bluuebunny/crossref_metadata_2025_split huggingface dataset:
COMPUTERIZED PLATELET AGGREGOMETRY
Authors: Szabados T , Hermán F , Kollányi G , Hadházy P , Magyar K 
Published: August 2018

One of the commonly used in vitro tests for assessing platelet function is a photodensitometric assay first established by Born. However the collection and analysis of aggregation data are tedious and time consuming. The single- and dual-channel aggregometers have limited utility for the analysis of large numbers of plasma samples in clinical laboratories or in studies on the mode of action of drugs.We now report on a multichannel platelet aggregome-ter system consisting of an IBM XT personal computer and three microprocessor-controlled 4 channel aggregometers. The system collects, displays and analyzes 12 different aggregation curves simultaneously, and - like other computerized systems - (1) significantly increa ses the efficiency and ease in performing the experiment, analyzing and presenting the data; (2) provides systematic storage and rapid retrieval of the data;(3) saves an enormous amount of time; (4) because of multichannel capability eliminates the effects of time-related changes in PRP on the dose-response curves.However our system has some advantages over the computerized aggregometer systems used so far: (1) since the aggregometers contain built in microprocessors they can be utilized to measure and analyse platelet aggregation without being coupled to a computer; (2) a special program helps to check the validity of the calculated parameters under visual control; (3) the individual points of the dose-response curves can be checked at any time during the experiment.
GUARANA (Paullinia cupana) INHIBITS AGGREGATION IN WHOLE BLOOD
Authors: Chamone D A F , Ivany-Silva M , Cassaro C , Bellotti G , Massumoto C , Hoshikawa-Fujimura A Y 
Published: August 2018

Guarana, a methylxanthine obtained from the seeds of Paullinia cupana has been largely used in the Amazon region by native indians during centuries as stimulant. We evaluated the effect of guarana on ex-vivo and in vitro platelet aggregation induced by adenosine-5-diphosphate (ADP) in human and rat whole blood with an impedance (Chrono-Log, model 500) and in their platelet rich plasma (PRP) with an optical aggregometer (Chrono-Log, model 440). Ex-vivo studies were carried out after single oral intake of guarana. Seven healthy volunteers (5 male and 2 female) aged 19-26 years who had taken no drugs for 10 days before, ingested 8gm of crude powder of guarana. Blood samples were drawn before and 1 hour after guarana intake. We observed a significative inhibition of platelet aggregation in whole blood meanwhile PRP was un changed as compared to basal values. In vitro studies were performed in whole blood and PRP from human volunteers and male Wis-tar rats. The combined effect of guarana and adenosine was also studied. A control aggregation was always run with saline. The results demonstrated an inhibition statistically significative (p < 0.001) of platelet aggregation in whole blood. Differently from whole blood the PRP with the same concentration of guarana did not result in inhibition of ADP induced aggregation when eva luated with the impedance method. The blood incubation with adenosine and guarana resulted in synergistic inhibitory effect that was much more strinking in whole blood than in PRP. Guarana fails to inhibit aggregation of rat platelets.Our results demonstrate that guarana prevents platelet aggregation in whole blood which depends on red blood cells, probably involving adenosine.
AGONIST INDUCED CALCIUM MOBILISATION IN PLATELETS OF PATIENTS WITH "ASPIRIN-LIKE" DEFECTS
Authors: Kingston P R , Bruckdorfer K R , Hutton R A 
Published: August 2018

A group of patients have been described with a condition often referred to as an'Aspirin-like" defect, which is characterised by easy bruising and prolonged bleeding following dental extraction or surgery. Initial studies eliminated a deficiency in coagulation factors, plasma factors, platelet glycoproteins or platelet storage granules as being the cause of this condition and demonstrated a diminished aggregatory and/or secretory response in platelet-rich-plasma to ADP and collagen. Aggregation responses in isolated platelets to thrombin, arachidonic acid and ionomycin are within the normal range, however the response to AD? is dimished. Recent studies have concentrated on the various mechanisms involved in platelet aggregation amongst which is the change in intracellular calcium concentration with accompanied secretion.Using the fluorescent indicator quin2 we have monitored the intracellular calcium changes induced by various agonists in the presence and absence of extracellular calcium in five patients with an "Aspirin-like" defect. In the presence of ImM extracellular calcium, thrombin and ionomycin caused a rapid elevation in intracellular calcium to greater than 1μM within 30 seconds of stimulation. In the absence of extracellular calcium, thrombin and ionomycin caused rises in intracellular calcium of 200nM and 300nM respectively. All the responses observed were within the normal range and indicate that both influx of extracellular calcium and mobilisation of calcium from internal stores has occurred. Therefore the defect that causes this "Aspirin-like" condition is not commonly associated with a defect in calcium mobilisation.
ENHANCED PLATELT RELEASE REACTION, INCREASED PLATELET AGGREGATION (PA) AND PLATELET THROMBOXANE B2 (TXB2) GENERATION IN MYOTONIC DYSTROPHY
Authors: Zahavi M , Zahavi J , Bornstein N M , Korczyn A D 
Published: August 2018

Plasma β-thromboglobulin (βTG) level, 14C 5-hydroxytryptamine (5Ht) release, PA induced by 1-epinephrine (0.2-1.0 μM) and ADP (0.5, 1.0 and 1.5 μM), and platelet TXB2 generation and eTG release after stimulation with the 2 PA inducers, was studied in platelet rich plasma of 16 patients with myotonic dystrophy (MYD) and 14 apparently healthy controls. Plasma βTG values were higher (p< 0.011) and 5Ht release was increased (p<0.035) in the patients than controls. The extent of PA induced by 1-epinephrine at 1 and 3 min. or by ADP at 3 min. was significantly raised in the MYD patients. These results indicate that the platelet α2 receptor activity as well as the release reaction is increased in MYD patients. Platelet TXB2 generation and βTG release after stimulation with ADP 1.5 μM or 1-epinephrine 1 μM were significantly more pronounced in the patients than controls (p<0.01). Furtheremore, the increased TXB2 generation correlated positively with the extent of PA induced by ADP (r=0.829 and p<0.05) or 1-epinephrine (r=0.929 and p<0.01) (Spearman one tailed test). There was no such correlation in the healthy controls. These results suggest that the enhanced PA in the MYD patients is presumably mediated through the increased platelet TXB2 synthesis. The enhanced platelet function and Ca++ TXB2 synthesis in MYD patients might be due to an increase in Ca++ uptake related to a generalized membrane abnormality.Further studies of this model inclu�jng assessment of platelet α2 receptor activity and changes in Ca++ ion fluxes through the platelet membrane, may lead to a better understanding of the basic cellular abnormalities in MYD.
EFFECTS OF PLASMA MEMBRANE GLYCOPROTEIN OF PLATELETS ON THE AGGREGATION ACTIVITY OF THE STORED CELLS
Authors: Imura J , Suzuki N , Higashi K , Tubokura M , Shirasawa K 
Published: August 2018

Platelet aggregation activity is gradually reduced depending on the duration of storage. Platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa) complex is intimately related to the aggregation activity. We intended, therefore, to analyse a sequential change in plasma membrane glycoprotein of stored platelets by means of flow cytometry (FCM). On this occasion, aggregation activity of the platelets was also studied. Concentrated human platelets (150×104 cell/μl) were stored in the bag containing citrate-phosphate-dextrose at 22 °C for 24 to 72 hours. The bags were either kept in a flat position without agitation or continuosly stirred by a tumbler agitator (6rpm), or a flat bed rotator (30rpm). At the beginning of each experiment, fresh platelets separated from healthy donor were used as control group. The remaining platelets were washed twice with the 0.38% sodium-citrate dissolved in the lOmM of PBS. After incubation of the suspended platelets in the Tyrode's buffer solution at 37 °C for 30 minutes, they were fixed with 1% paraformaldehyde at 4 C for 2 hours, and were then incubated with anti-human GP IIb/IIIa mouse monoclonal antibody (5μg/ml) at 37 °C for 1 hour. The platelets, thus treated with primary antibody, had undergone further incubation with fluorescein-conjugated goat anti-inouse IgG immunoglobulin at 37°C for 1 hour. After analysis of labelled platelets on the Coulter EPICS V, the positive rate was estimated by counting 5×104 cells. In each group, aggregation activity of platelets induced by ADP (100μM) was measured by an aggregometer.The positive rate was significantly decreased in the stored platelets compared with those in the control group. In addition, the positive rate was more decreased in the non-agitated group than in the agitated group. No difference, however, occurred in the rate from the agitated groups. Moreover, the aggregation activity in each group was well compatible with the positive rate from FCM.It is finally suggested that the decrease in aggregation activity of the stored platelet is due to the reduction in the functioning receptor sides of GPIIb/IIIa on the platelet surface.
BENEFIT OF A CELL PENETRABLE SH-COMPOUND FOR PLATELET PREPARATION AND STORAGE
Authors: Bergmann I , Siepmann T , KLeinert P , Pescarmona G P , Lôsche W , Till U 
Published: August 2018

The SH-containing drug 2-mercaptopropionylglycine (2-MPG) which inhibits platelet aggregation in a reversible manner was used as a cytoprotective agent in some experiments on storage of human platelets for transfusion.In vitro-experimentss Blood of 6 healthy volunteers was collected in ACD (control) and in ACD containing 2-MPG. Platelets were stored as PRP at room temperature. Daily, samples of stored platelets were spun down, resuspended in fresh homologous plasma adjusted to pH 7.4, and rewarmed to 37° C for 1 hour, thus simulating in vitro the in vivo-conditions after platelet transfusion. Among various platelet function tests the morphological index according to KUNICKI et al. (Transfusion 15, 414, 1975) indicated a significantly higher integrity of the platelets prepared and stored in presence of 2-MPG compared to controls.In vivo-experiments: Platelets of 2 healthy volunteers were labelles with 111-In after storage of 2 days and retransfused. When platelets had been prepared and stored in presence of 2-MPG the recovery was increased and the survival prolonged compared to the controls.The results indicate that 2-MPG may have benefit for the preparation and storage of platelets for transfusion.
PRIMARY STRUCTURE OF ANTI-LIPOPOLYSACCHARIDE FACTOR ISOLATED FROM AMERICAN HORSESHOE CRAB
Authors: polyphemus Limulus , Muta T , Miyata T , Tokunaga F , Nakamura T , Iwanaga S 
Published: August 2018

In 1982, a protein component that inhibits the limulus coagulation cascade was found in the hemocyte lysates from Japanese and American horseshoe crabs and named anti-lipopolysaccharide (LPS) factor. This protein specifically inhibited the LPS-mediated activation of limulus factor C and had a strong anti-bacterial effect on the growth of Gram-negative R-type bacteria. Moreover, it had a hemolytic activity on the red blood cells sensitized with LPS.In the present study, the complete amino acid sequence of anti-LPS factor purified from the Limulus (L) polyphemus hemocytes was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, clostripain, and Staphylococcus aureus V8 protease. Upon sequencing the peptides by the automated Edman method, the following primary structure was obtained:During the sequence analysis, two species of the protein, which differed from each other at one locus, were found and characterized. L. polyphemus anti-LPS factor was a basic protein consisting of a single polypeptide chain of 101 residues and a calculated molecular weight of 11,786 or 11,800. The hydrophobic NH2-terminal sequence and positive charges found in the disulfide loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein (J. Aketagawa, et al. (1986) J. Biol. Chem. 261, 7357-7365), and the sequence similar to that observed in the EF-hand structure was found to contain in the COOH-terminal portions of these proteins, although its function is unknown.
LIPOPOLYSACCHARIDE-SENSITIVE SERINE-PROTEASE ZYMOGEN (FACTOR C) OF LIMULUS HEMOCYTES: IDENTIFICATION AND ALIGNMENT OF PROTEOLYTIC FRAGMENTS PRODUCED DURING THE ACTIVATION SHOW THAT IT IS A NOVEL TYPE OF SERINE-PROTEASE
Authors: Tokunaga F , Miyata T , Nakamura T , Morita T , Iwanaga S 
Published: August 2018

Limulus clotting factor, factor C, is a lipopolysaccharide (LPS)-sensitive serine-protease zymogen present in the hemocytes. It is a two-chain glycoprotein (M.W. = 123,000) composed of a heavy chain (M.W. = 80,000) and a light chain (M.W. = 43,000) T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521 .On further studies of this zymogen, a single-chain factor C (M.W. = 123,000) was identified by Western blotting technique. The heavy chain had an NH2-terminal sequence of Ser-Gly-Val-Asp-, which was consistent with the NH2-terminal sequence of the single-chain factor C, indicating that the heavy chain is located in the NH2-terminal part of the zymogen. The light chain had an NH22-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with LPS resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72.amino acids) and B chains derived from the light chain was formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar structuraly to those a family of repeats in human β2 -glycoprotein I, complement factors B, Clr, Cls, H, C4b-binding protein, 02, coagulation factor XIII b subunit, haptoglobin a chain, and interleukin 2 receptor. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence of -ASP-Ala-Cys-Ser-Gly-Asp-SER-Gly-Gly-Pro-.These results indicate that limulus factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine-protease by hydrolysis of a specific Phe-Ile peptide bond. The correlation of limulus factor C and mammalian complement proteins was also suggested.
DOMAIN OF BINDING ACTIVITY WITH PLASMIN KRINGLE IN SYNTHESIZED C-TERMINAL PEPTIDES , OF α2-PLASMIN INHIBITOR
Authors: Iwamoto M , Sugiyama N , Sasaki T , Abiko Y 
Published: August 2018

The inhibitory reaction between plasmin and α2-plasmin inhibitor (α2-PI) proceeds with two steps, a very fast reversible reaction followed by a slower irreversible transition. The first step is dependent on the interaction between lysine binding site (LBS) of plasmin and the corresponding complementary site of α2-PI (kringle binding site(KBS)). It has been reported that KBS is located in a C-terminal tryptic fragment (T-11; J. Biochem. 99, 1699 (1986)).In order to investigate which amino acid residues of T-ll play important roles in binding of plasmin kringle, we tested inhibitory activity of synthesized peptides on the apparent rate constant in the reaction between α2-PI and plasmin. 50% inhibition concentrations of T-ll, peptide I, II, III and IV were 7, 18, 13, 35 and 250pM respectively, indicating that Leu9-Lysl0 is an important part for binding of T-ll to LBS. Peptide III lost its activity by depletion or amidation of the C-terminal lysine residue.In the system consisted of α2-PI and miniplasmin which lacked kringle 1-4, peptide I did not inhibit the interaction between them. Furthermore, peptide II competitively inhibited the binding of tranexamic acid to kringle 1-3 (Ki 0.85μM).These findings suggest that the C-terminal part is involved in the high affinity binding of α2-PI to plasmin kringle and that LyslO in T-ll and C-terminal carboxyl residue play crucial roles in binding to LBS of kringle.
vWF-MULTIMER PATTERNS IN PLATELETS,PLASMA AND MEGAKARYOCYTES FROM RAT VISUALIZED BY IMMUNO-STAINING OF WESTERN-BLOTTED AGAROSE GELS
Authors: Brosstad F , Bjørnsen S , Prydz H 
Published: August 2018

An established rat megakaryoblast line defferentiates when grown at Tow serum concentrations into megakaryocyte-like cells with apparent release of thrombocyte-like cytoplasmic fragments. To study the nature of intracellular vWF,cells grown at high(20%) and low(2%) serum concentrations were harvested, centrifuged and washed three times.They were then resuspended in 2% SDS in 0.15 M Tris-HCl, pH 8.6 at 100°C for 10 min.After centrifugation the supernatants were subjected to SDS-agarose flat bed electrophoresis(1% gel) followed by Western-blotting as were rat plasma and identically treated rat platelet SDS-extracts.The nitrocellulose membranes were then blocked by gelatine and incubated with goat anti-human vWF serum.As secondary antibody a peroxidase-conjugated rabbit anti-goat serum was used with o-dianisidine as chromogene. From the patterns obtained it was evident that:l) goat antihuman vWF antiserum effectively bound to rat vWF,2) vWF-multimer patterns of rat platelets and rat plasma were similar, demonstrating a pattern indisdinguishable from that of human plasma. 3)vWF-pattern of megakaryoblasts differed markedly from the above pattern, demonstrating only the presence of monomeric and protomeric species of vWF.Cells grown at 2% serum concentrations contained markedly more vWF per cell than cells grown at 20%, but the same pattern was seen in both cases.
FUNCTION OF PLATELET 47K PHOSPHOPROTEIN
Authors: Imaoka T , Asaji T 
Published: August 2018

Secretion of platelet granule constituents is closely associated with the phosphorylation of a cytosol polypeptide that we called P47 of Mr 47,000 (Imaoka, T. and Haslam, R.J., J. Biol. Chem. 258, 11404, 1983), by protein kinase C. Since the identity and function remains to be known, we purified protein kinase C, unphosphorylated and phosphorylated P47 to homogeneity from human platelets. Then precise phosphorylation reaction of P47 in vitro and a biological function of P47 were studied. Protein kinase C catalysed the phosphorylation reaction of P47 protein, platelet myosin light chain, histone III-S with Km of 0.8±0.2, 4.2±0.5, 4.7±0.7μM, and Vmax of 0.312, 0.189, 0.874 nmole/min/mg, respectively. Some data previously obtained in this laboratory and others utilizing histone III-S as substrate are consistent with the synergistic effect by diacylglycerol (DG) in the presence of Ca++ , phosphatidylserine (PS). Using P47 as substrate, the enzyme required both Ca++ and PS, but not DG for activity.
125I labelled unphosphorylated P47 had an ability to bind with platelet membrane fraction in the presense of phosphatidylserine. Effect of diacylglycerol was inhibitory in this PS dependent P47 binding with membrane. Unphosphorylated P47 had a inhibitory activity in platelet actin polymerization. Molar ratio to inhibit actin polymerization was 1:8(P47:actin). These activities were Ca++ independent. Purified 32P-labelled P47 lost the binding ability with membrane, also the inhibitory activity in actin polymerization.Therefore, we propose the hypothesis that unphosphorylated P47 may loosely bind with the inside of plasma membrane of platelet and inhibit actin polymerization as a modulator, when stimulated, protein Kinase C rapidly phosphorylate P47 and induce the activation of cytoskeletal network and subsequently release reaction. On the other hand, whether DG in fact can act as a second messenger remain uncertain, (supported by MESC of Japan)
ANALYSIS OF THE VON WILLEBRAND FACTOR (vWF) GENE IN 6 PATIENTS WITH SEVERE TYPE III VON WILLEBRANDS DISEASE
Authors: Standen G , Moodie P , Pannekoek H , Verweij C L , Peake I R 
Published: August 2018

DNA from 6 unrelated patients with severe type III von Willebrands disease (vWF antigen < 0.01u/dl) was studied with a cDNA probe for the 3' end of the vWF gene. DNA was extracted from peripheral blood leucocytes using standard techniques and was digested with a range of restriction enzymes. DNA fragments were separated by electrophoresis in 0.7% agarose and were southern blotted onto hybond-N (Amersham). The probe used was pvWF1100, a 1.1kb PstI fragment derived from the 2.28kb vWFcDNA insert of pvWF2280 isolated from a human endothelial cell cDNA expression library (Verweij et al, Nucleic Acids Res 13 (1985) 4699-4717). The probe corresponds to nucleotides 7083 to 8191 of the vWF cDNA (first nucleotide of initiator methionine as 1).When digested with Bglll and probed with pvWF11000, normal DNA showed two invariant bands (13 and 4.9kb) and polymorphic bands of 9 and/or 7.4kb. This pattern was also seen in 5 of the 6 severe vWD patients DNA suggesting that in this 3' area of the gene they had no major deletions or rearrangements. In the 6th case however the band of 4.9kb was not seen and did not appear to be replaced by any novel fragments, suggesting a partial deletion including some of the 3' end of the gene. This patient had the clinically severest form of the condition in that the patient had developed, some 10 years ago, an antibody (inhibitor) to vWF as detected by the ability of the patients plasma to inhibit restocetin cofactor activity in normal plasma. His parents were related (his mother was his father's second cousin) and had levels of vWFAg, considerably lower than those of factor VIII activity. This situation has been previously reported in carriers of recessive severe vWD. vWD was also present in a second family member, but in a less severe form (vWFAg 3u/dl). This patient and all other members of the family have, to date, given normal restriction fragment patterns with the vWF probe and several enzymes, including BgIII.