The platelet as a model system for the acute actions of nuclear receptors

Platelets are circulating cell fragments which play a critical role in thrombosis, and whose activity is associated with the progress of cardiovascular diseases, diabetes, inflammation, and cancer cell metastasis. Recently, a number of nuclear receptors have been found present in human platelets, including the receptors for sex steroids, and glucocorticoids, along with peroxisome proliferator-activated receptors (PPAR)s and retinoid X receptors (RXR)s. Although the platelet contains no nucleus, selective ligands for these receptors activate their respective platelet nuclear receptors and regulate platelet aggregation and activation. The human platelet, because of its abundance and accessibility therefore represents an excellent model system to study the rapid non-genomic mechanism of nuclear receptors. Moreover, since targeting platelets is a major clinical therapeutic area, analysis of platelet nuclear receptors may provide clues for new drug targets as well as provide important information regarding the physiological roles of nuclear receptors in the circulation.     

Platelet aggregation and vasopressin receptors in patients with diabetes mellitus

Plasma vasopressin, vasopressin-induced platelet aggregation, and platelet vasopressin receptors were investigated in 10 normal subjects and 14 diabetic patients free of microangiopathy. Basal plasma vasopressin concentration was identical in two groups. Platelet aggregation induced by vasopressin as well as by epinephrine was not significantly altered in the diabetic patients. However, exploration of platelet V1-vasopressin receptors revealed in the diabetic group a dramatic reduction in the number of binding sites without alteration of the receptor affinity for tritiated vasopressin. Thus vasopressin-induced platelet aggregation in uncomplicated diabetes mellitus remains normal despite a decrease in the number of vasopressin receptors presumably due to alterations of the platelet membrane structure.     

PGE1 and PGE2 modify platelet function through different prostanoid receptors

There is evidence that the overall effects of prostaglandin E(2) (PGE(2)) on human platelet function are the consequence of a balance between promotory effects of PGE(2) acting at the EP3 receptor and inhibitory effects acting at the EP4 receptor, with no role for the IP receptor. Another prostaglandin that has been reported to affect platelet function is prostaglandin E(1) (PGE(1)), however the receptors that mediate its actions on platelet function have not been fully defined. Here we have used measurements of platelet aggregation and P-selectin expression induced by the thromboxane A(2) mimetic U46619 to compare the effects of PGE(1) and PGE(2) on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, were also determined. We also investigated the ability of the selective prostanoid receptor antagonists CAY10441 (IP antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist) to modify the effects of the prostaglandins on platelet function. The results obtained confirm that PGE(2) interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE(1) interacts with EP3 and IP receptors, but not EP4 receptors. In both cases the overall effects on platelet function reflect the balance between promotory and inhibitory effects at receptors that have opposite effects on adenylate cyclase.     

Scavenger receptors: Implications in atherothrombotic disorders

Scavenger receptors are modified lipoprotein binding receptors, expressed on the surface of a variety of cells including endothelial, macrophages and platelets. The most extensively studied class B scavenger receptors comprise of CD36 and SR-BI and have been found to bind to native and modified LDL. Interaction of modified LDL to CD36 accelerates foam cell formation, the key step in atherosclerotic plaque deposition. Recently scavenger receptors have also been implicated in thrombosis. Platelet CD36 serves as a sensor of oxidative stress and modulator of platelet reactivity under hyperlipidemic conditions thus, inducing prothrombotic signals. In contrast, targeting platelet SR-BI corresponds to reduce platelet hyperreactivity in hyperlipidemia suggesting that targeting these receptors could be a promising strategy for the treatment of atherothrombotic disorders.     

Adenosine and blood platelets

Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. Adenosine released from different cells or generated through the activity of cell-surface ectoenzymes exerts its effects through the binding of four different G-protein-coupled adenosine receptors. In platelets, binding of A₂ subtypes (A_2A or A_2B) leads to consequent elevation of intracellular cyclic adenosine monophosphate, an inhibitor of platelet activation. The significance of this ligand and its receptors for platelet activation is addressed in this review, including how adenosine metabolism and its A₂ subtype receptors impact the expression and activity of adenosine diphosphate receptors. The expression of A₂ adenosine receptors is induced by conditions such as oxidative stress, a hallmark of aging. The effect of adenosine receptors on platelet activation during aging is also discussed, as well as potential therapeutic applications.     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

A new platelet receptor specific to type III collagen. Type III collagen-binding protein

Platelet interaction with type III collagen is mediated by several platelet receptors that recognize specific sequences in collagen. We previously described an octapeptide KP*GEP*GPK within the alpha(1)III-CB4 fragment that binds to platelets and specifically inhibits platelet aggregation induced by type III collagen. In this study, we demonstrated that the octapeptide prevented platelet contact and spreading on type III collagen and subendothelium under static and flow conditions. Platelets adhered to the immobilized octapeptide, and anti-bodies directed against other platelet collagen receptors (glycoprotein (GP) Ia/IIa, GP IV, p65, p47) did not impair this adhesion. The platelet octapeptide receptor was identified by ligand blotting as a protein doublet with molecular masses of 68 and 72 kDa and does not correspond to any other already known platelet collagen receptors (GP Ia, GP IV GP VI, and p65). Our results indicate that a specific type III collagen receptor, expressed on the platelet surface, is involved in the first stages of platelet type III collagen interaction.     

Human blood platelets possess specific binding sites for C1q

Although platelet interactions with C1q are implied by the inhibitory effect of C1q on collagen-induced platelet aggregation, specific receptors have not as yet been identified. To address the question of platelet receptors for free C1q, direct radioligand binding studies were performed by using human blood platelets and purified, 125I-labeled C1q, and a monoclonal antibody (II1/D1) (IgM, lambda) directed against C1q receptors on peripheral blood leukocytes. Washed platelets bound both purified 125I-labeled C1q and II1/D1 in a specific and saturable manner under physiologic ionic strength conditions. At equilibrium, approximately 4000 molecules of C1q bound per platelet with an apparent dissociation constant of 3.5 X 10(-7) M. Maximum C1q binding was achieved in 5 min and correlated well with inhibition of collagen-induced platelet aggregation. Equilibrium binding of 125I-labeled II1/D1 to washed platelets required an incubation period of 15 to 30 min and II1/D1 concentrations approaching 50 micrograms/ml. Approximately 2000 molecules of II1/D1 bound per platelet, with an apparent dissociation constant of 2.8 X 10(-8) M. II1/D1 binding could be inhibited by the collagenous tail of C1q (c-C1q), suggesting that platelet receptors for these ligands are either the same or in close proximity. The data demonstrate that human blood platelets possess specific and saturable binding sites for free C1q that may function as collagen receptors, and may antigenically resemble C1q receptors on peripheral blood leukocytes.     

Inhibition of collagen-induced platelet reactivity by DGEA peptide

Direct interactions between collagen, the most thrombogenic component of the extracellular matrix, and platelet surface membrane receptors mediate platelet adhesion and induce platelet activation and aggregation. In this process two glycoproteins are crucial: integrin alpha2beta1, an adhesive receptor, and GPVI, which is especially responsible for signal transduction. Specific antagonists of the collagen receptors are useful tools for investigating the complexity of platelet-collagen interactions. In this work we assessed the usefulness of DGEA peptide (Asp-Gly-Glu-Ala), the shortest collagen type I-derived motif recognised by the collagen-binding integrin alpha2beta1, as a potential antagonist of collagen receptors. We examined platelet function using several methods including platelet adhesion under static conditions, platelet function analyser PFA-100TM, whole blood electric impedance aggregometry (WBEA) and flow cytometry. We found that DGEA significantly inhibited adhesion, aggregation and release reaction of collagen activated blood platelets. The inhibitory effect of DGEA on static platelet adhesion reached sub-maximal values at millimolar inhibitor concentrations, whereas the specific blocker of alpha2beta1 - monoclonal antibodies Gi9, when used at saturating concentrations, had only a moderate inhibitory effect on platelet adhesion. Considering that 25-30% of total collagen binding to alpha2beta1 is specific, we conclude that DGEA is a strong antagonist interfering with a variety of collagen-platelet interactions, and it can be recognised not only by the primary platelet adhesion receptor alpha2beta1 but also by other collagen receptors.     

Adaptor proteins of blood platelets

Adaptor proteins play a pivotal role in the regulation of signal transduction events elicited after the engagement of cell surface receptors. Platelets exhibit a number of integral membrane receptors capable of initiating a cellular response. These include collagen receptors, von Willebrand factor receptors, the fibrinogen receptor, and a number of G-protein coupled receptors, such as those for thrombin and ADP. The primary function of platelet receptors is the translation of externally applied signals into appropriate responses leading to platelet activation being a prerequisite for normal hemostasis. Multitude of signalling pathways described in platelets is based on the interaction of compounds of many different categories, such as transmembrane receptors, protein kinases, protein phoshatases, G-proteins, transmembrane and cytosolic adaptor proteins, phosphoinositides, cyclic AMP or GMP. Adaptor proteins lack intrinsic effector function, but contain distinct molecular domains, which mediate protein-protein and protein-lipid interactions. These molecules thus serve as a scaffolding, around which effectors and their substrates are assembled into three-dimensional signaling complexes. Adaptor proteins integrate receptor-mediated signals at intracellular levels and couple signaling receptors to cytosolic signaling pathways. While the function of adaptor proteins is well established in immune cells, the knowledge about their role in platelet activation is still at the onset Over the last decade numerous adaptor proteins have been identified in platelets and shown to be involved in accurate assembly of intracellular signaling complexes. Collagen-induced platelet intracellular signaling through GPVI resembles the functional response of B- and T-cell antigen receptors and is the best described in the literature. This review focuses on the structure and functional role of the most extensively studied adaptor proteins during platelet activation induced by physiological agonists.     

Effects of D- and L- enantiomers of adenosine, AMP and ADP and their 2-chloro- and 2-azido- analogues on human platelets.

Aggregation of human platelets by ADP and the inhibition of this effect by adenosine are apparently mediated by different receptors. One of the criteria for receptors is that they show stereospecificity for their ligands. We have synthesized L-enantiomers of D-adenosine, AMP and ADP, together with their corresponding photolysable 2-azido analogues so that platelet receptors could be tested for stereospecificity. All of the L-enantiomers were completely inactive as aggregators or inhibitors of platelet function. None of the L-enantiomers changed levels of platelet cAMP. 2-Azido-L-adenosine, AMP and ADP are proposed as useful controls in photoaffinity experiments for non-specific labelling.     

Primary platelet adhesion receptors

Thrombotic diseases such as heart attack and stroke remain a major health concern in the Western world despite existing anti-thrombotic drugs. Current studies are revealing structure-function relationships of primary platelet adhesion receptors mediating adhesion, activation and aggregation, and the molecular mechanisms underlying platelet thrombus formation. Platelet adhesion is relevant not only to thrombotic disease, but there is increasing evidence of a specific role for platelets in vascular processes such as inflammation and atherogenesis. This review focuses on recent advances in understanding the molecular basis for platelet thrombus formation, in particular the receptors, glycoprotein (GP)Ib-IX-V and GPVI, that initiate platelet adhesion and activation at high shear stress.     

Thrombin interaction with platelets. Influence of a platelet protease nexin

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.     

Physiological implications of adenosine receptor‐mediated platelet aggregation

Adenosine is an important mediator of inhibition of platelet activation. This metabolite is released from various cells, as well as generated via activity of ecto‐enzymes on the cell surface. Binding of adenosine to A₂ subtypes (A_2A or A_2B), G‐protein coupled adenosine receptors, results in increased levels of intracellular cyclic adenosine monophosphate (cAMP), a strong inhibitor of platelet activation. The role and importance of adenosine and its receptors in platelet physiology are addressed in this review, including recently identified roles for the A_2B adenosine receptor as a modulator of platelet activation through its newly described role in the control of expression of adenosine diphosphate (ADP) receptors. J. Cell. Physiol. 226: 46–51, 2010. © 2010 Wiley‐Liss, Inc.     

Platelet and vascular smooth muscle thromboxane A2/prostaglandin H2 receptors

Thromboxane A2 (TxA2) and prostaglandin H2 (PGH2) aggregate platelets and contract vascular smooth muscle. Inasmuch as both compounds produce the same effects and presumably through the same receptor, their receptors have been referred to as TxA2/PGH2 receptors. Pharmacological studies of stable agonists and antagonists of the TxA2/PGH2 receptors have shown different rank order potencies for these compounds in platelets compared with blood vessels. These studies have provided evidence to support the hypothesis that the platelet TxA2/PGH2 receptor is different from the one found in vascular tissue. The vascular receptor has been named [TxA2/PGH2]tau and the platelet receptor has been named [TxA2/PGH2]alpha. In the past few years several radiolabeled antagonists and agonists have been developed and used in radioligand-binding studies, primarily in platelets. One of these ligands, 125I-labeled PTA-OH, a TxA2/PGH2 receptor antagonist, has been extensively used to characterize the human platelet TxA2/PGH2-binding site. It has been found to have a Kd of approximately 20 nM and a Bmax of 2500 binding sites/platelet. Through the combination of pharmacological and biochemical approaches, it should be possible to characterize platelet and vascular TxA2/PGH2 receptors.     

Differential effects of concanavalin A and succinyl concanavalin A on the macromolecular events of platelet activation

Concanavalin A is capable of activating platelets in a concentration-dependent manner as judged by [14C]serotonin secretion from prelabeled platelets. In contrast, succinyl concanavalin A does not induce platelet secretion. Concanavalin A treatment also results in a number of alterations in platelet macromolecules which are presumably associated with the process of platelet activation. These include the phosphorylation of 20 and 47 kDa platelet proteins, the increased polymerization and association of new proteins with the platelet cytoskeleton and the association of the platelet membrane glycoprotein IIb/III complex with the platelet cytoskeleton. Succinyl concanavalin A treatment results in none of these macromolecular events. This difference is observed despite the demonstration that both lectins bind to the platelet surface. Gel overlay experiments also indicate that concanavalin A and succinyl concanavalin A bind to the same receptors. These differences in the biological effects of concanavalin A and succinyl concanavalin A on platelets may be due to decreased receptor crosslinking by the succinylated derivative. The formation of multiple linked interactions between surface receptors may be an important event in the activation of platelets by concanavalin A.     

Lipid rafts are critical membrane domains in blood platelet activation processes

Among the various hematopoi;etic cells, platelets are critical for maintaining the integrity of the vascular system. They must be rapidly activated by sequential and coordinated mechanisms in order to efficiently prevent haemorrhage upon vascular injury. Several signal transduction pathways lead to platelet activation in vitro and in vivo, among them, several are initiated via receptors or co-receptors containing immuno-receptor tyrosine-based activation motifs (ITAM) which trigger downstream signalling like the immune receptors in lymphocytes. However, in contrast to immune cells for which the role of lipid rafts in signalling has largely been described, the involvement of laterally segregated membrane microdomains in platelet activation has been investigated only recently. The results obtained until now strongly suggest that early steps of platelet activation via the collagen receptor GpVI or via FcgammaRIIa occur preferentially in these microdomains where specific proteins efficiently organize key downstream signalling pathways. In addition, lipid rafts also contribute to platelet activation via heterotrimeric G-protein-coupled receptors. They are sites where the phosphoinositide (PI) metabolism is highly active, leading to a local generation of lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate. Here, evidence is accumulating that cholesterol-enriched membrane microdomains are part of a general process that contributes to the efficiency and the coordination of platelet activation mechanisms. Here we will discuss the biochemical and functional characterizations of human platelet rafts and their potential impact in platelet physiopathology.     

Lipid rafts are critical membrane domains in blood platelet activation processes

Among the various hematopo?̈etic cells, platelets are critical for maintaining the integrity of the vascular system. They must be rapidly activated by sequential and coordinated mechanisms in order to efficiently prevent haemorrhage upon vascular injury. Several signal transduction pathways lead to platelet activation in vitro and in vivo, among them, several are initiated via receptors or co-receptors containing immuno-receptor tyrosine-based activation motifs (ITAM) which trigger downstream signalling like the immune receptors in lymphocytes. However, in contrast to immune cells for which the role of lipid rafts in signalling has largely been described, the involvement of laterally segregated membrane microdomains in platelet activation has been investigated only recently. The results obtained until now strongly suggest that early steps of platelet activation via the collagen receptor GpVI or via FcγRIIa occur preferentially in these microdomains where specific proteins efficiently organize key downstream signalling pathways. In addition, lipid rafts also contribute to platelet activation via heterotrimeric G-protein-coupled receptors. They are sites where the phosphoinositide (PI) metabolism is highly active, leading to a local generation of lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate. Here, evidence is accumulating that cholesterol-enriched membrane microdomains are part of a general process that contributes to the efficiency and the coordination of platelet activation mechanisms. Here we will discuss the biochemical and functional characterizations of human platelet rafts and their potential impact in platelet physiopathology.     

The role of heterotrimeric G proteins in platelet activation

Activation of platelets plays a central role in hemostasis as well as in various thromboembolic diseases like myocardial infarction or stroke. Most platelet activating stimuli function through receptors which couple to heterotrimeric G proteins of the Gi, Gq and G12 families. Recent studies have elucidated the roles of individual G proteins in the regulation of platelet functions like shape change, aggregation and granule secretion. The signaling pathways mediated by heterotrimeric G proteins operate synergistically to induce a full activation of platelets. This review summarizes recent progress in the understanding of upstream regulation of platelet activation through G protein-coupled receptors.     

Receptors that activate platelets.

This review highlights the increasing knowledge of the biochemistry, pathology, and cell and molecular biology of platelet receptors. A receptor for ADP has been identified using the affinity label FSBA as aggregin, a 100-kDa membrane protein responsible for shape change, aggregation, and exposure of fibrinogen binding sites. A variety of putative receptors for collagen have been described, with GPIa/IIa and GPIV receiving the most attention recently. A thromboxane A2 receptor has been identified using receptor antagonists and photoaffinity labels. The alpha 2-adrenergic receptor has been cloned and expressed. The platelet thrombin receptor has been tentatively identified as GPIb. Following binding of thrombin to this receptor, activation of calpain occurs, with cleavage of aggregin leading to exposure of GPIIb/III alpha and platelet aggregation. Isolation, expression, or both of the ADP, collagen, and thrombin receptors as single gene products of the human platelet responsible for activation, and more complete understanding of stimulus-response coupling, should allow for greater specificity of drugs with selective therapeutic actions.