bluuebunny/crossref_metadata_2025 huggingface dataset:
SYNVINOLIN (SYN) IN TYPE Ha HYPERLIPOPROTEINAEMIA : NORMALIZED PLATELET HYPERREACTIVITY ASSOCIATED WITH AN IMPROVED RESPONSIVENESS AGAINST PGI2 AND ENHANCED PGI2 RECEPTORS
Authors: Schrör K , Löbel P , Steinhagen-Thiessen E
Published: August 2018

Patients with familial hyperlipoproteinaemia (HLP) are at high risk for the premature development of atherosclerosis. This study was designed to investigate the effect of long-term treatment (8 months) with the HMG-CoA-reductase inhibitor SYN (20-40 mg/day) on platelet reactivity and PGIo receptors in 12 HLP patients (B) as compared with 11 untreated HLP patients (A) and 11 healthy subjects (C). Treatment with SYN reduced the plasma cholesterol to 234 ± 12 mg/dl as compared to 307 ± 21 (A) and 195 ± 14 mg/dl (C), respectively. Collagen (0.6 pg/ml) induced platelet thromboxane (TXB2) formation was 50±6 ng/ml in group A and significantly reduced to 32 ± 3 (B) which was not different from the 28±3 ng/ml in group C. Similar results were obtained by measuring pTatelet ATP secretion and aggregation. SYN treatment significantly improved the reduced number of PGI2 receptors (determined according to Schillinger & Prior, 1980) and normalized the iloprost (ILO, 30 nM) induced cAMP stimulation in platelet-rich plasma while the kg remained unchanged:

These data demonstrate that SYN-treatment of HLP patients at doses that reduce plasma cholesterol by about 25% results in sig-ficant improvement of platelet function. This includes both normalization of platelet hyperreactivity against proaggregatory agents as well as platelet hyporeactivity aganist PGI2. The last effect might involve an increase of the (reduced) number of PGI2 receptors in HLP type IIa.
INITIAL STUDIES IN MAN WITH A NOVEL THROMBOXANE RECEPTOR BLOCKING DRUG GR32191
Authors: Thomas M , Lumley P , Ballard P , O'Brien J R
Published: August 2018

In-vitro GR32191 is a potent and specific thromboxane receptor blocking drug on platelets, and vascular and airways smooth muscle (Lumley et al this meeting). We have undertaken studies in healthy male subjects (n) to examine the effects of oral GR32191 upon platelet aggregation ex-vivo and template bleeding time. Platelet aggregation was monitored in whole blood by counting platelets electronically. Concentration-effect curves to U-46619 and ADP were constructed prior to and following drug or placebo. The degree of rightward displacement of a curve due to treatment was expressed as a concentration-ratio (CR) which was calculated at the 50% aggregation level (ECso post-treatment ECso pre-treatment). Plasma concentrations of GR32191 were determined by h.p.l.c. After single doses of GR32191 mean peak CR's of 8 and 80 were achieved with 0.125 and 0.25mg/kg (n=4) and values of 74 and 234 with 0.5 and lmg/kg (n=4). Peak effects were seen within 2 hours of dosing while activity was still present between 8 and 24 hours. ADP-induced aggregation was unaffected by drug (CR<2) and placebo was without significant effect upon the sensitivity to either aggregating agent (CR<2). GR32191 was rapidly absorbed and the plasma elimination half-life was about 2 hours. GR32191 17.5mg 12-hourly for 10 days (n=6) produced a progressive antagonism of U-46619 induced aggregation which resulted in a large continuous blockade in all subjects (range of 12htrough CR's 85 to 287). However, plasma concentrations of GR32191 did not accumulate on repeated administration. In a double-blind, placebo-controlled, cross-over study (n=16), a statistically significant (p= 0.002) increase in bleeding time was seen following treatment with GR32191 40mg twice daily for 7 days (pre-treatment mean 3.79 min, post-placebo mean 3.47 min, post-GR32191 mean 5.42 min). Rectal bleeding (n=l) has occurred with GR32191 but otherwise tolerability has been good. No drug related changes have been seen in routine laboratory safety screens. Clinical studies are in progress.
R 68 070, A COMBINED TXA2-SYNTHETASE/TXA2-PROSTAGLANDIN ENDOPEROXIDE RECEPTOR INHIBITOR. REDUCES CEREBRAL INFARCT SIZE AFTER PHOTOCHEMICALLY INITIATED THROMBOSIS IN SPONTANEOUSLY HYPERTENSIVE RATS
Authors: Van Reempts J , Van Deuren B , Borqers M , De Clerck F
Published: August 2018

The effects of R 68 070, an oxime-alkane carboxylic acid derivative combining specific thromboxane A2 (TXA2) synthetase inhibition with TXA2/prostaglandin endoperoxide receptor blockade in one molecule, were investigated in a model of photochemically induced stroke in spontaneously hypertensive rats.

Each experimental group was compared with an untreated control group. All animals were anesthetized with halothane in N20/02 and artificially ventilated. After incision of the scalp and stereotaxic positioning of a fibre optic light source, halothane was discontinued. When physiological variables reached normal values, a focal cortical infarction was produced by injection of 10 mg.kg-1 rose bengal and 20 min irradiation of the brain through the intact skull. Four hours later the brains were perfusion fixed and damaged areas measured on consecutive histologic sections. Infarct size was calculated by numerical integration.

R 68 070 (40 mg.kg-1 p.o.,-3 h) significantly reduced the cerebral infarct size to 2.32 mm3 compared with 5.78 mm3 in controls (median values; n = 5; p < 0.05). At 2.5 mg.kg-1 the lesion was reduced from 11.75 mm3 in the control group to 7.82 mm3 in the treated group (n = 5; p = 0.095). Serum TXB2 levels were reduced by > 80 %.

Production of damage in this model is based upon photodynamic generation of singlet molecular oxygen, resulting in peroxidative endothelial cell injury and subsequent platelet thrombus formation. Protection with R 68 070 can be explained by the anti-thrombotic effect of the compound. The relative contribution to this protective effect of synthetase and receptor blockade by R 68 070 are being investigated.
HYPERACTIVITY OF PLATELETS TO 5-HYDROXYTRYPTAMINE IN PATIENTS WITH CARDIOVASCULAR DISEASES
Authors: De Crée J , Roels V , Verhaegen H
Published: August 2018

Controversy still exists about the exact role of platelets in the pathogenesis of atherosclerosis. However, patients with cardiovascular (CV) diseases have been reported to have enhanced platelet activity and to show hyperaggregability in response to common aggregators. Very little is known on 5-hydroxytryptamine (5-HT)-induced platelet aggregation in these patients. In a previous preliminary study we observed an increased sensitivity of platelets in response to 5-HT in patients with CV diseases. We further showed that ketanserin, a selective 5-HT receptor antagonist both on platelets and vascular tissue, abolished the 5-HT dependent hyperreactivity of platelets in patients with CV diseases. In a prospective study we investigated platelet aggregation in response to 5-HT at 2 x 10-5Mol in 405 patients with various CV diseases as compared with an age-matched control group of 110 apparently healthy donors. Evaluation of the results was based on the presence of a second irreversible aggregation in response to 5-HT. The control subjects responded to 5-HT with a shape change and a weak, reversible aggregation, except for 9 of the 110 volunteers, where a second irreversible wave occurred (8%). In contrast, it was found that 119 out of 405 patients with CV diseases had a biphasic irreversible aggregation (29%) (Chi-square test : p < 0.0001). From these 405 patients 129 patients suffered from an acute myocardial infarction (AMI), between 4 and 14 days after the onset of symptoms, 78 patients suffered from ischemic heart disease (IHD), 99 patients from peripheral arterial obstructive disease (PAOD) and 99 patients from diabetes, without clinical symptoms of atherosclerosis. A secondary irreversible platelet aggregation to 5-HT was observed in 36% of patients with AMI, in 22% of patients with IHD, in 25% of patients with PAOD and in 31% of patients with diabetes, all the subgroups being significantly different from the control subjects (p <0.01). These findings suggest that platelets may play a role in the propagation and manifestations of CV diseases and that in diabetes the enhanced platelet activity may be a contributing risk factor in de development of atherosclerosis. Finally, since 5-HT is a potent mediator of vasospasm, treatment with ketanserin might be of therapeutic value in atherosclerosis, where platelet activation is thought to be involved.
CHANGES IN PLATELET ACTIVITY, FIBRONECTIN AND TISSUE PLASMINOGEN ACTIVATOR DURING ARTERIOGRAPHY IN PATIENTS WITH ISCHAEMIA EXTREMITIES INFERIES
Authors: Augustyniak W , Cierniewski C S , Slawincki V , GOCH J H , Polanowaka R
Published: August 2018

Platelets and endothelial cells may be activated to release their contents during arteriography, which is one of the most frequently used examinations of patients with peripheral vascular diseases. This technique involves lumbar aortic puncture and catheterization with administration of contrast medium in arteriosclerosis obliterans (AO) extremities inferies. In this report we have studied 26 patients aged 37 to 73 years with AO who were subjected to arteriography performed according to the Seldinger method. Concentrations of β-thromboglobulin (β-TG), platelet factor 4 (PF4) and plasma fibronectin (Fn) were determined by radioimmunoassay and tissue plasminogen activator (t-PA) activity was measured by a solid-phase fibrinolytic assay using polystyrene test tubes coated with 125I-fibrin. The levels of these parameters were analysed in samples of both the arterial and vein blood which had been taken before and after the injection of contrast medium and obtained results are given in the Table.

Our results suggest that after the injection of contrast medium there is a significant increase in the t-PA activity in the arterial blood and the β-TG concentration in the vein blood.
Effect of Platelet Activating Factor(PAF) on the collagen induced platelet aggregation in whole blood.
Authors: Oura Y , Sakiyama N , Ueshima R , Higuchi M , Kakishitha E , Nagai K
Published: August 2018

Studies of platelet aggregation are generally performed in p1ate1et-rich plasma(PRP) by the transmittance method. Recently, impedance aggregometry has been introduced which shows the platelet aggregability in whole blood. We compared the impedance aggregometry in whole blood with the transmittance method in PRP, with regard to collagen induced platelet aggregation. The aggregation rate in whole blood increased with increasing concentration of collagen, but remained unchanged in PRP. The factors which influence the platelet aggregation rate in whole blood were studied. CV-3988, that is the specific antagonist of PAF, acetylsalicylic acid (ASA) and phosphocreatine / creatine phosphokinase (CP/CPK) were used in order to evaluate the contribution of PAF, thromboxane and ADP in whole blood. CV-3988 dose-dependently inhibited platelet aggregation induced by collagen in whole blood, but did not inhibit the aggregation in PRP. ASA(10mM) inhibited the aggregation in whole blood incompletely too, but completely in PRP. And the inhibition of CP/CPK(CP/CPK : 1.5mM/50U/ml) was very weak in whole blood compared to that of other antagonists. The inhibitory effect of CV-3988 was investigated on the collagen induced platelet aggregation in whole blood which was pretreated with ASA ( 1 OmM ) and CP/CPK (1.5mM/50U/ml), resulting in a collagen induced aggregation in whole blood that was not completely inhibited. We conclude that there are some other different factors, which influence platelet aggregation in whole blood, in addition to thromboxane, ADP and PAF.
48740 RP INHIBITS PLATELET SHAPE CHANGE INDUCED BY PAF-ACETHER IN VITRO.
Authors: Jung G , Sedivy P
Published: August 2018

Platelet shape change (PSC) is the first measurable physiological response to platelet pro-aggregant agonists such as PAF-acether (PAF) (1). The laser thromborheometer (2) was used to assess the PSC (detected as decrease in light scattering as 2 of maximal velocity) and the platelet aggrega tion (detected as increase in the light transmission as 2 of maximal velocity and maximal amplitude) in citrated platelet -rich plasma (cPRP) (1 volume 3.8 2 sodium citrate ± 9 volumes blood) from 12 volunteers of either sex. PAF concentrations ranging from 0.1 to 1 yM induced PSC (range of velocities : 13.2-69.8 2 of maximal velocity, n = 12) and platelet aggregation (range of velocities : 9.3-22.2 2 of maximal velocity and maximum amplitude of aggregation always superior to 20 2, n = 12). These effects were inhibited by 48740 R.P., 3-(3- pyridy1)-1H,3H pyrrolo [1 , 2-c] thiazo1e-7-carboxamide (100200 yM) a selective and competitive PAF-antagonist (3). For instance, 100 yM 48740 RP decreased by 76.4 ± 3.5 (mean ± S.E.M., n = 3) PSC evoked by 0.1 yM of PAF. Furthermore, 48740 RP was also able to reduce PSC in heparinized platelet - r ich plasma (hPRP). The antagonist effects of 46740 RP could be overcome by increasing PAF concentrations in the cPRP and hPRP. During this study, 6 volunteers were found, in whom even concentrations of PAF up to 10.5 pM failed to induce an amplitude of aggregation in cPRP greater than 20 2 whereas PSC was in the range of 19.1-79.4 2 of maximal velocity. These responses to PAF which were antagonized by 48740 RP were not due to an alteration in plasma properties ; thus, they are proposed to result from undetermined changes occuring at the level of the platelet (e.g.) decreased number of PAF-operationa1 receptors or alteration of the excitation - response pathways). In conclusion, 48740 RP is an antagonist of PAF-evoked aggregation and PSC in cPRP from volunteers responding either normally or poorly to PAF. (1) LAPETINA E.G. et al. J. Biol. Chem. 258, 7241 (1983). (2) JUNG G. et al. Nouv. Rev. Fr. H6matol. 27, 103 (1985). (3) SEDIVY P. et al. Thromb. Haemost. 54, 185 (1985).
A PLASMA FACTOR FROM A PATIENT WITH A BLEEDING TENDENCY CAUSES PLATELET SECRETION IN THE ABSENCE OF EXTRACELLULAR CALCIUM
Authors: Cockbill S R , Heptinstall S , Burmester H B
Published: August 2018

A woman with a history of bruising and bleeding but with a normal platelet count and normal clotting factors, had platelets that appeared grey when stained and viewed under the microscope. Unlike the grey-platelet syndrome, the abnormality was only evident when the blood had been collected into EDTA andnot when citrate or heparin was used as anticoagulant. When we examined the EDTA-blood further we found that large quantities of beta-thromboglobulin andserotonin (5HT) were present in the plasma with only small quantities in the platelets. Hie reverse was the case for blood collected into citrate or heparin. LDH (a cytoplasmic marker) levels in EDTA-plasma were not raised.Platelet-rich plasma (PRP) was prepared from blood collected into heparin and labelled with ^-4C-5HT. Incubating the PRP with EDTA (4mM) or EGTA (3mM, sufficient to chelate all the plasma calcium but not the magnesium) caused extensive release of 14C-5HT from the platelets. When Ca++ was removed by passing the PRP through an ion-exchange resin, extensive 14C-5HT release also occurred. Hie release reaction induced by exposing the platelets to a low-Ca++ environment could be prevented by agents that increase cAMP levels.In a series of cross-over experiments, we discovered that the platelet secretion that occurred on removing Ca++ was caused by a plasma factor. Platelets from a healthy donor which had been labelled with 14C-5HT were resuspended in the patient's heparinised plasma. Incubating the platelet suspension with EGTA resulted in extensive release of 14C-5HT. Heparinised plasma from the patient was also passed through a column of Protein A-Sepharose to remove the immunoglobulin fraction. EGTA-challengof control platelets resuspended in the eluted plasma did not cause any release of 14C-5HT, suggesting that the plasma factor responsible may be an immunoglobulin.We have yet to discover whether this new abnormality (that on initial investigation could be confused with the grey-platelet syndrome) has any relevance to the in vivo bleeding situation in the patient in whom it was discovered.
PLATELET RELEASE DURING EXERCISE IN PACEMAKER PATIENTS
Authors: Myreng Y , Gjesdal K , Kjeldsen S E , Lande K , Eide I , Grendahl H
Published: August 2018

Seven pacemaker patients with complete AV-block were studied during one to three week periods of either ventricular (VVI), or atrio-ventricular pacing (DDD) or triggered ventricular pacing (VAT)

At rest, the plasma p-thromboglobulin (BTG), arterial adrenaline and nor-adrenaline as well as atrial rate were not significantly different during standard and physiological pacing. Following ergometer bicycling, BTG increased significantly (p <0.004), and more so in VVI pacing (from 38 ±15 to 73 ±15 ng/ml) compared to DDD (27 ±6 to 44 ±13),p<0.05. Arterial plasma adrenaline at baseline was 60 ±27 in VVI and 58 ± 27 pg/ml in DDD, with increases during exercise to 353 ±197 and 338 ±164 respectively. Neither was any noradrenaline cone significantly different: 253 ±74 to 2962 ±2031 in VVI versus 226 ±143 to 2848 ±2032 during DDD at rest and during exercise, respectively. The atrial rate was 73 ±11 at baseline increasing to 125 ±15 ppm, versus 69 ±12 to 127 ±15 ppm (ns). Physiological pacing is thus associated with lower platelet release at baseline (ns,p<0.07) and exercise (p<0.05), despite no significant difference in arterial catecholamine concentration or the related atrial rate. Increased blood turbulence during atrio-ventricular asynchrony may contribute to the increased release in VVI-pacing
ASPIRIN EFFECT ON PLATELET ANTIPLASMINS PRELEASE
Authors: Woods A I , Lazzari Ji A
Published: August 2018

It was shown platelet antiplasmin activity which is released by col 1anen(Col)and thrombin, probably associated with a-oran u 1 es . Lys i s area produced by eunl obul i ns (eun) of PPP and PRP whether or not previously treated with ADP(5uM), Col(20ug/ml) and aspirin(ASA)(00up/nl)in plas-mi nooen- f ree-f i bri n plates were s tudi ed. Res u 1 ts (mm2 ) were:eunPPP:151.3±39.8;+ADP:155.7±47;+Co1: 155.5±150.8;
+ ASA: 160±142. -EugPRP: 142*42;+ADP: 145*72;+Col : 136*43 ; -⋋ASA: 138+47.-EugPRP produced slightly lower lysis area than euaPPP(p:ns).ADP , Co 1 and ASA neither modify the lysis area of eugPPP nor of eugPRP.Washed piatelets(WP)whether or not treated with ASA were resuspended in eu^PPP and then treated with Co 1 ( 2 and 20ug/ml ). Resul ts (mm2 )were : euoPPP:109.8+35;+3xl05WP/ul:66.4±24;+106WP/u1:63±17;+2x lO WP/ul:58.4±13.- Wi th Col(20ug/ml):euoPPP:101.3±28;
+ 3xl05WP/ul:48.4±14; + 10cWP/ul :46.2±16;+2xl06WP/ul :44.5± i2;+ 2x106WP/u1+ ASA:45.2±8.-With Col(2ug/nl):eugPPP:10 3± 25;+2xl06WP/ul : 49±10 ;+2x1 Of>WP/ul+AS A: 50 . 9±13 . WP in eug PPPshowed lower lysis area than eugPPP(d<0.00 1). The higher number of piatelets ,the lower lysis area.Col-treated WP showed greater inhibition of lysis area than non-treated WP.ASA could not inhibit this effect

Conclusion:In euoPRP there was a slioht antiplasmin activity related with platelets not comoletely eliminated from eug fractionation.WP in euoPPP showed hioher antiplasmin activity.High and low doses of Col produced antiplasmin release.ASA could not inhibit its release with both doses of Col.We must consider the possibility that platelet antiplasmins could be released by different metabolic pathways other than cycl oox.ygenase way or they are not stored in the a-granules.
NON-REVERSIBLE BINDING OF 5'-p-FLUOROSULFONYLBENZOYL ADENOSINE TO WASHED INTACT PLATELETS
Authors: Bowry S K , Tepel M
Published: August 2018

ADP is an important in vivo mediator of platelet aggregation. The identification and estimation of a finite number of receptor sites has been made difficult by the reversible nature of the action of ADP on platelets and because ADP can be degraded by enzymes on the platelet surface. Analogues of ADP have therefore been commonly used to study the ADP receptor. We investigated the validity of using 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), an affinity analogue of ADP that inhibits ADP-induced aggregation, for studying the ADP receptor on intact washed platelets. Binding of labelled FSBA, like for ADP, was saturable. Scatchard plot analyses of equilibrium binding data indicated, positive cooperativity; the apparent affinity (2.4 × 10−6) correlated well with the amount of ADP required to cause aggregation and with the value obtained from aggregation studies. However, the platelet bound FSBA was not displaceable with excess unlabelled FSBA indicating non-reversibility of ligand binding. As the minimal criteria for receptor identification (specificity, saturability and reversibility) are not fulfilled, the usage of FSBA to study the ADP receptor must be treated with caution. As FSBA is able to bind covalently to at least four polypeptides in isolated platelet membranes, a finite number of receptors cannot be postulated using FSBA. Furthermore, positive cooperativity, as indicated by the Scatchard plots, may be explained in terms of specific non-receptor binding. As the ADP receptor cannot be identified with any degree of certainty, the possibility therfore exists that the mechanism by which ADP initiates aggregation by rearrangement of platelet membrane proteins without binding to a single protein receptor.
ANALYSIS OF THE FUNCTIONAL ROLE OF PLATELET MEMBRANE GLYCOPROTEINS WITH MONOCLONAL ANTIBODIES
Authors: Nagata H , Nomura S , Oda K , Kokawa T , Yasunaga K
Published: August 2018

Eight monoclonal antibodies were obtained which recognized platelet surface antigens of these, 5 (NNKYl-32, NNKY2-5, NNKY2-6, NNKY2-11, NNKY2-18 ) recognized GP IIb-IIIa complex, 2 (NNKY5-4, NNKY5-5 ) recognized GP lb and 1 (NNKYl-19) recognized CD 9 antigen. They were used to research the platelet membrane antigens.

Monoclonal antibodies that recognize CD 9 antigen, which exists on the surface of platelets, acute lymphoblastic leukemia cells, eosinophils and other tissue, are known to act as an aggregating agent to platelets and NNKYl-19 was fond to induce platelet aggregation accompanied by ATP release. NNKY5-4 had no effect on platelet functions. NNKY5-5 inhibited aggregation induced by ristocetin but had no effect on aggregation induced by ADP, collagen, thrombin, and NNKYl-19. NNKYl-32, 2-5, 2-6, 2-11, and 2-18 inhibited aggregation induced by ADP, collagen, thrombin, and NNKYl-19, although slight release of ATP was recognized when NNKYl-19-induced aggregation was completely inhibited by NNKYl-32. Mutual inhibition of binding to platelet membranes between the 3 groups of monoclonal antibodies was not recognized

NNKYl-19-induced aggregation was associated with a lag time that was plo-longed in inverse proportion to antibody concentration. Aspirin had almost no effect on NNKYl-19-induced aggregation. A TXA2 receptor antagonist, a calci-um-channel blocking drug and EDTA inhibited NNKYl-19-induced aggregation. These results indicate that GP I b, GP IIb-IIIa complex and the cyclooxygenase pathway are not involved in NNKYl-19-induced platelet activation, that the target of NNKYl-19 on the platelet membrane is same as that of TXA2, and that the mechanism of activation by NNKYl-19 is related to calcium flux.
PLATELET ACTIVATION INDUCED BY A MONOCLONAL ANTIBODY AGAINST THE PLATELET GP Ilb/IIIa COMPLEX
Authors: Modderman P W , Huisman J G , van Mourik J A , Kr A E G , Borne v d
Published: August 2018

A receptor for fibrinogen on the platelet GP Ila/lIIb complex is induced by ADP, thrombin and other agonists. To study functional domains on GP Ilb/IIIa, the effects of anti-GP Ilb/IIIa monoclonal antibodies (Mab’s) on platelet function were determined. One of these Mab’s, 6C9, induced platelet aggregation. The antibody binds to the intact GP Ilb/IIIa complex only, not to free GP lib or free GP Ilia. Its epitope is different from that of C17, a Mab that inhibits ADP-induced fibrinogen binding and platelet aggregation. 6C9 induces fibrinogen-mediated aggregation rather than agglutination since 6C9-induced platelet interactions were blocked by treatments that also inhibited the effects of ADP etc., without inhibiting binding of 6C9 itself. 6C9 induces binding of 125I-fibrinogen (35.000 ± 7.300 molecules/platelet, Kd = 1.3 ± 0.4 µM) to unstirred platelets. Binding of fibrinogen was 60 to 80% inhibited by apyrase, which indicates that 6C9-induced fibrinogen binding is largely mediated via ADP released from platelets. In addition, 6C9 induced aggregation of platelets in the absence of extracellular fibrinogen. Mediation of this process by platelet fibrinogen or other a-granule proteins, released upon activation by 6C9, was implicated by the finding that aggregation of washed platelets, but not of platelets to which fibrinogen was added, could be blocked by PGI2. Platelet release was also assessed directly by measuring β-thromboglobulin (α-granules) and (14C) serotonin (dense granules) in the medium of unstirred platelets incubated with 6C9. F(ab')2 fragments of 6C9 only aggregated platelets in the presence of fibrinogen and did not release (14C) serotonin. Moreover, release induced by intact 6C9 was inhibited by anti-GP Ilb/IIIa Mab C17 but not by C17 F(ab’)2, although the latter inhibited ADP-induced platelet aggregation. These data indicate that binding of antibodies to specific sites on GP Ilb/IIIa may induce Fc-dependent platelet activation.

This study was supported by the Foundation for Medical Research MEDIGON (grant no. 900-526-057.
CONTACT ACTIVATED PLATELETS BIND VON WILLEBRAND FACTOR TO GPIIb-IIIa
Authors: Estry D W , Mattson J C , Chediak J
Published: August 2018

Using a rabbit polyclonal anti-von Willebrand factor (vWF) antibody, normal human adherent platelets extensively bind vWF in a diffuse pattern as detected by immunogold electron microscopy. This pattern differed significantly from the zonal pattern observed for direct fibrinogen-gold labelling in contact activated platelets. In order to determine if contact activated platelets bind vWF to GPIIb-IIIa or GPlb, the extent and pattern of bound vWF in platelets from patients with Glanzmann’s thrombasthenia (GT) and Bernard Soulier Syndrome (BSS) was determined. Virtually no bound VWF was detected by immunogold labeling in GT platelets previously characterized as being deficient in GPIIb-IIIa. On the other hand, BSS platelets, lacking GPlb, demonstrated extensive labeling of vWF in a pattern identical to that seen in normal platelets. This data is consistant with vWF binding to GPIIb-IIIa in contact induced adhesion and spreading.
THE ROLE OF THE GP IIb/IIIa COMPLEX AND vWF IN PLATELET -COLLAGEN INTERACTION
Authors: Andreas Ruf , Morgenstern Eberhard , Patscheke Heinrich , Santoso Sentot , Müller-Eckhardt Christian , Heimburger Norbert
Published: August 2018

Our recent studies showed that collagen fibrils (CF) are internalized by platelets in citrated plasm. This phenomenon was not observed in EDTA-PRP. In order to investigate whether collagen internalization is mediated by a receptor we studied the effect of monoclonal antibodies against the receptor molecules GP IIb/IIIa (Gi5) and GP Ib (AN51, Da-kopatts GmbH, Hamburg). Washed human platelets from healthy donors were incubated with lOμg of Gi5 or 4μg of AN51 per ml of platelet suspension (2 × 108 platelets per ml). After 10 min. at 37° C we added AOpg collagen (Hormonchemie, München) per ml of platelet suspension. After an additional incubation period of 10 min. the reaction was stopped by glutaraldehyde fixation (40μl of a 3% glutaraldehyde and of a 0.2 % tannin solution in phosphate buffer). To investigate the role of vWF in the internalization process we added 1Oμg/ml ferritin-labeled anti-vWF Fab-fragments to the platelet suspension two min. in advance of collagen stimulation (40μg/ml; 10 min). The values in percent of the inhibition of the internalization phenomena were obtained by statistical evaluation of these phenomena observed on ultrathin serial sections. Washed platelets interacted with CF in the same manner as platelets in citrated plasm, but aggregates were only formed after stirring. Washed platelets which had internalized CF were found to be completely degranulated in contrast to platelets which had only contacted CF without internalization. These platelets were hardly degranulated. The membrane system containing the internalized CF surrounded the contractile sphere of the platelets and always displayed an opening to the platelet surface.We found that Gi5 inhibited collagen internalization up to 95% and anti-vWF to approximately 50%, whereas AN51 showed no effect on this process. Futhermore, Gi5 inhibited the platelet function aggregation and the release reaction which was enhanced by CF internalization. These findings show that the GP Ilb/IIIa complex is strongly involved in the internalization of CF by platelets and suggest an involvement of vWF released from the stimulated platelets in this process by binding to the GP Ilb/IIIa complex.

Supported by DFG, Grant MO 124/2-4
FIBRIN PROTOFIBRIL AND FIBRINOGEN BINDING TO ADP-STIMULATED PLATELETS:IS THERE A COMMON MECHANISM
Authors: Hantgan R R , Lewis J C
Published: August 2018

We have investigated the molecular basis of platelet:fibrin binding by studying interactions between platelets and protofibrils, soluble two-stranded polymers of fibrin which are intermediates on the fibrin assembly pathway. We have taken advantage of the well-characterized anticoagulant properties of the fibrinogen degradation product, fragment D, to prepare solutions of short protofibrils, composed of fewer than twenty fibrin monomer molecules per polymer. Fibrin protofibrils bound to ADP-activated platelets in a time- and concentration-dependent process which was effectively blocked by excess unlabelled fibrinogen, i.e. the binding was specific and appeared to involve a common receptor. ADP-stimulated cells bound approximately 3 micrograms of fibrin protofibrils/108 platelets, compared to 4 micrograms of fibrinogen/108 cells, following a 30 min incubation period at room temperature. The apparent first order rate constant for fibrin protofibril binding was found to be five-fold slower than that measured for fibrinogen. Two monoclonal antibodies directed against the glycroprotein Ilbtllla complex inhibited the binding of fibrin protofibrils and fibrinogen in a similar, concentration-dependent manner, providing strong evidence for a common receptor. Neither fibrin protofibrils nor fibrinogen bound to -Glanzmann’s thrombasthenic platelets, further supporting the hypothesis that fibrinogen and fibrin bind to a common platelet receptor present on the glycoprotein IIb:IIIa complex. The specificity of these interactions was examined with transmission electron microscopy, which clearly showed thin fibers of lengths up to 150 nm attached to the surface of normal, stimulated platelets. Immunogold electron microscopy using rabbit anthihuman fibrinogen as the first stage antibody verified identity of the surface bound molecules and the immunogold distribution paralleled that observed with the fibrin/fibrinogen molecules alone. Contacts between the ends of the fibers and the platelets were frequently observed, but lateral contacts were also evident
REVERSIBLE EXPOSURE OF BINDING SITES FOR FIBRINOGEN ON PLATELET MEMBRANE GLYCOPROTEIN IIB-IIIA
Authors: Akkerman J W N , Mommersteeg M E
Published: August 2018

When human, gel-filtered platelets are stimulated with Platelet Activating factor (PAF, 500 nM) in the presence of 1 μM 125I-fibrinogen (*F) 15.360 ± 4.850 (n = 5) mol*F bind per platelet after 10 min incubation at 22°C. Under similar conditions 10 μM adrenaline and 10 μM ADP induce the binding of 17.020 ± 4.550 and 20.500 ± 3.910 mol*F/platelet, respectively. Stimulation with PAF in the absence of fibrinogen followed by addition of *F at different intervals (Δt) thereafter, reveals an exponential decrease in bound *F reaching 29 ± 8% of initial binding at Δt = 30 min. Similar patterns are seen with ADP and adrenaline. This decrease is enhanced by raising cyclic AMP (5 μM forskolin) or reducing the cell's energy supply (30 mM deoxyglucose - 1 mM CN- ). Thus, exposed binding sites disappear in the absence of fibrinogen

Following a first stimulation (with PAF; *F absent), a second stimulation (ADP or adrenaline; *F present) restores the binding to the range found with the second stimulator under optimal conditions. Thus, following a first disappearance of accessible binding sites a second type of binding can be induced

Repeated stimulation with different agonists (PAF-A t-adrenaline-Δ t-ADP- Δ t-collagen, 5 μg/ml) induces repeated exposure and disappearance of binding sites without exhaustion of accessible binding sites. Re-exposure of binding sites is complete with adrenaline and ADP as second and third stimulator, whereas collagen induces 60-70% of the first binding induced by PAF. When the 10 min incubation with *F is increased to 60 min, re-exposure results in binding of 40.000-50.000 mol*F/ platelet, which is close to the number per platelet of glycoprotein IIb-IIIa complexes, the binding sites for fibrinogen

These data support the concept that exposure and disappearance of fibrinogen binding sites is a reversible process and that the maintenance of the exposed state is under tight metabolic control
CHARACTERIZATION OF FIBRINOGEN RECEPTORS ASSOCIATED WITH GLTCCPROIEIN IIb/IIIa (GPIIb/GPIIIa) COMPLEX BY TRIGRAMIN, A UNIQUE LOW MOLECULAR WEIGHT PEPTIDE PROBE
Authors: Huang Tur-Fu , Lukasiewicz H , Holt C J , Niewiarowski S
Published: August 2018

Trigramin (Mr weight 10 kDa), an acidic, cysteine rich peptide purified into homogeneity from Trimeresurus gramineus snake venom contained a single protein chain with EAGE at the NH2 terminal end. It inhibited platelet aggregation induced by various agents without affecting release reaction. It blocked competitively the binding of 125I-fibrincgen to AEP stimulated and chymotrypsin treated platelets (Ki= 2 × 10−8). 125-I trigramin bound to intact and to AEP stimulated platelets in a saturable manner (approx. 16,000 sites per platelet). However, AEP increased 5 fold, the binding affinity of trigramin to platelets (to Kd = 4 × 10−8M) suggesting that AEP is changing the conformation of receptors associated with GPIIb/GPIIIa complex. The binding of trigramin to thran-basthenic platelets was markedly reduced. The binding to normal platelets was significantly inhibited by EDTA and by monoclonal antibodies directed against GPIIb/GPIIIa complex but not by the antibodies directed against GPIIb or GPIIIa molecules. The binding of 125I-trigramin to AEP-stimulated platelets was inhibited by RGES (IC50 = 125 μM) and by YHHLGGAKOAGDV (C-terminal fragment of fibrinogen gamma chain, IC50 = 250 μM) suggesting that these or similar peptide sequences are required for interactions of various ligands with GPIIb/GPIIIa complex
EFFECT OF FIBRINOGEN RECEPTOR BLOCKERS ON HUMAN PLATELET AGGREGATION, ATP SECRETION AND THROMBOXANE B2 PRODUCTION
Authors: Kerry R , Findlay V , Ambler J , Wallis B J
Published: August 2018

The sequences of those parts of fibrinogen involved in the binding of fibrinogen to activated platelets are known and mimetics of these sequences, namely Fib(γ)402-411 and Fib(Aα)572-575 are selective fibrinogen receptor blockers. By competing with fibrinogen, these agents prevent the platelet: fibrinogen interaction and hence represent agents capable of selectively preventing platelet:platelet interaction (i.e. aggregation). In the experiments described here these peptides are used as tools to elucidate the interdependence of platelet aggregation, secretion and thromboxane (TxB2) production using a range of aggregating agents

Both peptides gave quantitatively similar results but Fib(Aα)572-575 was ten times more potent than Fib(γ)402-411. The results are summarised in the table:

Experimentally aggregation and secretion were measured simultaneously in a lumi-aggregometer. Thromboxane B2 was subsequently determined by RIA in the same samples. The concentration of Fib(γ)402-411 (ImM) and Fib (Aα)572-575 (100 μM) employed were those that gave half maximal inhibition of an aggregatory response.

The results show that a) PAF induced thromboxane production and ATP secretion are dependent on platelet aggregation, b) collagen induced ATP secretion is dependent on platelet aggregation while thromboxane production is independent of aggregation and c) thrombin induced ATP secretion and thromboxane production can both occur independently of platelet aggregation.

These observations throw further light on the interdependence of platelet responses and show fibrinogen receptor blockers to be true anti-aggregatory agents.
PLATELET DYSFUNCTIONS DUE TO AN ANTI-GP Ilia AUTOANTIBODY IN A HEMORRHAGIC PATIENT WITH MALIGNANT LYMPHOMA
Authors: Kubota T , Tanoue K , Kitagawa H , Murohashi I , Aoki N , Yamazaki H
Published: August 2018

A 38-aged woman developed a moderate purpura and enanthema. Before 3 years when she had no bleeding tendency, she was diagnosed with retroperitoneal malignant lymphoma (follicular, large cell type) by a laparotomy and successfully treated with surgery and radiation. Laboratory tests at the time of the present admission revealed normal coagulation tests, platelet counts of 14-0,000/μl and prolonged bleeding time. Platelet aggregation by 1-10μM ADP showed a immediate lag time lasting 30-40 sec, then followed by an aggregation. Aggregation by epinephrine showed a loss of the first wave with a normal second wave. Collagen-induced aggregation showed an abnormally long lag time. Crossed immunoelectrophoresis (CIE) of \% Triton X-100 solubilized platelets from the patient revealed a two-peaked immunoprecipitate of GP IIb/IIIa complex, of which the abnormally slow peak was thought to be the GP IIb/IIIa complex coated with an antibody. CIE of her platelets also showed abnormally decreased amounts of both albumin and fibrinogen in the platelets, suggesting intravascular release reaction from her platelets stimulated with the antibody. She gradually developed EDTA-induced pseudo-thrombocytopenia with true platelet counts of 200,000-250,000/μl. Then, the patient developed true thrombocytopenia ranging from 9,000 to 37,000/μl and was found to have the left tonsillar swelling, which was subsequently diagnosed as a relapse of the malignant lymphoma by a biopsy and treated successfully by radiation. After the disappearance of the tumor, the patient's platelet counts increased to 160,000/μl. At this time, aggregation patterns by ADP, epinephrine or collagen were completely normalized. CIE of her platelets showed a normal shape of GP IIb/IIIa complex as well as normal amounts of albumin and fibrinogen. The purified IgG from the patient in the thrombocytopenic stage inhibited dose-dependently ADP- or epinephrine-induced aggregation of normal PRP. Western-blot analysis showed that the patient's IgG reacted with a protein of approx. 100kDa, corresponding to GP Ilia, in unreduced SDS-PAGE of normal platelets.
A NEW MONOCLONAL ANTIBODY TO PLATELET MEMBRANE GLYCOPROTEINIV: EXPRESSION OF GLYCOPROTEIN IV ON PLATELETS, MEGAKRYOCYTES AND ERYTHROBLASTS
Authors: kariyone S , kambayashi H , satoh T , uchida T , ohto H , maeda H
Published: August 2018

A New monoclonal antibody, designated as TP85, was produced by fusion of the X63.Ag8.653 murine myeloma cell line with splenocytes from BALB/C mice immunized with washed human platelets. TP85 monoclonal antibody precipitated a single petide of 97,000 daltons in both reduced and nonreduced states by immune precipitation of 125I-labelled solubilized membrane. An isoelectric point was around pH 5.0. The antibody was IgG2b in isotype, as determined by the Ouchterlony immunodiffusion method.

The reactivity of TP85 was examined by using indirect immuno-fluorescent assays and ABC immunoperoxidase method. TP85 reacted with platelets, megakaryocytes and erythroblasts from normal human and patients with myeloproliferative disorders, but not with lymphocytes, granulocytes and other bone marrow cells. In a panel of cultured cell line, only 5-10% cells of K652 myeloid/ erythroid cell line were positive, but other cell lines, including common ALL(Reh), T lymphoid(Molt-3, CCRF-CEM), B lymphoid(Raji ,Daudi), myeloid(KG-l, HL-60) and monoblastoid(U937, THP-l) cell lines, were all negative.

TP85 did not inhibit platelet aggregations induced by ADP, collagen, epinephrine and ristocetin while TP80 which was an antibody for GPIIb/IIIa inhibited these aggregations except induced by ristocetin.

It is concluded that glycoprotein IV, which is immunoprecipitated by TP85, may exist not only on platelet membrane, but also on megakaryocytes and erythroblasts, and has no role on platelet aggregation.
DISTRIBUTION OF INTRAMEMBRANOUS PARTICLES (IMP) ON PLATELETS IN CLOTS UNDER ISOMETRIC TENSION
Authors: White James G , Cohen Isaac
Published: August 2018

Development of isometric tension in platelet-rich clots is a manifestation offibrin binding to the cells, as well as to platelet contractile activity. However, the nature of the platelet-fibrin association through which the force of contraction is transmitted remains unknown. A previous report suggested that clustering of the intramembranous particles (IMP) visible in replicas of freeze-fractured platelets in clots might represent sites of fibrin attachment and force transduction across the platelet membrane. In the present study platelet clots were prepared at various stages during development of isometric tension. Clots fixed in glutaraldehyde were combined with osmic acid and further processed for thin sections, or with glycerol, frozen in liquid nitrogen and freeze-fractured in a Balzer's device. Thin sections revealed the longitudinal orientation of platelets and fibrin strands in the long axis of tension. Close associations between fibrin strands and extended portions of platelets were readily identified. However, no clearly discernible repetitive associations could be identified. Replicas of freeze fractured platelet clots under isometric tension revealed aspects of fibrin strands substructure and the platelet secretory pathway. However, no specific associations between fibrin and IMP were apparent. The usual 2 to 1 ratio of IMP on the inside of the outer layer (E face) compared to the outside of the interior layer (P face) of resting platelets was preserved on thrombin activated platelets in isometric clots. No evidence of IMP clustering could be identified. Experiments to identify the origin of IMP clustering revealed it occurred only on severely damaged platelets. Thus, clustering of IMP in replicas of freeze fractured platelets does not appear related to fibrin binding to platelets or to transduction of contractile force from the interior of the platelet to fibrin strands and the development of tension in isometric clots.
MEGAKARYOCYTIC ORIGIN OF PLATELET HLA CLASS I ANTIGEN
Authors: Kieffer N , Titeux M , Henri A , Breton-Gorius J , Vainchenker W
Published: August 2018

The existence of HLA class I antigens on human platelets is well established. However, several authors have suggested that platelet HLA antigens are not integral membrane components but are acquired from soluble plasma sources and adsorbed to the platelet surface.

In the present study, we used the monoclonal antibody W6/32, directed against a monomorphic epitope of the HLA class I antigen for the immunochemical characterization of platelet HLA. Immunoprecipitation experiments, performed after in vitro metabolic radiolabeling of human platelets revealed a band of molecular weight 44,000 identical to that precipitated from metabolic labeled U937 or HEL cells. When the same antibody was tested by indirected immunofluorescence in a double labeling technique on in vitro cultures of human megakaryocytes, performed in the absence of human serum in the culture medium, megakaryocytes identified by an anti-vWF MoAb revealed a membrane staining with W6/32 identical to that observed on other bone marrow cells, e.g. macrophages. Our results provide evidence that platelet HLA has a megaka-ryocytic origin and that residual biosynthesis of HLA antigen does still occur in circulating platelets. However, our results do not exclude the ability of human platelets to adsord circulating HLA class I antigen from plasma.
LUMI-AGGREGOMETER STUDIES OF THE INITIAL ATP-SECRETION FROM COLLAGEN-ADHERENT PLATELETS
Authors: Malmgren R
Published: August 2018

We have earlier, with the use of a lumi-aggregometer and sub-aggregating doses of collagen (0.2-0.8 ug/ml PRP), been able to detect the initial, aspirin-insensitive secretion of ATP from the collagen-adherent platelets, and to correlate this secretion to the doses of collagen, and onset and degree of subsequent shape change of non-adherent platelets (Malmgren, Thromb Res 4:445, 1986). The present study shows, that 200 ATU of hirudin,which reduced near-maximal aggregation and ATP-secretion induced by high collagen doses (2.5 ug/ml PRP) from 3.35 ± 0.2 uM to 2.85 ± 0.1 uM, did neither reduce the secreted amount of ATP that were 82.5 ± 15 nM in control samples and 90 ± 27.5 nM in hirudin-treated samples, nor reduce platelet shape change when platelets were challenged with 0.31 ug collagen /ml PRP. (200 ATU hirudin completely abolished an equal degree of platelet shape change induced by 0.01 U thrombin). Assuming that 3 % of the platelets in PRP were actually adhering to the collagen fibrils, the secreted amount corresponds to 14.6 ±0.04 pmoles ATP/106adheringplatelets, amounts which closely represented 100 % of their dense granule content. The finding confirms that hirudin does not inhibit platelet adhesion and also indicates, that thrombin-mediated activation of secretory pathways appears not to be involved during the initial phase of platelet-collagen interactions.

Dipyridamole (DPA) and dibutyryl cAMP (DBcAMP) inhibited ATP-secretion and platelet aggregation in a dose-dependent manner at high collagen concentrations, but only DBcAMP caused a dose-dependent reduction of ATP secretion (IC50 =10-4 M) induced by sub-aggregating doses of collagen. DPA was devoid of effect in this respect and thus did not inhibit platelet adhesion.

Yohimbine, dihydroergotamine and phentolamine reduced ATP-secretion induced by sub-aggregating collagen doses in the mentioned rank order of potency, and with IC50 values in the micromolar range. Ketanserin, ritanserin and propranolol were devoid of effect. The findings suggest that the initial collagen-plate-let interaction involve alfareceptor-mediated mechanisms that may encompass adhesion, while DBcAMP probably interacts with secretory mechanisms connected to phosphatidylinositol turnover.
A SENSITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY(ELISA) FOR SERUM LAMININ
Authors: Sugihara T , Takamatsu J , Kamiya T , Saito H , Kimata K , Kato K
Published: August 2018

Laminin, a large glycoprotein, is a major and specific component of basement membrane. There were little or no circulating laminin in normal persons, although some recent reports have showed increased values in various diseases(ex. diabetes mellitus and liver disease) by a radioimmunoassay(RIA) using laminin fragment. The minimum detectable sensitivity of RIA was reported to be 20 ng/ml of serum sample. We describe here a more sensitive immunoassay system, and also the concentrations of laminin in sera from healthy subjects and patients. A sandwich ELISA method for measurement of laminin was established by use of purified antibodies to mouse laminin. The assay system consisted of poly-stylene balls with immobilized antibody F(ab’)2 fragments and the same antibody Fab1 fragments labeled with β-D-galactosidase from E.Coli. The assay was highly sensitive and can detect as small as 0.5 ng/ml of serum laminin.

Coefficients of variation in within-run and between-run precision studies for serum laminin were good. Serum laminin levels in healthy subjects of various ages ranged from 1.5 to 3.9 ng/ml(n=60). Fifty eight patient sera (collagen disease(n=18), hepatic disease(n=20), and renal disease (n=20)) were examined. Significant differences between the normal sera and 3 diseases sera were observed as shown below

It is concluded that circulating laminin apparently exists in normal persons and there are higher laminin level in some diseases which appears to involve basement membrane-rich organ