Extracellular vesicles as next generation immunotherapeutics.

Extracellular vesicles (EVs) function as a mode of intercellular communication and molecular transfer to elicit diverse biological/functional response. Accumulating evidence has highlighted that EVs from immune, tumour, stromal cells and even bacteria and parasites mediate the communication of various immune cell types to dynamically regulate host immune response. EVs have an innate capacity to evade recognition, transport and transfer functional components to target cells, with subsequent removal by the immune system, where the immunological activities of EVs impact immunoregulation including modulation of antigen presentation and cross-dressing, immune activation, immune suppression, and immune surveillance, impacting the tumour immune microenvironment. In this review, we outline the recent progress of EVs in immunorecognition and therapeutic intervention in cancer, including vaccine and targeted drug delivery and summarise their utility towards clinical translation. We highlight the strategies where EVs (natural and engineered) are being employed as a therapeutic approach for immunogenicity, tumoricidal function, and vaccine development, termed immuno-EVs. With seminal studies providing significant progress in the sequential development of engineered EVs as therapeutic anti-tumour platforms, we now require direct assessment to tune and improve the efficacy of resulting immune responses - essential in their translation into the clinic. We believe such a review could strengthen our understanding of the progress in EV immunobiology and facilitate advances in engineering EVs for the development of novel EV-based immunotherapeutics as a platform for cancer treatment.

Microlyse: a thrombolytic agent that targets VWF for clearance of microvascular thrombosis.

Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (ie, tissue plasminogen activator and urokinase plasminogen activator [uPA]), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi sevenfold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13-/- mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease.

Semisynthesis of A6-A11 lactam insulin.

Insulin replacement therapy is essential for the management of diabetes. However, despite the relative success of this therapeutic strategy, there is still a need to improve glycaemic control and the overall quality of life of patients. This need has driven research into orally available, glucose-responsive and rapid-acting insulins. A key consideration during analogue development is formulation stability, which can be improved via the replacement of insulin's A6-A11 disulfide bond with stable mimetics. Unfortunately, analogues such as these require extensive chemical synthesis to incorporate the nonnative cross-links, which is not a scalable synthetic approach. To address this issue, we demonstrate proof of principle for the semisynthesis of insulin analogues bearing nonnative A6-A11 cystine isosteres. The key feature of our synthetic strategy involves the use of several biosynthetically derived peptide precursors which can be produced at scale cost-effectively and a small, chemically synthesised A6-A11 macrocyclic lactam fragment. Although the assembled A6-A11 lactam insulin possesses poor biological activity in vitro, our synthetic strategy can be applied to other disulfide mimetics that have been shown to improve thermal stability without significantly affecting activity and structure. Moreover, we envisage that this new semisynthetic approach will underpin a new generation of hyperstable proteomimetics.

Chemoselective Methionine Labelling of Recombinant Trastuzumab Shows High In Vitro and In Vivo Tumour Targeting.

A highly effective 2-step system for site-specific antibody modification and conjugation of the monoclonal antibody Herceptin (commercially available under Trastuzumab) in a cysteine-independent manner was used to generate labelled antibodies for in vivo imaging. The first step contains redox-activated chemical tagging (ReACT) of thioethers via engineered methionine residues to introduce specific alkyne moieties, thereby offering a novel easy way to fundamentally change the process of antibody bioconjugation. The second step involves modification of the introduced alkyne via azide-alkyne cycloaddition 'click' conjugation. The versatility of this 2-step approach is demonstrated here by the selective incorporation of a fluorescent dye but can also be applied to a wide variety of different conjugation partners depending on the desired application in a facile manner. Methionine-modified antibodies were characterised in vitro, and the diagnostic potential of the most promising variant was further analysed in an in vivo xenograft animal model using a fluorescence imaging modality. This study demonstrates how methionine-mediated antibody conjugation offers an orthogonal and versatile route to the generation of tailored antibody conjugates with in vivo applicability.

Current and future strategies to monitor and manage coagulation in ECMO patients.

Extracorporeal membrane oxygenation (ECMO) can provide life-saving support for critically ill patients suffering severe respiratory and/or cardiac failure. However, thrombosis and bleeding remain common and complex problems to manage. Key causes of thrombosis in ECMO patients include blood contact to pro-thrombotic and non-physiological surfaces, as well as high shearing forces in the pump and membrane oxygenator. On the other hand, adverse effects of anticoagulant, thrombocytopenia, platelet dysfunction, acquired von Willebrand syndrome, and hyperfibrinolysis are all established as causes of bleeding. Finding safe and effective anticoagulants that balance thrombosis and bleeding risk remains challenging. This review highlights commonly used anticoagulants in ECMO, including their mechanism of action, monitoring methods, strengths and limitations. It further elaborates on existing anticoagulant monitoring strategies, indicating their target range, benefits and drawbacks. Finally, it introduces several highly novel approaches to real-time anticoagulation monitoring methods including sound, optical, fluorescent, and electrical measurement as well as their working principles and future directions for research.

18F Site-Specific Labelling of a Single-Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography.

Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non-site-specific bio conjugation approach with N-succinimidyl-4-[18F]fluorobenzoate (S[18F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine-18 PET radiotracer, based on this antibody, using site-specific bio conjugation to engineer cysteine residues with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). ScFvanti-LIBS and control antibody mut-scFv, with engineered C-terminal cysteine, were reduced, and then, they reacted with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Radiolabelled scFv was injected into mice with FeCl3-induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non-injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer 18F-scFvanti-LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine-18 variant of the scFvanti-LIBS against activated platelets using site-specific bio conjugation.

Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro.

Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of SRGs but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create shear rate gradients during blood perfusion. VWF-GPIbα interactions were increased at sites of shear rate gradients compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRGs but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFV-A1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRGs on VWF dependent platelet aggregation and developed the shear-gradient sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of shear rate gradients. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal haemostasis.

Cobalt-Directed Assembly of Antibodies onto Metal-Phenolic Networks for Enhanced Particle Targeting.

The orientation-specific immobilization of antibodies onto nanoparticles, to preserve antibody-antigen recognition, is a key challenge in developing targeted nanomedicines. Herein, we report the targeting ability of metal-phenolic network (MPN)-coated gold nanoparticles with surface-physisorbed antibodies against respective antigens. The MPN coatings were self-assembled from metal ions (FeIII, CoII, CuII, NiII, or ZnII) cross-linked with tannic acid. Upon physisorption of antibodies, all particle systems exhibited enhanced association with target antigens, with CoII systems demonstrating more than 2-fold greater association. These systems contained more metal atoms distributed in a way to specifically interact with antibodies, which were investigated by molecular dynamics simulations. A model antibody fragment crystallizable (Fc) region in solution with CoII-tannic acid complexes revealed that the solvent-exposed CoII can directly coordinate to the histidine-rich portion of the Fc region. This one-pot interaction suggests anchoring of the antibody Fc region to the MPN on nanoparticles, allowing for enhanced targeting.

Applications of Nanotechnology in the Diagnosis and Therapy of Stroke.

Stroke is a leading cause of death and disability worldwide. The classification of stroke subtypes is difficult but critical for the prediction of clinical course and patient management, and limited treatment options are available. There is an urgent need for improvements in both diagnosis and therapy. Strokes have rapidly evolving phases of damage involving unique compartments of the brain, which imposes severe limitations for current diagnostic and treatment procedures. The rapid development of nanotechnology in other areas of modern medicine has ignited a widespread interest in its potential for the field of stroke. An important feature of nanoparticles is the relative ease in which their structures and surface chemistries can be modified for specific and potentially multiple, simultaneous purposes. Nanoparticles can be synthesized to carry and deliver therapeutics to specific cellular or subcellular compartments; they can be engineered to provide enhanced contrast for imaging based on the detection of changes in the blood flow; or possess ligand-specific chemistries which can facilitate diagnosis and monitor the treatment response. More specifically for a stroke, nanoparticles can be engineered to release their payload in response to the distinct extracellular processes occurring around the clot and in the ischemic penumbra, as well as aid in the detection of pathological hallmarks present at various stages of stroke progression. These capacities allow targeted release of disease-modifying agents in the affected brain tissue, increasing treatment efficacy, and limiting unwanted side effects. While nanospheres, liposomes, and mesoporous nanostructures all emerge as future prospects for stroke treatment and diagnosis, much of this potential is yet to be clinically realized. This review outlines aspects of nanotechnology identified as having potential to revolutionize the field of stroke.

Ligand-Functionalized Poly(ethylene glycol) Particles for Tumor Targeting and Intracellular Uptake.

Drug carriers typically require both stealth and targeting properties to minimize nonspecific interactions with healthy cells and increase specific interaction with diseased cells. Herein, the assembly of targeted poly(ethylene glycol) (PEG) particles functionalized with cyclic peptides containing Arg-Gly-Asp (RGD) (ligand) using a mesoporous silica templating method is reported. The influence of PEG molecular weight, ligand-to-PEG molecule ratio, and particle size on cancer cell targeting to balance stealth and targeting of the engineered PEG particles is investigated. RGD-functionalized PEG particles (PEG-RGD particles) efficiently target U-87 MG cancer cells under static and flow conditions in vitro, whereas PEG and cyclic peptides containing Arg-Asp-Gly (RDG)-functionalized PEG (PEG-RDG) particles display negligible interaction with the same cells. Increasing the ligand-to-PEG molecule ratio improves cell targeting. In addition, the targeted PEG-RGD particles improve cell uptake via receptor-mediated endocytosis, which is desirable for intracellular drug delivery. The PEG-RGD particles show improved tumor targeting (14% ID g-1) when compared with the PEG (3% ID g-1) and PEG-RDG (7% ID g-1) particles in vivo, although the PEG-RGD particles show comparatively higher spleen and liver accumulation. The targeted PEG particles represent a platform for developing particles aimed at balancing nonspecific and specific interactions in biological systems.

Functional Brush Poly(2-ethyl-2-oxazine)s: Synthesis by CROP and RAFT, Thermoresponsiveness and Grafting onto Iron Oxide Nanoparticles.

Brush polymers are highly functional polymeric materials combining the properties of different polymer classes and have found numerous applications, for example, in nanomedicine. Here, the synthesis of functional phosphonate-ester-bearing brush polymers based on poly(2-oxazine)s is reported through a combination of cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazine and reversible addition-fragmentation chain transfer (RAFT) polymerization. In this way, a small library of well-defined (D ≤ 1.17) poly(oligo(2-ethyl-2-oxazine) methacrylate) P(OEtOzMA)n brushes with tunable lower critical solution temperature (LCST) behavior and negligible cell toxicity is prepared. Upon deprotection, the phosphonic acid end-group of the P(OEtOzMA)n brush enables the successful grafting-onto iron oxide nanoparticles (IONPs). Colloidal stability of the particle suspension in combination with suitable magnetic resonance imaging (MRI) relaxivities demonstrates the potential of these particles for future applications as negative MRI contrast agents.

Self-Assembled Metal-Phenolic Networks on Emulsions as Low-Fouling and pH-Responsive Particles.

Interfacial self-assembly is a powerful organizational force for fabricating functional nanomaterials, including nanocarriers, for imaging and drug delivery. Herein, the interfacial self-assembly of pH-responsive metal-phenolic networks (MPNs) on the liquid-liquid interface of oil-in-water emulsions is reported. Oleic acid emulsions of 100-250 nm in diameter are generated by ultrasonication, to which poly(ethylene glycol) (PEG)-based polyphenolic ligands are assembled with simultaneous crosslinking by metal ions, thus forming an interfacial MPN. PEG provides a protective barrier on the emulsion phase and renders the emulsion low fouling. The MPN-coated emulsions have a similar size and dispersity, but an enhanced stability when compared with the uncoated emulsions, and exhibit a low cell association in vitro, a blood circulation half-life of ≈50 min in vivo, and are nontoxic to healthy mice. Furthermore, a model anticancer drug, doxorubicin, can be encapsulated within the emulsion phase at a high loading capacity (≈5 fg of doxorubicin per emulsion particle). The MPN coating imparts pH-responsiveness to the drug-loaded emulsions, leading to drug release at cell internalization pH and a potent cell cytotoxicity. The results highlight a straightforward strategy for the interfacial nanofabrication of pH-responsive emulsion-MPN systems with potential use in biomedical applications.

Psammomys obesus: a Natural Diet-Controlled Model for Diabetes and Cardiovascular Diseases.

PURPOSE OF REVIEW: This review specifically summarises and reports terrestrial mammals of the gerbil subfamily, known as Israeli sand rats or Psammomys obesus (P. obesus) as a diet-controlled, unique, polygenic rodent model for research in the areas of obesity, type 2 diabetes, and cardiovascular diseases. The animal model closely mimics phenotypic and pathophysiological resemblance with human populations. RECENT FINDINGS: The physiological status and biochemical composition in P. obesus can be manipulated effectively by controlling its nutritional intake, making it a natural model for cardiovascular and diabetic research. Humans exhibit remarkable disparity in physiology and pathology, which are inter-dependent factors. However, variations in these factors in most animal models currently being used for cardiovascular/diabetes research are insignificant. Consequently, it is a necessity to identify and develop animal models exhibiting physiological variations mimicking human pathological conditions. We have compiled research developments conducted with this rodent model manifesting pathophysiology, closely mimicking that in human beings, thereby enabling better translation of novel therapeutic and diagnostic discoveries.

Triggered and Tunable Hydrogen Sulfide Release from Photogenerated Thiobenzaldehydes.

Hydrogen sulfide (H2 S) has been identified as an important cell-signaling mediator and has a number of biological functions, such as vascular smooth muscle relaxation, neurotransmission, and regulation of inflammation. A facile and versatile approach for H2 S production initiated by light irradiation and controlled by reaction with an amine or an amino acid was developed. The donor was synthesized in a one-pot reaction, and simple crystallization led to a yield of approximately 90 %. The synthetic strategy is scalable and versatile, and the H2 S donors can be expressed ina number of different molecular and macromolecular forms, including crystalline small-molecule compounds, water-soluble polymers, polystyrene films, and hydrogels. The H2 S donors based on polystyrene film and hydrogel were used as cell-culture scaffolds. The H2 S donor based on water-soluble polymer was applied in photocontrolled inhibition of P-selectin expression on human platelets and subsequent regulation of platelet aggregation. This study provides the simplest controllable H2 S source to study its biological functions. The developed materials are also new therapeutic platforms to deliver H2 S, as there is no accumulation of toxic byproducts, and the donor materials from polystyrene films and hydrogels can be readily removed after releasing H2 S.

Towards effective and safe thrombolysis and thromboprophylaxis: preclinical testing of a novel antibody-targeted recombinant plasminogen activator directed against activated platelets.

RATIONALE: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. OBJECTIVE: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. METHODS AND RESULTS: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet-integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5-scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5-scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5-scuPA as compared with the same dose of a mutated, nontargeting scFv-scuPA or vehicle. The ability of scFvSCE5-scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. CONCLUSIONS: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.

Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?

The ecto-nucleoside triphosphate diphosphohydrolase CD39 represents a promising antithrombotic therapeutic. It degrades adenosine 5'-diphosphate (ADP), a main platelet activating/recruiting agent. We hypothesized that delayed enrichment of CD39 on developing thrombi will allow for a low and safe systemic concentration and thus avoid bleeding. We use a single-chain antibody (scFv, specific for activated GPIIb/IIIa) for targeting CD39. This should allow delayed enrichment on growing thrombi but not on the initial sealing layer of platelets, which do not yet express activated GPIIb/IIIa. CD39 was recombinantly fused to an activated GPIIb/IIIa-specific scFv (targ-CD39) and a nonfunctional scFv (non-targ-CD39). Targ-CD39 was more effective at preventing ADP-induced platelet activation than non-targ-CD39. In a mouse carotid artery thrombosis model, non-targ-CD39, although protective against vessel occlusion, was associated with significant bleeding on tail transection. In contrast, targ-CD39 concentrated at the thrombus site; hence, a dose ∼10 times less of CD39 prevented vessel occlusion to a similar extent as high-dose non-targ-CD39, without prolonged bleeding time. An equimolar dose of non-targ-CD39 at this low concentration was ineffective at preventing vessel occlusion. Thus, delayed targeting of CD39 via scFv to activated platelets provides strong antithrombotic potency and yet prevents bleeding and thereby promotes CD39 toward clinical use.

Targeted antithrombotic protein micelles.

Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site-specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one-pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single-chain antibody (scFv), which binds to the ligand-induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.

A versatile approach for the site-specific modification of recombinant antibodies using a combination of enzyme-mediated bioconjugation and click chemistry.

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

Single-chain antibody conjugated to a cage amine chelator and labeled with positron-emitting copper-64 for diagnostic imaging of activated platelets.

Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.