Diminished thrombus formation and alleviation of myocardial infarction and reperfusion injury through antibody- or small-molecule-mediated inhibition of selectin-dependent platelet functions.
OBJECTIVE
P-selectin ctin has been implicated in important platelet functions. However, neither its role in thrombus formation and cardiovascular disorders nor its suitability as a therapeutic target structure is entirely clear.


METHODS
Platelet aggregation was assessed in complementary in vitro settings by measurements of static aggregation, standardized aggregometry and dynamic flow chamber assays. Degradation of aggregates was also analyzed under flow conditions using video microscopy. In vivo, platelet rolling in cutaneous venules was assessed by intravital microscopy in wild-type mice treated with selectin-blocking compounds as well as in P-selectin-deficient mice. FeCl3-induced arterial thrombosis was studied by intravital microscopy in untreated mice or mice treated with an inhibitor of selectin functions. Finally, inhibition of selectin functions was studied in an ischemia/reperfusion injury model in rats.


RESULTS
Antibody- or small-molecule-mediated inhibition of P-selectin functions significantly diminished platelet aggregation (p<0.03) and platelet-neutrophil adhesion in vitro (p<0.01) as well as platelet aggregate sizes under flow (p<0.03). Established aggregates were degraded, either via detachment of single platelets following addition of efomycine M, or via detachment of multicellular clumps when P-selectin-directed Fab-fragments were used. In vivo, selectin inhibition resulted in a greater than 50% reduction of platelet rolling in cutaneous venules (p<0.01), producing rolling fractions similar to those observed in P-selectin-deficient mice (p<0.05). Moreover, inhibition of selectin functions significantly decreased the thrombus size in FeCl3-induced arterial thrombosis in mice (p<0.05). In an ischemia/reperfusion injury model in rats, small-molecule-mediated selectin inhibition significantly reduced myocardial infarct size from 18.9% to 9.42% (p<0.001) and reperfusion injury (p<0.001).


CONCLUSIONS
Inhibition of P-selectin functions reduces platelet aggregation and can alleviate platelet-related disorders in disease-relevant preclinical settings.
Oostingh
Gertie J
GJ
University of Würzburg, Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, Würzburg Germany.
Pozgajova
Miroslava
M
Ludwig
Ralf J
RJ
Krahn
Thomas
T
Boehncke
W-Henning
WH
Nieswandt
Bernhard
B
Schön
Michael P
MP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology / Q000627:therapeutic use*; D001157:Arterial Occlusive Diseases / Q000097:blood / Q000139:chemically induced / Q000517:prevention & control*; D002712:Chlorides; D004353:Drug Evaluation, Preclinical; D042783:Endothelial Cells / Q000166:cytology; D005290:Ferric Compounds / Q000633:toxicity; D005343:Fibrinolytic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D018056:Hemorheology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology / Q000627:therapeutic use*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008970:Molecular Weight; D009203:Myocardial Infarction / Q000097:blood* / Q000503:physiopathology; D015428:Myocardial Reperfusion Injury / Q000517:prevention & control*; D009504:Neutrophils / Q000166:cytology; D009844:Oligosaccharides / Q000494:pharmacology; D019007:P-Selectin / Q000276:immunology / Q000502:physiology*; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects* / Q000502:physiology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D051381:Rats; D011917:Rats, Inbred Lew; D000080506:Sialyl Lewis X Antigen; D013927:Thrombosis / Q000097:blood / Q000139:chemically induced / Q000517:prevention & control*
D000911:Antibodies, Monoclonal; D002712:Chlorides; D005290:Ferric Compounds; D005343:Fibrinolytic Agents; D007140:Immunoglobulin Fab Fragments; D018942:Macrolides; D009844:Oligosaccharides; D019007:P-Selectin; D010975:Platelet Aggregation Inhibitors; D000080506:Sialyl Lewis X Antigen; C453219:efomycine M; C024555:ferric chloride