Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased — and, under conditions of decreased platelet adhesion, PDI inhibition reduced — fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet–secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features

Immune cell pathology in rabbit hemorrhagic disease

Aim: The aim of this research was to study the effect of rabbit hemorrhagic disease virus (RHDV) on the host immune response by examining the cellular composition/pathology of lymphoid organs and serum levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). Materials and Methods: Nine adult rabbits were inoculated with 1 ml of 10% infected liver homogenate, and three rabbits served as controls. The rabbit hemorrhagic disease (RHD)-induced animals were studied on 3 consecutive days post-infection. Diagnosis of RHD was made through routine hemagglutination tests and the polymerase chain reaction. Blood smears and tissue samples from bone marrow (BM), spleen, lymph nodes, and liver were analyzed for cell composition and cytopathology. Serum levels of TNF-α and IFN-γ were measured by enzyme-linked immunosorbent assay. Results: RHD showed a decreased absolute cell count of blood as well as lymph nodes, spleen, and BM cell populations with marked left shift. This was seen as a progressive rise in immature and blast cells. Quantitative cellular changes were accompanied by an increase in specific inflammatory cytokines. Immunocytopathological alterations were evidenced by: Vacuolized, hyperactivated tissue macrophages, finding of Dohle bodies in neutrophils, and activated lymphocytes with increased nuclear-cytoplasmic ratio. Cytoplasmic eosinophilic viral inclusions found in tissue (liver, spleen, and BM) macrophages were shown for the 1st time in RHD. Megakaryocytic emperipolesis was a common feature of RHD. Conclusion: These studies suggest that RHDV induces pathology in leukocytes due to hyperactivation with left shift (toward immature stages of the different cell lineages). Macrophages are increased in number and show an expressed cytopathic effect often accompanied by viral eosinophilic cytoplasmic inclusions. They also developed a secretory activation (increased levels of pro-inflammatory cytokines)

Mice deficient in the anti-haemophilic coagulation factor VIII show increased von Willebrand factor plasma levels - Table 1

<p>Mice deficient in the anti-haemophilic coagulation factor VIII show increased von Willebrand factor plasma levels</p> - Table

Bleeding phenotype of the <i>F8</i>^<i>-/y</i> mice.

<p>(<b>A</b>) Patella diameter before and 24h after joint bleeding induction to the right knee (n = 7,6) of C57BL/6J and <i>F8</i>^<i>-/y</i> with the left knee (n = 6,6) set as a control. (<b>B</b>) Representative rotational thromboelastometry (ROTEM) graphs of a C57BL/6J and a <i>F8</i>^<i>-/y</i>, illustrating clotting time (green), clot formation time (pink) and clot firmness (blue). (<b>C</b>) Clotting times and (<b>D</b>) clot formation times of C57BL/6J, <i>F8</i>^<i>-/y</i>, and <i>F8</i>^<i>-/y</i> blood samples reconstituted with 2.5 U/ml of human recombinant FVIII (n = 3). All data were expressed as means ± SEM. Statistical comparisons were performed using one-way ANOVA or two-way ANOVA * p< 0.05, ** p<0.01, ***p<0.001.</p

Increased VWF levels in the plasma of <i>F8</i>^<i>-/y</i> mice.

<p>(<b>A</b>) VWF antigen levels in plasma of <i>F8</i>^<i>+/y</i> (WT) vs <i>F8</i>^<i>-/y</i> mice (n = 9). (<b>B</b>) VWF multimer analysis of plasma samples from <i>F8</i>^<i>+/y</i> vs <i>F8</i>^<i>-/y</i> mice (n = 5). (<b>C</b>) ADAMTS13 levels in plasma of <i>F8</i>^<i>+/y</i> vs <i>F8</i>^<i>-/y</i> mice (n = 7). (<b>D</b>) VWF antigen levels in plasma of <i>F8</i>^<i>-/y</i> mice, 2h after tail vein injection of recombinant FVIII (Kogenate) at a dose of 1.5 U per 30g body weight or with 0.9% NaCl solution as a vehicle control (n = 11,10,12). All data were expressed as means ± SEM. Statistical comparisons were performed using the Student’s <i>t</i>-test or one-way ANOVA, * p< 0.05, ** p<0.01.</p