Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

SummaryThe aim of this study was to determine if there is a correlation between the activity of a MoAb as an agonist and its ability to bind to the Fc platelet receptor, FcγRIIa. A polymorphism at amino acid 131 [arginine (Arg) or histidine (His)] of FcγRIIa was first shown to be determinant for MoAb-IgG1 binding on monocytes. To clarify the role of this polymorphism in platelet activation by MoAb-IgG1 we (i) established the FCγRIIA polymorphism at the gene level by adapting the denaturating gradient gel electrophoresis method, (ii) analyzed the binding affinity of the MoAbs to FrγRIIa on platelets from homozygous Arg, homozygous His, and heterozygous Arg/His donors, and (iii) characterized the different reactivities of platelets according to the FCγRIIA polymorphism. Among 167 Caucasian donors we found 46% heterozygous Arg/His, 36% homozygous His and 18% homozygous Arg. ALB6, an anti CD9, P256 an anti GPIIb-IIIa, and AP3 an anti-GPIIIa were chosen according to their ability (ALB6, P256) or not (AP3) to activate platelets. These 3 MoAbs-IgG1 bind to FcγRIIa with a stronger affinity for the Arg-form of FcγRIIa, a result which was confirmed with the use of diverse MoAbs directed against various antigens. The different abilities of MoAbs to bind to the two FcγRIIa forms were well correlated to the different platelet responses induced by ALB6 and P256. However, low concentrations of ALB6, which allow full activation of platelets from homozygous Arg donors, as did P256, did not induce any activation of platelets from homozygous His donors, whereas P256 is able to induce a low aggregation. The results further define the respective roles of the antigen and the Fc receptor, depending on the MoAb, and the role of the FcγRIIa polymorphism in platelet activation induced by MoAbs. In addition, the results obtained with MoAbs unable to induce platelet activation provide evidence that the binding of a MoAb on FcγRIIa does not predict its ability to activate platelets.

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Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽

10.1182/blood.v93.12.4222 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4222-4231 ◽

Cited By ~ 119

Author(s):

Anna Shcherbina ◽

Eileen Remold-O’Donnell

Keyword(s):

Platelet Activation ◽

Caspase 3 ◽

Human Platelet ◽

Thrombus Formation ◽

Shape Change ◽

Vascular Wall ◽

Human Platelets ◽

Granule Secretion ◽

Platelets Function

Abstract Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

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Role of arachidonic acid metabolism in human platelet activation and irreversible aggregation

American Journal of Hematology ◽

10.1002/ajh.2830190404 ◽

1985 ◽

Vol 19 (4) ◽

pp. 339-347 ◽

Cited By ~ 19

Author(s):

Gundu H. R. Rao ◽

James G. White

Keyword(s):

Arachidonic Acid ◽

Platelet Activation ◽

Acid Metabolism ◽

Human Platelet ◽

Arachidonic Acid Metabolism ◽

Irreversible Aggregation

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The role of extracellular Ca2+ and Na+ in paf-acether-induced human platelet activation

Biochemical Pharmacology ◽

10.1016/0006-2952(89)90109-3 ◽

1989 ◽

Vol 38 (20) ◽

pp. 3415-3421 ◽

Cited By ~ 1

Author(s):

Chantal Lalau Keraly ◽

Daniele Delautier ◽

Jacques Benveniste

Keyword(s):

Platelet Activation ◽

Human Platelet

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The central role of the P 2T receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity

British Journal of Haematology ◽

10.1046/j.1365-2141.2000.02208.x ◽

2000 ◽

Vol 110 (4) ◽

pp. 925-934 ◽

Cited By ~ 188

Author(s):

Robert F. Storey ◽

Heather M. Sanderson ◽

Ann E. White ◽

Jane A. May ◽

Kathryn E. Cameron ◽

...

Keyword(s):

Platelet Activation ◽

Human Platelet ◽

Procoagulant Activity

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Platelet 12-Lipoxygenase Is Required for Dense Granule Secretion and Platelet Aggregation: Role of 12-hLO In Platelet Hemostasis and Thrombosis

Blood ◽

10.1182/blood.v116.21.3203.3203 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3203-3203

Author(s):

Patrick Apopa ◽

Megha Patel ◽

Olivier Boutaud ◽

Michael Holinstat

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Reactivity ◽

Dense Granule ◽

Clot Formation ◽

Dense Granule Secretion ◽

Granule Secretion ◽

12 Lipoxygenase

Abstract Abstract 3203 Platelet activation plays a central role in regulating hemostasis. Uncontrolled activation of circulating platelets can result in the formation of occlusive thrombi and stroke. Following activation, metabolism of arachidonic acid by 12-lipoxygenase (12-hLO) may play a significant role in regulating the degree and stability of platelet reactivity. Using specific inhibitors for 12-hLO which do not interact with other lipoxygenases or enzymes in the COX-1 pathway, we were able for the first time to asses the involvement of 12-hLO in platelet reactivity. In order to assess the role of 12-hLO in platelet activation and thrombosis, dense granule secretion, platelet aggregation, alpha granule secretion, and platelet adhesion and clot formation under flow were measured. Inhibiting 12-hLO results in a complete inhibition of dense granule secretion with only a partial attenuation of alpha granule secretion indicating a novel regulatory scheme for modulating positive autocrine reinforcement of platelet reactivity and clot formation. Addition of the 12-hLO metabolite, 12-HETE (as low as 250 nM), resulted in a significant (25%) increase in PAR1-mediated dense granule secretion compare to agonist alone indicating that 12-HETE may be the crucial metabolite formed by 12-hLO metabolism of arachidonic acid. Importantly, platelet aggregation and adhesion are also significantly attenuated in the absence of 12-hLO. In fact, collagen-mediated platelet aggregation was shifted over 25 fold to the right in the absence of 12-hLO. These studies support the role of 12-hLO in hemostasis and may be a good target for anti-platelet therapy. Disclosures: No relevant conflicts of interest to declare.

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Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽

10.1182/blood.v93.12.4222.412k34_4222_4231 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4222-4231 ◽

Cited By ~ 2

Author(s):

Anna Shcherbina ◽

Eileen Remold-O’Donnell

Keyword(s):

Platelet Activation ◽

Caspase 3 ◽

Human Platelet ◽

Thrombus Formation ◽

Shape Change ◽

Vascular Wall ◽

Human Platelets ◽

Granule Secretion ◽

Platelets Function

Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

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Abstract 57: 12-Lipoxygenase is a Critical Enzyme Regulating FcyRIIa-Mediated Platelet Activation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.57 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Jennifer Yeung ◽

Benjamin Tourdot ◽

Theodore R Holman ◽

Steven E McKenzie ◽

Michael Holinstat

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Underlying Mechanism ◽

Human Platelets ◽

Regulatory Interaction ◽

Platelet Factor ◽

Essential Enzyme ◽

12 Lipoxygenase ◽

Pkc Activation

Introduction: FcγRIIa plays a key role in platelet activation mediated by immune complexes containing IgG and platelet factor 4 (PF4) bound to heparin. One of the primary mechanisms by which FcγRIIa mediates it effects in the platelet is through its ITAM motif. 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of platelet function through GPVI, which also signals in part through an ITAM motif. We therefore hypothesize that 12-LOX can play an important role in regulation of FcγRIIa-mediated platelet activation resulting in aggregation and thrombosis. Identifying the importance of this regulatory interaction is essential for understanding the role of 12-LOX in regulation of hemostasis and thrombosis, and identifying a possible target for prevention of FcγRIIa -induced platelet activation. Objectives: Determine the requirement of 12-LOX activation in FcγRIIa -mediated platelet activation and identify the underlying mechanism by which 12-LOX activation regulates FcγRIIa. Methods: FcγRIIa-mediated Platelet aggregation was measured in washed human platelets and mouse platelets in the presence of a 12-LOX inhibitor or in FcγRIIa +/+ mice crossed with 12-LOX -/- mice. PLCγ and PKC activation were measured in the absence of 12-LOX activity. Additionally, Rap1, calcium, and αIIbβ3 activity was measured in the presence or absence of 12-LOX activity following stimulation of FcγRIIa with CD9 or goat anti-mouse IV.3 antibody (IV.3). Results: Both human and FcγRIIa +/+ mouse platelets were able to fully aggregate following stimulation with CD9 or IV.3. Pharmacological inhibition of 12-LOX or absence of 12-LOX significantly delayed and attenuated platelet aggregation. Additionally, PLCγ, calcium, Rap1 and activation of αIIbβ3 were inhibited. Conclusions: These observations support our hypothesis that 12-LOX is an essential enzyme in the regulation of FcγRIIa-mediated platelet activation. Further investigation of this regulatory pathway will help to confirm 12-LOX as a viable target for inhibition of FcγRIIa and other ITAM-mediated platelet activation.

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