bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphoinositide metabolism in resting and thrombin-stimulated human platelets: Evidence for metabolic homogeneity
Authors: Tysnes Q-B , Verhoeven A J M , Aarbakke G M , Holmsen H
Published: August 2018

On the basis of differences in specific radioactivity (SA), separate pools of phosphoinositides have recently been proposed in platelets. Human platelets were labelled for 60 min with [32p]p- and subsequently transferred to a phosphate- and Ca2+free Tyrode’s solution by gel-filtration. Thereafter, the platelets were either incubated at 37°C for 120 min, a condition which induces increase in specific labelling of the diester phosphate of phosphatitylinositol (PI), or stimulated with 0.5 U/ml of thrombin. The changes in SA of both diester and monoester phosphates of the phosphoinositides were detrmined. Immediately after the gel filtration, the SA of the diester phosphate of phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) were both similar to that of PI and amounted to 4% or the SA of the monoester groups of PIP and PIP2- Whereas the SA of the monoester phosphate essentially remained constant and the same for PIP and PIP2 during the entire incubation, the SA of their diester phosphates increased gradually in parallel to that of PI, and reached 20% of the monoster groups after 120 min. The effect of thrombin was studied at 15, 60 and 180 sec after the addition. The absolute radioactivity of both diester and monoester phosphates of all phosphoinositides increased conciderably after an initial decrease. However, for the monoester groups, the changes in radioactivity were parallelled by the changes in mass for both PIP and PIP2. Thrombin therefore induced no changes in SA of the monoester phosphates. In contrast, the SA of the diester phosphates increased 5-fold and remained similar for all three phosphoinositides during the 180 sec of stimulation.

In conclusion, our results demonstrate close metabolic equilibrium between all three phosphoinositides.

Thrombin-induced changes in SA of PIP and PIP2 are purely secondary to changes in specific labelling of the diester phosphate.
Formation and metabolism of inositol 1,4,5 trisphosphate in human platelets
Authors: Smith J B , Dangelmaier C A , Daniel J L
Published: August 2018

[3H]Myo-inositol (1,4,5)trisphosphate ((1,4,5)IP3), when added to lysed platelets, was rapidly converted to [3H]inositol ,3,4,5)tetrakisphosphate which was in turn converted to [3H]inositol (1,3,4)trisphosphate. This result demonstrates that platelets have the same metabolic pathways for interconversion of inositol polyphosphates that are found in other cells. Labelling of platelets with [32P]orthophosphate, followed by h.l.p.c. was used to measure thrombin-induced changes in the three inositol polyphosphates. Interfering compounds were removed by a combination of enzymatic and nonenzymatic techniques. [32P]-(1,4,5)IP3 was formed rapidly and reached its maximal level at about 4 sec. It was also rapidly degraded and was no longer detectable after 30-60 sec. Formation of
(1.3.4.5)IP4 was almost as rapid as that of (1,4,5)IP3 and remained at detectable levels for a longer time. (1,3,4)IP3 was formed after an initial lag and this isomer reached its maximal level that was ten-fold higher than that of (1,4,5)IP3 at 30 sec. Comparison of the intracellular Ca2+ concentration as measured with fura-2 indicates that agents other than (1,4,5)IP3 are responsible |or the sustained maintenance of a high level of intracellular Ca2+. It is proposed that either (1,3,4)IP3 or (1.3.4.5)IP4 may also be Ca2+ -mobilizing agents.
ADRENALINE POTENTIATES Ca2+ INFLUX AND INTRACELLULAR MOBILIZATION INDUCED BY PAF AND THROMBIN IN QUIN 2 LOADED PLATELETS
Authors: Powling M J , Hardisty R M
Published: August 2018

In a medium containing 1 mM extracellular calcium (Ca2+o), sub-threshold concentrations of thrombin (0.01-0.02 u/ml) and PAF (0.1-0.2 nM) induced shape change but not aggregation and raised [Ca2+i] about 200 nM above the basal level of 80 nM. Addition of 0.5 uM adrenaline (which produced no effect alone) 15-30 seconds before those concentrations of second agonist induced rapid aggregation and substantial ATP secretion. In parallel, the rate and magnitude of the Ca2+i flux were both increased: each approximately doubled in response to thrombin while in response to PAF the rate increased about 2.5 fold and the maximum response by about 50%. In the presence of Fab fragments of Ml48, a monoclonal antibody against GPIIb/IIIa, adrenaline produced the same effect on the Ca2+i response although aggregation and ATP secretion were completely abolished.

In the presence of 2 mM EGTA ([Ca2+o] = <100 nM) the threshold concentrations of PAF and thrombin raised the [Ca2+i] from 60 nM only to about 110 nM. Under these conditions pre-incubation with 0.5 uM adrenaline did not induce aggregation or ATP secretion, nor was there a significant increase in the maximal extent of Ca2+i flux.

The rate of Ca2+i mobilization, however, was approximately doubled in response to thrombin and increased by about 50% in response to PAF.

Blockade of cyclo-oxygenase with aspirin did not inhibit the adrenaline-induced potentiation of Ca2+i mobilization whether Ca2+o was 1 nM or <100 nM.

It appears that adrenaline may exert its synergistic effects on low doses of other agonists by accelerating Ca2+ influx and intracellular mobilization.
POSSIBLE ROLE FOR THE CA2+-DEPENDENT PROTEASE (CALPAIN I) AS AN IRREVERSIBLE ACTIVATOR OF CA2+/CALMODULIN-MEDIATED REACTIONS IN THE HUMAN PLATELET
Authors: Wallace Robert W , Tallant E Ann , Brumley Lynn M
Published: August 2018

Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and 125I-CaM. Ten proteins of 245, 225. 175, 150, 90. 82(2), 60 and 41(2) kilodaltons (kDa) bind 125I-CaM in a Ca2+-dependent manner; the binding is blocked by both trifluoperazine and nonradiolabeled CaM. The 225 and 90 kDa proteins are labeled by antisera against myosin light chain kinase (MLCK); the 60 kDa and one of the 82 kDa proteins have been identified as the CaM-dependent phosphatase (calcineurin) and caldesmon. The other proteins are presumed to be other Ca2+/CaM regulated enzymes and proteins which may be important in platelet function. Most of the CaM-binding proteins are degraded upon addition of Ca2+ to a platelet homogenate; the degradation may be blocked by either EGTA, leupeptin or N-ethylmaleimide which suggests that the degradation is due to a Ca2+-dependent protease. Activation of intact platelets under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca2+) also results in limited proteolysis of CaM-binding proteins including those labeled with anti sera against MLCK and the phosphatase. In vitro studies utilizing purified phosphatase and calpain I indicate that the phosphatase is irreversibly activated upon Ca2+-dependent proteolysis. The proteolytically-activated enzyme is insensitive to either Ca2+ or Ca2+/CaM; in addition, its activity in the absence of Ca2+ is even greater than the activity of the unproteolyzed enzyme in the presence of Ca2+ and CaM. Proteolytic stimulation of the phosphatase is accompanied by degradation of the 60 kDa subunit of the enzyme (subunit A) to 56, 52 and 45 kDa fragments, sequentially; proteolysis results in the loss of CaM binding to the enzyme. These results suggest that the Ca2+-dependent protease may have a physiological role in platelet activation as an irreversible activator of Ca2+/ CaM-dependent reactions. Supported by NIH grant HL29766.
RELATIONSHIP BETWEEN ELEVATION OF CYCLIC-3∲,5∲-GMP (cGMP) AND AGGREGATE FORMATION IN HUMAN PLATELETS
Authors: Edgecombe M , Scrutton M C , Kerry R
Published: August 2018

Maximal 3- to 5-fold increases in platelet cGMP levels as measured by specific radioimmunoassay are observed on stimulation of aspirin-treated platelet-rich plasma with saturating doses of ADP, adrenaline, 5HT, PAF, thrombin, 9,11-epoxymethano-PGH2 (U44069), collagen, ristocetin and the Ca2+-ionophore ionomycin. The dose/response curves for the elevation in cGMP induced by these agents are either superimposable on, or lie to the right of, those describing the rate or extent of the aggregatory response as measured by an increase in light transmittance. The increase in cGMP induced by ADP is totally inhibited by addition of PGI2 or forskolin with dose/response relationships superimposable on those observed for inhibition of the aggregatory response. No increase in cGMP is observed if platelets are stimulated by PAF or ionomycin in an unstirred system or when aggregation induced by ADP is prevented by addition of a monoclonal antibody which recognises the glycoprotein IIb/IIIa complex.

Addition of the fibrinogen γ-chain C-terminal decapeptide (γ402-411) or α-Chain tetrapeptide ARG-GLY-ASP-SER prevents aggregation and the increase in cGMP induced by PAF. The γ-chain decapeptide also completely prevents the increase in cGMP induced by ristocetin, but the a-chain tetrapeptide is ineffective in this respect. Both peptides inhibit to some extent aggregate formation induced by ristocetin.

The data demonstrates a strong correlation between aggregate formaticfn and the increase in the platelet cGMP levels and support the previous postulate that platelet-platelet contact causes, activation of guanylate cyclase. No relationship is apparent between the effects of the various agents tested on cGMP levels and their known ability to increase cytosolic Ca2+ concentration. (Supported by SERC and Ciba-Geigy.)
COMPARISON OF THE PLATELET AGGREGATION INDUCED BY THREE THROMBIN-LIKE ENZYMES OF SNAKE VENOMS AND THROMBIN
Authors: Teng C M , Ko F N
Published: August 2018

Acutin was isolated from Agkistrodon acutus venom and batroxobin and thrombocytin were isolated from Bothrops atrox venom. These three thrombin-like enzymes had different specificity for platelet activation and fibrinogen clotting. The clotting activities were 700, 170 and 7 μ/mg for batroxobin, acutin and thrombocytin, respectively. They induced aggregation and ATP release of washed rabbit platelets. The aggregating activities were 102, 104 and 105 times less than that of thrombin for thrombocytin, acutin and batroxobin, respectively basing on the clotting unit. The platelet -activating potency was correlated with their effectiveness on the retractility and elasticity of the clots. Platelet aggregation induced by thrombin or thrombocytin could be inhibited by heparin with antithrombin III while that by acutin or batroxobin could not. The thrombin-like enzymes did not induce aggregation of thrombin-degranulated platelets even fibrinogen was added. Indomethacin showed weak inhibition on the aggregation while the ADP - scavenging system, creatine phosphate/creatine phosphokinase, or apyrase inhibited the aggregation induced by the three thrombin-like enzymes but not that by thrombin. In the presence of EGTA, only thrombin could induce ATP release from platelets. It is concluded that the aggregation induced by thrombin-like enzymes is different from that of thrombin and mainly due to ADP released from platelets.
AUSTRALIAN SNAKE VENOMS AND THEIR EFFECT UPON HUMAN PLATELETS
Authors: Marshall L R , Herrmann R P
Published: August 2018

The in vitro effect of Australian snake venoms on human citrated plasma has been documented and the majority induce coagulation, in keeping with the common clinical presentation of D.I.C. following envenomation. The effect of these venoms upon platelets in vitro has hitherto not been studied extensively and clinical evidence is conflicting, some cases with thrombocyto-paenia have been reported. Twenty Australian venoms were tested, 19 elapids and one hydrophiid (Enhydrina schistosa). Four crotalid snake venoms from the Americas and S.E. Asia were also tested. All of the venoms (1 mg/ml) were investigated for t^eir ability to aggregate both fresh washed platelets (200 × 109/l) resuspended in modified Ardlie’s buffer pH 7.35 and formaldehyde fixed platelets (200 × 109/l) in phosphate buffered saline pH 7.35 using a dual channel Chronolog aggregometer. Samples were taken for electron microscopy (EM).

All elapid venoms induced aggregation in fresh platelets, some only minimally and often after a long lag phase. EM studies revealed only clumping without degranulation of the platelets. This was in marked contrast to the crotalid venoms where rapid aggregation and gross degranulation occurred. The hydrophiid venom failed to induce aggregation of the fresh platelets, however upon addition of normal plasma gross aggregation and degranulation was demonstrated. Aggregation of fixed platelets was negligible in the presence of the majority of elapid and the hydrophiid venoms. The crotalid venoms however did induce aggregation, although to a lesser extent than with the fresh platelets.

The elapid venoms, along with the others studied, required metabolically active platelets to exert their maximal effect. Crotalid and hydrophiid venoms were more active against platelets than the elapid venoms. The hydrophiid venom’s action on platelets was unique in that a plasma co-factor appeared to be required and this is the subject of further investigations.
INHIBITION OF HUMAN PLATELET AGGREGATION BY THE THROMBOXANE MIMETIC, U46619
Authors: Stratton L , Hornby E
Published: August 2018

Recent publications suggest that high concentrations (10-100μM) of the stable analogues of PGH2, U46619 and U44069 stimulate adenylate cyclase (Avdonin et al.. 1985) and cause an elevation in cAMP in intact platelets (Best et al., 1979). The effect of high concentrations of U46619 on responses to ADP [1.0-30μM] and vasopressin [1-10nM] was investigated in human platelet rich plasma preincubated with the thromboxane antagonist, GR32191, [10μM] (Lumley et al., this meeting) and aspirin [0.1mM]. Concentration-effect curves to ADP were not affected by preincubation with O.lmM U46619 in the presence of GR32191 [10μM]. Under the same conditions, aggregation to vasopressin was inhibited. U46619 at 30μM had no effect on the vasopressin concentration-effect curve, but at 0.1mM, it resulted in an eight-fold rightwards shift and also reduced the maximum response by 30%. This was equivalent to the inhibition of vasopressin-induced aggregation achieved with prostacyclin at a concentration of 1nM in the same experiments. At 0.3mM, U46619 caused total inhibition of aggregation to vasopressin, equivalent to that seen with prostacyclin at 3nM. The failure of U44619 to inhibit ADP-induced aggregation may reflect inhibition of adenylate cyclase by ADP (Cusack et al. 1982). Aggregation induced by vasopressin is independent of inhibition of adenylate cyclase (Thomas et al.. 1983) and under these conditions, U46619 may induce increases in cAMP which exert an inhibitory action on platelet function. These results indicate that experiments using U44619 as an aggregating agent should be interpreted with caution. The high concentrations of U46619 required to induce aggregation in the presence of thromboxane antagonists may also cause stimulation of adenylate cyclase, thereby making an additional contribution to the inhibition observed. Avdonin P.V. et al.f Thromb. Res., 40, 101-112, 1985.

Best L.C. et al.. Biochim, Biophys. Acta, 583. 344-351, 1979.

Thomas M.E. et al.. Thromb. Res., 32, 557-566, 1983.

Cusack et al.. Br. J. Pharmac., 76, 221-227, 1982.
ABNORMALITIES OF PLATELET AGGREGATION AND ENHANCED FACTOR X ACTIVATOR ACTIVITY OF WASHED PLATELETS IN SICKLE CELL DISEASE
Authors: Chamone D A F , Hoshikawa-Fujimura A Y , Massumoto C , Bellotti G , Arashiro F , Jamra M
Published: August 2018

The occurence of microvascular occlusion is one of the most prominent pathologic features of sickle cell anemia. The mechanism of vaso occlusion has generally been attributed to the abnormal shape and reduced deformability of the sickled erithrocy tes. However, the involvement of vascular endothelium, platelets and their interactions with coagulation factors may also be of pathogenic significance in microvascular occlusive crises.

We investigated the interaction between vascular endothelium, platelets and blood coagulation factors in 23 patients with Sickle Cell Disease (SCD) and in normal volunteers.

Factor X activator activity in washed platelets was performed according to Semeraro and Vermylen (1977), thromboxane B2 (TXB2) and 6-keto-PGF1β were determined using specific radioimmunoassays.. PAF-acether from platelets was determined according to Chignard et al (Nature, 1979, 279:799). Platelet aggregation was performed with a Chrono-Log Aggregometer (Model 440) on platelet rich plasma (PRP) using the Born method. Prostacyclin release from endothelium was performed according to Mon-cada et al (Lancet i:18, 1977).

Our results showed that platelets from patients with SCD ha ve enhanced factor X activator activity (p < 0.0001), produce mo re PAF-acether than controls (p < 0.02) and showed hyperaggregability in these patients as compared to normal volunteers (p < 0.00001).

We concluded that platelets from homozygous sicklers have enhanced factor X activator activity as well as increased capacity for PAF-acether production. These abnormalities may contribute to the incidence of vaso occlusive crises in these patients.
RIBAVIRIN: STUDIES OF THE EFFECTS OF THE ANTIVIRAL DRUG ON PLATELET FUNCTION
Authors: Cosgriff T M , Canonico P G , Hodgson L , Parrish D , Chapman T , Huggins J W , Gong Z-J , Xiang L-B , Hsiang C-H
Published: August 2018

Ribavirin is a broad-spectrum antiviral drug which is presently undergoing testing in patients with AIDS-related complex. It has also been shown to have activity against respiratory syncytial virus, Sicilian sandfly fever virus, influenza A and B viruses, as well as several hemorrhagic fever viruses. It has proved effective in clinical trials in Lassa fever and shows promise as therapy for hemorrhagic fever with renal syndrome. Because platelet dysfunction may contribute significantly to hemostatic impairment in viral hemorrhagic fever, the effects of ribavirin on platelet function were measured in rhesus monkeys after daily injections of 100 mg/kg IM for 14 days. Drug administration led to a significant increase in platelet count associated with megakaryocyte hyperplasia but had no effect on aggregation of platelet-rich plasma (PRP) in response to either collagen (1.6 μg/ml) or ADP (10 μM/ml). Aggregation in whole blood was also unaffected. Addition of ribavirin to human PRP in concentrations up to 0.5 mg/ml had no effect on aggregation in response to collagen, ADP, or epinephrine (5 μg/ml). Preliminary data from Chinese patients treated with ribavirin for hemorrhagic fever with renal syndrome also reveal no evidence of drug-induced platelet dysfunction as indicated by normal aggregation and release reactions to collagen (3 μg/ml) and ADP (10 μM/ml). Bone marrow studies of megakaryocyte number and ploidy are presently underway to further characterize drug-associated thrombocytosis
TRANSITORY INFLUENCE OF FIBRINOGEN/FIBRIN DEGRADATION PRODUCTS (FDPs) ON PLATELET AGGREGATION
Authors: Nelson S D , Moriarty A J , Hughes R , Balnave K
Published: August 2018

This paper describes a pilot study to investigate the influence of FDPs on platelet aggregation in a small cohort of patients (N = 12) undergoing systemic thrombolytic therapy with streptokinase (600,000 I.U. or 1,500,000 I.U. delivered over 30 minutes) for acute myocardial infarction.

Serial pre- and post-therapy blood samples were anticoagulated with sodium citrate, and whole blood aggregation studies carried out over 24 hours using a Crono-log 540 aggregometer and the standard adenosine diphosphate (ADP), adrenalin (A), collagen (C) and ristocetin (R) aggregating agents.

Results, in the form of mean percentage voltage change from baseline voltage change, measured at 8 minutes after addition of aggregant, are presented for the cohort at times in Figure 1. Serial aggregometry tracings for one representative patient are shown in Figure 2.

Clearly comparison of the 1 hour and 18 hour results for each aggregating agent shows a variable but consistent return towards baseline (at FDP < 8 μg/ml) as the FDP concentration drops. This implies that, provided the same platelet population is involved, there is no generalised permanent platelet defect consequent on systemic STK therapy. Coulter counter measurements do not indicate the increase in platelet number that would suggest a large influx of new platelets.
PREPARATION OF A REPRESENTATIVE PLATELET POPULATION BY A SINGLE STEP SLOW CENTRIFUGATION
Authors: Denfors I , Wadenvik H , Kutti J
Published: August 2018

Circulating platelets differ with respect to size, density and functional ability. In several experimental settings it is of vital importance that a representative platelet-rich plasma (PRP) is extracted from the anticoagulated blood. We therefore investigated the effect of centrifugation time and gravitational force on platelet yield and platelet volume distribution in PRP obtained by a single step slow centrifugation.

Methods. From each of 12 healthy male blood donors, 357 ml of venous blood were mixed with 63 ml citrate phosphate dextrose in a plastic bag. 20 ml aliquots of the anticoagulated blood were transferred into 30 ml screwcap polycarbonate tubes (Nalgene Labware). PRP was prepared by a single step slow centrifugation in a microprocessor-controlled bench centrifuge (Hettich Rotanta/RP). The gravitational force was calculated at the bottom of the tube. Centrifugations were performed at 180, 200, 220, 240, 260 g for 10 min, at 180, 200, 220, 240, 260 g for 15 min and at 120, 140, 160, 180, 200 g for 20 min. Three hours after blood collection, the platelet count, mean platelet volume (MPV) and platelet distribution width were determined in PRP and anticoagulated blood with an impedance cell counter (Coulter Counter Model S-Plus VI). Student's t-test was employed for comparison of mean values.

Comments. Very precise centrifugation conditions can be provided by using a microprocessor-controlled centrifuge and a "fully representative" platelet population can be isolated with a single step slow centrifugation method.
MEASURING WHOLE BLOOD PLATELET AGGREGATION AND ATP-RELEASE WITH A CHRONO-LOG WHOLE BLOOD LUMI-AGGREGOMETER: EFFECT OF STORAGE TIME OF BLOOD SAMPLES
Authors: Sieders F C , Houwelingen A C v , Hornstra G
Published: August 2018

The influence of storing blood for either one or two hours after blood sampling, on whole blood platelet aggregation and ATP-release was measured with a Chrono-log whole blood lumi-aggregometer, in 21 healthy male volunteers. Storage of blood samples, gently revolving at 37 °C in an incubator for one hour, caused a significant increase in aggregation and release as compared with results obtained immediately after sampling. After two hours' storage, the values had returned to their initial levels.

Significant positive correlations were seen between values obtained before and after storage of blood, and between various aggregation and release parameters. In this study, bleeding time nor hematocrit values were significantly correlated with the aggregation and release parameters. The considerable influence of storage time on whole blood platelet aggregation and ATP-release underlines the importance of performing these determinations immediately after sampling, or possibly after a standardized storage time. Otherwise, comparison of results -obtained either in clinical situations or in trials - will increase variability as a result of which false conclusions may be obtained. This will be illustrated in a small trial using paracetamol.
THROMBASTHENIC-LIKE DEFECT IN PLATELETS OF THE AFRICAN GREEN MONKEY
Authors: Lewis J C , Greene M J , Taylor R G , Hantgan R R
Published: August 2018

African green monkeys, an animal model widely used in cardiovascular research, have platelets which ultrastructurally and functionally are similar to man. Through clinical screening of animals in a controlled breeding colony a congenital defect has been identified in platelets from an adolescent AG monkey, AJ403. The defect was characterized as abortive primary aggregation with ADP in the range 1.25-20.0 μM and PAF at concentrations to 5 × 10-6 M. Ultrastructurally, platelets within the aggregates had pseudopods, centralized granules and evidence of degranulation. Granule content release was verified using the luciferin-luciferase reaction in the Lumi-aggregometer. Platelet-platelet binding and close apposition, typically found with full primary and secondary aggregation, were not observed. Using a standardized in vitro assay at 37°C for 15-45 minutes, adhesion to formvar coated srufaces with AJ403 was 10-20% of control. Adherent cells had few pseudopods, minimal elaboration of the hyalomere, and limited cytoskeletal polymerization. Since the aggregation and adhesion patterns paralleled those described for Glanzmann's thrombasthenia, fibrinogen binding was quantitated using 125I fibrinogen and was ultrastructurally evaluated using fibrinogen-gold conjugates. Following ADP stimulation in the presence of fibrinogen at 0.08, 0.16 and 0.24 mg/ml, normal platelets respectively bound 64, 81 and 106 × 103 molecules fibrinogen/cell whereas platelets from AJ403 bound 28, 43 and 52 × 103 molecules/cell. This correlated with the number of gold/fibrinogen conjugates bound to the platelet surface which with AJ403 were less than 40% of control. Our observations suggest a parallel of AJ403 with Type II Thrombasthenia in man.
INTRACELLULAR CA++ MOBILIZATION IN GRAY PLATELET SYNDROME. ELECTRONMICROSCOPIC STUDIES ON AEQUORIN LOADED PLATELETS
Authors: Mori K , Suzuki S , Sugai K , Akutsu Y , Ishikawa M , Sakai H , Hiwatashi K
Published: August 2018

Light microscopic examinations on platelets in Gray Platelet Syndrome(GPS) showed peculiar gray colored platelets due to deficiency in ^-granules on the peripheral blood smear by May-Grunwald-Giemsa stain. Besides α-granule deficiency, however, several morphological abnormalities, especially abnormal features of dense tubular system(DTS) etc., were recognized in the transmission electronmicroscopic examinations. In the platelet function tests, release abnormalities rather than storage-pool deficiency were noted. We were strongly interested in the relationships between these morphological and functional abnormalities, because DTS in the platelets have been thought to be main storage sites of intracellular Ca ion.

We examined the intracellular Ca++ mobilization using aequo-rin loaded platelets by means of Lumi-aggregometer(Salzman's method) under the stimulation of A-23187 and thrombin, and also morphological changes of platelets during the process of platelet aggregation by light |ipd transmission electronmicroscope.

Intracellular Ca concentration increased dose-dependently after addition of A-23187 in both normal and GPS platelets. Namely, besides the first peak of emission which located at the same site as normal control, the slowly appearing second peak were recognized on the trace line by the addition of A-23187 and also abnormal by thrombin in GPS platelets. Transmission electronmi-crographs showed insufficient contraction of platelet-aggregates and malformation or wide appearance of pseudopods by the addition of A-23187 and thrombin. Most of the contractile gels, which were usually seen in the center of the platelets, were slightly enlarged and eccentric in the position. Delayed intracellular Ca mobilization were also noted even in the buffer solution containing EGTA.

From above mentioned results, intracellular Ca++ mobilization were abnormal and these low and delayed mobilization were thou -ght to be related with prominent abnormal morphology, especially abnormalities of DTS in the GPS platelets.
ACQUIRED STORAGE POOL DISEASE IN DECOMPRESSION SICKNESS
Authors: Yamazaki H , Tanoue K , Kuroiwa K , Suzuki H , Shibayama M , Mano Y
Published: August 2018

The presence of hemostatic abnormalities has been reported in decompression sickness. It is suggested that platelets recognize air bubbles in the blood stream as a foreign surface and adhere to them with ensuing platelet aggregation. It is important to determine if consumption of platelets occurs in vivo to understand a role of platelets in the genesis of decompression sickness. To analyse the problem, platelet behavior was studied in 34 rabbits with decompression sickness which was brought about by the exposure to 6 ATA (atmospheres absolute) for 40 min followed by rapid decompression. All
rabbits died within one hr after the decompression due to apnea which always preceded the cardiac arrest. Platelet counts decreased significantly during the time course of decompression. A regression line can be drawn between the changes in platelet count (Y) and the time after decompression (X): Y=100.2 - 0.8X, r= -0.876, p<0.001. Platelet counts measured just before the apnea were 56 to 72% (65.7 ± 6.1%) of the precompression value. Kinetic studies with 111 In-oxine-labeled platelets revealed shortened survivals of the circulating platelets and autoradiograms indicated the accumulation of radioactivity in the lungs after the decompression. Although there was no change in the mode volume of platelets after the decompression, the transient appearance of smaller or fragmented platelets suggested a random over-destruction of platelets. Whole and releasable adenine nucleotide contents of platelets decreased significantly after the decompression. There were no significant changes in cytoplasmic adenine nucleotide contents. Therefore, in decompression sickness, the circulating platelets behaved similarly to those of acquired storage pool disease. Platelet thrombi were found in the pulmonary artery, compatible with the accumulation of the labeled platelets. These findings suggest that circulating air-bubbles interact with circulating platelets, causing the platelet release reaction, and these activated platelets participate in the formation of thrombi in decompression sickness.
THE EFFECT OF RED BLOOD CELLS ON PLATELET AGGREGATION IN NORMAL SUBJECTS AND MYELOFIBROTIC PATIENTS
Authors: Balduini C L , Bertolino G , Polino G , Gamba G , Sinigaglia F , Ascari E
Published: August 2018

We investigated the effect of red blood cells (RBC) on "in vitro" platelet aggregation by the use of the "Electronic Whole Blood Aggregometer" (Chrono-Log Corporation). Preliminary experiments, studying platelet aggregation in the same PRP by the simultaneous use of the optical method and the electronic method, demonstrated that the maximum rate of impedance changes corre lated well with both the rate and the extent of platelet aggregation as measured by the optical method. The refore, the maximum rate of impedance increase was cho sen for the measurement of platelet aggregation in the presence of different concentrations of RBC. RBC, both at 40 and 60%, significantly inhibited platelet aggregation stimulated by low and high concentration of ADP and epinephrine. Platelet aggregation stimulated by co llagen was slightly reduced only by the higher RBC con centration. The effect of RBC on platelet aggregation was also investigated in idiopathic myelofibrosis, a pathological condition characterized by both platelet and RBC alterations. While on the basis of PRP studies 5 out of 17 patients had hypo-aggregation and 12 had normal aggregation, whole blood studies evidentiated hypo-aggregation in 3 patients, normal aggregation in 4 and spontaneous platelet aggregation (SPA) in 10. SPA was a consequence of platelet abnormality, since it occurred also when platelets from patients were sti rred with normal RBC.

In conclusion, RBC may exert different effects on the aggregation of normal and pathological platelets.
LIPID COMPOSITION OF PLATELETS FROM PATIENTS AFFECTED BY Ila HYPERLIPOPROTEINEMIA
Authors: Prisco D , Rogasi P G , Paniccia R , Panetta A , Coppo M , Gensini G F
Published: August 2018

Platelets from patients with familial hypercholesterolemia (IIa HLP), a condition associated with high prevalence of atherosclerosis and of its thrombotic complications, are known to be hyperresponsive to aggregating stimuli and to synthesize increased amounts of thromboxane A2. In order to search if these functional alterations can depend on a different platelet lipid composition, we studied 12 young patients ( aged 20 to 34 years) affected by Ila HLP and 12 suitable controls with similar dietary habits. Lipids were extracted from washed platelets with a chloroform/methanol (2/1) mixture. After silicic acid chromatography and thin-layer chromatography different lipid fractions were eluted and fatty acid methylesters were prepared by acid transmethanolysis. The esters were extracted with hexane and analyzed by gas-liquid chromatography. Different fatty acids were identified on the basis of retention time with respect to standard methylester mixtures and silver nitrate thin-layer chromatography. Cholesterol and lipid phosphorus were assayed by colorimetric methods. Both cholesterol and phospholipid content of platelets were higher in patients than in controls with a significant increase of cholesterol/ phospholipid molar ratio (p<0.05). The percentage content of the phospholipid fractions was not different from that of controls. On the contrary the proportion of saturated fatty acids esterified in the different phospholipid fractions was significantly increased (minimum p<0.05). In addition thromboxane A2 production by platelets from patients with Ila HLP was higher than in controls (<0.001). Our results indicate that lipid composition of platelets from patients with Ila HLP is altered and may be responsible for the enhanced platelet activity described in these patients.
PLATELET HETEROGENEITY IN MYELOPROLIFERATIVE DISORDERS: IS THERE A RELATION TO PLATELET FUNCTION?
Authors: Wehmeier A , Scharf R E , Schneider W
Published: August 2018

Morphological abnormalities and impaired platelet function are common in myeloproliferative disorders (MPD). To characterize abnormal platelet populations in MPD, we analyzed platelet volume in relation to platelet density. Platelet function and platelet secretion were evaluated in comparison to morphometric abnormalities. For morphometric analysis, blood was anticoagulated with citrate (.38%)/glutaraldehyde (.125%) and platelet density subpopulations were prepared by equilibrium centrifugation as described by Martin (Br.J.Hematol. 54: 337, 1983). The platelet volume distribution was analyzed from these fractions using the impedance method. Platelet adhesion was performed according to Hellem II. Platelet aggregation in response to ADP, collagen and adrenalin was tested in PRP. β-thromboglobulin and platelet factor 4 plasma levels were determined by radioimmunoassay. The platelet density distribution was unimodal and symmetrical with the mean modal density being 1.066±0.003 g/cm3 in 5 control subjects and 1.063±0.005 g/cm3 (x±SD) in 10 MPD patients. The patient group was heterogenous with respect to the platelet density/volume relation: One group of MPD patients (n=5) showed an increase of platelet volume with increasing density, comparable to the controls. However, in the other 5 patients, MPV was about the same in all density subfractions. There was a significant difference in modal density between the patient group that morphometrically resembled the controls ( 1.065±0.005) and those with an abnormal density/volume relation ( 1.061±0.0009 g/cm3, p±0.001). This difference is partially due to a subpopulation of light but rather large platelets that was prominent in those MPD patients with an abnormal density/volume relation. Impaired platelet adhesion and impaired aggregation in response to ADP was more often in the patient group that morphometrically resembled the control subjects. There was no difference in platelet secretion or in response to the other aggregating agents between the two patient groups. We conclude that abnormal platelet subpopulations can be identified in MPD by analysis of the platelet density/volume relation. However, these data do not indicate any consistent relation between abnormal platelet subpopulations and platelet dysfunction.
EVALUATION OF THROMBOPOIESIS IN ESSENTIAL THROMBOCYTHAEMIA BEFORE AND AFTER α-INTERFERON TREATMENT
Authors: Gugliotta L , Macchi S , Catani L , Mattioli Belmonte M , Gaggioli L , Tura S
Published: August 2018

The α-interferon (α-IFN) has been shown efficacious in controlling thrombocytosis in chronic myeloproliferative disorders. In order to better understand the mechanisms by which this effect is produced, the main parameters of thrombopoiesis have been evaluated in 8 patients with Essential Thrombocythaemia (ET)just before and at the end of induction therapy with α-IFN. The patients, 2 males and 6 females, 17-54 years old, at diagnosis or at least 3 months off cytotoxic drugs, received α-IFN (Roferon A-Roche) s.c. at a daily dose of 3 × 106 IU for 6-13 weeks. The baseline platelet count of 993±266×109/1 fell, after treatment, to a value of 377±96×109 /1. The histological analysis of the bone marrow showed that the number of megakaryocytes (MK), initially 5-15 times the normal value (N), decreased to a value of 3-6 × N, while the MK volume resulted always high. The "in vitro" study of megakaryocytopoiesis, by the plasma-clot culture technique, documented a significant decrease of the number of MK colonies (from 73±18 to 29±15; p <0.0001). The half life-span of autologous platelets labelled withulIn-oxine remained unchanged (97±22 and 101±25 hours before and after therapy, respectively), while the platelet function abnormalities (hypo-aggregation, storage pool deficiency, etc) appeared less severe after treatment.

It is concluded that in ET the α-IFN therapy is able to normalize the platelet count mainly or exclusively by a decrease of platelet production.

The work was supported in part by grant of National Research Council (CNR), special Project Oncology No 85.02200.44.
HETEROGENEITY OF MEGAKARYOCYTE PLOIDY PROFILE IN PATIENTS WITH ITP
Authors: Bussel J , Rabellino E
Published: August 2018

Thrombocytopenia in patients with ITP is ascribed to peripheral platelet destruction mediated by antiplatelet antibodies. Patients with a chronic severe form of disease may also have im paired platelet production. Profile of DNA content and distribu tion in marrow megakaryocytes (MK) is a sensitive parameter of megakaryocyte development. In these studies MK derived from clinical aspirates were analyzed by flow cytometry for DMA content and platelet glycoprotein GP IIb/IIIa. Both whole unsenar-ated and MK-enriched marrow were stained using double label staining for GP IIb-IIIa by immunofluorescence (green) and for DNA with propidium iodide (red). 3 different profiles were seen in ITP patients as indicated in the Table:

Profile I represented the commonest pattern and was identical to that seen in normals. Profiles II and III demonstrated peak ploidy values of 8C and a bimodal 2C/16C pattern respectively. In order to investigate the relationship of ploidy profile to platelet production, we compared the peak platelet response to IVGG, a treatment which stops platelet destruction, among the 3 groups. The average platelet response to IVGG was 221,000/ul for group I, much greater than both group II (119k) and group III (96k)(I vs II + III, p < 0.05). Heterogeneity of clinical cases of ITP may be caused by differences in ploidy profile of patient marrow megakaryocytes.These profiles may be caused by a heterogeneity of the target antigen of the antiplatelet antibodies in different patients with differing crossreactivity to and effects on megakaryocytes. This heterogeneity appears to be reflected in different responses to therapies suggesting that it may be useful in designing treatment strategies.
FURTHER EVIDENCES OF VON WILLEBRAND FACTOR INVOLVEMENT IN THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)
Authors: Chediak J , Eldridge J , Sobel D , Maxey B , Baron J , Telfer M C
Published: August 2018

Laboratory findings of TTP include severe thrombocytopenia and marked anemia due to intravascular hemolysis. The pathogenesis of the thrombocytopenia is poorly understood. Possible mechanisms include the presence of a platelet aggregating factor (JCI 76:1330, 1935), a calcium dependent protease (Blood 63:310a, 1986), abnormal prostaglandin production or metabolism, and an excessive consumption of high molecular weight forms of von Uillebrand factor (VUF). Von Willebrand factor proteins from 12 patients (pts) diagnosed as having TTP were studied. They include 8 females and 4 males; nine were studied during the acute presentation and seven pts also during the remission period. Three pts died during the acute event. None of the pts had a recurrence. Control subjects include both normal individuals and thrombocytopenic pts due to a variety of underlying diseases including marrow aplasia and immune thrombocytopenia, but excluding pts with DIC or suffering infection. Plasmas were tested for VWF antigen and Ristocetin Cofactor (RiCof) activity. The electrophoretic mobility (CIE) of VWF:Ag was also assessed and the ratio VWF:RiCof to VWF:Ag was determined. Statistical analysis including p values will be reported. Results:

These results suggest that during the acute event there is an excessive consumption of large VWF multimers and the ratio VWF:RiCof/VWF:Ag could be used to corroborate the diagnosis of TTP and by doing sequential measurements to monitor the response to therapy.
PLATELET AGGLUTINATING PROTEIN P37 FROM A THROMBOTIC THROMBOCYTOPENIC PURPURA PLASMA FORMS A COMPLEX WITH IMMUNOGLOBULIN G
Authors: Siddigui F A , Lian E C -Y
Published: August 2018

A 37-KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP), has been shown to be present in a subset of TTP patients. The platelet agglutination induced by PAP p37 has been demonstrated to be inhibited by igG from normal adults. To elucidate the mechanism of inhibition of IgG, the interaction between PAP p37 and IgG was studied. The complex formation was demonstrated by the binding of fluid-phase IgG to adsorbed PAP using enzyme-linked immunosorbent assay. The binding was specific, concentration dependent and saturable. The IgG covalently cross-linked to Sepharose 4B bound 125I-PAP but not to 125I-fibrinogen. Sucrose density gradient ultracentrifugation of a mixture of 125I-PAP and IgG also revealed the fluid phase complex formation with a sedimentation value of 19S. Complexes of molecular weight ranging from 180,000 to over 350,000 daltons were also detected by molecular sieve chromatography. The specific complex formation between PAP p37 and IgG is likely to account for the in vitro inhibition of TTP plasma-induced agglutination and , at least partly, the in vivo successful treatment with IgG-containing normal plasma.
RELAPSING THROMBOTIC THROMBOCYTOPENIA PURPURA (TTP): A CLINICAL STUDY OF 38 PATIENTS
Authors: Eldor A , Rose M
Published: August 2018

A dramatic change in the prognosis of TTP has taken place since the introduction of plasma therapy in 1977. However, plasma therapy does not always result in a complete cure since TTP manifestation may reappear. We describe 38 patients who were treated for TTP in 15 hospitals in Israel and New York City since 1977. 37 patients received plasma (transfusions and exchanges) and 30 survived. 12 patients (37%) developed relapsing TTP. Infections, pregnancy and surgery frequently preceded the initial episodes and the relapses. To evaluate the clinical course of relapsing TTP, a scoring system was developed based on 4 parameters: neurological findings, renal function impairment, platelet count and hemoglobin value at presentation. Each parameter was marked on a scale of 0 to 2: 0= no abnormality, 1= moderate impairment, 2= severe impairment. The severity of each episode was the sum of the 4 scores. The cases were divided into 3 groups: Group A= patients who died during 1st episode; Group B= patients who had only one episode; Group C= patients who relapsed. The following are the average severity scores:

The differences between Groups A and B; A and C; B and C; and B and the relapses are significant (p<0.005)(Chi-square test). The differences between Group C first episode* and subsequent relapses are not significant (paired t-test). Hence, relapsing TTP was manifested by a milder clinical course, however two relapses were fatal, demonstrating that this is not a benign disorder. Therapy consisted of plasma, plasmapheresis, steroids, platelet-inhibitor drugs and 11 patients underwent splenectomy. Addition of vincristin seemd to shorten the duration of the TTP episode. No differences were observed in the treatment of Group A, B and C patients. However, during relapses, patients received less therapy probably due to their milder clinical course. Splenectomy did not prevent relapses, but prolonged the remission duration. Based on our data, we recommend that the severity score be used in determining the intensity of treatment.
INCREASED SENSITIVITY OF A PLATELET-SUBPOPULATION TO CRYOPRESERVATION
Authors: van Marwijk-Kooy M , Dullemond-Westland A C , van Prooijen H C , Riemens M I , Akkerman J W N
Published: August 2018

When cryopreserved human platelets are stimulated with a high dose of collagen (40 μg/ml), about 50% of the cells fail to aggregate, as measured by counting the disappearance of single platelets. This suggests the presence of a subpopulation with increased susceptibility to cryopreservation-induced cell damage. In an attempt to identify this subfraction fresh platelet suspensions were separated into four size-dependent fractions by counterflow centrifugation. Large platelets (MPV 11.1 μm3 ) better preserved their responsiveness than small platelets (4.2 μm3) with intermediate responses for fractions of 5.7 and 8.0 μm3. In contrast, the partial loss of secretion granules (βTG and PF-4) seen during cryopreservation was greater in the large platelets than in the small cells and did not parallel the differences in aggregability.

In a second separation technique the unresponsive fraction was separated from the cells that aggregated with collagen. This fraction consisted of intact cells (LDH), and normal secretion granules (βTG-PF-4) but failed to show changes in Pl-cycle and protein phosphorylation, two essential steps in the activation mechanism of platelets. In accordance with this, the total cryopreserved suspension showed a partial reduction in these second messengers with a 35% lower phosphatidic acid formation, 50-55% less protein phosphorylation and a 50-75% reduction (depending on stimulus concentration) of the liberation of intracellular Ca2+ ions, compared with fresh platelets.

Hence, cryopreservation disturbs the activation mechanism of a platelet subpopulation consisting of about 50% of the cells, probably as a result of membrane damage, whereas the other half of the population recovers relatively unharmed from the freeze-thawing procedure.

Supported by the Praeventiefund (grant 28953), The Hague.
PLATELET-MEDIATED REDUCTION OF PERIPHERAL TISSUE PERFUSION IN THE CAT : SEROTONIN AND PROSTANOIDS ARE CAUSAL VASOACTIVE MODULATORS
Authors: Jageneau A , Loots W , Nevelsteen A , De Clerck F
Published: August 2018

In cats, a femoral artery was ligated in order to mobilize a collateral circulation in the hind leg, leaving the contralateral artery intact. Graded infusions of collagen (50 to 400 μg/kg) in the aorta above the bifurcation reduced the blood flow, mainly in the collateral circulation (> 80 %). Plasma 5-HI and TXB2 increased in local arterial blood. Ketanserin or ritanserin (S2-receptor blockade, 0.63 mg/kg I.V.) and dazoxiben or R 68070 (TXA2 synthetase inhibition, 5 and 1.25 mg/kg I.V.) reduced (> 50 %, p < 0.05) the collagen-induced loss of tissue perfusion. Indomethacin (10 mg/kg I.V.) and prazosin (0.1 mg/kg I.V.) were ineffective. The effect of TXA2 synthetase inhibitors was annihilated by indomethacin, suggesting that their activity is linked to a reorientation of arachidonic acid metabolism to vasodilating prostanoids rather than to an inhibition of the production of vasoconstrictive cyclo-oxygenase products. By contrast, combined treatments with ketanserin/indomethacin or ketanserin/R 68070 remained effective. Reserpine/PCPA treatment also prevented the collagen-induced changes in perfusion. This study demonstrates 1) oversensitivity of collaterals to platelet products; 2) serotonin to be the causal vasoconstrictive mediator; 3) effective modulation by S2-receptor blockade and by vasodilating prostanoids produced after inhibition of TXA2 synthetase.