Hiromu Fukui

Hemostatic effect of a heat-treated factor VIII concentrate (Haemate P) in von Willebrand's disease

SummaryA heat-treated factor VIII (F VIII) concentrate (Haemate P) has been administered to patients with various types of von Willebrands disease (vWD). The 4 activities of F VIII/vWF as well as change in the multimeric structure of vWF were then studied. In 4 patients with type I vWF who were given a Ristocetin cofactor (Rcof) dose of 42–78 U/kg, there was a clear reduction of the bleeding time and an increase of F VIII:C, F VIII:Ag, Ag, Rcof and vWF: Ag for several hours. The recovery of Rcof. after 1 h was 50–75%. Although the multimeric composition of vWF in these patients was similar to that of normal plasma, the density of each multimer band was very low. After infusion, however, the density of all multimer bands increased for several hours, to decrease again after 24 h. In 4 patients with type II A vWD who received a dose of Rcof of 55–76 U/kg, the 4 activities of F VIII/vWF increased similarly as was the case in type I. All patients had only 3–4 smaller multimer bands. New larger and intermediate multimers appeared for several hours after infusion of the preparation. Two patients with type III vWD who received doses of Rcof of 52 and 65 U/kg showed also a similar increase in the 4 activities of F VIII/vWF after infusion. All the multimers lacking in these patients appeared for several hours after infusion.

Laboratory evidence of DIC under FEIBA treatment of a hemophilic patient with intracranial bleeding and high titre factor VIII inhibitor

Abstract A 17-year-old hemophilia A patient with an intracranial bleeding and a high titre of factor VIII inhibitor was treated with repeated infusion of an activated prothrombin complex concentrate (FEIBA). A good effect was achieved clinically, but laboratory tests on 8th day of the treatment revealed no shortening effect on non-activated partial thromboplastin time and on r-value of thrombelastograph. A prolongation of prothrombin time and thrombin time, a decrease in fibrinogen and antithrombin III, and an increase in serum FDP were also observed. These findings returned to normal after FEIBA was stopped. It was suggested that repeated and massive infusion of FEIBA might cause a decrease in natural inhibitors which lead to DIC.

A monoclonal antibody (NMC-VIII/10) to factor VIII light chain recognizing Glu1675–Glu1684 inhibits factor VIII binding to endogenous von Willebrand factor in human umbilical vein endothelial cells

The monoclonal antibody NMC‐VIII/10 is a neutralizing antibody which recognizes the Glu1675–Glu1684 sequence of the factor VIII light chain and inhibits factor VIII (FVIII) binding to immobilized von Willebrand factor (vWf). In this study we immunohistochemically determined, using human umbilical cord tissue, whether or not NMC‐VIII/10 has an inhibitory effect on FVIII binding to endogenous vWF in endothelial cells. Tissue sections were reacted with purified FVIII followed by peroxidase‐conjugated monoclonal antibody (C5) recognizing the 54 kD fragment of the FVIII heavy chain. The labelling pattern of bound FVIII was similar to that of endogenous vWF and appeared as a fine granular deposit in the endothelial cells. Addition of purified vWF completely inhibited the binding of FVIII to endothelial cells. Furthermore, FVIII did not bind to endothelium in the presence of 0·25 M CaCl2, and similarly, thrombin‐treated FVIII did not bind to the vascular site. These findings suggested that FVIII was bound to endogenous vWF in the endothelial cells. The binding reaction was completely inhibited by NMC‐VIII/10, confirming that the monoclonal antibody recognizes the specific epitope responsible for FVIII binding to endogenous vWF.

In vitro characterization of various heat-treated prothrombin complex concentrates(PCC)

All of 6 heat-treated prothrombin complex concentrates (PCC) tested contained adequate levels of factor IX but factor VII content was low. Levels of factors II, X, protein C and protein S were variable and antigen levels were always greater than those of functional activities. On crossed-immunoelectrophoresis factor IX showed variable anodal shift in all concentrates tested and in some activated factor IX was demonstrated by immunoblotting technique. These findings suggested some activation and/or denaturation during production and/or heating. Modest amount of factor VIII clotting activity by solid-phase amidolytic method and of factor VIII antigen was demonstrated in some concentrates but none contained more than a trace factor VIII inhibitor bypassing activity. The results suggested that heat-treated PCC should provide safe therapeutic products for hemophilia B.

Antiplatelet Glycoprotein lb Monoclonal Antibody (OP-Fl) Totally Abolishes Ristocetin-Induced von Willebrand Factor Binding, but Has Minimal Effect on the Botrocetin-Induced Binding

We describe here a new antiplatelet glycoprotein (GP) Ib monoclonal antibody (MoAb) designated OP-F1 (IgG1 kappa). Both OP-F1 and a well-characterized anti-GPIb MoAb, AP-1, totally abolished ristocetin-induced von Willebrand factor (vWF) binding to platelets and desialylated vWF binding to platelets at an IgG concentration of 2-8 micrograms/ml. AP-1 also blocked snake venom botrocetin-induced vWF binding at a similar IgG concentration, whereas OP-F1 had a minimal effect on botrocetin-induced binding. At a higher IgG concentration (150 micrograms/ml), OP-F1 inhibited botrocetin-induced binding by 50%. AP-1 (IgG) did not interfere with binding of [125I]OP-F1 (IgG) to platelets. Thus, the epitope involved in the binding of OP-F1 or AP-1 appears to be quite different. These results suggest that the vWF binding site(s) on the GPIb molecule generated by these inducers is in close proximity but not completely identical.

Urinary fibrin-fibrinogen degradation products and intraglomerular fibrin-fibrinogen deposition in various renal diseases

Abstract The typing of the urinary fibrin-fibrinogen degradation products (FDP) and the deposition of fibrin-fibrinogen, factor XIII-subunit A (XIII-A) and factor XIII-subunit S (XIII-S) in the glomeruli were investigated in the children with several kinds of renal diseases. In acute glomerulonephritis with diffuse proliferative lesion, the urinary FDP were composed of fragments X,Y,D and E. There was no D-dimer fragment detected in the urine, although, mild deposition of fibrin-fibrinogen, XIII-A and XIII-S in the glomeruli was observed. The urinary FDP may be derived from plasma fibrinogen or serum FDP. In nephrotic syndrome with diffuse proliferative or focal global sclerosing lesion and in purpura nephritis with mesangial proliferation with crescents, the urinary FDP consisted of fragments X and Y, and lacked in D-dimer. In these cases, the major part of fibrin-fibrinogen in the glomeruli might be fibrinogen or non-cross-linked fibrin, and FDP excretion might arise from glomerular permeability or from lysis of fibrinogen or non-cross-linked fibrin. In one patient with hemolytic uremic syndrome and in another patient with systemic lupus erythematodes nephropathy, intense deposition of fibrin-fibrinogen, XIII-A and XIII-S within the glomerular capillaries or mesangial area were observed. In the urine of those cases, D-dimer was detected, which seems to reflect the lysis of cross-linked fibrin in the glomeruli.

A deletion involving intron 13 and exon 14 of factor VIII gene in a haemophiliac with anti-factor VIII antibody

SummaryA deletion mutation in the factor VIII gene of a severe haemophiliac patient was found along with a high level of factor VIII inhibitor in the blood plasma among seventy Japanese haemophilia A patients. The 6 kbp long deletion involved a region from somewhere between PstI and SstI sites at nucleotide positions 2659 and 2991 of exon 14 and intron 13, respectively (nucleotide positions were defined as in Wood et al., 1984).

NONSENSE MUTATION IN FACTOR VIii GENE OF A SEVERE HAEMOPHILIAC PATIENT WITH ANTI-FACTOR VIII ANTIBODY

SummaryA nonsense mutation was found in exon 23 of the factor VIII gene of a haemophiliac patient with anti-factor VIII antibody. Genomic DNA of lymphocyte cells from the patient analyzed by Southern blot analysis with various segments of factor VIII cDNA revealed that the TaqI site in exon 23 was erased in the patient gene. The 0.3 kbp nucleotide sequence of the exon 23 was cloned and sequenced, and the substitution of nonsense (TGA) codon for the arginine (CGA) codon was found to be the possible cause of the factor VIII deficiency.

RFLPs of factor IX gene in Japanese haemophilia B families and gene deletion in two high-responder-inhibitor patients

SummaryThe factor IX genes in four Japanese families with haemophilia B were analysed for the restriction fragment length polymorphisms (RFLPs) of TaqI, XmnI and DdeI, using subcloned intragenic DNA fragments as probes (probes VIII and XIII). The factor IX genes in 12 patients with haemophilia B and three high-responder-inhibitor cases showed no size difference using a cDNA probe (cVII) when restricted by TaqI, EcoRI and HindIII. Complete gene deletions were observed in two other high-responder-inhibitor cases.

Localization of intrarenal cross-linked fibrin in children with various renal diseases.

The localization of intrarenal cross-linked fibrin was examined by the effect of monochloroacetic acid treatment on the kidney sections. In acute glomerulonephritis or in mild diffuse or focal proliferative type of nephritis, cross-linked fibrin was observed mainly within glomerular capillary walls. Extension of cross-linked fibrin deposit over the mesangium or sclerotic area was seen in moderate to severe proliferative type of nephritis or in membranoproliferative glomerulonephritis. In hemolytic uremic syndrome or disseminated intravascular coagulation syndrome, cross-linked fibrin was detected within glomeruli and vessels.