Protection of ß2GPI Deficient Mice from Thrombosis Reflects a Defect in PAR3-facilitated Platelet Activation.

Background: Antibodies to ß2-glycoprotein I (ß2GPI) cause thrombosis in antiphospholipid syndrome, however the role of ß2GPI itself in regulation of coagulation pathways in vivo is not well understood. Methods: We developed ß2GPI-deficient mice (Apoh -/- ) by deleting exon 2 and 3 of Apoh using CRISPR/Cas9 and compared the propensity of wild-type (WT) and Apoh -/- mice to develop thrombosis using rose bengal and FeCl 3 -induced carotid thrombosis, laser-induced cremaster arteriolar injury, and inferior vena cava (IVC) stasis models. We also compared tail bleeding times and assessed platelet activation in WT and Apoh -/- mice in the absence and presence of exogenous ß2GPI. Results: Compared to WT littermates, Apoh -/- mice demonstrated a prolonged time to occlusion of the carotid artery after exposure to rose bengal or FeCl 3 , and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Similarly, significantly smaller thrombi were retrieved from the IVC of Apoh -/- mice 48 hours after IVC occlusion. The activated partial thromboplastin time (aPTT) and prothrombin time, as well as aPTT reagent- and tissue factor-induced thrombin generation times using plasma from Apoh -/- and WT mice revealed no differences. However, we observed significant prolongation of tail bleeding in Apoh -/- mice, and reduced P-selectin expression and binding of fibrinogen to the activated α2bß3 integrin on platelets from these mice after stimulation with low thrombin concentrations; these changes were reversed by exogenous ß2GPI. An antibody to PAR3 blocked thrombin-induced activation of WT, but not Apoh -/- platelets, as well as the ability of ß2GPI to restore the activation response of Apoh -/- platelets to thrombin. ß2GPI deficiency did not affect platelet activation by a PAR4-activator peptide, or ADP. Conclusions: In mice, ß2GPI may mediate procoagulant activity by enhancing the ability of PAR3 to present thrombin to PAR4, promoting platelet activation at low thrombin concentrations. Key Points: ß2GPI deficient mice are protected from experimental arterial, venous, and microvascular thrombosis.ß2GPI deficient mice display prolonged tail bleeding times and reduced PAR3-facilitated platelet activation by low concentrations of thrombin.

Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation.

Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EVs are elevated in cancer, and studies suggest that EV may stimulate thrombosis in patients with cancer through expression of tissue factor. However, limited data also implicate EV in the activation of the contact pathway of coagulation through activation of factor XII (FXII) to FXIIa. To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with the activation of FXII, EV induced the cleavage of high molecular weight kininogen (HK) to cleaved kininogen. We also observed that EVs from patients with cancer stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or Escherichia coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EVs and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by a deficiency in FXII, HK, or prekallikrein. Moreover, pretreatment of EVs with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates the binding of contact factors to EV and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.

Stress Granule-Mediated Oxidized RNA Decay in P-Body: Hypothetical Role of ADAR1, Tudor-SN, and STAU1.

Reactive oxygen species (ROS) generated under oxidative stress (OS) cause oxidative damage to RNA. Recent studies have suggested a role for oxidized RNA in several human disorders. Under the conditions of oxidative stress, mRNAs released from polysome dissociation accumulate and initiate stress granule (SG) assembly. SGs are highly enriched in mRNAs, containing inverted repeat (IR) Alus in 3' UTRs, AU-rich elements, and RNA-binding proteins. SGs and processing bodies (P-bodies) transiently interact through a docking mechanism to allow the exchange of RNA species. However, the types of RNA species exchanged, and the mechanisms and outcomes of exchange are still unknown. Specialized RNA-binding proteins, including adenosine deaminase acting on RNA (ADAR1-p150), with an affinity toward inverted repeat Alus, and Tudor staphylococcal nuclease (Tudor-SN) are specifically recruited to SGs under OS along with an RNA transport protein, Staufen1 (STAU1), but their precise biochemical roles in SGs and SG/P-body docking are uncertain. Here, we critically review relevant literature and propose a hypothetical mechanism for the processing and decay of oxidized-RNA in SGs/P-bodies, as well as the role of ADAR1-p150, Tudor-SN, and STAU1.

Successful surgical outcome of infected aneurysmal drug-coated stent endocarditis.

A 52-year-old man developed pyrexia after primary angioplasty following anterior myocardial infarction Noninvasive evaluation revealed staphylococcal septicemia with suspicion of an aneurysm or abscess at the stented segment. Coronary angiography confirmed the presence of a large aneurysm. The infected left anterior descending artery aneurysm was de-roofed and ligated, and a saphenous vein bypass graft was placed, with an excellent outcome.

Characterization of RNA damage under oxidative stress in Escherichia coli.

We have examined the level of 8-hydroxyguanosine (8-oxo-G), an oxidized form of guanosine, in RNA in Escherichia coli under normal and oxidative stress conditions. The level of 8-oxo-G in RNA rises rapidly and remains high for hours in response to hydrogen peroxide (H2O2) challenge in a dose-dependent manner. H2O2 induced elevation of 8-oxo-G content is much higher in RNA than that of 8-hydroxydeoxyguanosine (8-oxo-dG) in DNA. Under normal conditions, the 8-oxo-G level is low in RNA isolated from the ribosome and it is nearly three times higher in non-ribosomal RNAs. In contrast, 8-oxo-G generated by a short exposure to H2O2 is almost equally distributed in various RNA species, suggesting that although ribosomal RNAs are normally less oxidized, they are not protected against exogenous H2O2. Interestingly, highly folded RNA is not protected from oxidation because 8-oxo-G generated by H2O2 treatment in vitro increases to approximately the same levels in tRNA and rRNA in both native and denatured forms. Lastly, increased RNA oxidation is closely associated with cell death by oxidative stress. Our data suggests that RNA is a primary target for reactive oxygen species and RNA oxidation is part of the paradox that cells have to deal with under oxidative stress.