bluuebunny/crossref_metadata_2025_split huggingface dataset:
RELEVANCE OF FREE t-PA ASSAY FOLLOWING VENOUS OCCLUSION IN PATIENTS WITH VENOUS THROMBOEMBOLIC DISEASE
Authors: Alessi M C , Juhan-Vague I , Valadier J , Philip-Joet C , Holvoet P , Collen D 
Published: August 2018

Out of 100 consecutive patients with a history of venous thromboembolism, 80 were subjected to 10 min venous occlusion (VO). Blood samples were collected on citrate, immediately cooled on ice, centrifuged and frozen. Euglobulin fibrinolytic activity (EFA) was measured on fibrin plates, plasminogen activator inhibitor (PAI) activity according to Verheijen et al., and total t-PA antigen with an ELISA. In addition, free t-PA was assayed in plasma collected on the monoclonal antibody MA-2G6, which is directed against the active site of t-PA.The assays allowed to differentiate three groups of patients. Good responders (GR) to VO with at least a doubling of the EFA after venous occlusion. Poor responders (PR) with less than a two-fold increase in EFA could be classified either as having a high PAI level resulting in rapid inhibition of t-PA (PR-I) or as having a deficient release of t-PA from the vessel wall (PR-II).It is concluded that patients with a history of venous thromboembolism may have an abnormal fibrinolytic response to venous occlusion which is due either to an increased inhibitor level or to a deficient t-PA release. Both abnormalities are recognized by the free t-PA assay.
LEUCOCYTES AND PLATELETS DEPOSITION CN ENDOTHELIAL SURFACE INDUCED BY PARTIAL VENOUS OCCLUSION.AN ULTRASTRUCTURAL STUDY IN THE DOG
Authors: Andrade J Aguiar , Resende C , Monteiro A C , Adolfo A , Pina Cabral J M 
Published: August 2018

After partial venous occlusion of dog's femoral vein until 60% of its initial cross section for 90 min. we have observed a decrease in plasminogen activator content (t-PA) in the pre-occlusion segment and an increase of prostacyclin like activity (PLA) in the post -occlusion segment (in press).In this work and in order to try to find an ultra-structural basis for these findings, we carried out observations of the constricted segments with transmission and scanning E.M. (n=10).We observed that : -1) endothelial cells were maintained in place, with minimal ultrastructural changes, namely a small increase in vaccuolization ;-2) there was massive subendothelial deposition of leucocytes, predominantly polimorphonuclears, which permeate internal elastic lamina and were also found in subjacent structures; -3) platelets were rarely seen and platelets agregates were never found. Autologous Indium labelled platelets injection confirmed "in vivo" these findnigs (n=10).We conclude that in partial venous occlusion, platelet deposition and endothelial cell descamation are not initiating factors in changes observed at t-PA and PLA vessel wall levels, although the role of white blood cells will need further investigation.
ISOLATION OF THE GENOMIC REGION CODING FOR TYPE-1 PLASMINOGEN ACTIVATOR INHIBITOR
Authors: Strandberg L , Lawrence D , Ny T 
Published: August 2018

The type-1 Plasminogen Activator Inhibitor (PAI-1) has recently been identified as a member of the Serine Protease Inhibitor family (SERPINS). This family of proteins contain many serine protease inhibitors but also functionally unrelated proteins like ovalbumin and anginotensinogen. PAI-1 inhibits both u-PA and t-PA and might therefore be an important regulator of the fibrinolytic system.In order to study the evolution of the Serpin family as well as PAI-1 gene expression we have isolated the genomic region carrying the PAI-1 gene. A cDNA sequence for PAI-1 was used as probe to screen a human genomic library. When 2 million independent phages were screened, 13 positive clones were isolated. Characterisation of these clones showed that they could be divided into 3 overlapping groups covering a genomic region of approximately 30 kb. The gene was localized and characterized by restriction enzyme analysis, southern blotting using cDNA and oligomer probes, and DNA sequencing.
MEASUREMENT OF PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI-1) IN PLASMA WITH AN ELISA BASED ON TWO MURINE MONOCLONAL ANTIBODIES
Authors: Declerck P J , Alessi M C , Verstreken M , Kruithof E K O , Juhan-Vague I , Collen D 
Published: August 2018

An enzyme-linked immunosorbent assay (ELISA) for quantitation of plasminogen activator inhibitor 1 (PAI-1) was developed, based on two murine monoclonal antibodies (MA-7D4B7 and MA-7F5), raised against purified PAI-1 from HT-1080 fibrosarcoma cells, which react with non-overlapping epitopes. MA-7D4B7 was coated on microtiter plates and bound PAI-1 antigen was quantitated with MA-7F5 conjugated with horseradish peroxidase.In normal plasma, collected on citrate at pH 7.4, the PAI-1 level is 27 ± 16 ng/ml (mean ± SD, n=ll), with a corresponding value of 19 ± 11 ng/ml (n=12) in plasma collected on acid citrate pH 4.5, which inhibits the release of PAI-1 from platelets. The lower limit of the assay in plasma is 2 ng/ml; the intra-assay, inter-assay and inter-dilution coefficients of variation are 5.2%, 8.0% and 7.1% respectively.This ELISA was used to measure PAI-1 levels in plasma (collected on citrate, pH 7.4) of non-pregnant women and of women at different stages of pregnancy. A progressive increase is observed : before: 20±9 ng/ml, n=7; first trimester: 25 ± 12 ng/ml, n=5; second trimester: 40 ± 25 ng/ml, n=ll; third trimester: 98 ± 46 ng/ml, n=13. A correlation coefficient of 0.70 is found between the duration of pregnancy and the PAI-1 level.Preliminary data indicate that the PAI-1 antigen level is increased in several disease states, including myocardial infarction and deep vein thrombosis.Thus, this newly developed ELISA allows a direct measurement of the fast-acting inhibitor of plasminogen activator in plasma. Application of this assay to plasma of non-pregnant and pregnant women substantiates previous results obtained with the use of functional assays. In order to quantitate PAI-1 antigen circulating in plasma, blood should be collected under conditions that prevent platelet stimulation.
A SIMPLE AND RAPID PLASMINOGEN ACTIVATOR INHIBITOR (PAI) ASSAY IN PLASMA, USING A UNIQUE CONCENTRATION OF TISSUE PLASMINOGEN ACTIVATOR (tPA) AND PAI-DEPLETED PLASMA
Authors: Contant g , Anglescano E , Martinoli J L 
Published: August 2018

Interferences of plasmin inhibitors are a main drawback in the functional assay of PAI in plasma. To overcome them, an acidification step, prior to residual tPA assay has been frequently used.A simple chromogenic method, based on the inhibition of purified tPA by plasma prediluted in PAI-depleted plasma, is described. The residual tPA was subsequently measured in the presence of fibrinogen fragments used as Stimulator, plasminogen and chromogenic substrate (CBS 30.41). The assay can discriminate between PAI and plasmin inhibitors since the concentration of these proteins remains constant because brought in excess by the PAI-depleted plasma. Parallel dose-response curve were obtained when PAI-depleted plasma was used instead of buffer, for the preparation of the reference curve and for the dilution of samples. The residual tPA activity measured was directly proportional to the PAI activity of the sample using a single concentration of tPA. Samples containing 0 to 30 IU/ml of PAI could be measured using a 1:40 dilution.Values for 27 normal volunteers at rest were 5.1 IU/ml (range : 0 to 11.8 IU/ml). PAI activity was decreased in afternoon samples (4.6 IU/ml) compared to those of the morning (5.8 IU/ml). After stasis, when a concomitant release of tPA was observed, the values obtained (3.1 IU/ml) were significantly lower than the values obtained before (4.1 IU/ml).The activity measured is probably related to PAI-1 (endothelial ceil type PAI), but PAI-2 related activity (placental PAI) can be also estimated since pregnancy plasma showed high levels of inhibitor (mean : 15.1 IU/ml, range : 9.5 to 28.1 IU/rni).The present method is simple and rapid to perform. It can be routinely used in the screening of various thrombotic disorders.
DETERMINATION OF PA INHIBITOR 1 (PA INHIBITOR ACTIVITY AND PA INHIBITOR 1 ANTIGEN) IN PLASMA BEFORE AND AFTER VENOUS OCCLUSION
Authors: Juhan-Vague I , Valadier J , Alessi M C , Ansaldi J , Ailluad M F , Kruithf EK O 
Published: August 2018

PA Inhibitor activity (PAIact-Verheijen’s method-U/ml) and PA Inhibitor 1 antigen (PAIlAg- Kruithof’s radioimmunoassay -ng/ml) were evaluated, on blood samples before (B) and after (A) venous occlusion (VO) (10 min) in order to analyse, in B-VO samples, the ratio (R=PAIlAg/PAIact) ' between the immunological and enzymatic material, and the change of PAIlAg levels after VO.The B-VO values (m ± SD) were determined from 111 plasmas : -86 with normal ( 12U/ml) PAIact levels = 3.7 ± 2.5 ; PAIlAg = 17.4 ± 10.1 ; R = 7.1 ± 6.2 (range 1.5 - 24 )-25 with high PAIact levels (15 post surgery, 10 obese patients)= 30.9± 19.3; PAIlAg = 75.3 ± 44.7 ; R = 2.7 ± 1.5 (range 1.3- 4.4). The correlation between the 2 dosages was r = 0.82 (p 0.01). In the 2 kinds of patients with high PAIact levels, a parallel high PAIlAg level was found. As the range of the ratio R was very large, patients were divided in 2 Groups : GrI : Normal R ( 7), n=81 (normal PAIact : n=57 ; high PAIact : n=24) and GrII : high R, n=30. The results for PAIact/PAIlAg/R were = GrI :12.6± 16.3/ 31.5±34.4/ 3.2 ± 1.5. GrII : 2.6±4.1/ 27.9 ±30.1/ 13.8±6.1. In GrII the platelet origin of inactive PAIlAg from in vitro activation of platelets could be demonstrated (high level of BTG lug/ml) in 50 % of cases. No in vitro platelet activation could be demonstrated in GrI. These results point out the necessity of a good preparation of the plasma (mainly 0-4°) for PAIlAg determination.The B and A-VO values were analysed from 60 subjects. Plasmas with in vitro activation of platelet determined by BTG levels had been discarded. No platelet activation occured with the VO. The results were not corrected with the PCV. PAIlAg B-VO/A-VO = 19.2 ± 15.1/ 26.9 ± 18.3. 36 subjects (60 % of total) had an increase in PAIlAg A-VO 20 % (mean increase = 62 %) . There was no correlation between basal values of PAIlAg and the increase after VO, and between PAIlAg increase and t-PA-Ag release. It is concluded that a weak increase of PAIlAg may occur after VO ; this increase is not parallel with t-PA release. The physiopathological significance of this increase of PAIlAg as yet to be evaluated.
DIFFERENTIAL EFFECT OF TEMPORARY Ca2+ DEPRIVATION UPON ADP-AND COLLAGEN-INDUCED PLATELET AGGREGATION IN HUMAN WHOLE BLOOD
Authors: Watts I S , Keery R J , Lumley P 
Published: August 2018

In platelets exposed to chelating agents at 37°C and recalcified, aggregation to ADP, adrenaline, collagen and thrombin were either abolished or markedly reduced (1). This phenomenon has been suggested to be caused by disruption, by Ca2+ deprivation, of the fibrinogen receptor (glycoprotein lib Ilia complex) on the surface of the platelet (2). This effect was dependent on the exposure time of Ca2+ chelators and was reduced at lower temperatures. We have investigated the effect of exposure of human platelets to EGTA upon aggregation to a range of agonists at both 37 and 25°C. EGTA (5 mM) was incubated with citrated (12.9 mM) whole blood for either 0, 1, 5, 15 or 30 min. One minute following recalcification (5 mM CaCl2), full aggregation concentration-response curves were constructed to ADP and adrenaline (0.1 to 3 μM), PAF (3 to 100 nM), collagen (0.1 to 4 μg/ml) and U46619 (0.03 to 1 μM), using a platelet counting technique (3). At 25°C, EGTA produced little or no significant loss of sensitivity to any agonist. At 37°C marked rightward shifts of the ADP, adrenaline and PAF aggregation curves occurred which were related to the time of incubation with EGTA (e.g. ADP concentration ratios (CR) of 2.0 (0.8-4.8), 4.7 (2.1-10.7), › 186.6 (135.2-257.6) (95% confidence intervals n=4) obtained at 1, 5 and 15 min respectively). Following 30 min incubation aggregation to all three agonists was abolished (up to ADP 300 pM; adrenaline 100 μM; PAF 10 μM). In contrast, whilst collagen and U46619 concentration-effect curves were displaced to the right following 30 minutes exposure to EGTA (CR =13.6 (6.1-30.4) and 9.0 (4.3-18.7) respectively, n=4) full aggregation curves could still be established. Furthermore, a 60 min incubation with EGTA caused little further effect. Our findings suggest platelet agonists such as collagen and U46619, but not ADP, adrenaline and PAF, can evoke expression of a population of fibrinogen receptor sites involved in platelet aggregation that are inaccessible to EGTA.(1) Zucker, MB & Grant, RA (1978). Blood, 52, 505. (2)Shattil, SJ et al. (1985). Blood, 66, 92. (3) Lumley, P & Humphrey, PPA (1981). J. Pharm. Methods, 6, 153.
PHOSPHOLIPASE A2 ACTIVATION BY A MECHANISM SEPARATE TO THAT RESPONSIBLE FOR PHOSPHOLIPASE C STIMULATION IN ALPHA-THROMBIN-STIMULATED HUMAN PLATELETS
Authors: Crouch Michael F , Lapetina Eduardo G 
Published: August 2018

The ability of cell surface receptor occupation to increase the activity of phospholipase A2 has been thought to be due to the prior activation of phospholipase C and an increase in the intracellular Ca2+ concentration. However, recent evidence from our and other laboratories has suggested that this may not be the case, but rather stimulation of phospholipase A2 may be under the control of separate receptor-activated events. We have investigated this further by comparing the ability of prostacyclin (PGI2) and epinephrine to alter platelet responses to thrombin and examining the resulting phospholipase A2 activities.Alpha-thrombin stimulated aggregation of human platelets, the formation of inositol phosphates and phosphatidic acid, mobilizaton of Ca2+ from internal stores and Ca2+ influx, protein phosporylation (47 kDa and 20 kDa) and arachidonic acid (AA) release. Each of these responses was partially inhibited by prostacyclin (PGI2) except that of AA release, which was abolished. In combination with epinephrine and PGI2, alpha-thrombin-induced aggregation, phosphatidic acid formation and protein phosphorylation were restored, but the release of AA only reached 50% of its control value. Epinephrine alone had no effect on any of these responses, either in the presence or absence of PGI2. Thus, alpha-thrombin-induced activation of phospholipase A2 is more sensitive to the effects of PGI2 than is phospholipase C, and supports the possibility that there are distinct control mechanisms for receptor activation of these enzymes. We are presently examining the role of Gs in the inhibition by PGI2 of platelet phospholipase A2 and of Gi in the thrombin stimulation of this enzyme
THROMBIN-INDUCED STIMULATION OF HUMAN PLATELETS LEADS TO AN INCREASE IN CYTOPLASMIC CALCIUM
Authors: Davies T A , Drotts D , Weil G , Simons E R 
Published: August 2018

Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are detected with indo 1. They are extremely rapid and are maximimal by 1015 seconds. Suspension studies, which reflect an average value over 3 × 107 cells/ml, indicate a thrombin dose-dependent increase in cytoplasmic calcium. However, flow cytometry indicates that subpopulations of cells are responding differently. The responses of these populations seem to be dependent on thrombin concentration. A maximal overall response is found when more than 50 % of the cells respond. This may explain multiple stimulations of the same suspension of cells at low thrombin dosesElevated extracellular Ca++ increases the maximal cytoplasmic response and significantly retards its subsequent decrease. When extracellular Ca++ is removed via addition of EGTA just before stimulation, the initial response is halved and the subsequent slow decrease is more marked and returns to lower (near-basal) levels. Intracellular Ca++ can be chelated with s - (0-am inophenoxy) - e thane-N, N, N',N'-tetraacet ic acid (BAPTA), which has a twofold greater affinity far Ca++ than does indo, but which does not fluoresce (at indo sensitive wavelengths) upon binding of Ca++. Cells loaded with both indo and BAPTA exhibit no rise in cytosolic Ca++ or altered membrane potential when stimulated with αthrombin, unlike cells loaded with indo alone. When Ca++ (2 mM) is added back to the extracellular buffer, these cells will take up the Ca++ at a constant rate as seen by a rise in indo fluorescence. When cytosolic Ca levels reach those of the resting platelets loaded with indo alone, these cells recover the αthrombin-indue ed Ca response of control platelets. However, they no longer depolarize partially under these same Ca++ replenished conditions. This implies that some of the Ca++ required for the platelet thrombin response comes from non-replenishable internal stores.
The aggregation of bovine platelets is not dependent on thromboxane B2 production
Authors: Gentry P A , Bondy G S 
Published: August 2018

Bovine and human platelets appear to be equally sensitive to inhibition by several environmental toxins even though the bovine is not generally recognised as a species prone to thromboembolic and hemorrhagic disease. During the examination of the mechanism of action of the toxins it became necessary to establish parameters for normal bovine platelet function.Bovine platelets suspended in homologous plasma demonstrate various responses to different agonists. Bovine platelets aggregate effectively and irreversibly to fibrillar form collagen and adenosine-diphosphate (ADP), undergo reversible aggregation in response to platelet activating factor (PAF), exhibit a minimal aggregation response to serotonin (5HT) and arachidonic acid (AA) and are refractory to acid-soluble collagen (calf skin). A synergistic aggregation response is observed with the combination of ADP and 5HT, an additive response with ADP and PAF, while the addition of AA has no effect on the ADP induced aggregation response. A biphasic response has not been observed with any of the agonists or with the combinations of agonists. There is no direct correlation between the maximum aggregation response induced by ADP and PAF and the amount of thromboxane B2 (TXB2) released from the stimulated platelets. Similarly when acetylsalicylic acid (ASA) is added to the platelet suspensions, TBXg release can be completely inhibited without any observable effect on the aggregation response. Platelet aggregation was also unaffected by indomethacin and dipyridamole. Verapamil is a weak inhibitor of ADP induced aggregation but is a potent inhibitor of PAF stimulated platelets. Bovine platelets appear to have some distinct characteristics since they exhibit some responses which are not only different from human platelets but are also different from platelets obtained from other large animals such as the sheep and horse.
DIACYLGLYCEROL AND PHORBOL ESTER REDUCE AEQUORIN-INDICATED [Ca++] ELEVATIONS INDUCED BY THROMBIN OR ADP
Authors: Ware J A , Smith M , Salzman E W 
Published: August 2018

Platelet aggregation and secretion induced by phorbol ester (PMA) or diacylglycerol (DAG) are preceded by an increase in [Ca++] that is detected byaequorin, but not by quin2, fura-2, or indo-1, suggesting that these indicatorsreflect different aspects of Ca++ homeostasis, possibly different functional Ca++ pools. Addition of two conventional agonists in subthreold concentrations synergistically enhances the [Ca++] rise and aggregation.However, if PMA or DAG is the first agonist the subsequent quin2-indicated [Ca++] rise after thrombin is reduced.Whether aequorin-indicated [Ca++] is similarly affected is unknown. We studied gel-filtered platelets loaded with aequorin or a fluorophore and added PMA, DAG, thrombin or ADP, alone or in combination. Either PMA or DAG alone caused a concentration-dependent increase in [Ca++] detectable with aequorin but not with the fluorophores; simultaneous addition of thrombin or ADP with DAG or PMA produced a larger [Ca++] rise than either alone. However, addition of DAG or PMA as a first agonist reduced subsequent aequorin-indicated [Ca++] rises following thrombin or ADP in a concentration and time-dependent manner. Inhibition of ADP or thrombin-induced [Ca++] rise was not always accompanied by inhibition of aggregation or secretion. Combination of subthreshold concentrations of ADP and thrombin produced an enhanced [Ca++] rise and aggregation. However, this synergistic effect was inhibited by preincubation with DAG or PMA. Neither this effect nor DAG-induced [Ca++] rise was inhibited by the protein kinase C inhibitor H-7. In genera^ preincubation of platelets with an agonist enhances Ca rise and aggregation in response to a second agonist; in contrasl protein kinase C activators, which themselves elevate [Ca++] as shown by aequorin, inhibit aequorin-indicated Ca rises after ADP or thrombin, and limit synergism between these two agonists.
INSULIN RECEPTORS ARE NOT COUPLED TO THE PHOSPHOINOSITIDE OR ADENYLCYCLASE MESSENGER SYSTEMS IN HUMAN PLATELETS
Authors: Pfliegler G , Arnout J , Kienast J , Wittevrongel K , Vermylen J 
Published: August 2018

Insulin receptors have been found not only on its “target Cells” but also on several other cell-types, including human platelets. From studies on adipocytes and liver cells it seems that they are coupled both to the adenylate cyclase-cyclic AMP and the polyphosphoinositide messenger systems. Circulating blood cells might faithfully reflect the insulin receptor state of target organ tissues. Impaired platelet function has an important role in the pathogenesis of vascular and thrombotic complications in diabetes mellitus, and insulin seems to act directly on platelets. A reduction in the number and binding capacity of platelet insulin receptors in diabetic patients (Udvardy et al. 1986) suggested a (patho)physiological role for these receptors. In our studies, insulin (1 × 10-9 - 1 × 10-6 M) did not affect basal platelet cyclic AMP levels, as measured following incorporation of [3H] adenine. Insulin did not prevent PGI2 (25-75 nmol/L) induced cyclic AMP formation in platelets. Insulin did not modify the basal levels of inositol phosphate (IP), IP2 or IP3 in platelets, as measured following incorporation of [3H] inositol. Insulin did not affect formation of IP, IP2 or IP3 by thrombin. No changes in cytosolic free Ca2+ (Quin 2 method) were detected in the presence of insulin. Sodium nitroprusside on the other hand, which is known to mimic several effects of insulin on adipocytes, inhibited IP formation induced by threshold concentrations of thrombin.On the basis of our results the insulin receptors in human platelets seem to be “non-functional” insofar as their occupancy is not accompanied by the stimulation or inhibition of phospho-inositide breakdown or cyclic AMP formation. Similarly, “silent” muscarinic-cholinergic receptors have recently been reported in human erythrocytes (Sehar et al. 1986).