A rapid and sensitive immunofluorescence assay utilizing the flow cytometry to detect in platelet associated IgG (PAIgG) and platelet binding IgG (PBIgG) in patients with idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE) and leukemia cases received multiple platelet transfusion are described. Using this assay the percentages of PAIgG positive platelets of normal individuals were 2.7%. Increased levels were found in 22 cases of 23 patients with ITP in a range of 2.5-86.6%. The correlation of platelet counts and the percentages of PAIgG positive platelets was found to be significant (p<0.01). Elevated levels of platelet associated complement C₃ (PAC₃) in 21 out of 23 and platelet associated IgM (PAIgM) in 10 out of 11 were also found. Between PAIgG and PAC₃, PAIgM, the good correlation were demonstrable (r=0.833, r=0.900). In SLE cases, the percentages of PBIgG and PBC₃ positive platelets were elevated as well as in ITP cases, but PAIgG levels were decreased compared with ITP cases. Further, this assay was useful to predict the effects of platelet transfusion. Finally, in comparsion to other PAIgG assays, this new assays provide specific information in the nature of the immunoglobulin deposited on the platelet surface in ITP cases.

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Prevention of functional impairment of platelet during storage was tried by using modified poly (vinylchloride) bags, and the mechanism of functional impairment is discussed. Platelet concentrates were stored in glow-discharge treated (GD) and/or hydrogel (PEO) coated bags. Platelets stored in bags in which plasticizer (DEHP) leakage was prevented by GD, maintained functions (aggregability and hypotonic shock response) well. DEHP was added to fresh PRP and the effects of DEHP were examined. Cytoplasmic Ca²⁺ (Cyt-Ca²⁺), membrane-associated Ca²⁺ (M-Ca²⁺) and membrane potential (MP) increase during storage. Although MP and Cyt-Ca²⁺ were not affected by DEHP, M-Ca²⁺ had increased after incubation at 22°C for 24 hours. The degree of increase of Cyt-Ca²⁺ in platelets stored in PEO bags was lower than that in platelets in non-coated bag. The relationship between Ca²⁺ distribution in resting platelets and function is not clear. However, the results in this paper suggest the possibility that modification of bag is effective on prevention of platelet change during storage.

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Platelet cyclo-oxygenase (PCO) deficiency is known to be a disease with a moderate bleeding tendency due to impairment of platelet thromboxane A₂ production. However, the hemostatic control in a bleeding episode or during operation has been poorly documented. We studied the effect of DDAVP infusion (0.4μg/kg) on bleeding time, pletelet function and coagulation factors in a PCO deficiency patient. After DDAVP infusion, VIII: C, vWF: Ag and RCoF markedly elevated and bleeding time was normalized without improvement of “in vitro” platelet aggregation (Table 1, Fig. 1). It seemed that DDAVP could be used as a hemostatic agent in PCO deficiency.
This patient suffered from hypermenorrhea requiring a lot of blood tansfusion and total hysterectomy for relieving severe hypermenorrhea was considered.
Before operation, we studied the function of the mixed platelet rich plasma (PRP) in vitro which was composed of the patient PRP and the normal control PRP. The existence of more than half of the normal PRP almost normalized the mixed platelet aggregation induced by various agonists and malondialdehyde production (Fig. 2). According to these results, we transfused platelet concentrates (PC) so that the platelet count of the patient would increase to 2 folds of that before.
After PC transfusion, “in vitro” platelet aggregation and bleeding time of the patient were normalized (Table 2). No abnormal bleeding was found during and after the operation. From this experience, it seemed that “in vitro” study on the function of the mixed platelets was available for the determination of the PC amount requiring for hemostatic control before operation in PCO deficiency.

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Using a newly developed damped oscillation type rheometer, viscosity change of blood during clotting was measured and fundamental examination for an application of its method to a clinical field was performed. The rheometer consists of a cylindrical tube suspended from a torsion wire and filled with blood. The tube is excited in torsional oscillation and subsequent damped oscillation is observed. From damped oscillation curves measured at a certain interval, the change of logarithmic damping factor (Δ) which is directly related to viscosity/or fluidity of blood is determined. Change of Δ of blood during clotting occurred before the dynamic viscoelasticity of blood during clotting, which was measured using a forced oscillation type rheometer, began to increase. The present method can sensitively follow the initial change of viscosity of blood during clotting. Effect of tube materials on the change of Δ of blood during clotting was examined. With expanded polytetrafluoroethylene (Gore tex^®) and polydimethylsiloxane (Silastic^®), the decrease of Δ for blood proceeded over 60min, while, with glass tube, the decrease of Δ rapidly occurred within 10min, suggesting that the present method is available for in vitro analysis of the interaction between the surface of material and blood as well as analysis of mechanism of blood coagulation. With glass tube, Δ for PRP and whole blood gradually increased after reached the lowest value, depending on the value of hematocrit and the number of platelet. This is due to the separation of clot from the glass surface. The present method would also useful for the measurement of the process of clot retraction.

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The acute clinical course of DIC in the field of obstetrics does not allow to await the resuts of coagulation tests before starting therapy. We, therefore, established a clinical scoring system with the aim of early start of therapy. In our scoring system, which was found to be in good agreement with that of the Japanese Ministry of Health & Welfare, more weight was given to the underlying disease and to clinical findings than to laboratory parameters (coagulation tests).
In the next place the results of coagulation test in the cases of abruptio placentae were analyzed. The distribution of coagulation test's values was shown in Fig. 4. The characteristics in the distribution of coagulation test's values are following: higher FDP, lower Fbg, prolonged PT and not so decreased platelet count are found. We made a coagulation test's score in obstetrical DIC. The difference between this DIC score and the coagulation test's score in MHW's DIC score was analyzed.
Using above mentioned clinical Symptom score and this obstetrical coagulation test's score, we analyzed the therapeutical usefulness in obstetrical DIC cases and the difference between FOY and AT-III concentrate.

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A human melanoma cell line (Bowes) secretes tissue-type plasminogen activator (t-PA) into a culture medium. We previously reported that the secretion of t-PA was affected by changing the concentrations of Na⁺ or K⁺ in the culture medium and by the addition of monensin, an ionophore for monovalent cations. In this study we investigated the effects of exogenous ATP and Ca²⁺ on the t-PA secretion in order to elucidate its basic mechanism. The cells, arter 10min preincubation with 1mM EGTA in MEM, were incubated in MEM containing various concentrations of ATP (0-5.0mM) and Ca²⁺ (1.8-9.0mM) for 6 or 24 hours. The increase in the extracellular ATP or Ca²⁺ concentration resulted in depression of t-PA secretion in a dose-dependent manner. Intracellular t-PA was increased in the combination of ATP and Ca²⁺ concentrations: 0.5-2.5mM ATP with 9.0mM Ca²⁺ at 6h, or 0.5-2.5mM ATP with 4.3 or 5.3mM Ca²⁺ at 24h. The protein synthesis was suppressed by the addition of ATP (0.5-5.0mM) to the medium, while it was only slightly affected by extracellular Ca²⁺ with 0-0.1mM ATP. At 5.3mM Ca²⁺ the suppressive effect of ATP on the protein synthesis became obvious. The DNA synthesis was stimulated by Ca²⁺ without ATP, but a remarkable increase in the DNA synthesis was observed at 0.1-2.5mM ATP, where Ca²⁺ had no additional effect on the DNA synthesis. However at 5.0mM ATP, the DNA synthesis was inhibited. The RNA synthesis was enhanced by 0.1 or 0.5mM ATP but suppressed above 1.0mM, while Ca²⁺ had little effect. Intracellular cAMP level was not changed by Ca²⁺ and ATP after 5min incubation in the experimental medium. Extracellularly applied dibutyryl cyclic AMP did not influence the t-PA secretion or the intracellular t-PA level. These results suggest that ATP and Ca²⁺ co-operatively suppress the t-PA secretion system without participation of cAMP.

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Two molar potassium thiocyanate exract of psoriatic scales demonstrated fibrinolytic activity only in the pesence of plasminogen (0.54+0.51IU/mg protein). The extract also showed high level of activity on Ile-Pro-Arg-p-nitroanilide (pNA) (3.98±1.43nmol/min/mg protein). Sephacryl S-200 gel chromatography revealed that plasminogen activator (PA) activity positioned at approximately MW 60K. This PA activity was totally adsorbed to benzamidine-Sepharose column and dissociated with 2M KSCN. Enzymographic analysis demonstrated 62K, 43K and 33K PA in the adsorbed fraction. By the following high performance anion-exchange chromatography on Mono Q, three peaks of PA activities were separated. Among them, the major peak of PA activity was 62K. Futhermore, anti-urokinase (UK) IgG-Sepharose affinity chromatography of S-200 PA fraction showed that only 62K PA is adsorbed to this column and represents 70% of the total psoriatic PA activity. In order to demonstrate the localization of UK-type PA antigen in psoriatic epidermis, we performed an immunofluorescence study. UK-type PA was mainly found in upper layer of psoriatic epidermis, although diffuse fluorescence was seen in whole psoriatic epidermis.

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The sialic acid of platelet was determined in Platelet Concentrates during a five day period. Fresh platelet contained 9.49μg sialic acid per mg protein. The content of sialic acid decreased gradually during the storage at 22°C with a final loss of 40% of initial value after five days.
The role of sialic acid on several functions of platelet was investigated by neuraminidase treatment. Aggregabilities induced by both ADP and collagen, PF-4 release stimulated by collagen, and adhesiveness to collagen were significantly increased after the treatment under the condition of which a half of sialic acid was removed, whereas hypotonic shock recovery showed a significant diminution. The result suggests that the sialic acid of platelet works in a different manner on each function.

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Heparinlike glycosaminoglycans on the endothelium are available for interactions with antithrombin. We have tested the hypothesis that heparan sulfate on the endothelial cell surface binds antithrombin III and accelerates the thrombinantithrombin III reaction. Purified porcine ¹²⁵I-antithrombin III was bound to cultured porcine aortic endothelial cells. The binding reached equilibrium by 15min at 37°C and was saturable with approximately 10nM of antithrombin III concentration at half maximal binding. The inhibition of thrombin by antithrombin III was enhanced in the presence of endothelial cells. Removal of heparan sulfate by the treatment of cells with heparitinase completely abolished both the binding and accelerated thrombin-inhibitory effect of antithrombig III. On the other hand, chondroitinase treatment did not affect either binding or thrombin inhibition. Displacement of antithrombin III bound to endothelial cells by heparin with lower affinity to antithrombin III was less potent than that by unfractionated or higher affinity heparin. Our results suggest that heparan sulfate on the endothelial cell surface is involved in the antithrombin III binding to endothelial cells and the acceleration of the effect of antithrombin III by the cells.

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Only few studies on the relationship between vitamin D₃ and platelet functions have been described. We studied the effects of 1α, 25-dihydroxyvitamin D₃ [1α, 25(OH)₂ D₃] on aggregation of platelet rich plasma (PRP) from wistar rats. Collagen-induced aggregation in PRP was inhibited for 1-12 hours after the administration of 1α, 25(OH)₂ D₃, while there was no inhibition of ADP-induced aggregation in PRP. Inhibited platelet aggregation recovered to normal after 16 hours from administration and correlated with the dosage of 1α, 25(OH)₂ D₃ administrated. Serum 1α, 25(OH)₂ D₃ of the rats studied and MDA production were also determined. There was a good relationship between the serum of 1α, 25(OH)₂ D₃ and inhibition rate of platelet aggregation, but there was no change of MDA production after the administration of 1α, 25(OH)₂ D₃.
These results show that 1α, 25(OH)₂ D₃ transiently inhibited platelet aggregation induced by collagen. The mechanizm of aggregation inhibition remains to be clarified.

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The fibrinolysis in 6 patients with lupus anticoagulant was studied. The intrinsic fibrinolytic activity by kaolin activated euglobulin with plasminogen free fibrin plate was low level in all patients. Although we could not obtain common results in fibrinolytic factors-plasminogen, α₁-AT, α₂-M, α₂-PI and Cl-inactivator with immunodiffusion method, and in fibrinolytic activity by euglobulin from plasma with ELT and plasminogen rich fibrin plate.
In two patients with Budd-Chiari syndrome, the IgG fraction of plasma through Sephadex G 200 had the inhibitory activity of fibrino (geno) lysis on the fibrin plate method and the immunoelectrophoresis, as well as high lupus anticoagulant activity.
Those findings suggest that fibrinolysis plays a role on formation of thrombus in patient with lupus anticoagulant.

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It was reported that immediate acidification is necessary for measurement of t-PA activity in blood samples to avoid its inactivation by t-PA inhibitor (PAI).
In this study, the immunological activity and enzymatic activity of t-PA were determined by ELISA method and parabolic rate assay method, respectively, and the interaction between t-PA and PAI was analysed by gel filtration method with Sephadex G-200. The following results were obtained.
1. Standard t-PA, obtained from Bio pool AB, showed one peak with Mr=60kDa by gel filtration, in which both immunological and enzymatic activities were found consistently.
2. By the same method, the plasma sample from a pregnant woman (PAI rich plasma) showed one peak with Mr=120kDa in which only immunological activity of t-PA was found, and no enzymatic activity was found.
3. On the other hand, the mixture of standard t-PA and the plasma showed an increased peak with Mr=120kDa showing only immunological activity of t-PA, and showed a decreased peak with Mr=60kDa showing enzymatic activity of t-PA.
From these results, it was presumed that activity of t-PA (Mr=60kDa) was immediately inhibited with PAI in human blood, by forming a t-PA -PAI complex (Mr=120kDa), which was undissociable under the gel filtration method. Accordingly, the Mr of PAI seemed to be 60kDa. PAI might play an important role in regulation of fibrinolysis, and its clinical significance remained to be elucidated.

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