The Effect of Halofenate – Free Acid on Aggregation – the Release Reaction, Coagulant Activity, and Lipid Metabolism of Human Platelets

1977 ◽  
Vol 38 (03) ◽  
pp. 0612-0619 ◽  
Author(s):  
Huzoor- Akbar ◽  
N. G Ardlie
Keyword(s):  
Free Acid ◽  
Platelet Rich Plasma ◽  
Lipid Synthesis ◽  
Human Platelets ◽  
Second Phase ◽  
Inhibitory Effects ◽  
Platelet Factor ◽  
Release Reaction ◽  
Dense Granules ◽  
Coagulant Activity

SummaryHalofenate – free acid (HFA), the major metabolite of the hypolipidemic drug, halofenate, inhibited platelet aggregation induced by collagen and sodium arachidonate and blocked the second phase of aggregation caused by ADP, thrombin and epinephrine in human platelet-rich plasma. The aggregation of washed platelets by thrombin and collagen was also blocked. HFA also inhibited the release by thrombin and collagen of 5-hydroxytryptamine from dense granules of platelets and the release by thrombin of β-glucuronidase from platelet α-granules. These inhibitory effects were concentration and time-dependent. HFA decreased platelet factor 3 activity by 31 % and also inhibited the incorporation of 14C-acetate and U-14C-glucose into platelet lipids by 89 % and 56 % respectively. Thrombin-induced lipid peroxidation and prostaglandin formation was investigated by measuring the by-product malonyldialdehyde, and this was found to be inhibited by HFA. It is suggested that the effect of HFA on aggregation is attributable to inhibition of the release reaction which may in turn be a consequence of the effects of the drug on platelet lipid synthesis.

Inhibition of ADP-Induced Responses in Human Platelets by Agents Elevating the Cyclic AMP Level: Comparison of Aggregation and Shape Change

1984 ◽  
Vol 52 (03) ◽  
pp. 333-335 ◽  
Author(s):  
Vider M Steen ◽  
Holm Holmsen
Keyword(s):  
Inhibitory Action ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Early Step ◽  
Stimulus Response ◽  
Sequential Relationship

SummaryThe inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses.Human platelet-rich plasma was preincubated for 2 min at 37° C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors.These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.

Human High Molecular Weight Kininogen - A Secreted Platelet Coagulant Protein

1981 ◽  
Author(s):  
A H Schmaier ◽  
J Kuchibhotla ◽  
R W Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Ionophore A23187 ◽  
High Molecular Weight Kininogen ◽  
Contact Phase ◽  
Platelet Factor ◽  
Coagulant Activity

Platelets have been shown to contain a number of secret- able coagulant proteins, which participate as substrates or cofactors in plasma coagulation reactions. Since we have previously demonstrated that high molecular weight kininogen (HMWK) is immunochemically present in platelet extracts, we posited that HMWK is secreted during activation of platelets. Fresh normal platelets were washed by a combination of albumin-gradient and gel-filtration procedures. In 11 experiments the supernates of freeze-thaw lysates of normal human platelets contained a mean of 5.7 Units (range 3.16 to 8.14) of HMWK coagulant activity/3 × 1011 platelets. This coagulant activity was neutralized by a goat antiki- ninogen antibody. Using a 125I-HMWK tracer in PRP, the supernate of washed activated platelets contained 0.082% radioactivity as the starting PRP, suggesting that 14% of the total HMWK coagulant activity could be accounted for by plasma contamination. In four experiments, ionophore A23187 (15μM) induced a net secretion of 39% of the total platelet HMWK (range 16 to 49%). Platelet HMWK secretion by A23187 was concentration dependent (1 to 15 μM) . At A23187 (15μM) platelets released 75% 14C-5HT (range 61 to 99%) and 81% low affinity platelet Factor 4 (range 60 to 99%). Ninety-five percent of A23187-induced secretion of HMWK could be blocked by platelet pretreatment with metabolic inhibitors. LDH determinations indicated that only 5% (range 0 to 10%) of total secreted platelet HMWK could be attributed to lysis. Collagen and PGH2 also caused secretion of platelet HMWK coagulant activity. This study indicates that human platelets contain functional HMWK which may be secreted locally to modulate the reactions of the contact phase of plasma proteolysis.

Inhibition of PF4 and ßTG Release from Platelets by Piracetam

1979 ◽  
Author(s):  
R.L. Henry ◽  
R.M. Nalbandian ◽  
G.E. Herman ◽  
T. Ho
Keyword(s):  
Platelet Aggregation ◽  
Normal Individual ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Complete Inhibition ◽  
Clot Retraction ◽  
Platelet Factor ◽  
Storage Pool ◽  
No Inhibition ◽  
No Release

Platelet factor four (PF4) and beta-thromboglobulin (βTG) were released from human platelets alpha granules by ADP and epinephrine and measured by radioimmunoassay. Both release materials are antiheparins but PF4 is reported to be more potent. However, PF4 is released at about 1/4 the level of βTG in nanog rams/ml. Total release occurred with 5 ugm/rnl ADP in platelet-rich-plasma adjusted to 200,000 platelets/mm3 and with 1.25 × 10-5M epinephrine. No further release was found by freeze-thawing procedures. In one case, no release occurred although full aggregation proceeded normally with both mediators. Only minimal amounts were recorded after freeze-thawing indicating a storage pool deficiency of PF4 and βTG in an apparantly normal individual. Complete inhibition of PF4 and βTG release was obtained concurrently with elimination of the 2nd epinephrine wave by 6.4 × 10-4 M Piracetam. In contrast to aspirin, no inhibition of ADP, Collagen, or Ristocetin aggregation or release occurred with Piracetam. In previous work it was determined that Piracetam even at 6.4 × 10-3 M did not modify thrombin, prothrombin, or activated partial thromboplastin times. In addition, clot retraction was not modified in concentrations of Piracetam as high as 1.28 × 10-2 M known to eliminate the 2nd wave of platelet aggregation by epinephrine.

scholarly journals Thrombocytin, A Novel Platelet Activating Enzyme from Bothrops Atrox (MARAJOENSIS) Venom

1977 ◽  
Author(s):  
S. Niewiarowski ◽  
E.P. Kirby ◽  
G.J. Stewart ◽  
R. Turna ◽  
M. Wiedeman ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Activate Factor ◽  
Factor X ◽  
Electron Microscopic ◽  
Platelet Factor ◽  
Small Vessels ◽  
Platelet Aggregates ◽  
Bothrops Atrox ◽  
Induced Aggregation

Thrombocytin (TCN) was purified from Bothrops atrox (BA) venom by precipitation with 1.2% Na-salicylate and chromatography on heparin-agarose column using increasing concentrations of lysine as eluent. It was homogeneous on SDS electrophoresis and had an apparent MW of 36,000. Immunoelectrophoresis with polyvalent anti-BA venom serum gave one cathodic arc indicating an isoelectric point higher than pH 8.6.TCN at a concentration of 1 yg/ml caused aggregation of human platelets, release of low affinity platelet factor 4 and serotonin, and stimulated platelets to retract fibrin.TCN was essentially free of fibrinogen clotting and fibrinolytic activities.TCN action on platelets was not mediated by the formation of thrombin since TCN did not activate Factor X or prothrombin and its action was not inhibited by hirudin.TCN is a serine protease since it was inhibited by DFP and it hydrolyzed a synthetic peptide, chromozyme UK (BZ-Val-Gly-Arg-pNA·HCl).TCN-induced aggregation of human platelets was completely inhibited by soy bean trypsin inhibitor, heparin, prostaglandin E1 and apyrase. Washed human platelets were 2-4 times less sensitive to TCN as compared to platelets in freshly prepared platelet rich plasma (PRP); their sensitivity to TCN gradually deteriorated during incubation of PRP at room temperature for 3 hours. Electron microscopic observations revealed formation of platelet aggregates characterized by pseudopod formation, centralization and partial loss of platelet granules. Infusion of TCN (3 yg) into the main artery of bat wing resulted in the formation of platelet aggregates seen on arterial and venous side which occasionally occluded small vessels.

Reduction of cAMP and cGMP inhibitory effects in human platelets by MRP4-mediated transport

2012 ◽  
Vol 108 (11) ◽  
pp. 955-962 ◽  
Author(s):  
Alessandra Borgognone ◽  
Fabio Pulcinelli
Keyword(s):  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Thrombus Formation ◽  
Physiological Mechanism ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Dense Granules ◽  
Dependent Inhibition ◽  
Novel Target ◽  
Camp And Cgmp

SummaryCyclic nucleotide-dependent inhibition of platelets represents the most important physiological way to limit thrombus formation. cAMP and cGMP increase in platelets as a consequence of prostacyclin and nitric oxide production by endothelial cells and act through PKA and PKG, respectively. The cytosolic concentration of cyclic nucleotides in platelets is regulated by AC- and GC-dependent synthesis and PDE-dependent degradation. In some cells cyclic nucleotides are eliminated also through MRP4/5/8-dependent efflux. As only MRP4 is expressed in platelets, at high levels in dense granules, we determined its role in the elimination of cyclic nucleotides from platelet cytosol. We studied the effects of MRP4 inhibition on cAMP/cGMP effects in platelets. Cyclic nucleotide inhibitory effects triggered by cAMP and cGMP-elevating agents on platelet aggregation are strongly enhanced by MRP4 inhibition and so is cyclic nucleotide-dependent phosphorylation of the common substrate VASP. MRP4 inhibition decreases cAMP concentration in platelet granules and both cAMP and cGMP compete with an established substrate of MRP4 (fluo-cAMP) for entrance in granules. Here we provide the first evidence of the transport of cyclic nucleotides mediated by MRP4 as part of their physiological mechanism of elimination in human platelets, which might represent a novel target to increase cyclic nucleotide-dependent inhibition.

Platelet sialic acid and platelet survival after aggregation by ADP

Blood ◽  
1980 ◽  
Vol 56 (5) ◽  
pp. 876-880
Author(s):  
MA Packham ◽  
MA Guccione ◽  
RL Kinlough-Rathbone ◽  
JF Mustard
Keyword(s):  
Sialic Acid ◽  
Human Platelets ◽  
Platelet Surface ◽  
Release Reaction ◽  
Dense Granules ◽  
Total Sialic Acid ◽  
Platelet Survival ◽  
Rabbit Platelets ◽  
Induced Aggregation

Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP- induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.

The Effects of Citrate and Extracellular Calcium Ions on the Platelet Release Reaction Induced by Adenosine Diphosphate and Collagen

1979 ◽  
Vol 42 (02) ◽  
pp. 778-793 ◽  
Author(s):  
Stanley Hepinstall ◽  
Patricia M Taylor
Keyword(s):  
Calcium Ions ◽  
Sodium Citrate ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Extracellular Calcium ◽  
Release Reaction ◽  
Calcium Dependent ◽  
Platelet Release ◽  
Dependent Mechanism

SummaryThe ADP-induced release of 3H-serotonin from human platelets in heparinized platelet rich plasma is markedly stimulated by the addition of sodium citrate. The aggregation and release that is induced by collagen is less affected by citrate. Data is presented that supports the view that the effects of citrate on both ADP-and collagen-induced release are largely via alteration of the concentration of ionized calcium in plasma.Collagen can induce release of 3H-serotonin via extracellular calcium-independent and -dependent mechanisms. The possibility that the calcium-dependent mechanism is aggregation-dependent and that the calcium is required for platelet aggregation rather than directly involved in the release reaction is discussed.

scholarly journals Platelet release reaction and intracellular cGMP

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 524-531 ◽  
Author(s):  
A Weiss ◽  
NL Baenziger ◽  
JP Atkinson
Keyword(s):  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Serotonin Release ◽  
Secretory Process ◽  
Release Reaction ◽  
Intracellular Cgmp ◽  
Exogenous Addition ◽  
Human Thrombin ◽  
Platelet Release

Abstract Enchanced cAMP concentrations inhibit the aggregation and release reaction of isolated human platelets and platelet-rich plasma to all known inducing agents. An opposing role for cGMP in this phenomenon has been proposed by some but not by others, and the function of cGMP in this secretory process is unclear. To further elucidate the role of cGMP in the release reaction, the effect of increased concentrations of this cyclic nucleotide on 14C-serotonin release was evaluated utilizing isolated human platelets and highly purified human thrombin or commercially available bovine thrombin. Several recently described stimulators of guanylate cyclase, including sodium nitroprusside, sodium azide, nitrosoquanidines, and ascorbic acid, were found to markedly augment platelet cGMP levels. Enhanced platelet cGMP concentrations produced by these drugs or by the exogenous addition of cGMP and its analogues neither caused these cells to secrete nor modulated the thrombin-induced serotonin release reaction. The inhibition of serotonin release by increased cAMP concentrations was not counteracted by increased cGMP levels. Platelet cGMP concentrations were unaltered by thrombin. These data indicate that cGMP is not an obligatory signal or a modulator of the thrombin-induced platelet release reaction.

scholarly journals Further Evidence of the Involvement of Thrombin in Platelet Release by ADP and Other Agents

1977 ◽  
Author(s):  
N. G. Ardlie ◽  
Huzoor- Akbar
Keyword(s):  
Calcium Concentration ◽  
Chelating Agent ◽  
Coagulation Factors ◽  
Free Calcium ◽  
Second Phase ◽  
Inhibitory Effects ◽  
Buffer Solution ◽  
Release Reaction ◽  
Free Calcium Concentration ◽  
Platelet Release

There is evidence of the involvement of coagulation factors in platelet aggregation and the release reaction caused by ADP and collagen but this has been challenged. This report concerns further experiments which can explain the apparently conflicting observations of various laboratories and which provide additional evidence of the involvement of coagulation factors and thrombin in the platelet release reaction caused by ADP and other agents.Washed platelets suspended in a buffer solution responded poorly to ADP with no second phase aggregation or release of [3H]5HT. In contrast, washed platelets suspended in dialysed plasma underwent second phase aggregation and released radioactivity. This response depended on calcium. Dialysed plasma deficient in factors XI or X did not restore second phase aggregation or the release reaction. Hirudin and heparin inhibited second phase aggregation and release by ADP and epinephrine. However, the inhibitory effects of heparin and hirudin on ADP,epinephrine and collagen were not observed when citrate was present. To explore the possibility that a reduction of the free calcium concentration accounts for the inhibition of action of these antithrombin agents by citrate experiments with EGTA were carried out. This alternative chelating agent also prevented the inhibitory actions of heparin and hirudin.These observations support the view that platelets and clotting function cooperatively in platelet reactions involved in haemostasis. We suggest that small amounts of thrombin formed prior to fibrin clotting mediate platelet reactions in haemostasis initiated by collagen and ADP and that secondary aggregation does not represent a citrate artifact.

scholarly journals Factors Affecting the Factor X Activator Activity of Human Platelets

1977 ◽  
Author(s):  
J. Vermylen ◽  
N. Semeraro
Keyword(s):  
Factor Viii ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Activate Factor ◽  
Factor Viii Inhibitor ◽  
Factor X ◽  
Factors Affecting ◽  
Coagulant Activity ◽  
Viii Inhibitor

Several recent studies have indicated that patients with a thrombotic tendency have enhanced platelet coagulant activity. It therefore is of importance to attempt to identify factors which modify platelet coagulant activities. Recent work in our laboratory has provided evidence that human platelets possess the capacity to directly activate factor X.Adenosine-5'-diphosphate, collagen, acetylsalicylic acid and prostaglandin E1 do not modify this activity. All endotoxins studied however clearly enhanced this activity.Furthermore, infusion of ‘activated’ prothrombin concentrates in haemophiliacs with or without factor VIII inhibitor enhanced the activity of this platelet activator of factor X. The hypothesis has been put forward that this may be the mechanism through which ‘activated’ prothrombin concentrates ‘bypass’ factor VIII of IX inhibitors. Platelet isolated from platelet-rich plasma to which the ‘activated’ prothrombin concentrate had been added at a concentration approaching the maximal concentration achieved following in vivo infusion, also showed an increase in platelet coagulant activity. Work is in progress to identify the component in ‘activated’ prothrombin concentrates which enhances platelet coagulant activity.

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