The phorbol ester 4β-phorbol-12β myriste-13α acetate (PMA), a potent tumor promoter, is known to be a agent which causes platelet aggregation and release reaction. But the exact mechanism of PMA induced platelet aggregation remains unclear. In this paper, to search for a possible mechanism of PMA induced platelet aggregation, the following anti-platelet agents were applied; acetylsalicylic acid (ASA), phenidone, OP. 3708, verapamil, TMB-8, cilostazol, monoclonal antibody (Ab) to GPIIb/IIIa, W-7, H-7 and PGI₂. Futhermore in congenital platelet disorders (platelet cyclooxygenase (PCO) deficiency, Bernard-Soulier syndrome, Hermansky-Pudlak syndrome, thrombasthenia, familiar defect of A23187 induced platelet aggregation), PMA induced platelet aggregation was studied.
In order to clarify the usefullness of PMA induced platelet aggregation in clinical cases, aggregation study at various PMA doses was performed in chronic myeloproliferative disorders.
Among anti-platelet agents, TMB-8, verapamil, PGI₂, W-7 and H-7 had inhibitory effects on PMA induced platelet aggregation. In congenital platelet disorders, PMA induced platelet aggregation was markedly decreased only in thrombasthenia. These data need further confirmation, but they suggest that PMA (especially at low concentrations) induced platelet aggregation was not mediated by cyclooxygenase and lipoxygenase products, release of dense granules and GPIb. In most patients with chronic myeloproliferative disorders, PMA induced platelet aggregation was decreased at low PMA concentrations. And it was significantly decreased at low PMA concentrations (<0.1μg/ml) in essential thrombocythemia. This may be related to bleeding tendency in essential thrombocythemia.
Platelet aggregation study induced by PMA, as well as other platelet agonists, may be a useful method for the elucidation of the mechanism of various bleeding and/or thrombus formation tendency in clinical cases.

View full abstract

The ultrastructure of megakaryocytes in 2 patients with familial gray platelet syndrome were investigated and the findings in these patients were compared with those of the other reported cases.
Light microscopic examinations on megakaryocytes revealed a deficiency of granules in the cytoplasm as Raccuglia described. However, maturation of megakaryocytes and liberation of platelets from megakaryocytes appeared normal.
Electronmicroscopic examinations showed these megakaryocytes had normally developed endoplasmic reticulums, Golgi zones and many small granules which were thought to be precursors of α-granules around the Golgi apparatuses. However, mature megakaryocytes had a deficiency of α-granules in the cytoplasm. Extensively developed demarcation membranes (DM) were observed in some megakaryocytes and electron dense materials which were thought to be secreted materials from immature α-granules were seen in the gaps of these DM. Liberation of abnormal platelets with deficiency of α-granules were also noted from their megakaryocytes. These findings were identical to those reported by Breton-Gorius et al. and impaired packaging of α-granule contents in the maturation process of their megakaryocytes were suggested. In addition, small areas of cytoplasmic sequestration, masses of dense tubular systems, membrane complexes and clumps of dense materials as seen in peripheral platelets were also noted in their megakaryocytes.

View full abstract

In this report, we describe a case of thrombosed valve in early postpartal period. The patient was a 35-year-old woman who received an operation of aortic valve replacement (Björk-Shiley valve) for her rheumatic heart disease 13 years ago. Since then she was constantly on oral anticoagulants. Two years ago she intentionally stopped to take oral warfarin because of her wish to have a baby. She became pregnant early 1985, since then low-dose heparin injection combined with oral dipyridamole had begun. On Oct. 3, 1985 she gave a birth to a 3, 080g baby. But on the 8th postpartal day, she began to complain of increasing dyspnea. Her chest X-ray film showed remarkable pulmonary congestion. She died on 12th day following the delivery due to heart failure. The postmortem examination revealed a thrombosed Björk-Shiley valve with very limited leaflet motion.

View full abstract

Effects of membrane fraction of human platelets on polymerization of actin have been examined. Freshly prepared membrane fraction inhibited the polymerization, whereas the membrane fraction which was once frozen after preparing fresh membrane fraction accelerated the polymerization. Liposomes which were composed of lipids obtained from the platelet membrane showed higher acceleration of the polymerization. When the membrane fraction was pretreated with thrombin or phospholipase A₂, acceleration of actin polymerization was observed by the fraction. Pretreatment with phospholipase C, however, showed no effect on the polymerization. Liposomes formed by PtdIns (4, 5) P₂ showed highest acceleration. From these findings we concluded that not only changes in free calcium ion level, but also some conformational change or electric charge of the membrane would be responsible to the induction of actin polymerization.

View full abstract

We reported the effects of serine protease inhibitors on the interaction of platelet cytoskeletal proteins. Serine protease inhibitors such as PMSF, FOY-007 or FUT-175 and aspirin inhibited the incorporation of myosin heavy chain into the 1% Triton X-100 insoluble residue during the aggregation induced by ADP. FUT-175 also suppressed the incorporation of actin binding protein and fibrinogen into the cytoskeletons. This may result in the inhibition of not only the second aggregation but also the primary wave of aggregation. Anti-hydrolytic activity of FUT-175 against trypsin was recovered in cytosol fraction at least 2×10^-6M, when platelets were incubated with this agent, 10^-4M. This seems to indicate that FUT-175 has unique site (s) of action in cytosol which is related to the inhibition of integration of cytoskeletons during the process of platelet aggregation besides it's action on arachidonate metabolism. In fact, zymogram of platelet cytosol fraction with Tosyl-L-lysine α-naphthyl ester as substrate, demonstrated 5 active bands (pl. 9.2, 8.1, 6.25, 5.9, 5.3, 4.4). Some of these cytosomal serine proteases might act as an important role in the formation of cytoskeletons during the process of platelet reaction.

View full abstract

The effects of antiplatelet agents on the disorders of lipid and arachidonic acid (AA) metabolism were studied in hypercholesteromic rats (HC). HC rats received an atherogenic diet (containing 2% cholesterol, 0.5% cholic acid, 10% lard, 5% cane sugar, 0.2% methylthiouracil, and 82.3% basal diet) for 6 weeks, 350, 000units/kg of vitamin D₂, and also olieve oil (5ml/kg) for 4 days. Antiplatelet agents, OKY 046 and ONO 3144, were administered to HC rats (100mg/kg/day). After 6 weeks, plasma lipids were measured by conventional methods, and both TXA₂ generation in the platelet and PGI₂ generation in the isolated aorta were measured by RIA.
The results were as follows: 1) Plasma cholesterol, triglyceride and β-lipoprotein were increased in HC rats. 2) Vascular UGI₂ generation was decreased, and AA induced platelet TXA₂ generation, and TXA₂/PGI₂ induced by collagen or AA were elevated in HC rats, while platelet TXA₂ generation and TXA₂/PGI₂ were decreased in OKY and ONO treated HC rats, 3) Atherosclerotic changes in intima and media were observed in 5/12 HC rats, 1/6 OKY treated HC rats and none of 4 ONO treated HC rats.
These results might suggest that vascular PGI₂ generation was decreased and platelet TXA₂ generation was increased in these experimental atherosclerotic rats, however the administration of antiplatelet agents OKY 046 and ONO 3144 seemed to prevent atherosclerosis through the repairment of AA metabolism.

View full abstract

Lipoxygenase activities were estimated in platelet subcellular fractions as well as in intact platelets obtained from normal subjects and 7 patients with platelet-lipoxygenase deficiency. From a washed platelet suspension (intact platelets), 12, 000g supernatant of sonicated platelets (F-I), cytosol fractions (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. 12-Hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with platelets or each fraction from them was measured by reversed-phase high-performance liquid chromatography analysis.
In normal subjects, 12-HETE production by subcellular fractions was 87.1% (F-I), 31.4% (F-II) and 17.2% (F-III) of that by intact platelets, and was not significantly affected by the addition of CaCl₂. Of the 7 patients, one patient showed no detectable lipoxygenase activity in any subcellular fractions and in intact platelets; the other patients showed reduced activities in all subcellular fractions as well as in intact platelets. In some of these patients, it appeared that lipoxygenase activities were not fully expressed in intact platelets because much more amounts of 12-HETE was produced by their subcellular fractions than by intact platelets especially in the presence of CaCl₂.

View full abstract

The binding of anti-human platelet glycoprotein (GP) IIb/IIIa monocolonal antibodies to human umbilical vein endothelial cells (HUVE) was studied. Scatchard analysis using 125 I-anti-platelet GPIIb/IIIa monoconal antibody showed that the maximum binding capacity was 8×10⁴/cell and Kd was 40.2nM. The binding rates of 125 I-anti-platelet GP IIb/IIIa monocolonal antibodies to HUVE treated with TTP sera and ITP sera were 11.6±2.8% and 15.2±3.3%, compared with the 24.0±7.5%, observed for HUVE treated with normal sera. A combination analysis of SDS-PAGE and western blotting of washed platelet and HUVE lysates showed that two proteins were similar or identical molecular masses.
These findings showed that TTP sera and ITP sera may contain antiplatelet GP IIb/IIIa antibodies and that there are GP IIb/IIIa on HUVE.

View full abstract

A 38-year-old woman was admitted to our hospital on Nov. '85 because of mild bleeding tendency. There was past history of malignant lynphoma of the stomach at age 34. Then she had gastrectomy and splenectomy with subsequent X-ray therapy.
Coagulation tests on admission revealed; platelet count 259, 000/μl; bleeding time (Duke), 8.5min.; whole blood clotting time, prothrombin time, partial thromboplastin time were normal. There was the abnormality of platelet function to aggregate in response to ADP, epinephrine and collagen, whereas ristocetin-induced agglutination was normal (Fig. 1). It was characteristic that the remarkable lag time was observed in aggregation by ADP, epinephrine and collagen. Furthermore, crossed immunoelectrophoresis (1) of the patient's platelets showed biphasic peak of GPIIb/IIIa incontrast to the monophasic peak of normal GPIIb/IIIa (Fig. 2).
She was admitted again on Aug. '86 because of the aggravation of bleeding tendency with severe thrombocytopenia. At that time, the relapse of malignant lymphoma was proved by the biopsy of the swallen tonsil. Surprisingly, the X-ray therapy for cervical area normalized the platelet count and platelet function.
Taken together, the platelet dysfunction of this patient was thought to be closely related to the complication of malignant lymphoma. It is possible that the lymphoma cells produce autoantibody reactive with platelet membrane.

View full abstract

Blood coagulation was studied in patients with idiopathic thrombocytopenic purpura (ITP), essential thrombocythemia (ET), polycythemia (PC), myelodysplastic syndrome (MDS), and aplastic anemia (AA), and prolongation of APTT was observed in ITP, ET, MDS and PC with thrombocythemia, but shortening of APTT was seen in AA. Circulating anticoagulant was detected in only 4 ITP patients by cross mixing experiment and the effect of high-dose platelin on kaolin clotting time. The prolongation of APTT was statistically improved after treatment of ITP with steroid and high-dose gammaglobulin therapy (HDGT). In ITP patients with prolonged APTT, megakaryocytes were increased, serological tests were more positive and weight of spleen was increased compared with patients with normal APTT. In terms of effectiveness on therapy, steroid therapy and HDGT were more effective in patients with prolonged APTT than in patients with normal APTT. Homogenate of more than 4×10⁵/μl platelets prolonged APTT, PTT and recalcification time but shortened kaolin clotting time. Phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin, phosphatidic acid) also prolonged APTT at high concentration.
In ITP and MDS, phospholipids from destroyed platelets might be increased and prolong APTT, and it was suspected that increased platelets also prolonged APTT in ET and PC. In these diseases, APTT might reflect the function of reticuloendothelium system and the number of destroyed platelets.

View full abstract

The purpose of the present study is to clarify the inhibitory mechanism of taurine on platelet aggregation in myocardial infarction. In vitro, platelet aggregations induced by 4.6μM ADP and calcium ionophore in the dose range of 1.7 to 10.0μM A23187 were measured by Born's method with and without 10mM taurine in normal subjects and in cases with old myocardial infarction. Platelet malondialdehyde formation induced by 125μM arachidonic acid was measured by TBA method. In order to estimate the effect of taurine on the change of cytoplasmic Ca²⁺ in platelet activated by 0.4U/ml thrombin, the intensity of quin 2 fluorescence was measured by Rink's method with and without 10mM taurine.
Platelet aggregations induced by 4.6μM ADP and by 2.5 and 5.0μM 23187 were significantly inhibited with 10mM taurine in cases with old myocardial infarction, and also the increase of cytoplasmic Ca²⁺ in platelet activated by thrombin was significant. In normal subjects, on the other hand, taurine did not inhibit them. In both of cases with old myocardial infarction and normal subjects, platelet malondialdehyde formation was not significantly affected by incubation with 10mM taurine. These results suggest that the inhibitory mechanism of taurine on platelet aggregation is not due to the effect of taurine on arachidonic metabolism, but the inhibition of cytoplasmic Ca²⁺ change in platelets of the cases with old myocardial infarction.

View full abstract

In the fibrinogen-fibrin conversion, remarkable turbidity changes can be observed in experimental conditions of both a neutral pH and low ionic strength. The relationships between fibrin structure and turbidity changes were studied. In the region, which slope is designated as α_I, of the initial small turbidity changes on the turbidity-time curve obtained experimentally, the hydrolysis of fibrinogen by thrombin was stopped by adding a potent thrombin inhibitor MD-805. Then dimer and trimer of fibrin monomers, detected from the reaction mixture by a SDS (0.1%)-agarose (2%) gel electrophoresis, were regarded as nuclei of polymerization of fibrin monomers.
On the other hand, the region, which slope is designated as α_II, of the most remarkable turbidity change, e. g., α_II/α_I=10, on the turbidity-time curve could be attained neither by increase of the equilibrium constants in the simplified model for the polymerization of fibrin monomer nor by the formation of infinitely long fibrin polymer. Thus, the remarkable turbidity changes in the fibrinogen-fibrin conversion was deduced not only by the contribution of the growth of protofibrils but also by the lateral association of protofibrils.

View full abstract

Leukocyte cathepsin G (CLP) and elastase (ELP) from human blood were partially purified, and enzymatic properties of CLP and α₂-macroglobulin-CLP complex (α₂M-CLP) were studied in comparison with those of ELP and α₂M-ELP. Furthermore, some synthetic substrates and synthetic reversible inhibitors for CLP were newly synthesized and specificities for CLP were compared with those of α-chymotrysin. The results obtained were as follows.
1) CLP was extracted with 2M NaClO₄ for 1hr, and delipidated by CCl₄ after dialysis in tris-HCl buffer (0.1M, pH 7.5) containing 0.5M NaCl. Then, CLP was partially purified by Ultrogel AcA 44 gel chromatography.
2) The amidolytic activity of CLP increased with the addition of NaCl, KCl, MgCl₂ and CaCl₂ as was that of ELP, while the addition of NaClO₄, KSCN and KI enhanced the activity of ELP, but inactivated that of CLP.
3) The amidolytic activity of CLP or α₂M-CLP was reduced with the addition of dimethyl sulfoxide in the reaction mixture, whereas that of ELP or α₂M-ELP increased.
4) Kinetic parameters of Suc-Ile-Pro-Phe-pNA synthesized by the authors were 1.3mM at Km value and 7.5s^-1 at k_cat value.
5) Some derivatives of Suc-Tyr-Leu-Phe-pNA were synthesized, and Ki value for CLP, ELP and α-chymotrysin was compared.

View full abstract

Purification and function of blood Plg. Act. have not been studied sufficiently. Therefore, we evaluated the separation of blood Plg. Act. using by chromatography of hydroxyapatite (H-AP). H-AP varies in each characteristics according to the size and the degree of production by heat and large particle of H-AP calcined by 1200°C are selected for the open column.
Blood Plg. Act. are adsorbed on H-AP column smoosely from human plasma instead of activation of coagulation and kallikrein system. Plg. Act. was eluted out by 0.05M phosphate buffer containing 0.2M Argine and 0.3M Nacl. H-AP column adsorbed blood Plg. Act. more effective than Fibrin Celite column or Lysine Sepharose column.
This method is considered an effective application in the separation and purification of blood Plg. Act. as an affinity column.

View full abstract

Urokinase (UK) immobilized material is known to have excellent antithrombogenicity. This antithrombogenicity has been ascribed to the fibrinolytic activity of plasmin activated by UK.
We found that platelet adhesion was not recognized on the surface of UK immobilized polyurethane catheter indwelled in vein. To investigate the interaction between platelet adhesion and fibrinolytic activity, inactivated immobilized UK was prepared by chemical modification with (p-amidinophenyl) methanesulfonyl fluoride hydrochloride. Polyurethane, UK immobilized polyurethane and inactivated UK immobilized polyurethane were incubated with human blood in Chandler loop and their surface were observed by scanning electronmicroscopy. Platelet adhesion on the surface of the material was recognized for polyurethane but not observed for UK immobilized polyurethane and inactivated UK immobilized polyurethane.
We concluded that the antithrombogenicity of UK immobilized material depends not only on fibrinolytic activity but also inhibition of platelet adhesion which is not mediated with plasminogen activation by urokinase.

View full abstract

Until recently, prenatal diagnosis of hemophiliacs was based on determining the fetal sex by amniocentesis under ultrasound guidance. However, chorion biopsy in the early pregnancy and applications of fetoscopy in the mid of pregnancy have led to an improvement of prenatal diagnosis of the hemophilia A which allows determination of fetal sex and measurements of FVIII/vWF complex, but several diagnostic errors still remain.
Results: 1) The titers of FVIII: C (one step method) and vWF: Ag (Laurell's method) as well as FVIII: Ag (ELISA method) in the cord blood were generally lower than those of maternal blood during pregnancy. 2) A 37-year-old woman who had two sons with hemophilia A was pregnant again after divorce from the former husband. The pregnancy continued because the fetus had been suspected to be female by sex determinatin. At 27 weeks, the fetus was proved to be male by prenatal diagnosis using Y-specific DNA probe and FVIII in the fetal blood obtained by fetoscopy was extremely low, thereby the fetus was affected by hemophilia A. 3) A 23-year-old woman was diagnosed as a hemophilia A carrier at 10 weeks of pregnancy. The fetus was suspected to be female by sex determination using chorion biopsy, and at 22 weeks, the fetus was resuspected to be female by using amniotic cell. A normal male was born at 34 weeks and FVIII in the cord blood was fortunately within normal limit.

View full abstract

Fibrin gels formed in the presence of thrombin have exhibited to be able to adhere plastics. This adhesion became a strong with increment of fibrinogen concentrations of the system, whereas changes in the thrombin concentrations gave a constant value. No significant effect of NaCl, CaCl₂ on the fibrin adhesion was observed. However, the adhesion is if anything slightly stronger for a fine than for a coarse gel. In the presence of plasmin, the adhesive force of fibrin became a weak with the degradation of fibrin deposits, indicating that the amount of fibrin clots itself is play a role on the force of adhesion.
An experiment of the fibrin adhesion during gelation process has showed that concomitant with increase in γ-chain cross-linking as judged by SDSPAGE analysis, there is increase in the adhesive force. During this process, no cross-linking of α-chain occurred. These results have suggested that the γ-chain cross-linking is also important to the fibrin adhesion. This has been also supported by the experiment using Batroxobin which induces γ-chain cross-linking alone.

View full abstract

Vitamin K deficiency has occurred in patients receiving broad-spectrum antibiotics. The mechanism of hypoprothrombinemia in patients treated with antibiotics is still unknown, although it is suggested to result from impared synthesis of vitamin K by suppression of intestinal bacteria or interference of vitamin K utilization in the liver. We studied the serum vitamin K concentration in patients receiving broad-spectrum antibiotics for 7 days or more, in order to clarify the mechanism of antibiotics induced vitamin K deficiency. The coagulation abnormalities compatible with vitamin K deficiency were seen in 7 patients in whom predisposing factors for vitamin K deficiency were male, poor dietary intake and therapy of antibiotics with N-methyl-tetrazol thiol. In these patients, serum vitamin K₁ and vitamin K₂ (menaquinone-7) levels was extremly lower when compared to normal adult levels. On the other hand, vitamin K₁ levels in 5 of 6 patients with normal coagulation status moderately decreased to the levels under minus 1 standard deviation of normal adults, and serum vitamin K₂ in 3 patients with normal coagulation status and very poor dietary intake was not detected.
These results may suggest that vitamin K deficiency in patients receiving antibiotics is induced by poor oral intake, altered absorption of vitamin K in the intestine and/or impaired synthesis of vitamin K by suppression of intestinal bacteria.

View full abstract

We have examined the thrombolysic effect of human tissue-type plasminogen activator (t-PA) purfied from established human melanoma cell line and two chain urokinase (u-PA) from human urine administered intravenously and intracoronarily in a open chest-canine acute coronary thrombosis model. Two hrs after the total occlusion had been occurred, the 30min administrations of t-PA and u-PA were initiated. Intravenous administration of t-PA in the doses of 30×10³IU/kg and 100×10³IU/kg induced 60% and 100% reperfusion rate without systemic fibrinolysis. On the other hand, administration of u-PA was associated with the severe reduction of α₂-PI, plasminogen and fibrinogen levels. Intracoronary administration of t-PA and u-PA also showed the similar results in smaller doses than intravenous administration. Accordingly, it is concluded that infusion of t-PA induces the effective coronary thrombolysis without severe systemic fibrinolysis in dogs.

View full abstract

For the analysis of thrombophilic state in patients of dialysis, SLE and diabetes mellitus (DM), we examined the molecular markers of coagulation and fibrinolytic system in plasma, i. e. Fibrinopeptide A (FPA), fibrinopeptide Bβ1-42, 15-42, (Bβ1-42, 15-42), tissue type plasminogen activator (t-PA). The mean values of normal adults (n=20) were as follows; FPA 1.69±0.23ng/ml, Bβ1-42 3.12±0.48pmole/ml, Bβ15-42 3.41±0.53pmole/ml, t-PA 8.25±2.34ng/ml. In the patients on dialysis (n=30), a tendency of increase in the plasma concentrations of FPA and Bβ1-42 was showed, but the low levels of the plasma concentrations of t-PA were observed. The levels of Bβ15-42 in plasma were almost normal. According to the clinical course of dialysis, we divided the patients into two groups, less and more than 5 years. In the former group, the mean plasma concentration of Bβ1-42 was 35.57±23.67pmole/ml, Bβ15-42 was 5.44±1.29pmole/ml. In the latter group, the plasma concentration of Bβ1-42 was 29.56±27.24pmole/ml, Bβ15-42 was 6.47±1.26pmole/ml. On the contrary, in the caces of SLE patients less than 5 years (n=10) and DM patients more than 10 years (n=10), the concentrations of Bβ1-42 was within normal value, but Bβ15-42 was apparently increased. As a result of these examinations, it was suggested that these patients were in hypercoagulable state, but the fibrin formations could not be complete in the cases of dialysis patients.

View full abstract

We found that there was an antigen common to the biliary plasminogen activator, bilokinase (BK) in hepatocytes by histochemical and immunological studies. It was also demonstrated that the cultured hepatocytes excreted a plasminogen activator into the medium. These observations suggest that there is close relation between hepatic and biliary fibrinolytic systems.
The present study was carried out to clarify whether a pathological state of liver affects to biliary fibrinolysis.
Cirrhosis was produced in rats by the methods of Tanner et al. and McLean et al. with some modifications. The cirrhosis was raised at 5 week period, showing 2 times increased level of proline and marked development of collagen fibers in the liver tissues. Of the parameters measured, fibrinogen was found to be elevated significantly. PT and APTT were slightly shortened and ELT tended to prolongation.
Fibrinolytic autography showed clear lysis areas at the sites corresponding to the biliary canaliculi. BK activity in bile was higher in the cirrhotic rats than of control rats.
Thus, dynamics of BK might change in association with the pathophysiology of the liver.

View full abstract