P-selectin and leukocyte microparticles are associated with venous thrombogenesis

Objectives: P-selectin inhibition has been found to limit venous thrombosis. We hypothesize that elevated levels of P-selectin will amplify thrombosis, mediated by procoagulant microparticles (MPs). Methods: Male mice (Mus musculus, n659), 20 to 25 grams, underwent IVC ligation to induce thrombosis. Groups consisted of wild type (WT) C57BL/6 controls, mice with high circulating levels of soluble P-selectin (<^>CT), P-selectin gene-interrupted knockout mice (PKO), and E- and P-selectin gene-interrupted mice (EPKO). Additional groups were used to evaluate the ability of a P-sel antagonist (rPSGL-Ig) and an antibody directed against PSGL-1 to downregulate the effects of P-sel in ACT mice and WT mice administered soluble P-sel at time of thrombosis. Animals were sacrificed on days 2 and 6 after IVC ligation. Thrombus mass (TM), vein wall morphometrics, and serum leukocyte/platelet microparticles (MPs) were evaluated by means of double-stained fluorescence-activated cell scanning analysis, and soluble P- and E-sel protein determination by ELISA. Results: At days 2 and 6 in phase I of the experiment, significant differences (P <.01) in TM were noted between groups, with <^>CT animals having the largest thrombi (50% and 57% increase in TM compared to WT at days 2 and 6) while EPKO mice had the smallest thrombi. Statistically, greater levels of neutrophils and total inflammatory cells were noted in the vein walls of <^>CT animals at day 2 compared with WT and PKO animals. A significant difference was noted between <^>CT and EPKO for neutrophils, monocytes, and total inflammatory cells, also at day 2. At day 6, the only statistically significant difference was found for monocytes, with a higher number in the <^>CT animals than in WT animals. The evaluation of MPs revealed that the <^>CT mice had a mixed leukocyte (MAC-1) and platelet (CD41) MP population that was also present in WT and PKO mice on day 2 and day 6. EPKO mice revealed a primarily platelet-derived MP population. Of interest, the <^>CT mice with the highest TM showed a high amount of mean channel fluorescence for MAC-1 (phycoerythrin) antibody, indicative of leukocyte MPs. <^>CT mice revealed statistically higher levels of soluble P-selectin at days 2 and 6. In phase 2, an antibody directed against PSGL-1 was more effective than rPSGL-Ig in decreasing TM and limiting leukocyte-derived MP fluorescence. Conclusions: This study demonstrates that high circulating levels of P-selectin are associated with increased thrombosis, whereas a lack of P-selectin and E-selectin is associated with a lessening of thrombosis. Additionally, leukocyte MPs are associated with venous thrombus formation. These data suggest the importance of selectins to venous thrombogenesis and show that P-selectin and leukocyte-derived MPs should be good targets to limit venous thrombus formation.