Platelet Granules and Organelles
Platelets possess secretory granules and mechanisms for cargo release to amplify responses to stimuli and influence the surrounding environment. Platelet granule structures include 𝛼- and dense granules, lysosomes, and peroxisomes. 𝛼-Granules and the dense bodies are the main secretory granules that release cargo (e.g., fibrinogen and adenosine diphosphate [ADP]) upon platelet activation.
Platelet granule secretion begins with a dramatic increase in platelet metabolic activity, set off by a wave of calcium release and marked by increased adenosine triphosphate (ATP) production. After platelet stimulation by agonists, a "contractile ring" develops around centralized granules, the granules fuse with the surface membranes, and then they extrude their contents. Granule secretion in platelets is a graded process that depends on the number, concentration, and nature of the original stimulus/stimuli, either strong (e.g., thrombin and collagen) or weak (e.g., ADP and epinephrine).
What the 𝛼-granules have include:
- Other plasma proteins (small amount)
- Growth factors
- 𝛼IIb𝛽3 [platelet receptor]
𝛼-Granules, with a cross-sectional diameter of approximately 300 nm and numbering approximately 50 per platelet, are the predominant platelet granules. They are approximately spherical in shape, with an outer membrane enclosing two distinct intragranular zones that vary in electron density. The larger, electron-dense region is often eccentrically placed and consists of a nucleoid material that is rich in platelet-specific proteins such as 𝛽-thromboglobulin. The second zone, of lower electron density, lies in the periphery adjacent to the granule membrane and contains tubular structures with adhesive GPs such as von Willebrand factor (vWF) and multimerin, along with factor V. Platelets take up plasma proteins and store them in their 𝛼-granules.
Three proteins, 𝛽-thromboglobulin, PF4, and thrombospondin, are synthesized in megakaryocytes and highly concentrated in 𝛼-granules. The first two, 𝛽-thromboglobulin and PF4, show homology in amino acid sequence and share the additional features of localization in the dense nucleoid of 𝛼-granules, heparin-binding properties, and membership in the CXC family of chemokines. Together, they constitute approximately 5% of total platelet protein, and they can serve as useful markers for platelet activation in serum or plasma. Thrombospondin may comprise up to 20% of the total platelet protein released in response to thrombin, and likely participates in multiple biologic prcesses.
vWF is also synthesized by megakaryocytes and is present in the tubular structures of the 𝛼-granule peripheral zone, similar to its localization within Weibel-Palade bodies of vascular endothelial cells. Factor V and multimerin, a factor V/Va-binding protein, co-localize with vWF in platelets but not in endothelial cells. Fibrinogen is also found in 𝛼-granules, but is incorporated actively from plasma and not synthesized by megakaryocytes. In fact, small amounts of virtually all plasma proteins, such as albumin, immunoglobulin G (IgG), fibronectin, and 𝛽-amyloid protein precursor, may be taken up into the platelet 𝛼-granules. 𝛼-Granules also contain many growth factors, including platelet-derived growth factor, transforming growth factor-𝛽1 (TGF-𝛽1), and vascular endothelial growth factor. These signaling molecules may contribute to the mitogenic activity of platelets.
Platelet 𝛼-granules serve as an important reservoir for 𝛼(IIb)𝛽3 that contributes significantly to the surface fibrinogen receptors present on activated platelets. The 𝛼-granule membrane protein, P-selectin (granule membrane protein-140) is translocated to the plasma membrane after platelet activation. Finally, a number of additional proteins have been located to the surface of 𝛼-granules alone, including CD9, platelet endothelial cell adhesion molecule-1 (PECAM-1), Rap 1b, GPIb-IX-V, and osteonectin.
The platelets and megakaryocytes of patients with gray platelet syndrome have decreased numbers of 𝛼-granules and reduced levels of some proteins. It is proposed that there is incorrect targeting of 𝛼-granule proteins to the 𝛼-granule in the megakaryocyte in this disease.
Dense bodies contain a large reservoir of ADP, a crtitical agnoist for platelet activation that amplifies the effect of other stimuli. In addition to this nonmetabolic pool of ADP, the dense bodies are rich in ATP, pyrophosphate, calcium, and serotonin (5-hydroxytryptamine), with lesser amounts of guanosine triphosphate (GTP), guanosine diphosphate (GDP), and magnesium. The adenine nucleotides are synthesized and segregated by megakaryocytes, whereas serotonin is incorporated into dense granules from the plasma by circulating platelets. There is more ADP than ATP in dense bodies, and both can lead to adenosine monophosphate (AMP). In turn, AMP can be dephosphorylated to adenosine or cyclized to produce cyclic AMP, an inhibitor of the platelet-stimulatory response. The dense granule membrane contains P-selectin and granulophysin.
Compared ADP/ATP within the metabolic/cytoplasmic pool (at least two different pools: metabolic pool and cytoplasmic pool), ADP/ATP in storage pool (dense bodies) contains approximately two-thirds of the total platelet nucleotides, mainly in the form of ADP and ATP, and is metabolically inactive, does not rapidly incorporate exogenous adenine or phosphate, and equilibrates slowly with the metabolic pool. Nucleotides in this pool (storage pool) are extruded fromt the platelet during the release reaction and cannot be replenished after release.
The ATP (metabolic pool) that is broken down to provide energy for the release reaction is not rephosphorylated; rather, it is irreversibly degraded to hypoxanthine, which diffuses out of the cell.
Lysosomes are small, acidified vesicles, approximately 200 nm in diameter, that contain acid hydrolases with pH optima of 3.5 to 5.5, including 𝛽-glucuronidase, cathepsins, aryl sulfatase, 𝛽-hexosaminidase, 𝛽-galactosidase, heparitinase, and 𝛽-glycerophosphatase. Additional protein found in lysosomes include cathepsin D and lysosome-associated membrane proteins (LAMP-1/LAMP-2, which are expressed on the plasma membrane after activation). Lysosomal constituents are released more slowly and incompletely (maximally, 60% of the granules) than 𝛼-granules or dense-body components after platelet stimulation, and their release also requires stronger agonists such as thrombin or collagen.
Peroxisomes are rare, small granules, demonstrable with alkaline diaminobenzidine as a result of their catalase activity. The structure may participate in the synthesis of platelet-activating factor.
Mitochondria in platelets are similar, with the exception of their smaller size, to those in other cell types. There are approximately seven per human platelet, and they serve as the site for the actions of the respiratory chain and the citric acid cycle. Glycogen is found in small particles or in masses of closely associated particles, playing an essential role in platelet metabolism.
Approximately one-third of the total platelet mass appears to pool in the spleen. The splenic pool exchanges freely with the platelets in the peripheral circulation. Administration of epinephrine, which evacuates platelets from the spleen, increases the peripheral platelet count 30% to 50%, and platelet counts in asplenic patients are not affected by epinephrine. Some studies suggest that the splenic pool consists of the youngest, largest platelets. Pathophysiologi states can result in 80% to 90% of platelets being sequestered in the spleen, resulting in thrombocytopenia.
Other organs that have pool of platelets (accounting for about 16% of total platelets) include the lungs and the liver and so on.
The life span of platelet has been estimated to be 8 to 12 days in humans. In steady state, when platelet production equals destruction, platelet turnover has been estmated at 1.2 to 1.5 x 1011 cells per day.
PS: Details of various platelet products can be found in thread "Platelet Transfusion for Patients w/ Cancer" at http://www.tomhsiung.com/wordpress/2013/04/platelet-transfusion-for-patients-with-cancer-part-one/
Platelet Adhesion and Activation
Part I – Adhesion
- Adhesive ligands: vWF, collagen, fibronectin, thrombospondin, laminin (perphaps)
- Platelet surface receptors: GPIb/V/IX complex, GPVI, 𝛼IIb𝛽3, 𝛼2𝛽1, 𝛼5𝛽1, 𝛼6𝛽1
- Interaction of GPVI with collagen activates platelet intergrins
- At low shear conditions, fibrinogen is the primary ligand (interacting with 𝛼IIb𝛽3), but other ligands may also be involved
Platelet adhesion to exposed subendothelium is a complex multistep process that involves a diverse array of adhesive ligands (vWF, collagen, fibronectin, thrombospondin, and perphaps laminin) and surface receptors (GPIb/V/IX, GPVI, integrins 𝛼IIb𝛽3, 𝛼2𝛽1, 𝛼5𝛽1, and 𝛼6𝛽1). The specific ligand/receptor palyers in primary platelet adhesion are largely dependent on the arterial flow conditions present. In high shear conditions, platelet tethering is dependent on the unique shear-dependent interaction between GPIb/V/IX and subendothelial vWF, derived either from plasma or released by endothelial cells and/or platelets. A tether forms between GPIb and vWF that either halts platelet movement or reduces it such that other interactions can proceed. Subsequent interactions are mediated by GPVI binding to glycineproline-hydroxyproline sites on collagen and perhaps to exposed laminin. The interaction of GPVI with collagen strongly activates platelets such that 𝛼IIb𝛽3, can engage in high-affinity interactions with ligands. At low shear conditions, fibrinogen is thought to be the primary ligand supporting platelet plug formation through its interaction with 𝛼IIb𝛽3, although thrombus formation can take place in the absence of vWF and fibrinogen, so other ligands may also be involved.
Following initial platelet adhesion, subsequent platelet-platelet interactions are intially mediated by two receptors, GPIb/V/IX and 𝛼IIb𝛽3, and their respective contributions are dependent on the flow conditions present. In high shear stress conditions, GPIb/V/IX receptor and vWF ligand action are predominant, with fibrinogen playing a stabilizing role.
Platelet Glycoprotein Ib Complex-von Willebrand Factor Interaction and Signaling
The interaction of the platelet GPIb "complex" (the polypeptides GPIb𝛼, GPIb𝛽, GPIX, and GPV) with its primary ligand. vWF, is the receptor-ligand pairing that initiates platelet adhesion followed by a cascade of events leading to pathologic thrombosis or physiologic hemostasis. A unique aspect of this receptor-ligand interaction is that it requires the presence of high arterial shear rates to take place, thus explaining the predisposition of platelet-rich "white clots" in the arterial circulation over clots found in the venous circulation, with its relatively lower shear forces, in which clot formation takes place independent of the GPIb complex.
The binding site for vWF is present in the N-terminal 282 residues of GPIb. Important to the interactions are a cluster between residues Asp 252 and Arg 293 containing sulfated tyrosine residues and important anionic residues, a disulfide loop between Cys 209 and Cys 248, and an N-terminal flanking sequence of the leucine-rich repeats (LRG). Mutations involving single amino acid residues within these LRGs account for some cases of the congential bleeding disorder Bernard-Soulier syndrome, in which the GPIb complex binds poorly, or not at all, to vWF.
Unlike other receptors, GPIb does not require platelet activation for its interactions with vWF. In vitro, the interactions of vWF and binding with the GPIb complex occur with generally very low affinity in the absence of shear. The presence of the vancomycin-like antibiotic ristocetin or viper venom proteins, such as botrocetin, promotes the interactions. Mobilization may uncoil vWF to promote interactions with GPIb. The addition of shear, in a parallel-plate flow system, results in platelet interaction with subendothelial vWF that occurs in a biphasic fashion. Likewise, the rate of translocation of platelets from blood to the endothelial cell surface, which is dependent, increases linearly up to wall shear rates of 1,500 s-1, whereas the translocation rate remains relatively constant with the wall shear rate between 1,500 and 6,000 s-1. Thus, the presence of shear is important for promoting the interactions between the GPIb complex and vWF. Studies of real-time thrombus formation in the absence of platelet GPIb complex and in blood from individuals with severe (type 3) von Willebrand disease indicate that GPIb and vWF interaction are required for platelet surface interaction at high shear rates (>1,210 s-1), whereas GPIb deficiency results in poor platelet adhesion at lower shear. Shear accelerates thrombus formation likely by promoting this receptor-ligand interaction.
When the GPIb complex interacts with its vWF ligand under conditions of elevated shear stress, signals are initiated that activate integrin 𝛼IIb𝛽3. The pathways involved lead to a) elevation of intracellular calcium; b) activation of a tyrosine kinase signaling pathway that incorporates nonreceptor tyrosine kinases such as Src, Fyn, Lyn, and Syk, phospholipase C(gamma)2, and adaptor protein such as SHC, LAT, and SLP-76; c) inside-out signaling through the 𝛼IIb𝛽3 integrin followed by platelet aggregation; and d) activation of protein kinase C (PKC), protein kinase G (PKG), and phosphoinositide 3-kinase (PI3K), …… and so on.
Once vWF binds to GPIb-V-IX, signaling complexes form in the vicinity of the GPIb𝛼 cytoplasmic tail consisting of cytoskeletal proteins such as 14-3-3ζ as well as signaling protein like Src and PI 3-kinase. This process leads to Syk activation, protein tyrosine phosphorylation, and recruitment of other cytoplasmic proteins with pleckstrin homology domains that can support interactions with 3-phosphorylated phosphoinositides and ultimately activation of integrin 𝛼IIb𝛽3.
Glycoprotein Ib Complex Interaction with Thrombin and Other Molecules
The GPIb complex serves as an 𝛼-thrombin binding site on platelets, although the physiologic relevance of the interactions is not clear. The density of GPIb complexes (~20,000/platelet) far exceeds the number of thrombin binding sites reported on platelets (~6,000/platelet). Studies have identified interaction of the GPIb complex with ligands other than vWF. These include a study a reversible association of GPIb with P-selectin, which is examined in more detail in the section "Platelets and Endothelium." The interaction of platelet GPIb with the neutrophil adhesion receptor 𝛼(M)𝛽2 (Mac-1) is discussed in the section "Platelets and White Blood Cells." Additionally, GPIb reportedly interacts with high-moecular weight kininogen, factor XII, and factor XI.
Platelet-Collagen Interaction and Signaling
- Receptors: GPVI, 𝛼2b𝛽1
- Ligands: collagens
Collagens, one of the most thrombogenic substance in vessels, are very important activators of platelets in the vascular subendothelium and vessel wall, and thus are prime targets for therapeutic intervention in patients experience a pathologic arterial thrombotic event such as MI or stroke. Platelets have two major surface receptors for collagen, the immunoglobulin superfamily member GPVI and the integrin 𝛼2𝛽1. The former is considered to be the primary palyer in platelet adhesion. In additon to these two surface receptors, the GPIb complex can also be considered an indirect collagen receptor because its subendothelial vWF ligand essentially acts as bridging molecule between platelets and collagen by fixing itself to the latter, which, in turn, acts as scaffolding for the multimers. Collagen adhesion also results in indirect activation of the protease-activated receptor 1 via MMP-1. Other molecules, such as CD 36, may also sustain collagen interaction.
Glycoprotein VI receptor
GPVI is the main receptor involved in collagen-mediated platelet activation. Studies of mice lacking platelet GPVI show that they lose collagen-induced platelet activation due to a defect in platelet adhesion. Thus, GPVI appears to serve as teh initial receptor involved in platelet adhesion, and it activates integrin binding. GPVI alone supports adhesion to insoluble collagens, and works with 𝛼2𝛽1 to promote platelet adhesion to soluble collagen microfibrils. GPVI can also be engaged by collagen-related peptides (arranged in triple helical structures with sequences similar to collagen) and teh snake venom convulxin, which elicit signals through GPVI.
Synergism between GPVI pathways and those related to other adhesion receptors such as GPIb-V-IX and soluble agonists released by activated platelets are likely necessary for the full repertoire of platelet-collagen signaling. Exposure of platelets to collagen surfaces likely results in GPVI clustering that in turn triggers the tyrosine phosphorylation of the FcRγ chain. The GPVI/FcRγ-chain complex leads to platelet activation through a pathway that has many aspects in common with signaling by immune receptors, such as the Fc receptor family and the B- and T-cell antigen receptors.
The first platelet collagen receptor identified was the integrin 𝛼2𝛽1 receptor, also known as platelet GPIa/IIa and lymphocyte VLA-2.
When compared to vWF, collagen is a more efficient substrate when it comes to supporting stable platelet adhesion and thrombus formation. The fact that initial platelet tethering to collagen under high shear flow first requires interaction between vWF and platelet GPIb serves to underscore the importance of the two major collagen receptors, GPVI and 𝛼2𝛽1, in promoting platelet adhesion and activation under shear conditions.
In addition to GPVI, the α2β1 receptor also propagates signaling. The use of α2β1 selective ligands has demonstrated calcium-dependent spreading and tyrosine phosphorylation of several proteins when interaction with platelets takes place.
Physiologic Inhibition of Platelet Adhesion
Negative regulation of platelets is essential to set the stimulus threshold for thrombus formation, determine final clot size and stability, and prevent uncontrolled thrombosis. The mechanisms behind the negative regulation of platelet activation are described later, and in this respect, roles of players such as nitric oxide and prostacyclin have been well characterized. Platelet activation can also be inhibited by signaling through the adhesion moleculde PECAM-1 (CD31). Expressed on a number of blood cells and endothelial cells, PECAM has a wide array of regulatory functions in processes such as apoptosis and cell activation. Following homophilic interactions and/or clustering, PECAM-1 is tyrosine phosphorylated in its cytoplasmic tail ITIM domain. Phosphorylation of PECAM-1 recruits and activates the SH2 domain-containing protein-tyrosine phosphatase, SHP-2. Studies suggest that the PECAM-1/SH-2 complex functions to counteract platelet activating, most particularly for collagen by inhibiting GPVI/FcRγ chain signaling.
Part II – Activation
PAR Thrombin Interactions
- See Figure 16.9
Platelet thrombin receptors/platelet protease-activated receptors/PARs and signaling
PARs are G-protein-coupled receptors that use a unique mechanism to convert an extracellular protein cleavage event into an intracellular activation signal. In this case, the ligand is already part of the receptor per se, by virtue of the fact that it is represented by the amino acid sequence SFLLRN (residues 42 through 47) and is unmashed as a new amino terminus after thrombin cleaves the peptide bond between Arg 41 and Ser 42 (Figure 16.9). This "tethered ligand" then proceeds to irreversibly dock with the body of its down receptor to effect transmembrane signaling, as shown in Figure 16.9.
Thrombin signaling in platelet is mediated, at least in part, by four members of a family of G-protein-coupled PARs (PAR-1, -2, -3, and -4). Human platelets express PAR-1 and PAR-4, and activation of either is sufficient to trigger platelet aggregation. PAR-1, -3, and -4 can be activated by thrombin, whereas PAR-2 can be activated by trypsin, tryptase, and coagulation factors VIIa and Xa. Presumably, other proteases are capable of recognizing the active sites of these receptors and can thus also trigger PAR signaling.
Once activated, PAR-1 is rapidly uncoupled from signaling and internalized into the cell. It is then transported to lysosomes and degraded. Platelet presumably have no need for a thrombin receptor recycling mechanism, becuase once activated, they are irreversibly incorporated into blood clots. Conversely, in cell lines with characteristics similar to megakaryocytes, new protein synthesis is needed for recovery of PAR-1 signaling, and in endothelial cells, sensitivity to thrombin is maintained by delivery of naive PAR-1 to the cell surface from a preformed intracellular pool.
Platelet ADP (Purinergic) Receptors and Signaling
Evidence that ADP plays an important role in both the formation of the platelet plug and the pathogenesis of arterial thrombosis has been accumulating since its initial characterization in 1960 as a factor derived from red blood cells that influences platelet adhesion. ADP is present in high concentratons (molar) in platelet-dense granules and is released when platelet stimulation takes place with other agonists, such as collagen; thus, ADP serves to further amplify the biochemical and physiologic changes associated with platelet activation and aggregation. Inhibitors of this ADP-associated aggregation include commonly used clinical agents, including ticlopidine, clopidogrel, prasugrel, and ticagrelor, proven to be very effective antithrombotic drugs.
Adenine nucleotides interact with P2 receptors that are ubiquitous among different cell types and have been found to regulate a wide range physiologic processes. They are divided into two groups, the G-protein-coupled superfamily named P2Y and the ligand-gated ion channel superfamily termed P2X. Two G-protein-coupled (P2Y) receptors contribute to platelet aggregation. The P2Y1 receptor initiates aggregation through mobilization of calcium stores, and the P2Y12 receptor is coupled to inhibition of adenylate cyclase and is essential for a full aggregation response to ADP with stabilization of the platelet plug.
PS: ADP >>> P2Y12 >>> inhibition of adenylate cyclase >>> decreased cAMP production >>> decreased intensity of aggregation
Inhibition of either P2Y1 or P2Y12 receptors is sufficient to block ADP-mediated platelet aggregation, and coactivation of both receptors is therefore necessary, through the G proteins Gq and Gi, respectively, for ADP to activate and aggregate the platelet.
Although considered a weak agonist in comparison to collagen or thrombin, ADP clearly palys an important role in thrombus stabilization, likely by contributing to the recruittment of additional platelets to growing thrombi. Aggregation is often reversible when platelets are stimulated by ADP alone. In addition, low concentrations of ADP serve to amplify the effects of both strong and weaker agonists, the latter inlcuding serotonin and adrenaline, among others.
Platelet Activation by Soluble Agonists
Epinephrine is unique among platelet agonists because it is considered to be capable of stimulating secretion and aggregation, but not cytoskeletal reorganization responsible for shape change. Platelet responses to epinephrine are mediated through 𝛼2-adrenergic receptors, and these responses have been found to vary among individuals, with some donors with otherwise normal platelets manifesting delayed or absent responses.
Arachidonic acid, thromboxane A2, and thromboxane receptors
After platelet stimulation by a number of agonists, arachidonic acid is generated directly by phospholipase A from its membrane phospholipid precursors (PC, PS, and PI) and indirectly by PLC generation of DAG followed by DAG lipase action. Most platelet agonists are believed to activate this pathway. Three known eicosanoid subsetsl of biochemical compounds are known to be derived from the formation of arachidonic acid – the prostanoids, leukotrienes, and epoxides. Although all three of these pathways are present in platelets, most arachidonic acid ends up being metabolized to thromboxane A2 (TxA2).
TxA2 is produced in platelets from arachidonic acid through the generation of PGH2 by the enzyme cyclo-oxygenase, which is irreversibly inhibited by aspirin through acetylation of a serine residue near its C terminus. PGE2 and PGI2 act to inhibit platelet activation by generating intracellular cAMP, whereas TxA2 activates platelets. Platelets primarily synthesize thromboxane, and endothelial cells mainly synthesize prostaglandins such as PGI2.
Like ADP and epinephrine, TxA2 is also capable of activating nearby platelets after its release into plasma. It has a very short half-life of 30 seconds before its conversion to the inactive metabolite thromboxane B2 prevents widespread platelet activation beyond the vicinity of thrombus formation. Both arachidonic acid and analogs of TxA2 have been found to activate and aggregate platelets by mediating shape change and phosphorylation of signaling enzymes. The thromboxane receptor (TP) is a member of the seven-transmembrane G-protein-coupled receptor family and has been localized to the plasma membrane. Two isoforms of the receptor have been identified in platelets TP𝛼 and TP𝛽 – and they activate platelets through ghe Gq pathway.
Physiologic Inhibition of Platelet Activation
One of the many remarkable features of platelets is their ability to remain in a physiologic resting state and resist becoming activated while navigating the heart, arterial, and venous circulations. Indeed, the pathologic consequences associated with widespread inappropriate platelet activation are life- and limb-threatening in the settings of well-characterized clinical disorders, such as thrombotic thrombocytopenic purpura and heparin-induced thrombocytopenia. The mechanisms responsible for maintaining the fine balance of keeping platelets in a resting state until they encounter a genuine need to undergo adhesion, activation, and aggregation at the site of vascular injury are nearly as diverse as those responsible for mediating these physiologic phenomena.
Some general mechanisms involved in physiologic inhibition of platelet activation include phenomena such as a) generation of negative-regulating molecules by the platelet (e.g., cAMP), endothelium (e.g., PGI2, nitric oxide, heparan sulfate), and at distant sites (e.g., antithrombin); b) barrier of endothelial cells that prevents direct contact of circulating platelets with collagen; c) ecto-ADPase (CD39) expression by endothelial cells that metabolizes ADP secreted from platelets; d) tendency for blood flow to wash away unbound thrombin and other soluble mediators from the site of platelet plug formation; e) brief half-life of certain key platelet activators such as TxA2; f) tight regulation of the affinity state of receptors such as 𝛼IIb𝛽3; g) downregulation of signaling receptors to limit their actions; and h) inhibitory pathways mediated by ITIM-containing and/or contact-dependent adhesion receptors, such as PECAM, CECAM-1, JAM-A, and potentially others.
Receptor downregulation and desensitization
Signaling through G-protein-coupled receptors present on the surface of platelets is limited by their phosphorylation, which triggers desensitization, that is, uncoupling from G proteins, and internalization via Claritin-mediated endocytosis (for detail about G-protein coupled receptors please refers to thread "G Protein-Coupled Receptors and Second Messengers" at http://www.tomhsiung.com/wordpress/2014/09/g-protein-coupled-receptors-and-second-messengers/). G-protein kinases and 𝛽-arrestin are central to these processes. In addition, G-protein-coupled receptors interact with a myriad of other molecules that finely tune their signaling, including regulators of G-protein signaling (RGS) and GPCR-associated sorting proteins.
Generation of the prostaglandins from arachidonic acid metabolism, such as PGI2 and PGE2 (at high concentrations), results in inhibition of platelet activation and aggregation, and counterbalances the actions of thromboxanes derived from the same pathway. While PGI2 and PGD2 inhibit platelet function at low doses, PGE2 displays a biphasic reponse, and inhibits platelet function only at higher concentrations, likely via the EP4 receptor. The inhibitory effects are mediated via G-protein-coupled receptors (IP and EP receptors, respectively) that couple to the 𝛼 subunits of Gs to regulate adenylate cyclase-mediated generation of cAMP. cAMP levels in platelets are also governed by the activity of phosphodiesterase, the enzyme responsible for cAMP metabolism. This enzyme activity is inhibited drugs such as the weak antiplatelet agent dipyridamole, the bronchodilator theophylline, and sildenafil, used to treat erectile dysfunction in men.
NO is generated by endothelial cells and platelets from L-arginine in response to shear stress forces and other platelet agonists, such as thrombin and ADP. The bulk of the evidence suggests that at high concentrations NO functions to inhibit platelet activation through the cyclic guanosine monophosphate (cGMP) second messenger generated by guanylate cyclase activation. Elevations in cGMP, by modulating phosphodiesterase activity, can raise intraplatelet cAMP. Paradoxically, low levels of NO may elicit platelet activation pathways. Endothelial NO synthase activity is enhanced during platelet activation, presumably as an additional means for limiting platelet aggregation.
Platelet Aggregation: 𝛼IIb𝛽3 (GPIIb/IIIa) Receptor and Its Signaling Mechanisms
Platelet aggregation is a complex phenomenon that is the end result of a series of adhesion- and activation-related processes. Essential components of this process include an agonist, calcium, and the adhesive proteins fibrinogen and vWF. Divalent cations, such as calcium and magnesium, are required for platelet aggregation in trace amounts, and these alter the specificity of the integrin 𝛼IIb𝛽3 for its ligands. Fibrinogen and vWF play dominant roles in platelet aggregation through binding to 𝛼IIb𝛽3, and also by the ability of the former to generate polymerized fibrin as support for the platelets in a thrombus.
The signaling pathways of 𝛼IIb𝛽3 are complex. Central concepts of the signaling pathway include inside-out signaling, which involves the processes termed affinity and avidity modulation, and outside-in signaling, in which messages are transmitted to the inside of the platelet via the events occurring outside the membrane through 𝛼IIb𝛽3 activation. Primary platelet agonists such as ADP, thrombin, and matrix proteins collagen and vWF affect platelet aggregation through a process known as inside-out signaling. In the inside-out signaling, agonist-dependent intracellular signals stimulate the interaction of key regulatory ligands (such as talin) with integrin cytoplasmic tails. This leads to conformational changes in the extracellular domain that result in increased affinity for adhesive ligands such as fibrinogen, vWF, and fibronectin. In the outside-in signaling, extracellular ligand binding, initially reversible, becomes progressively irreversible and promotes integrin clustering and further conformational changes that are transmitted to the cytoplasmic tail. This results in the recruitment and/or activation of enzymes, adaptors, and effectors to form integrin-based signaling complexes.
Brief Review of Physiology of Platelet
Following injury to the blood vessel, platelets interact with collagen fibrils in the exposed subendothelium by a process (adhesion) that involves, among other events, the interaction of a plasma protein ,vWF, and a specific glycoprotein (GP) complex on the platelet surface, GP Ib-IX-V (GPIb-IX). This interaction is particularly important for platelet adhesion under conditions of high shear stress. After adherence to the vessel wall via vWF and the long GP Ib-IX-V receptor, other platelet receptors interact with proteins of the subendothelial matrix. Hereby collagen provides not only a surface for adhesion but also serves as a strong stimulus for platelet activation.
Activated platelets release the contents of their granules (secretion), including ADP and serotonin from the dense granules, which causes the recruitment of additional platelets. These additional platelets form clumps at the site of vessel injury, a process called aggregation (cohesion). Aggregation involves binding of fibrinogen to specific platelet surface receptors, a complex composed of GPIIb-IIIa (integrin 𝛼IIb𝛽3), an integrin that normally exists in a resting (low-affinity) state but that transforms into an activated (high-affinity) state when stimulated by the appropriate signal transduction cascade. GPIIb-IIIa is platelet specific and has the ability to bind vWF as well. Although resting platelets do not bind fibrinogen, platelet activation induces a conformational change in the GPIIb-IIIa complex that leads to fibrinogen binding.
Moreover, platelets play a major role in coagulation mechanisms; several key enzymatic reactions occur on the platelet membrane lipoprotein surface. During platelet activation, the negatively charged phospholipids, especially PS, become exposed on the platelet surface, and essential step for accelerating specific coagulation reactions by promoting the binding of coagulation factors involved in thrombin generation.
A number of physiologic agonists interact with specific receptors on the platelet surface to induce responses, including a change in platelet shape from discoid to spherical, aggregation, secretion, and thromboxane A2 production. Other agonists, such as prostacyclin, inhibit these responses. Binding of agonists to platelet receptors initiates the production or release of several intracellular messenger molecules, including products of hydrolysis of phosphoinositide (PI) by phospholipase C, TxA2, and cyclic nucleotides. These induce or modulate the various platelet responses of Ca2+ mobilization, protein phosphorylation, aggregation, secretion, and thromboxane production.