Braking platelet activation: deactivating the receptors

Glycoprotein (GP) VI and Fc receptor FcγRIIa are 2 ITAM (immunoreceptor tyrosine-activation motif)–bearing platelet surface receptors. In this issue of Blood, Gardiner and colleagues show that activation of either receptor results in simultaneous proteolytic cleavage of GPVI ectodomain and FcγRIIa cytoplasmic tail, providing distinct mechanisms for their down-regulation on platelet activation.

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Src family kinases: at the forefront of platelet activation

Blood ◽

10.1182/blood-2014-01-453134 ◽

2014 ◽

Vol 124 (13) ◽

pp. 2013-2024 ◽

Cited By ~ 140

Author(s):

Yotis A. Senis ◽

Alexandra Mazharian ◽

Jun Mori

Keyword(s):

Platelet Activation ◽

Src Family Kinases ◽

Cytoskeletal Proteins ◽

Receptor Complex ◽

Platelet Surface ◽

Surface Receptors ◽

Von Willebrand ◽

Activation Motif ◽

Willebrand Factor ◽

Primary Activation

Abstract Src family kinases (SFKs) play a central role in mediating the rapid response of platelets to vascular injury. They transmit activation signals from a diverse repertoire of platelet surface receptors, including the integrin αIIbβ3, the immunoreceptor tyrosine–based activation motif–containing collagen receptor complex GPVI-FcR γ-chain, and the von Willebrand factor receptor complex GPIb-IX-V, which are essential for thrombus growth and stability. Ligand-mediated clustering of these receptors triggers an increase in SFK activity and downstream tyrosine phosphorylation of enzymes, adaptors, and cytoskeletal proteins that collectively propagate the signal and coordinate platelet activation. A growing body of evidence has established that SFKs also contribute to Gq- and Gi-coupled receptor signaling that synergizes with primary activation signals to maximally activate platelets and render them prothrombotic. Interestingly, SFKs concomitantly activate inhibitory pathways that limit platelet activation and thrombus size. In this review, we discuss past discoveries that laid the foundation for this fundamental area of platelet signal transduction, recent progress in our understanding of the distinct and overlapping functions of SFKs in platelets, and new avenues of research into mechanisms of SFK regulation. We also highlight the thrombotic and hemostatic consequences of targeting platelet SFKs.

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The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis

Blood ◽

10.1182/blood-2008-08-174318 ◽

2009 ◽

Vol 113 (20) ◽

pp. 4942-4954 ◽

Cited By ~ 84

Author(s):

Yotis A. Senis ◽

Michael G. Tomlinson ◽

Stuart Ellison ◽

Alexandra Mazharian ◽

Jenson Lim ◽

...

Keyword(s):

G Protein ◽

Platelet Activation ◽

Drug Target ◽

Tyrosine Phosphatase ◽

Underlying Mechanism ◽

G Protein Coupled Receptors ◽

Bleeding Tendency ◽

Platelet Surface ◽

Surface Receptors ◽

G Protein Coupled

Abstract Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase–linked and G protein–coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein–coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.

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Diacylglycerol kinase ζ is a negative regulator of GPVI-mediated platelet activation

Blood Advances ◽

10.1182/bloodadvances.2018026328 ◽

2019 ◽

Vol 3 (7) ◽

pp. 1154-1166 ◽

Cited By ~ 3

Author(s):

Alyssa J. Moroi ◽

Nicole M. Zwifelhofer ◽

Matthew J. Riese ◽

Debra K. Newman ◽

Peter J. Newman

Keyword(s):

Platelet Activation ◽

Bleeding Time ◽

Thrombus Formation ◽

Negative Regulator ◽

Platelet Surface ◽

Rotational Thromboelastometry ◽

Surface Receptors ◽

Glycoprotein Vi ◽

Granule Secretion

Abstract Diacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ζ isoform of DGK (DGKζ) has been reported to inhibit T-cell responsiveness by downregulating intracellular levels of DAG. However, its role in platelet function remains undefined. In this study, we show that DGKζ was expressed at significant levels in both platelets and megakaryocytes and that DGKζ-knockout (DGKζ-KO) mouse platelets were hyperreactive to glycoprotein VI (GPVI) agonists, as assessed by aggregation, spreading, granule secretion, and activation of relevant signal transduction molecules. In contrast, they were less responsive to thrombin. Platelets from DGKζ-KO mice accumulated faster on collagen-coated microfluidic surfaces under conditions of arterial shear and stopped blood flow faster after ferric chloride–induced carotid artery injury. Other measures of hemostasis, as measured by tail bleeding time and rotational thromboelastometry analysis, were normal. Interestingly, DGKζ deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGKζ as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo.

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TULA-2 Deficiency Enhances Platelet Functional Responses to CLEC-2 Agonists

TH Open ◽

10.1055/s-0038-1676358 ◽

2018 ◽

Vol 02 (04) ◽

pp. e411-e419 ◽

Cited By ~ 2

Author(s):

John Kostyak ◽

Benjamin Mauri ◽

Carol Dangelmaier ◽

Akruti Patel ◽

Yuhang Zhou ◽

...

Keyword(s):

Platelet Activation ◽

Tyrosine Phosphatase ◽

Negative Regulator ◽

Functional Responses ◽

Protease Activated Receptors ◽

Surface Receptors ◽

Downstream Signaling ◽

Glycoprotein Vi ◽

Thromboxane Production ◽

Activation Motif

AbstractPlatelet activation is essential for hemostasis. Central to platelet activation are the signals transmitted through surface receptors such as glycoprotein VI, the protease-activated receptors, and C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a HemITAM (hem-immunoreceptor tyrosine activation motif)-bearing receptor that binds podoplanin and signals through spleen tyrosine kinase (Syk). T-cell ubiquitin ligand-2 (TULA-2) is a protein tyrosine phosphatase that is highly expressed in platelets and targets phosphorylated Y352 of Syk. We wanted to determine whether TULA-2 regulates Syk phosphorylation and activity downstream of CLEC-2. To that end, we used TULA-2 knockout mice and wild-type (WT) littermate controls. We found that TULA-2 deficiency enhances the aggregation and secretion response following stimulation with an excitatory CLEC-2 antibody or the CLEC-2 agonist rhodocytin. Consistently, Syk phosphorylation of Y346 is enhanced, as well as phosphorylation of the downstream signaling molecule PLCγ2, in TULA-2 knockout platelets treated with either CLEC-2 antibody or rhodocytin, compared with WT control platelets. Furthermore, the kinetics of Syk phosphorylation, as well as that of PLCγ2 and SLP-76, is enhanced in TULA-2 knockout platelets treated with 2.5-μg/mL CLEC-2 antibody compared with WT platelets. Similarly, thromboxane production was enhanced, in both amount and kinetics, in TULA-2−/− platelets treated with 2.5-μg/mL CLEC-2 antibody. TULA-2 acts as a negative regulator of CLEC-2 signaling by dephosphorylating Syk on Y346 and restraining subsequent Syk-mediated signaling.

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Pathophysiology of thrombosis

10.1093/med/9780198755777.003.0018 ◽

2017 ◽

Author(s):

Lina Badimon ◽

Felix C. Tanner ◽

Giovanni G. Camici ◽

Gemma Vilahur

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Dependent Manner ◽

Platelet Surface ◽

Surface Receptors ◽

Circulating Cells ◽

Cerebrovascular Events ◽

Undifferentiated Cells ◽

Protein Receptors ◽

Von Willebrand

Ischaemic heart disease and stroke are major causes of death and morbidity worldwide. Coronary and cerebrovascular events are mainly a consequence of a sudden thrombotic occlusion of the vessel lumen. Arterial thrombosis usually develops on top of a disrupted atherosclerotic plaque because of the exposure of thrombogenic material, such as collagen fibrils and tissue factor (TF), to the flowing blood. TF, either expressed by subendothelial cells, macrophage- and/or vascular smooth muscle-derived foam-cells in atherosclerotic plaques, is a key element in the initiation of thrombosis due to its ability to induce thrombin formation (a potent platelet agonist) and subsequent fibrin deposition at sites of vascular injury. Adhered platelets at the site of injury also play a crucial role in the pathophysiology of atherothrombosis. Platelet surface receptors (mainly glycoproteins) interact with vascular structures and/or Von Willebrand factor triggering platelet activation signalling events, including an increase in intracellular free Ca2+, exposure of a pro-coagulant surface, and secretion of platelet granule content. On top of this, interaction between soluble agonists and platelet G-coupled protein receptors further amplifies the platelet activation response favouring integrin alpha(IIb)beta(3) activation, an essential step for platelet aggregation. Blood-borne TF and microparticles have also been shown to contribute to thrombus formation and propagation. As thrombus evolves different circulating cells (red-blood cells and leukocytes, along with occasional undifferentiated cells) get recruited in a timely dependent manner to the growing thrombus and further entrapped by the formation of a fibrin mesh.

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Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1

Blood ◽

10.1182/blood.v95.4.1301.004k24_1301_1308 ◽

2000 ◽

Vol 95 (4) ◽

pp. 1301-1308 ◽

Cited By ~ 14

Author(s):

James P. McRedmond ◽

Patrick Harriott ◽

Brian Walker ◽

Desmond J. Fitzgerald

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Cell Activation ◽

Fc Receptor ◽

Thrombin Inhibitor ◽

Platelet Response ◽

Intact Cells ◽

Platelet Surface ◽

Tethered Ligand ◽

Protease Activated Receptor 1

Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A2production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S741A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1.

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Further Characterization of Wheat Germ Agglutinin Interaction with Human Platelets: Exposure of Fibrinogen Receptors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661676 ◽

1986 ◽

Vol 56 (03) ◽

pp. 323-327

Author(s):

Marilyne Lebret ◽

Francine Rendu

Keyword(s):

Platelet Activation ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Lectin Binding ◽

Human Platelets ◽

Cyclooxygenase Inhibitors ◽

Platelet Surface ◽

Surface Receptors ◽

Fibrinogen Binding ◽

Exogenous Calcium

SummaryIt was previously shown that (i) Wheat germ agglutinin, (WGA)-induced platelet activation occurred when only 17% of the lectin binding sites were occupied on the platelet surface and (ii) WGA caused the release of a platelet constituent which in turn participates in the observed effect. We now further define the platelet activation induced by WGA: the lectin induces a binding of fibrinogen to specific surface receptors. 125I-fibrinogen binding increases with the WGA concentration from 5 to 15 ug/ ml. Binding occurs without addition of exogenous calcium; its analysis demonstrated 54000 sites with a Ka = 0.8 × 106 M-1, Addition of 1 mM Ca2+ enhances the 125I-fibrinogen binding and reveals a second class of sites with higher affinity (9200 sites, Ka = 0.17 x 108 M-1). This 125I-fibrinogen binding is totally abolished by EDTA, ATP and arginine, and inhibited by 75% by CP/CPK; cyclooxygenase inhibitors and PGE1 also reduce the fibrinogen binding. Thus the WGA-induced fibrinogen binding is (1) release-dependent and (2) responsible for the aggregation process but not for the agglutinating effect of the lectin.

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Immunological Evidence for Prothrombin in Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661534 ◽

1986 ◽

Vol 55 (02) ◽

pp. 268-270

Author(s):

R J Alexander

Keyword(s):

Electrophoretic Mobility ◽

Platelet Activation ◽

Double Diffusion ◽

Human Platelets ◽

Secreted Proteins ◽

Platelet Surface ◽

Activated Platelets ◽

Similar Protein ◽

Diffusion Assay ◽

Supernatant Solution

SummaryAn attempt was made to isolate from plasma the platelet surface substrate for thrombin, glycoprotein V (GPV), because a GPV antigen was reported to be present in plasma (3). Plasma fractionation based on procedures for purification of GPV from platelets revealed a thrombin-sensitive protein with appropriate electrophoretic mobility. The protein was purified; an antiserum against it i) reacted with detergent-solubilized platelet proteins or secreted proteins in a double diffusion assay, ii) adsorbed a protein from the supernatant solution of activated platelets, and iii) inhibited thrombin-induced platelet activation, but the antiserum did not adsorb labeled GPV. The purified protein was immunochemically related to prothrombin rather than to GPV. Other antibodies against prothrombin were also able to adsorb a protein from platelets. It is concluded that 1) plasma does not contain appreciable amounts of GPV, and 2) platelets contain prothrombin or an immunochemically similar protein.

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THROMBOXANE-A2 MEDIATES THE ACTION OF INOSITOL (1.4.5) TRISPHOSPHATE (IP3) IN SAPONIN-PERMEABILISED PLATELETS

10.1055/s-0038-1644519 ◽

1987 ◽

Author(s):

K S Authi ◽

B J Evenden ◽

E J Hornby ◽

N Crawford

Keyword(s):

Phospholipase A2 ◽

Shape Change ◽

Thromboxane A2 ◽

Thromboxane B2 ◽

Cyclooxygenase Inhibitors ◽

Storage Site ◽

Platelet Surface ◽

Surface Receptors ◽

Thromboxane Receptor ◽

Thromboxane Production

Inositol trisphosphate (IP3) has now been identified as an important intracellular second messenger that can initiate the release of Ca2+ from intracellular stores in a variety of cells, including platelets. We have studied the effects of IP3 on washed platelets permeabilised with saponin (12-14 μg/mi) which allows penetration into the cell of low M.Wt polar molecules. The permeabilised cells show normal responses to the agonists thrombin and collagen. The addition of IP (1-20 μM) after saponin treatment induces shape change, aggregation and secretion of preloaded [14C] 5HT. Concomitant with these responses, thromboxane is produced in a dose related manner. With 20 μM IP3 thromboxane B2 increases from basal levels of 5-4 ± 3-0 ng/ml to 140 ± 23 ng/ml. Both thromboxane production and the platelet responses induced by IP3 are inhibited by pretreatment with the cyclooxygenase inhibitors, indomethacin (EC50 50 μM) and aspirin (EC50 30 μM). Aggregation and secretion responses to IP3 are also inhibited by thromboxane B2 receptor agonists; EPO 92 (R. Jones, Edinburgh) and AH 23848 (Glaxo Ltd.). If Ca2+ EGTA buffers age used with permeabilised platelets to "lock" the cytosolic [Ca2+] at 0.1 μM, thromboxane production is reduced to the basal level. Intact platelets were labelled with Ca2+ (4h incubation) and after washing, resuspension and saponisation, IP3 induced the release of 20% of the cell associated Ca2+. The release was unaffected by pretreatment with antimycin and oligomycin indicating an gndoplasmic reticulum-lige storage site for the sequestered Ca2+. This IP3 -induced Ca2+ release was also not affected by pretreatment with either cyclooxygenase inhibitors or thromboxane receptor antagonists (EPO 92 and AH 23848). We believe these studies indicate that the action of IP3 in sagonised platelets involves release of intracellularly stored Ca2+, activation of phospholipase A2 and cyclooxygenase, and production of thromboxane A2. The release of thromboxane mediates and/or attenuates platelet responses by acting upon platelet surface receptors.

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The (Patho)Biology of SRC Kinase in Platelets and Megakaryocytes

Medicina ◽

10.3390/medicina56120633 ◽

2020 ◽

Vol 56 (12) ◽

pp. 633

Author(s):

Lore De Kock ◽

Kathleen Freson

Keyword(s):

Platelet Activation ◽

Knockout Mice ◽

Missense Variant ◽

Surface Receptors ◽

Src Signaling ◽

Normal Platelet ◽

Fibrinogen Binding ◽

Cellular Environment ◽

Proplatelet Formation

Proto-oncogene tyrosine-protein kinase SRC (SRC), as other members of the SRC family kinases (SFK), plays an important role in regulating signal transduction by different cell surface receptors after changes in the cellular environment. Here, we reviewed the role of SRC in platelets and megakaryocytes (MK). In platelets, inactive closed SRC is coupled to the β subunit of integrin αIIbβ3 while upon fibrinogen binding during platelet activation, αIIbβ3-mediated outside-in signaling is initiated by activation of SRC. Active open SRC now further stimulates many downstream effectors via tyrosine phosphorylation of enzymes, adaptors, and especially cytoskeletal components. Functional platelet studies using SRC knockout mice or broad spectrum SFK inhibitors pointed out that SRC mediates their spreading on fibrinogen. On the other hand, an activating pathological SRC missense variant E527K in humans that causes bleeding inhibits collagen-induced platelet activation while stimulating platelet spreading. The role of SRC in megakaryopoiesis is much less studied. SRC knockout mice have a normal platelet count though studies with SFK inhibitors point out that SRC could interfere with MK polyploidization and proplatelet formation but these inhibitors are not specific. Patients with the SRC E527K variant have thrombocytopenia due to hyperactive SRC that inhibits proplatelet formation after increased spreading of MK on fibrinogen and enhanced formation of podosomes. Studies in humans have contributed significantly to our understanding of SRC signaling in platelets and MK.

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