bluuebunny/crossref_metadata_2025_split huggingface dataset:
INCORPORATION OF PLASMINOGEN ACTIVATOR INHIBITOR INTO FIBRIN, AN ALTERNATIVE REGULATORY PATHWAY OF FIBRINOLYSIS
Authors: Murayama H , Bang N U 
Published: August 2018

A plasminogen activator inhibitor (PAI-1) Mj, 50 kd is normally found in plasma at low concentrations. Plasma levels increase sharply upon stimulation of endothelial cells with endotoxin or monokines and activated platelets secrete significant quantities of PAI-1. It is possible that high levels of PAI-1 may be achieved at the local sites of intravascular thrombi. Semipurified PAI-1 was therefore prepared from human platelets to study its affinity for fibrin (F). Approximately 50% PAI-1 adsorbed to F monomer immobilized on sepharose and desorbed under conditions of acidic pH and high ionic strength suggesting hydrogen bonding as the mode of interaction. Wells of 96-well microtiter plates were each coated with 50 yg [125I] plasminogen (P)-free fibrinogen and clotted with thrombin in the presence and absence of different concentrations of PAI-1. After extensive washing of the wells, they were incubated with 5 mU of tissue plasminogen activator (t-PA) and 5 mU of P for 6 h. Appropriate calibration curves utilizing different concentrations of t-PA and different concentrations of PAI-1 added to the supernatant rather than to F established that 8-15% of 21-166 mU PAI-1 incorporated into crosslinked (XL) F or noncrosslinked (NXL) F. Incorporated PAI strikingly inhibited fibrinolysis (FL). Percent inhibition of FL of XL or NXLF (Mean±S.D., N=5) plotted in the presence of 166, 83, 42 and 21 mU of PAI were: 83±3.3, 59.5±1.8, 29.7±5.2 and 15.2±6.14 for XLF and 78±5.3, 31±8.1, 14.5±10.5 and 0 Tor NXL F. As demonstrated by radioautography on SDS PAGE PAI-1 incorporated into F readily formed complexes with [125I] urokinase (u-PA). In these experiments, no evidence for crosslinking of PAI-1 into F has been obtained to date. In experiments utilizing agarose immobilized proteins, it was evident that not only F but also fibrinogen binds PAI-1; PAI-1 associated with F as well as fibrinogen is capable of forming complexes with [1251] u-PA.In contrast, fibronectin, collagen, gelatin and albumin did not bind PAI-1. Thus, PAI-1 in analogy with alpha-2 plasmin inhibitor may modulate physiological fibrinolysis through incorporation into fibrin.
STUDIES ON THE EFFECT OF CILOSTAZOL ON THE PROSTAGLANDINS PRODUCTION DURING ADP-INDUCED PLATELET AGGREGATION WITH ENDOTHELIAL CELLS
Authors: Chijiwa T , Shiragiku T , Kato S , Igawa T , Kimura Y 
Published: August 2018

It has been clinically reported that cilostazol has a potent inhibitory effect on platelet aggregation without changing the prostacyclin level. This study was undertaken to elucidate this clinical effect by a technique developed by us in which platelet aggregation could be evaluated in the presence of cultured endothelial cells. Human umbililical cord vein endothelial cells (HUVEC) were coated (cultured for a few days supplemented with 10% fetal claf serum) on the inner surface of glass cuvette, and platelet aggregation was traced by the stimulation of citrated-PRP with 7.5 μM ADP in this cuvette. The 6-keto-prostaglandin F1α (6-k-PGF) and thromboxane B2 (TXB) produced in the supernatant of the stimulated PRP were measured by radioimmunoassay (Amersham). The cyclic adenosine monophosphate (cAMP) level in platelets and HUVEC was measured by radioimmunoassay (YAMASA). Cilostazol showed a potent inhibitory effect on platelet aggregation in the presence of HUVEC by the suppresion of TXB production, but not by the suppression of 6-k-PGF production. Cilostazol stimulated cAMP production in both platelets and HUVEC. On the other hand, aspirin also showed an inhibitory effect on platelet aggregation in the presence of HUVEC, but suppressed production of both TXB and 6-k-PGF.As a result, the clinical effect of cilostazol was confirmed by the fact that TXA2 production in a platelet/HUVEC coexisting system was specifically suppressed.
REGULATION OF PLATELET cAMP FORMATION BY PROTEIN KINASE C
Authors: Hopple Sara , Bushfield Mark , Murdoch Fiona , MacIntyre D Euan 
Published: August 2018

Exogenous synthetic 1,2-diacylglycerols (e.g. 1,2-dioctanoylglycerol, DiC8) and 4β Phorbol esters (e.g. phorbol myristate acetate, PMA) routinely are used to probe the effects of protein Kinase C (PKC) on cellular responsiveness. Such agents act either independently or synergistically with elevated [Ca2+]i to induce platelet activation, but also inhibit agonist-induced inositol lipid metabolism and Ca2+ flux. These findings led to the concept that activated PKC can function as a bi-directional regulator of platelet reactivity. Therefore, DiCg and PMA were utilized to examine the effects of activated PKC on receptor-mediated stimulation and inhibition of adenylate cyclase, as monitored by cAMP accumulation. All studies were performed using intact human platelets in a modified Tyrodes solution, and cAMP was quantified by radioimmunoassay. Pretreatment (2 min.; 37°C) of platelets with PMA (≤ 300 nM) but not DiCg (200 μM) attenuated the elevation of platelet cAMP content evoked by PGD2 300 nM) but not by PGE1 (≤300 nM), PGI2 (≤100 nM) or adenosine (≤ 100 μM).These effects of PMA were unaffected by ADP scavengers, by Flurbiprofen (10 μM) or by cAMP phosphodiesterase inhibitors (IBMX, 1 mM) but were abolished by the PKC inhibitor Staurosporine (STP, 100 nM). In contrast, DiC8 (200 μM), but not PMA ( ≤ 300 nM), reduced the inhibitory effect of adrenaline (5 μM) on PGE1 (300 nM)-induced cAMP formation. This effect of DiCg was unaltered by STP (100 nM). Selective inhibition of PGD2-induced cAMP formation by PMA most probably can be attributed to PKC catalysed phosphorylation of the DP receptor. Reduction of the inhibitory effect of adrenaline by DiC8 could occur via an action at the α2 adrenoreceptor or Ni. These differential effects of PMA and DiC8 may result from differences in their distribution or efficacy, or to heterogeneity of platelet PKC.
LACK OF CORRELATION BETWEEN INTRAPLATELET cAMP INCREASE AND SYSTEMIC CIRCULATORY EFFECTS
Authors: Haarmann W , Weisenberger H 
Published: August 2018

Compounds inhibiting platelet function by acting on platelet cAMP metabolism usually also have effects on the circulatory system, i.e. they decrease systemic blood pressure (bp) and are positive inotropic. For several compounds selected because of their distinct platelet inhibitory effects, the influence on these parameters in animals and on the cAMP metabolism in human platelets was determined.Inotropic effects and bp were measured via an indwelling catheter in anestetised cats after i.v. application of the test compounds. The inhibition of platelet PDEs was measured in freeze-thaw homogenates of human platelets using 3H-cAMP as substrate. Intraplatelet cAMP changes were measured by prelabelling the ATP pool with 3H-adenine and isolation of 3H-cAMP. Linear regression analysis of the drug concentrations causing a doubling of intraplatelet cAMP levis and the % difference in bp or the % difference in dp/dt, resp., by i.v. application of 0.3 mg/kg test compound yielded the following results:cAMP vs % diff. bp : r=0.02, N=18cAMP vs % diff. dp/dt: r = 0.02 , N = 15In contrast to a good correlation between intraplatelet cAMP levels and inhibition of platelet function tests, no obvious relationship was seen between cAMP and decrease in bp and positive initropic effects. It is not known whether the lack of correlation could be due to a different drug access to platelets and the bp regulatory system.A biochemical parameter, i.e. intraplatelet cAMP increase by inhibition of PDEs correlates reasonably well with the inhibition of platelet function tests. This parameter is not useful, however, to predict the effects on the heart and the circulatory system.
COMPARISON BETWEEN IN VITRO AND IN VIVO AGGREGATION OF PLATELETS
Authors: TAPPARELLI C , GFELLER P , SANJAR S , MORLEY J 
Published: August 2018

The extensive utilisation of in vitro tests of platelet aggregation presumes that corresponding effects occur in vivo. Platelet aggregometry in vitro and in vivo have been compared using a range of agonists in order to test this assumption.PRP from citrated peripheral blood of man, rat and guinea-pig was exposed to ADP, adrenaline, serotonin, collagen, thrombin and PAF in a Born aggregometer to define the time course and amplitude of these responses. In rat and guinea-pig, these aggregatory stimuli have also been used to define the time course and amplitude of intrathoracic accumulation of 111 -Indium labelled platelets, using an automated monitoring system (AIMS 8000).Concordance between these tests was evident for ADP, collagen, thrombin and PAF in both species; but, substantial discrepancy was observed between in vitro and in vivo responses to serotonin and adrenaline, since sustained aggregation followed injection of these agonists in the rat.For these platelet stimuli, in vivo aggregometry in rat and guinea-pig may more faithfully reflect the behaviour of human platelets than in vitro studies.
PLATELET AGGREGATION DOES NOT CONFORM TO SIMPLE PARTICLE COLLISION THEORY
Authors: Jamaluddin Moideen P 
Published: August 2018

Platelet aggregation kinetics, according to the particle collision theory, generally assumed to apply, ought to conform to a second order type of rate law. But published data on the time-course of ADP-induced single platelet recruitment into aggregates were found not to do so and to lead to abnormal second order rate constants much larger than even their theoretical upper bounds. The data were, instead, found to fit a first order type of rate law rather well with rate constants in the range of 0.04 - 0.27 s-1. These results were confirmed in our laboratory employing gelfiltered calf platelets. Thus a mechanism much more complex than hithertofore recognized, is operative. The following kinetic scheme was formulated on the basis of information gleaned from the literature.where P is the nonaggregable, discoid platelet, A the agonist, P* an aggregable platelet form with membranous protrusions, and P** another aggregable platelet form with pseudopods. Taking into account the relative magnitudes of the k*s and assuming aggregation to be driven by hydrophobic interaction between complementary surfaces of P* and P** species, a rate equation was derived for aggregation. The kinetic scheme and the rate equation could account for the apparent first order rate law and other empirical observations in the literature.
VARIATION OF FUNCTIONAL PLATELET PARAMETERS DURING STORAGE AND DURING VENOUS OCCLUSSION MEASURED
Authors: Spannagl M , Valet G , Schramm W 
Published: August 2018

Information on platelet function would be of great importance for many clinical situations in addition to platelet count and bleeding time. It was the purpose of this study to test Di0c6 (3,3-dihexyl-oxacarbocyanine: transmembrane potential), AO (acridine orange: granular content), and ADB (1,4-di-acetoxy-2,3-dicyanobenzene: intracellular esterase-activity and pH) stained platelets after 1 and 5 hours storage (anticoagulated with EDTA, Heparin and Sodium-Citrate) and 2, 6, 12 and 20 minutes after venous occlussion (immediately diluted in HEPES-Buffer (1:50)). A fresh whole blood sample diluted in Buffer served as control. All blood samples were gained from normal persons. Platelets were finally diluted 1:200 in HEPES buffered saline and stained. Cell volume, green and blue fluorescence were then measured in a Fluovo-Metricell-II flow cytometer.- The mean platelet volume increased to 111% (1 h) and 117% (5 h) of control during storage. The volume remained stable during venous occlussion.
- The transmembrane potential (Di0c6) decreased to 52% of control after 5 hours storage. We saw an increase to 141% after 20 minutes venous occlussion.- The granular content (A0) decreased to 81% of control during storage. There was no variation during VOT.- Esterase activity (ADB) remained constant during storage and had the lowest coefficient of variation (CV = 51%). There was an increase to 132% after 20 minutes venous occlussion.- We saw most increase in volume and decrease in Di0c6 and A0 dye content after storage in EDTA compared to Citrate and Heparin.The present results show that the dyes of functional platelet parameters are sensitively picked up by flow cytometry. The methodology seems attractive for clinical purposes because measurements can be performed in diluted blood samples within less than five minutes after venipuncture.
PLATELET IONIZED CALCIUM MOBILIZATION (AEQUORIN METHOD) IN PATIENTS WITH PRIMARY PLATELET DYSFUNCTION
Authors: Nagayama R , Hattori A , Fuse I , Takeshige T , Takizawa S , Shibata A 
Published: August 2018

Intracellular calcium level of platelets of the patients with primary platelet dysfunction (thrombasthenia, platelet cyclo-oxygenase deficiency, familial defect of A23187 induced platelet aggregation, Hermansky-Pudlak syndrome. Bernard-Soulier syndrome, each 1 case, and other 3 cases of platelet release mechanism defect of unknown etiology) were measured with the photoprotein Aequorin according to the method by Johnson et al. The peak level and the lag time to the peak were evaluated. Activation was done by 4 or more different concentrations of either thrombin (0.125-1.0μ/ml), A23187 (0.25-2.0μM), ATA2 (0.05-0.4μM) or occasionally arachidonate (0.25-100μM).In case of stimulation by thrombin, the maximum [Ca2+] level in thrombasthenia was much lower than those in normal. The lag time was prolonged in Bernard-Soulier syndrome. In case of stimulation by STA2 the maximum [Ca2+] level was very much lower in thrombasthenia and was lower in a familial defect of A23187-induced platelet aggregation and a case of platelet release mechanism defect than normals. In case of stimulation by A23187, the maximum [Ca2+] level was much lower in thrombasthenia and PCO deficiency and platelet release mechanism defect (2 cases) in which the lag time of A23187 induced platelet aggregation was also prolonged. In PCO deficiency, arachodonate less than μM produced a dose-dependent rise in intracellular calcium level and that (1-25μM) caused a rise of a consistent level although it didn't induce aggregation. Arachidonate (25-100M) caused both higher rise and aggregation. Mobilization was normal in response to STA2 and thrombin but decreased to A23187 in this deficiency.These findings suggest that [Ca2+] mobilization was deteriorated by many mechanisms such as defect of PGG2H2-and Tx-forma-tion, membranous abnormality (lack of glycoproteins) and storage pool deficiency, and further that atachidonate even at low concentration may cause mobilization without conversion to PGG2H2 or Tx.
STORAGE OF LEUKCCYTE POOR PLATELET CONCENTRATES PREPARED FROM BUFFYCOATS IN VARIOUS TYPES OF PLASTIC BAG
Authors: Pietersz R N I , Reesink H W , Dekker W J A 
Published: August 2018

Platelet concentrates (PC) prepared from buffycoats in a quadruple bag CPD-SAG M system proved to have a good survival in vivo after storage for 72 h at 22°C (Vox Sang 1985, 49: 81-85). Storage of these PC for 7 days at 22°C on a linear platelet reciprocator, was studied using 6 different types of bag made of polyvinylchloride (PVC) with either DEHP and/or TOTM as plasti-cizer. From Biotest the 76 standard PVC (n=14) and 763 thin PVC (n=16)bag, from NPBI the PSV 3277 standard PVC (n=15) and DPL-110 TOTM (n=14) and from Terumo the old (n=18) and new (n=14) molded Teruflex bags were examined.The Biotest 763, the NPBI DPL-110 and the Terumo new molded Teruflex are recommended for platelet storage.The plasma volumes averaged 63 ml with a range of 39 to 81 ml. Platelet concentrations were comparable in all types of bag with an overall mean of 0.89±0.33 (SD)×109 /ml. The number of contaminating leukocytes and erythrocytes never exceeded 107 per unit. The initial pH was between 6.98 and 7.19. After 7 days storage the pH in all bags remained above the critical value of 6.0 with a range of 6.56 to 7.38. No decrease of PO2 was observed during storage, on the contrary a significant (p<0.05) increase in the PSV 3277 and new molded Teruflex bags was measured.The PCO2 diminished equally in all bags also indicating adequate gas exchange. Good morphology scores of the platelets (greater than 200) were observed in 98% of the PC after 5 day storage and in 83% after 7 days.These data indicate that PC can be equally well be stored in normal PVC bags as in special platelet storage bags, provided that the PC are leukocyte poor.
MEGAKARYOCYTIC DEVELOPMENT IN LIQUID CULTURES OF CRYOPRESERVED LEUKOCYTE STEM CELL CONCENTRATES FROM CHRONIC MYELOGENOUS LEUKEMIA PATIENTS
Authors: Berthier R , Duperray A , Valiron O , Prenant M , Newton I , Schweitzer A 
Published: August 2018

The proliferation and differentiation of human megakaryocytes in liquid culture has been obtained using cryopreserved light density blood cell concentrates from chronic myelogenous leukemia (CML) patients. These cryopreserved leukocytes concentrates contain a large number of viable granulo-monocytic, erythroid and megakaryocytic committed stem cells. A high number of spontaneous megakaryocytic colonies was observed in semisolid cultures plated with the CML leukocytes concentrates. A liquid culture system using RPMI 1640 supplemented with 20% human plasma (HP) has been defined where maturing megakaryocytes make up 20 to 60% of the total cells after 14 days of incubation. The same cell suspension cultured in medium supplemented with 20% foetal calf serum (FCS) showed poor megakaryocytic cell development. The megakaryocytic nature of the cells produced in HP supplemented cultures was confirmed by cytological studies and indirect immunofluorescence labeling using monoclonal antibodies (MoAb) against membrane platelet GPIb and Ilbllla, and intracellular antigens like fibrinogen and von Willebrand factor.Ploidy of the cultured cells was studied after labeling with propidium iodide and the DNA fluorescence determined using the fluorescence activated cell sorter (FACSIV). Peaks of 8N, 16N and 32N cells were observed from HP supplemented cultures representing about 20% of the cells reacting with a GP11b111 a MoAb, while very few cells greater than 4N were observed in FCS supplemented cultures. The megakaryocytes produced in HP cultures could be further enriched by cell sorting on the FACSIV after labeling with an anti-IIbIIIa MoAb. Depending on the initial megakaryocytic concentration of the cells cultured, one to 2 é 106 megakaryocytes per hour could be harvested. Thus, cryopreserved CML blood stem cell concentrates seem to offer a reproducible source of human megakaryocytes which retain their capacity to proliferate and differentiate in liquid cultures supplemented with human plasma. These megakaryocytes can be used for the study of platelet glycoprotein biosynthesis as well as the regulation of megakaryocytopoiesis.
EVALUATION OF THE PROPOSED FUNCTIONS OF PROTEIN KINASE C IN PLATELET SIGNAL TRANSDUCTION BY THE USE OF A DIACYLGLYCEROL KINASE INHIBITOR
Authors: de Chaffov de Courcelles D , De Clerck F , Roevens P 
Published: August 2018

Protein kinase C is suggested to play a major role in propagation as well as in termination of excitatory signal transduction in the platelet. Most of its properties were discovered by the use of synthetic diacylglycerol analogs or phorbol esters that directly stimulate protein kinase C. It is, however, unknown to what extent activation of the protein kinase C by these exogenously added compounds can be compared to that after receptor activation. To evaluate the role of protein kinase C in excitatory signal transduction, we transiently elevated the endogenous diacylglycerol level after receptor activation by the use of a diacylglycerol kinase inhibitor (R 59 949). On addition of the agonist vasopressin to platelets prelabeled with [32P] orthophosphate, 32P-phosphatidic acid (PA) formation was inhibited by R 59 949 in a dose-dependent manner (IC50 α 10−6 M). Vasopressin induced formation of 32P-phosphatidylinositol-4’-phosphate (PIP) and the phosphorylation of the 40 k Da protein (major substrate of the protein kinase C) were increased in the presence of the compound. In platelets prelabeled with [3H]-inositol, the agonist-induced formation of all the water-soluble inositol phosphates was inhibited in the presence of the diacylglycerol kinase inhibitor and Li+. Vasopressin induced increase in intracellular Ca2+ was lower in the presence of R 59 949. The platelet shape change induced by a threshold concentration of vasopressin was reduced by the compound. By contrast, the rate and the maximum of the first-wave aggregation was enhanced in the presence of R 59 949.These data evidence that protein kinase C, stimulated by endogenously generated diacylglycerol after receptor activation, plays a major inhibitory role on the primary steps of signal transduction since its activation reduces i) phospholipase C activity and ii) the increase in intracellular Ca2+ and the concomitant shape change reaction. The inhibitory role of protein kinase C on signal transduction is largely independent of its stimulatory role on platelet aggregation. Our data further confirm that stimulation of protein kinase C induces the formation of PIP but questions the role of the kinase in the breakdown process of inositoltrisphosphate.
A MONOCLONAL ANTIBODY (PL/IM 430) THAT BLOCKS THE ACTIVE TRANSL0CATI0N OF Ca2+ INTO HUMAN PLATELET INTRACELLULAR MEMBRANE (ER) VESICLES
Authors: Hack N , Wilkinson J M , Crawford N 
Published: August 2018

In earlier studies [1] we identified a number of important biological properties associated with highly purified human platelet intracellular membrane (ER), isolated by continuous flow electrophoresis. These included a high affinity Ca2+Mg2+ ATPase and protein phosghorylation both of which are involved inthe active uptake of Ca into ER vesicles. The stored Ca2+ could be released with inositol(1,4,5)trisphosphate, (IP ), (approx. 50% release in 30 s 1/2 max. for release - 0.253 μM IP3,) [2]. To probe the structure-function relationship of proteins in these ER vesicles, a panel of monoclonal antibodies (Mabs) has been raised, using the ER membrane preparation as immunogen. Four of these Mabs recognise a single 100 kDa polypeptide by immunoblotting. This protein is present in platelet membranes and can also be identified in cultured human monocyte, macrophage and endothelial cell lines. None of the M^bs showed any significant effect upon the ER membrane Ca2+ Mg2+ ATPase activity but one, PL/IM 430 (of IgGl subclass), inhibited the Ca2+sequestration by the vesicles significantly (approx. 70% inhibition at 10 μM IgG). This inhibition was independent of the ATP concentration over a range2of 0-2 mM ATP, but was2dose-dependent for external free Ca 2between 30-300 nM Ca2+, giving maximum inhibition at 300 nM Ca with 10 pM IgG2+ Binding of the antibody substantially lowers the Vmax for Ca2+for Ca2+ uptake but is without effect upon the Km. PL/IM 430 therefore appears to recognise a 100 kDa polypeptide closely involved with Ca2+ trnslocation but at a site which i, s without effect upon the Ca2+Mg− ATPase associated with the Ca pump.We are grateful to the Wellcome Trust and the British Heart Foundation for financial support for these studies.[1] Hack, N., Croset, M. and Crawford, N. (1986) Biochem. J. 233, 661-668.[2] Authi, K. S. and Crawford, N. (1985) Biochem. J. Z3O, 247-253