Platelet dense granules were examined quantitatively using mepacrine-labelling method in normal subjects and in patients with congenital platelet dysfunction. A mean number of mepacrine-positive granules per platelet (MGN: mean granule number) was 5.24—0.44 (SD) in 10 normal subjects. The number of positive granules was distributed in unimodal guassian distribution and was proportional to the sizo of platelets. In this study distribution curve and platelet granule number/platelet volume (MGN/MPV) ratio were referred for evaluation. The MGNs were 1.25 and 1.38 in two cases of Hermansky-Pudlak syndrome, 1.51 in a case of Wiskott-Aldrich syndrome. In these cases the decrease of dense granules was confirmed by ultrastructural studies. Although the reduction in MGN of platelets from a patient with Wiskott-Aldrich syndrome is partly due to smaller volume of these platelets, the decreased MGN/MPV ratio reflects a net decrease of dense granules. Platelets from patients with cyclooxygenase-deficiency and with intracellular Ca transport defect, in which platelet dysfunctin is supposed to be due to impaired release mechanism, had normal MGN value and normal distribution of mepacrine-positive granules. In a patient with Bernard-Soulier syndrome MGN per platelet was increased, but MGN/MPV ratio, that is, granule numder per μ³ was within normal range. Mepacrine-labelling method was proved to be very useful for diagnosis of storage pool disease. This method could be also applied to thrombocytopenic patients in which aggregation studies and/or biochemical quantification of adenine nucleotides are not feasible.

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In order to make a molecular design for ideal antithrombogenic material, it is important to find an interaction between the material and platelets.
Homogeneous hydrophilic poly-2-hydroxyethylmethacrylate (PHEMA), hydrophobic poly-styrene (PSt) and HEMA-St ABA type block copolymer containing 0.608 mole fractions of HEMA which has hydrophilic and hydrophobic lamella microdomain and has lamellar structure of 300-500 Å in widths were prepared as experimental polymers.
Morphological changes of rat blood platelets adhered to the polymer surfaces in the microsphere columnar with Hanks' platelet solution (Ca⁺⁺ Mg⁺⁺free) were observed by transmission and scanning electron microscopes.
For the inhibition of platelet activation which was assessed by observing its deformities and degranulation, the surface of the material which has lamellarly-shaped microdomain was superior to the homogeneous one.
It was noted that the platelets have the ability to discriminate high dimension structure which forms the polymer surface, and the mode of platelet adhesion was found to be greatly influenced by the chemical structure of the polymer, its domain shape and the microstructural surface of the polymer.

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Thrombus formation in carotid artery of adult wister rats (WR), young (8-9W) stroke-prone spontaneously hypertensive rats (SHRSP) or adult (18-19W) SHRSP was studied.
Electrode of anode was attached to the carotid artery and cathode was held the neighbor skeletal muscle. Three-mA direct current was discharged. Arterial occlusion was judged by the flow of evans blue dye which was injected through the femoral vein. Thrombus formation was quantified by a score of luminal setnosis of arterial transverse section. By 2min discharge, WR showed complete occlusion in all and 18±1.7 (mean±SE) in score at 15min after the discharge, in 50% (13±1.3) at 5min. By 1min discharge, occlusion in 25% (11±3.5) was observed at 15min.
Young SHRSP by 2min discharge showed occlusion in 85% (14±2.5) at 15min, but adult SHRSP did not have occlusion in all (5±1.5) at 15min. Pretreatment of tranylcypromine (i. v. or i. p.), PGI₂ synthetase inhibitor, did not influenced in the thrombus formation of adult SHRSP.
From the results, arterial thrombus by an electric current depended on discharge time and period to the sacrifice from the disharge. Thrombus formation was markedly less in adult SHRSP than in WR, and was not restored by administration of PGI₂ synthe tase inhibitor.
These results suggest that effects of synthetized PGI₂ in arterial wall may be less effects than platelet aggregability and others to diminished arterial thrombus formation in adult SHRSP.

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Experimental vascular iujuries induced by activation of platelets were observed under electron microscopy. Twenty eight rabbits were divided into two groups. In the first group, 0.7mg/kg of sodium arachidonate (AA) or 20-40mg/kg of ADP was injected into right carotid artery. AA was injected for 5 seconds and ADP was injected for one minute. In the second group, blood in the carotid arteries was washed out with saline throughout from 10 seconds before to one minute after the injection of AA or ADP. Immediately after, and 5 and 60 minutes after the injection, middle cerebral arteries in the subarachnoid space were isolated and observed under electron microscopy.
Immediately after the AA injection, deendothelialization, edematous changes in subendothelial layer and convolution of internal elastic lamina were seen in both groups. Adhesion of platelets on the deendothelialized inner surface was observed frequently. Sixty minutes after, abundant platelet thrombi with fibrin formation were seen. The results suggest the direct effect of AA, as a detergent agent, caused vascular injuries. Immediately after the ADP injection, small aggregates of platelets were seen in the vascular lumen in the first group, whlie the vascular wall showed completely normal finding in the second group. After 5 minutes, remarkable intracytoplasmic vesicular formation in endothelial cells was observed. Edematous changes in subendothelium and even in smooth muscle cells and sporadic deendothelialization were also seen. Adhesion of platelets on the deendothelialized site was seen. After 60 minutes, platelet thrombi with fibrin formation were observed. On the other hand, such injuries were not seen in any of the second group after the ADP injection. The results suggest that the presence of blood, especially aggregation of platelets, must be necessary to produce these vascular injuries.

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L-asparaginase, a unique anti-tumor agent that depletes L-asparagine, has been used as one of the effective drugs for human leukemias, especially for ALL.
However, this agent has been shown to exhibit various side effects which may cause serious problems clinically.
Although coagulation disturbance due to hypofibrinogenemia, among many side effects by the agent, has been well known, the mechanism involved has not been clearly defined.
In this study, we investigated the mechanism which may induce hypofibrinogenemia by administrating this agent to 10 cases of leukemias (7 cases of ALL, 1 case of AML, 1 case of AUL and 1 case of CML-BC).
1) In 9 out of 10 cases, hypofibrinogenemia (fibrinogen value is below to 200mg/dl) was observed but it did not have any significant correlation to doseor time-dependency.
2) PT and APTT in these cases were prolongedin parallel with the decrease in the fibrinogen value. FDP values were less than 20μg/ml.
3) The activities of other coagulation factores did not necessarily correspond to the decrease in the fibrinogen value.
4) The decrease in fibrinogen was followed by a decrease in serum albumin level.
5) In some cases, dysfunction of fibrinogen was observed at the stage of hypofibrinogenemia.
6) It was speculated that the mechanisms of hypofibrinogenemia after administration of L-asparaginase were due not only to inteference with protein synthesis but also to specific inhibiton of synthesis of fibrinogen and disturbance of its function.

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Anticoagulant properties of culture medium conditioned by human umbilical vein endothelial cells were investigated. In the presence of conditioned medium, both prothrombin time and activated partial thromboplastin time of normal plasma were prolonged by 4 and 10 seconds respectively compared with those in the presence of nonconditioned medium. The prologation of coagulation time was not affected by the presence or absence of serum in the culture medium, indicating that endothelial cells release the anticoagulant activity into the culture medium. The concentrated serum-free conditioned medium, not only directly inhibited Factor Xa activity but also enhanced the inactivation of Xa activity by antithrombin III. On the other hand, acidic glycosaminoglycans (GAG) isolated from conditioned culture medium showed Xa inhibition only in the presence of antithrombin III.
These results suggest that anticoagulant properties of endothelial conditioned medium are, at least in part, attributed to GAG (s) released by these cells.

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Highly active heparan sulfate was isolated from bovine kidney by affinity chromatography on antithrombin III-Sepharose.
High-affinity heparan sulfate (A-HS) accounted for only about 1% of the total HS. There were no significant differences between A-HS and nonaffinity heparan sulfate (NA-HS) in mobility by electrophoresis, in susceptibility to heparitinase and in chemical properties.
The anticoagulant activity of A-HS was 64units/mg by APTT method heparin as the standard, while NA-HS showed only 1unit/mg. In the assay, high-affinity heparin (A-Hep), prepared by antithrombin III-Sepharose affinity chromatography, had 4.5-fold higher specific activity than A-HS.
A-HS preferentially potentiated the inhibitory activity of antithrombin III for factor Xa rather than for factor IIa. On the other hand, the reversed results were obtained for A-Hep. A-HS showed its highest specific activity in potentiation of antithrombin III at the molecular weight of 13, 500.
Anti-Xa activity of A-HS measured by prothrombin/thrombin conversion assay was 3.8-fold lower than anticoagulant activities described above.
These results suggest that A-HS might contain an antithrombin III binding region smilar to that of A-Hep in the molecule despite the differences in the chemical properties of the two glycosaminoglycans.

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Suc-Tyr-Leu-Val-pNA and Suc-Ala-Tyr-Leu-Val-pNA were reported as the synthetic substrates specific for spleen fibrinolytic proteinase (SFP) and also leucocyte elastase-like proteinase (ELP). The present paper deals with reversible and irreversible synthetic inhibitors of ELP which were designed, based on the amino acid sequence of the substrates, and the application of one of the inhibitors for the purification of ELP. Among the various stereoisomers of Suc-Tyr-Leu-Val-pNA and Suc-Ala-Tyr-Leu-Val-pNA examined, Suc-L-Tyr-D-Leu-D-Val-pNA was the most effective and highly specific inhibitor. Based on these properties of the inhibitor to ELP, the affinity chromatographic purification of the enzyme was performed employing the inhibitor-Sepharose. The ELP preparation partially purified by delipidation, salting out and gel chromatography was applied on the affinity column equilibrated with Tris-HCl buffer (0.1M, pH 8.0) containing 0.5M NaClO₄. Adsorbed protein was eluted by the buffer containing 8M urea. It was noteworthy that the ELP preparation thus obtained showed a single band by SDS-PAGE and contained neither collagenase nor cathepsin G.
Concerning with irreversible inhibitors of ELP, chloromethyl ketone derivatives of Tyr-Leu-Val- and Ala-Tyr-Leu-Val- type peptides were synthetized. Among the compounds tested, Boc-L-AIa-L-Tyr-L-Leu-L-Val-CH₂Cl showed the strongest inhibitory effect on the enzyme.
The reversible and irreversible inhibitors reported here inhibited the hydrolysis of specific synthetic substrates and fibrin by either ELP or SFP.

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Modern attempts to re-evaluate Chinese traditional herb medicine conducted in recent China are regarded to be very much promising and therefore of noteworthy. The present paper is to introduce the results by the authors that some Chinese herb extracts which have been believed traditionally to improve the circulation disturbance in patients, do show very characterisitc type of inhibition of plasmin or thrombin enzymatically. The author's first screening test of 12 kinds of the herbs demonstrated luckily that several herb extracts potently inhibited plasmin or thrombin with dose dependency. Among them, Chi-shao (a kind of Paeonia Radix) was found to be the strongest inhibitor of plasmin. Therefore, mode of inhibition by Chi-shao was studied by using synthetic chromogenic substrate in the presence or absence of synthetic inhibitors such as EACA or t-AMCHA, which led to the following conclusions.
When the synthetic substrate are used to measure the enzyme activities, it is reasonably deduced that the plasmin-inhibition is the immediate and noncompetitive action, while the thrombin-inhibition, the slow and uncompetitive. When ca. 20mM of the synthetic inhibitor, AMCHA is present, inhibition of plasmin clearly reversed (disappeared). It is to be reminded that “mM order” concentration of AMCHA can inhibit active amidolytic activity while “ μM order” AMCHA can inhibit the fibrinolytic activity only indirectly through the binding of AMCHA to the “kringle part” of the plasmin molecule. Therefore the peculiar phenomena above mentioned may be well understood by assuming that the active ingredient of Chi-shao exerts two kinds of inhibition; one is towards the active center, while the other is towards the lysine binding site (LBS) of “kringle part” of plasmin. The similarity of the mode of action between Chi-shao inhibitor and α₂ plasmin inhibitor is to be emphasized.

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We have found urokinase cofactor (UK-cofactor) in normal plasma which directly increase the hydrolysis of S-2444 (Glu-Gly-Arg-pNa) by urokinase (UK) or the hydrolysis of plasminogen by UK. In the present paper UK-cofactor was purified and UK-cofactor activity was examined using purified plasminogen. UK-cofactor was purified as follows: Human plasma was passed through lysine-Sepharose, and precipitated by ammonium sulfate (50% saturation). Next, the precipitate formed was dissolved and passed through plasmin-Sepharose, chromatographed on DEAE Sephadex A-50. The eluted protein was gel filtrated on Sephadex G-75, chromatographed on hydroxyapatite. Finally, hydroxyapatite eluted protein was applied to Amberlite CG-50. Hydrophobic-ionic chromatoraphy was performed 0.2 M acetic, pH 4.0 buffer. Elution was done by pH gradient. The purified UK-cofactor showed single band on SDS-PAGE and PAGE. UK-cofactor did not activate plasminogen (Plg) to plasmin, while it increased the hydrolysis of S-2444 by UK and the hydrolysis of plasminogen by UK. UK-cofactor activity was analyzed using S-2251, fibrin plate and SDSPAGE. The activity of UK+UK-coactor+Plg or UK+Plg was measured by S-2251 and fibrin plate. The values of UK+UK-cofactor+Plg were 1.790 and 25.0mm², that of UK+Plg were 0.174 and 12.2mm². On SDS-PAGE, marked plasmin band was observed in UK+UK-cofactor+Plg, while small plasmin band was observed in UK+Plg.

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The inhibitor of activated protein C (APC), designated protein C inhibitor (PCI), was purified from human plasma. The inhibitor was a single chain polypeptide with molecular weight of 57, 000, and with microheterogeneity in pI: six pIs exist between 8.6 and 7.4. PCI was different from already established seven plasma protease inhibitors chemically and immunologically. It seemed to be also different from the recently discovered heparin cofactor II judging from amino acid composition, amino-terminal sequence and pI. PCI reduced an amidolytic activity of APC noncompetitively by forming a 1:1 molar complex with the enzyme, and also blocked the prolongation of plasma clotting time (APTT) by APC. PCI inhibited thrombin, Factor Xa, trypsin and chymotrypsin as well. The values of Ki of PCI against APC, thrombin and Factor Xa were 5.8×10^-8 M, 6.7×10^-8M and 3.1×10^-7M, respectively. The rate of inactivation of APC by PCI was enhanced about 30 fold in the presence of a relatively higher concentration of heparin (5-10units/ml). The rabbit antiserum to PCI completely abrogated the inhibitory activity to APC in plasma, thus suggesting that the PCI purified in this study is the sole inhibitor to APC in plasma. The purification and characterization of PCI is important to elucidate the mechanism of regulation of APC in blood coagulation and fibrinolysis in future studies.

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The arachidonic acid (AA) metabolism of leukocytes as well as platelets was studied in 5 patients with myeloproliferative disorders. When the leukocyte preparations from normal controls, whose polymorphonuclear neutrophil (PMNN) purity was more than 90%, were incubated with AA and A23187, the distinct peaks of leukotriene B₄ (LTB₄) and hydroxyeicosatetraenoic acids (HETEs) were constantly detected using reversed phase high-performance liquid chromatography. However, we found the lack of LTB₄ synthesis as well as the deficient production of 5-HETE in two patients with chronic myeloid leukemia (CML). Although the PMNN purity of their leukocyte preparations was 55% and 56%, it was likely that such an abnormality of CML leukocytes was derived from the 5-lipoxygenase deficiency of patient's PMNNs, because the formation of 5-lipoxygenase metabolites was observed in experiments with normal PMNNs mixed with CML leukocytes and leukocytes of the CML patient had good response to A23187. Platelet lipoxygenase deficiencies were found in one patient with CML and one with polycythemia vera (PV). Thrombin-induced malondialdehyde formation by their platelets was increased. On the other hand, the 5-lipoxygenase deficiency of PMNN was found neither in two patients with PV nor in one with essential thrombocythemia.

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Platelet-derived growth factor (PDGF) was partially purified from platelet lysate by isoelectric focusing and gel filtraction. Platelet lysate had three growth promoting factors for a human tumor cell line (K 562) which consisted of acidic (pI 3.1-3.5), neutral (PI 6.5-7.0) and basic (pI 8.8-10.4) substances. The activity of acidic growth factor in platelet lysate was higher than the activities of the other two factors. This acidic factor had a molecular weight of about 30, 000 daltons and was extremely stable to treatments with heat (90°C 10min), alkali or acid, but was inactivated by pepsin digestion.
Growth promoting activity was studied in vivo by means of a biological assay. The intramuscular administration of acidic factor purified by DE 52 chromatography had a marked potentiating effect on the growth of Lewis lung carcinoma in Balb/c mice.
It was, therefore, suggested that not only did PDGF enhance the growth of primaly tumor cells, but also that the PDGF released by platelet aggregation during the process of formation of the tumor thrombus activated the growth of metastatic tumor cells.

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In recent years, accumulating evidence has appeared suggestive of the fact that platelets are deeply involved in the lodging of tumor cells during the process of bloodborne metastases. One of the most significant observations inferring a role for platelets in the development of metastases is that the abiility of certain tumor cells to form metastases is related to their ability to promote aggregation of host platelets.
In the present paper, attempts were made to characterize the platelet aggregating material obtained from established human cancer cell lines.
The following retults were obtained
1) The platelet aggregating activities of tumor cells (HMV-1, M7609) were diminished by treatments with trypsin but not with collagenase or neuraminidase. Aggregating activity was preserved with a preparation of membrane from these tumor cells, although it was abolished by heating (100°C 15min) or sonication.
2) The existence of divalent cation was required for the platelet aggregation induced by HMV-1 tumor cells.
3) By SDS PAGE (autoradiography), membrane proteins with MW of 10, 000-20, 000 daltons which specifically bound to platelets were commonly found in cells with platelet aggregating activity (HMV-1, M7609).
It was therefore, conculuded that platelet aggregation induced by human tumor cells is evoked by direct interaction of platelets with aggregating proteins (MW 10, 000-20, 000 daltons) on the cell membrane.

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Endothelial cells were isolated from human umbilical cord and cultured by the modified method of Jaffe et al. The supernatant over the monolayer of endothelial cells cultured for 5-7 days and growing confluent, was tested as the material of this experiment. The supernatant demonstrated an inhibitory effect for ADP induced platelet aggregation, and this effect was increased by addition of arachidonic acid (10μM), and was diminished by the pretreatment with acetylsalicylic acid (100μM) for 1hr. By means of thin layer radio-chromatography of the supernatant, it was identified that this antiaggregating effect was developed by prostacyclin released from endothelial cells. Suspended endothelial cells were incubated with several drugs in order to evaluate the prostacyclin generation from endothelial cells, and assayed by RIA as 6-keto PGF_1α. Prostacyclin synthesis increased in 25 folds by addition of arachidonic acid. Nitroglycerin increased prostacyclin synthesis of endothelial cells, but hydralazine and verapamil did not increase it. Through these experimental results, it was concluded that effect of these vasodilators was possibly developed by the increase of prostacyclin synthesis from endothelial cells.

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PGI₂ and TXA₂ synthesis in glomeruli isolated from rat kidney was investigated and following results were obtained. PGI₂ and TXA₂ were measured by RIA kit (New England Nuclear). TXB₂ production was increased by addition of arachidonic acid, but synthesis of 6-keto-PGF_1α was slightly increased. The recovery of added TXB₂ in presence of indomethacin was almost 100%. On the contrary, the recovery of added 6-keto-PGF₁ was remarkably reduced. From these results it was concluded that 6-keto-PGF_1α might be farther metabolized in glomeruli. TXA₂ synthesis was inhibited by addition with aspirin (ASA), Indomethacin and OKY 1581. Dipyridamole slightly inhibited TXA₂ production in vitro and in vivo, and increased PGI₂ production in vivo, while Trapidil inhibited TXA₂ production in vivo and more remarkably increased PGI₂ production in vivo.

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Acid soluble protein (ASP) isolated from bone marrow or spleen cells prolonged the clotting times of human or mouse plasma in proportion to the amount added. The anticoagulant property of ASP was demonstrated in both APTT and PT, and the property was thought to be due to the inhibition of Xa generation or to the inhibition of prothrombinase (Xa·V·phospholipid·Ca complex) formation.
ASP had an anticoagulant activity, as heparin did, however, ASP and heparin neutralized the activity with each other.
In a modified APTT system, the anticoagulant effect of 1 unit of heparin was neutralized by 300-900μg ASP.
ASP also neutralized the antithrombin activity of heparin in the thrombinplasma system.
On the contrary, ASP significantly enhanced plasma or fibrinogen clotting caused by small amounts of thrombin (0.1unit/ml).
ASP could precipitate soluble fibrin monomer complexes (SFMC), as protamine sulfate did.
Endotoxin has been shown to cause strong cytotoxic and necrotic damages of bone marrow cells, therf ore, these findings suggest that DIC manifestation in endotoxemia and also in other inflammatory diseases accompanied by destruction of tissues or cells are controlled by the ASP.

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It is well known that human placenta contains tissue thromboplastins, which activate the blood coagulation systems.
The anticoagulant in the placenta, however, has not yet been fully clarified, because tissue thromboplastins dominate the anticoagulant activity.
We isolated an inhibitor, of the intrinsic and extrinsic coagulation systems, from the human placental microsome, of which thromboplastin activity was removed by delipidation and Con-A chromatography.
The inhibitor was purified by the following method.
The inhibitor was adsorbed on DEAE-Sephacel ion exchange column, and was eluted with stepwise increase of molar concentraion of NaCl.
The active fraction was further purified by gel filtration with Sephacryl S-300 and Sephadex G-100.
The final product showed a single band on polyacrylamide gel electrophoresis and was estimated to be approximatey 45, 000 daltons in molecular weight.
The heat and trypsin treatment suggested that the inhibitor would be a protein.
The acting point of this inhibitor on coagulation systems is unknown at present time, but the inhibitor was none of any well-known anticoagulants, such as AT-III, α₂-M, α₁-AT, C₁-INA, heparin.

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Most of complicated or semiquantitative factor XIII assay techniques are unsuitable for the purpose of quantitative assay in routine laboratories. However, now assay system developed by us requires only simple procedures and show high sensitivity and reproducibility. The principle of our method is as follows: Haet-defibrinated plasma is treated with casein, calcium, thrombin and dansylcadaverine (DC) and then maleimid is added to stop the incorporation reaction. Thereafter, the reaction mixture is gel-filtrated by Toyopearl (HW-40) mini column for the separation of DC-casein from free DC. With the excitation and emmition wavelengths fixed at 355nm and 525nm respectively, the fluorescence intensity is measured. The values of fluorescence intensity subtracted substrate blank are proportional to factor XIII activity. Comparative study was made between our method and Lorand's method and Laurell's method In twenty cases of normal subject and twenty cases of DIC, our method and Lorand method showed a good correlation. Our assay technique is simple and it requires only a couple of hours for a whole course of the assay. Moreover, the detectable limit, if necessary, can be brought to lower range than one percent by increasing the sample amount, elongating reaction time and subtracting plasma blank.

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This paper describes about measurement of Bβ 15-42 peptide derived from Bβ chain of fibrinogen or fibrin using a radioimmunoassay kit (IMCO). The Bβ 15-42 peptide was iodinated by chloramin T method, and the reaction mixture was passed through a Sephadex G-10 column. The yield of radioactivity was 68.3% with specific activity of 68μci/μg. This radioimunoassay system showed that coefficiency of variation was 7.7-11%, sensitivity was 0.16pmol/ml and recovery rate was 60.5%. The reactivity of Bβ 1-42 and fibrinogen with the antiserum to Bβ 15-42 was similar to that of Bβ 15-42. On the other hand, fibrinopeptide A and B showed no reactivity with the antiserum. When nomal plasma was incubated with urokinase in vitro, Bβ 15-42 immunoreactivity increased with a dependency on incubation time and dose of urokinase.
In 17 normal volunteers (male 8, female 9), values of Bβ 15-42 peptide were 0.30±0.56pmol/ml (mean±S. D.).
A patient with venous thrombosis who was treated with urokinase (48×10⁴ IU/day) and dextran sulfate (3000mg/day) showed high levels of Bβ 15-42 peptide.
These results suggested that the measurement of plasma levels of Bβ 15-42 peptide was important to know the degrees of fibrino (geno) lysis in vivo, but was not possible to distinguish fibrinogenolysis from fibrinolysis.

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In order to know the very early haematological changes which link closely with DIC of dengue haemorrhagic fever, haematological examinations at acute fever stage were performed on 52 children with unknown fever origin without haemorrhagic symptom. Serological examination on 30 cases revealed four cases of dengue virus (DV) infection, of which two were recent primary DV infection, and the other two were secondary. No haematological change was observed on the primary DV infected cases. However decrease of platelet counts (6×10⁴/μl at acute fever stage, and 20×10⁴/μl at 2 weeks after fever; 12×10⁴/μl and 21×10⁴/μl) and increase of factor VIII clotting activity (210%; 185%) were observed on the secondary cases, suggesting a participation of immunological mechanisms to the changes.

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A single polypeptide chain-high molecular weight urokinase purified from human kidney tissue culture was not decomposed autocatalytically, although it was easely traesformed to lower molecular weight forms (mol. wts. 31, 000-36, 000) by trypsin, plasmin, papain, and especially by elastase treatments. The new enzymes porduced had almost the same pyro-Glu-Gly-Arg-pNA amidolytic activity, but showed lower Glu-plasminogen-activating activity than those of the parent enzyme. Using immunological and fibrin-Sepharose column techniques, it was confirmed that the new enzymes lacked the light chain corresponding part of the urinary HMW-UK molecule and had much lower affinity for the fibrin molecule.
The Lys-plasmin was also found to be very sensitive to papain treatment, and subsequent production of a new enzyme (mol. wt. 45, 000-52, 000) was observed.
The urinary trypsin-plasmin inhibitor, UTI₆₇ could be also fragmented by papain and pronase tratments. Five new inhibitors (mol. wts. 6, 500-26, 000) purified from papain-treated UTI₆₇, exhibited much higher auti-plasmin/antitrypsin activities than those of the parent UTI.
It is possible that such enzymatic modifications of enzyme and inhibitor molecules could play a significant role in vivo in the regulation of plasma and tissue fibrinolytic systems.

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The direct clotiysis by urokinase (UK) may be due to exogenous clotlysis. On the other hand, the clot formed in the presence of UK may undergo endogenous clotlysis. To elucidate the optimal dosage of UK in the exogenous and endogenous clotlysis, the Chandler's loop method and thromboelastography (TEG) were used.
Fibrinogen, plasminogen, α₂-macroglobulin, α₁-antitrypsin, antithrombin III, α₂-plasmin inhibitor, C₁-inactivator, hematocrit and platelet count were measured. The correlation between these factors and clotlysis time on TEG were analysed.
In the loop method, UK agents from six companies (A-F) were used. previously formed Chandler's artificial clots were placed into loops containing 200, 120 and 60IU/ml of UK in saline solution, and these loops were rotated for 4-24 hours. Percentage reduction of clot weight was measured (Fig. 1).
In TEG, clotlysis time on TEG was measured using 5, 10, 20, 25, 30, 40, 50 and 100IU/ml of UK.
The artificial clots in 200IU/ml of UK solution showed mean clotlysis 55.8% in 4 hours by the Chandler's technique. By TEG, the clot formed in the presence of 10-20IU/ml of UK revealed clotlysis in 46-120min after clotting.
C₁-INA and α₂-PI showed correlation between clotlysis time on TEG (Fig. 2).

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OP-Rheometer System was developed to measure the viscosity at lower shear rate, 0.2-40 sec^-1 and viscoelasticity of blood at 0.3-1.0Hz in the view point of clinical medicine. This system consisted of the mechanical unit, the control unit, the data processing unit and printer unit. The dynamic or constant flow data were automatically printed out after data processing.
Outer cylinder was connected to the tortion wire assembly and supported by a pair of magnetic bearings. The angular displacement of outer cylinder due to blood viscosity by rotation or oscillation of inner cylinder, was detected by the pair of linear variable differential transformers.
In clinical application of this rheometer, we reconfirm the viscosity and viscoelasticity of blood in diabetic patients showed higher level thsn those of normal subjects.

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