Neutrophils in Deep Venous Thrombosis

Neutrophils are involved in venous thrombogenesis, through P-selectin and neutrophil endothelial traps (NETS). In order to determine the importance of neutrophils to thrombogenesis, neutrophil depletion was performed in our rat model of stasis-induced deep venous thrombosis (DVT). Animals were treated with control serum or rabbit anti-rat PMN serum administered perioperatively and sacrificed at two and seven days. At two days, neutropenic rats had 1.6-fold larger thrombi (P = 0.04) and 1.4-fold higher femoral venous pressures by manometry (P = 0.008) without a difference in thrombus neovascularization. By seven days, DVT sizes were similar, but vein wall injury persisted in neutropenic rats with a two-fold increase in vein wall stiffness by microtensiometry (P < 0.05), as well as a 1.2-fold increased thickness (P = 0.04). Vein wall and intrathrombus uPA by Western immunoblotting, as well as intrathrombus MMP-9 gelatinase activity was significantly less in neutropenic rats than controls (P < 0.001). Conversely, MMP-2 was significantly elevated in neutropenic inferior vena cava (IVC) at two days after DVT.¹ P-selectin inhibition has been found to limit venous thrombosis in mice. Animals with high circulating levels of sP-selectin (^CT) were compared to selectin gene-deleted animals (PKO, EPKO) and wild-type (WT) mice. ^CT mice showed a significant 50% increase in thrombosis in our IVC ligation model while EPKO mice had the smallest thrombi. A significant difference was noted between ^CT and EPKO for neutrophils, monocytes, and total inflammatory cells at day two. Microparticle (MP) analysis revealed that in the ^CT, WT and PKO mice, a mixed leukocyte (MAC-1) and platelet (CD41) MP population was present. EPKO mice (with the smallest thrombi) revealed primarily a platelet-derived MP population, suggesting the importance of leukocyte-derived MPs in venous thrombogenesis. Of interest, the ^CT mice with the highest TM showed an elevated level of mean channel fluorescence for MAC-1 antibody, indicative of leukocyte derived MPs.² Through processes that also involve the initial activation of leukocytes and platelets, neutrophils initiate and amplify thrombosis through the formation of NETS, which are extracellular fragments of DNA containing histones and antimicrobial proteins.^3,4 In vitro and in vivo, NETs provide a scaffold and stimulus for thrombus formation.⁵ In order to investigate if plasma DNA (surrogate for NETS) is elevated in patients with DVT and to determine correlations with other biomarkers of DVT, we studied patients presenting to our diagnostic vascular laboratory. From December 2008 to August 2010, patients were divided into three distinct groups: (1) DVT positive, patients symptomatic for DVT confirmed by ultrasound (n = 47); (2) DVT negative, patients with leg pain but negative by ultrasound (n = 28); and (3) control healthy non-pregnant volunteers without signs or symptoms of active or previous DVT (n = 19). Blood was collected for biomarkers and the Wells score risk of DVT was assessed. Results showed that circulating DNA was significantly elevated in DVT patients, compared with both DVT-negative patients (57.7 vs. 17.9 ng/mL; P < 0.01) and controls (57.7 vs. 23.9 ng/mL; P < 0.01). There was a strong positive correlation with C-reactive protein (P < 0.01), D-dimer (P < 0.01), VWF (P < 0.01), Wells score (P < 0.01) and MPO (P < 0.01), along with a strong negative correlation with ADAMTS13 (P < 0.01) and the ADAMTS13/VWF ratio. The logistic regression model showed a strong association between plasma DNA and the presence of DVT (ROC curve 0.814) suggesting a role for DNA in venous thrombogenesis.⁶

References:

1. Varma MR, Varga AJ, Knipp BS, Sukheepod P, Upchurch GR, Kunkel SL, Wakefield TW, Henke PK. Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg. 2003;38:1090-1098

2. Myers DD, Hawley AE, Farris DM, Wrobleski SK, Thanaporn P, Schaub RG, Wagner DD, Kumar A, Wakefield TW. P-selectin and leukocyte microparticles are associated with venous thrombogenesis. J Vasc Surg.2003;38:1075-1089

3. Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers DD, Wrobleski SK, Wakefield TW, Hartwig JH, Wagner DD. Extracellular DNA traps promote thrombosis. Proceedings of the National Academy of Sciences. 2010;107:15880-15885

4. von Brühl M-L, Stark K, Steinhart A, Chandraratne S, Konrad I, Lorenz M, Khandoga A, Tirniceriu A, Coletti R, Köllnberger M. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. The Journal of Experimental Medicine. 2012;209:819-835

5. Massberg S, Grahl L, von Bruehl M-L, Manukyan D, Pfeiler S, Goosmann C, Brinkmann V, Lorenz M, Bidzhekov K, Khandagale AB. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nature Medicine. 2010;16:887-896

6. Diaz JA, Fuchs TA, Jackson TO, Kremer Hovinga JA, Lämmle B, Henke PK, Myers Jr DD, Wagner DD, Wakefield TW. Plasma DNA is elevated in patients with deep vein thrombosis. J Vasc Surg. 2013;1:341-348