Takeshi Ohshiro

Chromogenic assay of endotoxin in platelet poor or rich plasma

In order to determine which sample preparation, platelet rich plasma (PRP) or platelet poor plasma (PPP), is more suitable for clinical endotoxin assay, we investigated the binding of endotoxins to platelets by comparing the amount of endotoxin in PRP with that in PPP, using a newly developed colorimetric assay with chromogenic substrate (Boc-Leu-Gly-Arg-pNA). When purified endotoxins were added to human whole blood, the amount of endotoxin recovered in PPP was significantly lower than that in PRP for all endotoxins tested except that from E. coli 0111:B4 and their ability to bind to platelets was varied depending on the species of bacteria from which they were purified. However, the amount of endotoxin in PRP obtained from surgical patients (n = 50) was almost same as that in PPP with a correlation coefficient r = 0.95, indicating that natural endotoxins circulating in human blood may not bind to platelets and that PPP can be used for endotoxin assay as well as PRP.

Studies on liposome-encapsulated heparin

In order to prolong the anticoagulant activity of heparin in vivo, attempts were made to encapsulate heparin into liposomes. Liposome-encapsulated heparin (lipo-heparin) prepared was large multilamellar vesicles (0.5-4.0 micron in diameter). The activity of lipo-heparin was 1.6-5.2 X 10(3) U/g lipid with recovery rate ranged between 0.4 to 1.3% and stable in saline at 4 degrees C for at least two weeks. When intravenously administered into rats, the anticoagulant activity of lipo-heparin was significantly prolonged (approximately three times), as compared with that of untreated heparin. Furthermore, the activity of lipo-heparin could be neutralized by protamine sulfate. From these observations, it was concluded that liposome-encapsulation of heparin results in the prolonged anticoagulant effect in vivo and lipo-heparin may be applicable for clinical use, after further studies on side effects of liposomes are completed.

Fluorescent Ca2+-indicator quin 2 as an intracellular Ca2+ antagonist in platelet reaction

Preincubation of fluorescent Ca2+-indicator quin 2 resulted in inhibition on platelet aggregation and secretion in the absence of extracellular Ca2+. And the mechanism of the inhibition was studied. The inhibition by quin 2 of thrombin stimulated aggregation and ATP secretion of human platelets was dose and incubation time dependent and the inhibition was overcome by an addition of CaCl2 to the suspending buffer. Combination of quin 2 and Ca2+-blockers exerted the complete inhibition of the reaction in the presence of extracellular Ca2+. The inhibitory effect was observed when the intracellular concentration of quin 2 exceeds 3 mM, regardless of the initial dose or the preincubation time. The cellular content of ATP was not reduced by loading platelets with quin 2 in the concentration which exerted an inhibitory effect on the platelet reaction. From these observations, it was postulated that the inhibition is due to chelation of intracellular Ca2+ by quin 2 and the application of this agent as an intracellular Ca2+ antagonist was proposed. Also, we discussed the limitations in the use of quin 2 system as an intracellular Ca2+ indicator.

Pathogenesis of early thrombus formation in experimental vein graft

When inferior vena cava of rabbit was replaced by 3 cm long woven Tetron (polyethylene terephthalates) graft under bolus injection of heparin (50 U/kg), the graft was completely occluded at 1.5 +/- 0.35 h after the bolus injection of heparin. In order to elucidate the pathogenesis of this early thrombus formation, the same venous grafting was performed in rabbits receiving anticoagulants and/or anti-platelet agents and the thrombus formation was analyzed by scanning electron microscopy as well as by measuring the weight of dehydrated thrombus. The grafts in rabbits receiving an additional bolus heparin were patent until the anticoagulant effect disappeared and the thrombus formed in these grafts was composed of platelet aggregates anchored to synthetic fibers and of erythrocytes trapped into fibrin network. The patency of the graft was maintained at least 5 hours in rabbits receiving intravenous injection of aspirin (20 mg/kg) or oral administration of ticlopidine (100 mg/kg/day x 5 days prior to the grafting). The weight of dehydrated thrombus of the graft in aspirin and ticlopidine treated rabbits was 25 +/- 5 and 12 +/- 4 mg respectively, which were significantly lower than that of control group (59 +/- 9 mg). Ultrastructural studies revealed in these grafts piles of erythrocytes with fibrin network which were layered over the synthetic fibers without bridges of platelet aggregates. Also, the treatment with anti-platelet agents, especially ticlopidine, resulted in inhibition of organization of fibrin network. These observations indicate that thrombus in venous graft is formed by anchorage of platelet aggregates to synthetic fibers followed by activation of coagulation to form network of polymerized fibrin entrapping erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Interrelationship between secretion, protein phosphorylation and intracellular Ca2+ concentration in platelets stimulated by thrombin or thromboxane A2 analogue

The interrelationship between ATP-secretion, protein phosphorylation and intracellular Ca2+ concentration ([Ca2+]i) was studied in both 32P and quin 2 loaded human platelets stimulated by thrombin or thromboxane A2 analogue (STA2). In platelets stimulated by thrombin, the degree of 47,000 dalton polypeptides (P47) phosphorylation was observed in completely dose-related manner, regardless of the amount of [Ca2+]i. In the same condition, the degree of myosin light chain (P20) phosphorylation, however, was well correlated with ATP secretion and [Ca2+]i, when platelets were stimulated by lower dose of thrombin. The similar results were obtained in platelets stimulated by STA2. These findings suggested that P20, but not P47, phosphorylation in activated platelets is mediated by a rise of [Ca2+]i and is well correlated with the secretory reaction. It was unlikely that P47 phosphorylation plays any role in promoting platelet activation.

Prevention of hemoglobinuria by administration of haptoglobin

SummaryThis report describes the role of haptoglobin in preventing hemoglobinuria.In rabbits, human haptoglobin administered i.v. worked preventively against the development of hemoglobinuria in spite of the presence of a large amount of hemolysate containing free hemoglobin. More specifically: (1) the kidney did not present a dark brown color, (2) the deposition of iron in the kidney was significantly less than that in the same organ of rabbits given hemolysate only, and (3) the microangiographic and histological findings of the kidney were close to normal patterns.From the above it may be concluded that the administration of haptoglobin is effective for the prevention of hemoglobinuria and subsequent renal damage.

Analysis of platelet phospholipids by high performance liquid chromatography II—studies on hitherto unknown peak of phospholipid of human platelets.

With our proposed method to analyze platelet phospholipids, utilizing a normal phase high performance liquid chromatography (HPLC), it was able to quantify the amount of major platelet phospholipids (Thromb. Res. 36, 335-344, 1984). A prominent peak of unknown nature (PX) was identified close to the peak of phosphatidylcholine in the HPLC analysis of platelet phospholipids, and the attempts were made to elucidate the nature of PX. By applying several additional authentic phospholipids and those treated by acids on both the HPLC and thin layer chromatography, it was concluded that the major component of PX is 1-lyso phosphatidylethanolamine plasmalogen (PEP), artificially degraded from PEP by the acidic HPLC solvent system. No further degradation of 1-lyso PEP was observed and the absorbance of possibly co-migrated 2-lyso PE in activated platelets was negligible because it was devoid of C-2 double bonds sensitive to the absorbance used in the assay. Therefore, the relative amount of PEP may be detected based on the number of double bonds in platelet PEP. The amount of PEP thus measured was significantly decreased in thrombin-stimulated platelets, suggesting a possible participation of PEP in stimulus-linked platelet reaction.

[47] Clinical applications of urokinase-treated material

Publisher Summary This chapter focuses on the clinical applications of urokinase-treated material. Urokinase-treated material has effective antithrombogenic ability; urokinase-treated Evatate tube is utilized for intravenous catheters and urokinase-treated polyvinyl chloride tube for drainage tubes. One of the materials is an ethylene–vinyl acetate copolymer (Evatate), which is used for intravenous catheters, 1.5 mm in diameter. Another material is polyvinyl chloride (PVC), which is used for drainage tubes, 9 mm in diameter. The chapter discusses the immobilization procedure of urokinase. The surface of Evatate is subjected to saponification and aminoacetalization followed by peptide linking of urokinase via maleic anhydride–methyl vinyl ether copolymer (Gantrez), which is also linked. Urokinase activity is measured by the MCA method. Immobilized enzyme is stable; the stability of immobilized urokinase is investigated. The chapter also discusses the advantages of immobilized urokinase. One of the advantages in immobilized urokinase is its insusceptibility to the influence of urokinase inhibitor. Later, the antithrombogenicity of urokinase-treated tubes is discussed. The Chandler loop method tests the antithrombogenicity of a urokinase-treated tube.

Metabolism of liposome-encapsulated heparin

In order to elucidate the metabolism of liposome encapsulated heparin (LIPO-HEP), LIPO-HEP containing 3H-heparin (3H-HEP) and/or 14C-phosphatidylcholine (14C-PC) was intravenously administered into rats and the radioactivity as well as the biological activity in plasma and certain organs was investigated. The amount of 3H-radioactivity in plasma was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin. The amount of 14C-radioactivity in plasma of rats receiving LIPO-HEP, however, was not proportional to the amount of 3H-radioactivity in the same rats, indicating the dissociation of liposome and heparin in plasma. Incorporation of 3H-radioactivity into various organs examined, i.e., liver, spleen, lung, was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin, e.g. 4.7 and 11.8 times higher in the liver and the spleen, respectively at 150 min after the injection. Thereby, in contrast to the untreated heparin, LIPO-HEP was selectively incorporated into the reticuloendothelial system (RES) and it may be suggested that prolonged biological activity in LIPO-HEP is due to a gradual release of heparin from the liposomes entrapped in RES, and that it is not due to prolonged circulation in blood.

Imaging platelet deposition on Dacron bifurcation grafts in man: Quantification by a dual-tracer method using 111In-labeled platelets and 99mTc-labeled human serum albumin

A dual tracer technique using 111In-labeled platelets and 99mTc-labeled human serum albumin was applied to evaluate the thrombogenicity of Dacron bifurcation arterial grafts. The level of platelet accumulation over the whole of the graft was estimated from the ratio of 111In-platelet radioactivity deposited on the vascular wall to these radioactivity circulating in the blood pool, i.e., the platelet-accumulation index (PAI). Furthermore, the PAI value was calculated for each pixel in digitized images and the PAI distribution image (PAI image) was reconstructed. Eighteen patients with DeBakey knitted Dacron bifurcation grafts and 11 normal volunteers were studied. Of the 18 patients, 11 had no graft occlusion (group I) and the remaining 7 (group II) had occlusion. The mean PAI value (±SD) over the whole of the graft in group I was 32.6%±33.7% as compared to -8.8%±4.5% in the control group (P<0.01). In group I, the PAI value over the entire graft decreased with the age of the fraft (r=-0.763; P<0.01). In contrast, in group II, platelet accumulation did not diminish with time and persisted beyond the time of which platelet accumulation was no longer found in group I. Moreover, analysis of the PAI images revealed enhanced platelet accumulation on the proximal part of the graft to be more frequent in group II than in group I (6/7 vs 0/11; χc2 = 10.55; P<0.005). The method used for platelet imaging in the present study may be useful in the study of platelet reactions on Dacron arterial prostheses.