An Important Role for P-Selectin in Thrombotic Microangiopathy

Background: Endothelial activation is a key initiating event in thrombotic microangiopathies (TMA), which include thrombotic thrombocytopenic purpura (TTP), a life-threatening disorder in which a deficiency in the von Willebrand factor (VWF)–cleaving protease ADAMTS13 leads to systemic microvascular thrombosis. Endothelial activation by a variety of stimuli leads to the externalization of P-selectin and VWF from Weibel-Palade bodies to the endothelial surface. Surface-bound VWF binds platelets and promotes thrombosis. The thrombogenicity of VWF is enhanced by its shear-induced self-association into fibers and strings, which capture platelets more efficiently. Previously, we reported that endothelium-bound VWF strings are anchored to the endothelial surface by P-selectin (PMID: 14630802). Later studies refuted this finding, suggesting that P-selectin is not required for the formation of VWF strings and thrombi on the endothelium (PMID: 17166247). Thus, the role of P-selectin in VWF-mediated microvascular thrombosis remains controversial, and the therapeutic potential of P-selectin blockade in TMA has not been investigated.

Aims: To study the role of endothelial P-selectin in VWF-mediated microvascular thrombosis initiated by endothelial activation and assess the role of P-selectin blockade in the treatment of TMA.

Methods: P-selectin was inhibited in ADAMTS13–KO mice with rat anti-mouse P-selectin antibodies. The results were compared those from ADAMTS13 and P-selectin double KO mice. Platelets and leukocytes were labeled with fluorescent antibodies. Endothelium was activated systemically by IV injection of a protease-activated receptor–1 agonist peptide TRAP-6, which does not activate platelets. Microvascular thrombosis was monitored by intravital imaging of the cremaster microcirculation.

Results: At baseline, the cremaster microcirculation of ADAMTS13-KO mice showed sparse platelet adhesion to the vessel wall and more rolling leukocytes in venules compared to WT mice. Endothelial activation by TRAP-6 led to substantial platelet adhesion, formation of platelet-VWF strings, and a dramatic increase in rolling and firmly adhered leukocytes on venular endothelium. Adherent platelets and leukocytes increased continuously, forming larger aggregates that eventually occluded the vessels after 15 min. In contrast, platelet and leukocyte adhesion in TRAP-6–challenged WT mice was transient and no thrombi formed. In ADAMTS13-KO mice, P-selectin blockade instantly reduced the number of rolling and adherent leukocytes. When challenged with TRAP-6, P-selectin–inhibited mice displayed markedly fewer adherent platelets, leukocytes, platelet aggregates, and leukocyte aggregates. Platelet aggregates on the vessel wall failed to enlarge and frequently embolized downstream. Vessel occlusion was completely prevented. In mice doubly deficient in ADAMTS13 and P-selectin, leukocyte adhesion was absent before or after TRAP-6 treatment. A limited number of platelets adhered to the vessel wall after treatment, and microvascular thrombosis and vessel occlusion were effectively prevented.

Conclusion(s): We have established and characterized a new intravital microscopy model of selective endothelial activation. Using this model, we showed that P-selectin plays an important role in supporting microvascular thrombosis in congenital TTP. Congenital deficiency of P-selectin or its pharmacological blockade reduced the adhesion of both platelets and leukocytes to the endothelium and prevented thrombosis after endothelial activation. Decreased platelet adhesion appeared to be associated with a large reduction in endothelial-bound VWF, suggesting that P-selectin not only provides a receptor for leukocyte adhesion and rolling, it also helps to anchor VWF to the endothelial surface, where it can self-associate into a highly adhesive surface for platelets. The precise mechanism of its involvement in TMA requires further study. Nevertheless, these data suggest that blocking P-selectin may be another strategy for treating TMA.