Paul C. J. Armstrong

Novel Single-Chain Antibody-Targeted Microbubbles for Molecular Ultrasound Imaging of Thrombosis Validation of a Unique Noninvasive Method for Rapid and Sensitive Detection of Thrombi and Monitoring of Success or Failure of Thrombolysis in Mice

Background— Molecular imaging is a fast emerging technology allowing noninvasive detection of vascular pathologies. However, imaging modalities offering high resolution currently do not allow real-time imaging. We hypothesized that contrast-enhanced ultrasound with microbubbles (MBs) selectively targeted to activated platelets would offer high-resolution, real-time molecular imaging of evolving and dissolving arterial thrombi. Methods and Results— Lipid-shell based gas-filled MBs were conjugated to either a single-chain antibody specific for activated glycoprotein IIb/IIIa via binding to a Ligand-Induced Binding Site (LIBS-MBs) or a nonspecific single-chain antibody (control MBs). Successful conjugation was assessed in flow cytometry and immunofluorescence double staining. LIBS-MBs but not control MBs strongly adhered to both immobilized activated platelets and microthrombi under flow. Thrombi induced in carotid arteries of C57Bl6 mice in vivo by ferric chloride injury were then assessed with ultrasound before and 20 minutes after MB injection through the use of gray-scale area intensity measurement. Gray-scale units converted to decibels demonstrated a significant increase after LIBS-MB but not after control MB injection (9.55±1.7 versus 1.46±1.3 dB; P <0.01). Furthermore, after thrombolysis with urokinase, LIBS-MB ultrasound imaging allows monitoring of the reduction of thrombus size ( P <0.001). Conclusion— We demonstrate that glycoprotein IIb/IIIa–targeted MBs specifically bind to activated platelets in vitro and allow real-time molecular imaging of acute arterial thrombosis and monitoring of the success or failure of pharmacological thrombolysis in vivo. # Clinical Perspective {#article-title-50}Background— Molecular imaging is a fast emerging technology allowing noninvasive detection of vascular pathologies. However, imaging modalities offering high resolution currently do not allow real-time imaging. We hypothesized that contrast-enhanced ultrasound with microbubbles (MBs) selectively targeted to activated platelets would offer high-resolution, real-time molecular imaging of evolving and dissolving arterial thrombi. Methods and Results— Lipid-shell based gas-filled MBs were conjugated to either a single-chain antibody specific for activated glycoprotein IIb/IIIa via binding to a Ligand-Induced Binding Site (LIBS-MBs) or a nonspecific single-chain antibody (control MBs). Successful conjugation was assessed in flow cytometry and immunofluorescence double staining. LIBS-MBs but not control MBs strongly adhered to both immobilized activated platelets and microthrombi under flow. Thrombi induced in carotid arteries of C57Bl6 mice in vivo by ferric chloride injury were then assessed with ultrasound before and 20 minutes after MB injection through the use of gray-scale area intensity measurement. Gray-scale units converted to decibels demonstrated a significant increase after LIBS-MB but not after control MB injection (9.55±1.7 versus 1.46±1.3 dB; P<0.01). Furthermore, after thrombolysis with urokinase, LIBS-MB ultrasound imaging allows monitoring of the reduction of thrombus size (P<0.001). Conclusion— We demonstrate that glycoprotein IIb/IIIa–targeted MBs specifically bind to activated platelets in vitro and allow real-time molecular imaging of acute arterial thrombosis and monitoring of the success or failure of pharmacological thrombolysis in vivo.

Antiplatelet Actions of Statins and Fibrates Are Mediated by PPARs

Objectives—Statins and fibrates are hypolipidemic drugs which decrease cardiac events in individuals without raised levels of cholesterol. These drugs inhibit platelet function, but the mechanisms by which this pleiotropic effect is exerted are not known. Methods and Results—We used a range of approaches to show statins inhibit human platelet activation in vitro while engaging PPAR&agr; and PPAR&ggr;. The effects of simvastatin were prevented by the PPAR&ggr; antagonist GW9662 or the PPAR&agr; antagonist GW6471. In a small-scale human study fluvastatin activated PPAR&agr; and PPAR&ggr; in platelets and reduced aggregation in response to arachidonic acid ex vivo. The effects of fenofibrate were prevented by PPAR&agr; antagonism with GW6471. Fenofibrate increased bleeding time in wild-type, but not in PPAR&agr;−/− mice. The inhibitory effect of fenofibrate, but not simvastatin, on aggregation was prevented by deletion of PPAR&agr; in murine platelets. PKC&agr;, which influences platelet activation, associated and immune-precipitated with PPAR&ggr; in platelets stimulated with statins and with PPAR&agr; in platelets stimulated with fenofibrate. Conclusions—This study is the first to provide a unifying explanation of how fibrates and statins reduce thrombotic and cardiovascular risk. Our findings that PPARs associate with PKC&agr; in platelets also provide a mechanism by which these effects are mediated.

IN THE PRESENCE OF STRONG P2Y12 RECEPTOR BLOCKADE, ASPIRIN PROVIDES LITTLE ADDITIONAL INHIBITION OF PLATELET AGGREGATION

Summary.  Background: Aspirin and antagonists of platelet ADP P2Y12 receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y12 receptors can inhibit thromboxane A2 (TXA2)‐dependent pathways of platelet activation independently of aspirin. Objectives: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y12 receptor blockade. Methods: With the use of platelet‐rich plasma from healthy volunteers, determinations were made in 96‐well plates of platelet aggregation, TXA2 production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP‐6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1–10 μmol L−1), aspirin (30 μmol L−1), PAM + aspirin or vehicle. Results: PAM concentration‐dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 μmol L−1. In further tests of PAM (3 μmol L−1), aspirin (30 μmol L−1) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA2 and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. Conclusions: P2Y12 receptors are critical to the generation of irreversible aggregation through the TXA2‐dependent pathway. As a result, strong P2Y12 receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.

GPIIb/IIIa inhibitors: From bench to bedside and back to bench again

From the discovery of the platelet glycoprotein (GP) IIb/IIIa and identification of its central role in haemostasis, the integrin GPIIb/IIIa (αIIbβ3, CD41/CD61) was destined to be an anti-thrombotic target. The subsequent successful development of intravenous ligand-mimetic inhibitors occurred during a time of limited understanding of integrin physiology. Although efficient inhibitors of ligand binding, they also mimic ligand function. In the case of GPIIb/IIIa inhibitors, despite strongly inhibiting platelet aggregation, paradoxical fibrinogen binding and platelet activation can occur. The quick progression to development of small-molecule orally available inhibitors meant that this approach inherited many potential flaws, which together with a short half-life resulted in an increase in mortality and a halt to the numerous pharmaceutical development programs. Limited clinical benefits, together with the success of other anti-thrombotic drugs, in particular P2Y12 ADP receptor blockers, have also led to a restrictive use of intravenous GPIIb/IIIa inhibitors. However, with a greater understanding of this key platelet-specific integrin, GPIIb/IIIa remains a potentially attractive target and future drug developments will be better informed by the lessons learnt from taking the current inhibitors back to the bench. This overview will review the physiology behind the inherent problems of a ligand-based integrin inhibitor design and discuss novel promising approaches for GPIIb/IIIa inhibition.

Reduction of platelet thromboxane A2 production ex vivo and in vivo by clopidogrel therapy

1 Hulot JS, Bura A, Villard E, Azizi M, Remones V, Goyenvalle C, Aiach M, Lechat P, Gaussem P. Cytochrome P450 2C19 loss-offunction polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects. Blood 2006; 108: 2244–7. 2 Shuldiner AR, O Connell JR, Bliden KP, Gandhi A, Ryan K, Horenstein RB, Damcott CM, Pakyz R, Tantry US, Gibson Q, Pollin TI, Post W, Parsa A,Mitchell BD, Faraday N, HerzogW, Gurbel PA. Association of cytochrome P450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy. JAMA 2009; 302: 849–57. 3 Giusti B, Gori AM, Marcucci R, Saracini C, Sestini I, Paniccia R, Valente S, Antoniucci D, Abbate R, Gensini GF. Cytochrome P450 2C19 loss-of-function polymorphism, but not CYP3A4 IVS10 + 12 G/A and P2Y12 T744C polymorphisms, is associated with response variability to dual antiplatelet treatment in high-risk vascular patients. Pharmacogenet Genomics 2007; 17: 1057–64. 4 Collet JP, Hulot JS, Pena A, Villard E, Esteve JB, Silvain J, Payot L, Brugier D, Cayla G, Beygui F, Bensimon G, Funck-Brentano C, Montalescot G. Cytochrome P450 2C19 polymorphism in young patients treated with clopidogrel after myocardial infarction: a cohort study. Lancet 2009; 373: 309–17. 5 Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, Brandt JT, Walker JR, Antman EM, Macias W, Braunwald E, Sabatine MS. Cytochrome p-450 polymorphisms and response to clopidogrel.NEngl J Med 2009; 360: 354–62. 6 Giusti B, Gori AM, Marcucci R, Saracini C, Sestini I, Paniccia R, Buonamici P, Antoniucci D, Abbate R, Gensini GF. Relation of cytochrome P450 2C19 loss-of-function polymorphism to occurrence of drug-eluting coronary stent thrombosis. Am J Cardiol 2009; 103: 806–11. 7 Simon T, Verstuyft C, Mary-Krause M, Quteineh L, Drouet E, Meneveau N, Steg PG, Ferrieres J, Danchin N, Becquemont L. Genetic determinants of response to clopidogrel and cardiovascular events. N Engl J Med 2009; 360: 363–75. 8 Gilard M, Arnaud B, Cornily JC, Le Gal G, Lacut K, Le Calvez G, Mansourati J, Mottier D, Abgrall JF, Boschat J. Influence of omeprazole on the antiplatelet action of clopidogrel associated with aspirin: the randomized, double-blind OCLA (Omeprazole CLopidogrel Aspirin) study. J Am Coll Cardiol 2008; 51: 256– 60. 9 O DonoghueML, Braunwald E, Antman EM,Murphy SA, Bates ER, Rozenman Y, Michelson AD, Hautvast RW, VerLee PN, Close SL, Shen L, Mega JL, Sabatine MS, Wiviott SD. Pharmacodynamic effect and clinical efficacy of clopidogrel and prasugrel with or without a proton-pump inhibitor: an analysis of two randomised trials. Lancet 2009; 374: 989–97. 10 PenaA, Collet JP, Hulot JS, Silvain J, BarthelemyO, Beygui F, FunckBrentano C, Montalescot G. Can we override clopidogrel resistance? Circulation 2009; 119: 2854–7.

Aspirin and the in vitro linear relationship between thromboxane A2-mediated platelet aggregation and platelet production of thromboxane A2

Summary.  Background: Currently, ‘aspirin resistance’, the anti‐platelet effects of non‐steroid anti‐inflammatory drugs (NSAIDs) and NSAID‐aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A2 formation is non‐linear and TXA2 generation must be inhibited by at least 95% to inhibit TXA2‐dependent aggregation. This relationship, however, has never been rigorously tested. Objectives: To characterize, in vitro and ex vivo, the concentration‐dependent relationships between TXA2 generation and platelet activity. Method: Platelet aggregation, thrombi adhesion and TXA2 production in response to arachidonic acid (0.03–1 mmol L−1), collagen (0.1–30 μg mL−1), epinephrine (0.001–100 μmol L−1), ADP, TRAP‐6 amide and U46619 (all 0.1‐30 μmol L−1), in the presence of aspirin or vehicle, were determined in 96‐well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. Results: Platelet aggregation, adhesion and TXA2 production induced by either arachidonic acid or collagen were inhibited in concentration‐dependent manners by aspirin, with logIC50 values that did not differ. A linear relationship existed between aggregation and TXA2 production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R2 0.92; n = 224). The same relationships were seen in combinations of aspirin‐treated and naïve platelets, and in blood from individuals taking an anti‐thrombotic dose of aspirin. Conculsions: These studies demonstrate a linear relationship between inhibition of platelet TXA2 generation and TXA2‐mediated aggregation. This finding is important for our understanding of the anti‐platelet effects of aspirin and NSAIDs, NSAID–aspirin interactions and ‘aspirin resistance’.

Association of MicroRNAs and YRNAs With Platelet Function

RATIONALE Platelets shed microRNAs (miRNAs). Plasma miRNAs change on platelet inhibition. It is unclear whether plasma miRNA levels correlate with platelet function. OBJECTIVE To link small RNAs to platelet reactivity. METHODS AND RESULTS Next-generation sequencing of small RNAs in plasma revealed 2 peaks at 22 to 23 and 32 to 33 nucleotides corresponding to miRNAs and YRNAs, respectively. Among YRNAs, predominantly, fragments of RNY4 and RNY5 were detected. Plasma miRNAs and YRNAs were measured in 125 patients with a history of acute coronary syndrome who had undergone detailed assessment of platelet function 30 days after the acute event. Using quantitative real-time polymerase chain reactions, 92 miRNAs were assessed in patients with acute coronary syndrome on different antiplatelet therapies. Key platelet-related miRNAs and YRNAs were correlated with platelet function tests. MiR-223 (rp=0.28; n=121; P=0.002), miR-126 (rp=0.22; n=121; P=0.016), and other abundant platelet miRNAs and YRNAs showed significant positive correlations with the vasodilator-stimulated phosphoprotein phosphorylation assay. YRNAs, miR-126, and miR-223 were also among the small RNAs showing the greatest dependency on platelets and strongly correlated with plasma levels of P-selectin, platelet factor 4, and platelet basic protein in the population-based Bruneck study (n=669). A single-nucleotide polymorphism that facilitates processing of pri-miR-126 to mature miR-126 accounted for a rise in circulating platelet activation markers. Inhibition of miR-126 in mice reduced platelet aggregation. MiR-126 directly and indirectly affects ADAM9 and P2Y12 receptor expression. CONCLUSIONS Levels of platelet-related plasma miRNAs and YRNAs correlate with platelet function tests in patients with acute coronary syndrome and platelet activation markers in the general population. Alterations in miR-126 affect platelet reactivity.

Evidence That Links Loss of Cyclooxygenase-2 With Increased Asymmetric Dimethylarginine Novel Explanation of Cardiovascular Side Effects Associated With Anti-Inflammatory Drugs

Background— Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. Methods and Results— Transcriptome analysis of wild-type and cyclooxygenase-2−/− mouse tissues revealed 1 gene altered in the heart and aorta, but >1000 genes altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-L-arginine. Cyclo-oxygenase-2−/− mice had increased plasma levels of ADMA and monomethyl-L-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide is cardio-protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage. Conclusions— We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction.

Dual antiplatelet therapy in cardiovascular disease: does aspirin increase clinical risk in the presence of potent P2Y12 receptor antagonists?

Aspirin is now widely accepted as the first-line antithrombotic platelet therapy for at-risk individuals. During the last decade or so it has also become established that co-administering antagonists of the ADP receptor P2Y12 with aspirin further reduces the risk of acute thrombotic events. By the nature of its evolution, this therapeutic approach assumes that P2Y12 receptor antagonists will be added to aspirin, and this therefore dominates the design of clinical trials. This strategy has resulted in the generation of a large body of clinical evidence showing the benefit of aspirin plus P2Y12 receptor antagonists, largely from studies with clopidogrel and more recently from those with prasugrel and ticagrelor, but with obvious limitations in terms of residual ischaemic event rates and bleeding complications. It is our hypothesis, however, that when administered in the presence of potent P2Y12 receptor antagonists, aspirin could actually increase total cardiovascular risk, although this has never been tested in large outcome studies. Clearly, this potentially negative interaction could be of relevance to millions of patients.

GPVI and GPIbα Mediate Staphylococcal Superantigen-Like Protein 5 (SSL5) Induced Platelet Activation and Direct toward Glycans as Potential Inhibitors

Background Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects. Methodology/Principal Findings In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro. Conclusions/Significance These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo.