bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
MEGAKARYOCYTES FROM THE MARROW OF A PATIENT WITH BERNARD-SOULIER SYNDROME LACKED GP Ib AND WERE DEFICIENT IN GP IX
Authors: Hourdillé P , Belloc F , Heilmann E , Pico M , Nurden A T
Published: August 2018

Although it is recognized that glycoprotein (GP) Ib/IX complexes are deficient in the platelets of patients with the Bernard-Soulier syndrome (BSS), the nature of the genetic defect remains unknown. We have looked for these GPs in permeabilized megakaryocytes (MK) of a BSS patient employing immunofluorescence (IF) or immunocytochemical procedures combined with electron microscopy. The study involved the use of monoclonal antibodies AP-1 (anti-GP Ib), AP-2 (anti-GP IIb-IIIa) and FMC 25 (anti-GP IX), gifts from Drs. T. Kunicki and M. Berndt respectively. Bound IgG were revealed by biotinylated anti-mouse IgG followed by Texas Red-streptavidin and MK identified in IF by a double-staining procedure using a polyclonal antibody to fibrinogen (Fg). Platelet morphology was typical of BSS with a high percentage of "giant" platelets. Flow cytometry confirmed that platelets of all sizes were negative for AP-1 and FMC 25 but normally bound AP-2. MK from a marrow aspirate obtained by sternal puncture were concentrated on a Percoll gradient. Electron microscopy showed the MK to be of normal size with a normal granule distribution. However, a striking feature was an irregular distribution of the demarcation membranes which often had a vacuolar appearance. Whereas all permeabilized MK from normal individuals were strongly fluorescent with AP-2, AP-1 and FMC 25, those from the BSS patient were negative for AP-1, weakly positive for FMC 25 and normal for AP-2. Incubation of intact cells with AP-1, with bound antibody located by anti-IgG bound to gold particles, confirmed the absence of GP lb from the surface membranes of BSS MK. Our results show that the platelet membrane GP defect in BSS results from an abnormal synthesis and/or stability of both GP lb and GP IX in the MK.
BLOOD PLATELET FUNCTIONS: INFLUENCE OF AGING
Authors: Véricel E , Croset M , Courpron Ph , Dechavanne M , Lagarde M
Published: August 2018

Aging has been associated with a high incidence of vascular diseases and it is suggested that platelet activation could contribute in the development of these diseases. The purpose of this study was to compare platelet functions from young adults (< 35 years) and elderly people (> 70 years). Aggregation of platelet rich plasma induced by arachidonic acid or epinephrine was significantly increased in the elderly. Similarly, an increase of platelet aggregation (platelets isolated from their plasma) induced by various agents (thrombin, U46619, arachidonic acid ...) was also noted. The same tendency was observed in whole blood aggregation. Platelet endogenous arachidonic acid metabolism under stimulation was evaluated. Production of thromboxane B2, measured by GLC, was significantly higher in the elderly people (510 ± 207 vs 242 ± 83 ng/10*platelets, p<0.02). On the other hand, platelet vitamin E, quantified by HPLC, was significantly decreased in elderly people (0.92 ± 0.21 vs 1.41 ± 0.55 nmoles/109 platelets, p<0.05). To further assess platelet and vascular function in vivo, we measured excretion of thromboxane B2 (TXB2), 2,3-dinor-TXB2 (M-TXB2), 6 keto-PGFlcr and 2,3-dinor-6 keto-PGFlα (M-6-k-PGFl±) in urine. These four metabolites were nearly all significantly increased in the older population (TXB2: 24.3 ± 2k.6 vs 3.1 ± 1.2 p<0.05; M-TXB2: 51.5 ± 43.2 vs 25.1 ± 14.5 NS; 6-k-PGFlα: 37.5 ± 29.3 vs 19.1 ± 4.2 p<0.05; and M-6- k-PGFlα: 193.6 ± 118.6 vs 116.4 ± 42.4 ng/mmole creatinine p<0.05). We conclude that changes in platelet functions reveal an enhanced platelet activity which may reflect a prethrombotic state in elderly people. The mechanisms of these modifications remain to be determined but the increased of specific peroxidation observed might be linked to the decrease of platelet vitamin E.
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.
REACTIONS OF DRUG-DEPENDENT ANTIBODIES WITH METABOLITES OF QUININE (Qn) AND QUINIDINE (Qd)
Authors: Christie D J , Diaz-Arauzo H , Cook J M
Published: August 2018

In many cases of drug-induced immunologic thrombocytopenia (DITP), a metabolite, rather than the native drug, is suspected of provoking the destructive drug-dependent antibodies (DDAB) responsible for this severe hemorrhagic disorder. However, this has not previously been investigated for Qn- and Qd-DDAB. We report evidence that the native drugs, and not their metabolites, are the provocative agents in Qn and Qd DITP. Reactions of Qn- and Qd-DDAB with platelets were studied with the native drugs and four of their metabolites: the N-oxide and 10,11-diol derivatives (quinuclidine ring modifications), the des-methyl derivatives (aromatic quinoline ring modification), and 2'-quininone and 2'-quinidinone (2'-oxo derivatives) (also quinoline ring modifications on Qn and Qd, respectively). Five antibodies were studied:two Group 1 DDAB (specific for compounds with native configuration at asymmetric carbon positions), two Group 2 DDAB (similar to Group 1 DDAB but also known to require the methoxy group on the quinuclidine ring for full activity), and one Group 3 DDAB (reactive with the native drug, its stereoisomer, and several nonmetabolic analogs of both compounds) . Using a complement-dependent 51Cr-lysis assay, the reactions of all DDAB with platelets and the four metabolites were similar to 100-fold weaker when compared to reactions obtained with the native drug, with these exceptions:Group 2 DDAB failed to react with the desmethyl and 2'-oxo metabolites and the Group 3 DDAB failed to react with 2'-oxo Qd. This observation shows that the activity of certain DDAB is critically dependent on the native quinoline ring structure. Importantly, none of the DDAB reacted more strongly with any of the metabolites tested when compared with reactions in the presence of the native drug. These findings indicate that DDAB react with platelets preferentially in the presence of the unaltered Qn and Qd molecules and suggest that, while the role of metabolites cannot be entirely ruled out, the native structure of the drug molecule is sufficient to stimulate production of the antibodies responsible for DITP.
STIMULATED MEGAKARYOCYTES ARE FOUND IN CHILDHOOD ITP BUT NOT IN ADULT ITP
Authors: Levine R F , Shoff P K
Published: August 2018

ITP is thought to be caused primarily by peripheral platelet destruction, but recent work has suggested that platelet production may also be impaired. Although the clinical course in children usually differs from that in adults, no distinctions have been established with regard to marrow characteristics. To evaluate megakaryocyte (mega) responses in this disease we examined mega size, ploidy, maturation and morphology in 8 children and in 8 adults with ITP and in 8 "normal" marrows (4 children, 4 adults). Marrows were prepared by a buffy coat wedge smear or by a cover slip squash method. Control values differed according to the type of marrow smear used. From 100-300 Feulgen-stained megas were examined in each specimen, as previously described. Wright-stained material was also examined. With the squash method megas from normal children and adults had similar characteristics. The megas of each child with acute ITP showed marked increases in size (volumes were 4X normal), ploidy (as high as 1024 N; medians were 64N, or 2 doublings higher than normals), and maturation stage (86% mature forms vs 43%). In contrast, none of the marrows of adults with acute or chronic ITP (1 with mild, 4 moderate, and 3 with severe thrombocytopenias) showed any stimulation of megas. Overall, their megas were normal in size, ploidy and maturation. Occasional dissociation of mega ploidy and maturation was seen, but not enough to alter the profiles of any one parameter. There were no obvious or suggestive signs of "damage" to the megas of children or adults with ITP. In conclusion, the megas of childhood ITP showed a pattern of marked stimulation of size, ploidy and maturation, as seen in animals injected with antiplatelet serum. The failure of adult marrows to response in these parameters to thrombocytopenia may be pathogenetically related to the chronicity of adult ITP.
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.
PLATELET STORAGE AT 22°C: A BIOCHEMICAL EVALUATION
Authors: Harmening Pittiglio D , Glatzel J W
Published: August 2018

Blood platelets are an important component of transfusion therapy, especially for the thrombocytopenic patient. As a result, platelet storage has become an important issue. This study evaluated biochemical parameters of platelet concentrates (PC) that were prepared from whole blood drawn in CPDA-1. Seventeen PC's were stored for 7 days at 22°C in polyolefin containers (PL 732) with horizontal flatbed agitation. Samples were taken on days 1,3,5, and 7 of storage and analyzed. This study demonstrated a change from aerobic to anaerobic metabolism during the 7 days of platelet storage. PC's which utilized oxygen during storage were associated with a higher pCO2 and bicarbonate values, lower lactic acid levels and better maintenance of pH indicating active aerobic metabolism. During the 7 days of PC storage, a block in oxidative phosphosylation occurred which was reflected in increases in the pO2as well as increases in the lactic acid production, resulting in a depletion of available bicarbonate and a fall in pH. This study indicates that the fall in pH was not associated with increased pCO2values . A 29% decrease in ATP levels associated with an increase in the level of organic phosphate and plasma calcium was observed during storage. The study confirmed that changes in morphology are associated with a decrease in pH and increases in LDH levels during room temperature storage. This study demonstrated that several interrelated variables affect the pH of PC storage, and that oxidative phosphorylation is crucial to platelet metabolism and important in preventing adverse changes in pH.
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.
EVIDENCE FOR INVOLVEMENT OF GTP-BINDING PROTEIN IN ARACKIDONIC ACID RELEASE BY PHOSPHOLIPASE A2 IN PERMEABILIZED HUMAN PLATELETS
Authors: Nakashima S , Tohmatsu T , Hattori H , Suganuma A , Nozawa Y
Published: August 2018

Platelet activation is accompanied by the active metabolism of membrane phospholipids. Phosphoinositide breakdown by phospholipase C generates second messengers; inositol trisphosphate and diacylglycerol. Recently, it is suggested that GTP-binding protein is linked to the activation of phospholipase C as is true for adenylate cyclase. Although it is known that the receptor stimulation by agonists leads to generation of arachidonic acid, its molecular mechanism has not yet been clear. However, several studies in neutrophils and mast cells using pertussis toxin, have shown the possibility that a GTP-binding protein may act as an intermediary unit component between the receptor and phospholipase A2. The present study was therefore designed to examine the effect of GTP and its analogue GTPγS on the arachidonic acid release in saponin-permeabilized human platelets. GTP or GTPγS alone caused a small but significant liberation of arachidonic acid in permeabilized cells but not in intact cells. GTP or GTPγS was found to enhance thrombin-induced [3H]arachidonic acid release in saponi n-permeabi li zed human platelets. The release of arachidonic acid has been ascribed to activity of phospholipase A2 and/or to sequential action of phospholipase C and diacylglycerol lipase. Inhibitors of phospholipase C (neomycin)/ diacylglycerol lipase (RHC 80267) pathway of arachidonate liberation did not reduce the level of the [3H]arachidonic acid release. The loss of [3H]arachidonate radioactivity from phosphatidylcholine was almost complementary to the increment of released [3H]arachidonic acid, suggesting thrombin-induced hydrolysis of phosphatidylcholine by phospholipase A2. Although phospholipase A2 usually are described as having a requirement for calcium, the effect of GTPγS was more evident at lower calcium concentrations (buffer>0.1 mM>1.0 mM). These data thus indicate that release of arachidonic acid by phospholipase A2 in saponin-treated platelets is closely linked to GTP-binding protein which may decrease the calcium requirement for phospholipase A2 activation.
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.
THE PLATELET AGGREGATING PROPERTIES OF TYPE IIB VON WILLEBRAND FACTOR (vWF): THE ROLE OF PLATELET ACTIVATION, FIBRINOGEN AND TWO DISTINCT MEMBRANE RECEPTORS
Authors: De Marco L , Mazzucato M , Del Ben M G , Budde U , Federici A B , Girolami A , Ruggeri Z M
Published: August 2018

Three preparations of purified von Willebrand factor (vWF), obtained from unrelated patients affected by type IIB von Willebrand disease, were found to have normal sialic acid content (between 129-190 nmoles/mg of vWF, as compared to 158 ± 17 nmoles/mg in four normal preparations) and to induce platelet aggregation in the presence of physiologic levels of divalent cations and without addition of ristocetin. A monoclonal antibody that blocks the vWF binding domain of the platelet glycoprotein (GP) Ib caused complete inhibition of IIB vWF-induced aggregation. On the contrary, a monoclonal antibody that blocks the receptor for adhesive proteins on the platelet GPIIb/IIIa complex failed to inhibit the initial response of platelets to high concentration of IIB vWF Moreover, IIB vWF caused agglutination of formalin-fixed platelets that was blocked only by the anti-GPIb antibody, suggesting that the binding of vWF to GPIb, even in the absence of ristocetin, results in platelet-platelet interaction that is followed by exposure of the GPIIb/IIIa receptors for adhesive proteins. Endogenous ADP, normally active platelet metabolism and fibrinogen binding to GPIIb/IIIa were necessary for maximal and irreversible platelet aggregation. In the absence of fibrinogen, however, aggregation was mediated by vWF binding to GPIIb/IIIa. A 52/48 kDa tryptic fragment containing the GPIb binding domain of normal vWF completely blocked the aggregation induced by all three IIB vWF preparations. The present study defines in detail the mechanisms involved in IIB vWF-induced platelet aggregation. Moreover, it establishes that the GPIb binding domain of normal and IIB vWF are closely related and that desialylation is not required for the direct interaction of IIB vWF with GPIb.
ABNORMALITY OF THE N-TERMINAL PORTION OF VON WILLEBRAND FACTOR (FRAGMENT Sp III ) IN TYPE IIA AND IIC VON WILLEBRAND DISEASE
Authors: Takahashi Y , Kalafatis M , Girma J P , Meyer D
Published: August 2018

A new analytical method was established, allowing the characterization of von Willebrand Factor (vWF) degradation fragments from minute amounts of plasma without the need for immunopurification of vWF. Ten ul of citrated plasma in 20 ul of 0.1 M Tris-HCl, pH 7.8, 0.1 M NaCl, 10 mM EDTA buffer were treated with S. aureus V-8 protease for 15 min to 3 h at 22°C. Following addition of 1 mM DFP, the cleaved fragments were separated by SDS-polyacrylamide or SDS-agarose gel electrophoresis and identified by 125 I-labeled polyclonal or monoclonal antibodies (MAbs) to vWF andautoradiography. Quantification of each fragmentwas estimated by counting radioactivity in slices of the dry gel. In normal plasma, S. aureus V-8 protease produced two dimeric fragments of vWF, with identical electrophoretic and antigeniccharacteristics to those produced from highly purified vWF, ie a C-terminal fragment of 220 kd (Spll) and a series of N-terminal fragments (Spill) with a major species of Mr 320 kd (SpIIIa) and two minor ones of 265 kd (SpIIIb) and 215 kd (SpIIIc). The method was applied to further characterize the molecular abnormalities of vWF in 15 patient plasmas with variant (type II) von Willebrand disease (vWD). In all cases with type IIA, IIB, IIC and IID tested, fragment Spll had the same mobility as in normal plasma. In 7 patients with type IIA, the amount and distribution ofthe three Spill species were clearly abnormal, with a marked decrease or absence of SpIIIa and an increase of SpIIIb and SpIIIc. Results slightly varied between patients but were consistent within one family. In 4 patients with type IIC, the band SpIIIb was lacking. In 2 patients with type IIB and 2 patients with type IID, there was no significant modification of Spill. In all cases the pattern of Spill was similar whether using polyclonal antibodies to vWF or MAbs to Spill.The pattern of normal or abnormal Spill remainedthe same in fresh or frozen plasma and was not modified by the presence of 5 mM EDTA at the timeof blood collection. Furthermore, the abnormaldistribution of Spill in type IIA or IIC vWD wasalready present after the shortest digestion time(15 min) indicating that the fragment Spill was produced from an abnormal vWF during the primary digestion with S. aureus V-8 protease. In conclusion, the molecular abnormality of vWF in type IIA and IIC vWD appears to reside in Spill, the N-terminal portion of the subunit (residues 1 to 1,365).
THROMBOSPONDIN: CELL BIOLOGY OF AN ADHESIVE GLYCOPROTEIN
Authors: Nachman R L , Silverstein R L , Asch A S
Published: August 2018

Thrombospondin (TSP), a multifunctional 450 KD glycoprotein is a secretory product of thrombin stimulated platelets. It is a major component of the platelets alpha granule constituting approximately 3% of total platelet protein. Thrombospondin does not circulate in appreciable concentrations ∽0 100 ng/ml); however, the tissue distribution is broad. In addition to its expression on the membrane of activated platelets, the protein is synthesized by fibroblasts endothelial cells, glial cell smooth muscle cells alveolar pneumocytes mononuclear phagocytes and various tumor cells. TSP is a major constituent of the extracellular matrix and has been demonstrated in the vessel wall, basement membrane and glandular connective tissue. Fibroblasts, smooth muscle cells and endothelial cells in tissue culture incorporate TSP into the extracellular matrix. Matrix TSP is under cell-cycle regulatory control. Mesenchymal cells in the proliferative phase synthesize greater amounts of TSP than non growing cells. Platelet derived growth factor induces smooth muscle cell and glial cell synthesis of TSP. Atheromatous lesions contain increased amounts of TSP compared to normal vessels emphasizing the potential role of TSP in the interaction of proliferating cells with the matrix. TSP binds specifically, saturably, and reversibly to mouse peritoneal macrophages and to cells of the monocyte-like human cell line U937. Binding was time dependent and was optimal in the presence of both Ca++ and Mg++. PMA stimulated U937 cells and activated macrophages bound TSP to an equivalent extent as resting cells. The TSP binding site on the surface of U937 cells and peripheral blood monocytes mediates the adhesive interaction between these cells and thrombin-stimulated platelets. Using a sensitive rosetting assay we found that monocytes were not rosetted by resting platelets while >90% were rosetted by thrombin-stimulated platelets. Monoclonal and polyclonal anti-TSP antibodies markedly inhibited rosetting as did TSP itself. Antifibronectin or non-immune control antibodies did not inhibit rosetting, nor did fibronectin, fibrinogen, the fibronectinadhesion tetrapeptide arg-gly-asp-ser (RGDS), or heparin. The TSP membrane receptor, an 88 KD glycoprotein, formely known as GPIV has been identified in platelets, endothelial cells, monocytes and a variety of tumor cells. TSP may thus serve as a molecular bridge linking activated platelets with monocytes at sites of early vascular injury. Such interactions involving the TSP receptor complex may be of critical importance in the regulation of thrombosis and the initiation of atherosclerosis.
REVERSIBLE DEFICIENCY OF INTACT THROMBOSPONDIN AND MEMBRANE GLYCOPROTEIN Ia IN PLATELETS OF A PATIENT WITH A BLEEDING DISORDER
Authors: Kehrel B , Ballesian L , Kokott R , Stenzinger W , Clemetson K J , De Loo J Van
Published: August 2018

A 52 year old female patient with a severe bleeding tendency since the age of two was studied. Her history revealed recurrent petechial bleedings, two severe postoperative haemorrhagic episodes and intensivemenstrual bleedings which required blood transfusions. Coagulation studies ruled out any coagulation disorder including von Willebrand's disease. Platelet count and morphology(using light and electron microscopy) were normal. The patient had prolonged bleeding times (up to 15 min). Her platelets aggregated normally in response to ADP, arachidonic acid, thrombin, ionophore A 23187, epinephrine and ristocetin. In contrast, platelet aggregation in the presence of collagen and wheat germ agglutinin could only be achieved with very high doses of these agonists. Repeated analyses of the patient's platelet proteins by two-dimensional 0'Farrel gel electrophoresis followed by silver staining or blotting onto nitrocellulose and staining with a mixture of peroxidase-coupled lectins showed that glycoprotein la and intact thrombospondin were absent. An immunoblot for thrombospondin showed several proteins with lower molecular weight than thrombospondin. Preincubation of the patient's platelets with thrombospondin normalized collagen-induced aggregation.At a recent follow-up examination the patient had no petechial bleedings. Platelet protein analysis revealed thatintact thrombospondin and glycoprotein la were present. These results suggest that both glycoprotein la and thrombospondin have essential roles in collagen-induced platelet aggregation.
ROLE OF POLYAMINES IN THE REGULATION OF SYNTHESIS AND SECRETION OF PLASMINOGEN ACTIVATOR FROM BOVINE AORTIC ENDOTHELIAL CELLS
Authors: Kuo Be-Sheng , Korner Gil , Bjornsson Thorir D
Published: August 2018

The effects of three polyamines, putrescine (PUT), spermidine (SPD) and spermine (SPM), were investigated on the synthesis and secretion of plasminogen activator (PA) and antiactivator (PAI) activities in confluent bovine aortic endothelial cells. PA activity was determined bythe fibrin plate method, and individual species with PA and PAI activities were separated and visualized using SDS-PAGE with zymography and reverse fibrin autography. Both control cells and cells treated with polyamines secreted PA activity in a time-dependent fashion. After 24-hour incubation, the three polyamines enhanced PA secretion in a dose-dependent manner (10-6 to 2.5 × 10-3 M), with a potency order of SPM > SPD> PUT, as estimated by the fibrin plate method. The maximum PA releases after PUT (0.5 mM), SPD (2.5 mM) and SPM (0.5 mM) were 1.7, 4.5 and 5.4 times control levels, respectively. Concentrations lower than 1 μM had essentially no effects. The enhancement of PA activity by polyamines was blocked by actino-mycin D and cycloheximide, while it was not affected by inhibitors of polyamine biosynthesis except that the enhancement by PUT (0.5 mM) was reduced by methylglyoxal bis(guanylhydrazone). These data suggest that polyamines directly stimulate PA synthesis and secretion through promotion of gene transcription and translation, and that this effect appears to be related to their position in the biosynthetic pathway of polyamines. The kinetic patterns of activities of ornithine decarboxylase and S-adenosy-methionine decarboxylase in confluent endothelial cells stimulated withfresh culture medium suggest that there is rapid turnover of intracellular polyamines. Multiple forms of secreted PA were observed and both tissue- and urokinase-type PA were enhanced by polyamines, while the PAI activity, as evaluted by reverse fibrin autograpy, was apparently reduced. These experimental results suggest that polyamines may play an important role in the regulation of the synthesis and secretion of plasminogen activators, and that this biological function could be modified by disease states and by agents that are associated with altered polyamine metabolism.
PHYSIOLOGICAL ROLE OF THE ENHANCED FIBRINOLYTIC ACTIVITY DURING CARDIOPULMONARY BYPASS IN OPEN HEART SURGERY
Authors: Kanamori Y , Yada I , Yuasa I , Kusagawa M , Deguchi K
Published: August 2018

Fibrinolytic activity is reportedly increased during cardiopulmonary bypass( CPB ), and this increase has been considered to be related to the bleeding complications in open heart surgery. The purpose of this study was to clarify the nature of the fibrinolytic activity duringCPB. Twenty patients with valve replacement or aortocoronary bypass surgery were examined. The following parameters were determined: fibrinogen, plasminogen, fibrinopeptide A( FPA ), fibrinopeptide B β 15-42 ( FPB β15-42 ), and tissue-type plasminogen activator ( t-PA ). For further characterization of the fibrinolytic activity, the fibrin plate method was used. Intrinsic fibrinolytic activity was determined by the assay of the fibrinolytic activity of the kaolin activatedeuglobulin. Extrinsic fibrinolyticactivity was estimated by the assayof Cl-inactivator resistant fibrinolytic activity as well as t-PA. Fibrinogen and plasminogen did not decrease except at the beginning of CPB. FPA was increased significantly during CPB. FPB3 15-42 was also increased to four times the preoperative value at 2 hrs of CPB. The intrinsic fibrinolytic system was activatedonly a short time after the startof CPB. The Cl-inactivator resistant fibrinolytic activity was activated gradually during CPB, reached a maximum level 1 hr after the start of CPB, and returned to the preoperative level within 1 hr after the end of CPB. The changes on t-PA paralleled the course of the Cl-inactivator resistant fibrinolytic activity, indicating that enhanced fibrinolytic activity during CPB is predominantly of extrinsic origin caused by t-PA. We conclude that thrombin activity continues during CPB despite the use of heparin, and thatthe enhanced fibrinolytic activity during CPB is essential because t-PA activates plasminogen predominantly at the sites where fibrin is formed, resulting in the dissolution of the microthrombi formed during CPB.
FRAGMENTATION OF CERULOPLASMIN BY THROMBINF
Authors: Szabo T
Published: August 2018

Copper containing proteolytic fragments of ceruloplasmin (CP), a 135 KDα2 -glycoprotein, have been shown to induce angiogenesis in the rabbit cornea assay.Neovascularisation plays an important role in different biological phenomena including chronic inflammation, wound healing, recanalisation of occluded blood vessels, tumor growth etc. Most of these events are associated with elevated CP levels. Neovascularisation occurs in diabetic macro- and microangiopathies as well. Serum CP concentrations in 92 diabetics were measured and compored to 50 healthy blood donors and 50 unselected hospitalized patients without malignancies. A marked CP elevation was observed in diabetics, especially in those with vascular complications. There was no correlation between the CP and the actual blood glucose concentrations, duration of the disease of the type of the treatment. Activation of the haemostatic processes has been found in the majority of diabetic patients with vascular diseases. In case CP is a substrate for thrombin, the generation of this specific serine protease may lead to the release of angiogenic peptides from CP on the site of vascular occlusion. For this reason, purified human CP was incubated with thrombin at pH 7.4, 37×C, and samples were removed at 0, 30, 60, 120, 240, min., and\after 2k hours for CP oxidase activity measurements and for SDS PAGE. Thrombin treatment did not affect the enzymic activity of CP. On SDS PAGE the band corresponding to the parent molecule eventually disappeared, and a Mr ll6 KD fragment together with three smaller peptides has been produced, with molecular masses 61, 4l and 20 KD, respectively. Based on these experiments, CP is to be considered as a new protein substrate for thrombin. The physiological relevance of this phenomenon needs further examination.