Plasma from five patients with megaloblastic anemia (including three patients with Hashimoto thyroiditis as an additional complication) were examined for the presense of an abnormality in von Willebrand factor (vWF) using a 0.1% sodium dodecyl sulfate (SDS) -1.5% agarose gel electrophoresis. Also, the plasma samples were evaluated for FVIII/vWF activities. A mild decrease of FVIII/vWF activities and a low ability of ristocetin-induced platelet aggregation and/or adhesiveness to glass beads were found in two patients with Hashimoto thyroiditis (out of three patients). The loss of the larger multimers of plasma vWF was demonstrated in these two patients. But, after supplemental therapy (both levothyroxine sodium and hydroxocobalamin in one case, and hydroxocobalamin only in another case), their multimeric compositions were found to be normal. Otherwise, there was no abnormality in the plasma vWF of two patients with megaloblastic anemia who did not have Hashimoto thyroiditis as a compliation. These findings suggest that the mechanism with which the autoimmune antibodies and/or vitamin B₁₂ deficiency may be associated might have a relationship with the pathophysiology in development of a kind of acquired von Willebrand syndrome.

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The monkey is classified as close to the human and is important as an experimental animal. The present study, a comparison of human and monkey platelet membrane glycopreteins and aggregation, was undertaken.
The major platelet membrane glycoprotein was similarly represented in humans and monkeys by SDS-polyacrylamide gel electrophoresis and PAS staining. Crossed immunoelectrophoresis showed that EDTA dissociated both human and monkey platelet GP IIb/IIIa complex. In monkey platelets, ADP-induced aggregation was slightly weaked than in humans. High concentrations of ristocetin were required to agglutinate monkey platelets. Human and monkey platelets were aggretated by collagen and thrombin in the same manner. Epinephrine did not induce aggregation in monkey platelets. All of five monoclonal antibodies against human platelet GP IIb/IIIa complex bound to monkey platelets, and inhibited monkey platelet aggregation induced by ADP and collagen. A monocolnal anti-human platelet GP Ib antibody which has no effect on either human platelet aggregation induced by thrombin or agglutination induced by ristocetin bound to monkey platelets. But an anti-GP Ib antibody which inhibits human platelet agglutination induced by ristocetin did not bind to monkey platelets. Both of two monoclonal antibodies against human CD9 antigen bound to monkey platelets and aggregated monkey platelets.
These rusults indicate that human and monkey platelets are similar in terms of antigenicity and function, especially GP IIb/IIIa complex. It is suggested that the monkey is a useful animal model for analysis of the effects of monoclonal anitbody against human platelets in vivo.

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Anti-human GPIIb/IIIa mouse monoclonal antibodies, NNKY2-11 and NNKY2-18, reacted with platelets of dogs, cats and monkeys as well as humans. These antibodies inhibited the platelet aggregations of humans and animals in vitro. In this report, we injected NNKY2-11 and NNKY2-18 intravenously into dogs and examined the effects of the monoclonal antibodies on dog platelets in vivo. When 100μg/kg of NNKY2-11 (IgG_2a) were injected into dogs, platelet aggregation was inhibited partially and no change in circulating platelet counts occurred. When 200-300μg/kg of the antibody was infused, the number of circulating platelets decreased rapidly to the level of 5, 000/μl, followed by the recovery to the original level in a time-dependent fashion. Infusion of NNKY2-18 (IgG 1) also caused the partial inhibition of aggregation and the decrease of platelet counts similarly. When NNKY2-11-F (ab′) 2 was injected into dogs, platelet aggregation was inhibited similarly to intact IgG, and thrombocytopenia was observed. When NNKY2-11-Fab was injected, a slight inhibition of aggregation was obtained, but thrombocytopenia was not observed.

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The relationship between the degree of ristocetin-induced platelet agglutination and the size of von Willebrand factor (vWF) involved in it was evaluated. Four hundred microliters of formalin-fixed platelet suspension and 50μl of purified vWF were placed in a cuvette in the platelet aggregometer. Agglutination curves were recorded by adding 50μl of various concentration of ristocetin. vWF molecules in each supernatant were examined by SDS-polyacrylamide-agarose gel electrophoresis (SDS-PAGE) and electron microscopy. By SDS-PAGE, no differences were seen in size distribution of vWF among each supernatant. By electron microscopy, although vWF in the supernatants after agglutination were somewhat folded, the numbers of nodules in vWF multimers in each supernatant after various degrees of agglutination were not different from that of vWF multimers with no agglutination. We conclude that there was no difference in the size distribution of vWF involved in the ristocetin-induced platelet agglutination.

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Binding ability of plasma von Willebrand factor (vWF) to purified factor VIII (F. VIII) in normal subjects and patients with von Willebrand disease (vWD Type IIA, IIB, IIC) was analysed in a solid phase assay. A vWF fraction, mainly containing large multimer-vWF, was obtained from gel filtration of cryoprecipitate, whereas another vWF fraction, containing small multimer-vWF, as cryo-supernatant. Binding ability of vWF in small and large multimer-vWF fractions was identical. Binding ability of vWF in the plasmas from vWD Type IIA, IIB and IIC was found to be within normal range. These results suggested that the vWF binding to F. VIII was independent from multimeric structure.

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The roles of platelet membrane glycoprotein (GP) Ib in human platelet aggregation induced by non-hemolytic influenza virus (IV) were studied. The anti-GPIIIa monoclonal antibody (TM83) which had been shown to inhibit fibrinogen-binding to platelets activated by ADP and thrombin also inhibited the aggregation induced by high and low hemagglutinin (HA) titers of IV, suggesting that the aggregation is also mediated by GPIIb/IIIa complex. The anti-GPIb monoclonal antibody (TM60) inhibited only the aggregation induced by low titers (HA: 5, 120 and 7, 680) of IV, but it could not inhibit the aggregation by high titers (HA: 10, 240) of IV. Removal of N-terminal 45 kDa fragment from GPIb by treatment with elastase resulted in the complete loss of ristocetin-induced aggregation but did not cause any decrease in IV-induced aggregation. The aggregation of chymotrypsin-treated platelets induced by IV or ristocetin gradually dereased with increasing of chymotrypsin. However the platelets pretreated with 10U/ml of chymotrypsin showed complete loss of ristocetin-induced aggregation but still had IV-induced aggregation, indicating that not only GPIb but also other components exhibited binding sites for IV. Treatment of the platelets with sialidase dose-dependently inhibited both high and low titers of IV-induced aggregation but had no effect on ristocetin-induced aggregation. IV-induced aggregation of sialidase-pretreated platelets was further decreased by TM60, while that of chymotrypsin-pretreated platelets was not inhibited.
These results indicated that IV binds to not only sialic acid residue of GPIb with the highest affinity but also other sialic acid of the surface membrane with low affinity.

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Two cases of congenital protein C deficiency combined with a congenital abnormal plasminogen with deep venous thrombosis were reported. First case (AA) was a 41 year-old male who had a suprasaggital venous thrombosis and left femoral venous thrombosis. Hemostatic tests of the family revealed that his elder daughter and a son had a molecular abnormality of plasminogen, and his younger daughter had the same hemostatic abnormalities (protein C deficiency and abnormal plasminogen). None of them showed thrombotic symptom. Second case (YO) was a 33 year-old male who had recurrent right femoral venous thrombosis. His father had an abnormal plasmionogen but normal level of protein C activity and antigen. The mode of inheritance was not clear because of unavailability of tests of their relatives. Although two proposita were found to be heterozygotes with abnormal plasminogen (normal Ala-601 [GCT] to abnormal Thr-601 [ACT]) by the analysis of DNA sequencing, molecular analysis of protein C deficiency remained to be cleared.

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