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.


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).


CONCLUSIONS
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.
Atherothrombosis and Vascular Biology, Baker IDI Heart and Diabetes Institute, PO Box 6492, St. Kilda Rd Central, Victoria 8008, Australia.
Xiaowei
X
Wang
Christoph E
CE
Hagemeyer
Jan David
JD
Hohmann
Ephraem
E
Leitner
Paul C
PC
Armstrong
Fu
F
Jia
Manfred
M
Olschewski
Andrew
A
Needles
Karlheinz
K
Peter
Ingo
I
Ahrens
D000818:Animals; D001666:Binding Sites, Antibody / Q000276:immunology; D004195:Disease Models, Animal; D016503:Drug Delivery Systems / Q000379:methods*; D039661:Integrin beta3 / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D045423:Microbubbles; D057054:Molecular Imaging / Q000379:methods / Q000639:trends; D015539:Platelet Activation / Q000276:immunology; D040201:Platelet Membrane Glycoprotein IIb / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000378:metabolism; D015912:Thrombolytic Therapy / Q000379:methods / Q000639:trends; D013927:Thrombosis / Q000175:diagnosis* / Q000000981:diagnostic imaging* / Q000378:metabolism; D013997:Time Factors; D017211:Treatment Failure; D016896:Treatment Outcome; D014463:Ultrasonography
D039661:Integrin beta3; D040201:Platelet Membrane Glycoprotein IIb; D057127:Single-Chain Antibodies