The effects of a new carbacyclin analogue (CS-570) and dibutyryl cAMP [db-cAMP (Actosin)] on the aggregation of the human platelet prostacyclin (PGI₂) generation of cultured human vascular endothelial cells were investigated. The endothelial cells were isolated from human umbilical cord veins and cultured by the modified method of jaffe et al.
CS-570 or db-cAMP inhibited ADP or collagen induced platelet aggregation and thromboxane B₂ (TXB₂) generation in dose dependent manners. They increased cAMP concentration, but they had no effect on cGMP levels. These results suggested that the inhibition of platelet aggregation by these reagents might be due to the elevation of cAMP levels and inhibition of TXB₂ generation.
In the endothelial cells, CS-570 and db-cAMP increased intracellular cAMP concentration, while CS-570 enhanced PGI₂ generation of endothelial cells and db-cAMP decreased it. Both of them had no effect on cGMP levels.
From these results, it was concluded that CS-570, which increased intrinsic PGI₂ generation of endothelial cells, was considered to be more beneficial for anticoagulant therapy than Actosin. Because Actosin brought the decrease of PGI₂ generation in the endothelial cells.

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Bleeding volumes of blood samples obtained from human, rat, rabbit and guinea pig were determined by the use of a Thrombostat, an apparatus simulating primary haemostasis. The optimum sucking pressure was 30mmHg for all the species. Constant data were obtained when determined during the time 1 and 3hrs. after blood drawing. Addition of ADP to collagen filter was necessary to complete haemostasis in this system where simultaneous addition of CaCl₂ did not affect the bleeding volume. The bleeding volume was highest in the rat, and then human, guinea pig and lowest in the rabbit in this order. PGE₁ (final concentration; 0.05μM or more in rats and 0.1μM or more in rabbits) increased the bleeding volume, especially phase II value, and inhibited ADP-and collagen-induced platelet aggregation in rat and rabbit blood. Ticlopidine, daily 200 and 400mg/kg for 3 days (po), increased the bleeding volume and decreased ADP-induced platelet aggregation in rats. warfarin, 1mg/kg for a day (po), increased prothrombin time, but caused no change in the bleeding volume and ADP-induced platelet aggregation in rats. Patients with thrombasthenia whose bleeding time were over 15 mins showed high values for the bleeding volume. These data suggest that this apparatus, Thrombostat was useful for determination of plateletinduced primary haemostasis.

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In order to evaluate the effect of small dose of aspirin (40mg/day) with or without dilazep (300mg/day) and ticlopidine (200mg/day) with dilazep for 4 weeks in patients with prosthetic heart valves under warfarin treatment, platelet survival time by malondialdehyde (MDA) method, platelet functions and some coagulation parameters were examined before and after the treatment.
Following results were obtained;
1) There was a negative linear correlation between platelet count and the recovery rate of platelet MDA production on the fifth day.
2) Although the platelet aggregability was suppressed significantly after administration of aspirin with or without dilazep, the suppression of the platelet aggregability was not found after administration of ticlopidine with dilazep.
3) Platelet count increased after aspirin with dilazep and after ticlopidine with dilazep as compared with that observed before treatment.
4) Platelet survival time was also prolonged after 4-weeks' administraton of aspirin with dilazep as compared with that observed before treatment.
These results suggest that the MDA recovery rate on the third and the fifth day may be useful parameter to evaluate the platelet survival time. The increase in platelet count and the prolongation of platelet survival time suggest that small dose of aspirin with dilazep may prevent thrombo-embolic complications in these patients with prosthetic heart valves under warfarin treatment.

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Progress in implantable artificial organs such as the artificial heart, artificial vessele and the artificial kidney depends greatly on the development of excellent antithrombogenic materials because prevention of thrombosis is indispensable for the successful utilization of these artificial organs. In order to design excellent antithrombogenic materials, it is important to clarify the effects of the functional groups of the material surfaces on the surface-induced activation of platelets.
For this purpose, poly (styrene) (PSt) (nonionic, hydrophobic polymer), and poly (p-dimethylaminoethylstyrene) (PMAS) (weakly cationic polymer) were prepared, and the adhesion of rat platelets to these polymer surfaces was examined. Albumin-coated as well as non-coated polymer surfaces were used in this experiment.
Platelets were collected from 5 rats by cardio-puncture, and were suspended in Hanks' balanced salt solution (Ca²⁺, Mg²⁺ free). The interaction of the platelets thus prepared with the polymer surfaces was studied by the microsphere column methed at 4°C and at room temperature. The morphological changes of the adhering platelets on the polymer surfaces were observed by scanning (SEM) and transmission electron-microscopes (TEM).
Platelets adhered to the albumin-coated as well as non-coated PMAS surfaces at room temperature; they were rough-surfaced, round in shape with a few long pseudopods. TEM observation revealed that the platelets on these PMAS surfaces had retained more organella such as α-granules than those on the non-coated PSt surface, which had a spreading form. Rounded platelets were observed on the PSt surface with albumin-coating. Similar results were obtained for experiments done at 4°C. Despite their distinct morphology, there was no significant difference in the percent retention of platelets between PSt and PMAS.
Thus, it is suggested that cytological changes of the platelets do not always coincide with adhesivity of the platelets to the material surface. Consequently, this study revealed that the albumin-coated as well as non-coated PMAS surfaces with a weakly cationic amino group have a high adhesivity to platelets but have lower tendency to activate platelets than the nonionic non-coated PSt surface.

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Effects of cyclic nucleotides on PGI₂ generaton were investigated with references to Ca⁺⁺ kinetics using cultured human vascular endothelial cell. Endothelial cells were isolated and cultured by the modified method of Jaffe et al. 6-keto PGF_1α and cyclic nucleotides concentration were measured by RIA. Ca⁺⁺ kinetics or cytosolic free Ca⁺⁺ concentration ([Ca⁺⁺]_i) was measured using ⁴⁵Ca or a new fluorescence indicator, Fura 2. Ca ionophore A23187 increased ⁴⁵Ca uptake and release, or [Ca⁺⁺]_i, and it also enhanced PGI₂ generation. The pretreatment of the cells with a Ca⁺⁺ immobilizer, TMB-8 decreased all of them. Dibutyryl cAMP or 8-bromoc GMP increased intracellular cAMP or cGMP concentration, respectively. While dibutyryl cAMP increased ⁴⁵Ca uptake or release, and decreased [Ca⁺⁺]_i, 8-bromo cGMP decreased ⁴⁵Ca uptake, ⁴⁵Ca release, and [Ca⁺⁺]_i. And administration of both these cyclic nucleotides analogues decreased PGI₂ generation. Through these results it was concluded that: 1) PGI₂ generation of cultured human vascular endothelial cells was brought about by the increase of [Ca⁺⁺]_i via the increase of Ca⁺⁺ uptake or the succeeded enhancement of the release of Ca⁺⁺ from the storage sites. 2) The direct evidence was shown that PGI₂ generation was suppressed by the decrease of [Ca⁺⁺]_i via the increase of intracellular cyclic nucleotides concentration.

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In our previous paper (1), the coagulation-fibrinolytic and kallikrein-kinin systems in the utero-placental circulation and amniotic fluid was studied in the full term pregnancy before and during labour. We observed thatc in amniotic fluid, hypercoagulability with secondary hyperfibrinolysis and the production of kinin increased significantly during labour. However, these systemic information based on multifactorial and stage dependent analysis have not been investigated.
In this study, the amniotic fluid was collected by amnioticentesis from 72 pregnant women in 17-20 weeks of gestation as a control group and full term women before and during labour (Table 2). We tried to find out the changes of plasminogen activator (urokinase and tissue plasminogen activator), α₂-plasmin inhibitor (TD-80), α₂PI-plasmin-complex (TD-80C), D-dimer, glandular kallikrein activity and quantity (anti-human urinary kallikrein antibody), kallikrein inhibitor and kininase activity (Table 1).
1. Coagulation-fibrinolytic system; The levels of α₂-PI was high (1.04±0.21μg/ml, M±SE) and unchanged during preg., but slightly decreased during labour (0.93±0.11μg/ml, M±SE). This finding indicated the fibrinolytic system was depressed on the whole, while the homeostatic plasmin activity in amniotic fluid was exhibited by multifactorial analysis.
1) Control group; The levels of UK, α₂PI-Pm-C and Bβ15-42 were slightly increased, and D-dieter increased significantly as compared to the full term preg. These finding suggested that the secondary fibrinolytic activity was markedly increased after the hypercoagulable state.
2) Full term preg; The low levels of UK, α₂PI-Pm-C, Bβ15-42 and Ddimer showed that the coagulable and fibrinolytic system was depressed.
3) During labour; The levels of UK, Bβ15-42, α₂PI-Pm-C and D-dimer were significantly increased and α₂PI wes slightly decreased. This pattern suggested the hypercoagulability with secondary hyperfibrinolytic activity.
2. Kallikrein-kinin system, The levels of kallikrein activity was unchanged during normal preg. but slightly increased during labour, and kall. quantity was markedly increased in full term preg. (20.9±4.9ng/ml, M±SE) and significantly increased during labour (32.3±5.8ng/ml, M±SE) as compared to the control group (≤4.0ng/ml, M±SE). The levels of kall. inhibitor and kininase activity were significantly decreased, and LMW-, HMW-kininogen (1) were markedly decreased in full term preg. before and during labor (kall. inhibitor 0.2±0.1%, kininase activity 592.8±103.1pg/min/ml, M±SE) as compared to the control group (kall. inhibitor 41.7±7.2%, kininase activity 2099.5±265.9pg/min/ml, M±SE). These findings suggested that with the significantly increased kall. quantity and decreased kall. inhibitor and kininase activity during labor exhibited the hyperproduction of kinin.

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We examined 1, 479 pilot plasmas of blood donors, 171 cases of cerebral infarction and 37 cases of SLE for their activities and antigen levels of plasminogen. M±SD of the activity of donor blood was 83.5±17.3% and that of antigen level was 111±19μg/ml, i. e. 86.7±14.8%. The mean activity-antigen ratio (AC/AG) was 0.96±0.15. It was found that 82 cases had plasminogen activity lower than 60%, and finally 50 cases (3.8%) were diagnosed as abnormal plasminogen as they had AC/AG less than 0.66. Zymogram and immunostaining after IEF of these specimens revealed that the phenotype of one case was homozygote (type 1′-1′) and those of 49 were heterozygotes (type 1-1′). The incidences of the abnormality were not statistically different between three separate prefectures. The incidence of this abnormality was 4.7% (8/171) in cases of cerebral infarction and that in SLE was 10.8% (4/37). It was concluded that abnormal plasminogen is regarded not as the primary causative factor but the predisposing one of thromboembolism. High risk of thrombosis develops when abnormal plasminogen is complicated with any form of vascular damages, such as trauma, operation and inflammation.

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This study describes the levels of urinary-fibrinopeptide A/creatinine (FPA/CRE), fibrin-derived peptide Bα15-42/creatinine (Bα15-42/CRE) and the kallikrein-kinin system in normal pregnancy, labor and puerperium. To understand the roles of u-FPA/CRE, Bα15-42/CRE and the kallikrein-kinin system in the kidney all components of the system and localization need to be considered.
Levels of u-FPA/CRE increased gradually from midterm of pregnancy. U-β15-42/CRE increased from early stage of preg, especially in late stage of pregnancy (FPA/CRE=0.9±0.6ng/mg, Bα15-42/CRE=12.2±3.7ng/mg). Levels of u-FPA/CRE and Bα15-42/CRE in the 2nd stage of labor (FPA/CRE=1.2α0.7ng/mg, Bα15-42/CRE=23.1±15.6ng/mg) were significantly increased when compared to late stage of pregnancy.
These pattern closely resembled that the intrarenal vascular hypercoagulation and hyperfibrinolytic activity and/or the increasing of renal plasma flow (RPF) and glomerular filtration rate (GFR) appeared during pregnancy and labor.
Levels of u-kallikrein activity/CRE and quantity/CRE significantly increased in midterm of pregnancy (u-kallikrein activity/CRE=2.3±0.9ng/min/mg, u-kallikrein quantity/CRE=406.2±217.5ng/mg), but gradually decreased (u-kallikrein activity/CRE=1.0±0.7ng/min/mg, u-kallikrein quantity/CRE=160.3±125.7ng/mg). Levels of u-kallikrein activity/CRE and quantity/CRE in the full term pregnancy and 2nd stage of labor were slightly decreased when compared to non-pregnant women. Levels of u-kinin/CRE during pregnancy markedly increased in 12-16 weeks of pregnancy (u-kinin/CRE=17.5±10.3ng/mg) and gradually decreased from midterm to late stage of pregnancy. Levels of u-kininase activity/CRE and kininase-II quantity/CRE gradually decreased from early stage to midterm pregnancy and slightly increased in late stage of pregnancy, but not significantly. Levels of u-kininase activity/CRE were markedly increased (u-kininase activity/CRE=23.4±12.5pg/min/mg), and u-kininase-II quantity/CRE were decreased (u-kininase-II quantity/CRE=4.4±1.4x10^-3IU/min/mg) in the 2nd stage of labor when compared to non-pregnant women.
These findings suggested that availability of renal kallikrein activity and quantity and kininase activity and kininase-II quantity in pregnancy, labor and puerperium were major determinants of levels of kinin in the kidney.

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The changes in FDP, FDP-D, crosslinked fibrin degradation products (D-dimer) and α₂plasmin inhibitor (α₂PI) during fibrinolytic therapy with urokinase (36-96x10⁴IU) were investigated in 10 patients with thromboembolic diseases (2 with acute myocardial infarction, 2 cerebral infarction, 3 deep vein thrombosis, 1 left ventricular thrombus, 1 thrombotic artificial heart valve and 1 pulmonary thromboembolism).
Immediately after urokinase infusion, D-dimer increased to 200-1000ng/ml with a marked decrease in α₂PI in all four patients who had undetectable D-dimer (<200ng/ml) before the infusion. In three patients D-dimer increased from 4500ng/ml to 6000ng/ml, from 1000ng/ml to 2000ng/ml and from 500ng/ml to 4000ng/ml. The elevation of D-dimer was associated with clinical effectiveness in these patients. In additional three patients, in whom D-dimer was assayed 24-48 hours after the infusion, D-dimer it was undetectable. Serum FDP, assayed by a currently used method, did not show any change in most patients. The results indicate that mesurement of D-dimer is useful to evaluate the in vivo thrombolysis during fibrinolytic therapy with urokinase.

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By an enzyme-linked immunosorbent assay (ELISA), plasmin-α₂ plasmin inhibitor (Plm-α₂PI) complex, an indicator of in vivo plasmin generation, was measured in plasma from 48 patients with disseminated intravascular coagulation (DIC), 10 patients with thrombotic thrombocytopenic purpura (TTP), one patient with primary fibrinolysis and 11 patients (15 episodes) with thromboembolic disorders during fibrinolytic therapy with urokinase. Urokinase was infused at an initial dose of 60, 000-960, 000 units, mostly by a bolus infusion followed by a drip infusion for 1-2 hours. In DIC, Plm-α₂PI complex was markedly elevated to 7.9±4.26mg/l (mean±SD), normal values being less than 0.8mg/l. Its level changed in parallel with the course of DIC, but remained high (1.9±1.49mg/l) in remission when FDP values became less than 10mg/l with normalization of fibrinogen concentration. Among various underlying disorders, DIC patients with acute promyelocytic leukemia had the highest levels (mean 10.8mg/l) and septic patients had the lowest (mean 3.4mg/l). In TTP, the elevation of Plm-α₂PI complex was modest (1.8±0.95mg/l). Plm-α₂PI complex was also elevated in a patient with primary fibrinolysis, and its level decreased following the administration of t-AMCHA. Following urokinase infusion, Plm-α₂PI complex increased in all patients except in one case who received a low dose of urokinase (60, 000 units). However, changes in cross-linked fibrin derivatives (XDP) were variable among the patients and were independent of the increase in Plm-α₂PI complex. These results indicate that the quantitative assay of Plm-α₂PI complex would be valuable for the assessment of hyperfibrinolysis in selected disease states. Simultaneous measurements of XDP and Plm-α₂PI complex would be useful to monitor the thrombolysis during fibrinolytic therapy.

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Biochemical activities of the human platelet vascular permeability factor (HVPF) and platelet-derived growth factor (PDGF) were studied on rabbit skin and on in vitro cell culture in the present experiments. HVPF was extracted from human platelet α-granules, while PDGF was obtained from the α-granule lysate.Commercial PDGF was used as a control of the lysate. Vascular permeability was estimated by ¹²⁵I-albumin or chromatoscanner method and its effects were observed by means of electron microscopy. Immediately after injection, all of the factors induced vascular permeability response followed by inflammatory cell infiltration to almost the same extent. At 1 hour, the injected skin site showed an increase of radio activity and bluing. Growth activity was determined by either counting the cell number or estimating the uptake of ³H-thymidine by K 562, Balb/c and NIH 3T3 fibroblast in the culture well. Following the addition of either factors, the cell had proliferated and the uptake of ³H-thymidine was increased in degree. Both HVPF and lysate were heat-stable at 100°C for 10 minutes, but were inactivated by pepsin or 2-mercaptoethanol.
In conclusion, HVPF and PDGF including the commercial one are quite similar in respect of the biological nature. Therefore, it is suggested that the factors play a role not only in the inflammation, but also in the repair of tissue injury.

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Red cell deformability was measured from the red cell filtration rate (RFR) in the patients of peripheral vascular diseases, using a 5μm Nuclepore microfilter and 20% Ht red cell suspension.
In the patients of ASO, TAO and Raynaud's syndrome, the red cell deformability was cleared to be reduced significantly, comparing with age-matched controls.
Furthermore, the effects of PGE₁, PGI₂ analogue (OP-41483) and TXA₂ blockade (OKY-046) on red cell deformability in peripheral vascular disease patients were studied.
PGE₁ was given intravenously 11 patients (80-120μg/day, 5-14 days), and reduced red cell deformability (23.8μl/sec) was improved (47.1μl/sec) significantly (p<0.01).
PGI₂ analogue was also given to 16 patients (20μg, 40min) intravenously, and pre-treated value was 37.9μl/sec, while post-treated value increased to 48.8μl/sec (p<0.01).
TXA₂ blockade was given orally (600mg/day, 6 weeks) to 13 patients and red cell deformability was improved significantly (from 46.5 to 58.8μl/sec, p<0.01).
The study showed that PGE₁, PGI₂ and TXA₂ blockade were significantly effective on reduced red cell deformability, besides the effectiveness as an anti-platelet agent and a vasodilating agent.
These prostanoids were suggested to play an important role in keeping a good microcirculation in the body.

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Factor XIa (F. XIa) is inactivated with either α₁ antitrypsin (α₁AT) or antithrombin III (AT III). To assess the contribution of α₁ AT and AT III to the inactivation of F. XIa, we assayed F. XIa-α₁AT complex and F. XIa-AT III complex in normal serum and in plasma of the patients with disseminated intravascular coagulation (DIC), in which F. XIa and plasma protease inhibitors were coexisting.
In serum, we could detect more F. XIa-AT III complex than F. XIa-α₁AT complex. Next, we measured the levels of both complexes in the patients with DIC originated from various triggers. F. XIa-α₁AT complex level was always higher than F. XIa-AT III complex level in each patient, irrespective of the administration of heparin and the levels of F. XI, α₁AT and AT III.
These results indicated that the contribution of α₁AT to the inactivation of F. XIa was greater than that of AT III in vivo.

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Dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells were transfected with expression plasmid constructed from human tissue-type plasminogen activator (t-PA) and DHFR gene. The transfected cells were selected with a medium containing methotrexate (MTX, 0-5μM), followed by isolation of several DHFR⁺ clones. It was definitely obserbed that the expression levels of t-PA were dose dependently elevated on the MTX resistance and the gene copy number. This recombinant t-PA had a similar specific activity and the same N-terminal amino acid sequence to human melanoma cell derived t-PA, and also showed similar characteristics in a neutralization test with a human t-PA, SDS-PAGE, PAS staining, zymography and human plasma clot lysis time assay. The MTX-selected high expression clone could keep constant production of t-PA without MTX for at shortest 5 months. These results strongly suggestted that CHO cells-DHFR gene amplification system was useful for producing a large amount of human glycoprotein with a complicated structure such as t-PA.

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A human melanoma cell line (Bowes) secretes tissue-type plasminogen activator (t-PA). In order to elucidate the mechanism of t-PA secretion, we analyzed intracellular distribution of t-PA by fractionating cell organelles of melanoma cells with discontinuous sucrose density gradient ultracentrifugation method, and investigated the effect of monensin, a secretion depressing agent, on the intracellular distribution of t-PA. The t-PA was determined by the radioactivity adsorbed to anti-t-PA IgG-Protein A-Sepharose. The distribution of plasminogen activator (PA) activity was similar to that of cellular protein concentration when they were separated by the ultracentrifugation. Enzymography showed that the major intracellular PA activity was present at 72kDa and the minor, activity at 50kDa. When t-PA secretion was depressed by monensin, intracellular PA was accumulated, especially the 72kDa component predominantly. After longterm (3h) simultaneous labeling with ³⁵S-methionine and ³H-mannose, the ³H/³⁵S ratio of t-PA increased in the presence of monensin. Pulse labeling experiments showed that intracellular t-PA was transported from the high density fraction to the low density one and reached a maximum concentration at 30min. Labeled t-PA began to appear in the culture medium in about 30min. These results suggest that t-PA secretion mechanism is similar to that of other secretory glycoproteins and that about 30 minutes are necessary from the ingestion of methionine to the secretion of t-PA.

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