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.     

Disappearance of factor VIII antibodies upon frequent administration of factor VIII.

20 haemophilia patients known to have antibodies against F VIII for at least more than three years were treated on a regular base with 25 U/kg b.w. F VIII every other day. All 5 patients with previous maximal anti F VIII antibody levels between 5 and 60 BU/ml showed a decrease of antibody level and normal F VIII recovery within 1-2 months. From 12 patients with previous antibody levels above 60 BU/ml, 8 showed a disappearance of antibodies within 2-26 months. In 3 patients in whom no previous highest inhibitor level was known, one was treated successfully. Another group of 6 young patients in whom an inhibitor against F VIII had just (less than 3 months) developed, was treated with F VIII as soon as an inhibitor was detected. The dose infused was 25 U/kg b.w. F VIII twice weekly. In 5 patients this regimen was successful within 1-7 months. In the 6th patient the dosage was increased to every other day. One year after the beginning of therapy no inhibitor was detectable. So our results show that regular administration of F VIII in intermediate or low dose can lead to rapid disappearance of anti F VIII antibodies especially in patients with moderate inhibitor levels.     

Quantitative determination of factor VIII antigen with an enzyme immunoassay

We describe an enzyme-immunoassay for the determination of factor VIII antigen. After representation of the isolation of proteins the enzyme-immunoassay is presented. The principle of the method is the following: Test plasma is mixed with rabbit antibody in excess and incubated at 37 degrees C. The incubation mixture is added to polystyrene tubes, which are coated with human factor VIII. The rabbit antibody is available to adhere to factor VIII coating the tube and can be detected with an enzyme-labeled antibody to rabbit IgG. This method is sensitive to 7.8 . 10(-3) U/ml factor VIII antigen; the variation coefficient is 10.9%.     

Role of factor VIII C2 domain in factor VIII binding to factor Xa.

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.     

Effect of factor VIII concentrate on malondialdehyde release by normal human platelets]

It is well known that Factor VIII concentrates affect platelet function both "in vitro" and "in vivo" but the mechanism of their action is poorly understood. Therefore the aim of the present work was to investigate a possible effect of these concentrates on prostaglandin synthesis by platelets, measured as the amount of released malondialdehyde (MDA), which is an index of Thromboxane A2 production. Our data suggest that Factor VIII concentrates have no effect on MDA release by platelets and on its inhibition by aspirin and heparin. In conclusion the effect of Factor VIII concentrates on platelets is not mediated by an increased synthesis of prostaglandins.     

Production of progestin-stimulatory factor(s) by enriched populations of rat T and B lymphocytes

We have previously demonstrated that a progestin-stimulatory factor(s) (PSF) is present in the supernate of concanavalin A-activated rat splenocytes. In the absence of FSH, PSF evokes dose-dependent increases in both progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) production by undifferentiated rat granulosa cells while basal estrogen production is unaffected. Because splenocytes are composed predominantly of T and B lymphocytes, we sought to determine if PSF production is restricted to one of these specific cell types. To accomplish this, dispersed splenocytes were prepared from the spleens of adult female rats. Macrophages were removed by standard adherence procedures, and highly enriched populations of T and B lymphocytes were obtained by negative selection panning utilizing monoclonal antibodies to T and B lymphocytes. Purity of the T- and B-cell populations (typically greater than 85-95%) was assessed by single-color flow cytometry. Enriched populations of T cells, B cells, or unseparated lymphocytes were then seeded into tissue culture flasks and stimulated with 2.5 micrograms/ml concanavalin A. Following a 48-h incubation at 37 degrees C, cytokine-rich fractions were prepared from the conditioned medium of these cultures by ammonium sulfate fractionation. The presence of PSF was assessed by the ability of the resulting fractions to specifically stimulate progestin production by undifferentiated rat granulosa cells in vitro. Granulosa cells were treated with increasing doses (ranging from 0-30% v/v) of supernatants from cultures of T cells, B cells, or unseparated splenocytes for 48 h. Media were removed and progesterone, 20 alpha-OH-P, and estrogen levels were quantified by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paradoxical bleeding as a complication of the treatment of hemophilia with factor VIII and factor IX preparations]

Paradoxical bleedings are complications occurring under replacement therapy in haemophiliacs by disturbancies of the primary haemostasis. They have been observed during treatment with factor-VIII- and prothrombin-complex concentrates of long duration and in high dosage. Clinical complications, for example delayed wound healing as well as spontaneous bleedings into the skin and from the mucous membranes, have been observed in one quarter of haemophiliacs under substitution therapy. In one third of these patients pathological parameters of primary haemostasis (prolonged bleeding time, reduced retention, retraction, ADP- and collagen-induced aggregation and the platelet factor 3 release) were found out. The following mechanisms or substances may be the cause for these disturbancies: 1. fibrinogen and factor-VIII split products 2. high content of proteins predominantly fibrinogen and factor-VIII-related antigen 3. antigen-antibody reactions 4. development of inhibitors against the Willebrand factor. For treatment of the paradoxical bleedings freshly prepared cryoprecipitate, prednison and Etamsylatum have been used.     

A case of Osler-Rendu disease with simultaneous thrombopenia and a factor VIII inhibitor]

Long-term determinations of haemostasis factors in a case of Osler's telangiectasia revealed the temporarily simultaneous existance of periods of thrombocytopenia, a decrease of prothrombine and a reduction of the fibrinogen and plasminogen level. These findings considered as signs of consumption coagulopathy coincided with an increased bleeding tendency of the patient. The correlation existing between the clinical symptoms and the observed disorders of coagulation may possibly be explained by the appearance of an intravascular coagulation during the late period of the haemorrhagic diathesis, which could be proved by the simultaneous increase of fibrinogen degradation products. Moreover, the patient's plasma was capable of strongly inhibiting factor VIII of a normal plasma. The possible influence of plasmatic disorders of coagulation which are caused by secondary reasons on the clinical picture of a haemorrhagic diathesis primarily based on vascular conditions is discussed.     

Characterization of recombinant human factor VIII

Recently, complete human factor VIII DNA clones have been obtained and subsequently expressed in baby hamster kidney cells (Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., and Lawn, R. M. (1984) Nature 312, 330-337). The recombinant factor VIII (rVIII) protein secreted from these cells has now been purified allowing its structural analysis and comparison to plasma-derived factor VIII (pdVIII). Analysis of purified rVIII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with relative mobilities (Mr) ranging from 80,000-210,000. The same pattern of polypeptides is also observed for pdVIII resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins associated with rVIII are recognized by pdVIII antibodies in a Western blot. When rVIII and pdVIII are subjected to isoelectric focusing they are resolved into a similar pattern of protein bands. Thrombin, factor Xa, and activated protein C, which modulate factor VIII activity by proteolysis, process rVIII in the same manner they do pdVIII. As is the case for pdVIII, thrombin activation of rVIII coagulant activity correlates with the generation of subunits with Mr of 73,000, 50,000 and 43,000. These subunits appear to form a metal-(perhaps Ca2+) linked complex. EDTA inactivates thrombin-activated rVIII and pdVIII, with the activity being regenerated after the addition of a molar excess of MnCl2. The results suggest that rVIII is structurally and functionally very similar to pdVIII.     

Production of an insulin-like growth factor by osteosarcoma

To test the possibility that osteosarcoma cells produce their own growth factors, we measured levels of insulin and somatomedin C (SMC), an insulin-like growth factor, in culture media of two cell lines derived from patients with that disease. SMC but not insulin levels increased three- to ten-fold over a period of 7 days paralleling the increases in cell number. Production of SMC was inhibited by cycloheximide.     

Plasma exchange and human factor VIII concentrate in managing haemophilia A with factor VIII inhibitors.

Plasma exchanges were combined with human factor VIII concentrate therapy in the treatment of major bleeding episodes in five patients with haemophilia A and factor VIII inhibitors. All patients had a good clinical response to combined treatment. Inhibitor levels showed satisfactory falls before rapid secondary increases of inhibitor levels took place. A sixth patient with von Willebrand''s disease and a factor VIII clotting activity inhibitor was successfully prepared for operation using plasma exchange. Postoperative haemostasis and healing were normal. In two patients the plasma exchanges were relatively more effective than the administered human factor VIII in reducing the levels of factor VIII inhibitor. Combined plasma exchange and human factor VIII treatment may offer a rapidly effective means of reducing factor VIII inhibitor levels in this group of patients, together with significant saving of costs.     

Platelet adhesiveness and aggregation in combined factor V and factor VIII deficiency and in combined factor VII and factor VIII deficiency.

Platelet aggregation and adhesiveness were studied in 3 patients with combined factor V and factor VIII deficiency and in 3 patients with combined factor VII and factor VIII deficiency. The first three patients belonged to three different kindreds whereas the second group belonged to the same kindred. Serotonin C14 uptake and release was also found to be normal in these patients. These studies indicate that platelet function is normal in combined defects of factor VIII. These findings were in agreement with the presence of a normal bleeding time and a normal factor VIII antigen level in all these patients.     

Human factor VIII: purification from commercial factor VIII concentrate, characterization, identification and radiolabeling

Human factor VIII was purified from commercial factor VIII concentrate with a 12% yield. The specific coagulant activity of purified factor VIII was 8,000 units/mg. In the presence of SDS the purified factor VIII consisted of a variety of polypeptides on polyacrylamide gels, ranging between Mr 80,000 and Mr 208,000. In the absence of SDS the purified factor VIII showed an apparent molecular weight of 270,000 upon Sephadex G200 gel-filtration. The purified factor VIII could be activated by thrombin, which resulted in the disappearance of Mr 108,000-208,000 polypeptides in favor of an Mr 92,000 polypeptide. Treatment with factor Xa also activated factor VIII, whereas treatment with activated protein C resulted in the inactivation of coagulant activity. Coagulant-active 125I-factor VIII was prepared using a lactoperoxidase radioiodination procedure. This 125I-factor had the same characteristics as unlabeled factor VIII. All polypeptides could be precipitated with monoclonal antibodies directed against factor VIII. With 125I-factor VIII a pIapp of 5.7 was found in the presence of urea.     

pH-dependent denaturation of thrombin-activated porcine factor VIII

Thrombin-activated porcine factor VIII (fVIIIaIIa) is a stable, active, 160-kDa heterotrimer at concentrations exceeding 2 x 10(-7) M in 0.7 M NaCl, 0.01 M histidine Cl, 5 mM CaCl2, pH 6.0, at 4 degrees C or 20 degrees C. Two of the subunits, fVIIIA1 and fVIIIA2, are derived from the heavy chain of the plasma-derived, heterodimeric fVIII precursor. The third subunit, fVIIIA3-C1-C2, is derived from the fVIII light chain. We now find that fVIIIaIIa undergoes a sharp decline in coagulant activity between pH 7 and 8. At pH 7.5, the activity of fVIIIaIIa at 3 x 10(-7) M decays within a few hours to a stable level that is approximately 70% of the value at pH 6.0, whereas at pH 8.0, greater than 99% of the activity is lost. The activity cannot be restored by readjusting the pH to 6.0. The loss of activity at pH 8.0 coincides with dissociation of the fVIIIA2 subunit since an inactive fVIIIA1/A3-C1-C2 heterodimer can be isolated by Mono S high performance liquid chromatography. After prolonged incubation at pH 8.0, the fVIIIA1 subunit also dissociates. The free fVIIIA2 fragment appears to be poorly soluble which may explain the irreversible loss of activity. Analytical velocity sedimentation of the pH-inactivated fVIIIaIIa preparation also is consistent with dissociation and precipitation of the fVIIIA2 fragment. We propose that denaturation of fVIIIaIIa by pH-dependent subunit dissociation may provide a major mechanism of inactivation of fVIIIaIIa under physiologic conditions.     

Subunit structure of thrombin-activated porcine factor VIII

Factor VIII (fVIII) is synthesized as a single chain having a domainal sequence A1-A2-B-A3-C1-C2. Analysis of the proteolyic cleavage of fVIII by thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies three fragments designated fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 with fragment(s) derived from the B domain being difficult to visualize. The appearance of these fragments is associated with the development of coagulant activity, but the activity is labile without further apparent proteolysis. In this study, porcine fVIII was reacted with thrombin until peak coagulant activity was obtained and then subjected to cation-exchange (Mono S) high-pressure liquid chromatography. Coagulant activity was recovered in a single peak that contained all three fragments and was stable for weeks at 20 degrees C in 0.65 M NaCl/0.01 M His-HCl/0.005 M CaCl2 at pH 6.0. Analytical ultracentrifugation of activated fVIII was done to test whether all three fragments were associated. The apparent molecular weight of activated fVIII from equilibrium sedimentation increased from 148,000 to 161,000 as the loading concentration was increased from 0.06 to 0.16 mg/mL. This agrees well with the summed apparent molecular weights of fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 calculated from SDS-PAGE analysis (148,000) or from the amino acid sequence of human fVIII (159,000). This establishes the major species in the preparation as a fVIIIA1/A2/A3-C1-C2 heterotrimer and additionally indicates either weak self-association of the trimer and/or incomplete association of the individual subunits to form the trimer. Velocity sedimentation of activated fViii revealed a single boundry (S020,w = 7.2 S).(ABSTRACT TRUNCATED AT 250 WORDS)