A method for the separation of platelets on the basis of their volume has been developed using counterflow centrifugal elutriation (CCE).
Human peripheral blood platelets were eluted into nine fractions and four types of pattern of the eluted platelets from CCE were observed (Fig. 3),
For measurenment of pletelet diameter a flow cytometry was used, and the diameters are given as the mean channel of foward scatter. The smallest-size platelets had a mean channel of 118±7.3. Each succesive fraction had a progressively lager mean channel. The mean channel for the largest-sized platelets, designated fraction 9, was 146±6.8 (Fig. 5).
The nine platelet fractions were separated into small platelets (fraction 2-4) and large platelets (fraction 7-9).
Transmission electron microscopy demonstrated that small platelets had a mean diameter of 2.1±0.48μm and large platelets had a mean diameter of 3.1±0.48μm. The large platelets possessed a number of granules and mitochondria in the cytoplasm than that of the small platelets (Table 1).
Platelet function was measured by ADP and collagen-induced aggregation. For analysis, height of aggregation curve (h), slope in aggregation curve (θ), and lag time in aggregation time were observed (Fig. 8). No significant difference between small and large platelets in aggregation was found (Table 2).

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The morphology of the mesenteric arterial wall, and the role of the arachidonate cascade in ADP-induced thromboformation, is studied in two groups of streptozotocin-induced diabetic rats, in comparison to a group of normal rats.
Light microscopy demonstrates the dilated state of the arteries of the diabetic animals, together with the diminished wall thickness in “young diabetics”, and an increased thickness in “old diabetics”, as compared to normal animals. Scanning electron microscopy shows the area of local deendothelialization in the arteries of “young diabetics” to be larger than in both other groups.
Combined use of a cyclooxygenase and a prostacyclin synthetase inhibitor, reveals that the decreased PGI₂ release from arteries of diabetic animals, can be related to a diminished activity of the prostacyclin synthetase.

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The influence of disseminated intravascular coagulation (DIC) on major organs was studied in dogs. Endotoxin was intravenously infused in dogs over 90 minutes at a dose of 2mg/kg to induce DIC. The parameter used to evaluate this influence were WBC, platelet counts, levels of fibrin degradation products, plasminogen, antiplasmin, antithrombin III, S-GOT, S-GPT, S-BUN and S-Creatinine, prothrombin time, microcirculations in the liver and kidney, and respiration rate over a period of four hours.
Immediately after the onset of DIC, microcirculation in the liver was markedly decreased, while S-GOT and S-GPT were increased. S-BUN and S-Creatinine were also elevated although microcirculation in the kidney remained normal.
These findings demonstrate that multiple organ failure (MOF) should be treated as soon as DIC is suspected or diagnosed.

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In 40 cases of full term pregnancey cesarean section performed before and during onset of labor. The amniotic fluid and blood samples from the uterine artery (U. A), uterine vein (U. V) and the peripheral vein (P. V) were collected and the changes of FPA, FPB β 15-42, HMW-kg, LMW-kg, glandular kallikrein and kinin were studied.
The levels of FPA in UV increased significantly during onset of labor as compared to the increase in UA and PV and although the UV level of FPB β 15-42 also showed the same as that of FPA pattern of increase during labor but the increase was not so sharp. Levels of HMW- and LMW-kg gradually decreased during onset of labor but no significant differences among UV, UA and PV were found, Level of kinin in PV increased significantly during labor but remained unchanged in UV. In the amniotic fluid, the levels of FPA, FPB β 15-42, and HMW-kg increased gradually while that of kinin increased significantly and decreases of glandular kallikrein, LMW-kg and kininase II were seen during labor.
These findings suggested that with the increased consumption of HMW- and LMW-kg and kinin during labor, the uteroplacental circulation exhibited a state of hypercoagulability with secondary hyperactivity of plasmin resulting in the increase in vascular permeability to the amniotic fluid and increased production of kinin.

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In many secretory cells, cyclic nucleotides and Ca²⁺ cooperatively or antagonistically control cell responses. Activation of platelets with thrombin caused a rapid breakdown of phosphoinositides and an increase of cytoplasmic free Ca²⁺ concentration, which resulted in shape change, secretion and aggregation. The hypothetical concept has recently been proposed that inositol trisphosphate, a degradation product of phosphatidylinositol 4, 5-bisphosphate (PIP₂), serves as a second messenger for mobilizing intracellular Ca²⁺. The effects of cAMP and cGMP on thrombin-induced human pletelet responses were investigated. Thrombin-induced serotonin secretion and aggregation were inhibited by pretreatment with dibutyryl cAMP (dbcAMP) or 8-bromo cGMP (8bcGMP) in a dose-dependent manner. However, shape change was not affected by 8bcGMP. Preincubation of platelets with dbcAMP or 8bcGMP was without effect on the basal level of inositol trisphosphate and free cytosolic Ca²⁺, measured by fluorescent indicator quin 2, but suppressed their thrombin-induced enhancements. Enhanced [³²P] incorporation into phosphatidylinositol 4-phosphate (PIP) and PIP₂ was observed with dbcAMP or 8bcGMP treatment, suggesting activation of PI- and PIP-kinases. These results indicate that cGMP as well as cAMP acts as a negative messenger to prevent platelet activation. The inhibitory effect can be explained at least in part by the repression of phospholipase activation, resulting in reduced formation of inositol trisphosphate.

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A new anticoagulant (MD 805) was used in patients with chronic renal undergoing regular hemodialysis, and the results were compared with those of heparin. MD 805 produced no significant increase in PF-4 of the kind observed on hemodialysis with heparin. Moreover, the arterio-venous difference in β-TG during hemodialysis with MD 805 was significantly lower than that during hemodialysis with heparin. MD 805, even at the small amount of 15mg/hr, exhibited a stable antithrombin effect without marked interindividual differences in coagulation time as monitored by APTT. Consequently, it caused no increase in the proteins released as a result of platelet activation in the hemodialysis circuit.

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It is known that in diabetic patients with microangiopathy, the potency of blood coagulation and thromboxane production are activated. A specific thromboxane synthetase inhibitor, OKY-046 was given to 15 diabetic patients to study its effects on platelets, blood coagulation-fibrinolysis and renal function.
Plasma thromboxane B₂ tended to decrease after administration of OKY-046. Plasma 6-keto PGF_1α indicated a significant increase after 1 month. Plasma thromboxane B₂/6-keto PGF_1α ratio showed a significant decrease after 2 weeks, with a gradual increase thereafter. Urinary thromboxane B₂ also showed a significant decrease after 2 months. As the renal function, urinary β₂-microglobulin (β₂MG) showed a significant decrease after 2 months. In view of urinary β₂MG and NAG levels which are said to be the early indices of nephropathy, OKY-046 was markedly effective in 3 cases, effective in 10 cases, ineffective in 2 cases and no aggravation. In view of blood β₂MG which is said to reflect glomerular filtration rate, the drug was markedly effective in 2 cases, effective in 1 case and ineffective in 12 cases with no aggravation.
We conclude that OKY-046 suppresses thromboxane production, activates PGI₂ production and reduces thromboxane B₂/6-keto PGF_1α ratio in diabetic patients, suggesting to have an effect to improve diabetic nephropathy.

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We reported that β-Lipoportein (Apo-B) was another activated factor X inhibitor different from antithrombin III. The anti-Xa activity of β-Lipoprotein vanished with the addition of pronase. However, a new anti-Xa activity appeared. Namely, human citrated plasma was incubated with 1% pronase, at 37°C for 2 hours, and then was applied to a column of Sephadex G-200. Anti-Xa activity was observed in the first peak (void volume). Antigenicity of β-Lipoprotein did not exist any more but α₂-macroglobulin (α₂-MG) did. Further isolation of this first peak was performed on anti-α₂MG immunoabsorbent column chromotography. α₂-MG was eluted with glycine-HCl buffer, pH 2.5, and migrated to the α₂-globulin region with a single band on immunoelectrophoresis. Eluted. α₂MG inhibited factor X.
We investigated whether α₂MG itself could inhibit activated factor X. A protease inhibitor, aprotinin (Trasylol^®), was added to the pronase-treated α₂MG. Protease activity of pronase-treated α₂MG against S-2222 was reduced in proportion tote concentrations of aprotinin. The anti-Xa activity of pronase-treated α₂MG was also reduced in accordance with the decrement of protease activity.
Therefore, it is concluded that α₂MG-pronase complex inhibits activated factor X, but α₂MG itself is not the inhibitor of activated factor X.

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Endotoxin caused cytotoxic damages of mouse bone marrow cells and an increase in thromboplastin (Tp) activity of membrane fragments of the cells. Activities of synthetic lipid A analogs were compared with those of natural lipid A. Injection of 50μg of 406 increased cytotoxicity of the cells from 16.1±0.7 (control) to 26.4±1.7%, and the activity was comparable to that of natural lipid A (27.4±1.2%, dye exclusion test). Compounds 404 and 405 also induced cytotoxicity, however, compound 403 had no activity, compounds 503 to 506, which have double acyl groups at C2′ and C3′ positions, increased cytotoxicity. Among these analogs, compound 506 had the highest activity (from control, 16.1±1.9 to 32.6±1.6%). Compound 406 or 506 caused dose-dependent induction of Tp activity in the cells. Tp activity in the cells increased by injection of as little as 6μg of compound 406, 506 or by natural lipid A. Tp activity in the cells induced by compound 406 decreased after incubation with either Concanavalin A or Polymyxin B.
These findings indicate that active principle of endotoxin, as to the induction of cytotoxicity and Tp activity in bone marrow cells, is the lipid A portion. Hydroxymyristic acid at C3′ position and two phosphate groups at Cl and C4′ positions were thought to be necessary for the induction of both activities. Present studies further supported our hypothesis that membrane perturbation due to endotoxin would result in the induction of Tp activity.

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An antifibrinolytic activity was found in the supernatant of washed platelet suspension in the process of platelet aggregation induced by thrombin, ADP and 5-hydroxytryptamine. The antifibrinolytic activity was closely associated with inhibitors in platelets, which specifically inhibited plasmin but not inhibited other proteases such as urokinase, thrombin and trypsin. One casein unit of plasmin activity was inhibited by the inhibitors released from approximately 10⁸ platelets during the aggregation with thrombin. By the activity staining analysis, it was found that there are two kinds of plasmin inhibitors with the molecular weights of ≅25, 000 and ≅17, 000. These two inhibitors were stable after the treatment of the inhibitors with acid or denaturants, though the inhibitor with higher molecular weight lost the activity by dithiothreitol- and heat-treatments and two inhibitors lost also the activity by alkali-treatment. These inhibitors have been purified from the supernatant of aggregated platelets by ammonium sulfate precipitation, ion exchange chromatography, gel filtration and affinity adsorption to plasmin.

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Fibrin adhesion substrate and factor-XIII plays an important role in fibroblast growth during wound healing process. The effect of fibrinolysis on the cell growth was investigated to propose that fibrinolysis resistance by factor-XIII instead of fibronectin crosslinking was required to keep cell adhesion and growth. 3T6 cells in the medium supplemented with normal FCS were not proliferated on the non-crosslinked fibrin with fibrinolysis probably induced by the cell-derived activator, but well grown on the crosslinked fibrin. 3T6 cells in the medium with plasminogen-free serum were well grown even on the non-crosslinked fibrin without possibility of fibronectin crosslinking. BHK and S-180 cells were well grown in every cases regardless of the crosslinking and fibrinolysis. Plasmin pretreatment of fibrin, however, did not influence on the growth of any cells. The results indicated that not degradated substrate surface but some released products in the medium might have the effect on the cell growth, although the susceptibility depends on cell lines.

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A 62 years-old uremic patient who was treated with hemodialysis by heparin developed severe thrombocytopenia during hemodialysis. On admisson, when acute renal failure by diabetic nephrophathy was confirmed, the dialysis was carried out immediately through Shaldon catheter fixed into the femoral vein.
In eight and nine consecutive treatments of hemodialysis, massive clot formation in the dialyzer occurred in association with severe thrombocytopenia, and elevations of FDP and platelet release proteins. After aspirin intake, thrombocytopenia restored gradually and no clot formation in the dialyzer occurred.
Heparin induced platelet aggregation was demonstrated from the mixture of patient's PPP with normal PRP, and the addition of aspirin in vitro prevented completly heparin induced platelet aggregation. It was also confirmed that IgG purified from patient's PPP induced the platelet aggregation. In conclusion, a patient with heparin induced thrombocytopenia was found in the treatment of dialysis, and aspirin combined with heparin was effective in the next consecutive dialysis treatments performed.

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