Vasodilating activity of nitroglycerine (NTG) has not been completely analyzed so far. Recently it is reported that its mode of action resembles Ca antagonists. We have reported that NTG enhances the PGI₂ generation in cultured vascular endothelial cells. This paper reports the effects of NTG on the PGI₂ generation with reference to cyclic nucleotide metabolism. Endothelial cells isolated from human umbilical veins were cultured by a modified method of Jaffe et al. The released 6-keto PGF_1α, in the medium and intracellular cAMP level were assayed by RIA, and 6-keto PGF_1α level was employed as a parameter of PGI₂ generation. PGI₂ generation induced by NTG in the presence of arachidonic acid was diminished when the endothlial cells were pretreated by phospholipase A₂ inhibitor mepacrine. This results suggests that the stimulation of PGI₂ generation by NTG was dependent on the activation of the phospholipase A₂ level. The PGI₂ generation induced by A-23187 or NTG was markedly reduced by preincubating the cells with TMB-8. Intracellular Ca⁺⁺ was observed to affect mainly the early steps of arachidonic acid cascade in the generation of PGI₂. The augmented PGI₂ generation by A-23187 or NTG disappeared in the Ca⁺⁺ free medium. In the presence of MIX, intracellular cAMP level was increased and PGI₂ generation was decreased. NTG had no effect on the content of cAMP in this experimental system. These results indicate that the increase in PGI₂ generation in the vascular endothelial cells by NTG may contribute to the vasodilating activity chiefly through the activation of phospholipase A₂ in the arachidonic acid cascade, without influencing on the level of intracellurar cAMP.

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Polymeric material which has microphase separated structure is a most promising candidate for antithrombogenic material.
In order to investigate the role of microphase separated structure on the interaction between polymers and platelets, HEMA-St ABA type block copolymers with microphase separated structure which is composed of hydrophilic 2-hydroxyethylmethacrylate (HEMA) and hydrophobic styrene (St) were prepared. By changing mole fraction of HEMA in copolymers, block copolymers with the following three types of microphase separated structure were obtained: (1) isolated hydrophilic islands in continuous hydrophobic phase (sea-island microstructure), (2) lamellar microstructure which is composed of alternative hydrophilic and hydrophobic phases, (3) isolated hydrophobic islands in continuous hydrophilic phase reversed sea-island microstructure.
Rat platelets suspended in Hanks' balanced salt solution (Ca⁺⁺, Mg⁺⁺ free) were used for the experiment. Interaction of platelets with polymer surface was studied by microsphere columnar method. The morphological changes of adhering platelets on polymer surfaces were investigated by transmission and scanning electron microscopy.
Platelets adhered to the surface of lamellar microstructure have smooth surface with round shape and short pseudopods, indicating that the morphological changes are less marked than those of platelets adhered to the surfaces of both sea-island and reversed sea-island microstructure. Although platelets attached to the surfaces of the three types of microphase separated structures retained organella such as α-granules and mitochondria relatively well, extended open canalicular system was observed in platelets adhered to the surfaces of sea-island and reversed sea-island microstructure. These findings suggest that the surface of lamellar microstructure has an inhibitory action on platelet activation as compared with the surfaces of sea-island and reversed sea-island microstructure.
Thus, the mode of platelet adhesion was found to be greatly influenced by the domain shape and size of microphase separated structure. It is concluded that the morphology of the microphase separated structure is an important element for the molecular design of excellent antithrombogenic material.

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Heparan sulfate glycosaminoglycans released from endothelial cells have been demonstrated to have an anticoagulant potency. In the present study, the effect of thrombin on the release of ³⁵S-labeled glycosaminoglycans on the cultured endothelial cell surface has been examined. Thrombin significantly enhanced the release of ³⁵S-glycosaminoglycans in a dose-dependent fashion. At 10unit/ml of thrombin, the amount of released glycosaminoglycans was twice as much as that released by Hanks' solution alone. The release by thrombin reached a maximum up to one hour of incubation at 37°C. When thrombin was modified by diisopropylfluorophosphate or co-incubated with hirudin, its effect was completely abolished. Characterizations of ³⁵S-glycosaminoglycans showed that they consisted of heparan sulfate. A major part of ³⁵S-glycosaminoglycans released by thrombin was trichloroacetic acid-precipitable. Thrombin did not cause the release of ⁵¹Cr from radiolabeled endothelial cells.
These results indicate that thrombin can release heparan sulfate proteoglycans on the endothelial cell surface without any damage on the cells. Its effect was dependent on the dose and proteolytic activity.

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In order to study a relationship between blood viscosity and miscelaneous blood components in patients with cerebrovascular disease (CVD), whole blood and serum viscosity, hematocrit, γ-globulin, total protein, albumin and lipids were measured in 113 patients with CVD (cerebral thrombosis 90, TIA 23) and 31 patients with non-CVD neurological diseases served as control.
1) Whole blood viscosity, especially at the shear rate of 18.75 and 376sec^-1, was correlated with hematocrit (r=0.52).
2) γ-globulin was correlated with plasma viscosity (r=0.52) and whole blood viscosity (r=0.36 at 18.75sec^-1 and r=0.31 at 75.2sec^-1). Thus serum γ-globulin was thought to be another factor for increasing blood viscosity.
3) Thrombosis of carotid system and TIA were associated with the increase of whole blood viscosity and hematocrit. However thrombosis of vertebrobasilar system were not associated with them.
4) It is suggested that the increased blood viscosity plays a role on thrombosis of carotid system but not of vertebrovasilar system.

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Erythrocyte participate in low blood filtrability by their decreased stroma ATP content and quality changes in the membrane constituents. This study was intended to clarify fundamental association of erythrocyte membrane proteins with blood filtrability by the observation of changes of erythrocyte membrane proteins following the storage of whole blood added ACD solution. Blood specimens were drawn from 12 healthy volunteers aged 30 to 43 years. Hemorheological and biochemical determinations were carried out immediately after blood collection, on 1, 3, and 6 day after storage at 4°C. Blood filtrability determined by the method of Reid et al. and whole blood glucose decreased on 1, 3, 6 day. Levels of total erythrocyte membrane protein and spectrin 1 decreased on 6 day. Deformability index was directly proportional to the concentration of spectrin 1 in connection with contents of spectrin 2 and 4.1 fraction on blood collection as shown in previous articles. The storage of blood specimens resulted in the gradual disappearance of the above mentioned relations. The index was in direct proportion to the level of syndein affected by whole blood glucose and glycophorin on 6 day. The results obtained indicate that much attention should be raised to syndein other than spectrin in clinical hemorheology dealing with erythrocyte membrane proteins.

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The amounts of TXB₂ and PGD₂ which were produced during spontaneous platelet aggregation (SPA) in 3 patients with primary thrombocythemia (PTH) were measured. After platelet counts in platelet-rich plasma (PRP) were adjusted to 120×10⁴μl with autologous platelet-poor plasma, SPA was induced by stirring at 1100 r. p. m. in a Lumi-aggregometer and the lag time was measured. The amounts of TXB₂ and PGD₂ produced in the aliquots of PRP were measured by HPLC, using 9-anthryl-diazomethane. As a control, the amounts of TXB₂ and PGD₂ which were produced by the reaction of platelets with arachidonate (0.2mM, f. c.) were measured by similar method, using the platelets from 4 normal subjects (platelet count: 30×10⁴μl).
The amounts of TXB₂ and PGD₂ in normal subjects were 153.0±18.9, and 2.88±0.36ng/10⁸ platelets (mean±S. D.), respectively (TXB₂/D₂=43.4 to 68.3). The amounts of TXB₂ and PGD₂ in PTH were as follows: case 1, 48.5 and 0.37; case 2, 56.2 and 1.14; case 3, 53.6 and 0.93 (ng/10⁸ platelets), respectively (TXB₂/D₂=131.1, 49.3, 57.6). In case 1, the amount of PGD₂ was decreased, and TXB₂/D₂ was significantly increased. In this case, it was also found that SPA occured in a shorter lag time than in the other cases. It was suggested that the increased TXB₂/D₂ may play some role in the mechanism of SPA in patients with PTH.

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Plasma thromboxane B₂ were investigated in various diseases; arteriosclerosis obliterans (ASO), thromboangitis obliterans (TAO), abdominal aortic aneurysm (AAA) and malignant diseases. TxB₂ changes due to arterial recostructive surgery and general surgery were invetigated.
The results obtained were as fallows.
1) TxB₂ level was significantly increased in the patients with TAO or malignant diseases, and was increased in ASO, AAA compared with healthy control.
2) TxB₂ was significantly increased during operations and postoperative periods in arterial reconstructive surgery and general surgery.
3) There was significant positive correlation between TxB₂ and β-TG (p<0.01).
4) TxB₂ was elevated during operations, so it suggests that platelet activity was vivid during operations.

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CS-570, a chemically stable prostacyclin derivative, inhibited decrease of platelet counts and fibrinogen level, increase of FDP, prolongation of prothrombin time and activated partial thromboplastin time and deposition of fibrin in the renal glomeruli dose-dependently in the endotoxin-induced DIC model of rats. Ticlopidine, dazoxiben (thromboxane synthetase inhibitor) and BM 13177 (thromboxane antagonist) also showed inhibition of changes in these parameters. CS-570, a potent platelet stabilizer and vasodilator, would probably have inhibited thrombocytopenia and coagulation disorders by physiological antagonism against TxA₂ produced by endotoxin. This would suggest beneficial effects of CS-570 in endotoxin-induced DIC in humans.

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It is known that the number of platelets decreases after burn injury. However, the mechanism of such a decrease and its influence on systemic organs have not been fully elucidated. In this study, we evaluated the efficacy of OP-41483 (a derivative of the PGI₂) in burned rabbits with special reference to its association with renal function. Third-degree burns, covering 35% of the total body surface area, were experimentally produced on the back of rabbits. Among the following five groups of rabbits (6 rabbits each), the time courses of renal function and platelet function were compared; Group I (no treatment), Group II (fluid therapy only), Group III (treated with OP-41483+fluid therapy; a: 50ng/kg/min, b: 75ng/kg/min, c: 100ng/kg/min).
The results show that in Group I, all rabbits died after 8 hours. Renal function testes in this group showed a decrease in creatinine clearance and increases in FeNa and CH₂O. Such changes were improved in Groups III-b, III-a and II (in order of the degree of improvement). Group III-c showed no improvement. As for the platelets the decrease in platelets was the smallest in Group III-b, where platelets showed no decrease until 8 hours after injury and fibrinogen registered a satisfactory course of increasing. No improvement was found in Group III-c by our tests, presumably owing to an overdose of OP-41483. The optimal dose of OP-41483 for improving the renal function of burned rabbits was 75ng/kg/min in our study.

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The enhancement of cytoplasmic Ca²⁺ level upon stimulation is known to trigger the rapid response of secretory cells, such as platelets. The role of extracellular Ca²⁺ on activation of phospholipases A₂ and C activities was studied.
In the presence of 1mM EGTA, the level of platelet aggregation by thrombin was reduced to 20% of that in the presence of 1mM CaCl₂. The EGTA-suppressed platelet restored aggregability by addition of extra Ca²⁺ concentration. Thrombin (0.3U/ml)-induced serotonin release assessed at 1min was independent of the concentrations of EGTA or CaCl₂. The increased phosphatidylinositol (PI) turnover was observed as inferred by a decreased level of PI and increased 1, 2-diacyl glycerol and phosphatidic acid in [³H] arachidonic acid (AA)-labeled cells stimulated with thrombin (0.1 and 1U/ml). The increment of radioactivity in arachidonate and its metabolites was accounted for by the loss of [³H] phosphatidylcholine (PC) independent of the extracellular Ca²⁺ The degradation products of phospholipase A₂, lysoPI and lysoPC were detected in stimulated cells with or without Ca²⁺ in the medium. Extracellular Ca²⁺ levels do not affect the degradation or resynthesis of phosphoinositides, PI, PI-4-monophosphate (DPI), and PI-4, 5-bisphosphate (TPI). These results indicate that the activation processes of phospholipases A₂ and C in thrombinstimulated platelets are independent of extracellular Ca²⁺ level.

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Flow cytometric analysis was done for the detection of rabbit anti-guinea pig platelet antibody on guinea pig platelets and megakaryocytes. Antiserum, absorbed by guinea pig erythrocytes, was intraperitoneally administered.
Antibody-binding to platelets was strongly detected 1.5 and 3h after injection of 2ml/kg body weight of antibody followed by severe thrombocytopenia after 3h. Apparent antibody-binding to megakaryocytes obtained by centrifugal elutriation was detected 12 and 36h later. The number of megakaryocytes was not changed. Injection of 0.4ml/kg of antibody caused mild thrombocytopenia with manifest antibody-binding to platelets, and no apparent binding to megakaryocytes.
These findings suggest that platelet-associated antibody caused thrombocytopenia and that serum antibody reached to megakaryocytes. But small amount of antibody had different attitude on antibody-binding to platelets and megakaryocytes.

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In order to clarify the relationship between the platelet shape and the disease abnormality, the circualting form and its change in response to agonists were examined in various primary platelet abnormalities. Patients consisted of thrombashenia (T) 3, essential athrombia (E) 2, Bernard-Soulier syndrome (BS) 1, May-Hegglin anomaly (MH) 2, Hermansky-Pudlak syndrome (HP) 1, platelet cyclo-oxygenase deficiency (CD) 1, release mechanism abnormality characterized by defective A23187-aggregation (A) 1, and aspirin-ingested volunteers (AS) . The circulating form was examined by our method using light microscopy. Shape change was induced by incubation of native platelet suspension from the subjects with various concentrations of either ADP (-10^-5M, 1min), thrombin (-2^-1U/ml, 30sec) or arachidonate (-2^-1mM, 2min).
Striking spherification and pseudopod formation were noted in circulating form in BS, a mild one in some of T and E, and a slight spherification in MH, whereas not in the others. Shape change induced by ADP was inhibited in none, and that by arachidonate was completely inhibited in CD (as previously reported ¹⁴), AS, HP and 2 out of 11 normal subjects. Thrombin-induced change was inhibited only in BS probably in agreement with its defect of glycoprotein Ib as a possible receptor of thrombin.
Thus the circulating form and its change response to some agonists differed among various platelet abnormalities. This indicates the necessities for clarifying the mechanism of shape change and for investigating clinical usefulness of shape (change) determination.

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In order to clarify the effect of the sympathetic nervous system on the prethrombotic state in atrial fibrillation, systolic and diastolic blood pressure, heart rate, platelet count and plasma norepinephrine were measured before and after postural change (sitting from bed rest) in cerebral infarction with and without atrial fibrillation (AfCI and CI).
For the study of platelet function, platelet aggregation induced by 4.6μM ADP, plasma β-thromboglobulin and platelet factor 4 were measured.
Systolic blood pressure and platelet count did not change significantly by sitting in AfCI, while significantly increased in CI (p<0.05 and p<0.01 respectively). On the contrary plasma norepinephrine significantly increased in AfCI (p<0.05), but did not change significantly in CI. There were no significant changes by sitting in diasolic blood pressure and heart rate in both AfCI and CI.
Increases in plasma norepinephrine by sitting had significant correlations with platelet aggregation induced by 4.6μM ADP (p<0.01), plasma β-thromboglobulin (p<0.01) and platelet factor 4 (p<0.05) in cerebral circulatory disorders. These results indicate that low cardiac function in atrial fibrillation may induce high response of the sympathetic nervous system by postural change and this would promote platelet consumption.

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Hemorrhagic diathesis is often caused by antineoplastic drugs, especially the Anthracyclines used in the treatment of malignancy. In this respect, we investigated the effect of Anthracyclines, including Adriamycin (ADM), Daunomycin (DM), and Aclacinomycin (ACR) on platelet function and metabolism.
All these drugs inhibited ADP, collagen, and epinephrine-induced platelet aggregation in dose-dependent manner and the intensity of inhibition was DM, ADM, and ACR in order. ATP and ¹⁴C-5HT release reactions were also reduced by Anthracyclines in proportion to their inhibitory effects on the second phase of ADP-induced aggregation.
Coenzyme Q₁₀ (Q₁₀), known to alleviate such adverse action as cardiotoxicity and to increase the antineoplastic efficacy of ADM was shown to enhance the antiplatelet effects of ADM.
Thin layer radiochromatographic analysis of platelet arachidonate metabolism revealed that ADM at a high concentration cauced slight inhibition of thromboxane synthesis. Furthermore, the liberation of radioactivity from ¹⁴C-arachidonate-labeled platelets in response to collagen was reduced by ADM. Cyclic nucleotides levels in platelets incubated with ADM or Q₁₀ did not show any significant change.
In conclusion, it is suggested that impairment of platelet functions by Anthracyclines is partially attributable to the decrease of phospholipase activity, but the precise mechanism remains unexplained.

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Monoclonal antibodies against von Willebrand factor (vWF) were produced by immunization of BALB/C mice with FVIII/vWF fraction and fusion of spleen cells with NS-1 murine myeloma cells. Antibody producing hybridomas were detected by an enzyme linked immunosorbent assay (ELISA). Five cloned monoclonal antibodies obtained in this method were all belonged to IgG₁ class, and inhibited platelet retention by glass beads (Bowie et al.). The inhibitory action was most remarkable in 3B11. The antibody titer against vWF: Ag ranged from 1×10⁵ to 1×10⁷ by an ELISA. 3B11 inhibited ristocetin induced platelet aggregation and ristocetin cofactor up to 1×10⁴. VIII protein and vWF protein were separately eluted from FVIII/vWF fraction by immunoadsorbent chromatography using monoclonal antibody. A new ELISA method was developed using each monoclonal antibody to quantitate vWF: Ag. The lower limit was 0.001u/ml. In order to detect vWF: Ag in tissues immunoperoxidase staining by avidin-biotin system using monoclonal antibody was performed. Positive staining was obtained in the endothelial cells of the neonatal umbilical cord, washed platelets and megakaryocytes.

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Danazol, a derivative of 17α-ethinyl testosterone which is a synthetic steroid with antigonadotropic properties has been used in the treatment of endometriosis. The efficacy of Danazol due to long-term administration resulted in a reduction of menstrual blood loss. The purpose of this investigation was to study not only the influence of Danazol on clotting and fibrinolytic activities for patients with endometriosis, but also to know that Danazol therapy may decrease menstrual blood loss in patients with hereditary hemorrhagic disease as reported by Soma et al.
Results: 1) There were steady rises in F VIII and F IX after 4 months administration of Danazol, while no rises of such factors were observed in women given androgen. 2) No significant increase of platelets and other clotting factors was observed during Danazol treatment, even though a transient decrease of fibrinogen was seen. 3) No significant changes of fibrinolytic activities such as α₁-AT, α₂-MG, AT-III and C₁-INA were observed. 4) Plasma levels of kallikrein inhibitor at the onset of menstruation in women treated with Danazol was higher than the control group. 5) However, in vitro changes of clotting as well as fibrinolytic activities depending upon concentrations of Danazol were not evident.
In conclusion, Danazol therapy on coagulation and fibrinolytic factors was different from the effect of Pill treatment. Therefore, it will be applicable to clinical use of the treatment of hemorrhagic disorders.

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The endothelial cell is a rich source of plasminogen activator that is associated with fibrinolytic activity in blood vessel. Addition of fucosterol or sitosterol to the culture medium of endothelial cells from bovine carotid artery gave rise to a marked increment in the intracellular and extracellular activities of plasminogen activator. Removal of fucosterol or sitosterol from the culture medium resulted in a decrease of plasminogen activator activity back to normal levels. No enhancement of plasminogen activator activity in cultured endothelial cells was observed by cholesterol, 5-androsten-3β-ol and other steroids and sex hormones including androsterone, testosterone, estrone and estradiol. Synthesis of plasminogen activator in cells induced with fucosterol or sitosterol was inhibited by protein synthesis inhibitor, cycloheximide. The plasminogen activators produced in cells were classified into urokinase-type activators with molecular weights of 31, 000 and 55, 000 and tissue-type ones with molecular weights of 81, 000 and 130, 000, which were identified with respective antibodies. The synthesis of each type of plasminogen activator in endothelial cells was stimulated by fucosterol or by sitosterol.

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Changes in euglobulin lysis time (ELT) and Bβ15-42 peptide were investigated on 14 patients with benign disease and 24 with malignant disease before and after operation. ELT prolonged significantly in patients with malignant disease compared to one with benign disease on the first and second postoperative day.
A significant increase of Bβ15-42 peptide was found in patients with malignant disease compared to patients with benign disease, though a definite increase was found in the latter group compared to healthy control. No significant difference in Bβ15-42 peptide was found between both patient groups postoperatively, though it increased with linear pattern.
The same investigation was performed on patient who had intermittent compression of the legs for 48 hours postoperatively. Shortning of ELT was found in them compared to control. No significant difference in Bβ15-42 peptide was found between both groups, though it increased with linear pattern in both postoperatively. It suggests that no influence on activity of plasmin was produced by leg compression.

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Two species of plasminogen activator (PA) inhibitors (PA-I₁ and PA-I₂) were purified from human cornified cell extract by use of Fast Protein Liquid Chromatographic columns, Mono Q (high performance anion-exchanger) and Mono P (high resolution chromatofocusing). Both PA-I₁ with a molecular weight (MW) of 66, 000 and PA-I₂ with MW 45, 000 showed inhibition against urokinase (UK). However, different inhibition mechanisms were demonstrated for these inhibitors. E-I complex between PA-I₁ and UK was found to be dissociable by SDS-polyacrylamide gel electrophoresis. Whereas, PA-I₂ formed undissociated complex with UK. Using affinity column technique with immobilized active site modified UK, involvement of active site serine for E-I complex formation was investigated. PA-I₁ demonstrated binding affinity with native UK, but it was not adsorbed either to anhydro-UK whose active site serine was converted to dehydroalanine, or DIP-UK whose active site was covered with diisopropylphosphate group. PA-I₂ was found to bind with anhydro-UK in addtion to native UK, but PA-I₂ did not show binding affinity with DIP-UK.

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Partially purified plasminogen activator (gPA) extracted from hypersensitivitytype murine lepromas in C57BL/6N mice after Sephacryl S-200 and DEAE Sepharose columns showed a unique substrate specificity for chromogenic substrates, distinct from that of urokinase-type PA (uPA), and tissue-type PA (tPA). It was revealed that gPA was with the highest substrate specificity for Ile-Pro-Arg-pNA (s-2288) (Km=1.4×10^-4), second highest for Val-Leu-Lys-pNA (s-2251) (Km=5.2×10^-4), and a fairly high affinity for Glu-Gly-Arg-pNA (s-2444) (Km=9.3×10^-4), whereas it was found compatible with the substrate specificity of macrophage PA (mPA) secreted by thioglycollate stimulated mouse peritoneal macrophages in culture. An electrophoretic enzymography demonstrated that gPA consists of two molecular forms (subspecies) with Mr=45, 000 and 24, 000, distinct from uPA (Mr=55, 000 and 33, 000) and tPA (Mr=72, 000), whereas identical with the enzymographic pattern of mPA (Mr=45, 000 and 24, 000). An immunoblotting technique further revealed that gPA is antigenically not related to uPA and tPA.
A PA with a characteristics identical to macrophage PA was found in inflammatory tissue during the development of hypersensitivity-type of murine lepromas, suggesting its possible immuno-modulatory role in the inflammation.

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After normal human subjects (78 in number, aged 18-34 years) had taken the local Japanese spirit “Shochu, ” rice wine “Sake” and beer, almost no change in enzyme parameters of plasma fibrinolysis was observed. However, plasma eluate from [N^α-(ε-aminocaproyl)-DL-homoarginine hexylester]-Sepharose affinity chromatography revealed extremely strong pyro-Glu-Gly-Arg-pNA (S-2444) amidolytic and fibrinolytic activities in comparison with those of the control. The highest activities were observed in the Shochu group (1, 133nmol pNA/dl, 470IU/dl plasma), followed by the Sake (811nmol pNA/dl, 415IU/dl plasma) and beer (707nmol pNA/dl, 313IU/dl plasma) groups, corresponding to an approximately 2.1, 1.7 and 1.5 times increase in plasma fibrinolysis compared to the control group (490nmol pNA/dl, 242IU/dl plasma).
Fibrinolytic enzyme isolated from the Shochu group was partially inactivted by rabbit anti HMW-UK serum and could be adsorbed on an anti HMW-UK IgG-Sepharose immunoadsorbent column. The final preparation eluted from the immunoadsorbent column with 6M urea buffer revealed molecular weights of approximately 30, 000 (main band), 50, 000 and 30, 000 (minor bands) by zymography, and an active site of approximately 8% (assuming the molecular weight of 30, 000) by p-nitrophenyl-p′-guanidinobenzoate titration. The enzyme not only hydrolyzed the UK-specific substrate S-2444 but also strongly activated human Glu-plasminogen to plasmin. This indicated that some of the enzyme appearing in the plasma after drinking was “UK-like plasminogen activator”.

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