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Clinical Trial
. 2018 Apr 2;128(4):1471-1484.
doi: 10.1172/JCI97488. Epub 2018 Mar 5.

Tie2 Protects the Vasculature Against Thrombus Formation in Systemic Inflammation

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Free PMC article
Clinical Trial

Tie2 Protects the Vasculature Against Thrombus Formation in Systemic Inflammation

Sarah J Higgins et al. J Clin Invest. .
Free PMC article

Abstract

Disordered coagulation contributes to death in sepsis and lacks effective treatments. Existing markers of disseminated intravascular coagulation (DIC) reflect its sequelae rather than its causes, delaying diagnosis and treatment. Here we show that disruption of the endothelial Tie2 axis is a sentinel event in septic DIC. Proteomics in septic DIC patients revealed a network involving inflammation and coagulation with the Tie2 antagonist, angiopoietin-2 (Angpt-2), occupying a central node. Angpt-2 was strongly associated with traditional DIC markers including platelet counts, yet more accurately predicted mortality in 2 large independent cohorts (combined N = 1,077). In endotoxemic mice, reduced Tie2 signaling preceded signs of overt DIC. During this early phase, intravital imaging of microvascular injury revealed excessive fibrin accumulation, a pattern remarkably mimicked by Tie2 deficiency even without inflammation. Conversely, Tie2 activation normalized prothrombotic responses by inhibiting endothelial tissue factor and phosphatidylserine exposure. Critically, Tie2 activation had no adverse effects on bleeding. These results mechanistically implicate Tie2 signaling as a central regulator of microvascular thrombus formation in septic DIC and indicate that circulating markers of the Tie2 axis could facilitate earlier diagnosis. Finally, interventions targeting Tie2 may normalize coagulation in inflammatory states while averting the bleeding risks of current DIC therapies.

Keywords: Coagulation; Hematology; Platelets; Vascular Biology; endothelial cells.

Conflict of interest statement

Conflict of interest: SMP is listed as an inventor on a patent (WO2007033216A3) filed by Beth Israel Deaconess Medical Center. SJH is listed as an inventor on a patent (WO2015066426A3) pertaining to the use of angiopoietin-based interventions for treating complications of a disease or disorder associated with dysfunction of the Angpt/Tie2 pathway held by Regeneron Pharmaceuticals Inc./University Health Network.

Figures

Figure 1
Figure 1. Discovery proteomics in septic DIC implicate endothelium and Angpt-2.
(A) Heatmap representation of SOMAScan plasma analytes in severe sepsis with DIC (n = 7) and sepsis (n = 7). Color scale indicates relative expression. (B) Volcano plot showing analytes that were increased or decreased in sepsis DIC compared with sepsis without DIC (non-DIC) with P values less than 0.05 indicated in red. (C) STRING analysis and visualization of high-confidence interaction clusters (k-means = 9 clusters indicated by node color) formed by plasma proteins and labeled on related functional categories. Solid line represents within-cluster, dashed gray line represents between-cluster interactions.
Figure 2
Figure 2. Angiopoietin levels in septic DIC.
Plasma SOMAScan levels of (A) Angpt-2, (B) Angpt-1, and (C) within-patient Angpt-1 to Angpt-2 ratio. **P < 0.005, Mann-Whitney U test. (D) Plasma Angpt-2 levels by ELISA and (E) scatter plot correlating ELISA versus SOMAScan (r2 = 0.84, P < 0.0001, n = 14). (F) Receiver operating characteristic (ROC) curve for plasma Angpt-2 to predict sepsis DIC from nonsevere sepsis within the selected cohort. Scatter plot and linear regression analysis between ELISA Angpt-2 and laboratory measurements of (G) platelet count (r2 = 0.37, P = 0.0208, n = 14) and (H) fibrinogen (r2 = 0.81, P = 0.0009, n = 9).
Figure 3
Figure 3. Disruption of the Angpt/Tie2 axis and endothelial activation precede the onset of DIC in endotoxemic mice.
Markers of DIC, including (A) whole-blood platelet count, (B) plasma D-dimer levels, (C) prothrombin time (PT), and (D) activated partial thromboplastin time (aPTT) depicted as time to clot (seconds). (E) Disease severity signs (murine sepsis score) and (F) levels of circulating thrombin-antithrombin (TAT) complex were measured in C57BL/6J mice challenged with LPS (10 mg/kg) or saline at specific time points. (G) Immunoblots for p-Tie2 (p-Tie2Y992) and total Tie2 from lung tissue isolated at 3 hours after LPS challenge. (H) Densitometric analysis of p-Tie2 relative to total Tie2 from G (LPS = red, n = 7) normalized to saline control from 3 independent Western blots. (I and J) Plasma Angpt-1 and Angpt-2 kinetics following LPS determined by ELISA (n ≥ 5 per group). Data represent the mean ± SEM (n ≥ 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Mann-Whitney U test (2 groups) or 1-way ANOVA (3 groups).
Figure 4
Figure 4. Endotoxemic mice develop increased thrombotic response before the onset of overt DIC.
(A) Representative binarized images of thrombus formation in response to laser injury in cremaster arterioles of mice exposed to saline or LPS (10 mg/kg) for 1–3 hours. Platelet accumulation (red, Dylight 649) and fibrin generation (green, Dylight 488) were visualized for 180 seconds following injury. Scale bar: 10 μm. Median integrated fluorescence intensity and area under the curve (AUC) were calculated for platelets (B and C) and fibrin (D and E). (C and E) Data are represented as the median AUC of individual thrombi (saline n = 36; LPS n = 34). *P < 0.05; ***P < 0.001, Mann-Whitney U test. PAR4-mediated platelet aggregation was measured in platelet-rich plasma obtained from C57BL/6J mice 3 hours after saline or LPS injection showing (F) quantification of aggregation and (G) a representative aggregation experiment of 3 independent experiments (saline = gray tracing; LPS = red tracing). (H) Platelet-factor 4 (PF4) levels were measured in plasma of C57BL/6J mice at 0 or 3 hours after LPS (10 mg/kg) (n = 9–11). Mann-Whitney U test.
Figure 5
Figure 5. Tie2 deficiency alone recapitulates sepsis-associated thrombotic response.
(AE) Platelet and fibrin accumulation was monitored in Tie2 heterozygous mice (Tie2+/–) and wild-type littermate controls (Tie2+/+) at the site of laser injury using anti-platelet Dylight 649–labeled and anti-fibrin Dylight 488–labeled antibodies. (A) Representative binarized images of a single thrombus (platelet = red; fibrin = green) at time of laser injury (0 seconds) and 30 to 120 seconds following laser injury. Median integrated fluorescence intensities and AUC were calculated for platelets (B and C) and fibrin (D and E). (C and E) Data are represented as the median for individual thrombi (Tie2+/–, n = 41; Tie2+/+, n = 40). (FJ) Platelet and fibrin generation was monitored in wild-type mice infused with eptifibatide (10 μg/g body weight) and exposed to saline or 10 mg/kg LPS. (F) Representative binarized images of thrombus formation, at time of laser injury (0 seconds) and up to 120 seconds following laser injury. Median integrated fluorescence intensities were calculated for platelets (G) and fibrin (I). (H and J) Data are represented as median individual thrombi (saline, n = 35; LPS n = 32). **P < 0.01, Mann-Whitney U test. Scale bars: 10 μm.
Figure 6
Figure 6. Tie2 activation suppresses prothrombotic actions of LPS on the endothelium.
FXa (A and B) and thrombin (C and D) generation was determined on human umbilical vein endothelial cells (HUVECs) incubated with Angpt-1 (200 ng/ml 30 minutes prior to LPS), anti–tissue factor antibody (α-TF Ab, 10 μg/ml) or lactadherin (Lact., 100 nM) and exposed to LPS complex (LPS [100 ng/ml], sCD14 [100 ng/ml], and LBP [10 ng/ml]) or vehicle control for 3 hours. Representative experiments (mean ± SEM) are depicted as absorbance (405 nm) or arbitrary fluorescence units (RFU) as a function of time. (B and D) The rate of the reaction for FXa and thrombin generation converted to units/ml and normalized to controls. Immunoblot analysis (E) and quantification (F) of TF expression relative to GAPDH (n = 3) in lysates of LPS-stimulated HUVECs with and without Angpt-1. Flow cytometric analysis of (G) TF exposure, quantified from dot plots of FITC fluorescence intensity versus forward scatter and quantified as percentage of TF-positive cells relative to control and (H) phosphatidylserine (PtdSer) exposure, quantified as percentage of annexin V–FITC–positive and PI-negative cells (lower right quadrant of I). (I) Representative flow cytometric quadrant analysis of PtdSer exposure (annexin V–FITC) and cell death (PI) (n = 4–6 per group). *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Bonferroni’s posttest.
Figure 7
Figure 7. Tie2 activation normalizes the septic thrombotic response to injury without increasing bleeding risk.
Representative binarized images of thrombus formation (A) following laser injury in mice injected with control adenovirus (CtlAdv) (top) or an adenovirus expressing Angpt-1 (AdAngpt-1) after LPS injection (1–3 hours) (platelets = red; fibrin = green). Scale bar: 10 μm. Median integrated platelet (B) and fibrin (D) fluorescence intensities and AUC (C and E) were calculated for individual thrombi, CtlAdv (n = 2; 33 thrombi), CtlAdv plus LPS (n = 3; 37 thrombi), AdAngpt-1 (n = 3; 38 thrombi), and Angpt-1 plus LPS (n = 3; 37 thrombi). (F) Time to bleeding cessation (seconds) in CtlAdv and AdAngpt-1 mice 3 hours after LPS challenge (n = 18 or 19 mice). (G) The number of re-bleeding events during 10-minute observation window stratified as follows: 0, 1–3, or 4 or more events. Representative immunoblot (H) and quantification (I) of fibrin in liver isolates relative to pan cadherin. (J) Relative change in TAT complex levels at 3 hours after LPS challenge in CtlAdV- and AdAngpt-1–treated mice. Angpt-2 levels in (K) plasma and (L) lungs of CtlAdV or AdAngpt-1 mice after LPS (n = 5 per group). *P < 0.05, **P < 0.01 by 1-way ANOVA. (M) Representative immunoblots of p-Tie2 (p-Tie2Y992) and total Tie2 levels in lung isolates.

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