Roles of protease-activated receptors in a mouse model of endotoxemia

Endotoxemia is often associated with extreme inflammatory responses and disseminated intravascular coagulation. Protease-activated receptors (PARs) mediate cellular responses to coagulation proteases, including platelet activation and endothelial cell reactions predicted to promote inflammation. These observations suggested that PAR activation by coagulation proteases generated in the setting of endotoxemia might promote platelet activation, leukocyte-mediated endothelial injury, tissue damage, and death. Toward testing these hypotheses, we examined the effect of PAR deficiencies that ablate platelet and endothelial activation by coagulation proteases in a mouse endotoxemia model. Although coagulation was activated as measured by thrombin-antithrombin (TAT) production and antithrombin III (ATIII) depletion, Par1–/–, Par2–/–, Par4–/–, Par2–/–:Par4–/–, and Par1–/–:Par2–/– mice all failed to show improved survival or decreased cytokine responses after endotoxin challenge compared with wild type. Thus, our results fail to support a necessary role for PARs in linking coagulation to inflammation or death in this model. Interestingly, endotoxin-induced thrombocytopenia was not diminished in Par4–/– mice. Thus, a mechanism independent of platelet activation by thrombin was sufficient to cause thrombocytopenia in our model. These results raise the possibility that decreases in platelet count in the setting of sepsis may not be caused by disseminated intravascular coagulation but instead report on a sometimes parallel but independent process.

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Abstract 463: TLT-1 Deficiency Affects Hypercholesterolemia and Progression of the Atherosclerotic Plaque in Apoe Null Mice

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a463 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Marieli Gonzalez ◽

Fiorella Reyes ◽

Deborah Marrero ◽

A V Washington

Keyword(s):

Platelet Activation ◽

Inflammatory Responses ◽

Plaque Rupture ◽

Atherosclerotic Lesion ◽

Soluble Form ◽

Chow Diet ◽

Fatty Streak ◽

Wild Type ◽

Platelet Surface ◽

Cholesterol Levels

Platelet activation at sites of inflammation triggers the secretion of molecules that induce the transition of atherosclerosis from fatty streak to an acute disease, featuring an increased vulnerability of the atherosclerotic lesion that results in plaque rupture and thrombosis. TLT-1 (Triggering Receptor Expressed in Myeloid cells (TREM)-like transcript-1) is a molecule exclusively found in the α-granules of megakarocytes and platelets and has a demonstrated effect in inflammatory responses. Upon platelet activation, TLT-1 is moved to the platelet surface, while its soluble form, s-TLT-1, is secreted and detected in serum. Studies using the C57Bl/6 treml1 - /- mouse demonstrated a predisposition to hemorrhage after an acute inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms. Because we have found that sTLT-1 levels are significantly elevated in apoE mice when compared to wild type, we hypothesized that TLT-1 may be playing an important role in the progression of atherosclerosis. To address this possibility, we generated apoE - /- / treml1 - /- double knockout mice [DN]. Assessment of lesions after 4 weeks high-fat diet (HFD) demonstrated that at early stages, TLT-1 deficiency accelerates fatty streak formation. After 20 weeks on HFD, lesions in both apoE - /- and [DN] mice progressed to an advance fibrous plaque stage. Although their lesion sizes were not substantially different, lesion compositions were. The mechanistic basis of these differences appears to be that the [DN] mice have significantly higher cholesterol levels when compared to apoE - /- mice. The increased cholesterol levels extend to the treml1 -/- mouse when compared to wild type mice at 4 weeks on HFD, this difference, however, gradually subsides as wild type mice cholesterol levels increase over 20 weeks. Interestingly, cholesterol levels in 50 week old mice on chow diet revealed minimal differences between test and control mice suggesting the higher cholesterol levels are related to increased dietary intake. Our work defines a surprising role for TLT-1 in the regulation of serum cholesterol levels during atherogenesis.

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Endothelial Dysfunction Is Associated with Mortality and Severity of Coagulopathy in Patients with Sepsis and Disseminated Intravascular Coagulation

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029619852163 ◽

2019 ◽

Vol 25 ◽

pp. 107602961985216 ◽

Cited By ~ 7

Author(s):

Amanda Walborn ◽

Matthew Rondina ◽

Michael Mosier ◽

Jawed Fareed ◽

Debra Hoppensteadt

Keyword(s):

Disseminated Intravascular Coagulation ◽

Protein C ◽

High Mobility ◽

Severity Of Illness ◽

Endothelial Activation ◽

Endothelial Damage ◽

Intravascular Coagulation ◽

Angiopoietin 2 ◽

Von Willebrand ◽

Relationship Of

The role of the endothelium in sepsis-associated disseminated intravascular coagulation (DIC) is multifaceted and may contribute substantially to disease severity and outcome. The purpose of this study was to quantify measures of endothelial function, including markers of activation (endocan, Angiopoietin-2 [Ang-2], and von Willebrand Factor), endogenous anticoagulants (tissue factor pathway inhibitor and protein C), and damage-associated factors (High Mobility Group Box 1 [HMGB-1]) in the plasma of patients with sepsis and DIC, and to determine the relationship of these factors with severity of illness and outcome. Plasma samples were collected from 103 adult patients with sepsis within 48 hours of intensive care unit admission. Biomarker levels were measured using commercially available, standardized methods. Disseminated intravascular coagulation was diagnosed according to the International Society of Thrombosis and Hemostasis scoring algorithm. Twenty-eight-day mortality was used as the primary end point. In this study, endothelial damage and dysfunction were associated with the severity of coagulopathy and mortality in DIC patients. Loss of the endogenous anticoagulant protein C and elevation in the vascular regulator Ang-2 were associated with the development of overt DIC. In addition to Ang-2 and protein C, endocan, a biomarker of endothelial activation, and HMGB-1, a mediator of endothelial damage and activation, were significantly associated with mortality. This underscores the contribution of the endothelium to the pathogenesis of sepsis-associated DIC.

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0733. Impact of endotoxin challenge on disseminated intravascular coagulation in obese minipigs

Intensive Care Medicine Experimental ◽

10.1186/2197-425x-2-s1-p55 ◽

2014 ◽

Vol 2 (S1) ◽

Cited By ~ 1

Author(s):

T Duburcq ◽

A Tournoys ◽

F Pattou ◽

T Hubert ◽

V Gmyr ◽

...

Keyword(s):

Disseminated Intravascular Coagulation ◽

Endotoxin Challenge ◽

Intravascular Coagulation

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Disseminated Intravascular Coagulation in Cancer: An Update

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0039-1687890 ◽

2019 ◽

Vol 45 (04) ◽

pp. 342-347 ◽

Cited By ~ 5

Author(s):

Marcel Levi

Keyword(s):

Disseminated Intravascular Coagulation ◽

Inflammatory Responses ◽

Traumatic Injury ◽

Distant Metastases ◽

Coagulation Factors ◽

Bleeding Complications ◽

Adequate Treatment ◽

Intravascular Coagulation ◽

Coagulation Proteins ◽

Thrombotic Complications

AbstractCancer often leads to the activation of coagulation, manifesting as disseminated intravascular coagulation (DIC) in its most extreme form. DIC is characterized by systemic intravascular coagulation activation (leading to deposition of intravascular platelets and fibrin) and simultaneous consumption of coagulation proteins and thrombocytes (which may cause bleeding complications). The clinical course of DIC in patients with malignancies is typically less intense compared with DIC complicating alternative clinical settings, including systemic inflammatory responses following infection or traumatic injury. A more slowly proceeding, less fulminant, and widespread hemostatic derangement can remain asymptomatic. Eventually, the ongoing consumption may result in low levels of platelets and coagulation factors, and bleeding complications (frequently localized at the site of the tumor or distant metastases) may be the first clinical manifestation of DIC. An alternative clinical scenario is dominated by thrombotic complications, ranging from clinically manifest vascular thrombosis to microvascular platelet plugs. The main principle of DIC management is adequate treatment of the precipitating disorder; however, there are clinical presentations that may require additional supportive strategies specifically aimed at the amelioration of the coagulopathy.

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Prevalence of Anti-Heparin Platelet Factor 4 Antibodies in Patients with Disseminated Intravascular Coagulation.

Blood ◽

10.1182/blood.v114.22.2094.2094 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2094-2094

Author(s):

Jawed Fareed ◽

He Zhu ◽

Josephine Cunanan ◽

Walter Jeske ◽

Debra Hoppensteadt ◽

...

Keyword(s):

Platelet Activation ◽

Disseminated Intravascular Coagulation ◽

Conflicts Of Interest ◽

Platelet Factor 4 ◽

Additional Analysis ◽

Plasma Samples ◽

Platelet Factor ◽

Intravascular Coagulation ◽

Activation Products ◽

Low Levels

Abstract Abstract 2094 Poster Board II-71 Disseminated intravascular coagulation (DIC) represents a complex syndrome with multiple pathophysiologic components. Most patients with DIC exhibit thrombocytopenic responses due to endogenous consumption of platelets. A systematic study on the prevalence on anti-heparin platelet factor 4 (AHPF4) antibodies and HIT syndrome in DIC patients has not been presented. To determine the prevalence of AHPF4 antibodies in patients with suspected DIC syndrome a total of 25 plasma samples were retrospectively analyzed utilizing the two commercially available methods (GTI, Brookfield, WI and Hyphen Biomedical, Paris, France). Out of 25 patients, 24 samples were positive for the AHPF4 antibody in the GTI method (OD>0.400), whereas only 16 were positive in the Hyphen Biomedical assay (OD>0.500). Interestingly, only 9 samples were positive in both of these assays. None of the positive samples in either the GTI or the Hyphen assay exhibited a positive 14C serotonin response. Additional analysis of these samples revealed that only 8 of these patients were previously exposed to heparin. Only 4 of the baseline samples were found to contain low levels of heparin as measured by anti-Xa method (< 0.2 U/ml). Additional analysis of these samples revealed the presence of platelet activation products such as platelet factor 4 (PF4), selectin and p-selectin. These studies suggest that circulating AHPF4 antibodies are non-functional and do not produce any thrombocytopenic responses. The elevated circulating PF4 levels and other cytokines may be contributory to the generation of these antibodies in the DIC patients. Disclosures: No relevant conflicts of interest to declare.

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Systemic Inflammation and Disseminated Intravascular Coagulation in Early Stage of ALI and ARDS: Role of Neutrophil and Endothelial Activation

Inflammation ◽

10.1023/b:ifla.0000049049.81688.fe ◽

2004 ◽

Vol 28 (4) ◽

pp. 237-244 ◽

Cited By ~ 52

Author(s):

Satoshi Gando ◽

Takashi Kameue ◽

Naoyuki Matsuda ◽

Atsushi Sawamura ◽

Mineji Hayakawa ◽

...

Keyword(s):

Systemic Inflammation ◽

Disseminated Intravascular Coagulation ◽

Early Stage ◽

Endothelial Activation ◽

Intravascular Coagulation

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Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving the survival of mouse polymicrobial sepsis

Blood ◽

10.1182/blood-2011-10-386185 ◽

2012 ◽

Vol 119 (20) ◽

pp. 4762-4768 ◽

Cited By ~ 57

Author(s):

Erik I. Tucker ◽

Norah G. Verbout ◽

Philberta Y. Leung ◽

Sawan Hurst ◽

Owen J. T. McCarty ◽

...

Keyword(s):

Disseminated Intravascular Coagulation ◽

Inflammatory Responses ◽

Bowel Perforation ◽

Multiple Organ ◽

Factor Xii ◽

Contact Activation ◽

Factor Xi ◽

Intravascular Coagulation ◽

Vehicle Treatment ◽

Severe Infections

Abstract Severe bacterial sepsis often leads to a systemic procoagulant and proinflammatory condition that can manifest as disseminated intravascular coagulation, septic shock, and multiple organ failure. Because activation of the contact proteases factor XII (FXII), prekallikrein, and factor XI (FXI) can trigger coagulation and inflammatory responses, the contact factors have been considered potential targets for the treatment of sepsis. However, the pathogenic role of contact activation in severe infections has not been well defined. We therefore investigated whether an anticoagulant antibody (14E11) that selectively inhibits prothrombotic FXI activation by activated FXII (FXIIa) modifies the course of bowel perforation-induced peritoneal sepsis in mice. Early anticoagulation with 14E11 suppressed systemic thrombin- antithrombin complex formation, IL-6, and TNF-α levels, and reduced platelet consumption in the circulation and deposition in the blood vessels. Treatment with 14E11 within 12 hours after bowel perforation significantly improved survival compared with vehicle treatment, and the saturating dose did not increase tail bleeding. These data suggest that severe polymicrobial abdominal infection induces prothrombotic FXI activation, to the detriment of the host. Systemic anticoagulation by inhibiting FXI activation or FXIIa procoagulant activity during sepsis may therefore limit the development of disseminated intravascular coagulation without increasing bleeding risks.

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A Zebrafish Model Of Antithrombin III Deficiency Displays Bleeding and Thrombosis Secondary To Disseminated Intravascular Coagulation

Blood ◽

10.1182/blood.v122.21.200.200 ◽

2013 ◽

Vol 122 (21) ◽

pp. 200-200

Author(s):

Yang Liu ◽

Colin A. Kretz ◽

Morgan L. Maeder ◽

Michael C. Huarng ◽

Catherine E. Richter ◽

...

Keyword(s):

Small Molecule ◽

Disseminated Intravascular Coagulation ◽

Antithrombin Iii ◽

Targeted Mutagenesis ◽

Wild Type ◽

Zebrafish Model ◽

Human Fibrinogen ◽

Intravascular Coagulation ◽

Laser Injury ◽

Homozygous Mutant

Abstract Thrombosis is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Identification of small molecule inhibitors of thrombosis in an in vivo model would facilitate novel and improved therapeutics for patients. The zebrafish is a powerful genetic model in which the hemostatic system is nearly entirely conserved with humans. Its external development, ability to generate thousands of offspring at low cost, and optical transparency all make it a powerful tool to screen for genetic and chemical modifiers of coagulation disorders. We generated a zebrafish model of antithrombin III (AT3) deficiency by targeted mutagenesis using zinc finger nucleases. Homozygous at3 mutants displayed a lethal phenotype due to intracardiac thrombosis between 2 and 7 months of age, yet embryos and larvae appeared grossly normal with no overt evidence of pathologic clotting. Induction of thrombosis at 3-4 days post fertilization (dpf) in homozygous mutant larvae by laser-mediated endothelial ablation resulted in diminished rates of posterior cardinal vein (PCV) occlusion, a bleeding phenotype. To prove functional conservation with mammals, we expressed recombinant zebrafish At3 and demonstrated that it binds human thrombin in vitro. Furthermore, while injection of wild type zebrafish and human cDNAs rescued the laser injury phenotype, zebrafish at3 with a mutation in the putative reactive site failed to do so. We hypothesized that the discrepant larval bleeding and adult thrombotic phenotypes could be accounted for by disseminated intravascular coagulation (DIC). Consistent with this, we observed reduced fibrinogen levels in at3 homozygous mutant plasma, and were able to rescue mutant larvae by injection of human fibrinogen prior to laser injury. To identify the location of consumed fibrinogen, we tagged human fibrinogen with FITC (fluorescein isothiocyanate), followed by infusion into larvae from heterozygous intercrosses at 3 dpf. at3 homozygous mutants displayed extensive PCV fluorescence, which was absent in wild type and heterozygous siblings. Pre-incubation with warfarin completely prevented this phenotype, co-injection of tissue plasminogen activator (TPA) partially prevented fluorescence accumulation, and post-injection of TPA reduced the signal, consistent with our hypothesis of DIC. Our data have uncovered the mechanism underlying the discrepant at3 mutant phenotypes and demonstrated conservation of At3 function in zebrafish. Loss of At3 protein results in DIC in zebrafish larvae secondary to unopposed thrombin activity. Mutants survive, only to succumb to lethal thrombosis as adults. Further study as to why larvae are able to tolerate excessive clot formation, as well as small molecule screens for novel anticoagulants using this model, could potentially lead to innovative therapeutic modalities for affected patients. Disclosures: No relevant conflicts of interest to declare.

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