bluuebunny/crossref_metadata_2025 huggingface dataset:
COMPARISON OF PLATELET PHOSPHOINOSITIDE HYDROLYSIS BY THROMBIN AND COMBINATIONS OF OTHER AGONISTS
Authors: McNicol A , MacIntyre D E
Published: August 2018

The activation of human platelets is mediated by the generation of two distinct second messengers, inositol 1,4,5 trisphosphate (IP3) and 1,2 Diacylglycerol (DG). Both may be derived from agonist-induced phosphoinositide (PI) hydrolysis. We have examined the effects of combinations of agonists on PI hydrolysis (monitored as {32P}-phosphatidate (PA) production). Supramaximal concentrations of PAF (180 nM), 5HT (10 μM) and the thromboxane analogue EP171 (100 nM), but not ADP (1 μM), elicited a 2-6 fold increase in (32P}-PA levels. In combination the above agonists stimulated formation of {32P}-PA to a level (6.6 fold) which was marginally, but significantly, more than each of the individual agonists. This is considerably less than that induced by IU/ml thrombin (approx. 15 fold), perhaps indicative of an additional mechanism for thrombin-induced PA formation. The serine protease trypsin (1 μM), elicits a 30 fold increase in {32P}-PA and potentiates the EP171-induced {32P}-PA formation from approx. 6 fold to approx. 35 fold. Thus, EP171 synergises with trypsin but not with other receptor directed agonists. Consequently, it is mechanistically possible that thrombin stimulates platelet PI hydrolysis via both receptor activated (typified by EP171) and proteolytic (typified by trypsin) processes.
ENZYMES THAT DEPHOSPHORYLATE INOSITOL PHOSPHATES IN HUMAN PLATELETS
Authors: Molina Luis , Vedia y , Lapetina Eduardo G
Published: August 2018

Inositol trisphosphate (IP3) is now recognized as a second messenger molecule that mobilizes Ca2+ from intracellular stores to the cytosol. The persistence of the action of IP3 depends on the specific phosphatase that converts IP3 to inositol bisphosphate (IP2). The activation of IP3 phosphatase is important in terminating the Ca2+ signal in stimulated cells. In platelets it has previously been shown that this enzyme is regulated by protein kinase C since it is stimulated by phorbol esters and 1,2-diacylglycerol (Molina y Vedia, L., and Lapetina, E.G. J. Biol Chem. 261, 10493-10495, 1986) and the cytosolic platelet enzyme is phosphorylated by brain protein kinase C, resulting in a 4-fold increase in IP3 phosphatase activity (Connolly, T. M., Lawing, W.J., Jr., and Majerus, P.W., Cell, 46, 951-958, 1986). We have studied the subcellular distribution of the phosphatases that hydrolyze IP3, IP2 and inositol monophosphate (IP) in human platelets. Three subcellular fractions were obtained from human platelets lysed by freezing and thawing: a cytosolic fraction, a membrane fraction and a mixed particulate fraction containing granules, mitochondria and membranes. These fractions have been characterized by specific marker enzymes. The highest specific activity of IP3 -phosphatase is associated with the membrane fraction and accounts for about 10-15% of the total activity. The mixed particulate fraction has 35-40% of the activity while about 50% is cytosolic. The Km of the membrane fraction enzyme is 100 μM. This enzyme is extracted by 1M NaCl and hydrodynamic studies revealed a molecular weight of 50 kDa. The NaCl extracted-enzyme has been further purified by hydrophobic and gel filtration chromatographies. This activity does not hydrolyses IP but hydrolyse IP2 at a lower rate. The enzyme that hydrolyses IP to inositol is confined to the cytosolic fraction, has a Km of 130 μM, is inhibited by Li+, and hydrodynamic studies show an apparent molecular weight of 91 kDa.
THROMBIN, PAF AND VASOPRESSIN-INDUCED RISE OF PLATELET INTRACELLULAR CALCIUM, AGGREGATION AND ATP SECRETION ARE REDUCED BY ANTIOXIDANTS
Authors: Doni M G , Deana D , Alexandre A
Published: August 2018

Physiological agonists induce a rapid increase of cytosolic free Ca2+ concentration ([Ca2+]i) in platelets, as well as shape change, aggregation and1 secretion. They activate a phosphodiesterase which specifically splits polyphosphoinositides, generating the second messengers diacylglycerol, a protein kinase C ativator, and inositol 1,4,5 triphosphate, which releases Ca2+ from the intracellular stores - [Ca2+]i rise and protein kinase C activation are responsible for aggregation and exocytosis.

We have now studied the effects of the synthetic antioxidants: butyl-hydroxytoluene (BHT), butyl- hydroxyanisole (BHA), nordihydroguaiaretic acid (NDGA) and the one electron donor 1-1' dimethylferrocene plus ascorbate (FC) on the increase of [Ca2+]i, aggregation and ATP secretion. Human platelets were loaded1 with 20μM quin-2-acetoxymethylester and incubated with aspirin (100μM). Changes in the [Ca2+]. were measured with a spectrofluorimeter. Platelet aggregation and ATP secretion (luciferin/luciferase system) were evaluated in parallel. In the presence of 1mM external Ca2+,0.15 u/ml thrombin induced a [Ca2+]. increase to about 1μM, that was inhibited by 50% and abolished by 25μM and 100μM BHT respectively; [Ca2+]. increase induced by 75 nM PAF, 1μM vasopressin, 10 μM ADP was also inhibited by BHA, NDGA and FC in a range of 30-150 μM. Shape change, aggregation and ATP secretion were also inhibited. In the absence of external Ca2+ (1mM ECTA), 80μM BHT inhibits the [Ca2+]i increase originating only from the intracellular stores. Tumor-promoting phorbol esters induce aggregation and secretion without raising [Ca2+]i; 80 μM BHT induced 80-90% inhibition of aggregation and ATP secretion by 0.12nM TPA. Our results suggest that some free radical dependent reactions are involved both in the processes of platelet activation leading to the increase of [Ca2+]i and in those leading to aggregation and exocytosis.
Evidence for Ca2+-independent phosphorylation of human platelet myosin
Authors: Daniel J L , Rigmaiden M
Published: August 2018

Phosphorylation of platelet myosin is thought to be required for activation of the contractile events occurring during platelet activation. At present the only known mechanism for Onitiating myosin phosphorylation is through a Ca2+-calmodulin-dependent activation of myosin light chain kinase. However, our previous studies using the fluorescent Ca2+-indicator quin2 indicated that both platelet shape change and myosin phosphorylation could be induced in an EGTA-containing media in the absence of a measurable change in cytosolic free Ca2+ concentration (Hallam, Daniel, Kendrick-Jones & Rink. Biochem. J. 232 (1985) 373). In order to confirm this finding, we fyave investigated the regulation of myosin phosphorylation usin^+a preparation of electrically-permeabilized platelets and Ca2+ buffers to control the internal Ca2+ concentration. Fifty percent myosin phosphorylation was obtained at 700 nM Ca2+. When thrombin (5 U/ml) was added to this system, this curve shifted both to the left and upward; 50% myosin phosphorylation was obtained at 400 nM Ca2+.A synthetic inhibitor of protein kinase C, H7, had no effect on myosin phosphorylation in the absence of agonist but did inhibit the thrombin-induced shift to left suggesting that protein kinase C may modulate myosin phosphorylation. We also compared the effects of H7 agonist-induced myosin phosphorylation and shape change in control and an quin2 loaded platelets. Comparable inhibition of both phosphorylation and the rate of shape change was observed with both quin2 and H7. Addition of H7 to quin2-loaded platelets resulted in complete inhibition of both agonist-induced shape change and myosin phosphorylation. These results indicate that both protein kinase C and Ca2+-dependent reactions are involved in complete expression of myosin phosphorylation in human platelets.
DISSOCIATION OF PHOSPHOLIPASE A2 ACTIVATION FROM CYTOSOLIC FREE CA2+ MOBILIZATION IN PLATELETS EXPOSED TO FLUORIDE
Authors: Kienast J , Arnout J , Pfliegler G , Deckmyn H , Hoet B , Vermylen J
Published: August 2018

Elevated cytosolic free Ca2+ concentrations ([Ca2+]i) are thought to be required for phosphol ipase A2 activity to liberate arachidonic acid (AA) from membrane phospholipids in platelets. The major AA metabolite formed during agonist-induced platelet activation is thromboxane A2 (TxA2). We have investigated the effect of sodium fluoride (NaF) on platelet TxAz formation in correlation to platelet functional changes (aggregation and release of ATP) and intracellular events specific for either agonist- or antagonist-induced platelet responses. A first peak in platelet TxAffi formation reaching 30 × control values was observed at 20 - 30 mM of NaF which also induced platelet aggregation and release of ATP in association with a rise in [Ca2+]i . Increasing the concentration of NaF resulted in a decrease in TxA2 release to a minimum of 12 × control values at 50 mM. A second, concentration-dependent rise in TxA2 formation was observed at higher concentrations of NaF with a maximum of 40 × control values at 100 mM. These concentrations, however, did induce neither aggregation nor a significant rise in [Ca2+]i but a rapid, transient increase in platelet cAMP levels. Activation of phospholipase C and protein kinase C was observed at all concentrations of NaF tested whereby the rate rather than the extent of activation of these enzymes was concentration-dependent. Our results demonstrate that fluoride at high concentrations can induce platelet TxA2 formation in the absence of elevated [Ca2+]i suggesting an additional, Ca2+-independent mechanism of phospholipase A2 activation possibly mediated by fluoride sensitive GTP-binding regulatory proteins.
TIME DEPENDENCE OF AEQUORIN-INDICATED CALCIUM SIGNALS IN STIMULATED AND UNSTIMULATED PLATELETS
Authors: Lages B , Weiss H J
Published: August 2018

The photoprotein aequorin (aeq) has been used as an indicator of intracellular calcium levels ([Ca]i) in platelets (pits). Aeq is believed to monitor aspects of [Ca]i somewhat different from those reported by quin-2, since it reports a higher basal [Ca]i than quin-2 and the presence, rather than the absence, of increased [Ca]i following epinephrine (epi) stimulation. To characterize further aeq-indicated pit Ca signals we measured their time dependence in both stimulated and untimulated pits. Aeq was loaded into pits by membrane permeabilization and resealing at 0°C. The maximum aeq signal (Lmax), obtained by Triton X-100 lysis of pits in the presence of 1 mM Ca, decreased over 40-60 min in a non-linear manner to about 60% of its initial value. Basal [Ca]i also decreased in a similar manner, even after correction for the changes in Lmax. The decreases in both responses were greater in pits isolated in the presence of 1 mM Ca than in the absence of Ca, indicating a sensitivity to external Ca. Epi (1-50 uM) induced an aeq signal in pits of most, but not all, subjects initially, but this response disappeared completely over 40-60 min in most subjects, without a concomitant loss of aggregation response. In contrast, the endoperoxide analog U44069 (10 uM) induced aeq signals in pits from all subjects, which decreased only slightly with time. These results suggest 1) that a portion of pit aeq may be localized in a region of high [Ca]i which may be partially accessible to external Ca, and this may account, in part, for the higher basal [Ca]i reported by aeq than by quin-2; and 2) that the epi-induced aeq signal, which is not prerequisite for aggregation, may be derived primarily from this labile aeq pool since the time courses for the decay of Lmax and basal [Ca]i and the loss of the epi-induced aeq signal are similar.
SUB-SECOND CALCIUM DYNAMICS IN ADP AND THROMBIN-STIMULATED PLATELETS; ASSESSED BY A C0NTINU0US-EL0W APPROACH
Authors: Gear A R L , Jones G D
Published: August 2018

There is now evidence that many platelet reactions begin within 1 sec of platelet stimulation. These include "shape change," aggregation and biochemical events such as protein phosphorylation. Our laboratory has devised quenched-flow approaches for following such early events (J Lab Clin Med 100, 866, 1982) and we have extended these to fluorimetric analyses of rapid calcium changes. A micro, flow-through cell, with a sensing volume of 0.1 μ1, is placed on line from the quenched-flow apparatus. Indo-1 loaded, human platelets are pumped through the system and reaction times from 0.25 sec can be followed. Ratioing emission changes at 400 and 480 nm, after excitation at 355 nm, provides an index of free calcium. ADP (10 μM) induced a rapid increase in Ca++ to about 1 μM by 1.5 sec, beginning near 0.3 sec. This was faster and greater than the first increase caused by thrombin (10U/ml). However, thrombin induced a second (> 5s) and larger increase in free platelet calcium. Control experiments where the Indo-1 loaded platelets were simply pumped through the 0.3 mm ID reaction tubing, revealed a slight increase above resting calcium values, indicating some shear-induced activation. The use of the continuous-flow fluorescent cell coupled to the quenched-flow apparatus enables following calcium dynamics under Theological conditions very close to those iui vivo.

Correlations with other early events, such as protein phosphorylation, become possible. Supported by NIH HL-27014.
ROLES OF cAMP AND cGMP IN THE SYNERGISTIC INHIBITORY EFFECTS OF NITROVASODILATORS AND PGE1 ON PLATELET AGGREGATION AND DEGRANULATION
Authors: Maurice D H , Haslam R J
Published: August 2018

Washed rabbit platelets labelled with [14C]5-HT and suspended in a modified Tyrode’s solution were used in this study. Incubation of the platelets with 0.1, 1 and 10 μM sodium nitro-prusside (SNP) for 30 s inhibited aggregation induced by 10 nM platelet-activating factor by 7, 13 and 45% and the release of [14C]5-HT by 16, 30 and 45%, respectively. In combination with 0.02 μM PGE1, which had little effect alone (7% inhibitions), these concentrations of SNP caused 58, 90 and 100% inhibitions of aggregation and 60, 73 and 81% inhibitions of [14C]5-HT release. Thus, SNP and PGE1 acted synergisti-cally. The changes in [3H]cGMP and [3H]cAMP caused by these compounds were measured in platelets labelled by preincubation with [3H]guanine and [3H]adenine. [3H]cGMP (initially 0.01% of platelet [3H]GTP) increased by amounts equivalent to 0.02, 0.10 and 0.63% of [3H]GTP with 0.1, 1 and 10 μM SNP. PGE1 had no effects on platelet [3H]cGMP. The above SNP concentrations also increased [3H]cAMP by amounts equivalent to 0.01, 0.02 and 0.04% of platelet [3H]ATP (basal value 0.02%). However, in the presence of 0.02 μM PGE1, 0.1, 1 and 10 μM SNP increased [3H]cAMP by amounts equivalent to 0.13, 0.26 and 0.39% of platelet [3H]ATP. Both with and without PGE1, 10 μM SIN-1 (the active metabolite of molsidomine) had effects similar to those of 1 μM SNP. Thus, the synergistic actions of PGE-, and SNP or SIN-1 on platelet function correlated with increases in [3H]cAMP, not [3H]cGMP. Preincubation of the platelets for 30 s with 200 μM 2∲,5∲-dideoxy-adenosine (an inhibitor of adenylate cyclase) reduced the above increases in [3H]cAMP and inhibitions of platelet reactions by about 60%, without affecting [3H]cGMP levels. Addition of specific inhibitors of platelet cAMP phosphodiesterase (e.g. cilostamide) enhanced the actions of PGE1 in a manner similar to SNP but had little effect on the actions of SNP. The results show that the nitrovasodilators studied have effects on platelet function and [3H]cAMP similar to those of inhibitors of cAMP phosphodiesterase, suggesting that their actions may be mediated by increases in cAMP caused by an effect of cGMP on the platelet cGMP-inhibited cAMP phosphodiesterase. (HSFO Grant T443H)
AGGREGATION RESPONSE OF PLATELETS DURING INHIBITION OF PHOSPHOLIPASE A2
Authors: Labow R S , Meek E , Adams G A , Rock G
Published: August 2018

Arachidonic acid (AA) is liberated from platelet membrane phospholipids during stimulation and promotes cellular aggregation through the synthesis of thromboxane A2. Two pathways; phospholipase A2 (PLA2) or phospholipase C (PLC) followed by the action of acylglycerolipases, are thought to be activated during platelet stimulation and supply the necessary AA. We have reported that mono (2-ethylhexyl)phthalate (MEHP), a physiological metabolite of the plasticizer di(2-ethylhexyl)-phthalate (DEHP), commonly used in a variety of medical devices and storage containers, inhibits PLA2, but not PLC in platelet lysates. The effects of MEHP on intact platelets were studied. PLA2 activity in intact platelets or lysates was assayed by incubating them with 2-14C-arachidonyl-phosphatidylcholine and measuring formation of free 14C-arachidonic acid in 10 min. Platelet lysates hydrolyzed 10% of the substrate while 2.6% was hydrolyzed by intact platelets. The amount of MEHP needed to inhibit 14C-AA liberation was 0.35 mM for platelet lysates and 0.7 mM for intact platelets. Platelet aggregation induced by collagen was inhibited by MEHP (1 mM), although responses to adenosine diphosphate, AA and ionophore were unaffected. Identical effects on platelet aggregation were found when indomethacin (0.1 mM) was added. Higher concentrations of MEHP blocked platelet aggregation induced by adenosine diphosphate or AA but not ionophore or synergistic pairs of these stimuli, indicating a more generalized membrane disruption at higher MEHP concentrations. These results suggest that MEHP acts in a similar manner to indomethacin to block PLA2-mediated liberation of arachidonate during platelet aggregation

(supported by MRC and NHRDP, Canada)
CHANGES IN PLATELET HALF-LIFE, SENSITIVITY TO PROSTANOIDS AND AGGREGATION INDUCED IN THE DOG BY BODY HYPOTHERMIA
Authors: Cunha-Ribeiro L M , Cunha S , Brandão T , Goncalves F S , Almeida-Dias A , Pina-Cabral J M
Published: August 2018

During body cooling, there is a progressive thrombocytopenia, which is reversible after rewarming and is not prevented by previous treatement with aspirin, ticlopidine or prostacyclin.In this work, in order to evaluate if hypothermia induces alterations of platelet function we studied, in the dog, the platelet aggregation (PA) and the inhibitory action of PGE1 and of a stable prostacyclin analog, l loprost, before hypothermia(37°C.) and after rewarming(37°C.).Platelet half-life was also studied in another group of dogs before induction of hypothermia and after rewarming and recuperation of the animals. PA has been evaluated by platelet counting in whole blood.P1atelet half-life was estimated by serial determinations of MDA following administration of aspirin.PA induced by ADP (30μM) decreased 40% after rewarming(n=8). Platelet sensitivity to PGE1 (35 nM -1.4 μM f.c.) and Iloprost (7nM-172nM f.c.) was also decreased after rewarming: inhibition index 2.08±1.082 versus 1.19±0.362 (n=8; p<0.01) and 2.48<1.250 versus 1.10±0.227 (n=8; p<0.005) respectively.Platelet half-life increased after hypothermia from 3.99±0.730 days to 4.48±0.846 days(n=8 p<0.05).In the control group(n=6) platelet half-life determined twice with one week interval, did not change significantly.

We conclude that body hypothermia decreases platelet reactivity to ADP; renders platelets less sensitive to the inibitory effect of prostanoids and increases platelet half-1 ife.These results are probably due to alterations in platelet membrane induced by cooling and rewarming the animals.
RED BLOOD CELL LYSIS MAY INFLUENCE PLATELET AGGREGATION IN WHOLE BLOOD AGGREGOMETER
Authors: Herman A G , Bult H
Published: August 2018

The electronic whole blood aggregometer (WBA) has the advantage that it enables the study of platelet aggregation in whole blood shortly after blood collection. Using the WBA varying results have been obtained with respect to the anti-aggregating activity of dipyridamole. As dipyridamole is an efficient inhibitor of adenosine uptake, we tested whether the degree of red blood cell lysis (and thus availability of adenine nucleotides) affected its efficacy. Citrate (10.7 mM) blood was stored in sealed tubes and used between 20 and 100 min after venipuncture. One ml was placed in a Chronolog Model 540 WBA together with 10 μl 0.9 % NaCl, dipyridamole (final conc. 3, 10 or 30 μM) or its solvent (final conc. 0.03, 0.1 or 0.3 %). After reaching a stable baseline and WBA calibration, aggregation was induced by injection of 10 μM ADP dissolved in 10 μl 0.9 % NaCl (one channel) or 10 μl distilled water (other channel). Maximum impedance increase in 10 min was measured, red blood cells were removed by centrifugation, and from microhematocrit and absorbance at 416 nm the volume of lysed packed red blood cells was estimated. ADP caused aggregation (12.9 ± 1.9 and 11.1 ± 0.8, Ohm) and there was red blood cell lysis (2.8 ± 0.5 and 0.8 ± 0.2 μl red blood cells, ADP resp. in H20 and 0.9 % NaCF, n = 6). Dipyridamole (30 μM) suppressed aggregation when compared with solvent, but only when ADP was given in H20 (reduction resp. 4.4 ± 1.4 and 1.9 ± 1.6 ohm). Moreover, there was a negative correlation between the degree of haemolysis and the aggregation response at 10 as well as 30 μM dipyridamole. This reduced aggregation with increasing haemolysis was not observed in the presence of the corresponding solvent concentrations. The red blood cell lysis was proportional to the plasma ATP (luciferine-luciferase method) concentration as an index of adenine nucleotide leakage. In conclusion, a certain degree of haemolysis caused by stirring and injection with a microsyringe is inherent to the WBA, but use of hypotonic vehicles should be avoided. Release of red blood cell constituents may affect platelet aggregation as such, or interfere with the activity of adenosine uptake inhibitors and possibly other drugs. It may help to explain some of the variable results obtained with dipyridamole.
GLANZMANN'S THROMBASTHENIA IN PREGNANCY: CLINICAL FEATURES AND PLATELET SEROLOGY
Authors: Picó M , Ribera A , Martin C , Zuazu J , Monasterio J
Published: August 2018

A 24-year-old woman with a diagnosis of Glanzmann's Thrombasthenia (GT) type I (confirmed in our Hemostasis Section by sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and with a history of several transfusions, was seen in our Service since the second month of her first pregnancy.

In the first visit a strong anti-Rh(D) antibody was found in her serum. In 28th week an ammiocentesis was done. Seventy two hours later, she was hospitalized because of disminution of foetal movements. Then, a platelet-allo-antibody, IgG+IgM type (title IgG 1/512, IgM 1/2) was detected in her scrum by immunofluorescence test, as well as an anti-HLA antibody (by lymphocytotoxicity test) which reacted with her husband lyin-phocites.

The platelet antibody wasn't EDTA or Paraformaldehide (PFA) dependent and reacted with all normal platelets of a panel of known platelet phenotypes and, also although significantly weakly, with GT type II platelets. The negative results were only observed with GT type I platelets. Therefore, it seemed to recognize an antigenic site located in GP IIb-IIIa. Besides, this antibody inhibited the normal platelet aggregation.

A caesarian was advised because of foetal suffering an was performed without immediate complications. The patient was protected by our hemotherapy established support and delivered a boy with a severe hemolytic disease (Hematocrit: 6%, bilirrubine: 1,9 mgs./lOCimls., platelets: 40.000/ mm3), who died 48h. later. His platelets had a direct positive test IgG type and in his serum the platelet antibody as in his mother's was detected.

We discuss the specificity of the antibody detected and also, its possible implication in the neonatal thrombocytopenia.
GRAY PLATELET SYNDROME AND IDIOPATHIC PULMONARY FIBROSIS OCCURRING IN THE SAME PATIENT : A FORTUITOUS ASSOCIATION?
Authors: FACON T , GOUPEMAND J , CARON C , ZANDECKI M , ESTIENNE M H , COSSON A
Published: August 2018

A 46 yr old Caucasian woman has been diagnosed as having a congenital deficiency of platelet a-granules (gray platelet syndrome - GPS) associated with an extensive idiopathic pulmonary fibrosis (IPF). The patient had a life long history of bleeding tendency including dental bleedings in childhood, intraperitoneal bleeding, metrorrhagias which led to hysterectomy, and post-operative hemorrhages. When aged 16, splenectomy was performed because of a mild thrombocytopenia but did not result in a subsequent improvement of the platelet count. The spleen was enlarged and showed an excess of fibrous tissue.

Evaluation of hemostasis (July 1986) revealed a moderate thrombocytopenia of 120 × 109/1 constrasting with a markedly prolonged Simplate bleeding time (>30 min.). When examined on stained blood films, the platelets presented a "ghost-like" gray appearance. The mean platelet volume (coulter S + IV) was increased to 14 μ3 (N : 6.5-9.5 μ3). Ultrastructural studies confirmed the lack of a-granules and showed normal presence of dense-bodies, mitochondria and peroxisomes. Platelet aggregation was decreased when induced by thrombin, ADP and collagen but normal in response to arachidonic acid and ristocetin. A severely decreased content of platelet proteins such as fibrinogen, vWF:Ag, BTG and PF4. was further demonstrated. A bone marrow biopsy performed on March 1986 gave no evidence of myelofibrosis (occasionally recorded in GPS) but the patient developed for these last 6 years a severe IPF requiring a permanent oxygen-therapy. Although the association GPS-IPF might be only considered as a fortuitous one, we hypothesize that these two events might be related to each other, possibly through the presence of megakaryocytes in pulmonary capillaries, in the same way as bone marrow fibrosis has been suggested as a possible consequence of the lack of a-granules in GPS (DR0UET et al. - Nouv. Rev. Fr. Hematol., 1981, 23 : 95).
ELECTRONMICROSCOPIC STUDIES ON PLATELETS AND MEGAKARYOCYTES IN GIANT PLATELET SYNDROME
Authors: Suzuki Sozo , Mori Kazuo , Sugai Koji , Akutsu Yasuyuki , Ishikawa Masaaki , Sakai Hideaki , Hiwatashi Katsuhide
Published: August 2018

Giant platelet syndrome are characterized morphologically by many giant platelets associated with several functional abnormalities in the peripheral blood. However, the mechanism of large platelet production has not yet been clarified. In 1981, we reported acase with Bernard-Soulier syndrome(BSS) in whom giant platelets were considered to be formed by fusion of two or three platelets in the circulating blood. We examined the ultrastructure of platelets and megakaryocytes in another case with BSS (29 year-old female) and a case with May-Hegglin anomaly (31 year-old male). Whole blood and bone marrow specimens were fixed with glutaraldehyde-osmium solution. Thin sections were prepared and stained with uranyl acetate and lead cytrate. Membrane systems of platelets and megakaryocytes in a case with BSS was investigated by staining of surface coating with ruthenium red.

In a case with BSS, most platelets were very large and similar in morphology to those in formerly reported case. Giant platelets contained several-fold increased number of α-granules and mitochondria. Typical dense bodies were also observed. Contents of ATP/ADP, platelet factor-4(PF-4), B-thromboglobulin(B-TG) and platelet factor-3 availability(PF-3) were increased. Disorganization of microtubules was recognized. Some giant platelet contained membrane systems similar to demarcation membranes(DM) in megakaryocytes, characteristically. In mature megakaryocytes, areas divided by DM similar in size to those in normal megakaryocytes were observed. Several of these areas appeared to fuse together to form the giant platelets containing many granules and remnants of DM. In a case with May-Hegglin anomaly, typical Dohle’s bodies were shown in neutrophilic granulocytes. Giant platelets in this case also contained large number of α-granules and some of them contained membrane systems similar to DM. Areas similar in morphology to these giant platelets were clearly noted in the cytoplasm of mature megakaryocytes.

In these cases, most giant platelets in the peripheral blood may be formed in the cytoplasm of megakaryocytes by fusion of several areas divided by DM, each of which may become normal sized platelets in normal megakaryocytes.
BERNARD-SOULIER SYNDROME: WHOLE BLOOD DIAGNOSTIC ASSAYS OF PLATELETS
Authors: Nichols W L , Kaese S E , Gastineau D A , Otteman L A , Bowie E J W
Published: August 2018

Diagnosis of Bernard-Soulier syndrome (BSS) is complicated by the difficulty of separating the giant platelets from other blood cells to pursue analyses of platelet function and structure. We report on the utility of three whole blood assay techniques for diagnosis of a patient with BSS. To our knowledge, these three techniques have not been simultaneously applied or compared for efficacy in laboratory diagnosis of BSS. (1) Whole blood platelet aggregation responses, studied with an electrical impedence aggregometer, were equivalent to those more laboriously obtained using platelet-rich plasma prepared by unit gravity sedimentation, studied with an optical light transmittance aggregometer. Platelet aggregation responses were normal with ADP or collagen stimulation, and absent with Ristocetin or bovine plasma stimulation. (2) Whole blood radioimmunoassay of platelet glycoprotein (GP) expression was performed using iodinated murine monoclonal antibodies HP1-1D (anti-GP IIb/IIIa) and 6D1 (anti-GPlb, kindly supplied by Dr. Barry Coller, Stony Brook, NY). After incubation with citrated whole blood, centrifugation was used to separate cell-bound antibody which was quantitated with a gamma counter. The patient’s whole blood had a normal level of cell-bound GP Ilb/IIIa, but a markedly reduced level of cell-bound GP lb (5% of normal mean; n = 20). (3) Whole blood smear immunocytochemical staining with the monoclonals (indirect immuno-alkaline phosphatase technique), and qualitative analysis by light microscopy, revealed a marked reduction of GP lb expression by the patient’s giant platelets, whereas GP Ilb/IIIa expression was normal. This latter technique might be especially valuable as a screening technique when the patient is not directly available for laboratory study. Together with the patient’s life-long history of thrombocytopenia and moderate bleeding diathesis, and other laboratory observations including markedly prolonged bleeding times and reduced whole blood prothrombin consumption, these data established diagnosis of BSS. We conclude that these three relatively simple assays of platelets in whole blood should be of particular value in the laboratory differential diagnosis of patients with congenital thrombocytopenias and giant platelet syndromes.
THE BLEEDING TENDENCY OF PROGRESSIVE RENAL FAILURE IS NOT ASSOCIATED WITH DEFECTIVE PLATELET AGGREGATION
Authors: Gordge M P , Faint R W , Rylance P B , Neild G H
Published: August 2018

The bleeding tendency of uraemia may be related to reduction by anaemia of erythrocyte/platelet interaction, toxic inhibition of platelet aggregation and abnormal von Willebrand Factor (vWF) mediated platelet adhesion. Our aim in this study was to determine at what stage of renal failure bleeding time becomes prolonged and to investigate the mechanisms involved.

We have measured bleeding time (Simplate II), plasma levels of fibrinogen and vWF, and ex-vivo platelet responsiveness in 31 patients with chronic renal failure (CRF) of various degrees of severity and compared them with values obtained in 22 healthy controls. No patient was dialysed, nephrotic or suffering from immunological renal disease. Patients were divided into mild (plasma creatinine <300 umol/1), n=10, moderate (300-600 umol/1), n=14, or severe (>600 umol/1), n=7, CRF.

Bleeding time became significantly prolonged only in severe CRF (p<0.005). Haematocrit fell as renal failure advanced, and correlated with bleeding time (r=0.40, p<0.05). Platelet counts were normal. Platelet aggregation in response to ristocetin (mediated by vWF) and ADP increased progressively (p<0.005 in severe CRF), as did spontaneous aggregation (p<0.005 in severe CRF). This was associated with an increase in plasma vWF and fibrinogen (p<0.005 in severe CRF). Collagen induced aggregation was slightly, but not significantly increased. Thromboxane (TxB2) generation in clotting blood was the only measurement that showed a reduced platelet response (p<0.025 in severe CRF).

In summary, a bleeding tendency develops late in the course of progressive CRF when plasma creatinine has risen to at least 600 umol/1. Platelet aggregation is enhanced rather than reduced and platelet interaction with vWF is not defective. Anaemia appears more important than abnormal platelet aggregation in mediating uraemic bleeding, although reduced serum TxB2 generation suggests a defect in platelet response to endogenous thrombin which may also contribute. Increased platelet aggregation and fibrinogen concentrations might promote glomerular thrombosis and contribute to the progression of CRF.
INCIDENCE AND CHARACTERISTICS OF MACROTHROMBOCYTOPENIA IN A MEDITERRANEAN AREA
Authors: Casals F J , Moreno R , Alfonso L , Alonso A , Guerrero R
Published: August 2018

Idiopathic Thrombocytopenia Purpura (ITP) is diagnosed by exclusion in presence of a chronic thrombocytopenia, with evidence of bone marrow megakaryocytes and exclusion of associated diseases shortened platelet survival (PS), increased platelet volume (PV) and platelet associated immunoglobulin G (PAIgG) support this diagnosis. In subjects from mediterranean area, the idiopathic macrothrombocytopenia (IM) has been reported. This entity could be erroneously considered as a moderate type of ITP.

To evaluate the incidence and characteristics of the IM we studied in 147 patients with not secondary megakaryocytic chronic thrombocytopenia, the PS by the 111-Indium-Oxine method, the PV distribution in citrated blood with the Coulter ZBI- Channelyzer C1000 and the PAIgG by the antiglobulin consumption assay.

In 20 cases we found a macrothrombocytopenia, along with normal PS in 12 (Group A) or a near normal PS but marked facts of idiopathic hyperesplenism in the other eight (Group B ).

25 % patients in the Group A and 57%in the Group B had increased PAIgG , more marked in the latter.

Out of 147 patients studied, we found 40 with moderate thrombocytopenia (>40×10 plts/l) and 20 subjects were studied in remission (>100×10 plts/l), in all cases the PS was shortened.

In conclusion we think that idiopathic macrothrombocytopenia and the idiopathic hypersplenic thrombocytopenia could be the broad spectrum of the same entity, with a high prevalence inthe mediterranean area. 14% of the patients studied had this alteration and this incidence increase to 35% when considering exclusively the moderate thrombocytopenias.
PLATELET SUBPOPULATION HETEROGENEITY IN IDIOPATHIC THROMBO-CYTOPAENIA AND ESSENTIAL THROMBOCYTHAEMIA STUDIED BY CONTINUOUS FLOW ELECTROPHORESIS: MORPHOLOGICAL AND BIOCHEMICAL CHARACTERISATION
Authors: Crook M , Machin S J , Crawford N
Published: August 2018

Using continuous flow electrophoresis (CFE) normal human platelets have been separated into a surface-charge dependent profile (roughly gaussian) extending over 15-20 fractions with an electrophoretic mobility range between 0.86 and 0.93 μmetres/sec Volt cm. The technique has also been used to study platelets from adult patients with Stable Essential Thrombocythaemia (platelet counts - 600-800 × 109/L) and chronic Idiopathic Thrombocytopaenia (counts 15-80 × l09/L). Neither group was receiving specific therapy during the investigations. The platelets from both patient groups showed significant anodal shifts in electrophoretic mobilities in comparison with the CFE profiles from normal adults. Sialic acid, a negatively charged sugar moiety, associated with membrane glycoproteins is a major contributor to the platelet surface electronegativity and both total platelet sialic acid and surface neuraminidase-labile sialic acid measurements were made using the thiobarbituric acid procedure. Platelets from the Idiopathic Thrombocytopaenic group showed (with respect to normal levels) significant increases in both total and neuraminidase-labile sialic acid (expressed in terms of platelet number). Platelets from the Essential Thrombocythaemic group did not differ significantly in sialic acid contents from platelets from normal subjects. Platelet sizes (Coulter counter volumes) were also measured across the normal and patient platelet electrophoretic profiles together with morphological monitoring. In platelets from ITP patients these more electronegative platelets were substantially larger and of more bizarre shapes than those of normal patients when viewed with scanning and transmission electron microscopy, whereas platelets from the Essential Thrombocythaemic group showed less significant volume increases. These findings of marked changes in platelet surface electrokinetic and analytical properties in patients with the two disorders suggest that studies in other disease states, involving disturbances of thrombokinetics, may be warranted and particularly should be followed through therapeutic programmes.
QUININE CAN INHIBIT OR ENHANCE PRODUCTION OF MURINE ANTI-HUMAN PLATELET ANTIBODIES
Authors: Christie D J , Lennon S S , Wischnack L L
Published: August 2018

Quinine (Qn) can provoke potent antibodies capable of destroying platelets and inducing life-threatening immunologic thrombocytopenia (DITP). A question critical to understanding the mechanism of DITP is why do platelets from all normal individuals express Qn-induced antigens while only relatively few individuals make the destructive drug-dependent antibodies? This problem was investigated in a murine animal model. BALB/c mice (6-12wk) were injected intraperitoneally, every other week, with saline or lOOOpg of Qn (this dose of drug gave serum levels comparable to human therapeutic serum levels). Two wk after the second injection, mice were immunized with a single dose of 108 human platelets and either 0 or 1000μg of Qn. One wk later, mouse serum was screened in the presence and absence of drug for anti-platelet activity by a complement-dependent 51Cr release assay. Results are shown in the table and represent data from at least 2 groups of five mice each.

Antibody titers were more than 10-fold lower in mice receiving 1000 |lg Qn than in mice injected with platelets alone. In contrast, mice repeatedly immunized (3-6 injections) with platelets and low doses of Qn (20μg) developed enhanced antibody activity (drug-dependent and nondrug-dependent) that consistently titered two-to four-fold higher than mice injected with platelets alone. Results were confirmed by an enzyme-linked immunosorbent assay which showed that the murine antibodies were IgG. These findings demonstrate that by varying the dose, Qn can either inhibit or enhance production of anti-human platelet antibodies in mice. This suggests the possibility that most individuals fail to respond to Qn because therapeutic doses of the drug inhibit antibody formation; yet in individuals capable of responding, even low doses of Qn (as found in tonic water) can enhance production of antibodies that provoke DITP.
MONOCLONAL ANTI-CYTOSKELETON ANTIBODY DERIVED FROM AN ITP PATIENT
Authors: Varon D , Linder S , Gembom E , Guedj L , Berrebi A , Eshhar Z
Published: August 2018

A monoclonal anti-platelet IcpI antibody was established by the fusion of splenocytes from Immune Thrombocytopenic purpura (ITP) patient with a human-mouse-hetercmyelcma cell line. The splenic lynphocytes were cultured with platelets and lipopoly saccharide for 9 days prior to fusion. Anti-platelet activity in the hybridcma supernatant was tested by the ELISA technique using plastic adherent platelets as antigen. Out of three hybridomas that demonstrated anti-platelets activity, one (4G9) appeared to be stable producer of IgM, was cloned and served for further characterizations. In immunofluorescence studies it was found that 4G9 stains both platelets and mononuclear cells, exhibiting mainly intracellular pattern. When different cell lines were fixed and stained, specific decoration of cytoskeletal elements was detected on normal and transformed fibroblasts and not in epithelial or carcinoma cell lines. To characterize the antigen reacting with 4G9 antibody, whole platelet lysate was electrophoreased on SDS-PAGE, transferred to nitrocellulose filter and reacted with 4G9 followed by peroxidase anti-Ig staining. Major band of 45 Kd corresponding to act in, and minor band of 16 Kd reacted with 4G9 but not with control antibodies. The anti-actin activity of 4G9 was confirmed by immunoblot using purified rabbit-muscle actin, and by ELISA, using act in coated plates. Whether this autoantibody plays a role in the pathogenesis of ITP, or whether it belongs to the family of natural autoantibodies is under investigation.
ANALYSIS OF PLATELET ANTIGENS FOR ANTI-PLATELET ANTIBODIES IN ITP USING FLOW CYTOMETRY
Authors: Nomura S , Nagata H , Sone N , Oda K , Kokawa T , Yasunaga K
Published: August 2018

Idiopathic thrombocytopenic purpura (ITP) is a syndrome caused by circulating antibodies reactive with the platelet membrane. The antigenic specificity of these antibodies is unknown. We have characterized new monoclonal antibodies that react with a determinant specific to GP Ib and GP Ib- Ia complex, and used flow cytometry to investigate platelets in ITP for antigenic determinants to which autoantibodies are directed. Forty cases of ITP were analyzed in detail by the platelet suspension immunofluorescence test(PSIFT) of von dem Borne et al. The monoclonal antibodies used were 5 against GP Ib-Ia complex (NNKY1-32, NNKY2-5, NNKY2-6, NNKY2-11, NNKY2-18) and 2 against GP lb (NNKY5-4, NNKY5-5). The reactivity of monoclonal antibodies was inhibited by the presence of autoantibody on platelets in some ITP patients. Differences in inhibition were found not only between monoclonal antibodies but also between cases.

These results suggest that some ITP patients have circulating antibodies to GP Ib or GP II b - Ia, and that heterogeneous antibodies are present on platelets. Moreover, the presence of these autoantibodies may aggravate or initiate a bleeding tendency.
THE EFFECT OF INTRAVENOUS IMMUNOGLOBULIN ON PLATELET KINETICS IN CHRONIC IMMUNE THROMBOCYTOPAENIC PURPURA (ITP)
Authors: Badenhorst P N , Kotze H F , duP Heyns A , Lotter M G , Wessels P , Roodt J P
Published: August 2018

Most patients with ITP respond to high doses of intravenous immunoglobulin (IVIg) with a transient increase in platelet count. The effect of IVIg on platelet kinetics was studied in 5 patients with chronic ITP. Autologous platelets were labelled with In-111 and mean platelet lifespan (MPLS) calculated; in vivo distribution and sites of platelet sequestration were determined with a scintillation camera and computer assisted image analysis. The studies were performed before and after treatment with 2 g/kg Sandoglobulin. Two groups of patients were identified: those with a splenic platelet sequestration pattern (spleen-liver In-111-activity ratio >1.4) and those with diffuse sequestration of platelets in the reticuloendothelial system (RES).

There was a significant difference in mean platelet counts before and after treatment (p<0.05). Patients with a splenic sequestration pattern responded better to IVIg: the MPLS lengthened and the high spleen-liver ratio decreased. In the diffuse RES sequestration pattern group, IVIg had almost no effect on platelet kinetics. We conclude that platelet kinetic studies identify a subgroup of patients with ITP who will respond to IVIg therapy.
COAGULATION STUDIES IN THROMBOTIC THROMBOCYTOPENIC PURPURA, WITH SPECIAL REFERENCE TO VON WILLEBRAND FACTOR ABNORMALITIES
Authors: Takahashi Hoyu , Tatewaki Wataru , Nakamura Tadao , Hanano Masaharu , Wada Ken , Shibata Akira
Published: August 2018

The profile of blood coagulation and fibrinolysis was studied in detail in 8 patients with thrombotic thrombocytopenic purpura (TTP), who had most of the characteristic findings such as fluctuating neurologic signs, schistocytic hemolytic anemia, marked thrombocytopenia, renal abnormalities and fever. Fibrinogen (2.2-3.6 g/L) and factor XIII levels were normal in all patients, while FDP values were slightly elevated (from below 5 to 4-0 mg/L). Anti thrombin III, alpha 2-plasmin inhibitor and plasminogen were normal in all patients except one with elevated FDP. Alpha 2-macroglobulin was normal as well. Plasmin-alpha 2-plasmin inhibitor complex measured by an enzyme-immunoassay was either normal or marginally elevated. Tissue-type plasminogen activator antigen was elevated to 5.2-14.5 μg/L. Protein C activity and antigen were either normal or elevated, while protein S antigen was decreased in 3 patients. Factor VIII activity and von Willebrand factor antigen (vWf:Ag) and ristocetin cofactor (RCcf) were either normal or elevated, but RCof/vWf:Ag ratio was decreased (mean 0.546 ± SD 0.1876). Crossed immunoelectrophoresis and SDS-agarose gel electrophoresis revealed that the hemostatically most active, high-molecular-weight vWf multimers were absent from or relatively decreased in TTP plasma. In addition, a vWf fragment with a faster mobility than the major vWf was demonstrated in some patients. Histidine-rich glycoprotein and fibronectin were decreased in 3 and 4 patients, respectively. Most of these abnormal findings were nearly normalized in remission. Although the pathogenesis of TTP is still uncertain, these results indicate that in contrast to disseminated intravascular coagulation, the intravascular generation of thrombin and plasmin was minimal in TTP, and suggest that the high-molecular-weight multimer vWf and fibronectin are consumed probably due to their participation in platelet thrombus formation in addition to platelet aggregating factor.
VASOCONSTRICTION IN RESPONSE TO HUMAN PLATELET-VESSEL WALL INTERACTIONS
Authors: Van Nueten J M , Janssens W J , De Clerck F
Published: August 2018

Human blood platelets, stimulated with thrombin, induced contractions of isolated basilar artery segments of the dog. These platelet-mediated vascular contractions were inhibited in a concentration-dependent way by flunarizine, a Ca2+-entry blocker, selective for vascular tissues. This inhibition increased gradually as a function of time after contact with flunarizine to reach its maximum after 60-90 min. Biochemical and pharmacological analyses, using the 5-HT2-serotonergic antagonist ritanserin, the thromboxane A2/prostaglandin endo-peroxide antagonist BM 13.177 and the fatty acid cyclo-oxygenase inhibitor suprofen, showed that 5-hydroxytryptamine and prostanoids (thromboxane A2, prostaglandine endoperoxides) were the main mediators involved. They further suggested amplification between 5-hydroxytryptamine and prostanoids at the vascular level.

(1) Incubation period; (2) Inhibition of platelet-mediated vascular contractions.

This study demonstrates that 5-hydroxytryptamine, acting in concert with thromboxane A2 and/or prostaglandine-endoper-oxides, is responsible for the vasoconstrictor effects of aggregating platelets. It further indicates that influx of calcium ions is involved in these vasoconstrictor responses.