bluuebunny/crossref_metadata_2025_split huggingface dataset:
PLATELET AGGREGABLLITY AND METABOLISM IN PATIENTS WITH UNSTABLE ANGINA PECTORIS
Authors: Terres W , Hamm C , Kupper W , Bleifeld W 
Published: August 2018

Several platelet products indicating platelet activation have been detected in blood and urine of patients (PTS) with angina pectoris (AP) at rest. Platelet activation mainly depends on local changes in the morphology or biochemical behaviour of the vessels. Whether platelet hyperaggregability is of additional importance in the pathogenesis of unstable AP is up to now unclear. In a prospective trial we therefore evaluated 32 patients (PTS) with coronary heart disease, 16 with AP at rest during the last 8 hours before blood collection and 16 age and sex matched controls with stable exertional AP. Platelet aggregation was measured upon stimulation with ADP (0.5, 1 and 10 μmol/l) and collagen (1and 5μg/ml), and c-AMP was determined in platelet rich plasma before, and, as an estimate of platelet adenylate cyclase activity, after stimulation of this enzyme with PGE 1 (10 μmol/l for 30 s). For all concentrations of both ADP and collagen no significant differences in the rates and extents of aggregation could be found between the groups. Correspondingly, the mean (±. 2 SEM) concentrations of c-AMP were similar, basally (4.1 ±.1.4 pmol/ml for PTS withunstable AP and 5.3 t 1.3 pmol/ml for PTS with stable AP)and after stimulation of platelet adenylate cyclase with PGE 1 (14.8 ± 4.1 vs. 17.2 ± 2.8 pmol/ml).Conclusion: No generalized platelet hyperaggregability could be detected in our PTS with unstable AP when compared to controls with stable exertional AP.
CORONARY THROMBOLYSIS IN DOGS WITH A NONGLYCOSYLATED VARIANT OF HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR LACKING THE FINGER AND GROWTH FACTOR DOMAINS
Authors: Cambier P , van de werf F , collen D 
Published: August 2018

A variant of human tissue-type plasminogen activator (t-PA-AΔFE3X), with deletion of the NH2-terminal fibronectin-like finger (F) and epidermal growth factor (E) domains, and with amino acid substitution of Gin for Asn at all known N-linked glycosylation sites was expressed in Chinese Hamster Ovary Cells and purified to homogeneity. The thrombolytic and pharmacokinetic properties of this variant were studied in a canine model with copper coil induced thrombosis of the left anterior descending coronary artery. Infusion of t PAΔFE3X at a rate of 5 pg/kg/min for 30 min in 3 dogs resulted in a plateau level in plasma of 0.66 ± 0.08 ug/ml and induced recanalization of the coronary artery within 24 ± 4 min (mean ± SEM). Bolus injections over 2 min of 0.15 mg/kg in 3 dogs resulted in peak antigen levels in plasma of 1.6 ± 0.72 μg/ml and caused reperfusion within 14 ± 6 min. Bolus injection of 0.075 mg/kg in 3 dogs gave plasma antigen levels of 0.81 + 0.20 μg/ml and induced lysis in 31 ±15 min. Further reduction of the bolus to 0.038 mg/kg yielded plasma peak levels of 0.43 ± 0.20 μg/ml but did not cause reperfusion within 3 hours. Bolus injection of 0.075 mg/kg of natural t-PA isolated from melanoma cell culture fluid (Mel-t-PA) resulted in plasma peak levels of 0.46 ± 0.09 μg/ml and caused recanalization within 3 hours in only 1 of 4 dogs. None of the injections was associated with systemic fibrinolytic activation and fibrinogen degradation. The disposition of t-PAΔ related antigen from plasma following bolus injection could be described by a sum of two exponential terms with t1/2α: 17 min and t1/2β: 100 min. No significant difference in disposition rates for the different bolus injections were observed. Corresponding values for t1/2α of Mel-t-PA are 3 min.It is concluded that the deletion mutant t-PAΔFE3X has a markedly slower disposition rate from plasma than intact t-PA, which renders it relatively more effective than natural t-PA after bolus injection.
ENDOTHELIUM CURTAILS THE TRANSFER OF PLATELET SEROTONIN INTO THE BLOOD VESSEL WALL
Authors: Bult H , Fret H , Herman A G , Jordaens F , Peeters F A M , Verbeuren T J 
Published: August 2018

Blood platelets are the principal source of serotonin (5-hydroxytryptamine, 5HT) in the circulation. As it is assumed that 5HT release by activated platelets can influence vascular tone, we investigated whether platelet derived 5HT can penetrate from the lumen into the vessel wall. Venous blood was collected from pentobarbitone anaesthetized dogs on 5.8 mM EDTA, platelet rich plasma was prepared and incubated with 3H-5HT and 14c-adenine. After washing, a platelet suspension (PS, 108/ml) was made in Ca2+ free Krebs'. A segment of each saphenous vein was removed and de-endothelialized for about half (2-3 cm) of its length. The PS was perfused (37°C, 3 ml/min) for 15 min (4 dogs) or 30 min (4 dogs) through the lumen in the absence (one V. saphena) or presence (contralateral vein) of 0.1 unit thrombin/ml. Thereafter, 1 cm of the area with and the area without endothelium was excised, and these segments were washed 24 times with 5 ml Krebs. The 3H and 14c content of wash fluid and solubilized tissue was measured. The 12th wash contained 1.0 unit thrombin in order to verify that the 3H was not derived from adhering platelets. The 14c data indicated that - at low shear stress -similar platelet numbers adhere to the intima with or without endothelium, but in the absence of endothelium thrombin-induced aggregates remain loosely attached to the intima. The wash procedure removed the majority of these adhering platelets, as well as their 3H. In all tissues 3H accumulated, eg 708 dpm after 15 min and 1673 dpm after 30 min perfusion followed by washout. Both values were 7 to 10 fold enhanced when the platelet release reaction was induced with thrombin. In addition, endothelial denudation doubled the 3h accumulation under both circumstances. Previous experiments with soluble 5HT indicated that 70 % of the 3h is present in the adrenergic nerve terminals of the vessel. Application of the PS to the outside of the vessel led to a larger 3h accumulation, but then there was no indication of a difference between area's with or without endothelium.In conclusion, removal of the endothelial metabolic barrier enhanced vascular accumulation of 5HT, but the occurrence of a release reaction appeared to be more important in determining the access of platelet derived 5HT to vascular smooth muscle cells and adrenergic nerve endings.
CONTRACTIONS TO PLATELETS IN AORTAS OF CONTROL AND CHOLESTEROL-FED RABBITS
Authors: Verbeuren T J , Van Diest M J , Herman A G 
Published: August 2018

Atherosclerotic aortas obtained from cholesterol-fed rabbits show a decreased responsiveness to noradrenaline, an increased responsiveness to low concentrations of serotoninand an unaltered responsiveness to prostaglandins. In vitro contractions induced by aggregating platelets are largely due to serotonin liberated during the aggregation. The present study was designed to compare the contractile responses to aggregating platelets inaortas obtained from control and cholesterol-fed rabbits.Male New Zealand rabbits were fed either a control or a 0.3% cholesterol diet during 16 weeks. Macroscopic and microscopic examination of the luminal surface of the aortas obtained from these animals revealed a substantial amount of fatty streaks in the tissuesobtained from the cholesterol-fed rabbits. Segments of the aortic arch of the rabbits were then mounted in organ chambers for isometric tension recording.In both the control and the atherosclerotic aortas increasing concentrations of platelets evoked contractions; the contractions obtained with the lower concentrations of platelets were significantly greater in the atherosclerotic tissues. The maximal responses and the ED50-values were comparable in both groups of blood vessels. No significant differences were observed when platelets obtained from control or hypercholesterolemic rabbits were compared. In the control and the atherosclerotic aortas the thromboxane receptorantagonist BM13505 at 2 x 10-5M did not significantly affect the contractionsto platelets obtained from either control or cholesterol-fed rabbits. The serotonin receptor antagonist ketanserin at 5 x 10-8M nearly abolished the responses to platelets in bothgroups of aortas.These experiments illustrate that (1) thecontractions induced by rabbit platelets in control and atherosclerotic aortas are mediated by serotonin and (2) the responses to platelets, as those to serotonin, are augmented in the atherosclerotic preparations.
CORNARY HEART DISEASE, DIET, SERUM LIPIDS, PLATELET FUNCTION AND PLATELET FATTY ACIDS IN TWO POPULATIONS WITH A HIGH AND A LOW INTAKE OF DIETARY FISH
Authors: Simonsen T , Vårtun Å , Lyngmo V , Nordθy A 
Published: August 2018

In the coastal areas of Northern Norway the intake of fish is by tradition high whereas in the inland area it is low. We have examined the mortality of CHD in the period 1975-84 in a coastal community (C) and an inland community (I). In addition we have examined 30 healthy male subjects aged 30-year, selected by random in the two communities. The examination included a dietary survey based on registration and weighing of all dietary items for one week, blood pressure, serum lipids, primary bleeding time, platelet aggregation induced by collagen and fatty acid composition of platelet total phospholipids.The age-adjusted mortality of CHD was significantly higher for age groups 30-70 year in C whereas the opposite was found above 70 years of age. The mean intake of fish per day was 134 g (0.9 g eicosapentaenoic acid-EPA) in C and 53 g (0.25 g EPA) in I. Serum triglycerides was higher in C (p<0.05) whereas totalcholesterol was similar. The primary bleeding time was not different in the two areas. Significantly lower concentrations of collagen was needed to induce 30 and 60% aggregation in platelet rich plasma in C than in I. No significant differences in the content of eicosapentaenoicacid (EPA) was observed in platelet total phospholipid fatty acids. This study has not confirmed that a high intake of fish as a singledietary variant, is associated with a low mortality of CHD. The lack of changes in plasma lipids, platelet fatty acid composition between representative groups from the two populationsindicate that other factors mask the possible beneficial effects of a high fish diet. Furthermore, the daily intake of large amounts of lean fish give only a very moderate increase in dietary intake of EPA.
TURNOVER OF THE PHOSPHOMONOESTER GROUPS OF POLYPHOSPHOINOSITIDES IN UNSTIMULATED HUMAN PLATELETS
Authors: Holmsen Holm , Verhoeven Adrie J M , Tysnes Ole-Bjørn , Aarbakke Grethe M , Cook Carol A 
Published: August 2018

The metabolic activity of the polyphosphoinositides in unstimulated human platelets was studied by 1) short-term labelling with 32P-Pi, 2) by replacement of PP. from pre-labelled platelets with unlabelled phosphate and 3) by depriving the cells of metabolic ATP. Under short-term labelling conditions, the 4- and 5-phosphates of phosphatidylinositol-4-phosphate (PIP2) and phosphatidylinositol 4,5-bisphosphate (PIP2) had the same specific radioactivity as the γ-phosphate of metabolic ATP. The specific radioactivity of the 1-phosphates of phosphatidylinositol, PIP and PIP2 was similar but only 4-13 % as compared to the γ phosphate of ATP. When 32P-Pi. pre-labelled platelets were incubated with up to 25 mA of unlabelled phosphate, the displacement of the P-label from PIP, PIP2 and metabolic ATP followed similar kinetics. Inhibition of ATP regeneration in 32P-Pi pre-labelled platelets resulted in a rapid fall in metabolic ATP with much slower fall in 32P-PIP. 32P-PIP increased initially and decreased thereafter in parallel with PIP2. However, ATP turnover was not abolished, as indicated by the marked (25 % of the control) incorporation of extracellular 32P-Pi. into PIP and PIP2 in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PIP and PIP2 to metabolic inhibition.We conclude that PIP and PIP2 are present as a single metabolic pool in human platelets. Turnover of the 4- and 5-phosphates of PIP and PIP2 in unstimulated platelets is as rapid as that of the γ- Phosphate of metabolic ATP, and accounts for about 7 % of basal ATP consumption.
EVIDENCE THAT ABNORMAL PLATELET AGGREGATION IN SPONTANEOUSLY HYPERTENSIVE RATS IS LINKED WITH PHOSPHOINOSITIDES TURNOVER AND PHOSPHORYLATION OF 47,000 DALTON PROTEIN
Authors: Huzoor-Akbar H , Anwer Khursheed 
Published: August 2018

We have shown earlier that abnormal platelet aggregation in spontaneously hypertensive rats (SHR) is not caused by prostaglandins (Thromb. Res. 41, 555-566, 1986). In this study platelets from SHR and normotensive (Wistar Kyoto, WKY) rats were used to examine the role of phosphoinositides (Pins) and protein phosphorylation in increased platelet activation in hypertension. Thrombin (0.05 U/ml) induced rapid hydrolysis of phosphatidylinositol-4,5-bis-phosphate (PIP2), phosphatidyl-inositol-4-phosphate (PIP), and phosphatidylinositol (PI) in (32p)-pO4 labeled platelets. However, significantly greater hydrolysis of PIP2 and PI was seen in SHR platelets than in WKY platelets (see Table). Thrombin also caused two- to three-fold increased accumulation of phosphatidic acid (PA) in SHR platelets than in WKY platelets (see Table).Thrombin caused phosphorylation of 18,000 Dalton (P18) and 47, Dalton (P47) proteins in SHR and WKY Platelets. Significantly increased phosphorylation of P47 was seen at 5, 15, 60 and 240 seconds of incubation with thrombin in SHR platelets (60%, 68%, 98% and 91%) than in WKY platelets (13%, 37%, 44% and 47%). The extent of P18 phosphorylation was same in both SHR and WKY platelets. Aspirin (500 uM) did not affect phosphorylation of P47 or P18 in SHR or WKY Platelets. These data lead us to suggest that increased turnover of Pins and increased phosphorylation of P47 are involved in abnormal platelet aggregation in SHR (Supported in part by the COHC grant #86-01-A and the Ohio University College of Osteopathic Medicine).
IMMUNOAFFINITY PURIFICATION OF PLASMA INHIBITOR FOR ACTIVATED PROTEIN C
Authors: Laurell M , Carlsson T , Stenflo J 
Published: August 2018

Activated protein C (APC) is an important regulator of blood coagulation in vivo. In plasma this serine protease is slowly inhibited by a specific inhibitor, activated protein C inhibitor (PCI), (Suzuki et al. (1983) J.Biol.Chem. 258 , 163-168). We have now made monoclonal antibodies against PCI by immunizing with the APC-PCI complex. Positive clones were identified by solid phase immunoassay with 125I labelled partially purified inhibitor. After subcloning and expansion in mice, one of the monoclonal antibodies was immobilized on Sepharose 4B and used in the purification of the inhibitor. A two step purification procedure was deviced starting with passage of fresh human plasma over the column. Following extensive washing the inhibitor was eluted with 50 mM triethylamine- HCI,0.5 M NaCl, pH 11.0, from the column together with a small amount of high molecular weight material. After gel filtration on a column packed with AcA 44 the inhibitor appeared homogenous on SDS - PAGE. Approximatly 0.5 mg inhibitor was obtained from 200 ml of fresh plasma. The apparent Mr of the inhibitor was 57000 kDa on SDS -PAGE. The purified protein formed a complex (Mr =110000 kDa) with human APC. At the same time a band (Mr = 54000 kDa ) appeared that represented the modified inhibitor. When analyzed on agarose gel electrophoresis (75mM barbital buffer, 2mM EDTA at pH 8.7 ) the PCI migrated to the β2- region, whereas the modified inhibitor had a slightly more anodal mobility. The APC-PCI komplex migrated to the α2- region.Two immunoradiometric assays were constructed with the monoclonal antibodies. One measured the complexes between APC and PCI while the other one measured the total amount of PCI present. These assays were used to study complex formation in buffer and plasma.
A SECOND INHIBITOR OF ACTIVATED PROTEIN C (APC)
Authors: van der Meer F J M , van Tilburg N H , van der Linden I K , Briét E , Bertina R M 
Published: August 2018

The inactivation of APC in plasma, as measured with the chromogenic substrate S2366, follows, in the absence of heparin, pseudo-first order kinetics. The ti of about 20 minutes is independent of the APC concentration (31-500 nM) and increases linearly with the dilution of the plasma. These observations suggest that the concentration of the APC inhibitor in plasma is much higher than 500 nM, which is much higher than the concentration of 88 nM reported by Suzuki for the protein C inhibitor (PCI).In the presence of heparin (5 IU/ml) the inactivation of APC becomes biphasic. Fast inactivation with an apparent ti of 8 minutes is followed by slower inactivation with a ti of 20 minutes.Removal of PCI from the plasma with α-PCI antibodies (kindly provided by Dr. Suzuki) has no effect on APC inactivation in the absence of heparin. However, in this plasma APC inactivation could not be stimulated by addition of heparin (absence of fast phase of APC-inactivation). These data suggest the presence of two APC inhibitors in plasma: the heparin dependent PCI (PCI-I), earlier reported by Suzuki, and a sofar unknown heparin-independent inhibitor (PCI-II).Further experiments showed that this PC I-11 has a molecular weight of 60 kD and is different from the APC-binding protein reported by Kisiel. Using Heparin-Sepharose affinity chromatography we could separate PCI -II from both PCI-I and the APC binding protein. In this PC I-11 preparation APC inactivation was accompanied by the formation of an APC-PCI -11 complex of about 105 kD as demonstrated by immunoblotting with a-PC antibodies after SDS-PAGE. The identity of PCI-II is unknown; however, it is different from antithrombin III, heparin cofactor II, α1-antitrypsin and β2-antiplasmin.The demonstration of the presence of two APC inhibitors in plasma will require a re-evaluation of the current functional assays for the APC inhibitor.
IMMUNOCHEMICAL IDENTIFICATION OF A THROMBOSPONDIN -LIKE ANTIGEN IN ARTERIAL THROMBOGENIC MICROFIBRILS
Authors: FAUVEL-LAFEVE F , LEGRAND Y J 
Published: August 2018

The biochemical structure of arterial microfibrils (MFS) is unknown. Presently, the most probable hypothesis is that elastin associated MFS contain several antigenic determinants with MW varying between 31 and 200 KD.From our previous studies we know that MFS extracted by 6 M GuCl contain a major glycoprotein with a 128 KD MV (GP128). GP 128 is essential for the reactivity of MFS towards blood platelets but due tc the high insolubility of the extracted material it was not possible to isolate and study this GP 128. We have used immunoblotting to determine if MFS contain determinants recognized by antibodies against connective tissue glycoproteins such as fibronectin, type VI collagen or anti-platelet thrombospondin (TSP). 'The results showed that MFS do not contain fibronectin or type VI collagen but that anti-TSP IgG reacted with GP 128. Furthermore, the Fab fragments from anti-TSP IgG inhibited platelet aggregation induced by MFS but not by collagen or ADP . In a second step,to raise antibodies against GP 128, we prepared blots from entire MFS, the nitrocellulose band corresponding to GP 128 was cut, dissolved in DMSO, and wrs injected to rabbits. Such obtained antibodies recognized only GP 128 in arterial MFS and also TSP in a platelet lysate confirming that GP 128 and TSP have a common antigenic structure. IgG from anti-GP 128 inhibit platelet aggregation induced by MFS but not by collagen or ADP. Previously reported observations showed that tissue TSP and endothelial cells derived GP 128 have a similar affinity for chromatography supports and have the same effect on platelet-MFS interactions. All these results led us to propose that TSP, GP 128, and MFS recognize a common determinant on platelet membrane. This assumption would be strenghened if GP 128 indeed is derived from tissue TSP.
PENETRATION OF FACTOR Xa INTO PHOSPHOLIPID LAYERS
Authors: LECOMPTE M F 
Published: August 2018

Negatively charged phospholipids (PL) at the platelet surface play a crucial role in the conversion of prothrombin into thrombin in the prothrombinase complex. Interactions of prothrombin and factor Xa (FXa) with PL are generally thought to occur through ionic interactions via Ca++ bridges. However we have shown by various independent approaches that prothrom bin (as well as FV and FVa) actually penetrates into the PL layer. We show that this is also the case, in the presence of Ca++ , for the other vitamin K-dependent, Gla-containing protein of the complex, FXa.Electrochemical surface measurements indicate a reversible increase in the capacitance of condensed PL monolayers (containing various concentrations of phosphatidylserine (PS)), upon interaction with bovine FXa (kindly provided by Dr. C.M. Jackson). Reduction of the FXa disulfide bridges at the contact of the electrode further demonstrates penetration Parallely, radioactive surface measurements allow to follow the adsorption of FXa which is quite reversible on PS gono-layers and to determine the association constant, 6.106 1/mol, very similar to the Ka of FX, obtained by various different techniques.Finally, penetration of FXa with PS containing PL vesicles was tested using the radiolabelled apolar probe (INA) which binds selectively to the lipid embedded domains of protein. FXa was radioactively labelled and the label preferentially found on the active site containing FXa heavy chain Moreover, interaction of FXa with PS vesicles decreases its Km for S 2222.Supported by grant INSERM n° 845016
ASSOCIATION OF HEPARIN AND FACTOR Xa: INFLUENCE ON THE RATE OF INHIBITION OF FACTOR Xa BY ANTITHROMBIN III-HEPARIN
Authors: Owen B A , Owen W G 
Published: August 2018

Association of heparin non-covalently with bovine factor Xa was analyzed by Superose-12 gel chromatography. In 0.05 M NaCl, 0.02 M Tris, pH 7.5, DEGR-Xa (factor Xa inactivated by dans-Glu-Gly-Arg-CH2Cl) was eluted as a single, sharp peak at Ve/Vt=0-65 (elution volume/internal volume). Mixtures of heparin and DEGR-Xa were eluted as two partially resolved peaks of protein at Ve/Vt=0.59 and 0.65. The fraction of DEGFUXa in the leading peak was directly proportional to [heparin], and at 100 yM heparin the leading peak contained more than half the total protein. When 0.02 M HEPES was substituted for Tris a single, slightly broadened peak at Ve/Vt=0.64 was obtained on chromatography of 100 μM heparin and 10 μM DEGR-Xa. In a buffer system comprising 0.02 M Tris, 0.02 M HEPES, 0.03 M NaCl, pH 7.5, two peaks were eluted at Ve/Vt=0.59 and 0.65. Therefore, Tris increases the affinity of DEGR-Xa for heparin.Solutions buffered with Tris or HEPES were compared for effects on the kinetics of inhibition of factor Xa by antithrombin III-heparin. Reaction mixtures containing 1 nM factor Xa, 30 nM heparin and 600 nM antithrombin III were assayed with S-2222 at intervals of 2-10 sec. Reagent concentrations were chosen (a) to assure pseudo-first-order kinetics, (b) to have [heparin]<< Kq for factor Xa-heparin, and (c) to bind virtually all available heparin to antithrombin III. The same second-order rate constant, Kobs=2.5×107 M−1s−1, was obtained in both buffer systems. We conclude that the association of factor Xa with heparin observed directly by gel chromatography does not contribute to the reaction rate of factor Xa with antithrombin III-heparin.