/L) enhances rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by twofold, whereas membrane-bound FV showed no protein S dependence during inactivation. The addition of human FV to the APC/protein S inactivation mixture increases by approximately twofold the rate of inactivation of rhFVIII. The effect of FV on the rhFVIII inactivation by APC is protein S-dependent, because FV alone has no effect on the inactivation rate of rhFVIII by APC. Western blotting using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of human FV showed that FV was completely cleaved by APC at the beginning of the rhFVIII inactivation process. These data suggest that FV fragments derived from the B region of the procofactor after incubation of the membrane-bound procofactor with APC, but not intact single-chain FV, stimulate APC activity in the presence of protein S. rhFVIII, FV, and rhFVIIIa were not inactivated by Glu20-->Ala-substituted rAPC (rAPCgamma20A), and membrane-bound factor Va was only partially inactivated. Our data suggest that (1) FV and FVa are the physiologically significant substrates for APC inactivation and (2) membranes-bound APC-treated FV is a cofactor for the APC inactivation of rhFVIII only in the presence of the intact form of protein S.

Factor VIIIa↗
PubMed1983

Laboratory evaluation of factor VIII and factor IX.

It appears the state of the art of evaluating factors VIII and IX is in its infancy. With the exception of the screening tests, the more specific methodologies are expensive, time-consuming, and require a certain amount of technical proficiency for proper performance. However, our ability to evaluate these molecules has advanced greatly since the early 1970s. Most probably the methods described in this paper will be available in the near future to all laboratories wishing to perform them, either in kit form or in a more simplified version. Meanwhile, the APTT, mixing tests, qualitative factor assays, and bleeding times may be performed by nearly any laboratory. The performance of these tests will help identify those patients who may have potentially life-threatening postoperative bleeding episodes or who are having a bleeding episode that requires diagnosis for effective treatment. It is therefore important that these tests be incorporated into our routine coagulation protocols.

Antigens↗
PubMed1995

Characterization of des-(741-1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa.

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

Animals↗
PubMedDate not supplied

Lack of von Willebrand factor, factor VIII related antigen and factor VIII coagulant response to human growth hormone infusion in type 2 diabetes mellitus.

Excessive growth hormone (GH) secretion and platelet hyperaggregation have been considered to be involved in the development of the vascular complications of diabetes mellitus (DM). Trying to find a common link between GH and platelet hyperaggregation, we measured von Willebrand or ristocetin cofactor activity (VIII R:Rcof), factor VIII-related antigen (VIII R:Ag) and factor VIII coagulant activity (VIII:C) in ten type 2 DM (NIDDM) and seven normal control (C) human subjects. These three parameters were measured before (time 0), and after a one-hour intravenous infusion of 0.1 U/kg bw of GH, (times 1, 4, 6, and 24 hours). The NIDDM subjects were nonobese and without clinical evidence of diabetic vascular complications. Despite remarkably high levels of GH reached during the infusion (average 280 ng/ml), there were no significant changes in the measured parameters in either NIDDM or C group. The baseline levels of factors VIII R:Rcof, VIII R:Ag and VIII:C were also not significantly different in the two groups. Changes in GH serum levels seem to have no effect on the factors VIII:C, VIII R:Ag or VIII R:Rcof levels in normals (C) or in NIDDM subjects without evidence of vascular complications. These results do not preclude the possibility that there may be a different response to GH in DM patients with advanced vascular complications and probable endothelial cell abnormalities.

Adult↗
PubMed1990

Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine.

The effective activation of factor X by factor IXa requires the co-factor activity of activated factor VIII (FVIII). Factor Xa formation is also dependent on the presence of negatively charged phospholipid. A phospholipid binding domain of FVIII has been reported to be present on the FVIII light chain. Recent observations on a subset of human FVIII inhibitors have implicated the carboxyl-terminal C2 domain of FVIII as containing a possible phospholipid binding site. The purpose of this study was to investigate directly the role of the C2 domain in phospholipid binding. Twenty-six overlapping peptides, which span the entire C2 domain of FVIII, were synthesized. The ability of these peptides to inhibit the binding of purified human FVIII to immobilized phosphatidylserine was evaluated in an enzyme-linked immunosorbent assay. Three overlapping synthetic FVIII peptides, 2303-2317, 2305-2332, and 2308-2322, inhibited FVIII binding to phosphatidylserine by greater than 90% when tested at a concentration of 100 mumols/L. A fourth partially overlapping peptide, 2318-2332, inhibited FVIII binding by 65%. These results suggest that the area described by these peptides, residues 2303 to 2332, may play an important role in the mediation of FVIII binding to phospholipid.

Amino Acid Sequence↗
PubMed1992

Inactivation of factor VIII by factor IXa.

Factor VIII (FVIII) is the nonproteolytic cofactor for FIXa in the tenase complex of blood coagulation. FVIII is proteolytically activated by thrombin and FXa in vitro to form a heterotrimer with full procoagulant activity. Activated protein C inactivates thrombin-activated FVIII through cleavage adjacent to position Arg 336 in the cofactor. We have investigated the interaction of FIXa and FVIII and subjected FVIII polypeptides to N-terminal amino acid sequence analysis. Contrary to previous reports, we were unable to demonstrate the activation of FVIII by FIXa. Incubation of these two proteins at equimolar or close to equimolar concentrations resulted in the inactivation of FVIII, coincident with cleavage of the FVIII heavy chain adjacent to Arg 336 and the light chain adjacent to Arg 1719. These cleavages were detected in the presence or absence of thrombin, indicating that FIXa does not stabilize thrombin-activated FVIIIa. APC cleaved FVIII at the same position in the heavy chain, and simultaneous incubation of FVIII, APC, and FIXa did not result in stabilization of the cofactor. We conclude that FIXa does not play a role in the stabilization or activation of FVIII.

Amino Acid Sequence↗
PubMed1992

Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs.

The development of factor VIII:C inhibitors remains one of the most serious complications of repeated transfusion in patients with haemophilia A. The proportion of patients affected has been reported to range from 3.6% to 25%, but these figures have been derived mainly from retrospective data and from total numbers of known haemophiliacs instead of number at true risk. The assessment here is based on a prospective study, started in 1976, on the incidence of inhibitor development in haemophiliacs born after 1970 whose FVIII or FIX activity was 5% or less, and who had received replacement therapy at least once. 46 of 63 children with haemophilia A and 13 of 17 with haemophilia B fulfilled the enrollment criteria. Inhibitors developed only in haemophilia A patients who had previously been treated with FVIII products--inhibitor concentrations were high in 12 and low in 3. Inhibitors developed in 24% (15/63) of all haemophilia A patients, and in 52% (14/27) of those with severe disease. The incidence of inhibitor development for all haemophilia patients was 39.1 per 1000 patient-years of observation. All inhibitors were first detected when patients were aged 0.08-5.2 years. The cumulative risk was 33% at age 6 years. The findings indicate that previous reports have underestimated the risk of acquiring FVIII inhibitors. Prospective, standardised studies, especially in children, are needed for the assessment of the true risk of this complication.

Age Factors↗
PubMed1975

Effect of oral contraceptives on factor VIII clotting activity and factor VIII related antigen in normal women.

The factor VIII clotting activity (VIIIc1 and factor VIII related antigen (VIIIRAg) were determined repeatedly in 24 pairs of age-matched normal women, one of each pair being on oral contraceptives. No significant differences in either parameter or in the VIIIc/VIIIRAg ration were found between the two groups ,although the mean factor VIII clotting activity and VIIIc/VIIIRAg ratios for women on oral contraceptives were very slightly higher than for those not on oral contraceptives.

Adult↗
PubMed1984

Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid.

The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino-naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.

Amino Acid Chloromethyl Ketones↗
PubMed1984

Studies on the procurement of coagulation factor VIII: selective precipitation of factor VIII with hydrophilic polymers.

Early work on the purification of factor VIII using polyethylene glycol (PEG) indicated that other polymers might also be used to precipitate factor VIII leaving fibrinogen in solution. Recently polyvinylpyrrolidone (PVP) has also been advocated for this purpose. In this study, different concentrations and molecular weights of hydroxyethyl starch, dextran, PEG, PVP, Ficoll, Percoll and albumin were examined for their ability to precipitate the factor VIII complex from cooled (not frozen) fresh CPD plasma. At optimal concentrations near quantitative recovery of VIIIR:Ag and VIII:CAg was achieved in 2 hr with minimal precipitation of fibrinogen or total protein. The best separations were observed with hydroxyethyl starch, albumin or Ficoll. PVP and PEG gave inferior purifications. Results for cryoprecipitate and intermediate-purity factor VIII concentrate were inferior to those obtained with plasma. Simple pre-concentration of plasma prior to chilling is an attractive alternative for large scale continuous production.

Chemical Precipitation↗
PubMed1989

The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII.

The interaction between human Factor VIII and immobilized multimeric von Willebrand Factor (vWF) was characterized. Equilibrium binding studies indicated the presence of multiple classes of Factor VIII-binding sites on vWF. The high-affinity binding (Kd = 2.1 x 10(-10) M) was restricted to only 1-2% of the vWF subunits. Competition studies with monoclonal antibodies with known epitopes demonstrated that the Factor VIII sequence Lys1673-Arg1689 is involved in the high-affinity interaction with vWF.

Amino Acid Sequence↗
PubMed1976

An immunological investigation of factor VIII associated antigen in combined factor V and factor VIII deficiency.

The behavior of factor VIII associated antigen of three patients with combined factor V and factor VIII deficiency has been evaluated in several immunological systems. Factor VIII associated antigen resulted to be normal or higher than normal in all three patients in the radial immunodiffusion and in the electroimmunoassay systems. In the bidimensional electrophoresis system only one factor VIII precipitate was evident and such factor VIII precipitate showed the same electrophoretic mobility as normal factor VIII antigen. These findings firmly establish the fact that the factor VIII defect in congenital combined factor V and factor VIII deficiency is of the hemophilia type.

Antigens↗
PubMed1983

Dissociation of factor VIII procoagulant antigen VIII:CAg and factor VIII related antigen VIIIR:Ag by EDTA - influence of divalent cation on the binding of VIII:CAg and VIIIR:Ag.

Assuming 1 U/ml in citrated plasma, the VIII:CAg concentration was found 1.66 U/ml in EDTA-plasma, 1.09 U/ml in heparinized plasma and 0.67 U/ml in serum. Addition of 10 mmol/l EDTA to citrated and heparinized plasmas increased VIII:CAg 1.5fold. There was no increase of VIII:CAg in serum. Gel filtration of plasmas on different anticoagulants showed an elution of VIII:CAg in the void volume Vo and in the later fractions. The VIII:CAg amount detected in the internal volume increased following the series heparin less than citrate less than EDTA. Serum VIII:CAg was eluted at 2.2 Vo. Presence of EDTA in the elution buffer or incubation of plasma with EDTA prior to chromatography caused a displacement of practically all VIII:CAg amount in the internal volume with a peak at 2.2-2.3 Vo. VIIIR:Ag was exclusively detected in the void volume. Removal of divalent cation by chelation likely exposes more antigenic determinants of VIII:CAg, which are otherwise masked by steric hindrance due to VIIIR:Ag in citrated and heparinized milieu. Moreover gel filtration of plasma in the presence of EDTA completely dissociates VIII:CAg from VIIIR:Ag. The VIII:CAg fragment, having an estimated molecular weight of 70,000, might also be present in serum.

Anticoagulants↗
PubMed2000

A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor.

The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

Animals↗