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.
Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in several animal models of disease, and APC has been used clinically in severe sepsis ...and wound healing. Clinical use of APC, however, is limited by its immunogenicity and its anticoagulant activity. We show that a class of small molecules termed “parmodulins” that act at the cytosolic face of PAR1 stimulates APC-like cytoprotective signaling in endothelium. Parmodulins block thrombin generation in response to inflammatory mediators and inhibit platelet accumulation on endothelium cultured under flow. Evaluation of the antithrombotic mechanism showed that parmodulins induce cytoprotective signaling through Gβγ, activating a PI3K/Akt pathway and eliciting a genetic program that includes suppression of NF-κB–mediated transcriptional activation and up-regulation of select cytoprotective transcripts. STC1 is among the up-regulated transcripts, and knockdown of stanniocalin-1 blocks the protective effects of both parmodulins and APC. Induction of this signaling pathway in vivo protects against thromboinflammatory injury in blood vessels. Small-molecule activation of endothelial cytoprotection through PAR1 represents an approach for treatment of thromboinflammatory disease and provides proof-of-principle for the strategy of targeting the cytoplasmic surface of GPCRs to achieve pathway selective signaling.
Background
Although divalent zinc (Zn2+) is known to bind factor (F)XII and affect its sensitivity to autoactivation, little is known about the role of Zn2+ in the binding of FXII to platelets, where ...FXII activation is thought to occur in vivo, and the function of Zn2+ during thrombus formation following vascular injury remains poorly understood.
Objectives
To evaluate the role of Zn2+ in platelet‐dependent FXIIa generation.
Methods
FXII binding to platelets and FXII activation by stimulated platelets were assessed using flow cytometry and a platelet‐dependent thrombin generation assay. The mouse cremaster laser injury model was used to evaluate the impact of Zn2+ chelation on thrombus formation in vivo.
Results
Our data demonstrate that stimulated platelets support FXII‐dependent thrombin generation and that FXII activation by platelets requires the presence of Zn2+. By contrast, thrombin generation by stimulated endothelial cells occurred independently of FXII and Zn2+. Using flow cytometry, we found that FXII‐fluorescein‐5‐isothiocyanate binds to the surfaces of stimulated platelets in a specific and Zn2+‐dependent manner, whereas resting platelets demonstrated minimal binding. Other physiologically‐relevant divalent cations are unable to support this interaction. Consistent with these findings, the Zn2+‐specific chelator ethylenediaminetetraacetic acid calcium disodium salt confers thromboprotection in the mouse cremaster laser injury model without causing increased bleeding. We observed an identical phenotype in FXII null mice tested in the same system.
Conclusions
Our results suggest a novel role for Zn2+ in the binding and activation of FXII at the platelet surface, an interaction that appears crucial to FXII‐dependent thrombin generation but dispensable for hemostasis.
Stroke leads to serious long-term disability. Electrical epidural cortical stimulation has made significant improvements in stroke rehabilitation therapy. We developed a preliminary wireless ...implantable passive interface, which consists of a stimulating surface electrode, receiving coil, and single flexible passive demodulated circuit printed by flexible printed circuit (FPC) technique and output pulse voltage stimulus by inductively coupling an external circuit. The wireless implantable board was implanted in cats' unilateral epidural space for electrical stimulation of the primary visual cortex (V1) while the evoked responses were recorded on the contralateral V1 using a needle electrode. The wireless implantable board output stable monophasic voltage stimuli. The amplitude of the monophasic voltage output could be adjusted by controlling the voltage of the transmitter circuit within a range of 5-20 V. In acute experiment, cortico-cortical evoked potential (CCEP) response was recorded on the contralateral V1. The amplitude of N2 in CCEP was modulated by adjusting the stimulation intensity of the wireless interface. These results demonstrated that a wireless interface based on a microcoil array can offer a valuable tool for researchers to explore electrical stimulation in research and the dura mater-electrode interface can effectively transmit electrical stimulation.
The optimal management of infected abdominal aortic grafts is complete surgical excision plus in situ or extra-anatomic revascularization in patients who can tolerate this morbid operation. In ...addition to using age and the presence of comorbidities for risk assessment, physicians form a global clinical impression when deciding whether to offer excision or to manage conservatively. Functional status is a distinct objective measure that can inform this decision. This study examines the relative impact of age and functional status on outcomes of infected abdominal aortic graft excision to guide surgical decision-making.
Current Procedural Terminology code 35907 was used to identify patients undergoing excision of infected abdominal aortic graft in the 2005 to 2017 American College of Surgeons – National Surgical Quality Improvement Program (NSQIP) database. Patients were stratified by the upper age quartile (75 years old) as a cutoff, and then by functional status, independent vs dependent (as defined by NSIQIP). The patients were then stratified into four groups: Younger (<75)/Independent, Younger (<75)/Dependent, Older (≥75)/Independent, and Older (≥75)/Dependent. Outcomes measured included 30-day mortality and major organ-system dysfunction.
There were 814 patients who underwent infected abdominal aortic graft excision: 508 patients (62%) were Younger/Independent, 89 patients (11%) were Younger/Dependent, 176 patients (22%) were Older/Independent, and 41 patients (5%) were Older/Dependent. There was no statistically significant difference in 30-day mortality for Younger/Dependent (odds ratio OR, 1.66; 95% confidence interval CI, 0.90-3.09; P = .536) or Older/Independent (OR, 1.31; 95% CI, 0.78-2.19; P = .311) patients when compared with Younger/Independent patients, which suggests that neither old age nor dependent functional status by itself adversely affects mortality. However, when both factors were present, Older/Dependent patients had three times higher mortality when compared with Younger/Independent patients (41.5% vs 13.4%, respectively; OR, 3.13; 95% CI, 1.46-6.71; P = .003). Furthermore, as long as patients presented with independent functional status, old age by itself did not adversely affect major organ-system dysfunction (ORs for Older/Independent vs Younger/Independent were 0.76 P = .454, 1.04 P = .874, and 0.90 P = .692 for cardiac, pulmonary, and renal complications, respectively). On the contrary, even in younger patients, dependent functional status was significantly associated with higher pulmonary complications (Younger/Dependent vs Younger/Independent: OR, 2.22; 95% CI, 1.33-3.73; P = .002) and higher rates of unplanned reoperation (OR, 2.67; 95% CI, 1.62-4.41; P < .0001).
Dependent functional status has significant association with adverse outcomes after excision of infected abdominal aortic grafts, whereas old age alone does not. Therefore, this procedure could be considered in appropriately selected elderly patients with otherwise good functional status. However, caution should be applied in dependent patients regardless of age due to the risk of pulmonary complications.
Uncontrolled pro-coagulant responses in sepsis can lead to disseminated intravascular coagulation (DIC), a complication associated with markedly increased mortality. Abnormalities of coagulation, ...fibrinolysis, and platelet function can lead to both microvascular thrombosis contributing to multi-organ dysfunction syndrome or hemorrhagic complications. To understand the inciting causes of DIC in sepsis, we first evaluated the time course of platelet function, coagulation parameters and markers of endothelial activation following LPS exposure. These studies demonstrated that endothelial dysfunction preceded derangement of platelet function or coagulation parameters. To evaluate the thrombotic response of the endothelium early in endotoxemia, we injured the microvascular endothelium of cremaster arterioles of mice using a laser 1-3 hours following LPS exposure. Platelet and fibrin formation at sites of injury were significantly increased following LPS exposure to respectively 190% (p=0.026) and 195% (p<0.001) of control values. No significant differences were observed in platelet counts, platelet function (aggregation and activation) or coagulation parameters (PT) in mice treated with LPS for 3 hours compared to vehicle controls. Plasma levels of the endothelial markers VWF, soluble VCAM and E-selectin, however, were significantly increased following LPS exposure, demonstrating early endothelial activation. Furthermore, even when platelet accumulation was inhibited using the anti-platelet drug eptifibatide, fibrin generation at sites of laser injury was still significantly (p<0.01) increased in LPS-treated mice. Together, these data show that endothelial activation precedes disruption of platelets and coagulation in endotoxemia. Endothelial dysfunction is associated with perturbation of the endothelial Ang1/Tie2 pathway, characterized by significantly reduced Tie2 function and Ang1 levels. Therefore, we assessed thrombus formation in Tie2+/- mice in the absence of LPS. Fibrin generation at sites of laser injury was significantly increased in Tie2+/- mice to 192% of littermate (Tie2+/+) controls (p<0.01). As loss of Tie2 mimics the LPS-induced phenotype, we next determined whether activation of the Tie2 pathway could reduce fibrin clot formation. Ang1 stimulates phosphorylation of Tie2, promoting protective signaling in endothelium. To determine whether the pro-thrombotic consequences of endotoxemia on Tie2 signaling could be reversed using Ang1, mice were injected with adenovirus expressing Ang1 or control adenovirus prior to LPS exposure. Dephoshorylation of Tie2 associated due to LPS exposure was reduced with Ang1-treatment. In our in vivo thrombosis model, Ang1 inhibited the increased fibrin accumulation at sites of laser injury in endotoxemic mice to baseline levels. Tail snip assays showed that even though elevated Ang1 levels normalized LPS-induced augmentation of thrombus formation, increased Ang1 did not affect bleeding times. These data indicate that Ang1 stimulation of Tie2 signaling can regulate pathologic clot formation in the setting of inflammation without bleeding risk. Together, our studies show that endothelial dysfunction drives a pre-DIC state in endotoxemia. Targeting these early endothelial responses might represent an novel approach for reducing thrombosis in sepsis without enhancing bleeding risks. Future studies will evaluate the mechanism involved.
No relevant conflicts of interest to declare.
Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in animal models of inflammation and APC has been used clinically in sepsis and wound ...healing. Clinical use of APC in sepsis, however, was terminated as it was compromised by APC’s anticoagulant activity, which is associated with bleeding and limits its dosing in patients. We used a small molecule approach to circumvent this problem. With support from the Molecular Libraries Program, we screened 302,457 compounds to identify small molecules that modulated PAR1-mediated platelet activation. One class of PAR1-targeted compounds, which we termed parmodulins, was found to act at the cytosolic face of PAR1, at the G-protein binding sites. When evaluated in endothelial cell cultures,parmodulin 1 (PM1) and parmodulin 2 (PM2) inhibited apoptosis induced by thrombin, TNF-α, or staurosporine, in a manner similar to APC. PAR1 knockdown using siRNA abolished these protective effects demonstrating that parmodulins elicit a cytoprotective pathway by acting through PAR1. To assess the mechanism of action of parmodulin cytoprotection, we first evaluated proximal signaling mechanisms. Parmodulins stimulated phosphorylation of PI3-kinase and Akt in endothelium. Inhibition of Gβγ blocked parmodulin-induced phosphorylation of Akt, indicating that parmodulins act at the cytosolic face of PAR1 by releasing Gβγ. Transcriptional profiling of over 30,000 genes and specific evaluation of NF-kB transcriptional activation showed that exposure to PM2 blocked TNF-α-induced transcriptional activation. In addition to interfering with inflammatory signaling, parmodulins also stimulated the upregulation of cytoprotective proteins such as stanniocalcin-1. Since our premise was that parmodulins could achieve cytoprotective effects without anticoagulant effects, we compared dose curves of APC versus PM2 in both apoptosis assays and standard clotting assays. APC prolonged the aPTT at concentrations lower than those required to achieve cytoprotection of the endothelium. The low APC concentration used in our study was similar to plasma concentrations measured in clinical studies. Hence, these data were consistent with the fact that clinical bleeding was observed at doses of APC used for sepsis. In contrast, despite inhibiting apoptosis as effectively as APC, PM2 had no effect on plasma aPTT at any concentration. Nonetheless, PM2 was able to inhibit LPS- and TNF-α-induced thrombin generation and FXa activation on endothelium owing to its cytoprotective effect. PM2 also prevented TNF-α-induced accumulation of platelets on endothelium in bioengineered microvessels. These data demonstrate that PM2 can reduce inflammation-induced endothelial pro-thrombotic phenotype even without directly inhibiting coagulation factors. To assess the endothelial protective effects of PM2 in vivo we evaluated leukocyte rolling in cremaster venules of mice. Infusion of PM2 significantly reduced surgery-induced leukocyte rolling flux compared to vehicle-treated mice. As selectins are critically involved in leukocyte rolling we monitored soluble E-selectin levels in LPS-induced inflammation. Treatment of mice with PM2 significantly reduced the LPS-induced release of soluble E-selectin. Previously, we demonstrated that PM2 blocks platelet accumulation in a mouse laser injury model of thrombus formation. We here show that infusion of PM2 also significantly reduces fibrin accumulation to 25% of control (p<0.001) at the site of laser injury. Together our data show that PM2 exerts endothelial-mediated anti-inflammatory, anti-coagulant and anti-thrombotic effects in vitro and in vivo. These results demonstrate that modulating PAR1 at the cytosolic face could represent an alternative approach to APC in the treatment of thromboinflammatory disorders like sepsis.
No relevant conflicts of interest to declare.
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