bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation Of In Vivo Platelet Activation In Sickle Cell Disease
Authors: Watson-Williams E J , Westwick J , Krishnamurthi S , Marks G , White J , Kakkar V V
Published: July 2018

This study was made to identify indicators of in vivo platelet activation in patients with sickle cell disease. Seven patients, homozygotes for sickle cell haemoglobin and seven age, sex and race matched controls were each studied 2 or 3 times in a six week period of normal health. Venous blood was drawn with minimal use of a tourniquet and, after 5ml was taken for other tests, 2.5ml was collected into ice- cold “Amersham BTG” tubes. The samples were centrifuged at 2,600g for 25 minutes at 4°C and the plasma immediately separated and stored at -20°C until assayed for Betathromboglo- bulin (BTG), platelet factor 4 (PF4), Thromboxane (TXB2) and 6-oxo-PGF1α. Circulating platelet aggregation was measured by the method of Wu and Hoak (1974).

BTG was greater than 60ng/ml in 17 of 20 samples from the patients but in only 4 of 17 samples from the controls. This difference remained significant when corrected for the platelet count. In vivo aggregation was above 25% on 8 of 16 occasions in patients and on 3 of 16 occasions in controls. Plasma PF4, TXB2 and 6-oxo-PGF1α levels were not significantly different in the two groups. BTG and in vivo platelet aggregation may be useful parameters with which to assess in vivo platelet activation in patients with homozygous sickle cell disease whilst not in crisis.
Changes In Membrane Physicochemical Properties Of Hyperreactive Platelets In Estrogen-Treated Prostatic Cancer
Authors: Jung S M , Isohisa I , Kinoshita K , Yamazaki H
Published: July 2018

Aggregation and physicochemical properties of platelets from 5 prostatic cancer patients, 20 prostatic cancer on estrogen (300 mg stilbesterol diphosphate/day p.o.), 16 of the same patients given 300 mg aspirin/day, 19 prostatic hypertrophy and 21 aged males were examined. Prostatic cancer on estrogen show higher platelet aggregabilities towards ADP, adrenaline, and collagen than the other groups tested. Mean platelet electrophoretic mobilities were determined by the automatic Laser Zee 3000 system and the value for prostatic cancer on estrogen was -1.632±0.0182 um/sec/V/cm (mean ±SE). significantly higher (p <0.001)than the values for prostatic cancer not on estrogen, prostatic hypertrophy, and aged males with values of -1.534±0.0328, -1.526±0.009, and -1.535±0.012 um/sec/V/cm respectively. Electrophoretic mobility was a linear function of whole platelet sialic acid (r=0.96,n=66). The result suggests that the electrophoretic mobility of platelets was defined by sialic acid amount and there may be a selective consumption of platelets with less surface negative charge in prostatic cancer on estrogen. Membrane glycoprotein patterns determined by SDS-polyacrylamide gel electrophoresis showed prostatic cancer on estrogen to have lower glycoprotein I and higher glycoprotein IV than other groups (p < 0.05). There may be factors affecting changes on platelet membranes or population in such patients. Prostatic cancer on estrogen and aspirin had increased mobilities in 14 out of 16 cases with loss of linear relationship between mobility but no changes in sialic acid or glycoprotein pattern from those before aspirin. In vitro incubation of PRP with aspirin resulted in dose dependent increase in mobility with concomitant aggregability loss. The effect suggests another kind of inhibitory mechanism on platelet aggregation in addition to cyclooxygenase inhibition.
Factor VIII-RAG From Human Endothelial Cells
Authors: Chan T K , Chan Vivian
Published: July 2018

Factor VIII related antigen (VIII-RAG) can be demonstrated in cultured human endothelial cells (EC) from umbilical cord by immunofluorescence. 75Se-Methionine tracer added onto culture medium showed that radioactive 75Se was incorporated into VIII-RAG as evidenced by Laurell Crossed Immunoelectrophoresis (CIE) and autoradiograph. This indicates that EC synthesizes VIII-RAG in culture, confirming the previous findings of Jaffe et al (1973) and Shearn et al (1977). 75Se- VIII in medium had similar CIE pattern as normal plasma VIII, normal ristocetin cofactor activity (RCoF), but no procoagulant (VIII-C) activity. However, 75Se-VIII in cell sonicate (EC-VIII) migrated faster on electrophoresis and had no RCoF or VIII-C activities. The EC-VIII was compared to factor VIII dissociated into subunits by cleavage of SH bonds. It can be concluded that VIII-RAG is synthesized as monomers in EC and polymers are formed on secretion. Hence, defect in VIII-RAG may arise from defective synthesis or defective assembly mechanism in the EC.
Intracellular Localization Of F.VIII R:Ag In Platelet And Endothelial Cells Using Peroxidase Fab Conjugates In Electron Microscopy
Authors: Jeanneau C , Sultan Y
Published: July 2018

In previous studies (Jeanneau et al Thromb. Haemost.1977, 38, 42) specific rabbit antibodies against human F.VIII R:Ag isolated by immuno absorption on insolubilized polymers from monospecific rabbit antisera against the F.VIII/ VW factor molecule and coupled to peroxidase showed the labelling of platelet and endothelial cell membranes but did not allow to visualize intra cellular localization of this antigen. Preparation of the peroxidase coupled Fab fragments of the same specific antibodies showed the intra cellular localization of F.VIII R:Ag in platelet and endothelial cells. Fab antibody fragments were prepared using papain digestion. Fab and Fc fragments were separated from the non digested antibody by filtration through Sephedex G 100. The Fab fragments were then separated from the Fc using CM 52 carboxymethyl cellulose.

In human washed platelets, staining was observed on the plasma membrane, the canalicular system and some granules. After thrombin activation the release of granules containing F.VIII R:Ag better visualized in the canalicular system. In patients with storage pool disease only some platelets showed normal SCS labelling, however no stained granules were observed.

Native human endothelial cells were obtained from umbilical cord veins, washed and resuspended in culture medium prior to incubation with peroxidase Fab conjugates. After fixation in glutaraldehyde and exposure to diaminobenzidine, the peroxidase staining was seen on the plasma membrane and in a large number of vacuoles. In some endothelial cells the Golgi apparatus appeared labelled demonstrating evidence of F.VIII R:Ag synthesis by endothelial cells.
Elevation Of Antithrombin III By Sulfinpyrazone Therapy In Chronic Renal Failure
Authors: Bern M , Green J
Published: July 2018

Sulfinpyrazone can reduce the incidence of thrombosis of A-V shunts in chronic renal failure. The drug is also reported to prevent acute deaths from coronary artery disease. This study was to determine mechanisms for these protective effects.

Patients on chronic hemodialysis served as the study models. Six patients on dialysis three times per week for 6 or more months received sulfinpyrazone 200 mgm t.i.d. p.o. for 14 days. Blood samples were obtained before dialysis was begun before and after the 14 days of drug therapy.

Results are shown as mean ± standard error of mean.

AT III levels rose significantly by functional and immune assays. Functional levels (by von Kaulla technique) rose
24.5 ± 3.1 sec. to 47.3 + 5.5 sec. (P>.005) Plasma protein AT III (by radial immunodiffusion) rose 31.2 ± 2.17 mg/dl to 37.9 ± 2.1 mgm/dl. (P>.01) Platelet factor 4 (by Abbot radioimmunology assay) fell from 46.4 + 13.6 ngm/ml to 9.5 ± 1.1 ngm/ml.(P>.005) The concentration of thrombin-anti-thrombin complex (by R. Rosenberg, Harvard Medical School, Boston) rose from 4.2 ± .09 to 8.4 ± 1.0 (P>.005)

Thus it appears that sulfinpyrazone elevates antithrombin concentration and function while simultaneously suppressing platelet release. These two effects may or may not be mutually dependent. The clinical efficacy of sulfinpyrazone may relate in part to the elevation of antithrombin III, probably by inhibiting its consumption, while also inhibiting platelet function.
The Reaction Mechanism Of “Limulus Test”
Authors: Morita T , Tanaka S , Nakamura T , Ohki M , Iwanaga S
Published: July 2018

The principle of the so-called “Limulus Test” for detection of bacterial endotoxins (LPS) is based on the LPS- induced coagulation reaction, using horseshoe crab amebocyte lysate. To establish the overall molecular events in such a reaction system, we have previously studied on the complete amino acid sequence of a clottable protein, coagulogen, and the enzymatic properties of active clotting enzyme. We also developed a new chromogenic substrate method for assay of LPS, using Limulus amebocyte lysate. During these investigations, we found that proclotting enzyme, which has been characterized as a LPS-sensitive protein, is not sensitive to LPS but that another unknown factor sensitive to LPS is involved in the reaction sequence which mediates the activation of the known proclotting enzyme. The evidences were as follows.

When amebocyte lysate prepared from Tachypleus tridentatus (Japanese horseshoe crab) was applied to a column of heparin-Sepharose CL-6B, two components beside coagulogen, all of which are associated with the coagulation system, were separated into the breakthrough fraction (fraction A) and the adsorbed fraction (fraction B). Among them a component eluted in fraction A was identified as the known proclotting enzyme, but this proenzyme did not show any clotting activity even after preincubation with LPS. The other component, tentatively named factor B, eluted in fraction B showed a very weak LPS-dependent amidase activity towards Boc-Leu-Gly-Arg-pNA. However, when a catalytic amount of factor B treated with LPS was added to fraction A, a strong clotting activity appeared, indicating that both components are essential to develop the clotting activity in the presence of LPS. The same experiments as made for the lysate from T. tridentatus were performed using the lysate from Limulus polyphemus. The data was the same as those described above. These results indicate that the LPS- mediated coagulation system in the amebocyte consists of a multi-enzyme system and that this cascade system may provide an extremely high sensitivity of the lysate to endotoxins.
Inhibitory Effect Of Gabexate Mesilate On Experimental Disseminated Intravascular Coagulation
Authors: Ohno H , Chang S W , Kambayashi J , Imaoka S , Kōsaki G
Published: July 2018

Gabexate mesilate:[Ethyl p-(6-guanidinohexanoyloxy)benzoate]methanesulfonate, which is clinically used as a potent trypsin inhibitor in Japan, inhibits competitively the hydrolytic reactions with synthetic substrates by thrombin and factor Xa. However, its virtual anticoagulant effect in vivo remains to be evaluated, because of its short halflife due to the presence of plasma esterase. In the present study, the inhibitory effect of gabexate mesilate on experimental disseminated intravascular coagulation was investigated in comparison with those of heparin and aprotinin.

125I-Fibrinogen was injected into rats along with t-AMCHA to prevent lysis of fibrin formed. Five minutes after a infusion of gabexate mesilate, heparin, aprotinin or saline, intravascular cogulation was triggered by a continuous infusion of thrombin or tissue thromboplstin. The deposition of fibrin in kidney was continuously monitored by scintillation detector. Platelet count and 125I-fibrinogen in blood were determined. The formation of fibrin induced by thrombin and tissue thromboplastin was completely inhibited by continuous administration of 120 μmol gabexate mesilate/kg/h or 200 U heparin/kg/h. On the other hand, aprotinin failed to prevent the deposition of fibrin even at dose of 50,000 U/kg/h. Also, other parameters corroborated the same aspect. The naturally occuring lysis of the microthrombi previously formed by thrombin was not blocked by the infusion of 120 μmol gabexate mesilate/kg/h or 200 U heparin/kg/h. However, aprotinin, at 5,000 U/kg/h, completely blocked the lysis. Using clotting assay, the biological half life of gabexate mesilate in human plasma was found to be ten times longer than that in rat plasma, suggesting that much lower dose would be effective in human. In conclusion, gabexate mesilate is a relevant compound as an antithrombotic agent.
THe Effect Of The Synthetic Thrombin Inhibitor, No. 805, On An Experimental DIC In Rabbits
Authors: Tamao Y , Hara H , Kikumoto R , Okamoto S
Published: July 2018

Our studies of synthetic thrombin inhibitors of arginine derivatives yielded very recently No. 805 (MCI-9038 or MD-805) having a specific stereostructure of 4-methyl-2- piperidinecarboxylic acid at its carboxamide portion which showed an extremely potent and selective inhibition of thrombin with a Ki of 0.019 μM.

The effect of No. 805 on an experimental DIC was studied. An experimental DIC in rabbits was generated by a constant i.v. infusion of 0.1 M lactic acid and crude bovine placental tissue thromboplastin. A rapid decrease in platelet number and a gradual decrease in fibrinogen content in plasma were observed over the experiment period of 5 hrs. The i.v. infusion of No. 805 suppressed the decrease in platelet number and fibrinogen content dose- dependently from 1.0 to 31.6 μg/kg/min and the suppression of ca. 50% was attained at a dose of 3.16 μg/kg/min showing a plasma level as low as ca. 0.3 μM, while heparin given at a dose of 100 or 1,000 u/kg i.v. (x2) did not show a notable suppression of the platelet number decrease but a significant suppression of the fibrinogen content decrease. Thus, No. 805 was shown to be effective on the experimental DIC at lower plasma level, exhibiting more dominant effects on platelets than heparin. This result was carefully compared with those on other animal thrombosis.
The Amino Acid Sequence Studies On Limulus Polyphemus Coagulogen
Authors: Miyata T , Umezu M , Iwanaga S
Published: July 2018

Coagulogen, which is a clottable protein found in the amebocyte lysate of Limulus polyphemus, consists of a single basic polypeptide chain having the total of 174 amino acid residues. Upon gelation of this protein, a large peptide, named peptide C, which has 28 amino acid residues, was released, and the resulting gel protein consisted of A and B chains, bridged by two disulfide linkages. This work is to determine the whole sequence of B chain and to compare with the primary structure of Tachypleus tridentatus (Japanese horseshoe crab) coagulogen previously established. The Limulus B chain was digested with trypsin and the resulting fragments were separated into 17 major peptides. For the sequence determination, manual Edman degradation was employed and resulting PTH amino acids were identified by HPLC. These analyses established the sequence of the isolated tryptic peptides, ending with the COOH-terminal serine, and the following whole sequence of Limulus coagulogen was presumed, comparing with that of Tachypleus coagulogen.

Tachypleus: NH2-A-D-T-N-A-P-I-C-L-C-D-E-P-G-V-L-G-R-T-Q-I-V- Limulus : NH2-G-D-P-N-V-P-T-C-L-C-E-E-P-T-L-L-G-R-K-V-I-V-
T-T-E-I-K-D-K-I-E-K-A-V-E-A-V-A-Q-E-S-G-V-S-G-R-G-F-S-I-F-S- S-Q-E-T-K-D-K-I-E-E-A-V-Q-A-I-T-B-K-D-E-I-S-G-R⋅G-F-S-I-F-G⋅
H-H-P-V-F-R-E-C-G-K-Y-E-C-R-T-V-R-P-E-H-S-R-C-Y-N-F-P-P-F-T- H-G-P-A-F-K-E-C-G-K⋅Y-E-C-R⋅T-V-T-S-E-D-S-R⋅C-Y-N-F-F-P-F-S⋅
H-F-K-L-E-C-P-V-S-T-R-D-C-E-P-V-F-G-Y-T-V-A-G-E-F-R-V-I-V-Q-
H⋅F⋅H⋅P⋅Z⋅C⋅P⋅T⋅S⋅T⋅S⋅B⋅C⋅Z⋅P⋅V⋅L⋅S⋅Y⋅T⋅V⋅A⋅G⋅Z⋅F⋅R⋅I-I-V-Q-
A-P-R-A-G-F-R-Q-C-V-W-Q-H-K-C-R-F-G-S-N-S-C-G-Y-N-G-R-C-T-Q- A-P-K.A-G-F-R⋅D-L-E(W)V⋅H⋅K⋅C-R⋅A-Y-G-S-N-F-C-Q-S-Z-R⋅C-T-Q-
Q-R-S-V-V-R-L-V-T-Y-N-L-E-K-D-G-F-L-C-E-S-F-R-T-C-C-G-C-P-C- Q-R-S-V-V-R-L-V-T-Y-D-L-E-K⋅ G-V-F-F-C-E-N-V-R-T-C-C-G-C-P-C-
R-S-F-COOH
R-S-C00H
Platelet Function Tests Compared In An Indobufen And Aspirin Study
Authors: O’Brien J R , Etherinoton M D , Shuttleworth R D , Adams Cheryll
Published: July 2018

To compare the effect of Indobufen with Aspirin, a double blind cross-over study was carried out on 9 controls and 9 arteriopaths with 14 days on each drug. Many tests were carried out twice before and twice during each drug period and once in the wash-out period. The results were analysed using Wallenstein and Fisher’s model.

In patients and controls both drugs strongly inhibited malondialdehyde (MDA) and Thromboxane B2 production in thrombin stimulated platelets. Only Indobufen resulted in an increase in the volume of native blood passing through a 5μ Nuclepore filter (“bleeding time” model). No other tests were affected by these drugs.

Before treatment the patients (Pts) differed from the controls (Cs) by having a higher fibrinogen (Pts 399 ± 85.5 mg %, Cs 325 ± 45.8, p<0.0l) and low MDA production (Pts 176 ± 86.3 ng/109 plats, Cs 290 ± 111.3 p<0.05).

The following correlations were significant for the combined baseline figures: (n = 18, p<0.01 if r > 0.590, p<0.05 if r > 0.468). (l) plat PF4 and plat βTG (r 0.517), (2) plat PF4 and plat 5HT (r 0.518), (3) plasma PF4 and plasma βTG (r 0.582), (4) plat 5HT v plasma 5-hydroxy- indoles (5HIs) (r -0.465), (5) plat 5HT and plat 5HT uptake (r 0.788), (6) Age and plat PF4 (r -0.633) (7) Age and plat 5HT (r -0.644), (8) Age and plasma 5HIs (r 0.770).

Conclusions: Indobufen has similar effects to Aspirin and inhibits the platelet prostaglandin/thromboxane pathway. The 9 selected arteriopaths differed from the controls principally in having a high fibrinogen and low MDA production. The many correlations may be relevant to platelet physiology. The contents of both the α granules (PF4, βTG) and the dense bodies (5HT) are, at least under some circumstances, related and both are influenced by age.
Aspirin Preferentially Inhibits Arachidonic Acid Metabolism In Collagen Vs. Thrombin-Stimulated Platelets
Authors: Deykin D , Vaillancourt R
Published: July 2018

The purpose of this study was to compare the effect of aspirin on the release of metabolites of arachidonic acid from thrombin and collagen stimulated platelets. Human platelets were incubated with tritium-labeled arachidonic acid and then isolated by gel filtration. The labeled platelets were stimulated with varied doses of either thrombin or collagen for 15 minutes. The platelets were then pelleted and the released metabolites of arachidonic acid were separated by high-performance liquid chromatography. In experiments with aspirin, the aspirin was added 5 minutes before either thrombin or collagen. The total release of radioactivity was comparable at 15 μg/ml of collagen and 1.0 units/ml of thrombin (approximately 10% of the total) and at 100 μg/ml of collagen and 5 units/ml of thrombin (approximately 30%). Aspirin (25 μg/ml) preferentially inhibited collagen-stimulated release of radioactivity (62% inhibition of release with 15 μg/ml of collagen vs. 25% inhibition of release with 1.0 units/ml of thrombin; 54% inhibition of release with 100 μg/ml of collagen vs. 8% inhibition of release with 5.0 units/ml of thrombin). At all concentrations of collagen or thrombin, cyclo-oxygenase activity was markedly reduced by aspirin. The selective effect of aspirin on collagen reflects primarily preferential suppression of HETE formation. We conclude that aspirin inhibits the formation of both lipoxygenase and cyclooxygenase-derived products in collagen-stimulated platelets.
Effect Of Aspirin On The Production Of PGI2 By Various Blood Vessels And Mda By Platelets
Authors: Takada M , Tsukada H , Tanaka H , Gonmori H , Kobayashi N , Maekawa T
Published: July 2018

This study was desinged to determine whether the differential inhibition of prostacyclin(PGI2) production by vessel wall and malondialdehyde(MDA) production by platelet might be possible by oral administration of aspirin(ASA). Rabbits weghing 2-3kg were used. MDA production by platelet was measured by the Stuart’s method with minor modification. The PGI2 production by vessel wall was assessed by the Moncada’s method with minor modification.

The PGI2 production by caval vein, pulmonary artery, pulmonary vein,femoral artery, femoral vein and coronary artery was 148±55%, 136±56%, 153±55%, 134±56%, 123±64%, 103±55% to that of aorta, respectively. The PGI2 production by these vessels was inhibited to 20-40% to their initial level 6h after the single oral administration of 0.3g ASA, and restored to the initial level by 24h, while the MDA production was inhibited more conspicuously and remained at less than 50% of the initial level even 48h.

The effect of daily oral administration of ASA on production of PGI2 by aorta and MDA by platelet was investigated. PGI2 production was suppressed to about 10% of the initial level 24h after the last dose of 3 to 7 daily administration of 0,3g of ASA. This indicates that the daily ASA administration results in the cummulative inhibition of PGI2 production. On the other hand, when administered every other day, the same amount of ASA exerted significantly less inhibition of PGI2 production, being at about 50% of initial level 24h after the last dose. MDA production was nearly completely inhibited over the observation periods.

These results suggests that the differential inhibition of vascular PGI2 production and platelet aggregation may be possible by the administration of ASA at an appropriate amount and proper interval.
The Effect Of Low Dose Aspirin On Human Venous Prostacyclin Production And Platelet Thromboxane Synthesis
Authors: Hanley S P , Bevan J , Cockbill S , Heptinstall S
Published: July 2018

This study vas undertaken to determine if there was a dose of oral aspirin that would inhibit platelet cyclooxygenase but that had no effect on venous cyclo-oxygenase. Varicose veins were surgically removed from 68 patients of whom 21 had taken no medicines pre-operatively. Three groups of patients took aspirin 300 mg 14 hours (n=10), 24 hours (n=10) and 48 hours (n=9) pre-operatively. Two other groups took 40.5 mg (n=9) and 81 mg (n=9) 14 hours preoperatively. Two 6 mm discs of venous tissue were incubated with plasma that contained 1 mM sodium arachidonate (NaAA) and the anti-aggregatory activity of the plasma was compared with that of synthetic PGI2 in aspirinised citrated PRP challenged with ADP. PGI2 production in this procedure was confirmed by specific radio-immunoassay for 6-keto PGF1α. All doses of aspirin except 40.5 mg markedly inhibited PGI2 synthesis. Little recovery was seen with 300 mg at 48 hour post-drug.

Five normal volunteers took aspirin 300 mg and 5 others 40.5 mg. Citrated PRP was prepared immediately before, 2, 24, 48, 96 and 192 hours after ingesting the drug. The amount of malondialdehyde (MDA, a marker for thromboxane synthesis) that was produced when 1 mM NaAA was added, was markedly reduced compared to the control at 2 and 24 hours by both 40.5 mg and 300 mg and thereafter it returned slowly to control values. This study indicates that 40.5 mg of aspirin inhibits platelet cyclo-oxygenase but not vessel wall cyclo-oxygenase.
Purification Of Vasoactive Peptides From Human Fibrinogen Degraded By Human Leucocyte Elastase
Authors: Wallin R , Belew M , Ohlsson K , Saldeen T
Published: July 2018

The presence of leucocytes around extravascular fibrin deposits suggests that the leucocyte elastases might be partly responsible for the extravascular degradation of fibrin. Our previous studies have shown that the degradation of fibrin(ogen) by plasmin leads to the release of 2 small peptides which markedly increase vascular permeability and induce oedema e.g. in the lungs. The results of this investigation show that small peptides released from fibrinogen after degradation by leucocytes elastases also increase vascular permeability.

Human fibrinogen (Kabi, Grade L) was made plasminogenfree by affinity chromatography on Lysine-Sepharose 4B prior to use. The human leucocyte elastases were isolated from extracts of lysosome-like granules of human leukaemic myeloid cells by a combination of gel filtration, affinity chromatography and preparative agarose gel electrophoresis. The fibrinogen (0.5 %) and the leucocyte elastases (in a molar ratio of 100:1) were incubated together for 48 h at +37°C and at pH 8.5. The mixture was then cooled to +4°C to stop the lysis and ultrafiltrated on a DIAFLO PM 10 membrane until the retentate was approximately 10 % of the starting volume. The peptides in the diffusate accounted for about 20 % of the starting material as estimated from absorbance measurements at 280 nm. The diffusate was concentrated by lyophilization and fractionated by chromatography on a column of Bio-Gel P-6. At least 8 fractions were obtained of which only two showed a significant activity in their ability to increase vascular permeability in rat skin. The active peptides in these two fractions were further purified to homogeneity by column zone electrophoresis at various pHs and their amino acid compositions established.
Sickle Cell Trait, Diabetic Retinopathy And Erythrocyte Adhesion
Authors: Abadie E , Wautier M P , Passa Ph , Wautier J L
Published: July 2018

Increased erythrocyte adhesion to endothelial cells has been reported in sickle cell anaemia, and we have found the same phenomenon in diabetes mellitus. In sickle cell disease, increased adherence correlated with the clinical severity of the disease, and in diabetes with the extent of vascular complications. Previous studies have shown that there is no statistical difference in the prevalence of retinopathy between diabetics with and without sickle cell trait (Hb AS). We have had the opportunity to study two patients with diabetes and Hb AS to determine whether the uncommon association of these two conditions potentiates the abnormal erythrocyte adhesion found in diabetes. Both patients were non insulin-dependent diabetics treated by diet. Concentrations of HbS were 53 % (patient 1) and 38 % (patient 2). Severe peripheral proliferative retinopathy (patient 1) and background retinopathy (patient 2) were demonstrated by fluorescein angiography. Adhesion was measured using 51 Cr labelled erythrocytes and cultured human endothelial cells. Results were expressed as an adherence ratio (AR), defined by the percentage adhering patients erythrocytes/percentage adhering control erythrocytes. AR was increased in patient 1 (AR : 1.66) and normal in patient 2. The mean AR in 29 diabetics was 2.32 (range 0.8 - 5.2). These results suggest that there is no additional effect of Hb AS on erythrocyte adherence to endothelial cells in diabetes.
A Plasma Factor Which Stimulates Prostacyclin Formation Is Increased In Diabetes
Authors: Johnson M , Reece A H , Harrison H E
Published: July 2018

Vascular prostacyclin (PGI2) generation is decreased in diabetes in experimental animals and in man. In this study, we have investigated the possibility that levels of a plasma factor (s) modifying PGI2 production are abnormal in diabetes. Aortic rings from diabetic or age-matched control rats were washed in Krebs buffer to reduce endogenous PGI2 formation. Addition of rat or human cell-free plasma stimulated PGI2 release by the “exhausted” vascular rings, and this activity was still present after freezing and thawing. The stimulation of PGI2 synthesis by control tissue was significantly greater (p<0.001) with plasma from diabetic animals (0.25±0.04ng/mg) than from controls (0.05±0.02ng/mg). Similarly, plasma from diabetic volunteers showed increased (p<0.05) PGI2-stimulatory activity. Diabetic tissue was less responsive than control tissue to stimulation by diabetic plasma, and the difference between diabetic and control plasmas was not apparent. This suggests that the abnormal vascular PGI2 synthesis in diabetes may be due to a defect in the vessel wall and not to lack of stimulatory plasma factors.
Hemorrhage And Exudation In The Microcirculation Induced In Rabbits With Human Neutrophil Lysosomal Enzymes
Authors: Wasi S , Movat H Z
Published: July 2018

The purpose of this study was to assess the nature of the lesions in the microcirculation induced in rabbits by lysosomal enzymes derived from human neutrophil leukocytes (PMNs). PMNs were isolated from ACD blood. Their lysosomal contents were specifically released upon incubation with immune precipitates of bovine serum albumin (BSA)-anti BSA. Several enzymes were purified by means of cation exchange chromatography (SP-Sephadex) and gel filtration (Sephadex G-200). Two parameters of microvascular injury were quantitated: hemorrhage with 59Fe-labelled erythrocytes and vascular permeability with 125I-labelled serum albumin. The crude lysosomal lysate induced both hemorrhage and exudation. Several enzymes were characterized with respect to their size, charge and inhibition patterns with specific proteolytic inhibitors. When crude lysosomal lysate was treated with diisopropylfluorophos- phate, MeO-Suc-Ala-Ala-Pro-Val-NMec, or heated, both hemorrhage and exudation were inhibited. Hemorrhage was also partially inhibited by p-hydroxy mercury benzoate and leupeptin. Hemorrhage and exudation were produced by at least two different enzymes: one more basic, with an approx molecular weight (MW) of 23,000-27,000 daltons and one more acidic with an approx. MW of 69,000-76,000 daltons. Small doses of the enzymes induced only increase in exudation and large doses induced exudation, followed by hemorrhage. With the aid of ultrastructural studies it was demonstrated that the increase in vascular permeability was due to leaky vessels through which electron dense tracers escaped whereas hemorrhage occurred through disruption of basement membranes and necrosis of endothelial cells. Proteases, when released from PMNs, can induce lesions of the microcirculation. Mild lesions are characterized by leaky vessels and severe lesions by vascular wall disruption.
Rat, In Contrast To Rabbit, Blood Leukocytes Do Not Develop Procoagulant Activity Upon Exposure To Endotoxin In Vitro Or In Vivo
Authors: Semeraro N , Colucci M , Mussoni L , Donati M B
Published: July 2018

Leukocyte procoagulant activity is thought to play a major role in disseminated intravascular coagulation (DIC) occurring during experimental endotoxemia in rabbits and, possibly, in other animal species. The rat is much more resistant than rabbit to endotoxin-induced DIC and Sanarelli- Shwartzman phenomenon. We have therefore investigated the procoagulant activity of rat, as compared to rabbit, blood leukocytes in response to endotoxin stimulation in vitro and in vivo. Isolated rabbit leukocytes, upon prolonged incubation with E.coli or S. minnesota endotoxin (10 ug/ml f.c.) developed strong procoagulant activity as measured by their capacity to markedly shorten the recalcification time of autologous plasma (from 380 ± 32 to 41 ± 5 sec, n=6; blank value > 600 sec) and to generate activated factor X (monitored by the chromogenic substrate S-2222) in the presence of a source of factor X and VII (rabbit serum BaSO4-eluate) and CaCl2. In contrast, rat leukocytes failed to produce procoagulant activity when incubated with endotoxins (up to 200 ug/ml f.c.) under the same experimental conditions (from 131 ± 9 to 125 ± 12 sec, n=6; blank value 155 ± 18 sec, n=6). When rabbits were given two spaced (20 h apart) endotoxin injections (25-100 ug/kg b.w.), blood leukocytes harvested 30-60 min after the second injection consistently showed strong procoagulant activity (80 ± 17 versus 413 ± 39 sec, n=9). Again, in contrast to rabbit, blood rat leukocytes obtained after two endotoxin injections (up to 2 mg/kg b.w.) were completely devoid of procoagulant activity. These findings add further direct support to the view that leukocytes are involved in endotoxin-induced DIC in rabbits. On the other hand the poor response of rat leukocytes to endotoxins described here might be relevant to explain the resistance of rats to DIC and Sanarelli-Shwartzman reaction.
Some Observations On The Alteration Of The Platelet Membrane Transport Systems In Atherosclerosis
Authors: Ulutin O N , Emekli N , Cizmeci G , Yardimci T U
Published: July 2018

We have studied the membrane transport systems in the platelets from patients with atherosclerosis with hypercoagulability.

No qualitative and quantitative differences were observed in the transport of amino acids in normal platelets or platelets from atherosclerotic individuals. Normally glucose is transported with both an active transport mechanism and diffusion. The active transport of glucose is impaired in the platelets from atherosclerotic patients and this was associated with the loss of a binding protein active in the process. Normal active transport of galactose was unaffected in atherosclerosis. There was an increase, however,in the amount of transported galactose that got incorporated into the platelet glycoproteins in the latter condition. Normal active transport for arachi- donic acid is not observed in platelets from atherosclerotic patients. Indobufen and aspirin did not affect membrane transport of glucose, galactose, and arachidonic acid. Platelet glycoprotein synthesis, however, was inhibited. In addition to the previously reported alteration in the distribution of membrane phospholipids the alterations in the transport functions of the platelet membrane associated with changes in certain carrier proteins provide further evidence for structural modifications of the platelet membranes in atherosclerosis.
The effect of a large dose of aspirin, ticlopidine, and thromboxane A2 synthetase inhibitors on experimental arterial thrombosis
Authors: Kobayashi N , Tsukada H , Takada M , Tanaka H , Gonmori H , Maekawa T
Published: July 2018

The key role played by platelets in the thrombogenesis has been well recognized. The antithrombotic therapy by inhibition of platelet function has gained considerable support with this recognition. In this report, the effect of a large dose of aspirin,ticlopidine and thromboxane A2 synthetase inhibitors(OKY, AVS) on experimental arterial thrombosis was investigated.

The experimental arterial thrombosis was produced by Maekawa’s method. The femoral artery of rabbit was exposed and a steel wire of 0.9 mm in diameter was placed alongside the femoral artery. Then the artery was loosely ligated together with the wire by silk thread. A stricture was made in the femoral artery by pulling out the wire. Ten mg of ellagic acid dissolved in distilled water was injected intravenously. Twenty four hours later, the rabbit again anesthesized and femoral artery with ligature was exposed and examined macroscopically for the presence of occluding thrombosis. In the untreated rabbits, the occluding thrombosis was found in 70 % of the rabbits.

In rabbits which ingested 0.3 or 1.0 g of aspirin daily for 7 days, rate of rabbits with thrombus was significantly reduced in spite of marked inhibition of vascular prostacyclin production. Ticlopidine which inhibits the primary aggregation of platelets inhibited significantly the production of the occluding thrombosis. AVS, one of thromboxane A2 synthetase inhibitors, also reduced the incidence of rabbits with the occluding thrombosis. The latter two agents had no inhibitory effect on vascular prostacyclin production.

From these results, it is concluded that the formation of our type of arterial thrombosis could be inhibited by the antiplatelet drugs independently of the production of vascular prostacyclin.
Role Of Thromboxane (TXA2 ) And Adp In Collagen-Induced Platelet Aggregation
Authors: Emms H , Lewis G P , Watts I S
Published: July 2018

TXA2 and prostaglandins (PGs) released during collagen- induced aggregation of rabbit platelets were bioassayed on rabbit aorta (RA) and rat stomach strip (RSS) respectively in a cascade system. Platelet aggregation was monitored simultaneously. The technique was used to examine modulation of aggregation and release of TXA2 and PGs by inhibitors of TX synthetase (l-(7-carboxyheptyl)imidazole) (CHI) and cyclo-oxygenase (CO) (indomethacin, Indo).

At low dose collagen (5-10 μg/ml) Indo and CHI completely inhibited platelet aggregation in a dose-related fashion. In contrast, at high dose collagen (50 μg/ml) these compounds gave only a partial (30-40%) inhibition of aggregation, at a dose which abolished CO and TX synthetase activity.

The substrate/enzyme combination of creatine phosphate (CP) 5mM and creatine phosphokinase (CPK) 10 units/ml (which converts ADP to ATP) completely inhibited aggregation induced by low dose collagen but only partially inhibited (20%) that induced by high dose collagen. Neither CP nor CPK alone was effective.

A mixture of both CP/CPK and CHI abolished the platelet aggregation induced by a high dose of collagen.

These studies support the concept that collagen-induced aggregation is dependent on at least two mechanisms.

Firstly, at low collagen concentration, subthreshold levels of released ADP and TXA2 act synergistically in inducing platelet aggregation since either CP/CPK or CHI abolished aggregation. Secondly, at high collagen concentration, sufficient ADP and TXA2 are released to induce aggregation independently of each other, since CP/CPK or CHI produced only partial inhibition of aggregation, whilst in combination aggregation was abolished. Furthermore, the results suggest that endoperoxides play no significant role in collagen-induced platelet aggregation.
The Effect Of Ergotamine On The Formation Of Metabolites Of Arachidonic Acid In The Guinea Pig Ear And In Human Platelets
Authors: Vincent J E , Zijlstra F J , Spierings E L G
Published: July 2018

The relationship between the vasoconstrictor action on one side and a possible effect on the prostaglandin (PG) formation in the vascular system on the other side of ergotamine was investigated. The action of this substance on the formation of thromboxane B2 (TXB2), PGE2, 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT) and 12L-hydroxy-5,8,10,14- eicosatetraenoic acid (HETE) in platelets was also determined.

In both experiments, the formation of the above mentioned substances from {1-14C} arachidonic acid (AA) was measured. The metabolites were separated by TLC.

In guinea pig ear slices, a comparatively large formation of PGs occurs. The main substance formed is PGF2α.6-keto F1α, PGE2, PGD2 and PGA2 are also produced. The addition of 10-5M ergotamine leads to an inhibition of the synthesis of all PGs. In the ear, it is likely that the PGs with vasodilator action are dominating. A reduction in PG formation will most probably lead to vasoconstriction.

In platelets, the synthesis of TxB2 is inhibited by 5.10-5 M ergotamine. The amounts of PGE2 and HETE are increased. This indicates that the thromboxane A2 synthetase is inhibited.

In migraine, platelet aggregation occurs and a partial release reaction. The inhibition of the formation of TxA2 by ergotamine will result in a decrease in this effect. The therapeutic action of this substance may be related, at least in part to the changes induced in the transformations of AA in both systems.
Prostacyclin-Like Activity Of Amniotic Fluid In Preeclampsia
Authors: Bodzenta A , Thomson J M , Poller L
Published: July 2018

Studies have been undertaken to compare amniotic fluid from patients with pre-eclampsia (PET) with normotensive controls in inhibiting platelet aggregation (PGI2-like activity) and accelerating coagulation tests. After 39 weeks of gestation, samples from PET patients showed significantly less PGI2-like activity (p<0.001) and significantly lower kallikrein levels (p<0.01) than normotensive controls. These findings suggest that the biosynthesis and release of these substances may be impaired in PET. In view of the association of PET with intravascular clotting, the highly significant reduction of PGI2-like activity appears important and warrants a clinical trial of prostacyclin administration in this disorder.
Regulation Of Adenylate Cyclase-Phosphodiesterase By Testosterone In Cultured Endothelium
Authors: Ahmed S S , Stewart G J
Published: July 2018

The effects of testosterone on the activities of the enzymes involved in cyclic adenosine 3’:5’-monophosphate (cyclic AMP) metabolism in primary bovine endothelial cell cultures were examined. Adenylate cyclase was measured under basal and 10 mM NaF stimulated conditions. In control cultures, the enzyme activity remained essentially unchanged for 48 hours and then showed a significant increase (p<0.05) through 96 hours. Addition of testosterone (1 × 10-7M) six hours after plating stimulated the enzyme activity causing a significant increase (p<0.05) at 72 hours of incubation. Addition of 10 mM NaF enhanced basal activity as well as testosterone stimulated activity of adenylate cyclase throughout the experimental period. Low and high affinity forms of cAMP phosphodiesterase were measured independently by varying the substrate concentration. At 250 μM substrate the low Km enzyme contributed very little (about 8%) to the activity whereas at 0.1 μM substrate the contribution of the high Km form was about 26%. The activity of both forms fell rapidly during growth phase of control endothelial cells. The addition of 1 × 10-7M testosterone at the time of plating caused little change from the control level for the first 48 hours, i.e. the activity of phosphodiesterase fell at the same rate in control and treated cultures. Further incubation resulted in marked inhibition of both forms of phosphodiesterase by testosterone. Testosterone added to subconfluent cultures at low concentration (1 × 10-9M) failed to stimulate adenylate cyclase but did inhibit low Km phosphodiesterase (p<0.05). A high concentration (1 × 10-7M) of testosterone stimulated adenylate cyclase activity and markedly decreased activities of high and low Km phosphodiesterase. This study showed that testosterone induced a major change in cyclic nucleotide metabolism, an important factor in regulating cell function (cell adhesion in this case).
Evolving Concepts About Structural Lesions Of The Coronary Arteries And Their Clinical Significance
Authors: James Thomas N
Published: July 2018

To understand the clinical significance of coronary lesions one must consider: (1) The wide spectrum of histological abnormalities which comprise “coronary disease”, and (2) The importance of what structures that artery supplies. Fibrous scarring, intimal proliferation, fibromuscular dysplasia, hemorrhagic necrosis, cholesterol or other lipid deposition, and varying degrees of inflammation all occur within so-called typical atheromata. To assume that this all began as a single process, or even if so, that it continued as a uniform one may be a fundamental error which prevents a better understanding of the pathogenesis of coronary disease. The end result seen at necropsy may offer few genuine clues as to what had preceded years or decades before. Clinical events that follow the occlusion of a coronary artery depend in a major way upon which structures which are located distal to that lesion. Widely differing clinical consequences can be expected, for example, when the focal ischemia after coronary occlusion does or does not involve the sinus node, or the AV node and His bundle, or the papillary muscles, or the interventricular septum. The vague interpretation that all ischemic myocardium causes the same nature of clinical events is misleading and precludes accurate clinical assessment.
Structure Of Plasma Fibronectin. Gene Multiplication
Authors: Petersen T E , Thøgersen H C , Skorstengaard K , Vibe-Pedersen K , Sahl P , McDonagh R , McDonagh J , Magnusson S , Sottrup-Jensen L
Published: July 2018

Plasma fibronectin (cold insoluble globulin) is a multiple domain glycoprotein composed of two nearly identical disulphide bridged polypeptide chains with molecular weight of 220,000. It is a substrate for factor XIIIa and binds to gelatin and heparin. After digestion of bovine fibronectin with plasmin, four fragments with Mr 29,000, 170,000, 23,000 and 6,000 have been isolated. The N-terminal 29,000 Mr fragment (259 residues) has 10 disulphide bridges within five mutually homologous domains, called “fingers”. The sequence <Gln-Ala-Gln-Gln-Ile-Val-Gln-Pro-Gln- contains the acceptor site for factor XIIIa at position 3. The 170,000 Mr fragment contains both the gelatin and the heparin binding site. After further digestion with chymotrypsin a fragment (Mr 45,000) which binds to gelatin, and a fragment (Mr 30,000) which binds to heparin, have been isolated. The Mr 45,000 fragment consists of at least one more “finger” plus two other mutually homologous domains each with two disulphide bridges. The 23,000 Mr fragment (178 residues) consists of three “finger“- domains and has an N-terminal sequence Val-Arg-. The Mr 6,000 (C-terminal fragment) is a dimer of two identical 26-residue peptides linked by two disulphide bridges. 820 of the expected approx. 1800 residues have been placed in sequence.
Immunological Determined Plasminogen Groups And Its Possible Biological Relevance
Authors: Sachs V , Dörner R , Szirmai E
Published: July 2018

Anti human plasminogen sera of the rabbit precipitate human plasma in the agar gel diffusion test by means of intra-basin absorption with plasminogenfree human plasma with three different types: type I is represented by one strong precipitation line, type II by two lines, a big one and a small one, and type III by three slight but distinct lines. The following frequencies of the different types have been observed in a sample of 516 human plasmas: type I 65%, type II 33% and type III 2%. Suppose the types are phenotypical groups of a diallelic system where the types I and III represent the homozygous genotypes and the type II the heterozygous the estimated gene frequencies are in good agreement with the expected values. There is also a good agreement of the distribution of plasminogen groups determined by electrofocussing from RAUM et al. and HOBART. The plasminogen groups possibly may have also a biological meaning because the plasmas of type III always have a lesser fibrinolytic activity than the plasmas of the other types.
Effect Of Heparin And Protamine On The Antithrombin Activity Of α2-Macroglobulin
Authors: Shanberge J N , Love J , Swanborg N
Published: July 2018

It has been shown that certain fractions of porcine mucosal heparin may form complexes with α2-macroglobulin but still retain their capacity to activate antithrombin III. These heparin fractions are not neutralized by protamine. The effect of heparin and protamine on the antithrombin activity of α2-macroglobulin has now been studied.

Plasma to which a tritium-labelied heparin was added was chromatographed on Sephadex G-200. The macromolecular fractions, in which there is heparin (indicated by radioactivity) and α2-macroglobulin (demonstrated by radial immunodiffusion), have no immediate inhibitory action on the thrombin clotting time (TCT) of fibrinogen but do inhibit the TCT of whole plasma. In addition, these fractions have progressive antithrombin activity (PATA) but less than that in fractions containing α2-macroglobulin obtained with non-heparinized plasma. Fischer et al stated that heparin complexes with thrombin to prevent its inactivation by α2-macroglobulin. However, when the heparinized plasma is “neutralized” with protamine sulfate prior to chromatography, the PATA of the macromolecular fractions is reduced further even through these fractions still have immediate inhibitory action on the TCT of plasma. When the same concentration of protamine is added to non-heparinized plasma, the PATA in the macromolecular fractions is not altered. Treatment of the macromolecular fractions from heparinized plasma with triethylaminoethyl cellulose removes the immediate inhibitory action on the TCT of plasma but does not affect the PATA of the fractions. How the heparin and protamine interact to decrease the PATA of α2-macroglobulin containing fractions is not as yet determined. Recently it was found that a similar complexing of heparin fractions with α1-antitrypsin may occur in the same way as with α2-macroglobulin.