The following are the supplementary

The following are the supplementary data related to this article.
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Introduction Thrombosis (arterial and venous) is a major cause of vascular obstructive diseases including myocardial infarction, cerebral infarction and pulmonary embolism. Glycoprotein (GP) IIb/IIIa complex, also known as αIIbβ3 integrin, is a receptor expressed on the membrane surface of activated platelets and is an important factor in mediating the platelet aggregation (Phillips et al., 1988, Topol et al., 1999). Cyclic arginine–glycine–aspartate (RGD) peptides, which have the adhesive property for GP IIb/IIIa and for αvβ3 integrin, have been utilized as a tracer for thrombus and for tumor angiogenesis in nuclear imaging (Cai and Conti, 2013, Zhou et al., 2011). Ultrasound molecular imaging using antibody- or peptide-bearing microbubbles (MBs) is a useful diagnostic tool for the non-invasive visualization of molecular dynamics in situ. In ultrasound molecular imaging, MBs whose surfaces have been modified with cyclic RGD have been utilized as a targeted contrast agent for tumor angiogenesis (Anderson et al. 2011) and for arterial thrombus (Hu et al., 2012, Wu et al., 2013). Although many kinds of targeted MBs have been developed, there are no clinically translatable MBs for thrombus imaging at present. Sonazoid is a clinically available perfluorobutane gas MB stabilized by a membrane of hydrogenated egg phosphatidylserine (PS) (Sontum 2008). We previously reported that lactadherin (milk fat globule epidermal growth factor 8 [MFG-E8]), an endogenous secreted glycoprotein bridging the integrin-expressing phagocytes and PS-presenting apoptotic nae inhibitor (Hanayama et al. 2002), is a useful molecule yielding the RGD motif on the surface of Sonazoid MBs via PS (Otani and Yamahara 2013). Recently, we reported that surface modification with lactadherin enhances the targeting of Sonazoid MBs to αvβ3 integrin-expressing neovasculature (Otani et al. 2018). However, the applicability of lactadherin-bearing Sonazoid MBs to thrombus imaging is still unknown. Recently, Hagisawa et al. (2011) reported that the surface modification with linear RGD peptide enhances the attachment of liposomal bubbles to erythrocyte-rich clots (Hagisawa et al. 2011). Then, we hypothesized that RGD-presenting, lactadherin-bearing Sonazoid MBs might have the potential to be targeted MBs for GP IIb/IIIa-expressing platelets and erythrocyte-rich clots. In the present study, we examined the targeting of Sonazoid MBs to the recombinant αIIbβ3 integrin and activated platelets using the custom-designed binding assay system. We further examined whether surface modification with lactadherin enhances the attachment of Sonazoid MBs to erythrocyte-rich human clots.
Methods
Results
Discussion Many kinds of platelet-targeted MBs have been developed by modifying the MB surface with a liner hexapeptide (Lys–Gln–Ala–Gly–Asp–Val) (Unger et al. 1998), an RGD analogue (Takeuchi et al. 1999), an antibody fragment specific for GP IIb/IIIa (Alonso et al. 2007), cyclic RGD peptide (Hu et al., 2012, Wu et al., 2013) and single-chain antibodies for GP IIb/IIIa (Wang et al., 2012, Wang et al., 2016). In the present study, we found that surface modification with lactadherin augmented the targeting of Sonazoid MBs to recombinant GP IIb/IIIa, activated platelets and erythrocyte-rich clots. Although further studies examining the targetability of lactadherin-bearing Sonazoid MBs in animal models with thrombus are required, our data indicate that lactadherin-bearing Sonazoid MBs have the potential to be thrombus-targeted MBs. Intravenous injection of MBs has been known to enhance the efficiency of ultrasound-mediated sonothrombolysis (Culp et al., 2011, Lu et al., 2016, Xie et al., 2009a). In addition, sonothrombolysis combined with MBs also has the potential to improve the efficacy of thrombolytic agents (Brown et al., 2011, Wu et al., 2015). These favorable effects of administration of MBs in sonothrombolysis are partly attributable to the ultrasound-mediated oscillation of MBs in adjunction to the surface of the thrombus (Chen et al. 2014). As illustrated in Figures 7a and 8b, lactadherin-bearing Sonazoid MBs attached to the erythrocyte-rich clot were located on the surface of the clot and were easily destroyed by the high-power ultrasound exposure. In this regard, the lactadherin-bearing Sonazoid MBs would have the potential to serve not only as thrombus-targeted MBs but also as enhancers of sonothrombolysis.