Coagulant cancer antigen 1 (CCA-1) is a protease which activates factor X. CCA-1 demonstrates cancer-specific expression on immunohistological staining of breast cancer cells with anti-CCA-1 monoclonal antibody, a specific inhibitor of the coagulant activity of CCA-1. In the present study, the intensity of cell surface expression of tissue factor (TF), one of the procoagulant activators arising from cancer, and CCA-1 was determined in human breast cancer-derived cells by flow cytometry using anti-TF antibody and anti-CCA-1 antibody, respectively, as the primary antibodies. The influence of cell-induced coagulant activity on the coagulation time of human normal plasma was analyzed further using the above-mentioned antibodies. As a result, CCA-1 expression was observed in 4 out of 6 cells at the high rate of 65-95%. The degree of anti-CCA-1 antibody-induced coagulant inhibition was observed to be proportional to the intensity of CCA-1 expression. The intensity of TF expression was low in all of the cells, being under the level of 13%. The cells with proven TF expression showed concentration-dependent coagulant inhibition with the addition of anti-TF antibody. Conventionally, TF has been regarded as a major factor underlying blood coagulation on the cell surface. The present study revealed that compared to TF, the intensity of expression and the degree of distribution of CCA-1 were greater on the surface of breast cancer cells. These findings suggested that CCA-1 also plays an important role in blood coagulation induced by breast cancer cells.

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We previously reported a new analytical immunoblotting method to detect reduced plasma von Willebrand factor (vWF) fragments using polyclonal anti-reduced vWF antibody and demonstrated that the reduced normal plasma vWF consists of at least five or six fragments, 240kDa for the main fragment and 216 (220, 210), 194, 164 and 144kDa for minor fragments. In this study, we describe the reduced fragments in patients with various types of von Willebrand disease (vWD) (type I: 7 patients, type IIA: 4 patients and type IIB: 3 patients) using this method. Of seven patients with type I vWD, one patient with an equally low level of ristocetin cofactor activity (vWF: RCo) and vWF antigen (vWF: Ag) showed normal relative proportions of each fragment. However, three of four patients with a low level of vWF: RCo compared to vWF: Ag showed a relatively increased proportion of the 164kDa fragment. The remaining two patients demonstrated a new 200kDa reduced fragment in addition to those of normal plasma vWF. In type II A vWD, all patients showed a relatively increased proportion of the 164kDa fragment. In type IIB vWD, two of three patients also showed a relatively increased proportion of the 164kDa fragment. These results suggest that plasma vWF with a low vWF: RCo compared with the vWF: Ag ratio tends to show an increased proportion of the 164kDa reduced fragment, even in type I vWD with a normal multimeric structure. Fragment analysis of reduced vWF in patients with vWD is useful to understand the abnormality at the molecular level, especially in type I vWD.

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Five patients with chronic adult idiopathic thrombocytopenic purpura (ITP) resistant to standard therapy were treated with pulsed high-dose dexamethasone therapy reported by Andersen (six cycles of dexamethasone, 40mg/day for 4 consecutive days every 28 days). Five female patients were enrolled, median age was 35 years and median platelet count was 18×10³/μl at the onset of this treatment. One patient achieved a excellent response with this treatment, but the other four patients were resistant. No serious adverse effects were observed. We conclude that pulsed high-dose dexamethasone therapy had a relatively limited effect in resistant, chronic adult ITP.

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Human antithrombin III (AT-III) is the major inhibitor of blood coagulation and hereditary deficiency of this glycoprotein is a well known risk factor for familial venous thromboembolic disease. In order to identify the genetic defect in Japanese patient with type Ia AT-III deficiency, we have analyzed seven exons of the AT-III gene using polymerase chain reaction (PCR) and direct sequencing techniques. Comparing the nucleotide sequence found in the patient with the nucleotide sequence of the AT-III gene which was reported by Olds et al (Biochemistry 1993), three differences in the sequence, at position 67 (5′ untranslated region), 2745 (exon2), and 7626 (exon4), were detected. The substitution from G to A was observed at position 7626 in exon 4. This nucleotide substitution is silent and has been previously reported as a polymorphism. The frequency of this polymorphism in the analysis of 100 alleles from 50 Japanese normal individuals was 0.46(G)/0.54(A). Neither of the substitutions, G to A at position 67 in the 5′ untranslated region or C to T at position 2745 in exon 2, was found in the 100 normal alleles. Although the C to T substitution at position 2745 was detected in all 5 patients who were diagnosed as AT-III deficiency by the coagulation test, the G to A substitution at position 67 was detected in only 3 out of 5 patients. These findings suggest that the C to T transition, substituting alanine 94 for valine, may cause AT-III deficiency in this family. Furthermore, the G to A transition at position 67 is probably a low frequency polymorphism.

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