scholarly journals Blockade of Lipid Receptor GPR31 Suppresses Platelet Reactivity and Thrombosis with Minimal Effect on Hemostasis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1064-1064
Author(s):  
Layla Van Doren ◽  
Nga Nguyen ◽  
Chris Garzia ◽  
Elizabeth Fletcher ◽  
Ryan Stevenson ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Calcium Release ◽  
Conflicts Of Interest ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
Human Platelets ◽  
P2y12 Inhibitor ◽  
Occlusion Time ◽  
Genetic Ablation ◽  
Human Platelet Aggregation

Objective: Platelet agonist-activated 12-lipoxygenase (12-LOX)/12-Hydroxyeicosatetraenoic acid (12-HETE)/G protein-coupled receptor 31 (GPR31) signaling has been proposed to regulate platelet reactivity. While inhibition or genetic ablation of 12-LOX supports an important role of 12-HETE in response to platelet agonists thrombin and collagen, the participation of GPR31 in platelet lipid signaling has not been examined. We developed a potent pepducin inhibitor, GPR-310, to test the downstream involvement of GPR31 in thrombin and collagen mediated platelet activation and thrombosis. Approach and Results: Treatment of mice with GPR-310 reversibly inhibited ex vivo platelet aggregation in response to thrombin and the PAR4 agonist, AYPGKF. There was significant protection (P<0.002) against FeCl3-induced carotid artery injury in mice by extending occlusion time from 100% occlusion at 27 min in the vehicle cohort to 20% occluded at 45 min in the GPR-310 cohort. GPR-310 treatment did not affect tail bleeding time. In human platelets, GPR-310 significantly (P<0.001) inhibited PAR4 agonist and collagen-mediated platelet aggregation and PAR4 calcium release. GPR-310 inhibited 12(S)-HETE- and PAR4-mediated RAP1 activation, with no effect on the PAR1-RAP1 signal. Accordingly, PAR1-mediated aggregation of human platelets was not affected by either GPR-310 or the 12-LOX inhibitor, ML355. GPR-310 caused a 5-fold shift in thrombin-mediated human platelet aggregation, comparable to a direct P2Y12 inhibitor, AZD1283. Dual GPR31 and P2Y12 inhibition showed synergy and protected against thrombin-mediated human platelet aggregation with a 19-fold shift. Blockade of GPR31 was more effective than the P2Y12 inhibitor in a thrombin-mediated clot retraction assay. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. Conclusions: GPR31 may serve as a new therapeutic target in platelet-dependent arterial thrombosis and aggregation in humans. Disclosures No relevant conflicts of interest to declare.

Differential Ability of Agonists to Express Distinct Pools of Fibrinogen (Gpllb/llla) Receptors which Can Mediate the Aggregation of Human Platelets

1989 ◽  
Vol 62 (03) ◽  
pp. 955-961 ◽  
Author(s):  
Ian S Watts ◽  
Rebecca J Keery ◽  
Philip Lumley
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Surface Membrane ◽  
Blood Platelet ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Platelet Surface ◽  
Human Platelet Aggregation ◽  
Differential Ability ◽  
Blood Platelet Aggregation

SummaryWe have investigated the effect of two procedures that modify human platelet surface membrane glycoprotein (Gp) IIb and IIIa complexes upon whole blood platelet aggregation to a range of agonists. (A) Irreversible disruption of complexes by temporary (30 min) Ca2+-deprivation with EGTA at 37° C. (B) Binding of a monoclonal antibody M148 to the complex. EGTA exposure abolished aggregation to ADP, adrenaline and PAF. In contrast, full aggregation curves to collagen and U-46619 could still be established. EGTA exposure reduced M148 binding to platelets by 80%. Excess M148 abolished aggregation to ADP, PAF, collagen and U-46619. However, upon removal of unbound antibody from platelets full aggregation curves to collagen and U-46619 but not to ADP and PAF could be re-established. Thus human platelet aggregation to ADP, PAF and adrenaline appears absolutely dependent upon surface membrane GpIIb/IIIa complexes. In contrast, collagen and U-46619 cause expression of an additional distinct pool of Gp complexes inaccessible to EGTA and M148 in unstimulated platelets which is intimately involved in aggregation to these agonists.

scholarly journals Human platelet aggregation by murine monoclonal antiplatelet antibodies is subtype-dependent

Blood ◽  
1993 ◽  
Vol 81 (7) ◽  
pp. 1792-1800 ◽  
Author(s):  
S De Reys ◽  
C Blom ◽  
B Lepoudre ◽  
PJ Declerck ◽  
M De Ley ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Platelet ◽  
Human Platelets ◽  
Antigen Recognition ◽  
Metabolic Inhibitors ◽  
Antigen Binding ◽  
Human Platelet Aggregation ◽  
Murine Monoclonal Antibodies

Abstract Twenty murine monoclonal antibodies (MoAbs) generated against different human platelet antigens induced clumping of human platelets in plasma and buffer. Whereas one MoAb could agglutinate platelets, clumping for 19 MoAbs was blocked by metabolic inhibitors, indicating that these induce platelet activation. Fifteen MoAbs were of IgG1, two of IgG2a, and two of IgG2b subtype. F(ab')2 fragments of these did not evoke an aggregatory response, but specifically inhibited aggregations by and binding of their respective intact MoAbs to platelets. Single-platelet counting technology indicated that the MoAbs bind through their antigen- binding and Fc domains mainly to the surface of the same platelet, rather than cause interplatelet-binding. Despite these similarities, the mechanism of action was nevertheless subtype-dependent. Aggregation induced by all IgG1 antibodies could consistently be prevented by blocking the Fc gamma II-receptor, whereas aggregations induced by all IgG2 antibodies still occurred with blocked Fc-receptor, provided functional complement was present. We therefore conclude that platelet activation by MoAb-binding is initiated by antigen recognition followed by an Fc domain-dependent step, which involves the Fc gamma II-receptor for IgG1-type MoAbs and complement-binding for IgG2-type MoAbs. Thus, antibodies of different subtypes can aggregate platelets via different pathways.

Adenosine 5’-O-(2-Thiodiphosphate) (ADP-β-S) Is A Partial Agonist At The ADP Receptor Of Human Platelets

1981 ◽  
Author(s):  
N J Cusack ◽  
S M O Hourani
Keyword(s):  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Human Platelet ◽  
Partial Agonist ◽  
Human Platelets ◽  
Simultaneous Addition ◽  
Prostaglandin Receptor ◽  
Human Platelet Aggregation ◽  
Adp Receptor ◽  
Stimulation Of

ADP induces human platelet aggregation and inhibits the stimulation of platelet adenylate cyclase by prostaglandin E1 (PGE1), but analogues of ADP in which the diphosphate group is modified retain only weak aggregating activity. However, ADP-β-S, an ADP analogue in which a terminal phosphate oxygen is replaced by sulphur, is known to be equipotent with ADP as an inhibitor of PGE1-stimulated adenylate cyclase in purified human platelet membranes. We therefore tested ADP-β-S on intact human platelets. ADP-β-S induced human platelet aggregation and inhibited PGE1-stimulated adenylate cyclase, but in botn cases was less potent than ADP and only achieved 75% and 50% respectively of the maximal effects of ADP. Aggregation induced by ADP-β-S was competitively inhibited by ATP (50 μM), a known ADP antagonist.Both these actions of ADP could be inhibited by the simultaneous addition of ADP-β-S (50 μM). Aggregation induced by a stable endoperoxide analogue (11 ,9 -epoxymethano PGH2), which acts at a prostaglandin receptor rather than at an ADP receptor, was not inhibited by the simultaneous addition of ADP-β-S (50 μM). The behaviour of ADP-β-S towards human platelets is therefore tnat of a partial agonist at the ADP receptor.

Thromboxane A2-Stimulated Human Platelet Aggregation Is Potentiated By Epinephrine Acting VIA Alpha Adrenergic Receptors

1981 ◽  
Author(s):  
G J Johnson ◽  
G H R Rao ◽  
J G White
Keyword(s):  
Platelet Aggregation ◽  
Adrenergic Receptors ◽  
Human Platelet ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Adrenergic Agents ◽  
Alpha Adrenergic Receptors ◽  
Human Platelet Aggregation ◽  
Induced Aggregation

Epinephrine (E) potentiates arachidonate (A)-induced aggregation of human platelets. A-insensitive dog platelets (AIP), that form thromboxane A2 (T) but do not aggregate when stirred with A alone, aggregate when exposed to E + A. Therefore, we studied the effect of E on T-stimu- lated human platelet aggregation. AIP stirred with A formed T which was confirmed by TLC. 1/100 to 1/200 volume of AIP was removed 30 sec. after A, and transferred to gel- filtered, aspirin-incubated human platelets. Recipient platelet aggregation was proportional to the volume of AIP transferred. The addition of the thromboxane synthetase inhibitor, Azo Analog I, abolished the aggregating activity of AIP. Transfer of an aliquot of AIP that was inadequate to aggregate human gel-filtered, aspirin-incubated platelets resulted in irreversible aggregation in the presence of ≥0.5nM E. E potentiated aggregation when added 3 min. before but not 3 min. after aliquot transfer. T-stimulated aggregation was abolished by the T-antagonist, 13 azapro- stenoic acid (APA), but E added after APA and before T restored aggregation. E potentiation of T-stimulated aggregation was abolished by prior exposure to equimolar yohimbine, dihydroergocryptine and phentolamine, agents that bind to alpha2 adrenergic receptors, but not by prazosin an alpha1 antagonist. Higher concentrations of E reversed the inhibitory effects of the alpha2 adrenergic agents. All of these agents in higher concentrations (1-100μM) also blocked aggregation induced by T alone. Therefore T-induced platelet aggregation is potentiated by E, in concentrations attained in vivo, by a mechanism linked to platelet alpha adrenergic receptors. Platelet alpha2 receptors have a close functional relationship to the postulated T receptor. E may initiate platelet aggregation in vivo when T is formed in quantities inadequate to alone induce aggregation.

scholarly journals Low concentrations of lipid hydroperoxides prime human platelet aggregation specifically via cyclo-oxygenase activation

1997 ◽  
Vol 325 (2) ◽  
pp. 495-500 ◽  
Author(s):  
Catherine CALZADA ◽  
Evelyne VERICEL ◽  
Michel LAGARDE
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Lipid Peroxides ◽  
Human Platelets ◽  
Lipid Hydroperoxides ◽  
Cellular Functions ◽  
Human Platelet Aggregation ◽  
Low Concentrations ◽  
Oxygenase Activity ◽  
Aggregation Of Platelets

There is mounting evidence that lipid peroxides contribute to pathophysiological processes and can modulate cellular functions. The aim of the present study was to investigate the effects of lipid hydroperoxides on platelet aggregation and arachidonic acid (AA) metabolism. Human platelets, isolated from plasma, were incubated with subthreshold (i.e. non-aggregating) concentrations of AA in the absence or presence of hydroperoxyeicosatetraenoic acids (HPETEs). Although HPETEs alone had no effect on platelet function, HPETEs induced the aggregation of platelets co-incubated with non-aggregating concentrations of AA, HPETEs being more potent than non-eicosanoid peroxides. The priming effect of HPETEs on platelet aggregation was associated with an increased formation of cyclo-oxygenase metabolites, in particular thromboxane A2, and was abolished by aspirin, suggesting an activation of cyclo-oxygenase by HPETEs. It was not receptor-mediated because the 12-HPETE-induced enhancement of AA metabolism was sustained in the presence of SQ29,548 or RGDS, which blocked the aggregation. These results indicate that physiologically relevant concentrations of HPETEs potentiate platelet aggregation, which appears to be mediated via a stimulation of cyclo-oxygenase activity.

Impairment of Human Platelet Aggregation and Serotonin Release Caused in Vitro by Echis Colorata Venom

1973 ◽  
Vol 30 (01) ◽  
pp. 191-198 ◽  
Author(s):  
Haim Biran ◽  
Alexander Dvilansky ◽  
Ilana Nathan ◽  
Avinoam Livne
Keyword(s):  
Platelet Aggregation ◽  
Functional Impairment ◽  
Human Platelet ◽  
Human Platelets ◽  
Endothelial Damage ◽  
Serotonin Release ◽  
Bleeding Tendency ◽  
Human Platelet Aggregation

SummaryAggregation of washed human platelets, induced by either ADP, thrombin or collagen, was decreased by Echis colorata venom (EVC). With ADP as an inducer, the inhibition of aggregation was proportional to the venom concentration, starting from 0.27 μg/ml and attaining full inhibition with venom concentration of 9 μg/ml. Higher concentrations were required for comparable venom effects when collagen or thrombin were used as inducers. Based on serotonin release measurements and platelet counting, it is concluded that the ECV-diminished aggregation is not due to platelet lysis. Thrombin-dependent serotonin release was inhibited by the venom to an extent proportional to the log ECV concentration at a range of 0.27 to 90 μg/ml. ECV effeces on serotonin release are apparently independent on its effects on aggregation, since similar results were obtained either with or without EDTA.Endothelial damage and defibrination are already known to be associated with the bleeding tendency caused by ECV. The present data disclose a functional impairment of platelets as an additional antihemostatic effect of this venom.

TRYPTOPHAN PYROLYSIS PRODUCTS FOUND IN COOKED FOODS INHIBIT HUMAN PLATELET AGGREGATION BY INHIBITING CYCLOOXYGENASE

1987 ◽  
Author(s):  
S Manabe ◽  
H Yanagisawa ◽  
S Ishikawa ◽  
Y Kitagawa ◽  
K Tohyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Cell Function ◽  
Human Platelet ◽  
Human Platelets ◽  
Heterocyclic Amines ◽  
Inhibitory Effects ◽  
Pyrolysis Products ◽  
Human Platelet Aggregation ◽  
Leukocyte Aggregation

Humans are exposed to numerous toxic compounds in foods. During the past decade, several carcinogenic heterocyclic amines have been reported to be present in the cooked foods. Recently, we reported that some of the carcinogenic heterocyclic amines isolated from foods were present in human plasma. In order to know the effects of the carcinogens isolated from foods on the cell function, we investigated the effects of the carcinogenic heterocyclic amines including Trp-P-1(3-amino-l,4-dimethyl-5H-pyrido❘4,3-b❘indole) and Trp-P-2(3-amino-1-methyl-5H-pyrido❘4,3-b❘indole) on human platelet aggregation and polymorphonuclear leukocyte aggregation. Only tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, had potent inhibitory effects on human platelet aggregation when platelets were preincubated with the carcinogens for 15 min. Other carcinogenic heterocyclic amines such as glutamic acid pyrolysates (Glu-P-1 and Glu-P-2) and 3H-imidazo ❘4,5-f❘quinoline-2-amines(IQ and MelQ) did show no effect on platelet aggregation even at 100 μM.The autoradiogram demonstrated that Tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, dose-dependently inhibited the formation of HHT,PGD2,PGE2 and TXB2 induced by sodium arachidonate in human platelets labeled with ❘ 14c❘ arachidonic acid. Moreover, Trp-P-1 and Trp-P-2 did not show significant effects on leukocyte aggregation induced by sodium arachidonate (0.75mM) even at lOOnM. It is concluded that Trp-P-1 and Trp-P-2 isolated from cooked foodstuffs have potent inhibitory effects on the cyclo-oxygenase pathway of the platelet. Therefore, human platelet function might be affected with daily foods containing tryptophan pyrolysis products in vivo.

Human Platelet Aggregation Without Thromboxane (Tx) Formation

1981 ◽  
Author(s):  
J Westwick ◽  
J B Smith ◽  
V V Kakkar
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Human Platelets ◽  
Synthesis Inhibitor ◽  
Thromboxane Receptor ◽  
Human Platelet Aggregation ◽  
Thromboxane Receptor Antagonist ◽  
Induced Aggregation ◽  
Carbocyclic Thromboxane A2

It is not known whether PG endoperoxides have to be converted to TxA2 in order to induce aggregation and secretion We have examined this crucial question by measuring human platelet aggregation, 14C-5HT release and TxB2 formation induced by collagen, arachidonic acid (AA), adrenaline, U46619 and PGH2 in presence of either 1) 1-2-(4-carboxy-phenoxy) ethyl imidazole hydrochloride, a potent and selective thromboxane synthesis inhibitor (TSI) or a carbocyclic thromboxane A2 (CTA) - a so called thromboxane receptor antagonist. TSI, 1.5 to 75μM produced a dose related inhibition (IC50 5μM n=5) of Tx and elevation of PGE2 synthesis in adrenaline (8μM) and collagen (1.5μg/ml) stimulated platelets. When 14C-SHT labelled platelets were stimulated with 4.5μM adrenaline in the presence of 0, 2.5, 15 and 300μM TSI a 35±2.8, 27±0.5, 24.5±5.0% release of 14C-5HT release resulted and a 10014, 9612, 9711 and 9114% of control aggregation occurred. Similarly when human platelets were stimulated with 0.8mMAA a 51±1.4% (mean ±SE) release of 14C-5HT occurred, while in the presence of 15, 75 and 300μM of UK, a 42±0.4, 32±1, 33±3% of 14C-5HT release resulted. Aggregation induced by the PG endoperoxide analogue U46619 (lμM) was not inhibited by 300μM TSI or l00μM indomethacin, although 99.5% inhibition of Tx formation resulted, and 50 to 60% inhibition of 14C-5HT release was produced both by TSI and indomethacin. However CTA (l-3±M) produced a dose related inhibition of both aggregation and release induced by U46619. CTA (l-10μM) was found to produce superimposable dose related inhibition of collagen induced aggregation and secretion of platelets, whether the platelets were pretreated with 300μM TSI, or not.These results suggest that Tx formation is not necessary for human platelet aggregation, although is contributory to 14C-5HT release induced by collagen, adrenaline, AA and U46619.Also caution must be employed when CTA is used to elucidate the role of TxA2, as it appears to be an effective PG endoperoxide antagonist.

Recombinant albumins containing additional peptide sequences smaller than barbourin retain the ability of barbourin-albumin to inhibit platelet aggregation

2005 ◽  
Vol 93 (05) ◽  
pp. 914-921 ◽  
Author(s):  
Brianna Wilson ◽  
Louise Eltringham-Smith ◽  
Sharon Gataiance ◽  
Varsha Bhakta ◽  
William Sheffield
Keyword(s):  
Platelet Aggregation ◽  
Fusion Proteins ◽  
Human Platelet ◽  
Human Platelets ◽  
Antibody Detection ◽  
Rabbit Serum ◽  
Rabbit Platelet ◽  
Human Platelet Aggregation ◽  
Pichia Pastoris Yeast ◽  
Proteins And Peptides

SummaryThe previously described fusion protein BLAH6 (Marques JA et al., Thromb Haemost 2001; 86: 902–8) is a recombinant protein that combines the small disintegrin barbourin with hexahisti-dine-tagged rabbit serum albumin (RSA) produced in Pichia pastoris yeast. We sought to determine: (1) if BLAH6 was immunogenic; and (2) if its barbourin domain could be productively replaced with smaller peptides. Purified BLAH6 was injected into rabbits, and anti-barbourin antibodies were universally detected in plasma 28 days later; BLAH6 was, however, equally effective in reducing platelet aggregation in both naïve and pre-treated rabbits. Thrombocytopenia was not observed, and complexing BLAH6 to αIIbβ3 had no effect on antibody detection. The bar-bourin moiety of BLAH6 was replaced with each of four sequences: Pep I (VCKGDWPC); Pep II (VCRGDWPC); Pep III (barbourin 41–54); and Pep IV (LPSPGDWR). The corresponding fusion proteins were tested for their ability to inhibit ADP-induced platelet aggregation. Pep III-LAH6 inhibited neither rabbit nor human platelets. Pep I-LAH6 and Pep IV-LAH6 inhibited rabbit platelet aggregation as effectively as BLAH6, but Pep IV-LAH6 did not inhibit human platelet aggregation. Pep I-LAH6 and Pep II LAH6 inhibited human platelet aggregation with IC50s 10– and 20-fold higher than BLAH6. Cross-immunoprecipitation assays with human platelet lysates confirmed that all proteins and peptides interacted with the platelet integrin αIIbβ3, but with greatly varying affinities. Our results suggest that the antiplatelet activity of BLAH6 can be retained in albumin fusion proteins in which smaller peptides replace the barbourin domain; these proteins may be less immunogenic than BLAH6.

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