Cytosolic Ca²⁺ concentration ([Ca²⁺] i) transiently increases upon activation of human platelets and the increase is thought to be due to Ca²⁺ release from internal stores and Ca²⁺ influx from extracellular milieu. The mechanism to restore [Ca²⁺] i to the resting level is poorly understood. The efflux across the plasma membrane and the sequestration into endoplasmic reticulum play major roles in [Ca²⁺] i restoration. In this study, we investigated the relationship between protein kinase C (PKC) and Ca²⁺ pump in human platelet membrane vesicles.
Human platelet membrane vesicles were prepared from platelet concentrates, which are able to accumulate Ca²⁺ in the presence of adenosine triphosphate (ATP) and release Ca²⁺ upon exposure to inositol trisphosphate or ionophore A23187. Membrane vesicles prepared from platelets pretreated with phorbol myristate actate (PMA) showed an increaed activity of PKC but no changes in Ca²⁺ uptake. Ca²⁺ uptake was not affected by incubation of membrane vesicles with partially purified PKC of rat brains in the presence of PMA. PKC inhibitor (H-7) prevented neither Ca²⁺ uptake nor Ca²⁺-ATPase.
These results indicate that Ca²⁺ pump in human platelet membrane vesicles may not be regulated by PKC.

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Using a monoclonal antibody against GPIb (TM60), we established an assay method for glycocalicin concentration in human plasma. The assay was based on the competitive inhibitory action of glycocalicin in plasma against TM60-binding to platelets. Washed human platelets (1.0×10) were adsorbed on the surface of an immunoplate using polylysine. Plasma was incubated with 4μg/ml of TM60 in the immunoplate. Amounts of bound TM60 on the platelets were measured by ELISA using peroxidase conjugated anti-mouse IgG. Plasma from normal volunteers contained 4.15±0.66 (n=13, mean±SD), while plasma from myeloproliferative disorders contained 5.15±0.63 (n=11, mean±SD). In the patient group, glycocalicin concentrations correlated with their platelet numbers in whole blood, suggesting that increases of glycocalicin in the patients were dependent on their platelet numbers.

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Antiplatelet and antithrombotic effects of a thromboxane (TX) A₂ receptor antagonist, ONO-3708, which does not alter prostaglandin generation, were studied to investigate the role of TXA₂ in thrombogenesis. TXA₂ synthesis of washed platelets stimulated with 1mM arachidonate was perfectly inhibited by 10μM dazoxiben, a TXA₂ synthetase inhibitor, but never by 10μM ONO-3708 when quantitated with high-performance liquid chromatography. Ten μM ONO-3708 perfectly inhibited platelet aggregation (PA) to 1mM arachidonate and 1μM STA₂, a stable analogue of TXA₂, in not only 5 responders but also 5 non-responders to dazoxiben on PA to arachidonate out of 10 normal volunteers. These results indicate that the difference between responders and non-responders to TXA₂ synthetase inhibitors is not attributable to the difference in TXA₂ receptor among them.
Threshold concentration of STA₂ to induce irreversible PA (0.52±0.40μM) was significantly (p<0.01) lower and IC₅₀ of ONO-3708 on PA to 1μM STA₂ (1.78±1.03μM)was significantly (p<0.01) higher in 24 patients with cerebral ischemia than in 10 normal controls (0.96±0.12μM and 0.40±0.21μM, respectively) or 10 patients with neurological disorders except for stroke (patient controls; 0.90±0.18μM and 0.45±0.30μM, respectively). It is postulated from these results that there is an abnormality in TXA₂ receptors of platelets in patients with cerebral ischemia.
Secondary PA to ADP, epinephrine and PAF, which was not influenced by 10μM dazoxiben, was inhibited by 10μM ONO-3708. This might imply the role of TXA₂ receptor on secondary PA to these agonists. Synergistic effect of ONO-3708 on PA to these agonists when added with dazoxiben was greater in whole blood, where prostacyclin is generated by leukocytes, than in platelet rich plasma. PA to collagen was perfectly inhibited by 10μM ONO-3708 alone. Filter bleeding time (Uchiyama and Didisheim), an in vitro test of shear-induced PA, remained unchanged by 10μM ONO-3708 or dazoxiben alone, but significantly (p<0.05) prolonged from 210±158sec to 325±178sec by ONO-3708 when added with dazoxiben. These results indicate that shear-induced PA is reduced when multiple pathways leading to PA are inhibited but not when only arachidonate-TXA₂ pathway is inhibited.

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Platelet aggregometry in whole blood in both 24 normal controls and 57 survivors of cerebral infarction (CI) was performed by our new method, i. e. screen filtration rate method (SFR). SFR was based on the phenomenon that platelet aggregates in blood samples cease the natural flow of specimen through the metal micromesh. Several concentrations of ADP were used as inducer of platelet aggregation. Platelet aggregability was graded according to the minimum concentration of ADP which induce irreversible platelet aggregation.
Platelet aggregation in whole blood in CI patients was more readily occurred than that in platelet rich plasma. Spontaneous platelet aggregation, which was considered to play a important role in causing a ischemic episode, was often observed in whole blood of survivors. We conclude that platelet aggregation in whole blood should be studied to disclose the mechanism of thrombus formation.

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The purpose of the present study in to clarify the effect of bunazosin on platelet aggregation in the patients with essential hypertension.
In vitro, the effects of 10^-3-10^-5M bunazosin on platelet aggregations induced by ADP, epinephrine and A23187 were studied in untreated patients with essential hypertension. The concentration of 10^-3-10^-4M bunazosin inhibited the platelet aggregations.
Before this oral administration of bunazosin study, the patients were treated with other antihypertensive drugs for 10±7 (M±SD) weeks. Platelet aggregation (Born's method) induced by 4.6μM ADP and 5.0μM epinephrine and whole blood aggregation (Impedance method) induced by 10μM ADP and 5.0μg/ml collagen were measured in 14 patients with essential hypertension before and after administration of 1.5mg/day or 3.0mg/day for 6±4 (M±SD) weeks. Blood pressure and heart rate were measured before and after administration of bunazosin. Platelet aggregation induced by both 4.6μM ADP and 5.0μM epinephrine had a tendency to decrease and whole blood aggregation induced by both 10μM ADP and 5.0μg/ml collagen was significantly decreased (p<0.05) after administration of bunazosin. Heart rate and systolic blood pressure were not significantly changed after administration of bunazosin in the patients treated with other drugs. On the other hand, diastolic blood pressure was significantly decreased (p<0.05) after administration of bunazosin in the same patients.
The present results suggest that the inhibitory effect of bunazosin on platelet aggregation may be not due to the block of α₂ receptor but the inhibition of cytoplasmic calcium increase relating to the platelet activation in essential hypertension.

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Kinin-liberating activities were investigated in scales from the inflammatory skin diseases, psoriasis vulgaris and psoriasis pustulosa, using competitive enzme immunoassay for kinins. Tris-buffered saline extracts of both psoriatic scales liberated the same level of kinins from heated plasma (approximately 11ng/mg protein). In psoriasis vulgaris, three peaks of kinin-liberating activities were shown at M_r 90, 000 (VK-I), 65, 000 (VK-II) and 35, 000 (VK-III) by Sephacryl S-200 gel chromatography. They were further separated by DEAE Sepharose chromatography at pH 7.5. Kinin-liberating activity of VK-I was eluted in nonadsorbed fraction. However, this fraction liberated kinins only from the heated plasma but not from the purified low molecular weight kininogen (LMW-Kng), indicating VK-I is a plasma kallikrein-like enzyme. Kinin-liberating activity of VK-II was separated into two peaks. VK-IIa in nonadsorbed fraction liberated kinins only from the heated plasma, whereas VK-IIb in adsorbed fraction liberated kinins from both heated plasma and LMW-Kng, showing VK-IIa is plasma kallikrein-like and VK-IIb is glandular kallikrein-like. Kinin-liberating activity of VK-III eluted at the same position to VK-IIb, and was found to be glandular kallikrein-like, as well. In psoriasis pustulosa, majority of kinin-liberating activity peaked at M_r 90, 000 by Sephacryl S-200 gel chromatography (PK-I). PK-I demonstrated essentially the same elution profile as VK-I by the following DEAE Sepharose chromatography. VK-I, VK-IIa and PK-I were inactivated by soybean trypsin inhibitor, but only partially by aprotinin, confirming plasma kallikrein-like characteristics. VK-IIb and VK-III were inhibited by aprotintn but not by soybean trypsin inhibitor, confirming glandular kallikrein-like characteristics.

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Immunoradiometric assay (IRMA) for protein C was devised by using murine monoclonal antibodies against human protein C. The antibodies, specific for the activation peptide region of the heavy chain (HpC 9) or the light chain of human protein C (HpC 16), were used. HpC 9 was iodinated with a chlormine-T method and HpC 16 was immobilized to polyacrylamide beads (Immunobeads, Bio-Rad). The standard curves obtained either with purified human protein C or with pooled normal plasma with a known concentration of protein C were identical. The intraassay and interassay coefficients of variation for pooled normal plasma were 3.0% and 6.5% respectively. Twenty two normal individuals had a mean protein C level of 2.99±0/45mg/l (SD) and 9 warfarin-treated patients showed 1.26±0.24mg/l. Good correlation was obtained between this method and radioimmunoassay for human protein C in 22 normals, 9 warfarin-treated individuals and 3 patients with hereditary protein C deficiency (r=0.96, p<0.01).

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Hepatitis infection in haemophiliacs has been a major problem in their replacement therapy, and factor VIII concentrates manufactured from pooled plasma were realized to be the cause of transmission of AIDS virus. However, several investigators have shown that some of these viruses are inactivated by chemical or physical treatment on factor VIII concentrates. After infusion of a heat-treated factor VIII concentrate (heat-treated at 65°C for 96 hours), a follow up study was carried out in 11 patients who had no infusion of blood nor blood preparations beforehand. During the observation period, there was detected no sign of hepatitis in all patients, and no positive seroconversion to AIDS virus was proved. These results were in a good agreement with the experimental data previously reported from in vitro as well as in vivo experiments where chimpanzees were employed for virus infection test. For the evaluation of the safety for viral infection of clotting factor concentrates in clinical use, these survey results in untreated patients are of a great importance.

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In our previous study, we have demonstrated that plasminogen activators (PAs) with Mr=24, 000 and Mr=48, 000 play an important role in tissue fibrinolysis during the development of hypersensitivity granulomas. In order to investigate the cellular source of these PAs, we cultured mouse peritoneal macrophages in different conditions. C57BL/6N mice were used with or without infection by Mycobacterium lepraemurium. Antigen stimulated peritoneal exudate macrophages were obtained by intraperitoneal injection of sonicated M. lepraemurium to the infected mice. PA activity in conditioned media was assayed by using two-stage colorimetric method and electrophoretic enzymography. Resident peritoneal macrophages secreted PA with Mr=48, 000. However antigen stimulated peritoneal exudate macrophages secreted a high activity of PA with Mr=24, 000 in addition to Mr=48, 000. In order to investigate secretion mechanism, in vitro stimulation of macrophages was carried out by lymphokines which were obtained from culture supernatants of splenic cells exposed with antigen or splenic T cells with Concanavalin A. The results clearly demonstrated that macrophages became secretable of PA with Mr=24, 000 by lymphokine stimulation. Further experiments showed that the effect of lymphokine to induce secretion of PA was partially represented by γ-Interferon. These data indicated that the immunological stimulation induced macrophages to secrete PA, the molecular weight of which was altered from resident macrophages.

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The in vitro and in vivo combined effects of urinary plasminogen activator (u-PA) and tissue plasminogen activator (t-PA) were examined. In in vitro experiments the additive combined effects of u-PA and t-PA were observed on human plasma clot lysis. In in vivo studies using a canine coronary thrombus model, an intravenous combined administration of t-PA and u-PA synergistically enhanced their thrombolytic activity, while the reductions of fibrinogen, plasminogen and α₂-plasmin inhibitor were additive.
The studies on plasma and blood dynamic viscosities revealed that whole blood dynamic viscosity decreased significantly in both u-PA and the combination groups, while plasma viscosity was not changed. This lowered blood viscosity was rather notable at the lower shear rate condition than at the higher shear rate. It is, therefore, presumed that this combination therapy may have a great advantage on thrombolysis in acute myocardial infarction.

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The effects of transforming growth factor-β (TGF-β) from human platelets on anchorage-dependent and -independent growth of normal rat kidney fibroblast (NRK), SV-transformed mouse fibroblast (SV 3T3) and methylcholanthren-induced mouse fibrosarcoma A (Meth A) were investigated. In the presence of epidermal growth factor (EGF), TGF-β stimulated anchorage-dependent and -independent growth of NRK, whereas the same growth factor inhibited the growth of SV 3T3 and Meth A.
The mechanism of synergistic effect by TGF-β and EGF was then analysed with regard to the expression of EGF receptors by using ¹²⁵I-labelled EGF. The binding sites for EGF in NRK were increased by treatment with TGF-β, but not in SV 3T3 and Meth A. On the other hand, intracellular turnover rate of EGF-EGF receptor complex in NRK was not affected by treatment with TGF-β, though it was significantly impaired by the same treatment in Meth A.

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The platelet life spans of 16 patients with hepatic cirrhosis and 4 healthy adults were determined using ⁵¹Cr labelled autologous platelets. The mean half life time of platelet of hepatic cirrhosis was significantly shortened to be 2.8 days compared with 4.3 days of healthy adults. In the patients with hepatic cirrhosis the increase of platelet pool was demonstrated by the marked lowering of platelet recovery. The radioactivity of labelled platelet in the liver was low and that in the spleen was mild to moderate. It is, therefore, considered that not only spleen but also abnormality of blood vessels play important roles for the increase of platelet pool of the patients with hepatic cirrhosis.

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To evaluate the effect of pacemaker implantation on the cerebral embolization, we studied changes of platelet function and blood viscosity in patients with pacemaker implantation. Platelet count, mean platelet volume, platelet aggregation by ADP, epinephrine and collagen, plasma TXB₂, plasma 6-Keto-PGF_1α, blood viscosity and fibrinogen were measured before, at 1 week, at 2 weeks and at 3 weeks after implantation in 7 patients with sick sinus symdrome, 7 with complete AV block and 2 with bradycardiac atrial fibrillation. The modes of pacemakers were VVI in 8 and DDD in 8 patients.
In results, 1) Platelet count decreased immediately after implantation, then recovered to the initial level, and stayed increased at 2 and 3 weeks after implantation. 2) Mean platelet volume stayed decreased at 1 week, 2 and 3 weeks after implantation. 3) Platelet aggregation by ADP and epinephrine increased at 2 weeks, and in some patients, stayed increased even at 3 weeks. Aggregation by collagen recovered to the initial level at 3 weeks. 4) Plasma TXB₂ increased at 2 weeks, but plasma 6-Keto-PGF_1α didn't change. 5) Fibrinogen and plasma viscosity increased at 1 week, and recovered to the initial level at 2 weeks. Casson's viscosity and yield shear stress revealed no change.
Platelet function, plasma viscosity and fibrinogen increased transiently after implantation, but in some patients, platelet function stayed increased at 3 weeks after implantation. Some reports mentioned that changes platelet function after operation were transient and returned to the initial level within 1 month after operation. Then, it is necessary to follow up platelet function when it increases up to 1 month after implantation, because either the pacing lead or flow change by pacing might accelerate platelet function.

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A new and exciting animal model for endogenous hypercholesterolemia has recently become available through the discovery of a strain of rabbits designated as Watanabe heritable hyperlipidemic (WHHL). In these animals, massive hypercholesterolemia results from a single genetic defect of LDL receptor, and severe atherosclerosis occurs despite the ingestion of a cholesterol-free diet. In comparison with values in healthy normal-weight normolipidemic rabbits, activities of vitamin K-dependent clotting factors, clotting factors of contact phase, clotting factor VIII activity (F VIII: C), and fibrinogen were found to be significantly higher in WHHL rabbits. These data suggest hypercoagulable state in WHHL rabbit. Furthermore, it is known that atherosclerosis of the aorta severely develops in ranging from 5 to 15 months of age, and coronary disease begins to develop at 5 months of age. Vitamin K-dependent cloting factors were already higher at 2 months of age in the WHHL rabbits, and were not increased in amounts with age. Clotting factors of contact phase and fibrinogen had increased at the age 5 and 8 months. In WHHL rabbits, F VIII: C had increased during these months and then decreased. However, such changes in F VIII: C were not seen in control rabbits. Clotting factors might increase in hyperlipemia and some clotting factors would change in degree with development of atherosclerosis.

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1) I mmunodifusion test with goat anti-human fibronectin showed cross-reaction between plasma fibronectins of human and mouse. 2) By the immunoturbidimetric assay, standard curves of human and mouse fibronectins were quite linear, and fibronectin concentrations in mouse plasma could be determined up to 200μg/ml. 3) Fibronectin levels in mouse plasma slightly decreased 8 hours after i. p. injection of 50μg of S. typhimurium endotoxin, however, dose response was not good. 4) Shortening of clotting time of human plasma by the addition of phospholipid was inhibited by incubation of phospholipid with fibronectin. 5) By prior incubation of tissue thromboplastin (simplastin) with fibronectin, about 50 to 80% of the thromboplastin activity was inhibited. 6) Fibronectin also inhibited the thromboplastin activity of bone marrow cells from mouse given endotoxin. 7) Thromboplastin-induced lethality in mice was inhibited by prior incubation of thromboplastin with fibronectin.
Fibronectin has been known to bind to ganglioside or phospholipid. Therefore, these results indicate that inhibitory effects of fibronectin on the thromboplastin activity is thought to be due to the binding of fibronectin to phospholipid portion of thromboplastin.

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Restriction fragment length polymorphisms serve as valuable means for carrier detection of hereditary diseases. Factor VIII gene was recently cloned and sequenced, and an intragenic Bcl I polymorphism was found to be useful for carrier detection. We searched carriers of hemophilia A using a probe F8A which can detect the Bcl I polymorphism. Southern blots of Bcl I-digested DNA revealed polymorphic 1.2kb and 0.9kb bands by hybridization with the F8A probe. Of 15 unrelated women, 8 had both bands, 2 had a single 1.2kb band, and 5had a single 0.9 band. Study of four families in which carrier detection was possible by the Bcl I plymorphism analysis is presented in this paper: daughters of the three families were determined to be carrier and a daughter of the other family to be non-carrier. There is an inherent limitation in applying this method to carrier detection: the mothers must be heterozygous for the restriction fragment length polymorphism. However, use of multiple restriction fragment length polymorphisms will reduce the limitation.

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We examined ultrastructurally the postmortem tissues to search for AIDS virus from a patient with hemophilia B who developed AIDS. Tissues fixed in 10% formalin were processed to prepare specimens for electron microscopic study. Round particles, 100-110nm in diameter, with a centric or eccentric nucleoid, were observed frequently in the intra and extracellular spaces of the lymph node, liver and spleen. These particles were morphologically identical with the mature AIDS virus. The budding virions on the plasma membranes were also found in the liver. Besides these findings, two types of cytoplasmic inclusion bodies were observed: One was “tubuloretucular structure (TRS)” seen in the liver, and the other “test tube and ring shaped forms (TRF)”, observed in the lymph node. These findings were the same as we previously described with another hemophiliac who died of AIDS, but not so many as the other specimens treated with glutaraldehyde.

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Inactivation of viruses in the defibrination processes of heat defibirinated human anti-haemophilic factor concentrate (RCG-5) and its thermal stability were evaluated.
About 10¹¹ infection units of each added marker virus (VSV, sindbis) were inactivated by the defibirnation process in which cryoprecipite solution was heated at 54°C for 5min followed by centrifugation. The results suggested that the heat defibrination process was effective as a virus inactivation process as well as removing fibrinogen which sometimes caused a side effect of hyper-fibrinogenemia.
RCG-5 was store at various temperatures and the first-order rate constants of the loss of VIII: C were measured. From the rate data it was showed that the stability of RCG-5 was similar to that of the reported lyophilized cryoprecipitate and of some intermediate purity factor VIII concentrate at higher temperature. The activation energy of decomposition was calculate to be 31.8kcal/mole.
These data suggseted that RCG-5 was a safer product for virus contamination and that it was predicted to be stable sufficiently for several years storage at lower temperature.

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