Morphological aspects of the platelet release reaction induced by ADP and/or Teleocidin (tumor promoter) in the presence or absence of aspirin were studied by transmission electron microscopy. Human platelet-rich plasma (PRP) treated with reagents at 37°C for 1-5min was fixed with aldehyde and embedded in Epon or Lowicryl K 4 M. Addition of ADP (10μM) resulted in the centralization of granules without granule release, while Teleocidin (100ng/ml) induced the swelling of the open-canalicular system and the release of α-granules without centralization of these granules. When both ADP and Teleocidin were added, the synergetic effect was predominant and all of the granules were discharged from the cytoplasm. The results were essentially the same both in the presence and absence of aspirin. Post-embedding immunocytochemical studies employing protein A-gold as a label revealed that several components of α-granules, e. g., β-thromboglobulin, platelet factor 4, but not the dense-granule component, serotonin, moved into the swollen open-canalicular system following the administration of Teleocidin. In the case of the administration of both Teleocidin and ADP, both α-granular and dense-granular components moved into the swollen open-canalicular system.

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Effects of bovine platelet α-granule acid extract (BPAE) on vascular permeability were examined in the rabbits subjected to either depletion of polymorphonuclear (PMN) leukocytes by nitrogen mustard (nitrogen mustard-N-oxide hydrochloride, 20-40mg/Kg, i. v.) or pretreatments with histamine antagonists of H₁-(chlorpheniramine maleate, 10mg/Kg, i. p.) and H₂-receptors (cimetidine, 50mg/Kg, i. v.). Increase in vascular permeability due to BPAE (0.1ml of 50 or 250μg/ml) was evaluated by leaked ¹²⁵I-albumin (30μCi, i. v.). and its activity was correlated with fine structures of the reaction. Simultaneously, inflammatory cell infiltration occurred in the vascular wall as early as 3-5 minutes. The vascular permeability activity was gradually increased during the observation periods, 1 minute- 3 hours. On the other hand, effects of BPAE on the vascular permeability in rabbits were strongly inhibited by depletion of PMN leukocytes, but not effectively inhibited by pretreatment with the histamine H₁- and H₂-receptor antagonists.
In conclusion, it is confirmed that the vascular permeability factor is present in BPAE as has been described in human platelets. Furthermore, it is suggested that BPAE plays a role in the vascular permeability response as a PMN leukocyte-dependent mediator.

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Vascular permeability factor (VPF) was extracted from human and bovine platelet α-granules and partially purified by ion exchange and gel filtration chromatographies. Human and bovine VPFs were cationic proteins, and these isoelectric points were 9.2 and 8.8, respectively, by electric-focusing technique. The human VPF was mainly localized into the fraction of approximate Mw. of 30, 000 by gel filtration (G-75). On the other hand, the bovine VPF was localized into the fraction, an estimated Mw. 140, 000 by gel filtration (S-300). After purification by G-75 gel filtration, permeability activity of bovine VPF was 1, 300μl/mg of protein. The stability of VPFs was also studied after a variety of treatments. These VPFs were activated at 50-60°C and extremely stable to a temperature up to 80°C. They were also stable on exposure to acid or alkaline (pH 1.3-12), but inactivated by pepsin or 2-mercaptoethanol treatment.

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Several reports have suggested that the hyper-aggregation of platelet participated in the genesis of diabetic microangiopathy. And also it has been observed that the plasma of diabetic patients have strongly enhanced the aggregation of normal platelets.
In this paper, PRP filtrates were prepared after the platelet aggregation in PRP by 2μM of ADP (Agg-Filtrate). These Agg-Filtrates of diabetic patients had greater ability to enhance the ADP-induced platelet aggregation in normal PRP, in comparison with that of normal volunteers.
Fractionation of these Agg-Filtrates by Sephadex G-200 yielded three protein peaks (I, II, III). Platelet aggregatien enhancing activity of the Agg-Filtrate from diabetics existed in the relatively lower molecular weight fraction named Peak III. It was not affected by the neuraminidase-treatment, but was decreased by the trypsin-treatment.
SDS-PAGE of the Peak III of diabetic patients showed PAS-negative additional band (MW≅42000).
On the other hand, the Peak III inhibited the increase of the c-AMP level by PGE₁ in the normal platelets.
Therefore, the author have assumed that platelets of diabetics were activated by it's own platelet-derived protein, which had the ability to inhibit the increase of the c-AMP level in the other platelets.

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Antibodies I and II (heterologous anti-human platelet membrane antisera) were produced in rabbit against human platelet membrane dissolved in 10mM Tris/HCl buffer, pH 7.4, containing 1% Triton X-100, 0.25M sucrose, 2mM CaCl₂, and in 38mM Tris/HCl buffer, pH 8.6, containing 1% Triton X-100, 0.1M glycine, 5mM EDTA, respectively. Antibody III (mouse monoclonal antibody) was raised against crude human platelet membrane of normal platelets. By crossed immunoelectrophoresis (CIE), it was shown that antibody I could bind to a single protein. Since the immunoprecipitate on CIE was not detectable by using platelets from a Glanzmann's thrombasthenic patient, antibody I was considered to be specific antibody against GP IIb/IIIa complex. Avidin biotin complex system (ABC)-immunoblot showed the strong binding of antibody II to electrophoretically separated GP IIb and GP IIIa. ABC-immunoblot revealed no binding of antibody III to platelet proteins after SDS-PAGE, but two immunoprecipitates were obtained with an apparent molecular weight of 130K and 110K under reduced conditions by double antibody immunoprecipitate, thus indicating that antibody III was a monoclonal antibody against GP IIb/IIIa complex. Fluorescence flow cytometric analysis showed that the activity of antibody I to sensitize human platelets was less than that of antibody II. In contrast, antibody I was more potent in inhibiting ADP-and collagen-induced platelet aggregation than antibody II. Antibody III inhibited ADP-and collagen-induced aggregation in a dose dependent manner. The addition of 6.3μg/ml of antibody III to platelets sensitized 68% of platelets, and caused complete inhibition of ADP-induced aggregation, while its addition of 3.1μg/ml sensitized 50% of platelets, and caused complete inhibition of collagen-induced aggregation. The results give further support to the proposal that platelet GP IIb/IIIa complex, not dissociated GP IIb and GP IIIa, plays an important role in platelet aggregation.

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To obtain a monoclonal antibody to F. VIII, we immunized BALB/c mice with purified F. VIII and fused the spleen cells with X-63 murine myeloma cells. The hybridomas were screened for F. VIII antibody production by four-layer IRMA. Consequently one hybridoma was cloned which secreted F. VIII monoclonal antibody (NMC-VIII/1) detectable by IRMA but lacking inhibitory activity to F. VIII: C. Mouse ascitic fluid produced by the hybridoma cells had an antibody titer of 1×10⁵. The immunoglobulin was of IgG₁ subclass. Immunoadsorbent beads coupled with the monoclonal antibody adsorbed only F. VIII and F. VIII/vWF. Two-site solid phase ELISA for F. VIII: Ag was developed using the monoclonal antibody. F. VIII: Ag in normal plasma, normal serum and hemophilia A plasma was assayed by this method (F. VIII: Ag ELISA), and the results were compared with F. VIII: Ag values measured by ELISA using homologous polyclonal antibodies (F. VIII: Ag IRMA) and with F. VIII: C values measured by one-stage clotting assay. In normal and hemophilic plasmas, F. VIII: Ag ELISA levels correlated well with E. VIII: Ag IRMA. A significant level of F. VIII: Ag was detected equally by both methods in 5 moderate to mild hemophiliacs. In normal sera, the average value of F. VIII: Ag ELISA was 14±5u/dl, which was much lower than F. VIII: Ag IRMA. The reduction of F. VIII: Ag ELISA occurred rapidly after the beginning of blood clotting.

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We examined ultrastructurally the postmortem tissues of a patient with hemophilia A who developed AIDS to search for LAV/HTLV-III. Round particles, 80-100nm in diameter, with a bar-shaped nucleoid, were observed frequently in the extracellular spaces of the lymph node, liver and spleen. These particles were morphologically identical with the mature LAV/HTLV-III which belonged to D type retrovirus. The budding of virions on the plasma membranes, the characteristic release from of retrovirus from cells, was also found in these tissues. In addition to these findings, two types of cytoplasmic inclusion bodies were observed: one is “tubuloreticular structures (TRS)” in the lymph node and liver, and the other is structures analogous to “test tube and ring shaped forms (TRF)” in the lymph node. These structures have been described previously by Sidhu et al. as ultrastructural markers of AIDS. The relationship of these inclusion bodies with LAV/HTLV-III infection and growth remains to be clarified.

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The effects of BM-1 (extracts from the mackerel) on platelet aggregation and serum lipids were investigated.
Daily 6g of BM-1 was orally administered to 8 healthy male subjects for 30 days.
Before and after taking BM-1, its effects on aggregation of platelets and serum lipids were studied. Platelet aggregation induced by ADP collagen was signficantly inhibited by BM-1. No significant difference between before and after administration BM-1 in the serum levels of lipids (cholesterol, phospholipid, and fatty acid) was found. However, the serum levels of HDL were significantly decreased by taking BM-1.
No eicosapentaenoic acid (EPA) in BM-1 was detected.
These results suggest that BM-1 may contain a factor, not EPA, inducing inhibition of platelet aggregation.

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Thrombospondin (TSP) was purified from bovine platelet-released substances by heparin-sepharose affinity chromatography and gel-filtration. Purified TSP was investigated whether to induce platelet aggregation in order to clarify the role of TSP in platelaet aggregation.
TSP induced aggregation of gel-filtrated platelets (GFP) in dose-dependent fashion in the presence of calcium and magnesium ions. The aggregation were dissociated with EDTA. Aminosugars inhibited and dissociated the platelets aggregation by TSP. GFP fixed with formalin were aggregated by TSP in the same manner of GFP. Aggregability of GFP fixed with calcium ions was larger than without calcium ions.
These results suggest that TSP may agglutinate native inactive platelets by interbridging the platelets probably by its lectin activity, and this agglutination may need calcium ions not only for TSP but also for the receptor of TSP on the platelet membrane surface.

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Detection and metabolism of inositol-1, 2-cyclic-4, 5-trisphosphate (cIP₃) were investigated in the thrombin-stimulated human platelets. The reaction of ³²P-labeled platelets with thrombin were stopped by addition of CHCl₃/MeOH. The water soluble fraction was applied on the Whatman Partisil-10 column chromatogrphy. The unknown radioactive peak of ³²P was detected in the fraction between inositol-1, 4-diphosphate (IP₂) and inositol-1, 4, 5-trisphosphate (IP₃) in the chromatography. The unknown radioactive peak moved to IP₃ position with the internal standard of ³H-cIP₃ by acidification of the sample. Furthermore, the unknwon radioactive peak of ³²P was collected and was treated with IP₃-5-phosphatase. The radioactive peak moved to the position of cIP₂. It indicates that in the thrombin-stimulated platelets, the radioactive compound of ³²P detected between IP₂ and IP₃ fraction in the chromatography is cIP₃.
Rabiolabeled inositolphosphates and inositol-cyclic-phosphates were incubated with the cytosol fraction of human pletelets. The 1, 2-ring of cIP₃ was not cleaved immediatly after the production from platelets and the cIP₃ did not convert to IP₃ in the metabolic process. It indicates that cIP₃ is metabolited as follow: cIP₃→cIP₂→cIP₁→IP₁.

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The third degree burn covering 35% of the total body surface area was made on the back of rabbits, and the animal were divided into 6 groups and the effectiveness of OP-41483, aspirin (ASA: high dose), ASA (low dose), OKY-046 and CV-4151 on arachidonate cascade after burn injury were studied. Examinations of thromboxane B₂ (TX) and 6-keto prostaglandin F_1α (PG) showed the increase of TX and TX/PG ratio in infusion therapy group. On the contrary, TX/PG ratio was decreased in OP-41483, ASA (large dose), OKY-046 and CV-4151 groups. Only OP-41483 prevented the decrease of platelet count after burn injury while other drugs did not prevented the decrease. The organ failure after burn injury, such as renal failure, has close relation with alterations of acachidonate cascade, so the drugs which decrease TX absolutely or relatively may effective to prevent the renal insufficiency after the burn injury. From the results of this experiment, TX synthetase inhibitors such as CV-4151 and OKY-046 prevented the increase of TX and increased PG and decreased TX/PG ratio extrmely.

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Intracellular calcium level in platelets of the patients with myeloproliferative disorders (CML 11 cases, PV 7 cases, ET 4 cases) was measured by Aequorin method. In the patients with CML, PV and ET, maximum intracellular calcium levels [Cai²⁺] was significantly lower and the reaction period reaching to the peak calcium level (the arrival time) was significantly prolonged than the normals after the stimulation of thrombin (0.5U/ml). In the case of stimulation of 2.5μg/ml collagen, maximum [Cai²⁺] level in patients with CML and PV was significantly lower and the arrival time in patients with CML and ET was significantly prolonged than the normals. No correlation was found between maximum [Cai²⁺] level and maximum aggregation rate or platelet counts in those patients. In the patients with CML, the increasing rate of intracellular calcium levels after the stimulation with A23187 (5μM) in the presence of various concentration of extracellular calcium was lower than the normal. [⁴⁵Ca²⁺] uptake into platelets of patients with CML after thrombin stimulation had a tendency to decrease compaired with normals. These results suggest that platelets with CML have some abnormalities of GP IIb, IIIa resulting in an impaired calcium influx mechanism.

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The factors affecting PGI₂ synthesis following the platelet aggregation induced by collagen (1μg/ml) in whole blood were examined.
Trapidil, possessing the anti-platelet aggregating activity and the enhancing effect of PGI₂ synthesis in endothelial cells, markedly promoted PGI₂ synthesis in whole blood, while aspirin showed no effect. The increment of PGI₂ synthesis ran parallel to the concentration of trapidil, whereas TxA₂ synthesis was decreased. OKY-046, the inhibitor of TxA₂ synthetase, also increased PGI₂ level and decreased TxA₂ level in whole blood in the same manner of trapidil. The level of PGI₂ in the suspension of leukocytes added trapidil (1mM), arachidonic acid (0.3M) and collagen (1μg/ml) was signficantly higher than that in the suspension added 1mM of aspirin in place of trapidil.
These results suggest that trapidil has the inhibitory activity of TxA₂ synthetase, resulting in the increment of PGI₂ in whole blood. This synthesis of PGI₂ was found in the leukocyte suspension in the presence of arachidonic acid.

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Antithrombin III (AT III) is one of the most potent serine protease inhibitors, and the functional abnormalities of this inhibitor have been proved to be the pathogenesis of thromboembolism in the traits of congenital abnormal AT III. These abnormalities are also considered to be caused by some environmental factors. In this paper we have evaluated the possibility of glucose to reduce AT III activity and the contribution of impaired AT III activity to hypercoagulable state in diabetic patients.
When human citrated plasma was aseptically incubated with glucose (0-2.0M) at 37°C for up to seven days, heparin cofactor activity of AT III gradually decreased, depending on the added glucose concentration or incubation period. Moreover AT III or these samples showed the increase of slow moving peak on modified crossed immunoelectrophoresis, and the increase of the bands with lower pis (isoelectric points) which was accompanied by the decrease of other hands on agarose gel isoelectric focusing.
In plasma of diabetic patients, a significant inversed correlation exists between the ratio of heparin cofactor activity to antithrombin III antigen and the levels of hemoglobin A₁c.
Glycosylated AT III was considered to be responsible for the functional and constitutional abnormalities of plasma AT III and it was suspected to be important as the pathogenesis of diabetic microangiopathy in diabetes mellitus.

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Coagulation studies were carried out in 55 newborn infants with intracranial hemorrhage diagnosed by using cerebral CT, spinal tap and/or the findings at autopsy, excluding the cases with intracranial hemorrhage caused by birth trauma. Decrease of plasma fibrinogen, hepaplastintest values and platelet counts, and prolongation of APTT were observed in a majority of these cases. Based on the findings of hemodynamics, blood gas analysis and coagulation status, it was estimated that the causes of intracranial hemorrhage were definite DIC in 14 cases, probable DIC in 6 cases, vitamin K dependent coagulation factors deficiency in 8 cases, rupture of cerebral tumor in 2 cases, cerebral anomaly in 2 cases and increased carotid artery pressure in 2 cases, respectively. On the other hand, the causes of intracranial hemorrhage in 14 cases were unknown, and the causes in 7 cases could not be estimated because of lack of sufficient data. Mortality of the cases with DIC and/or vitamin K dependent coagulation factor deficiency was significantly higher than that of cases without major coagulation abnormalities.
We suggest that abnormalities of hemostasis are responsible for the occurrence and/or exacerbation of intracranial hemorrhage in newborn infants.

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Urokinase-converting protease (UK-CP) which directly degrades HMW-UK to LMW-UK, and small fragment has been purified from human urine. UK-CP was not adsorbed to glass beads and barium carbonate, and was stable at pH 2.3-9.5. UK degrading activity of UK-CP was inhibited by BAEE (benzoyl arginine ethyl ester) and DFP, but not inhibited by aprotinin, EACA (ε-aminocaproic acid), STI (soybean trypsin inhibitor) and PCMB (p-chloromercuribenzoic acid). Antiserum against purified UK-CP gave one precipitin line with both the purified UK-CP and plasma. UK-CP migrated in the α₁-region against anti UK-CP serum in immunoelectrophoresis. The concentration of UK-CP in urine measured with Laurell's electrophoresis was approximately 1.4mg/liter. UK-CP showed specific binding for UK.
It is suggested taat UK-CP may be a new serine protease, or a new function of the known serine protease, playing a significant role on thn metabolism of UK.

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Recently, we reported that fibrin high-affinity urokinase (Fib-UK) of which molecular weight was about 100K was present in human urine and was very similar to UK-UK inhibitor complex in immunological and enzymatic nature (3). In the present paper, stabilities of UK, pro-UK and UK-UK inhibitor complex in plasma were examined with zymography. UK-UK inhibitor complex which was formed by incubating UK and plasma was more stable in plasma than UK, and resistant to DFP. Similarly, pro-UK was most stable in plasma and resistant to DFP. These results suggest that when UK is bound to UK inhibitor, UK changes its comformation and its stability in plasma increases like pro-UK.

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We administered UK 48×10⁴U.+dextran sulfate 3, 000mg (UK₄₈ group, n=2) and UK 60×10⁴U. (UK₆₀ group, n=6) during 6 hours repeatedly to 8 patients with pulmonary thromboembolism and investigated the changes of α₂ PI·plasmin complex (α₂ PI·C), FDP-D and D-D.
Fibrinolytic effect of UK was evaluated according to the degrees of improvement of isotopic perfusion defect, A-a DO₂ and scoring points of diagnostic criteria as previously reported.
In effective cases (n=4), which all belonged to UK₆₀ group, α₂ PI·C and FDP-D demonstrated rapid increase from 1.9±0.8(m.±S. E.) and 2.6±1.4 to 81.9±5.8μg/ml and 19.3±14.5μg/ml, respectively. Agglutination titre of D-D elevated promptly from 3.5±1.5 to 18.0±5.0. On the contrary, FDP-D and D-D revealed no significant elevation in ineffective cases, while α₂ PI·C changed from 3.4±2.3 to 58.0±7.2μg/ml. α₂ PI·C, FDP-D and D-D also demonstrated singificant increase when α₂ PI decreased less than 3mg/dl.
So, it was concluded that prominent fibrinolysis was introduced when α₂ PI decreased less than 3mg/dl by repetitious administration of UK 60×10⁴U. per 6 hours and FDP-D and D-D were quite useful parameters to evaluate fibrinolytic effect of UK.

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A 47-year-old female manifesting deep vein thrombosis of the left leg had severe pulmonary embolism after thrombolytic therapy with t-PA for 5 days. Dyspnea was improved and PaO₂ was restored from 39mmHg to 64mmHg soon after additional dosages of t-PA. Right ventricular pressure decreased from 71mmHg to 34mmHg after 1 month. Although t-PA level and D-dimer increased after giving 120.000u of t-PA, there were no obvious changes in fibrinogen, plasminogen, FDP nor α₂-PI levels. Efficacy of thrombolytic therapy wtih t-PA was demonstrated by digital subtraction pulmonary angiograms taken before and after treatment.

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