Platelets in PET studies
Platelets (thrombocytes) are nucleus-free cell fragments, with life-span of ∼7-10 days. Platelets are generated by megakaryocytes in the bone marrow. ∼30% of platelets are stored in the spleen. Ageing platelets lose sialic acid from their surface, and are then removed from circulation in the liver (Kile, 2015).
In their inactive state platelets are disk-shaped with diameter of 2-4 µm. Platelets contain mitochondria, lysosomes, and peroxisomes, and contractile filaments, like cells normally do. Additionally, platelets contain α-granules and dense-core granules. The α-granules are abundant, and contain fibrinogen (endocytosed from blood), clotting factors V, XI, XIII, prothrombin, and von Willebrand factor (vWF). The less abundant dense-core granules contain serotonin, ATP, ADP, GDP, polyphosphate, Ca2+, and Mg2+ (Holmsen & Weiss, 1979; Golebiewska & Poole, 2015). Platelets play an important role in haemostasis, tissue repair, angiogenesis, and antimicrobial responses, but also in inflammatory and vascular diseases, including asthma, inflammatory bowel disease, atherosclerosis, and thrombosis; and in graft rejection, and tumour metastasis (Golebiewska & Poole, 2015; Gros et al., 2015; Kapur et al., 2015; Lam et al., 2015).
The surface of platelets is covered by integrins, and negative charge prevents platelets from adhering to themselves or to endothelial cells. If platelet surface glycoproteins Ia/IIa and Ic/IIa get into contact with collagen, fibronectin, or laminin at the basal membrane of the vascular walls, normally hidden behind endothelial cells, an intracellular signalling cascade activates the platelets. Another activator is vWF, released endothelial cells as response to high shearing force or certain cytokines, binds to glycoprotein Ib/Ia on platelet membranes. Activated platelets release the contents of α and dense-core granules, leading to activation of additional platelets. Activated platelets transform from their initially compact shape to show long membrane protrusions. The membranes of α-granules contain substantial amounts of glycoproteins IIb/IIIa and VI, which are trafficked to the platelet surface when the granules are exocytosed. P-selectin from α-granules translocates to the platelet surface to recruit circulating leukocytes. Platelets contain chemokines, in particular CXCL4, CXCL7, and CXCR1/2, which recruit monocytes and neutrophils (Golebiewska & Poole, 2015). Platelets convert arachidonic acid into prostaglandin H2 (PGH2) and further into thromboxane A2 (TXA2), which also activates more platelets. Acetylsalicylic acid inhibits the formation of PGH2. Conformational change in glycoprotein IIb/IIIa (integrin αIIbβ3) enables it to bind fibrinogen, which leads to bridge formation between platelets. Aggregated platelets can plug holes in small vessels. Various factors can further lead to formation of proteases, mainly thrombin, which start blood coagulation cascade. Thrombin cleaves fibrinogen into parts which spontaneously polymerize into a gel that traps blood cells into a thrombus. Thrombin also cleaves factor XIII to factor XIIIa, which leads to formation of covalent bonds between the fibrin polymers. Endothelial cells release NO, ADP, PGI2, vWF, and other factors, in response to thrombin.
Platelet α-granules contain also anticoagulation factors, including tissue factor pathway inhibitor (TFPI), protein S, amyloid β-A4 (nexin-2), plasmin, and plasminogen. These are essential to sustain haemostasis (Golebiewska & Poole, 2015). Amyloid precursor protein (APP) is found in platelets (Bush et al., 1990), and macrophages could process it to amyloid β that is found in the plaques in Alzheimer’s disease and atherosclerosis.
Fibrinogen is normally present in plasma in concentrations 15-35 mg/L (Oswald et al., 1983). Fibrinogen is composed of two polypeptide trimers. Fibrinogen is produced in the liver, and taken up in blood by platelets and platelet precursors megakaryocytes in the bone marrow. Half-life of fibrinogen in plasma is ∼4 days.
Thrombopoietin is a glycoprotein consisting of 353 amino acids. The N-terminal domain, consisting of 155 amino acid residues, has similarities with erythropoietin (EPO), and its target is c-Mpl receptor. The C-terminal domain, consisting of 177 amino acid residues, is not required for the function of TPO, but it affects it bioavailability (Vadhan-Raj, 1998). For drug use, full-length recombinant rTPO can be produced, but the C-terminal domain is often modified to obtain better properties for medical use.
TPO is produced mainly in the liver, and bone marrow stromal cells produce TPO in paracrine manner in response to reduced number of blood platelets (Kaushansky, 2016). The liver removes senescent platelets from circulation. Desialyation of glycoproteins on platelets reveals galactose oligosaccharides, which are recognized by Ashwell-Morell receptors (AMRs) on hepatocytes, causing the liver to increase TPO production; also interleukin 6 stimulates TPO production in the hepatocytes (Kile, 2015; Kaushansky, 2016; Hoffmeister, 2018). Maintenance of haematopoietic stem cells requires TPO production from the liver (Decker et al., 2018).
TPO receptor (c-Mpl)
The receptor for thrombopoietin, c-Mpl, is member of the cytokine receptor superfamily. Receptor is found in haematopoietic cell lines, particularly in the cells of megakaryocytic lineage. TPO binding to cMpl induces increase in the number and size of megakaryocytes. Platelets contain c-Mpl, and can bind and remove TPO from the circulation, providing negative feedback for production of new platelets.
Platelets have been labelled using 68Ga for thrombosis and atherosclerosis imaging (Welch et al., 1977; Yano et al., 1985; Goodwin et al., 1993). Initial trials for platelet labelling with Co-complexes were not promising (Karanikas et al., 1999). Also 111In-labelled platelets have been tested (Davis et al., 1980; Minar et al., 1989).
Targeting activated platelets
Adenosine nucleotides bind to P2T purinoceptors and specific oligonucleotide receptors on platelets. Labelled adenosine oligonucleotides, such as a derivative of platelet aggregation inhibitor [18F]AppCHFppA, could be used for detecting atherosclerotic plaques (Elmaleh et al., 2006).
Thrombosis has several molecular targets that could be used in diagnostic imaging, for instance for myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. Thrombi contain red blood cells and platelets. Fibrin is present in all thrombi but not in circulating blood. Several fibrin-targeted radiopharmaceuticals have been developed (Ciesienski et al., 2013; Oliveira et al., 2015; Blasi et al., 2015). Tissue factor (TF) is a transmembrane glycoprotein which, when combined with factor VII, starts the cascade leading to generation of thrombin and fibrin network. TF is also overexpressed in many tumour cells, promoting angiogenesis and tumour growth. [18F]ASIS, [18F]FVIIai, and [64Cu]NOTA-FVIIai are active-site inhibited factor VII analogues, which have shown promise in imaging TF expression level (Erlandsson et al., 2015; Nielsen et al., 2016a and 2016b).
Glycoprotein IIb/IIIa receptor is abundantly expressed on platelets. It is a member of integrin family, and also known as αIIbβ3. When platelets are activated, αIIbβ3 reveals ligand-induced binding sites (LIBS) that can be targeted for instance using labelled antibodies for PET imaging (Alt et al., 2014; Ziegler et al., 2016; Yap et al., 2017). Activated αIIbβ3 binds blood fibrinogen motifs with RGD motif, resulting in cross-linking and growing of thrombus. [18F]GP1 is a promising radioligand for PET imaging of thromboembolism (Lohrke et al., 2017; Chae et al., 2019; Kim et al., 2019).
Platelets as confounding factors in imaging
Platelets bind and collect many radiopharmaceuticals from the plasma, because they contain numerous receptors and transporters, including TSPO, serotonin transporter and receptors, adenosine A2A receptors, P2Y- and P2X-receptors, glutamate receptors, monoamine oxidases MAO-A and MAO-B, fatty acid translocase/CD36, cannabinoid CB1 and CB2 receptors, ABC-transporters, IgE receptors, and lactoferrin receptor. Platelets accumulate catecholamines from the plasma (Chamberlain et al., 1990). Enzymes in platelets participate in the metabolism of radiopharmaceuticals. This may cause problems in processing of blood samples, and it can increase background activity in PET images of blood-rich organs.
Platelets express serotonin transporter (SERT), and are able to store high concentrations of serotonin in the dense-core granules. 5-HT2A receptors on platelets have similar affinity for drugs as the receptors in CNS. Before PET imaging was widely available the platelets were used as surrogates for studying serotonin dysfunction in the brain; however, changes in platelet 5-HT2As may not indicate similar changes in the brain (Cho et al., 1999).
- Wound healing
- Deep vein thrombosis
- Inflammation and infection
- Converting blood TAC to plasma TAC
- Metabolite correction
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Updated at: 2019-03-22
Created at: 2019-01-31
Written by: Vesa Oikonen