Platelet activation by a synthetic hydrophobic polymer, polymethylmethacrylate

Platelets adhere to artificial surfaces in the initial stage of thrombus formation, but the subsequent steps in signal transduction that lead to platelet activation by artificial surfaces are not understood. When 0.325-micron diameter beads composed of a hydrophobic polymer, polymethylmethacrylate (PMMA), were added to gel-filtered aequorin-loaded platelets suspended in media containing Ca2+, the platelets aggregated; addition of fibrinogen was not required. Platelet aggregation was preceded by an increase in cytoplasmic Ca2+ and was accompanied by phosphorylation of the 47-Kd substrate of protein kinase C (PKC), 5-hydroxytryptamine (5-HT) release, and accumulation of phosphatidic acid. All these effects were partially inhibited by apyrase and aspirin. Monoclonal antibodies (MoAbs) 7E3 and M148 and the synthetic peptides RGDS and fibrinogen gamma chain fragment 400–411, all of which bind to the platelet fibrinogen receptor glycoprotein IIb- IIIa (GPIIb-IIIa) and inhibit fibrinogen binding, prevented PMMA- induced aggregation but did not inhibit the Ca2+ increase. Chymotrypsin- treated platelets aggregated after addition of fibrinogen, but not PMMA. We conclude that platelets interact initially with PMMA at membrane sites other than those required for fibrinogen binding, leading to activation of membrane phospholipases and PKC, an increase in cytoplasmic Ca2+, release of 5-HT, ADP, and fibrinogen from storage granules, and to platelet aggregation.

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Platelet activation by a synthetic hydrophobic polymer, polymethylmethacrylate

Blood ◽

10.1182/blood.v78.7.1713.1713 ◽

1991 ◽

Vol 78 (7) ◽

pp. 1713-1721 ◽

Cited By ~ 21

Author(s):

JA Ware ◽

J Kang ◽

MT DeCenzo ◽

M Smith ◽

SC Watkins ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Thrombus Formation ◽

Gamma Chain ◽

Fibrinogen Receptor ◽

Hydrophobic Polymer ◽

Fibrinogen Binding ◽

Initial Stage ◽

Artificial Surfaces ◽

Induced Aggregation

Abstract Platelets adhere to artificial surfaces in the initial stage of thrombus formation, but the subsequent steps in signal transduction that lead to platelet activation by artificial surfaces are not understood. When 0.325-micron diameter beads composed of a hydrophobic polymer, polymethylmethacrylate (PMMA), were added to gel-filtered aequorin-loaded platelets suspended in media containing Ca2+, the platelets aggregated; addition of fibrinogen was not required. Platelet aggregation was preceded by an increase in cytoplasmic Ca2+ and was accompanied by phosphorylation of the 47-Kd substrate of protein kinase C (PKC), 5-hydroxytryptamine (5-HT) release, and accumulation of phosphatidic acid. All these effects were partially inhibited by apyrase and aspirin. Monoclonal antibodies (MoAbs) 7E3 and M148 and the synthetic peptides RGDS and fibrinogen gamma chain fragment 400–411, all of which bind to the platelet fibrinogen receptor glycoprotein IIb- IIIa (GPIIb-IIIa) and inhibit fibrinogen binding, prevented PMMA- induced aggregation but did not inhibit the Ca2+ increase. Chymotrypsin- treated platelets aggregated after addition of fibrinogen, but not PMMA. We conclude that platelets interact initially with PMMA at membrane sites other than those required for fibrinogen binding, leading to activation of membrane phospholipases and PKC, an increase in cytoplasmic Ca2+, release of 5-HT, ADP, and fibrinogen from storage granules, and to platelet aggregation.

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

10.1055/s-0038-1643514 ◽

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.

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SELECTIVE ANTAGONISTS OF PAF: INTERFERENCE WITH PLATELET FUNCTION AND EFFECT ON THROMBOGENESIS INDUCED BY SUBENDOTHELIUM.

10.1055/s-0038-1643488 ◽

1987 ◽

Author(s):

P Hadvary ◽

H R Baumgartner

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Blood Platelets ◽

Rabbit Aorta ◽

Constant Infusion ◽

Induced Aggregation

Platelet activating factor (PAF) is a very potent excitatory agonist of blood platelets but the physiological importance of this mediator in platelet thrombus formation is not known. We investigated the effect of two chemically unrelated selective inhibitors of PAF-induced platelet aggregation on thrombogenesis induced by rabbit aorta subendothelium (SE) using an ex vivo perfusion system.Ro 19-3704 is a highly potent inhibitor structurally related to PAF. This compound inhibits PAF-induced aggregation of rabbit platelets in platelet rich plasma in vitro competitively. Against 4 nM PAF, a concentration resulting in submaximal platelet aggre-gregation velocity, the IC50 was 70 nM. Inhibition was highly selective for PAF-induced aggregation, since aggregation induced by collagen (HORM, 5 yg/ml), ADP (1 yM) or thrombin (0.4 U/ml) was not inhibited even at a concentration as high as 10 yM. Bro-tizolam, a triazolobenzodiazepine reported to be a selective inhibitor of PAF-induced platelet activation, had in our system an IC50 of 200 nM. The selective benzodiazepine antagonist Ro 151788 was without effect on inhibition of PAF-induced platelet activation by brotizolam.Ro 19-3704 was given intravenously to rabbits as a bolus of 0.2 mg/kg followed by constant infusion of 0.02 mg/kg/min. This dosage provoked ex vivo a constant right shift ratio of the dose response curve for PAF-induced aggregation (RSR[PAF]) by a factor of 25 to 35. Brotizolam was given orally at a dose of 100 mg/ kg together with 300 mg/kg of Ro 15-1788 (to antagonize the central effects) 90 minutes before starting the perfusion experiment, resulting in a RSR[PAF] of 35 to 135. ADP induced platelet aggregation was not impaired by either compound. SE was exposed to the non-anticoagulated blood withdrawn from the carotid artery for 3 min at 2600 s-1 and for 20 min at 200 s-1 shear rate. Quantitative morphometric evaluation showed that SE coverage by platelets and by fibrin, thrombus area and thrombus height were all unchanged by the PAF antagonists at low and at high shear rates despite a very substantial inhibition of PAF-induced platelet aggregation. Therefore a major role of PAF in SE-induced thrombogenesis seems unlikely.

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On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation

10.1055/s-0038-1652598 ◽

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.

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FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

Cardiovascular Research ◽

10.1093/cvr/cvy311 ◽

2018 ◽

Vol 115 (11) ◽

pp. 1672-1679 ◽

Cited By ~ 1

Author(s):

Qi Ma ◽

Weilin Zhang ◽

Chongzhuo Zhu ◽

Junling Liu ◽

Quan Chen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Mitochondrial Protein ◽

Dependent Manner ◽

Clot Retraction ◽

Akt Phosphorylation ◽

Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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Impaired function of platelet membrane glycoprotein IIb-IIIa in end-stage renal disease.

Journal of the American Society of Nephrology ◽

10.1681/asn.v5136 ◽

1994 ◽

Vol 5 (1) ◽

pp. 36-46

Author(s):

M P Gawaz ◽

G Dobos ◽

M Späth ◽

P Schollmeyer ◽

H J Gurland ◽

...

Keyword(s):

Renal Failure ◽

Chronic Renal Failure ◽

Platelet Aggregation ◽

Platelet Function ◽

Uremic Toxin ◽

Bleeding Tendency ◽

Fibrinogen Receptor ◽

Fibrinogen Binding ◽

Uremic Patients ◽

Stage Renal Disease

Impaired platelet function and a bleeding tendency are well-recognized complications of chronic renal failure. Because the fibrinogen receptor GPIIb-IIIa plays a central role in platelet aggregation and adhesion to the subendothelium, it was reasoned that a defect in this receptor may underlie the impaired platelet function in uremia. To test this hypothesis, the function of this receptor in the platelets of 11 uremic patients was studied. Aggregation studies were performed with flow cytometric techniques with anti-GPIIb-IIIa conformation-specific monoclonal antibodies (mAb) (anti-LIBS1 and anti-PMI-1). Antifibrinogen and antithrombospondin mAb were used to characterize fibrinogen binding to GPIIb-IIIa and the release of alpha-granules, respectively. Platelets from patients with chronic renal failure showed significantly decreased binding of conformation-dependent anti-LIBS1 mAb after ADP, phorbol myristate acetate, or RGD-peptide stimulation compared with normal controls, suggesting a defect related to the ability of the fibrinogen receptor to undergo a conformational change. Moreover, antifibrinogen and antithrombospondin binding to activated platelets were reduced in uremic patients, implying impairment of both ligand-binding and alpha-granule release. Hemodialysis partially restored GPIIb-IIIa function, which may account for the observed effects of this therapy in restoring platelet aggregation. These findings indicate that platelets of patients with chronic renal failure reveal an aggregation defect at least partially due to an intrinsic GPIIb-IIIa dysfunction and the presence of a putative uremic toxin that inhibits fibrinogen binding to GPIIb-IIIa.

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Ex vivo evaluation of anti-GPVI antibody in Cynomolgus monkeys: Dissociation between anti-platelet aggregatory effect and bleeding time

Thrombosis and Haemostasis ◽

10.1160/th06-05-0266 ◽

2006 ◽

Vol 96 (08) ◽

pp. 167-175 ◽

Cited By ~ 27

Author(s):

Yutaka Matsumoto ◽

Hisao Takizawa ◽

Kazuhiro Nakama ◽

Xiaoqi Gong ◽

Yoshihisa Yamada ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

Inhibitory Effect ◽

Bleeding Tendency ◽

Fab Fragment ◽

Dose Escalation Study ◽

Cynomolgus Monkeys ◽

Induced Aggregation

SummaryRecent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (>80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0. 2 mg/kg with a slight prolongation of bleeding time (1. 3 times baseline value). Furthermore, at 18. 8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1. 9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5. 0 times at 0. 35 mg/kg, the lowest effective dose on platelet aggregation. Ina pharmacodynamic study,a bolus injection of OM2 at 0. 4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exerta potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.

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Black Sorghum Phenolic Extract Modulates Platelet Activation and Platelet Microparticle Release

Nutrients ◽

10.3390/nu12061760 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1760 ◽

Cited By ~ 3

Author(s):

Borkwei Ed Nignpense ◽

Kenneth A Chinkwo ◽

Christopher L Blanchard ◽

Abishek B Santhakumar

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conformational Changes ◽

Thrombus Formation ◽

Antiplatelet Agents ◽

Limited Information ◽

Platelet Activity ◽

Dietary Polyphenols ◽

High Antioxidant Activity ◽

Phenolic Extract

Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.

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Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

International Journal of Molecular Sciences ◽

10.3390/ijms21113932 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3932 ◽

Cited By ~ 2

Author(s):

Preeti Kumari Chaudhary ◽

Sanggu Kim ◽

Youngheun Jee ◽

Seung-Hun Lee ◽

Kyung-Mee Park ◽

...

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Platelet Activation ◽

Functional Role ◽

Thrombus Formation ◽

Receptor Activation ◽

G Protein Coupled ◽

Gpcr Desensitization ◽

Stimulation Of

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

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Platelet Activation and Biofilm Formation by Aerococcus urinae, an Endocarditis-Causing Pathogen

Infection and Immunity ◽

10.1128/iai.00469-10 ◽

2010 ◽

Vol 78 (10) ◽

pp. 4268-4275 ◽

Cited By ~ 29

Author(s):

Oonagh Shannon ◽

Matthias Mörgelin ◽

Magnus Rasmussen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Biofilm Formation ◽

Platelet Rich Plasma ◽

Protein Structures ◽

Prostaglandin E ◽

Bacterial Surface ◽

Fibrinogen Receptor ◽

Aerococcus Urinae

ABSTRACT The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons. Biofilm formation and platelet aggregation are believed to contribute to bacterial virulence in IE. Five A. urinae isolates from human blood were shown to form biofilms in vitro, and biofilm formation was enhanced by the presence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet-rich plasma from healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets, as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and on platelet activation since it was sensitive to both trypsin and prostaglandin E1. Plasma proteins at the bacterial surface were needed for platelet aggregation; and roles of the complement system, fibrinogen, and immunoglobulin G were demonstrated. Complement-depleted serum was unable to support platelet aggregation by Au3 and complement blockade using compstatin-inhibited platelet activation. Platelet activation by Au3 was inhibited by blocking of the platelet fibrinogen receptor, and this isolate was also shown to bind to radiolabeled fibrinogen. Removal of IgG from platelet-rich plasma by a specific protease inhibited the platelet aggregation induced by A. urinae, and blockade of the platelet FcRγIIa hindered platelet activation induced by Au3. Convalescent-phase serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregate platelets in an otherwise nonresponsive donor. Our results show that A. urinae exhibits virulence strategies of importance for IE.

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