We describe here a new enzyme-linked Immunosorbent assay (ELISA) for the measurement of platelet glycoprotein (GP) IIb/IIIa antigen. In this assay, rabbit antihuman platelet polyclonal antibody was used for the first antibody to capture “washed whole platelets” on the polystylene immunoplate. After capturing the platelets, peroxidase-labeled anti-human platelet GPIIb/IIIa monoclonal antibody (IgG), designated as 3F11, was used for the second antibody. Using this method, platelets from 30 normal volunteers were analyzed. The mean±2SD value of GPIIb/IIIa antigen was 105.2±30.1% and the lower detection limit was 0.01%. Since ELISA does not require any specific equipments, this assay can be used in usual laboratories for the accurate determination of platelet GPIIb/IIIa antigen.

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In the first paper of this issue, we have described the establishment of whole platelet-captured enzyme-linked immunosorbent assay (WPC-ELISA) for the determination of platelet glycoprotein (GP) IIb/IIIa antigen. Using this method, we now analyzed the amount of GP IIb/IIIa antigen in 10 patients with Glanzmann thrombasthenia (GTA) belonging to 9 different families and their parents. In 6 patients with type I GTA who show a total lack of clot retraction, the GPIIb/IIIa antigen level ranged from 0.01% to 0.6%, and their parents (n=8) 9.3-64.9%. In 3 out of 4 patients with type II GTA who showed almost normal clot retraction indicated the level ranging from 3.6% to 5.0%, and their parents (n=4) 11.1%-35.3%. Another patient with type II GTA showed a level of 150% GPIIb/IIIa antigen, indicating that this case is a variant form of GTA.

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It is recently known that interleukin-6 (IL-6) has a potent thrombopoietic activity, resulting of thrombocytosis by in vivo administration in experimental animals. However biochemical properties of platelets and megakaryocytes induced by IL-6 are little known. To study function of platelets or megakaryocytes induced by IL-6, adenine nucleotides and serotonin content were measured in the murine platelets and megakaryocytes after an administration of IL-6. Platelets and megakaryocytes were recovered in 3 and 5 days of injection. Megakaryocytes were isolated by immunobeads with monoclonal antibody against mouse platelet (Pm1), prepared by ourselves. 0.5μg or rhIL-6 were intramuscularly given in BDF1 mice for 5 days and their platelet counts were elevated to 150×10⁴ (120% of control) at 5th day of injection and returned to control level thereafter. 5-HT and ADP content in platelets and megakaryocytes were promptly increased to maximum at day 3 when platelet counts began increasing, whereas they were declined at day 5 when platelet counts reached maximum. In conclusion, the functions of platelets and megakaryocytes induced by IL-6 enhance higher, which may give a benefit for clinical use.

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Neomycin, an aminoglycoside antibiotic, binds more strongly to inositol polyphospholipid than to other membrane phospholipids. Therefore, this antibiotic is commonly used as a relatively specific inhibitor of inositol phospholipid metabolism in studies regarding the involvement of inositol phospholipids in various cell functions. The mechanism of action of neomycin is presumed to be inhibition of phospholipase C. However, the mode of inhibition remains obscure. Other aminoglycosides, including gentamicin and streptomycin, may also possess phospholipid binding affinity and platelet activity, but their effects are less potent than those of neomycin. The present study investigated both the direct action of aminoglycoside antibiotics and the activation of platelets rendered semi-permeable with saponin, which allows polar aminoglycoside antibiotics access to the innerleaf let of the plasma membrane, where the phosphoinositides are located. With semi-permeabilized platelets, relatively low concentrations of aminoglycoside antibiotics stimulated secretion, aggregation, arachidonic acid release and generation of inositol trisphosphate. All of the above-mentioned effects on semi-permeabilized platelets were fully inhibited by high concentrations of the aminoglycoside antibiotics. By acetylsalicylic acid pretreatment, aggregation, secretion and generation of inositol trisphosphate were fully inhibited. In contrast, arachidonic acid release was only slightly inhibited by this treatment. These data therefore suggest that the mechanism of action of aminoglycosids antibiotics on semipermeabilized platelets is not related to phospholipase C, but to phospholipase A₂. These results provide evidence that aminoglycoside antibiotics activate phospholipase A₂, which leads to the release of arachidonic acid in semi-permeabilized platelets. But whether themechanism of activation of phospolipase A2 by aminoglycoside antibiotics is direct or not is yet unclear.

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Appearance of the activated platelets and their behaviors during cardiopulmonary bypass (CPB) surgery were studied in 21 adult patients undergoing coronary artery bypass grafting or valve replacement. Sampled blood was immediately fixed with 1% paraformaldehyde. The isolated and washed platelets were incubated with 5μg/ml of 2 T 60 (anti-GMP-140 monoclonal antibody) and then with FITC-conjugated anti-mouse IgG for flow-cytometric analysis.
Mean fluorescence intensity of the 2 T 60-stained platelets was 19.5±5.9 at pre-CPB and increased significantly (p<0.01) during CPB and reached the maximal level (26.2±9.2) at 120min of CPB. After the end of CPB, it decreased and on the first postoperative day it returned to the pre-CPB value. Immunofluorescence histograms of 2 T 60-stained platelets showed a main population composed of the inactivated GMP-140-negative platelets (channel number less than 20) with a small population of the activated GMP-140-positive platelets on the right slope shoulder of the main (channel number greater than 20) before CPB. After the beginning of CPB, the activated platelets rapidly increased and formed another distinguishable population of activated platelets. In 8 of 21 patients, the peak height of the population of the activated GMP-140-positive platelets was higher than or equal to that of the inactivated GMP-140-negative platelets. However, the peaks of the activated platelets did not shift to the right side, i. e., the channel number of the peak remained at the same immunofluorescent intensity, suggesting that there is a certain limit in the activation of the circulating platelets during CPB and the platelets activated beyond the limit may be removed from the circulation.
In conclusion, platelets are activated during CPB and the platelets activated beyond a certain limit are removed from the circulation, resulting in thrombocytopenia after CPB.

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Activated prothrombin complex concentrate (APCC), a by-passing hemostatic preparation for hemophilia A patients with a high titer of factor VIII inhibitors, often has an unpredictable hemostatic effect and occasionally results in severe thromboembolic complications. A clinical trial was conducted on 3 hemophilic patients with a high titer of factor VIII inhibitors to evaluate the safety and efficacy of a recombinant factor VIIa (rFVIIa; NN-007), an alternative by-passing preparaton. At the same time thrombelastgram (TEG) was evaluated to be better than activated-partial thromboplastin time (APTT) as a monitor of hemostatic effects when using rFVIIa. TEG was more suitable than APTT, because r, r+k and ma values of TEG were normalized at least for 4 hours after the infusion, whereas APTT was variably shortened and was not always maintained at a normal level for 4 hours. Sixty five μg/kg to 80μg/kg of rFVIIa was administered on demand once to 12 times per one bleeding episode. Eighty bleeding episodes were assessed during the 36-week treatment period. The efficacy rate of the hemostatic effect was confirmed to be 97.5%. No adverse reactions including laboratory or clinical DIC were observed in a total of 123 infusions. No anamnestic response of the factor VIII inhibitor developed. Vital signs and laboratory findings showed no significant changes attributable to rFVIIa. These findings suggest that rFVIIa is safe and efficacious as a by-passing hemostatic preparation for hemophilia A patients with a high titer of factor VIII inhibitors.

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We studied the changes of thrombin-antithrombin III complex (TAT) and plasmin-α₂ plasmin inhibitor complex (PIC) levels during pregnancy, and evaluated the clinical implications of TAT and PIC levels in pregnant women with obstetric past history of miscarriage or stillibirth, those with various obstetric troubles for the first time in the present pregnancy, and those with toxemia.
In normal pregnancy the TAT levels increased at term (less than 20ng/mL), but the PIC levels did not (lower than 0.8μg/mL). Many patients with toxemia showed an increase in TAT and PIC, and the elevation of PIC was marked as compared with normal pregnancy. Four of 8 patients (50%) with missed abortion and intrauterine fetal death showed elevated TAT, whereas increased PIC was observed in two of 8 (25%). TAT and PIC levels of the patients with other obstetric troubles did not show significant change.
We conclude that pregnant women are in hypercoagulable state rather than in hyperfibrinolysis. Further studies are needed with more careful and frequent venipunctures to clarify whether TAT is a predictor for missed abortion and intrauterine fetal death, and elevated PIC is related to toxemia of pregnancy.

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