P-selectin and leukocyte microparticles are associated with venous thrombogenesis.
OBJECTIVE
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 CT 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.
Myers
Daniel D
DD
Department of Surgery, University of Michigan, Ann Arbor, 48109-0654, USA. ddmyers@umich.edu
Hawley
Angela E
AE
Farris
Diana M
DM
Wrobleski
Shirley K
SK
Thanaporn
Porama
P
Schaub
Robert G
RG
Wagner
Denisa D
DD
Kumar
Anjali
A
Wakefield
Thomas W
TW
D000818:Animals; D002678:Chimera; D007249:Inflammation; D007962:Leukocytes / Q000276:immunology*; D016177:Macrophage-1 Antigen / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D019007:P-Selectin / Q000235:genetics / Q000276:immunology*; D040201:Platelet Membrane Glycoprotein IIb / Q000276:immunology; D013927:Thrombosis / Q000276:immunology; D014680:Veins / Q000473:pathology; D020246:Venous Thrombosis / Q000276:immunology*
D016177:Macrophage-1 Antigen; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D040201:Platelet Membrane Glycoprotein IIb