bluuebunny/crossref_metadata_2025 huggingface dataset:
RESTING MYOCARDIAL ISCHEMIA AFTER INTRAVENOUS INFUSION OF BM 13.177, A THROMBOXANE RECEPTOR ANTAGONIST
Authors: Terres W , Kupper W , Hamm C , Bleifeld W
Published: August 2018

We conducted a double blind placebo controlled trial of BM 13.177, a thromboxane receptor antagonist, given intravenously in patients (PTS) with stenoses >70 % of the left anterior descending coronary artery and stable exertional angina pectoris (AP). The study had to be stopped after enrollment of 8 PTS, because 2 had developed resting AP after initiation of the study medication. Both proved to belong to the 4 PTS who had received BM 13.177 (12.5 mg/min). While in one PT, AP was mild, transient and associated with only slight decreases in coronary sinus blood flow (CSBF) and myocardial lactate extraction (MLE), in the other, AP was severe and persisted for 30 minutes in spite of antianginal therapy. Severe clinical symptoms in this PT were associated with a marked fall in MLE from +24 to -121 %. Two PTS under BM 13.177 and 4 on placebo underwent supraventricular stimulation. For both groups, no change in clinical symptoms, CSBF or MLE occured in comparison to a former control stimulation without medication. BM 13.177 led to an inhibition of ex vivo platelet aggregation induced by collagen 1 pg/ml (mean reduction in rate of aggregation by 41 %, p< 0.05), while aggregation was not influenced with collagen 5 μg/ml or ADP. This effect of BM on platelets is explained by its thromboxane receptor blocking properties. The induction of resting myocardial ischemia, however, in 2 of 4 PTS with formerly stable exertional AP may have been the result of either a coronary steal mechanism or an intrinsic stimulation of vascular thromboxane receptors, followed by coronary vasoconstriction.
INHIBITION OF GUINEA-PIG PLATELET FUNCTION IN VIVO AND EX VIVO USING THE THROMBOXANE A2 ANTAGONISTS, AH23848 AND GR32191
Authors: McCabe P J , Stratton L E , Hornby E J , Foster M
Published: August 2018

The thromboxane A2 antagonist, GR32191 (Lumley et al., this meeting) was tested as an inhibitor of platelet aggregation in the guinea-pig and compared with another Tx-antagonist, AH23848 (Brittain et al, 1985). Guinea-pigs were dosed with AH23848 or GR32191 at 0.01-1.0mg/kg. At intervals, blood was taken and PRP was prepared for ex vivo aggregation studies. Collagen concentrations causing half maximal aggregation (IC50) were calculated for test and vehicle-dosed groups. Inhibition was expressed as a concentration ratio (IC50 test/IC50 vehicle). For in vivo studies, 111In-labelled platelets (12μCi, 200μl) were injected into anaesthetised guinea-pigs and 24 hrs later oral doses of AH23848 or GR32191 (0.01-1.0mg/kg) or indomethacin (5mg/kg) were given. After one hour, blood was taken for platelet and radioactivity counting. The carotid artery was exposed under anaesthesia and a current of 2mA was applied for 60 sec. After 90 min, 1cm of the damaged and contralateral carotid vessels were removed for gamma-counting. Inhibition of accumulation of platelets on the injured artery was measured by comparison with the undamaged contralateral artery. Numbers of platelets deposited were calculated from the radioactivity of each section of artery and the radioactivity and platelet count in the blood. Oral doses of AH23848 or GR32191 inhibited ex vivo platelet aggregation induced by collagen. Maximum inhibition occurred one hour after dosing, and was still present at 6 hours for AH23848 (l.Omg/kg) and GR32191 (0.3mg/kg). GR32191 and AH23848 were active in vivo causing inhibition of platelet deposition at doses of 0.01-lmg/kg. The maximum inhibition of deposition was 58% for AH23848 (0.1mg/kg) and 63% for GR32191 (0.1mg/kg), with 50% inhibition at 0.02mg/kg for both. Indomethacin (5mg/kg p.o.) caused maximum inhibition of 58% at 5mg/kg p.o. suggesting that this represents the total thromboxane involvement in platelet deposition. GR32191 and AH23848 are thromboxane A2 antagonists with antithrombotic activity after oral dosing to guinea-pigs.

Brittain R.T. et al Circulation, 72, 1208-1218, 1985.
INHIBITION OF PLATELET ACTIVATION BY THE NOVEL THROMBOXANE RECEPTOR ANTAGONIST BM 13.505
Authors: Patscheke H , Stegmeier K , Hornberger W , Staiger Ch , Neugebauer G
Published: August 2018

The effects of BM 13.505 (4-[2-(4-Chlorobenzenesulfonylami-no)ethyl]-benzene acetic acid = BM) on human washed platelets and platelet-rich plasma (PRP) were studied in vitro and after oral application in 10 male volunteers ex vivo/in vitro. BM inhibited the shape change, aggregation and (1H)serotonin release when the platelets were activated by agents that stimulate via the thromboxane Az/prostaglandin H2 (TXA2/PGH2) receptor. Such agonists were collagen, methyl mercury chloride (methyl-Hg), arachidonic acid and the PGH2 analogue U 46,619. BM was 9 times more potent an inhibitor than sulotroban (= BM 13.177). The slope of the Schild plot for U 46,619-induced shape change was close to unity, which is consistent with competitive antagonism. The pA2 -value in PRP was 6.5. BM did not inhibit the primary platelet activation induced by ADP, PAF or serotonin in aspirin-treated platelets, indicating that the inhibition by BM was specific for the platelet TXA2/PGH2 receptor. BM also suppressed platelet activation by PGH2 which accumulated when the platelets were stimulated by collagen, methyl-Hg or arachidonic acid in the presence of the thromboxane synthase inhibitor dazoxiben. At concentrations beyond about 600 times the apparent KD (200 μM in PRP and 10 μM in washed platelets), BM induced a transient shape change. This effect was inhibited by sulotroban and might indicate a slight intrinsic activity of BM. BM 10 μM exerted no effect on the formation of 14C-labelled TXB2, PGE2, PGD2, PGF2α , HHT and 12-HETE from 14C-labelled arachidonic in washed platelet suspensions, irrespective of whether exogenous or endogenous (by methyl-Hg mobilized) arachidonic acid was metabolized. Volunteers who received 7 oral doses of 400 mg in 12 hour intervals reached peak plasma concentrations between 9.5 and 45 pM 1 hour after dosage. The bleeding time was prolonged by 90 %. Platelet activation by collagen, methyl-Hg and U 46,619 was inhibited for at least 9 hours after a single dose. No objective and subjective side effects were observed in any of the subjects. Thus, BM 13.505 is a specific, we11-tolerated and long-acting TXA2/PGH2 receptor antagonist. (Supported by the DFG, Grant Pa-263).
SERENDIPITOUS PROTECTION FROM PLATELET-MEDIATED DISORDERS IN STEROID-TREATED PATIENTS?
Authors: Holloway H , Moriarty A J , Nelson S D , Balnave K
Published: August 2018

The aim of this study is to investigate the influence of exogenous corticosteroids on platelet aggregation. This is widely recognised to be of significance in the pathogenesis of acute myocardial infarction and other vascular/haematological disorders.

Blood was taken from a cohort of patients (N = 10) undergoing treatment with ACTH or corticosteroids for varied systemic illness. Ex vivo measurements of platelet parameters were made with a Coulter Counter Model S Plus III on nonanticoagulated blood directly from the circulation. Subsequently, aliquots of anticoagulated blood in batches of five were processed at intervals over 24 hours. For comparison, a similar study was undertaken in normal volunteers not on any medication. The mean percentage changes ± standard deviations over the 24 hour interval in platelet count (PLT), mean platelet volume (MPV) and plateletcrit or biomass (PCT) in the respective groups were as follows:-

PLT: -(3.12 ± 2.68) vs -(14.77 ± 3.96)(p < 0.001);

MPV: 28.88±8.92 vs 46.68±12.54 (p <0.01);

PCT: 25.18 ± 8.16 vs 23.75 ± 8.97 (NS).

The in vitro alteration in PCT over 24 hours in both cohorts is similar and is due to platelet swelling. The change in MPV is partly due to swelling and partly due to aggregation as evidenced by the decrease in PLT. The significantly smaller drop in PLT and smaller rise in MPV in the steroid-treated group clearly demonstrate that platelets from these patients are less aggregable. A possible cause is that the inhibitory effect of steroids on phospholipase may lead to reduced levels of thromboxane A2. Thus patients on steroid therapy or with high endogenous steroid levels may enjoy serendipitous protection from a variety of platelet mediated disorders. The work may also explain in part why such patients are more prone to bleeding diatheses.
CHILDHOOD HEMOLYTIC UREMIC SYNDROME (HUS) : VON WILLEBRAND FACTOR (vWF) AND PLATELET AGGREGATING ACTIVITY (PAA) STUDIES
Authors: SCHLEGEL N , MOAKE J , LOIRAT C , HURTAUD M F , LEVY-TOLEDANO S , MATHIEU H
Published: August 2018

It has been suggested that a vWF High Molecular Weight Multi-mers (HMWM) decrease or a PAA were involved in the pathogenesis of HUS. We have studied 8 children (6 girls,_2 boys; 7 months-8_1/2 years old) with HUS : plasma creatinine /μmol/l; mean(range)/=306 (105-524), hemoglobin (g/100ml)-7(6.3-7.8), schistocytes (%)=8(1-18), platelets (x103/mm3)-57(10-115). The vWF was studied quantitatively (antigen ; vWF RAg assay) and qualitatively (multimeric pattern : immunoblotting and autoradiography). PAA studied by incubating the patient's platelet poor plasma (RPR) with washed normal platelets (aggregometer, % light transmission) and confirmed by Thromboxane B2 (TXB2) assay and [14C] Serotonine release study. The PAA was characterized by studying the in vitro effect of several platelet aggregation inhibitors, Immunoglobulins (Igs) and Fresh Frozen Plasma (FFP) on the platelet aggregation.

An increase of vWF RAg (%) was observed in 6 cases : mean:330, and possibly related with renal failure. A vWF HMWM decrease was found in 3 patients : 2/3 with associated infection(E.Coli, Pneumococcus), 1/3 with severe hemolysis. Two of these 3 patients had a favourable renal outcome and 1 a severe course (chronic hemodialysis, Arterial Thrombotic MicroAngiopathy at renal histology).An important PAA was evidenced only in 1 patient : post bone-marrow graft HUS during neuroblastoma(NB),arterial hypertension and chronic renal failure. This PAA was Ca++, TXB2 and cAMP dependent; it was moderately inhibited in vitro by Igs and FFP, but persisted after 5 days of Igs infusion (0.3g/Kg/day). Treatment with aspirin and dipyridamole (10mg/Kg/day each) suppressed the patient platelet auto-aggregation although the PAA persisted (follow up:10months). The PAA did not seem to be related with the NB (absence of GD2 ganglioside, specific marker of NB); it could be related with anti platelet antibodies. The coexistence of the two abnormalities could not be demonstrated in our patients.

In conclusion, a vWF HMWM decrease was found in 3 out of 8 children patients with HUS. Its presence was not correlated with the severity of the disease. We could demonstrate the presence of PAA during childhood HUS in only 1 post bone-marrow graft case. The PAA characterization is useful for therapeutic decisions and contributes to a better pathogenetic understanding.
PLATELET DEPOSITION AFTER CAROTID ENDARTERECTOMY DECREASES WITH TIME.
Authors: Meek A C , Jarvis P , Backhouse C M , McCollum CN , Greenhalgh RM
Published: August 2018

Platelets are deposited on the exposed media following carotid endarterectomy and will continue to accumulate until neointima covers this thrombogenic surface. Radiolabelled platelet uptake was measured to assess the time to intimal repair.

Autologous llllndium labelled platelets were infused 2 days and 2 months postoperatively in 10 patients undergoing unilateral carotid endarterectomy. Platelet accumulation was measured daily by gamma camera images counting radioactivity over the operated artery and comparing it to the contralateral side as Carotid Uptake Ratio (CUR).

Mean (±sem) counts per gamma camera cell over the operated side at 24 hours were 46.3± 4.3 compared to 38.6± 3.9 on the unoperated side (p<0.001). At 2 months this difference had disappeared with counts of 38.8± 3.2 and 39.1± 3.2 over the operated and reference arteries respectively. Early postoperative CUR at 1.22± 0.04 was significantly higher than 1.01± 0.06 at 2 months which equates to no radiolabelled platelet uptake (p< 0.01). Radioiabellea platelet uptake was visible on 8 of the 10 early scans, but this was seen in only 2 patients at 2 months, both of whom had a persistently high CUR indicating continued platelet accumulation at that time.

Early postoperative platelet deposition decreases in the weeks following carotid endarterectomy presumably due to the development of a neointima. Those cases with persistently high platelet accumulation may have luminal thrombus which could lead intimal hyperplasia and restenosis.
HUMAN POLYMORPHONUCLEAR LEUKOCYTE ACTIVATION INDUCED BY PLATELET ACTIVATING FACTOR (PAF).
Authors: Del Maschio A , Albors M , Bucchi F , Tomasiak M , Bertele V , Cerletti C , de Gaetano G
Published: August 2018

Human polymorphonuclear leukocytes (PMNs) loaded with the photoprotein Aequorin, were exposed to PAF in the presence of extracellular Ca2+ (1 mM). PMNs aggregation measured In the “Platelet Ionized Calcium Aggregometer” (P.I.C.A.) was dependent on the concentration of the stimulus. Ca2+ cytoplasmatic increase was monitored in parallel at concentrations of PAF which did not modify cellular integrity (10-7-10-5M). The intracellular Ca2+ flux (up to 19±3 µM) triggered by PAF was also concentration-dependent. In order to establish the role played by this intracellular messenger, we studied some cellular responses possibly related to Ca2+ mobilization: enzymatic release, oxygen radicals production, and arachidonic acid metabolism. PAF induced release of both lysozyme , and β-glucuronldase (15% to 20% of the total enzyme content at the maximal concentration). However PAF (10-712-10“Vl) stimulated the production of only small amounts of oxygen radicals as compared to Phorbol Myristate Acetate (PMA). Leukotriene B4 (LTB4), the main arachidonic acid metabolite in PMNs and the products of its catabolism (20-OH and 20-C00H LTBO were assayed by two different technics (HPLC and RIA) in the same cellular suspensions. PAF (10-4 M)-stimulated PMNs (0.5-2xl07 cells/ml) did not produce any detectable amount of these arachidonic acid metabolites. In contrast, calcium ionophore A 23187 (2 μM)-stimulated PMNs (in the same range of cellular concentration) produce up to 170 ng/ml of LTB4. In conclusion, cytoplasmatic Ca2+ increase in PAF-stimulated PMNs was not accompanied either by oxygen radicals production or by activation of arachidonic acid metabolism catalyzed by 5-1 ipoxygenase.
PLATELET-ACTIVATING FACTOR (PAF)-INDUCED INTRACELLULAR Ca MOBILIZATION IN HUMAN PLATELETS.
Authors: Matsuno K , Katabami F , Koyama M , Abe K , Sakurada K , Miyazaki T , Ozasa S , Saitoh H , Maekawa I , Matsumiya H
Published: August 2018

PAF-induced intracellular Ca2+ mobilization and platelet aggregation were investigated in human platelets. Cytosolic free Ca2+ concentration ([Ca2+]cyt) was measured by using fluorescent 45Ca2+ probe quin2 and fura-2, and photoprotein aequorin. Ca2+ uptake was measured after stimulation by PAF. Platelet aggregation was studied by recording the change in light transmission with platelet rich plasma (PRP) or washed platelet suspension (WPS).

These three Ca2+ -indicators could determine the elevation of [Ca2+ ]cyt that was stimulated by PAF in the presence of extra- cellular Ca2+ (quin2 method: 98.2nM to 289.7nM; fura-2 method: 102.OnM to 351.4nM; aequorin method: 4.1μM to 8.2μM). In the absence of extracellular Ca2+ , however, little elevation of [Ca2+ ]cyt was detected after stimulation by PAF. PAF could evoke the transient Ca2+ uptake.

New PAF specific antagonist, ONO-6240 inhibited PAF-induced platelet aggregation at a concentration from lμM dose-dependently, whereas it didn’t inhibit collagen- and thrombin-induced platelet aggregation at a concentration of lOOyM. ONO-6240 inhibited PAF- induced increase in [Ca2+ ]cyt in a dose-dependent manner as deter mined by these Ca2+ -indicators, as well as platelet aggregation.

These results suggest the increase in [Ca2+ ]cyt is responsible for platelet aggregation induced by PAF, and the increased Ca2+ is derived from external Ca2+ influx chiefly.
IS IT POSSIBLE TO IDENTIFY HUMAN PLATELETS IN VITRO AS BEING DESENSITIZED TO PLATELET ACTIVATING FACTOR (PAF-ACETHER,PAF) IN VIVO?
Authors: Perger Cs , von Felten A
Published: August 2018

PAF is suggested to be of pathophysiological importance in a variety of diseases. Since platelets exhibit a reduced sensitivity to PAF after a contact with this agent, this behavior may be used as indicator of PAF released into the circulation. In extrinsic asthma, platelets show a diminished reaction to PAF after exposition of the patients to the antigen compared to their own platelets before exposition (Beer and von Felten, Adv. Inflamm. Res. 10:323,1986). We were therefore looking for a test system indicating directly whether platelets had been in contact with PAF.

Preparation of PAF-desensitized platelets: Citrated PRP was placed in a cuvette of an aggregometer, and PAF was added in 10 portions at intervals of 10 sec (37oc, constant stirring) to a final concentration of 10 to 100 nM, depending on the individual sensitivity of each platelet preparation. Therby, only a minimal, completely reversible aggregation was registered without any release of serotonin (ST) or 3-thromboglobulin (BTG). Control platelets were pretreated with buffer instead of PAF. Both platelets preparations were kept at 37°C for 45 min. Whereas control platelets showed a secondary aggregation to PAF (5x conc. used for desensitization), PAF-pretreated piatelets were only reversibly aggregated.

Sensitivity of PAF-desensitized and control platelets to other platelet agonists: No difference in aggregation, ST-or BTG-relea-se was observed after stimulation with several concentrations of ADP, collagen and arachidonate (p>0.05,n= 41).

Binding of 3H-PAF to platelets: PAF-desensitized and control platelets were separated from plasma by filtration through sepharose CL-2B (Pharmacia) in hepes-buffered Tyrode’s solution. After incubation with 3H-PAF, platelets were washed on Whatman 934-AH filters (vacuum filtration). On desensitized and control platelets, we found 175±48 (mean±sd) and 231±70 3H-PAF molecules / platelet respectively after incubation with 5 nM ^h-PAF, 399±36 and 504±66 ^H-PAF molecules / platelet after incubation with 20 nM. In spite of a statistically significant reduction of PAF-binding after desensitization (p<0.01),the variability of PAF-binding between platelets of different individuals is too high to allow a discrimination of normal from PAF-desensitized platelets.
PROTEASE NEXIN II IN HUMAN PLATELETS.
Authors: Van Nostrand W E , Cunningham P P
Published: August 2018

Normal human fibroblasts secrete a protein into the culture medium named protease nexin II (PN II), which forms sodium do-decyl sulfate (SDS)-stable complexes with epidermal growth factor binding protein (EGF BP). These complexes then bind back to the same cells and are rapidly internalized and degraded. We recently purified PN II to apparent homogeneity and showed that it is a single chain polypeptide with an estimated molecular weight of 106 KDa. Other interesting properties of this protein include its ability to bind heparin and its stability to treatment with SDS or pH 1.5. In addition to EGF BP, PN II will also complex the gamma subunit of 7S nerve growth factor (NGF-gamma) and trypsin. Here we show that human platelets contain a protein which possesses the same properties as does PN II from cultured human fibroblasts. This platelet PN II (PL-PN II) is also a 106 KDa single chain polypeptide which can complex EGF BP, NGF-gamma and trypsin. PL-PN II also possesses the unusual stability to treatment with SDS or pH 1.5. In addition, both PN II and PL-PN II exhibit the same affinity for binding to heparin-Sepharose. Furthermore, rabbit polyclonal antiserum raised against PN II recognized PL-PN II on Western blot analysis demonstrating that both proteins are immunologically related. Treatment of fresh unactivated platelets with epinephrine or thrombin resulted in a release of PL-PN II into the supernatant suggesting that PL-PN II is stored in the platelet alpha granules.
MONITORING OF THE PLATELET ALPHA-GRANULE SECRETION IN THE AGGREGOMETER.
Authors: Patscheke H , Mathieu G
Published: August 2018

If platelets are stimulated to secrete and the aggregation is prevented by EDTA and no stirring, the optical density (OD) decreases as a consequence of secretion (Patscheke et al. Thromb. Res. 33: 314, 1984). The purpose of this study was to determine which secretory compartment causes the change in OD and to analyze the quantitative relationship between decrease in OD and granule discharge. Human washed platelets were stimulated with thrombin and A 23187 in a Lumi-Aggregometer (Chrono-Log) which permitted simultaneous recording of the change in OD and of the ATP release from dense granules. At various time intervals, platelet factor 4 (PF-4), [3H)serotonin (5-HT), 8-N-ace-tylglucosaminidase (NAG) and lactate dehydrogenase (LDH) were determined in the supernatant as parameters of the release from the alpha granules, dense granules, lysosomes and the cytoplasm, respectively. In order to prevent the platelet shape change (increase in OD) from interfering with secretion (decrease in OD), the platelets were pretreated with 0.1 nM PAF 2 min prior to the secretagogue. PAF induced the shape change but no release of platelet constituents. The results show that the decrease in OD closely correlates with the release of PF-4. The fractional effects were identical in concentration-effect and time-effect studies. However, neither the decrease in OD nor the release of PF-4 were correlated with the release of ATP and 5-HT from the dense granules or the lysosomal release of NAG. The release of ATP and 5-HT required significantly higher agonist concentrations than the decrease in OD and the release of PF-4 and even higher concentrations were required for the release of NAG. LDH liberation did not exceed 1 % with 1 U/ml thrombin, indicating the absence of lysis. Thrombin 1 U/ml caused a maximum decrease in OD of 11 % and 40 % release of PF-4. In a patient with gray platelet syndrome, the decrease in OD was absent while the release of 5-HT was normal. These results show that the decrease in OD is due to alpha-granule secretion. The turbidimetric method offers a valuable tool for kinetic measurements of alpha-granule secretion. By using a Lumi-Aggregometer, secretion from alpha and dense granules can be monitored simultaneously. (Supported by the Deutsche Forschungsgemeinschaft, Grant Pa-263).
PLATELET DERIVED GROWTH FACTOR (PDGF) BINDS TO HUMAN PLATELETS AND MODULATES PLATELET ACTIVATION
Authors: Bryckaert M C , Wasteson A , Tobelem G , Rendu F , Caen J P
Published: August 2018

PDGF which is released during platelet activation like the other ∝ granule components (fibrinogen, F VIII/vWF, PF4) could bind to platelet membrane Following this hypothesis, we have studied the binding of 125I pure human PDGF to washed human platelets activated by collagen. This binding was specific and time dependent and reached a plateau with 20 μg/ml of collagen. With 200 fold excess of unlabeled PDGF, the binding of 125I-PDGF decreased progressively to 10 .whereas unlabeled Epidermal Growth Factor did not compete with 125I-PDGF. Saturation curve and scatchard analysis have shown one class of sites 3,000 sites/cell with an apparent Kd = 10-8 M. The demonstration of PDGF binding to platelets led us to investigate the effects of PDGF on platelet function. PDGF inhibited the aggregation and 14C serotonin release induced by thrombin or collagen. This inhibition was dose dependent and more effective with human PDGF. A total inhibition of collagen-induced platelet aggregation was obtained with 50 ng/ml of human PDGF and 200 ng/ml of porcine PDGF. The aggregation and 14C serotonin release induced by arachidonic acid were not inhibited by PDGF. The metabolism of phosphoinositide was also investigated on washed human platelets prelabeled with 32P orthophosphate. We found that PDGF (200 ng/ml) induced a decrease of 32P associated with phosphatidylinositol 4 biphosphate (72 %) after 3 min, with a parallel increase of 32P-phosphatidylinositol 4 Phosphate (120 %) and 32P-phosphatidylinositol (120 %).In conclusion i) PDGF binds to activated platelets, ii) PDGF inhibits platelet aggregation and secretion, iii) PDGF modifies phosphoinositide metabolism. These results are in favour of a role of PDGF in a negative feed back control of platelet activation.
THE PROFILE OF IN VIVO PLATELET ACTIVATION IN NOCTURNAL ASTHMA
Authors: Morrison J EJ , Latif A B , Mason C , Bramley P , Criag T R
Published: August 2018

The precise pathophysiology of nocturnal asthma still remains to be elucidated. Activated platelets have the ability to release potent broncho- and vaso-constrictors and therefore, have been implicated in asthma. However, there is no information on the status of in vivo platelet activation in patients with nocturnal asthma

In a randomised controlled study five healthy volunteers and five asthmatics were investigated during a period of 24h after acclimatisation for one day. Both peak flow rate (PFR) and blood samples were obtained at 4 hourly intervals. Plasma levels of platelet factor 4 (PF4) and beta- thromboglobulin

(BTG) were measured by radioimmunoassay and adrenaline (A), noradrenaline (NA) and dopamine (DOP) by radioenzymatic analysis.

PFR (1/min) for the 24h period was significantly lower in asthmatics (401±15SEM, P<0.001) than in controls (598+4SEM) with an apparent circadian rhythm peak (442±73, P<0.05 Wilcoxon's test) at 4.00pm only in asthmatics. Although there was no significant differences in either PF4 and BTG (ng/ml) or A, NA and DOP (nmol/1) between asthmatics and controls an apparent circadian rhythm in all of these parameters was demonstrated in both groups. Peak values (mean+SEM) for PF4 (8.9±1.5) and BTG (44.4±3.8) occurred at 8.00am whereas the highest values for the catecholamines (A: 0.36±0.08, NA: 1.75±0.23, DOP: 0.78±0.16) were observed at 4.00pm indicating a lag of 8h between the peaks for catecholamines and the platelet specific proteins

These initial data demonstrate a clear difference in PFR between asthmatics and controls which is apparently not associated with changes in either PF4 or BTG but which may concur with circadian changes in plasma levels of catecholamines at least in asthmatic patients. Thus, in vivo platelet activation is probably not a contributing factor in nocturnal asthma. Finally, the phase lag between peak plasma levels of platelet proteins and catecholamines requires further investigation
DIFFERENTIAL NEUTRALIZATION OF UNFRACTIONATED HEPARIN (UH) AND LOW MOLECULAR WEIGHT HEPARINS (LMWHS) BY HUMAN PLATELET FACTOR FOUR (PF-4)
Authors: Racanelli A , Fareed J
Published: August 2018

Platelet factor 4 neutralization profiles of four newly developed LMWHs, PK 10169 (Pharmuka, Gennevilliers, France)
CY 216 (Choay, Paris, France), KABI 2165 (KabiVitrum, Stockholm, Sweden), OP 2123 (Opocrin, Corlo Italy) and an unfractionated porcine mucosal heparin were studied utilizing amidolytic (anti-factor Xa, anti-factor Ila) and clot based (APTT, Heptest ) assays. The neutralization studies were carried out in normal human pooled plasma in two experimental protocols

In one set of experiments, PF-4 (10μg/ml) was added to varying amounts of UH or LMWH (10-2.5 μg/ml). In the second set of experiments, the concentration of heparins was fixed at 5 μg/ml and varying amounts of PF-4(10-2.5 μg/ml) were added. The relative neutralization profiles of these heparins were assay dependant. The anticoagulant effects of UH and LMWHs as measured by APTT were equally neutralizable. Similarly the anti-factor Ila actions of both UH and LMWH were effectively neutralized, whereas the anti-factor Xa activity of the LMWHs was less neutralizable than the UH. Different levels of residual anti-Xa activity of the LMWHs were seen whereas the UH was effectively neutralized. Interestingly, although the anti Xa activity of the UH and Kabi 2165 as measured by amidolytic and clot based methods were similiar and more potent than the other LMWHs, however there were differences between their PF-4 neutralization profile. At a gravimetric ratio of 2:1 , the anti Xa activity of the UH was completely neutralized whereas the Kabi 2165 was least neutralized of all of the LMWHs. The slope of the PF-4 titration curves in the anti-factor Xa assays for all of the LMWHs were similar and varied significantly from the UH. PF-4 neutralization of UH and LMWHs was not dependant on the molecular weight or pharmacopoeial potency (USP or antifactor Xa). These results show that UH and LMWHs exhibit a dissociation between the anti-factor Xa and anti-factor Ila neutralization which may contribute to their sustained antithrombotic effects.
FAB FRAGMENTS OF MONOCLONAL ANTIBODIES SPECIFIC FOR PORCINE PLATELET MEMBRANE GLYCOPROTEINS GP IB ANDGP IIB/IIIA
Authors: Takami H , Nichols W L , Kaese S E , Miller R S , Katzmann J A , Bowie E J W
Published: August 2018

For further study of the porcine hemostatic mechanism, we have prepared murine monoclonal antibodies, and F(ab')2 and Fab fragments, specific for porcine platelet membrane glycoproteins GP lb and GP Ilb/IIIa. To avoid production of antibodies to von Willebrand factor (vWF), mice were immunized with platelets obtained from pigs with severe von Willebrand,s disease. One monoclonal antibody (PP3-4C), of IgG1 subclass, caused 85% inhibition of Ristocetin-induced platelet binding of 125I-vWF (porcine) at ≥12 µg IgG/ml. PP3-4C did not affect ADP or collagen-induced platelet aggregation nor inhibit 125I-fibrinogen (porcine) binding. Pepsin and papain digestion, respectively, were used to prepare PP3-4C F(ab')2 and Fab fragments. PP3-4C F(ab')2 at concentrations ≥12 µg/ml caused 80% inhibition of washed platelet agglutination in the presence of vWF and Ristocetin, whereas Fab fragments at concentrations ≥10 µg/ml caused 60% inhibition. Another monoclonal antibody (PP3-3A), of IgG1 subclass, completely inhibited ADP or collagen-induced platelet aggregation at an IgG concentration of 6 µg/ml. At 10 µg IgG/ml PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated porcine platelets. PP3-3A did not affect vWF-dependent Ristocetin-induced platelet agglutination, nor 125I-vWF binding to platelets in the presence of Ristocetin. PP3-3A did not bind to platelets which were treated with 10 mM EDTA at 37°C for 60 min. F(ab')2 and Fab fragments were isolated from PP3-3A pepsin or papain digests. Both types of PP3-3A fragments caused 100% inhibition of ADP-induced platelet aggregation, at concentrations ≥6 yg/ml. Immunoprecipitation of surface-radiolabeled porcine platelets and subsequent SDS-PAGE demonstrated that PP3-4C recognized a glycoprotein with molecular weight of 140,000 (under reducing conditions), and 165,000 (non-reduced). PP3-3A recognized glycoproteins with molecular weights of 115,000 and 100,000 (reduced), and 130,000 and 80,000 (non-reduced). Neither monoclonal antibody bound to human platelets. These monoclonal antibodies to porcine platelet membrane glycoproteins which are analogues of human GP lb and GP Ilb/IIIa will be useful for in vitro and in vivo studies to further understanding of mammalian hemostatic mechanisms.
INTERACTION OF MEMBRANE GLYCOPROTEIN GPIIb AND Ilia WITH CYTOSKELETAL PROTEINS DURING PLATELET ACTIVATION
Authors: Fujimura K , Fujimoto T , Takemoto M , Oda K , Maehama S , Kuramoto A
Published: August 2018

Experiments were designed and performed to analyse the cytoskeleton assembly and the interaction of glycoprotein (GP)IIb, IIIa and cytoskeletal proteins during platelet activation. A23187 stimulated 125I labeled platelets were solubilised with Triton X-100 solution and centrifuged. The insoluble fraction were analysed by two dimensional electrophoresis and the soluble fraction were fractionated with 5-25% sucrose gradient centrifugation and analysed by SDS PAGE. In Triton X-100 insoluble fraction, high molecular weight protein fraction(MW > 106) was present after stimulation which were consisted of actin binding protein(ABP), myosin heavy chain(MHC), actin and GPIIb and IIIa. And some of the ABP and MHC formed dimer. ABP and actin in this fraction were decreased with 1 mM CaCl2 treatment but the reduction of ABP was inhibited by leupeptm. In Triton X-100 soluble fraction after stimulation, some of the ABP, MHC, P235 protein, actin and small amount of GPIIb, IIIa were sedimented in the same high density fraction but most proteins were sedimented as a monomer form or GPIIb-IIIa complex form. The GPIIb, IIIa incorporation in high molecular weight protein fraction or high density fraction was absent in Ca++ chelating condition or the presence of competitive fibrinogen binding inhibitor which blocked the platelet aggregation. It is concluded that cytoskeletal proteins and GPIIb, IIIa are assembled each other and formed high molecular weight protein fraction or dimer formation during activation. In stimulated platelets these assembled cytoskeletal proteins containing GPIIb, IIIa were also found in Triton X-100 soluble fraction as a precursor of high molecular weight fraction in Triton X-100 insoluble fraction. The binding of fibrinogen to GPIIb-IIIa complex induce the linkage of GPIIb, IIIa to assembled cytoskeletal proteins.
FIBRINOGEN SEQUENCES INTERACTING WITH PLATELET GPIIbIIIa
Authors: Andrieux A , Charon M H , Hudry-Clergeon G , Marguerie G
Published: August 2018

Fibrinogen (Fg), fibronectin (Fn) and von Willebrand factor (vWF), interact with GPIIbllla on AD? stimulated platelets, and a common mechanism has been postulated for the binding of these adhesive proteins. Fg, Fn and vWF contain the tripeptide Arg-Gly-Asp and synthetic analogues to this sequence inhibit their interaction with platelet and their concomitant adhesive reactions. On the other hand, sequences corresponding to the Fg γ chain inhibit the binding of Fg, Fn and vWF to platelet and may also represent a potential recognition site. This raises the possibility that the γ chain sequence and Arg-Gly-Asp interact with the same site or represent primary and secondary sites for the Fg molecule. Within this context, the capacity of these sequences to interact with GPIIbllla and to block fibrinogen binding were compared. The smallest γ chain sequence that was active in inhibiting this reaction was the hexamer Lys-Gln-Ala-Gly-Asp-Val corresponding to the last six amino acid residues at the C-terminus of the γ chain. In parallel, peptides with the structure Arg-Gly-Asp-X were synthesized and tested in vitro. The activity of these peptides was dependent upon the hydrophobicity of the amino acid residue at position X. Arg-GLy-Asp-Phe corresponding to the sequence at position 95-98 in the Fg Aα chain was 5 to 10 times more active than Arg-GLy-Asp-Ser, present at position 572-575 in the Aα chain, and was 10 to 20 times more active than the γ chain hexamer. Both the Aα chain and γ chain sequences however, inhibited Fg binding by greater than 90%. When the γ chain sequence and the Arg-Gly-Asp-X sequence were coupled to Sepharose, GPIIbIIIa interacted with these sequences and was eluted from each column by either of the peptides. Finally direct binding experiments indicated that Arg-Gly-Asp-X and γ chain sequences are competitive antagonists. These results suggest that both sequences interact with the same site on GPIIbIIIa and comparison of the hydrophilicity of these peptides suggests that the binding domain on GPIIbIIIa exhibits hydrophobic properties.
DIFFERENT SITES FOR FIBRINOGEN AND FIBRIN RECEPTORS ON PLATELETS
Authors: Cohen I , White J G
Published: August 2018

Platelet stickiness and aggregation depend on availability of surface membrane glycoprotein Ilb-IIIa complex to bind fibrinogen. The development of isometric tension in platelet-rich clots is a manifestation of fibrin binding to the cells as well as platelet contractile activity. The possibility that fibrinogen and fibrin may bind to different portions of the GPIIb-IIIa complex has apparently not been considered. In order to determine whether the receptors for the fibrinogen and fibrin on the GPIIb-IIIa complex are identical, we investigated the effect of various monoclonal antibodies to the Ilb-IIIa complex and the tetrapeptide Arg-Gly-Asp-Ser (RGDS), a recognition site on fibrinogen for IIb-IIIa, on the development of isometric tension and ultrastructure of platelet-fibrin clots. Monoclonal antibodies A2A6 and 7E3 decreased the maximal tension, as well as the rate of tension development. Platelets and fibrin were oriented longitudinally in antibody treated clots, but the concentrations of fibrin and platelet aggregates determined by morphometry were significantly reduced. T10 and 10E5 increased tension, while AP2 and PAC1 had no substantial effect. Increasing concentrations of RGDS from 62.5 pM to 500 pM resulted in greater maximal tension and rate of tension development, reaching a five-fold increase when 500 pM RGDS was used. RGDS did not affect the Mg-stimulated platelet actomyosin ATPase activity. Morphometry revealed increased concentrations of oriented fibrin and platelet aggregates in RGDS-treated clots. Results of this study confirm the different topography of the epitopes on the Ilb-IIIa complex and provide evidence for different receptor sites for fibrinogen and fibrin on the IIb-IIIa complex
DIRECT BINDING OF FIBRINOGEN-GOLD PROBES DOES NOT DISCLOSE THE ENTIRE POPULATION OF BOUND FIBRINOGEN ON ADHERENT PLATELETS
Authors: Estry D W , Mattson J C
Published: August 2018

The binding characteristics of fibrinogen to adherent platelets were determined using both direct protein-gold labeling and an immunogold procedure. The binding of colloidal gold-fibrinogen was studied in whole mounts of contact activated, gel filtered platelets by transmission electron microscopy. There was a transition from minimal binding of fibrinogen in dendritic platelets to marked zonal binding in fully spread platelets. The pattern of direct fibrinogen binding in fully spread platelets appeared to orient itself with the underlying filamentous cytoskeleton, a pattern that is consistant with that previously reported by us and others. In contrast, when bound fibrinogen was assayed using a rabbit anti-fibrinogen antibody, the pattern of bound fibrinogen was diffuse and strong labeling was present in both early dendritic forms as well as late fully spread platelets. To further confirm these observations the direct labeling technique was combined with the immunogold labeling procedure using two different sized gold probes. Platelets previously incubated with fibrinogen-gold (20 nm) were fixed, incubated with rabbit anti-human fibrinogen and then with gold conjugated goat anti-rabbit IgG (10 nm). The morphologic organization of the direct label was unchanged. However, the immunogold technique demonstrated a diffuse binding pattern over the entire cytoplasmic veil including areas previously unlabeled by the direct technique. This suggested that in addition to fibrinogen receptors identified by direct labeling, there are other receptors that either already contain bound fibrinogen, compete for released fibrinogen more affectively, or represent membrane bound granule fibrinogen that is exposed rather than released and rebound and is therefore identified only by the immunogold labeling procedure.
IDENTIFICATION OF YUK(b) ALLOANTIGEN ON PLATELET GLYCOPROTEIN IIIa*
Authors: Santoso S , Shibata Y , Kiefel V , Mueller-Eckhardt C
Published: August 2018

Neonatal alloimmune thrombocytopenic purpura (NATP) is caused by IgG platelet alloantibodies (ab), produced by the mother and directed against antigens present on the platelets of the child. The specificity of the platelet-specific ab is anti-Pl(Al) in the majority of cases. Other specificities, i.e. anti-Pl(E2), anti-Bak(a), anti-Pen, and anti-Pl(A2) have also been found. Recently, Shibata et al (1986) have described a new platelet antigen system Yuk(a)/Yuk(b) involved in NATP. The Yuk(a) and Yuk(b) antigens are not expressed on thromb-asthenic platelets indicating that these antigens do exiŞt on glycoprotein (GP) lib and/or Ilia. In order to investigate the molecular localization of these antigens, we studied the interaction of anti-Yuk(b) purified ab with membrane components of platelets using immunoblot procedure and compared their immunochemical behaviour with that of other platelet specific ab (anti-Pl(A 1), -Lek(a), -Bak(a)).In the absence of disulfide reduction Yuk(b) ab reacted with an antigen of molecular weight (mol wt) 92 kDa with an electrophoretic mobility identical to GP Ilia. An identical result was obtained for P1(A1) ab. In contrast, the Bak(a) ab as well as Lek(a) ab detected an antigen of mol wt 134 kDa which comigrated with GP lib. After reduction with 2-mercaptoethanol binding of anti-Pi(Al) and anti-Yuk(b) was not observed. To further localize the Yuk(b) antigen on GP Ilia, immunoblotting experiments were performed with anti-Pl(Al) and anti-Yuk(b) of chymotrypsin treated platelets. While anti-Pl(Al) bound to GP IIIa and a 68 kDa component, anti-Yuk(b) bound only to GP IIIa when the platelets had been treated for 45 min with chymotrypsin.This discrepancy became even more pronounced by prolonged treatment of platelets (225 min) in that the reactivity of anti-Yuk(b) was entirely abolished, whereas binding of anti-Pl(Al) shifted completely from the 92 kDa to the 68 kDa component. Thus, unlike the P1(A1) antigen, the Yuk(b) determinant either resides on the 30 kDa fragment of GP Ilia or it is destroyed by chymotrypsin treatment.
BLEB FORMATION ON THE HUMAN PLATELET MEMBRANE SURFACE IS INDUCED BY SURFACE BOUND CALCIUM ION
Authors: Wang C T , Tsai W J , Chen J C , Shiao Y J , Lee J Y
Published: August 2018

the morphological sthudy by scanning electron microscopy (SEM) showed that blebs were found on cell surface of the gel-filtered human platelets when incubated in the modified Tyrode's buffer containing 8mM or higher of Ca++ for 5 min. EDTA immediately abolished blebs [Wang et al Thrombos. Haemostas., 54, 184 (1985)]. The phenomenon was further investigated. The bleb-forming cells respond ghto thrombin stimulation with as aggregation rate 30% lower than that of control. EDTA reversed the aggregation rate back to that of control. Two other divalent cations, i.e. Ba++ andSr++ at a concentration above8 mM could not induce bleb on cell jsurface. however, pseudopods were formed. In response to thrombin stimulation, the cell treasted with either Ba++or Sr++ exhibited a similar aggregation rate to that of control. Hence, calcium ions may be specifically responsible for including the bleb formation, which may retard cell function. The amount of Ca++ bound to cell surface was estimated by using 4 5CaCl2 as a probe. After incubation with 4 5CaCl2, the cell suspension was divided into two aliquots. One was immediately centrifuged through a layer of silicon oil.The other was added with EDTA prior to the centrifugation. The difference between radioactivity of the former and that of the latter represented the amount of surface bound Ca++. We found that 1200 pmole/108 cells was bound on cell ksurface by incubating the cells in 10 mM Ca++ for 5 min. The same quantity was also found in the cells incubated with 5 mM Ca++ for 60 min. However, the latter had no bleb on the cell surface as revealed by SEM. The result implies that the bleb formation is caused by an impact of large amount of Ca++ on cell surface.
MORPHOLOGICAL CHANGES OF BLOOD PLATELETS IN MAN DURING DEEP SATURATION DIVING
Authors: Klausen Hanne , Flood Per , Hjelle John O , Holmsen Holm
Published: August 2018

The small number of reports concerning blood platelets during deep saturation diving have shown that there may be a decrease in the number of circulating platelets. Gas bubbles are often present in theblood during decompression. In vitro, gas bubbles activate platelets, and alter the shape from discoidto spherical configuration with multiple, blunt spikes. This study was done to investigate if there are changes in the morphology of human blood platelets during deep diving. An 18 day long experimentalon-shore saturation dive to 360 msw was performed at NUTEC 1986. Bloodsamples were obtained from the 6 divers on 6 occations: pre-dive control, at 360 msw, 300 msw, 140 msw, 1 and 3 days after surfacing. Using 18 G Wasserman needles antecubital venous blood samples (3ml) were collected directly into the fixative agent (7 ml of 2% glutardialdehyde in cacodylate buffer) whilestill in the hyperbaric chamber. The samples were then decompressed,and the platelets separated from the blood by centrifugation at roomtemperature for 10 min at 190 g. Preparation to transmission electron microscopy included post-fixation in osmium tetroxide, staining uranyl-en-bloc and eventually lead, dehydration and embedding in Epon.Ultrathin sections were examined by a Philips 300 I electron microscope. The examination revealed alterations in both platelet size and shape in the course of the dive. The mean platelet area was increased during and immidiately after thedive, the greatest increase occuring at 360 msw. There was a high incidence of shape changed plateletswith spherical configuration and multiple, blunt spikes. This form was by far most abundant at 360 msw, and the morphology normalised towards surface. This rises the suspection of a pressure-related rather rhan bubble-related effect and the results indicate that plateletsin circulation can be activated bypressure itself
THE INTERACTION OF BLOOD ELEMENTS WITH ENDOCARDIAL ENDOTHELIUM DAMAGED BY LACTIC ACID
Authors: Carter G , Gavin B J
Published: August 2018

It has already been demonstrated that ischaemic metabolites, which could diffuse frcm a myocardial infarct in vivo, can cause substantial damage to the endocardial endotheliun and this could predispose to mural thrombosis.

To investigate the role of ischaemic metabolites in the pathogenesis of mural thrombosis, lactic acid (pH6.4) was passed through a two-way concentric catheter ligated into the left ventricle of isolated beating rat hearts that were perfused with oxygenated Krebs-Henseleit buffer (KHB) through an aortic cannula. After periods of 1, 2, and 4 hours, the lactic acid was followed for 10 minutes by 10 mis of whole blood from hepa-rinized donor rats. Ventricles were then flushed with KHB, fixed in 2.5% glutaraldehyde and post-fixed in 1% osmium tetrox-ide in cacodylate buffer.

Scanning and transmission electron microscopy showed that platelets adhered to exposed basal lamina, microfibrils and collagen but not to intact or damaged endothelial cells. However densely aggregated thrombi only farmed on regions of exposed connective tissue and never on basal lamina. Fibrin, leukocytes and red blood cells were associated with these platelet thrombi. Thus lactic acid and other ischaemic metabolites which could possibly diffuse in vivo from an infarct can contribute to endocardial damage which predisposes to mural thrombosis.