The distribution of anionic sites on the platelet membranes was studied under the different conditions by using cationized ferritin (CF).
Experiment I; Human platelet rich plasma (PRP) was fixed in 2.5% glutaraldehyde at room temperature for 30min, and followed by washing with 0.01 M phosphate buffered saline (PBS). Samples of PRP were incubated with CF at 37°C for 30min. After washing with same PBS twice, the specimen was prepared for electron microscopy. CF were observed as evenly distributed dense particles on the cell surface of the platelet.
Experiment II; PRP was washed with 0.01 M PBS containing sodium citrate, and then resuspended in the solution (suspension of washed platelets: SWP). After incubation of the SWP with CF at 37°C, each specimen obtained at 1-45min was similarly washed with the PBS and prepared for observation. In the incubation for less than 15min, a large amount of the particles were clustered on several sites of the cell surface. The platelet incubated for more than 30min, showed an uptake of most particles into the dilated open canalicular system (OCS). A small amount of clustered particles, however, were left on the surface behind.
Experiment III; Immediately after fixation, the SWP was washed with the PBS, and incubated with CF at 37°C for 30min. The specimen was washed with the PBS and prepared for observation. The cell surface was evenly studded with the dense particles.
In each experiment, the control specimen revealed no presence of binding site for the native ferritin particles on the surface of the plasma membrane or the membrane of OCS.
It is concluded that the anionic sites on the platelet surface may be mobile in the fluid state and glutaraldehyde fixation may have some effects on the arrangement of CF particles as well. Furthermore, when the CF particles are taken up into the OCS, the anionic sites on the membrane systems may also undergo electrical changes, probably due to the secretion reaction of platelets stimulated by CF particles.

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To choose a suitable dose of aspirin (ASA) in ASA therapy, two small doses of ASA were administered to healthy volunteers and to patients with thromboembolism. From examination of platelet aggregation and malondialdehyde (MDA) formation after administration of ASA for a week, it was found that the pharmacologically effective dose of ASA as an anti-platelet drug was less than 50mg, since about 90% of MDA formation was inhibited after administration of 50mg of ASA. Furthermore, it was also observed that the average inhibition in all subjects examined was 70% after administration of 10mg of ASA. However, as the effects of 10mg of ASA on platelet aggregation and MDA formation were different from case to case, it seems reasonable that a suitable dose of less than 50mg of ASA should be selective by measuring platelet aggregation and MDA formation of each patient after ASA therapy. Although changes in platelet aggregation and MDA formation after ASA therapy were different between healthy male and healthy female volunteers, there was no marked difference between male patients and female patients.

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Factor VIII complex bound to both collagen in suspension and collagen coupled Sepharose beads with preferential binding of the forms of FVIIIR: Ag with the FVIIIR: RCoF. The FVIIIR: Ag in the eluate from collagen affinity column showed less anodal migration in crossed immunoelectrophoresis compared to the FVIIIR: Ag in the breakthrough fractions, suggesting the presence of higher molecular weight forms of FVIIIR: Ag in the eluate.
The new immunoelectrophoretic methods were described for studying the interaction between collagen and FVIIIR: Ag. Affinity immunoelectrophoresis with a collagen wedge revealed a different pattern of adsorption between FVIIIR: Ag in normal plasma and the FVIIIR: Ag in commercial FVIII preparations or in VWD IIa plasma. By the method of crossed affinity immunoelectrophoresis, most of the FVIIIR: Ag of normal plasma was adsorbed by the 100μg/ml collagen spacer gel, while no FVIIIR: Ag of variant VWD IIa plasma bound to the same concentration of collagen. Our methods of affinity immunoelectrophoresis with collagen spacer gel were easy to perform and may be useful for screening variant VWD plasmas. The use of an affinity wedge is a convenient method for finding quickly an optimal concentration of an affinity reagent.

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The effect of a substanse in human milk on the several metabolic reaction in platelet and a few characterization of the substance were studied in this paper. The amount of thromboxane B₂ produced after the aggregation did not differ from that of resting platelets. The activity of adenylate cyclase in platelet received the non competitive inhibition by the substance. Although the activity of cAMP phosphodiesterase was also inhibited by the substance, the inhibitory degree of the substance on the activity was less than that on the activity of adenylate cyclase. The platelet aggregating activity of the substance was lost by the heating at 100°C for 5min. and the treatment by trypsin and N-ethylmaleimide.
It was elucidated that platelet aggregating substance in human milk induce the aggregation by the mechanism unrelating to production of thromboxane B₂ and is a high molecular protein, which need the SH group for occurence of the aggregating activity.

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Platelet aggregation and adenine nucleotides of platelet concentrates (PC) produced by Kanagawa Red Gross Center were investigated.
ADP-and epinephrine-induced aggregation of PC were markedly decreased. On the other hand, collagen-and ristocetin-induced aggregation were normal. Twenty units of PC were transfused to the thrombocytopenic patients (11 cases of acute leukemia, 1 case of aplastic anemia). Citrated PRP was obtained 1 hour and 24 hours after transfusion. ADP-and epinephrine-induced aggregation were improved after transfusion, while collagen-and ristocetin-induced aggregation had a tendency to decrease.
The contents of ATP and ADP of the platelets in PC were almost normal. The release of ATP and ADP induced by collagen were slightly lower than that of the fresh platelets. There were no appreciable changes in the contents and the release of adenine nucleotides before and after transfusion. No significant correlation was found between the platelet aggregabilities and adenine nucleotide contents. Impaired aggregation by ADP and epinephrine of PC was improved by adding normal fresh plasma.
Therefore the cause of impaired aggregabilities of PC may be attributed to the alteration of the interaction between aggregating agents and platelet membrane rather than the impairment of adenine nucleotide metabolism.

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Thromboxne A 2 (TXA 2) and prostacyclin (PGI 2) play an important role in the development of thrombosis.
In this study, plasma TXB 2 (stable metabolite of TXA 2) and 6-keto-PGF1a (stable metabolite of PGI 2) concentration in several diseases were measured by RIA.
Results were as follows.
1) Plasma TXB 2 concentration was 223.4±47.9pg/ml (Mean±SD) in normal controls and significantly increased in patients with DM (328.4±45.3), IHD (320.3±99.1), liver cirrhosis (321.2±112.5), pancreatitis (342.2±189.9) and DIC (557.0±207.5).
2) Plasma 6-keto-PGF1a concentrtion was 9.1±6.9pg/ml in normal controls. There was no significant difference in 6-keto-PGF1a concentration in several diseases.
3) There was a positive correlation between plasma TXB 2 and 6-keto-PGF1a concentration. (r=0.22)
4) TXB 2, generated by exogenous thrombin, showed significant increase in platelets of diabetics with microangiopathy, compared with normal controls.
5) TXB 2 generation by exogenous arachidonic acid also showed significant increase in platelets of diabetics.
6) There was an negative correlation between plasma TXB 2 concentration and serum HDL-cholesterol. (r=-0.31)
From these findings, plasma TXB 2 concentration seemed to be increasing in diseases with thrombotic tendency. Moreover, it was suggested that in diabetics with microangiopathy, not only cyclooxygenase but also phospholipase activity in platelets are significantly accelerated.

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Mean platelet volume (MPV) and platelet count (PLT) were measured by a Coulter Counter Model S-Plus and cardiac output (CO) was measured with the dye dilution method. We reported previously MPV was significantly increased (p<0.01) and PLT was significantly decreased (p<0.001) in patients with congestive heart failure as compared to normal subjects. In this study, MPV, PLT and CO were measured before and after treatment of patients with cardiovascular disease including low output group (% Cardiac index<60%). After the symptoms were improved, MPV was significantly decreased (p<0.001), while PLT was significantly increased (p<0.01) in patients with congestive heart failure. MPV and PLT exhibited a significant negative correlation in patients with cardiovascular disease (r=-0, 439, p<0.01). In low output group, MPV was significantly decreased (p<0.01) after treatment. These results indicate that the measurement of MPV and PLT is useful in understanding the cardiac function and clinical course in patients with cardiovascular disease.

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In 22 untreated male patients with essential hypertension (HT) and 6 male normotensives (NT), platelet function and erythrocyte membrane sodium transport (ouabain sensitive rate constant: K, ouabain sensitive sodium efflux: M) were studied after low (NaCl 5g/day) and high (25g/day) sodium diet individually for 7 days.
In HT, 24 hour-average of mean blood pressure was elevated (5mmHg, p<0.005) after high sodium diet, but not in NT.
In HT, platelet aggregation induced by ADP and epinephrine increased significantly after high sodium diet compared with after low sodium diet (Fig. 1). In contrast, plasma levels of beta-thromboglobulin and platelet factor 4 were significantly higher after low sodium diet than after high sodium diet (Fig. 2). This may be due to the elevated blood viscosity (p<0.005 at any shear rate) because of increased hematocrit and serum protein (p<0.001) after low sodium diet than after high sodium diet.
In NT, no sgnificant change was obsreved in platelet function.
In HT, K and M were reduced (from 0.281±0.067 to 0.265±0.064/h, from 3.238±0.571 to 3.071±0.540 mEq/1 cell h, p<0.02) after high sodium diet compared with after low sodium diet. But there was no significant correlation between the changes in platelet function and the reduction in erythrocyte membrane sodium transport.

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To clarify the role of platelets on cancer metastasis formation, we have studied the inhibitory effect of various antiplatelet agents on hematogenous metastases. We have revealed that platelets played an important role on the growth of metastatic foci as well as the lodgement of tumor cells.
Combined inhibitory effect of antiplatelet agents and cyclophosphamide on metastases was obtained in mice treated with each agents, particulary diltiazem. NK activity of spleen cells was inhibited in mice treated with any antiplatelet agents.
Relationship between inhibition of NK cell activity and enhancement of i. v. induced pulmonary metastases by trypsin-treated 3 LL cells was observed in mice treated with antiplatelet agents. PDGF showed the potent enhancing effect on tumor growth in each metastatic step.
In conclusion, antiplatelet therapy was effective on the inhibition of tumor growth in pulmonary metastatic foci, and its inhibition was increased by the combination with anticancer agents. Disadvantage of inactivation of host cells such as NK cells by antiplatelet therapy should by considered against circulating tumor cells. Inhibition of release of PDGF by antiplatelet therapy suggested the marked usefulness on the inhibition or prevention of tumor growth and metastasis formation.

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Effects of local antifibrinolytic therapy for acute respiratory failure following DIC was investigated. Acute lung injury with DIC was evoked in rats by the intraperitoneal injection of 10mg/kg endotoxin. An intraperitoneal injection of endotoxin was followed by a rapid increase in plasminogen activator (PA) release from alveolar macrophages (AM) and corresponding increase in fibrinolytic activity in bronchoalveolar lavage (BAL) fluid. Ultrasonic nebulization of 500mg/kg tranexamic acid was performed 1 hour after injection of endotoxin. Ultrasonic nebulization of tranexamic acid prevented not only increase in fibrinolytic activity in BAL fluid, but widening of alveolar-arterial oxygen difference (AaDO₂) and increase in protein BAL fluid. These results suggest increase in fibrinolytic activity in alveoli by AM may be one of the key phenomena in development of acute lung injury following DIC, and also suggest local antifibrinolytic therapy may be available for treatment of acute respiratory failure following DIC.

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We reported that experimental disseminated intravascular coagulation (DIC) could be induced by a 4-hr sustained infusion of endotoxin (100mg/kg) in rats. This experimental DIC was used to study the effects of defibrinogenation with batroxobin against DIC. One hour before the infusion of endotoxin, 200 batroxobin unit (BU)/kg of batroxobin was injected intraperitoneally. Immediately after the injection, fibrinogen level markedly decreased and fibrinogen and fibrin degradation products increased. The preventive effect against DIC was noted by the partial inhibition of the fall in the platelet counts and lessened number of renal glomeruli with fibrin thrombi, in the rats treated with 200BU/kg of batroxobin 1hr before the infusion of endotoxin (100mg/kg/4hr). From these results, it was shown that fibrinogen metabolism plays an important role in the DIC state.

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Some factors in plasma or thrombi are thought to be related to the progress of the pulmonary fibrosis following the diffuse pulmonary microthromboembolism. Among such factors which promote the proliferation of fibroblasts in vitro, thrombin, factor XIII, PDGF, MDGF and EDGF are included.
The roles of factor XIII or PDGF were investigated in the recovery stage after the diffuse pulmonary microthromboembolism induced by the intravenous injections of tissue thromboplastin (T. Tbp).
In the lungs of rabbits autopsied 4 or 8 weeks after the repeated injections of T. Tbp and PDGF for 3 days, focal interstitial infiltrations of nuclear cells (mainly neutrophils) were seen and alveolar septa were thickened. Foci of such lesions were increased 8 weeks after the same injections for 6 days.
Above results suggest that the interstitial changes may progress to the proliferation of fibroblasts and the increase of collagen fibers.

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(Multiple organ failure) MOF or (Disseminated intravascular coagulation) DIC often becomes a fatal complication for the patients suffering from endotoxin shock. And it is also pointed out that MOF and DIC share many common or simiar clinical features. To clarify the relationship between these two, we surveyed the changes in haematological indices of organ failure patient.
DIC was complicated in 10 of 14 patients with endotoxin shock and 9 of these were victimized. In the latter 9 cases, failure of more than three organs occured concomitantly. DIC observed in patients with organ failure is as follows: 10 in 13 cases of lung failure, 3 in 5 cases of heart failure, 10 in 12 cases of renal failure, 3 in 5 cases of liver failure and 3 in 4 cases of gastro-intestinal bleeding. Both organ failure and DIC usually began at the lung in endotoxin shock patients. From the viewpoint of haematology, hypercoagulable tendency preceded the lung failure. For example, decrease in antithrombin III and plasminogen level was observed prior to that in platelet.
In endotoxin shock patients, MOF and DIC have close relationship, which suggests that hypercoagulable state induced by endotoxin triggers an organ failure and results in MOF through chain reactions.

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In the extensive burn injuries, functional changes in organs and hematological alterations were occured. In this report, the relation between hematological alterations (blood coagulation and fibrinolysis) and functional changes of kidney were studied experimentally. For this purpose, heparin and OP-41483 were administered to the burned animals in order to make clear the effects of anticoagulation and anti-platelet therapy. The animals which were induced third degree burn (35% of the body surface area) on the back has been divided into five groups:
1st group: no therapy
2nd group: intravenous infusion only
3rd group: intravenous infusion with heparin
4th group: intravenous infusion with heparin and antithrombin III
5th group: intravenous infusion with OP-41438
Laboratory examinations of renal function, blood coagulation and fibrinolysis were carried out pre and postburn (8 and 24 hours). The results were as follows.
The burned animals which belonged to the 4th and 5th groups showed a very favorable prognosis. It appeared to be the proof that administration of anti-platelet agent like OP-41483 or heparin with antithrombin III concentrated agent was extremely effective in extensive burn injuries to prevent renal insufficiency.

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The purpose of the study was to investigate the effect of eicosapentaenoic acid (c20: 5, ω3; EPA) on the serum lipids and fatty acid profiles, platelet functions and coagulation systems.
Highly concentrated (75%) EPA ethylester, which was isolated and purified from sardine oil, was given to 17 patients suffering from hyperlipoproteinemia and/or hypertension for at least 16 weeks. Daily doses of EPA were 0.9g in 6, 1.8g in 7 and 2.7g in 4 patients. Initial mean total cholesterol level (TC) was 270.9±45.0mg/dl and reduction rates of TC were 4.4% at 4 week-period, 5.8% at 8 week-period, 3.1% at 16 week-period and 6.4% at 12 month-period of the treatment. Triglyceride level fell significantly, 20.9% at 4 week-period, 13.0% at 16 week-period and 18.6% at 12 month-period. HDL-cholesterol did not significantly change throughout the treatment. Changes in plasma main fatty acid profiles were shown in fig 1. plasma EPA increased from 1.84mol% to 5.41mol% after 4 weeks and 4.77mol% after 16 weeks, while other polyunsaturated fatty acids except arachidonate inclined to decrease. Above all, plasma linoleate fell significantly after taking EPA. No correlation between ΔTG and Δlinoleate suggests that exogenous EPA bound mainly to β-position of triglyceride and phospholipids in competition with other polyunsaturated fatty acids.
As for platelet functions, no significant changes in platelet adhesiveness and collagen induced aggregation were observed. 1μM ADP induced aggregation lowered from 46.6% in maximal amplitued before the treatment to 29.0% at 16 week-period, 7.1% at 6 month-period and 27.3% at 12 month-period. Over 5mol% of EPA in plasma inhibited ADP induced aggregation. Coagulability expressed by thromboelastogram tended to be reduced, however, prothrombin time, PTT, antithrombin III, α₂ macroglobulin and α₁ antitrypsin did not alter.
In conclusion, EPA reduced both serum lipids and ADP induced aggregation, which would be favourable for the prevention of atherosclerotic thrombotic diseases.

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¹¹¹In-labeled platelet scintigraphy was carried out in 20 patients suspected of thrombosis, to evaluate its efficacy for the detection of intracardiac and intravascular thrombi. Hemostatic studies including PT, aPTT, fibrinogen, FDP, platelet retention by Hellem II method, platelet aggregation induced by ADP (1μM, 10μM), collagen (2μg/ml) and arachidonate (2mM), and platelet survival were examined simultaneously. Two of these patients were also studied by fibrinogen scintigraphy labeled with ^99mTc. The platelets obtained from the patients were labeled with ¹¹¹In-oxine according to a method originated by Yui et al.
In the platelet scintigram, positive findings were demonstrated in 9 cases: 2 left ventricular, 3 left atrial, and 4 vascular thrombi. These were in a good accordance to the findings obtained by at least one from angiography, echography and surgery.
Platelet aggregation induced by 1μM ADP was elevated in 4 of the 7 patients with positive findings and 3 of the 7 patients with negative findings. Shortened platelet survivals were observed in two of the 10 patients; dissecting aortic aneurysm and infective prosthetic valve endocarditis with hepatosplenomegaly. There was no significant difference in the platelet function and the platelet survival between the patients with positive and negative findings.
It was difficult to detect thrombi by the fibrinogen scintigraphy labeled with ^99mTc in the two patients studied.
In conclusion, ¹¹¹In-labeled platelet scintigraphy seems to be very useful to detect thrombi.

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Antithrombotic therapy may be effective to prevent the reocclusion in vascular surgery. In this report, we investigated the process of thrombus formation in prosthetic graft, by means of platelet imaging. The objective cases were 18 patients who underwent the reconstructive operation of prosthetic graft for aortoiliac vascular disease, such as abdominal aneurysm, Leriche's syndrome, arterio-sclerosis etc. Platelet scintigraphy was performed in 48 hours after the administration of ¹¹¹In oxine labelled autoplatelet.
The results were as follows:
1. Platelet accumulation on the inner surface of prosthetic graft was serious in within 7 month after reconstruction, mild in 8 month to 12 month, and slight in beyond 1 year.
2. In patient who took the antiplatelet therapy since the replacement of velour knitted Dacron, platelet accumulation diminished under the administration of Ticlopidine (300mg/day).
The conclusions were as follows:
1. Platelet accumulation correlates with postoperative period more than the types of vascular lesion and prosthetic graft.
2. Platelet accumulation is influenced by the antiplatelet therapy.
3. We propose that the antithrombotic therapy must be continued for at least 1 year after reconstruction.

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Chages in blood coagultion and fibrinolysis by acute portal vein occlusion were investigated using adult mongrel dogs.
All five dogs died within 81-130 minutes (mean: 105 minutes) after the ligation of the portal vein, and portal system became to be in hypercoagulable state. Deposition of fibrin was observed in capillary vessels in the mucous membrane of the small intestine 1.0 minutes after the ligation of the portal vein. Thus histological examination proved DIC. Before ligation of portal vein, a high degree of activity of tissue plasminogen activator was noted in the small vessels, mainly of the submucosal layer of the small intestine. However a significant decrease of its activity was observed after the ligation of the portal vein, and finally it disappeared within 20 minutes. This suggested the progression of the process into irreversible DIC. After simultaneous occlusion of portal vein and superior mesenteric artery, the same changes were demonstrated later than portal vein ligation alone, and these five dogs died within 70-195 minutes (mean: 138 minutes).
However, by bypassing portal blood into the femoral vein through the heparinized hydrophilic catheter (Anthron^®) under complete ligation of the portal vein, these deteriorating changes were not observed, and these five dogs survived in good condition for long period. Thus, a safe method for preservation of portal bed circulation during temporary shut-down of the portal vein has been established.

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The enzymatic and protein-chemical properties of a biliary plasminogen activator, bilokinase (BK) were described by Oshiba and Ariga (JBC, 258, 622, 1983). In the present investigation, the immunological natures of BK were examined and compared with those of urokinase (UK). The tissue localizations of BK in hepato-biliary and other systems were studied using the immunofluorescent technique.
A precipitin line was clearly formed between BK and its anti-body whereas, no precipitin line formed between BK and UK-antibody, or between UK and BK-antibody.
The bovine BK-antibody inhibited bovine BK, but did not inhibit UK. On the contrary, the bovine UK-antibody inhibited bovine UK, but not bovine BK.
Bovine liver treated with bovine BK-antibody showed immunofluorescence in hepatic cells. Note that there are stained cells and unstained cells.
The following conclusions were obtained;—
1) BK and UK were immunologically distinct.
2) The localization of BK-antigen was clearly demonstrated in hepatic cells and in the mucosa of gall bladder with the immunofluorescent technique.
3) The presence of BK-antigen was also detectable in the outer and inner layers of blood vessels.
4) The site of BK production and the difference between BK and tissue plasminogen activators remain to be clarified.

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Following intrarectal administration of high molecular weight urokinase (HMW-UK; mol. wt. 53, 000, 124, 000IU/mg protein) or its functionally-active heavy chain (H-chain UK; mol. wt. 31, 800, 212, 000IU/mg protein) to mice, prolonged activation of plasma fibrinolysis was observed. The plasma levels of pyro-Glu-Gly-Arg-pNA amidolytic and euglobulin fibrinolytic activities reached a maximun (42.8±5.6nmoles/ml and 10.6±7.1mm/0.03ml, respectively) at 1 to 2hr after the administration of 300IU of either enzyme per mouse. Thereafter, both activites declined slowly. Absorption of the UK was confirmed by pooling the fractions which were eluted by 6M urea buffer following [N^α-(ε-aminocaproyl) -DL-homoarginine hexylester]-Sepharose affinity chromatography. Although the eluted fractions were shown to form a precipitation arc with appropriate antisera by immunodiffusion, the neutralization was not complete and approximately 40% of the activity remained. Activation of plasma fibrinolysis may not be due only to UK absorption but also be related to some enzyme indirectly released.

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A Chinese herb, Chi-shao (CS: a kind of Paeoniae Radix) was previously reported to inhibit plasmin. In this note, the active component was purified from CS extract: When active solution obtained by aceton fractionation (33-55%, v/v) was chromatographed with a Sephadex G-50 column, a target ingredient was eluted forming a broad peak with large tailing which indicated weak adsorption to the Sephadex bed. By the re-chromatography in the presence of a detergent, the component was eluted without tailing. The resulting chromatogram showed that the molecular weight of the component should be about 1, 200.
The purity was increased by almost 1, 000 times from the initial extract.
As to the mode of plasmin inhibition, combined effect of CS extract and synthetic inhibitor, tranexamic acid (tA) was examined: When an amidolytic assay (S 2251) was used, to interrupted the inhibition of plasmin by CS, whereas in fibrinolysis, CS and to were additive to each other.

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The activity of placental urokinase inhibitor (PUKI) was examined using a new fluorogenic substrate, Glutaryl-Gly-Arg-MCA, which is specific for urokinase. The rate of AMC release was determined by the increment of fluorescence from 5 to 10min, after the incubation of the assay mixture. The activity of PUKI was determined indirectly by measurement of residual urokinase after incubation with the inhibitor separated from placental extract using Lysine-Sepharose.
The residual urokinase activity was observed to be much higher in the presence of more than 0.3% albumin in the assay mixture, which was due to increased stability of urokinase. The residual urokinase activity was also quite labile at pH 7.5, but stable at pH 8.0. The relative fluorescence and residual urokinase activity was found to be linear in the range of 5-20 IU/ml (Fig. 1).
The usefulness of this method was evaluated by comparison with fibrin plate method. The correlation coefficient obtained from assays of 33 samples was 0.78 (Fig. 2).

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It has been not reported whether thrombin-like enzyme was contained or not in venom of Habu (Trimeresurus flavoviridis) which have the strongest toxic effect in the case of the Habu bite. The present study was taken to clarify the existence of thrombin-like enzyme in venom of Habu. As a starting material, the lyophilized crude venom of T. flavoviridis was used and ammonium sulphate fractionation gel filtration using sephadex G-150 and affinity chromatography using arginine-sepharose were procedured in order to separate and purify a thrombin-like enzyme from crude venom. Time required to convert from the purified rabbit fibrinogen to fibrin by the addition of this enzyme was determined as clotting time using Hema tracer. The enzyme was purified a 137-fold increase in specific activity and purified preparation showed a single band on SDS-PAGE. Molecular weight of this enzyme was 65, 000-70, 000 daltons by means of SDS-PAGE and gel filtration and isoelectric point of this enzyme was pH 4.5-5.5. Any enzymatic properties were studied on the differences between this enzyme and bovine thrombin and this enzyme could coagulate only the rabbit plasma.
From these results, it was concluded that thrombin-like enzyme entirely different from bovine thrombin, existed in T. flavoviridis venom.

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It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycos-aminoglycans such as heparan sulfate, which could be antithrombin cofactor on the endothelium. In the present study we have directly evaluated ¹²⁵I-labeled bovine thrombin binding to cultured porcine aortic endothelial cells. Scatchard plot revealed high affinity binding sites (3×10^-8M) for thrombin on endothelial cells. Specific binding of ¹²⁵I-thrombin to endothelial cell surface was approximately by 50% inhibited in the presence of protamine sulfate and after the removal of cell surface heparan sulfate by the treatment of cells with heparitinase. Whereas, removal of chondroitin sulfates by chondroitinase ABC did not affect the binding.
Our results suggest that cell surface heparan sulfate is involved in approximately a half of high affinity binding sites for thrombin on endothelial cells.

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