Endotoxaemia-augmented murine venous thrombosis is dependent on TLR-4 and ICAM-1, and potentiated by neutropenia[*]Financial support: This study was supported by: HL092129 (PKH), NIH 5 T32 HL076123–09 (TW and AO), HL089407 (TW), HL122388 (FAJ), K08HL131993 (YK), Elizabeth Anne Baiardi Research Fund, the Conrad and Caroline Jobst Foundation and the Mary Ann Tinker, M. D. Surgical Educational and Research Fund.
Received:17 March 2016
Accepted after major revision:19 October 2016
01 December 2017 (online)
Venous thromboembolism is a major cause of death during and immediately post-sepsis. Venous thrombosis (VT) is mediated by cell adhesion molecules and leukocytes, including neutrophil extracellular traps (NETs). Sepsis, or experimentally, endotoxaemia, shares similar characteristics and is modulated via toll like receptor 4 (TLR4). This study was undertaken to determine if endotoxaemia potentiates early stasis thrombogenesis, and secondarily to determine the role of VT TLR4, ICAM-1 and neutrophils (PMNs). Wild-type (WT), ICAM-1-/- and TLR4-/- mice underwent treatment with saline or LPS (10 mg/kg i.p.) alone, or followed by inferior vena cava (IVC) ligation to generate stasis VT. In vivo microscopy of leukocyte trafficking was performed in non-thrombosed mice, and tissue and plasma were harvested during early VT formation. Pre-thrombosis, circulating ICAM-1 was elevated and increased leukocyte adhesion and rolling occurred on the IVC of LPS-treated mice. Post-thrombosis, endotoxaemic mice formed larger, platelet-poor thrombi. Endotoxaemic TLR4-/- mice did not have an augmented thrombotic response and exhibited significantly decreased circulating ICAM-1 compared to endotoxaemic WT controls. Endotoxaemic ICAM-1-/- mice had significantly smaller thrombi compared to controls. Hypothesising that PMNs localised to the inflamed endothelium were promoting thrombosis, PMN depletion using anti-Ly6G antibody was performed. Paradoxically, VT formed without PMNs was amplified, potentially related to endotoxaemia induced elevation of PAI-1 and circulating FXIII, and decreased uPA. Endotoxaemia enhanced early VT occurs in a TLR-4 and ICAM-1 dependent fashion, and is potentiated by neutropenia. ICAM-1 and/or TLR-4 inhibition may be a unique strategy to prevent sepsis-associated VT.
Supplementary Material to this article is available online at www.thrombosis-online.com.
KeywordsTLR4 - cellular adhesion molecules - ICAM-1 - sepsis - LPS - venous thrombosis - venous thromboembolism
* Presented in part at the 2014 Vascular Research Initiatives Conference and Arteriosclerosis, Vascular Biology and Thrombosis 2014 Annual Scientific Sessions.
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