An ultrasound contrast agent targeted to P-selectin detects activated platelets at supra-arterial shear flow conditions.
OBJECTIVE
To evaluate targeting of a microbubble contrast agent to platelets under high shear flow using the natural selectin ligand sialyl Lewis.


METHODS
Biotinylated polyacrylamide Sialyl Lewis or biotinylated carbohydrate-free polymer (used as a control) were attached to biotinylated microbubbles via a streptavidin linker. Activated human platelets were isolated and attached to fibrinogen-coated culture dishes. Fibrinogen-coated dishes without platelets or platelet dishes blocked by an anti-P-selectin antibody served as negative control substrates. Dishes coated by recombinant P-selectin served as a positive control substrate. Microbubble adhesion was assessed by microscopy in an inverted parallel plate flow chamber, with wall shear stress values of 40, 30, 20, 10, and 5 dynes/cm2. The ratio of binding and passing microbubbles was defined as capture efficiency.


RESULTS
There was no significant difference between the groups regarding the number of microbubbles in the fluid flow at each shear rate. Sialyl Lewis-targeted microbubbles were binding and slowly rolling on the surface of activated platelets and P-selectin-coated dishes at all the flow conditions including 40 dynes/cm2. Capture efficiency of targeted microbubbles to activated platelets and recombinant P-selectin decreased with increasing shear flow: at 5 dynes/cm2, capture efficiency was 16.11% on activated platelets versus 21.83% on P-selectin, and, at 40 dynes/cm2, adhesion efficiency was still 3.4% in both groups. There was neither significant adhesion of Sialyl Lewis-targeted microbubbles to control substrates, nor adhesion of control microbubbles to activated platelets or to recombinant P-selectin.


CONCLUSIONS
Microbubble targeting using sialyl Lewis, a fast-binding ligand to P-selectin, is a promising strategy for the design of ultrasound contrast binding to activated platelets under high shear stress conditions.
Guenther
Felix
F
Department of Cardiology and Angiology, University Hospital of Freiburg, Freiburg, Germany. felix.guenther@uniklinik-freiburg.de
von zur Muhlen
Constantin
C
Ferrante
Elisa A
EA
Grundmann
Sebastian
S
Bode
Christoph
C
Klibanov
Alexander L
AL
D001158:Arteries / Q000000981:diagnostic imaging*; D001783:Blood Flow Velocity; D001792:Blood Platelets / Q000000981:diagnostic imaging* / Q000201:enzymology; D002448:Cell Adhesion; D003287:Contrast Media; D005340:Fibrinogen; D006801:Humans; D008024:Ligands; D045423:Microbubbles; D019007:P-Selectin; D015539:Platelet Activation / Q000276:immunology*; D011994:Recombinant Proteins; D033081:Shear Strength; D014463:Ultrasonography
D003287:Contrast Media; D008024:Ligands; D019007:P-Selectin; D011994:Recombinant Proteins; D005340:Fibrinogen
Stroke. 2007 Oct;38(10):2726-32
17823379
J Control Release. 2009 Dec 3;140(2):100-7
19666063
Blood. 2002 Dec 15;100(13):4485-94
12393554
J Am Coll Cardiol. 2006 Apr 18;47(8 Suppl):C48-56
16631510
Circulation. 1999 Jan 5;99(1):E1-E11
9884400
J Clin Invest. 1993 Mar;91(3):1157-66
7680663
Prog Cardiovasc Dis. 1975 Jul-Aug;18(1):89-103
1098111
Circulation. 2001 Sep 11;104(11):1280-5
11551880
Circ J. 2008 Jul;72(7):1092-9
18577817
Invest Radiol. 2008 Jan;43(1):49-55
18097277
Invest Radiol. 2004 Oct;39(10):619-25
15377941
Chemphyschem. 2004 Feb 20;5(2):175-82
15038277
Invest Radiol. 2002 Nov;37(11):587-93
12393970
Mol Pharm. 2006 Sep-Oct;3(5):516-24
17009850
Ultrasound Med Biol. 1999 Oct;25(8):1195-201
10576262
Arterioscler Thromb Vasc Biol. 2010 Feb;30(2):169-76
19815819
Biochem Biophys Res Commun. 1992 Oct 15;188(1):244-51
1384480
J Nucl Cardiol. 2007 Nov-Dec;14(6):876-84
18022115
Circulation. 2008 Jul 15;118(3):258-67
18574047
J Control Release. 2006 Sep 12;114(3):288-99
16887229
Biotechnol Bioeng. 2005 Dec 20;92(6):780-8
16121392
J Am Coll Cardiol. 2008 Jul 1;52(1):1-12
18582628
Eur Heart J. 2009 Nov;30(21):2566-74
19833636
J Exp Med. 2002 Oct 7;196(7):887-96
12370251
Invest Radiol. 2009 Nov;44(11):697-704
19809344
Semin Thromb Hemost. 2007 Mar;33(2):165-72
17340465
Am J Med. 2002 Dec 1;113(8):668-80
12505118
Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7390-7
7519775
Invest Radiol. 2006 Mar;41(3):354-62
16481920
J Biol Chem. 1996 Feb 23;271(8):4289-97
8626776
Arterioscler Thromb Vasc Biol. 2009 Jul;29(7):1031-8
19461053
Nature. 2008 Feb 21;451(7181):953-7
18288186
Circulation. 1981 Aug;64(2 Pt 2):II221-7
7249326
J Clin Invest. 2005 Dec;115(12):3378-84
16322783
J Med Chem. 2009 Aug 13;52(15):4725-42
19580288
Arterioscler Thromb Vasc Biol. 1995 Sep;15(9):1512-31
7670967
Radiology. 2007 Aug;244(2):337-55
17592037
Anal Biochem. 1996 Jul 1;238(2):184-90
8660609
Mol Imaging. 2008 Mar-Apr;7(2):59-67
18706288
Cardiovasc Res. 1996 Oct;32(4):733-42
8915191
J Control Release. 2004 May 18;96(3):473-82
15120903
MAGMA. 1999 Aug;8(3):177-84
10504045
Cancer Res. 1989 Feb 1;49(3):745-52
2910493
Contrast Media Mol Imaging. 2006 Nov-Dec;1(6):259-66
17191766
Acta Biochim Pol. 2002;49(2):303-11
12362971
Biophys J. 2000 Nov;79(5):2391-402
11053118
Stroke. 2007 May;38(5):1508-14
17379828
J Magn Reson Imaging. 2007 Sep;26(3):460-79
17729343
Ann N Y Acad Sci. 1994 Apr 18;714:185-9
7517114
Cardiovasc Res. 2008 Apr 1;78(1):148-57
18174291
Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):961-6
17215360