Differential Inhibition of Arachidonic Acid Mediated Aggregation of Platelets by Branded and Generic Argatroban Preparations

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5131-5131
Author(s):  
Christopher Aranda ◽  
Debra Hoppensteadt ◽  
Omer Iqbal ◽  
Bruce E Lewis ◽  
Jawed Fareed
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Serotonin Release ◽  
Final Concentration ◽  
Release Mechanism ◽  
Clinical Effects ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Inhibition Of Thrombin

Abstract Abstract 5131 Argatroban represents a parentral antithrombin agent which is used in the management of anticoagulation in heparin compromised patients. Its main mechanism of action is mediated via inhibition of thrombin and its generation. While its effect on platelet activation inhibition by thrombin have been reported, very little information on the effects of this drug on thromboxane formation and Arachidonic Acid mediated activation of platelets is available. Argatroban and its generic versions namely Slovastan, Argaron and Gartban, may modulate Arachidonic Acid mediated platelet aggregation and release processes. To test this hypothesis a branded version of Argatroban (Mitsubishi – Tanade, Tokyo, Japan) and three generic versions of Argatroban namely Slovastan, Gartban, and Slovastan were compared for their effects on Arachidonic Acid mediated aggregation of platelets in normal healthy male and female volunteers (and n = 4). Other agonists such as Epinephrine, Collagen, and ADP were also used. The effect of Arachidonic Acid on serotonin release was also measured using an Elisa technique for the measurement of serotonin. All of the generic and branded versions of Argatroban produce varying levels of the inhibition of the Arachidonic Acid mediated aggregation of platelets, ranging from the 24 to 36 percent in comparison to the control at a concentration of 1 mg/ mL (p value < 0.05). Interestingly all of the Argatrobans produced a relatively weaker inhibition of the Arachidonic Acid mediated aggregation of platelets 24–26 percent inhibition versus 36 percent at the final concentration of 1 mg/mL. No differences were noted in the aggregation profile of ADP, Collagen, and Epinephrine between the control and the Argatrboban at a final concentration of 1 mg /mL. No differences were noted between the generic and branded Argatroban on all of the other agonists induced aggregation. In the serotonin release assays, all of the generic and branded Argatroban produced a concentration dependent inhibition of serotonin release which was stronger with the branded version of Argatroban. These results indicate that besides the inhibition of thrombin Argatroban is capable of inhibiting platelet activation via other mechanisms. Moreover, the generic versions of Argatroban exhibit a weaker inhibition of Arachidonic Acid mediated platelet aggregation and release. These studies suggest that beside thrombin mediated aggregation Argatroban and its generic versions can modulate platelet activation and release reactions. Furthermore a difference is observed between the generic and branded product which may impact the safety inefficacy profile in these agents. Argatroban is used commonly in the management of patients with heparin induced thrombocytopenia where multiple mechanisms of platelet activation are contributory to the pathogenesis of this syndrome. Modulation of thrombanxane formation and platelet release mechanism by Argatroban may represent an additional mechanism of the clinical effects of this parentral antithrombin agent. Disclosures: No relevant conflicts of interest to declare.

On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation

1981 ◽  
Author(s):  
M Yamamoto ◽  
K Watanabe ◽  
Y Ando ◽  
H Iri ◽  
N Fujiyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Coagulation Factors ◽  
Marked Enhancement ◽  
Matrix Bound ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Plasma Heparin

It has been suggested that heparin caused potentiation of aggregation induced by ADP or epinephrine. The exact mechanism of heparin-induced platelet activation, however, remained unknown. In this paper, we have investigated the role of anti-thrombin III ( AT ) in heparin-induced platelet activation using purified AT and AT depleted plasma. When ADP or epinephrine was added to citrated PRP one minute after addition of heparin ( 1 u/ml, porcine intestinal mucosal heparin, Sigma Co. USA ), marked enhancement of platelet aggregation was observed, compared with the degree of aggregation in the absence of heparin. However, in platelet suspensions prepared in modified Tyrode’s solution, heparin exhibited no potentiating effect on platelet aggregation induced by epinephrine or ADP. Potentiation of epinephrine- or ADP-induced platelet aggregation by heparin was demonstrated when purified AT was added to platelet suspensions at a concentration of 20 μg/ml. AT depleted plasma, which was prepared by immunosorption using matrix-bound antibodies to AT, retained no AT, while determination of α1-antitrypsinα2- macroglobulin and fibrinogen in AT depleted plasma produced values which corresponded to those of the original plasma when dilution factor was taken into account. The activities of coagulation factors were also comparable to those of the original plasma. Heparin exhibited potentiating effect on ADP- or epinephrine-induced aggregation of platelets in original plasma, but no effect in AT depleted plasma. When purified AT was added back to AT depleted plasma at a concentration of 20 μg/ml, potentiation of platelet aggregation by heparin was clearly demonstrated.Our results suggest that effect of heparin on platelet aggregation is also mediated by anti-thrombin III.

Pyridoxal phosphate inhibition of platelet function

1980 ◽  
Vol 238 (1) ◽  
pp. H54-H60 ◽  
Author(s):  
E. Kornecki ◽  
H. Feinberg
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Pyridoxal Phosphate ◽  
Shape Change ◽  
Serotonin Release ◽  
Platelet Surface ◽  
Partial Inhibition ◽  
Induced Aggregation

The effect of pyridoxal phosphate (PLP) on human platelet function in vitro was studied. PLP inhibited adenosine diphosphate (ADP)-induced shape change, aggregation, and the potentiation by ADP of arachidonic acid-induced aggregation. This inhibition could easily be reversed by increasing concentrations of ADP or by removing PLP. The addition of sodium borohydride to PLP-treated platelets produced an irreversible inhibition of ADP aggregation. Thus it is possible that PLP inhibited ADP-induced platelet function by forming a Schiff base with platelet-surface amino groups. PLP also produced a partial inhibition of platelet aggregation to epinephrine, arachidonic acid, A23187, and a dose-dependent inhibition of [14C]serotonin release to epinephrine and arachidonic acid. PLP did not inhibit [14C]serotonin release to A23187, nor did it suppress arachidonic acid-induced malondialdehyde production. The conclusion is drawn that the partial inhibition by PLP of platelet aggregation observed to epinephrine, arachidonic acid, and A23187 resulted from PLP's inhibition of the effect of released ADP.

scholarly journals Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5′-[β-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction

1988 ◽  
Vol 250 (1) ◽  
pp. 209-214 ◽  
Author(s):  
S Krishnamurthi ◽  
Y Patel ◽  
V V Kakkar
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Human Platelet ◽  
Inhibitory Mechanism ◽  
Inhibitory Effects ◽  
Similar Concentration ◽  
Granule Secretion ◽  
Induced Aggregation ◽  
Inhibition Of Thrombin

The effect of guanosine 5′-[beta−thio]diphosphate (GDP[beta S]), reported to be an antagonist of GTP at the G-protein-binding site, on human platelet activation was examined. GDP[beta S] (0.3-3 mM) had significant inhibitory effects on platelet aggregation and 5-hydroxytryptamine (5HT) secretion induced by thrombin, collagen, the thromboxane mimetic U46619 and 1,2-dioctanoylglycerol (diC8) in intact platelets, as well as in saponin-permeabilized platelets. Similar inhibitory effects in intact platelets were also observed with ATP (over similar concentration ranges) and GDP and GTP (at 2- and 10-fold higher concentrations respectively). All four nucleotides also inhibited ADP-induced platelet aggregation in indomethacin-treated platelets under conditions where no 5HT secretion occurred. Inhibition of thrombin-induced aggregation and secretion by GDP[beta S] and ATP in intact platelets was accompanied by a reduction in the thrombin-induced rise in intracellular Ca2+ levels and 45 kDa-protein phosphorylation. The results suggest that at least some of the effects of GDP[beta S] may be unrelated to inhibition of G-protein-GTP interaction, but, instead, may be mediated via an extracellular site, common to all the nucleotides tested and perhaps via inhibition of the effects of endogenous/released ADP. The usefulness of GDP[beta S] as a tool in studying G-protein-GTP interactions in platelets is thus questionable.

PLATELET ACTIVATION INDUCED BY A MONOCLONAL ANTIBODY AGAINST THE PLATELET GP Ilb/IIIa COMPLEX

1987 ◽  
Author(s):  
P W Modderman ◽  
J G Huisman ◽  
J A van Mourik ◽  
A E G Kr ◽  
v d Borne
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Medical Research ◽  
Platelet Activation ◽  
Dense Granules ◽  
Fibrinogen Binding ◽  
Platelet Release ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

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.

scholarly journals Human platelet activation by C3a and C3a des-arg.

1983 ◽  
Vol 158 (2) ◽  
pp. 603-615 ◽  
Author(s):  
M J Polley ◽  
R L Nachman
Keyword(s):  
Platelet Aggregation ◽  
Analysis Of Variance ◽  
Platelet Activation ◽  
Human Platelet ◽  
Human Platelets ◽  
Serotonin Release ◽  
Release Reaction ◽  
C Terminus ◽  
Induced Aggregation ◽  
Synergistic Reaction

C3a liberated from C3 by treatment with C3 convertase (or by trypsin) induced aggregation of gel-filtered human platelets and stimulated serotonin release. At concentrations of 10(-10) M to 8 X 10(-12) M, C3a induced aggregation when added alone to platelets. However, at lower concentrations (2 X 10(-12) M) C3a did not aggregate platelets directly but exhibited highly significant synergism (two-way analysis of variance P less than 0.0001) with ADP in mediating platelet aggregation and release of serotonin. Removal of the C-terminus arginine from C3a abolished anaphylotoxin activity but did not affect the platelet-stimulating activity of the peptide. C3a and C3a des-arg were equally reactive in mediating platelet aggregation and release of serotonin. Further C3a and C3a des-arg exhibited synergism with ADP of equal significance in both aggregation and the release reaction. The concentrations of C3a required for the platelet-stimulating activity involve relatively small number of molecules per platelet (4,000-10,000 for the synergistic reaction with ADP). These data suggest the possibility of a C3a (C3a des-arg) receptor on human platelets. This premise is strengthened by the demonstration ultrastructurally of C3a on the platelet membrane subsequent to C3a stimulation.

Inhibition by Glaucocalyxin A of Aggregation of Rabbit Platelets Induced by ADP, Arachidonic Acid and Platelet-Activating Factor, and Inhibition of [3H]-PAF Binding

1992 ◽  
Vol 67 (04) ◽  
pp. 458-460 ◽  
Author(s):  
Zhang Bin ◽  
Long Kun
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Activating Factor ◽  
Northeastern China ◽  
Ethereal Extract ◽  
Ic50 Value ◽  
Rabbit Platelets ◽  
Ic50 Values ◽  
Induced Aggregation

SummaryGlaucocalyxin A is a new diterpenoid isolated from the ethereal extract of the leaves of Rabdosia japonica (Burm f) Hara var glaucocalyx (Maxim) Hara (Labiatae) collected in the northeastern China. When it was incubated with washed rabbit platelets, glaucocalyxin A inhibited ADP- or arachidonic acid-induced platelet aggregation with IC50 values of 4.4 μmol/1, 14.1 μmol/1 respectively. Glaucocalyxin A also inhibited PAF-induced aggregation of rabbit platelets which were refractory to ADP and arachidonic acid with an IC50 value of 13.7 μmol/1. Analysis of [3H]-PAF binding showed that glaucocalyxin A prevented [3H]-PAF binding to intact washed rabbit platelets with an IC50 value of 8.16 μmol/1, which was consistent with its inhibition of PAF-induced platelet aggregation.

Testosterone Potentiation of Ionophore and ADP Induced Platelet Aggregation : Relationship to Arachidonic Acid Metabolism

1981 ◽  
Vol 46 (02) ◽  
pp. 538-542 ◽  
Author(s):  
R Pilo ◽  
D Aharony ◽  
A Raz
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Unsaturated Fatty Acids ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryThe role of arachidonic acid oxygenated products in human platelet aggregation induced by the ionophore A23187 was investigated. The ionophore produced an increased release of both saturated and unsaturated fatty acids and a concomitant increased formation of TxA2 and other arachidonate products. TxA2 (and possibly other cyclo oxygenase products) appears to have a significant role in ionophore-induced aggregation only when low concentrations (<1 μM) of the ionophore are employed.Testosterone added to rat or human platelet-rich plasma (PRP) was shown previously to potentiate platelet aggregation induced by ADP, adrenaline, collagen and arachidonic acid (1, 2). We show that testosterone also potentiates ionophore induced aggregation in washed platelets and in PRP. This potentiation was dose and time dependent and resulted from increased lipolysis and concomitant generation of TxA2 and other prostaglandin products. The testosterone potentiating effect was abolished by preincubation of the platelets with indomethacin.

Decreased Platelet Vitamin C in Diabetes Mellitus; Possible Role in Peraggregatoon

1979 ◽  
Author(s):  
K.E. Sarji ◽  
J. Gonzalez ◽  
H. Hempling ◽  
J.A. Colwell
Keyword(s):  
Ascorbic Acid ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Vitamin C ◽  
Platelet Rich Plasma ◽  
Solution Ph ◽  
Dose Dependent Fashion ◽  
Insulin Dependent Diabetic ◽  
Induced Aggregation

To determine whether Vitamin C might relate to the increased platelet sensitivity in the diabetic, we have measured levels of platelet Vitamin C and studied the effects of Vitamin C on platelet aggregation. Ascorbic acid levels in washed platelets from diabetics were significantly lower than from normals (4s.2±3 μg/1010 platelets vs. 2s.s±2 μg/1010 platelets, p<.001). The effects of ascorbic acid on platelet aggregation in vitro were studied by adding ascorbic acid in buffered solution (pH 7.35) prior to-aggregating agents. Ascorbic acid in platelet-rich plasma consistently inhibited platelet aggregation with threshold concentrations of ADP, epinephrine, and collagen. With washed platelets, ascorbic acid inhibited arachidonic, acid-induced aggregation. When platelets were incubated at 37°C for 10 minutes with varying concentrations of ascorbic acid, rewashed, and aggregation with arachidonic acid tested, aggregation was inhibited in a linear dose-dependent fashion. Oral ingestion of ascorbic acid (2 gm/day) for seven days by normal non-smoking males produced a marked inhibition of aggregation. In a similar study, platelets from an insulin-dependent diabetic showed no change in aggregation. These results suggest that platelet levels of ascorbic acid may relate to the hyperaggregat ion of platelets from diabetics.

Impairment of Ristocetin-Induced Platelet Aggregation in Patients with Infective Endocarditis

1994 ◽  
Vol 22 (1) ◽  
pp. 63-66
Author(s):  
M Matsumoto ◽  
H Murakami ◽  
T Matsushima ◽  
J Tamura ◽  
H Sadakata ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Infective Endocarditis ◽  
Plasma Factors ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

An investigation was carried out on two patients with infective endocarditis associated with reduction of ristocetin-induced aggregation of platelets. The plasma of these patients reduced the aggregability with ristocetin of normal platelets. It is suggested that the patients had certain plasma factors which disturbed platelet aggregation with ristocetin.

Effects of Ethanol on Pathways of Platelet Aggregation In Vitro

1988 ◽  
Vol 59 (03) ◽  
pp. 383-387 ◽  
Author(s):  
Margaret L Rand ◽  
Marian A Packham ◽  
Raelene L Kinlough-Rathbone ◽  
J Fraser Mustard
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Secondary Phase ◽  
Primary Phase ◽  
Rabbit Platelet ◽  
Membrane Phospholipids ◽  
Rabbit Platelets ◽  
Induced Aggregation

SummaryEthanol, at physiologically tolerable concentrations, did not affect the primary phase of ADP-induced aggregation of human or rabbit platelets, which is not associated with the secretion of granule contents. Potentiation by epinephrine of the primary phase of ADP-induced aggregation of rabbit platelets was also not inhibited by ethanol. However, ethanol did inhibit the secondary phase of ADP-induced aggregation which occurs with human platelets in citrated platelet-rich plasma and is dependent on the formation of thromboxane A2. Inhibition by ethanol of thromboxane production by stimulated platelets is likely due to inhibition of the mobilization of arachidonic acid from membrane phospholipids, as ethanol had little or no effect on aggregation and secretion induced by arachidonic acid or the thromboxane mimetic U46619. Rabbit platelet aggregation and secretion in response to low concentrations of collagen, thrombin, or PAF were inhibited by ethanol. Inhibition of the effects of thrombin and PAF was also observed with aspirin-treated platelets. Thus, in addition to inhibiting the mobilization of arachidonate for thromboxane formation that occurs with most agonists, ethanol can also inhibit aggregation and secretion through other effects on platelet responses.

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