bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kininogen Deficiency Protects From Ischemic Neurodegeneration in Mice by Reducing Thrombosis, Blood-Brain-Barrier Damage and Inflammation
Authors: Langhauser Friederike , Gob Eva , Kraft Peter , Geis Christian , Schmitt Joachim , Brede Marc , Göbel Kirsten , Helluy Xavier , Pham Mirko , Bendszus Martin , Jakob Peter , Stoll Guido , Meuth Sven G , Nieswandt Bernhard , McCrae Keith R. , Kleinschnitz Christoph 
Published: October 2019

Abstract
Abstract 104

Background and objective:
High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. Recent studies have suggested an important role for the intrinsic coagulation pathway in pathologic thrombosis. We have previously described the production of kininogen deficient mice (Kng1−/−), and the prolongation of Rose-Bengal induced carotid artery occlusion in these animals with no effect on the bleeding time. Kininogen also plays a central role in enhancing vascular permeability through the release of the nonapeptide bradykinin following cleavage by plasma kallikrein, and has been implicated in mediating acute inflammatory responses. Since thrombosis and inflammation are hallmarks of ischemic stroke still unamenable to therapeutic interventions, we hypothesized that the severity of acute ischemic stroke would be ameliorated in kininogen deficient mice.

Results:
We investigated the consequences of kininogen deficiency in models of ischemic stroke. Kng−/− mice of either sex subjected to transient middle cerebral artery occlusion (tMCAO) developed dramatically smaller brain infarctions and less severe neurological deficits without an increase in infarct associated hemorrhage. This protective effect was preserved at later stages of infarction as well as in elderly mice, but was lost after replacement of exogenous kininogen in the deficient mice. The improved outcomes were attributable to markedly reduced thrombus formation in ischemic vessels of mKng1−/− mice, improved cerebral blood flow, and less severe blood-brain barrier damage and edema formation. Moreover, the ingress of inflammatory cells (monocytes and neutrophils), and levels of IL-1 were significantly decreased in the absence of kininogen, consistent with a reduced inflammatory response following ischemic injury. The survival of mice subjected to tMCAO was markedly improved compared to control animals.

Conclusion:
Mice deficient in kininogen were markedly protected from acute ischemic stroke in the transient middle cerebral artery occlusion model. Protection was mediated through decreased thrombus formation, diminished vascular permeability, and a dampened inflammatory response. The mechanisms underlying these altered responses are currently under investigation. However, since kininogen appears to be instrumental in pathologic thrombus formation and inflammation, but entirely dispensable for normal hemostasis, kininogen inhibition may offer a safe and selective strategy for combating the effects of stroke and other thromboembolic diseases.

Disclosures:
No relevant conflicts of interest to declare.
The 3'-5' Exonuclease, TREX1, Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) in Response to DNA Damage
Authors: Miyazaki Takuya , Kim Yong-Soo , Yoon Jeong-Heon , Wang Hongsheng , Morse Herbert C. 
Published: October 2019

Abstract
Abstract 1046

Background:
Recognition of nucleic acids plays a key role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). The major 3'-5' DNA exonuclease, TREX1, degrades single- and double-stranded DNA to minimize potential immune activation by persistent self or retroelement DNA. Mutations in the human TREX1gene have been linked to four diseases: SLE; Aicardi-Goutières syndrome (AGS); retinal vasculopathy and cerebral leukodystrophy (RVCL); and familial chilblain lupus (CBL). SLE, AGS and CBL have overlapping features characterized by expression of interferon-alpha and autoantibody production, but are distinct clinical conditions. RVCL is a non-inflammatory endotheliopathy.

Methods:
As part of our efforts to determine the molecular mechanisms underlying TREX1 involvement in the pathogenesis of these diseases, we identified a novel TREX1-binding partner protein by Mass Spectrometry and Co-Immunoprecipitation (Co-IP) assays. We examined the interaction domains and the effects of mutations in TREX1 associated with these diseases.

Results:
We found that TREX1 interacted with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear DNA repair enzyme involved in the DNA damage response. The interaction domain of PARP1 was mapped to the amino-terminal zinc finger domains. Subcellular localization analysis showed that TREX1 normally localized to the cytoplasm and cytomembranes, but translocated to the nucleus (with some bound to chromatin) in response to sublethal DNA damage. Co-IP assays revealed that the interaction was increased following DNA damage in concert with the post-translational PARlation of PARP1. Functional studies showed that TREX1 induced the PARlation of PARP1 in a TREX1 dose-dependent manner. Furthermore, siRNA knockdown of TREX1 promoted PARP1 cleavage and loss of PARP1 enzymatic activity, resulting in the induction of apoptosis. We further analyzed the effects of TREX1-PARP1 interaction in relation to the pathogenesis of AGS or RVCL. Co-IP assays revealed that the RVCL-related TREX1-mutant protein, V235fs, bound to PARP1 as well as wild-type TREX1, whereas the AGS-related R114H mutant protein did not bind PARP1.

Conclusion:
These findings demonstrate that TREX1 contributes to the regulation of PARP1 in the DNA damage response and that mutation-induced alterations in the function of TREX1 may be a factor in the development or progression of these autoimmune diseases by affecting PARP1 activity.

Disclosures:
No relevant conflicts of interest to declare.
Acyl-Protein Thioesterase 1 Functions in Palmitoylation/Depalmitoylation Cycles of G Proteins and Regulates Platelet Activation
Authors: Dowal Louisa M. , Dilks James R. , Fadel Nathalie A. , Aisiku Omozuanvbo R. , Merrill-Skoloff Glenn , Flaumenhaft Robert C. 
Published: October 2019

Abstract
Abstract 1062
Protein palmitoylation is a reversible post-translational modification that regulates both lipid-protein and protein-protein interactions. During the palmitoylation cycle, palmitoylation occurs through a thioester linkage to a cysteine residue. Depalmitoylation occurs primarily through cleavage of this bond by acyl-protein thioesterase 1 (APT1). We have previously demonstrated the presence of APT1 in platelets and showed that APT1 translocates to membranes in an activation-dependent manner. However, the function of APT1 in platelet activation is not known. To determine whether APT1 functions in platelet signal transduction we evaluated the effect of palmostatins, novel small molecule inhibitors of APT1, on platelet function. Palmostatins B and M both inhibited platelet aggregation and α -granule secretion induced through protease-activated receptor (PAR) 1 with an IC50 of 15 μM. To assess which signaling pathways were affected by APT1 inhibition, we screened palmostatins for their ability to inhibit activation induced by several agonists. Palmostatins blocked platelet aggregation induced by a PAR1 agonist, a PAR4 agonist, TxA2, or epinephrine. In contrast, palmostatins failed to inhibit aggregation induced by collagen, PMA, or ionophore. Palmostatins also inhibited α -granule exocytosis induced by a PAR1 agonist or TxA2, but not exocytosis induced by PMA or ionophore. These results suggested that palmostatins blocked proximal signaling events mediated through G protein coupled receptors (GPCRs). To evaluate this supposition, we tested the effect of palmostatin B on PAR1-mediated [Ca2+]i flux. Palmostatin B inhibited PAR1-induced Ca2+ signaling with and IC50 of 15 μM, the same concentration required for inhibition of platelet aggregation and α -granule secretion. We have recently described the platelet palmitoylome (Dowal et al., Blood, 118:e62-73) and found several components of the proximal G protein signaling pathway that are palmitoylated, including many Gα subunits. To directly assess the effect of APT1 inhibition on palmitoylation/depalmitoylation cycles on a target Gα subunit, we evaluated Gα q palmitoylation using acyl biotin exchange chemistry. Total Gα q palmitoylation decreased substantially with activation of platelets through PAR1. In the presence of palmostatin B, however, Gα q palmitoylation increased following PAR1 activation. These results demonstrate that Gα q is a substrate for APT1. Our studies demonstrate a role for palmitoylation/depalmitoylation cycles in proximal signaling pathways downstream of GPCRs and implicate APT1 as an essential regulator of G protein signaling in platelets.

Disclosures:
No relevant conflicts of interest to declare.
Role of Gas6 in Cancer-Induced Venous Thrombosis
Authors: Aghourian Meghedi , Lemarie Catherine A , Blostein Mark D 
Published: October 2019

Abstract
Abstract 108
Venous thrombosis is the most common morbid complication related to cancer and its treatments. The onset of cancer has been shown to predispose patients to hemostatic disorders mainly because of the pro-thrombotic milieu generated by the cancer. Growth arrest specific 6 (gas6) is a vitamin K dependent secreted protein that is expressed by different cell types. Gas6 and its receptor Axl are overexpressed in different cancers. Gas6 null mice have been shown to be protected against life-threatening thrombosis. Because of the association of gas6 with cancer and thrombosis, the objective of the present study is to elucidate the role of gas6 in cancer-induced venous thrombosis. Thus, we hypothesize that the absence of gas6 in a mouse model of cancer would reduce venous thrombosis. Wild type (WT) and gas6 null (−/−) mice received an intravenous injection of M27 murine lung cancer cells. After 2 weeks, WT and gas6−/− mice developed end stage cancer. Venous thrombosis was induced using 10% ferric chloride. Thrombus surface area was evaluated using ultrasonography, 30 minutes after ferric chloride application. We found that thrombi induced in mice with cancer were smaller than those induced in WT mice, by both ultrasound (p<0.05) and histologically (p<0.05). To elucidate the role of gas6 in cancer-induced thrombosis, we co-cultured murine endothelial cells, prepared from lungs of both WT and gas6−/− mice, with M27 cells. RNA was extracted from endothelial cells and we used a whole genome microarray analysis to identify differential gene expression induced by cancer in the presence or absence of gas6. Microarray analysis revealed that 28 genes were differentially expressed in WT or gas6−/− cells in the presence of cancer. Of interest, we found that thrombospondin 2 was downregulated by cancer cells in WT endothelial cells and even further decreased in gas6−/− cells. We confirmed these results using real-time qPCR and western blot analysis (p<0.05). These results suggest the possibility that gas6, through the downregulation of thrombospondin 2, has a pathophysiologic role in cancer-induced thrombosis.

Disclosures:
No relevant conflicts of interest to declare.
Thrombopoietin Can Induce Tolerogenic Responses Via the Modulation of Thymic and Splenic T Lymphocyte Populations in a Murine Model of Immune Thrombocytopenia (ITP)
Authors: Semple John W. , Zhang Xu , Hu Yu , Guo Li , Speck Edwin R. , Ni Heyu , Aslam Rukhsana 
Published: October 2019

Abstract
Abstract 1088
Clinical trials using thrombopoietin (TPO) mimetics have clearly shown their benefit in raising platelet counts in patients with immune thrombocytopenia (ITP), however, little is known whether TPO may affect immunologic responses. It has been demonstrated that, like other therapies, TPO treatment can rescue the peripheral CD4+CD25+FoxP3+ T regulatory cell (Tregs) deficiency that is commonly observed in patients with ITP (Bao W et al Blood 116;4639, 2010). Of interest, an earlier murine study also suggested that TPO could affect the production of T cells, particularly CD4+ T cells (Zhao JZ et al Haematologica 83:572, 1998). Using a murine model of ITP that demonstrates both antibody and T cell mediated thrombocytopenia, we studied T cell responses in ITP mice treated with TPO. ITP was induced in severe combined immunodeficient (SCID) mice by administration of 2×104 splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 and the mice where either treated or not with weekly subcutaneous injections of 1ug of murine TPO. Mice were bled weekly to count platelets and obtain sera for anti-platelet antibody production and at 3 weeks after the splenocyte transfer, they were sacrificed and their spleens and thymi were harvested. Splenocytes and thymocytes were isolated and examined by flow cytometry to quantify CD4+CD25+FoxP3+ (Tregs) and CD8+CD25+FoxP3+ (Tc-regs). We observed that TPO treatment caused an expected significant increase in platelet counts, however, the treatment also caused a significant reduction in the titre of serum anti-platelet antibodies. This was associated with significant changes in the proportions of both CD4+ Tregs and CD8+ Tc-regs within the thymus and spleen. TPO treatment rescued the peripheral splenic Treg and Tc-reg deficiencies and the cells ability to functionally suppress the proliferation of conventional CD4+CD25- T cells. The data suggests that in addition to its megakaryocyte-stimulating properties, TPO treatment has additional tolerance-inducing effects associated with anti-platelet antibody reduction and CD4+ Treg and CD8+ Tc-reg stimulation.

Disclosures:
No relevant conflicts of interest to declare.
The Immature Platelet Fraction Is Sensitive to the Platelet Size and a Useful Parameter for Screening for Macrothrombocytopenia
Authors: Miyazaki Koji , Koike Yukako , Danbara Mikio , Horie Ryouichi , Higashihara Masaaki 
Published: October 2019

Abstract
Abstract 1097
The immature platelet fraction (IPF) is a useful parameter indicating thrombopoietic activity to differentiate the causes of thrombocytopenia. We previously reported that the percentage of IPF (%IPF) is negatively correlated to the platelet count among ITP patients, and not among myelodysplastic syndrome (MDS) patients. We also noticed that some MDS patients exhibited extremely high %IPF values, which were dissociated from the percentages of reticulated platelets (%RP) measured by flow cytometry. Such discrepancies were also observed in hereditary macrothrombocytopenias, which are sometimes difficult to be distinguished from ITP, because ITP also exhibits increased number of reticulated platelets in a slightly larger size. Once misdiagnosed, a hereditary macrothrombocytopenia patient might be subjected to an invasive treatment such as splenectomy. In order to avoid such mistreatments, a clear marker to differentiate macrothrombocytopenia is desperately needed. In this study, we investigated the IPF values of 16 individuals from 12 families with various hereditary macrothrombocytopenia in order to clarify whether the IPF could be a useful marker to distinguish macrothrombocytopenia from ITP, and examined the IPF during EDTA aggregation and cold-storage to elucidate how platelet size may affect the IPF value.
The IPF values were about 5 times higher in MYH9 disorders (%IPF 48.0 ± 1.8) and about 1.5 times higher in other macrothrombocytopenias (%IPF 17.0 ± 2.2) than immune thrombocytopenic patients with similar platelet counts (%IPF 9.3 ± 0.4). These results suggested that the platelet size can affect the IPF value. However it still remains the possibility that some factors specific to these macrothrombocytopenias other than the platelet size might make an influence on the IPF, because the characteristic of large platelets in hereditary macrothrombocytopenia has not been fully understood, and no one knows whether large platelets are functionally identical to normal platelets except for the size. In order to exclude the possibility, we next examined the changes of IPF values during EDTA aggregation and cold-storage. The IPF was significantly increased during storage in a time dependent manner along with forming platelet clumps. The IPF was strongly influenced by a few tiny platelet aggregates rather than other platelet indices, such as mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW).
In conclusion, the IPF is susceptible to the platelet size, and could be a useful parameter for screening of macrothrombocytopenia from ITP.

Disclosures:
No relevant conflicts of interest to declare.
CPB2 Deficiency Protects Against Polymicrobial Sepsis
Authors: Shao Zhifei , Nishimura Toshihiko , Leung Lawrence L. , Morser John 
Published: October 2019

Abstract
Abstract 1110
Procarboxypetidase B-2 (pro-CPB2) (also termed thrombin-activatable fibrinolysis inhibitor, TAFI) is activated by the thrombin/thrombomodulin complex to generate a basic carboxypeptidase (CPB2). Fibrin and complement C5a have been shown to be physiological CPB2 substrates, but the role of CPB2 in sepsis is unclear. We hypothesized that in polymicrobial sepsis, the CPB2 KO mice would have exacerbated disease because there would be enhanced activation of C5a, leading to an augmented inflammatory response and tissue injury. To our surprise, in a polymicrobial sepsis model in which the cecum was ligated and punctured (CLP), CPB2 deficient mice had a significant improvement in survival compared to wild type (WT) mice (p = 0.021) (Fig. 1). The median survival time of 3 days in WT mice was extended to 4 days in CPB2 KO mice (p = 0.015). Lung edema as measured by wet/dry lung weight ratio was decreased from 4.4 in the WT mice to 2.5 in the CPB2 KO mice (p = 0.0001) at 48 hr after CLP (Fig. 2). Peritoneal lavage fluid levels of protein and the amount of cell infiltration into the peritoneum at 48 hr was lower in CPB2 KO than WT mice. There was less liver damage assessed as AST and ALT in plasma in the CPB2 KO mice at 48 hr than in WT mice while there was a trend to an increase in BUN suggesting kidney damage in WT mice. There was a greater decrease in the white blood cell count of WT mice than CPB2 KO mice with a drop in lymphocyte numbers accounting for most of that decrease. Inflammatory mediators such as IL-6 and total C5a were lower in plasma from CPB2 KO mice at 6 hr after CLP than in WT plasma. The data suggest that WT mice in this model underwent multiple organ failure leading to their death and the CPB2 deficient mice were protected from the consequences of CLP. Overall these data show that lack of CPB2 protects against polymicrobial sepsis with a concomitant reduction in inflammation. Further studies are underway to determine which substrate is key to the prolonged survival of CPB2 deficient mice in this model.

Disclosures:
No relevant conflicts of interest to declare.
Evaluation of Prosthetic Valve Function by Platelet Function Analysis and Von Willebrand Factor Indices
Authors: Wysokinska Ewa M. , Duello Katherine M. , Chen Dong , Blackshear Joseph L 
Published: October 2019

Abstract
Abstract 1128

Background
Dysfunctional or mismatched prosthetic heart valves can results in high shear stress related von Willebrand factor (VWF) impairment, and clinical bleeding. This mechanism is similar to that seen in aortic stenosis patients, where gastrointestinal bleeding related to VWF multimer loss is well described (Heyde syndrome). In this study, we compared the results of VWF testing of patients with normally functioning cardiac valve prostheses, dysfunctional prosthetic valves, severe native aortic stenosis, and normal controls.

Methods
A total of 75 patients, 31 female and 44 male with a median age of 79.5 years were recruited to this study. Thirty patients had severe aortic stenosis (mean gradient > 40 mm Hg); 39 patients had normally functioning prosthetic valves, 21 aortic (AVR) and 18 mitral valves (MVR); and 6 patients had with dysfunctional valves (4 aortic and 2 mitral). Whole blood and plasma samples were drawn from patients and tested for platelet function analyzer-100 collagen ADP closure time (PFA-CADP), VWF antigen (VWF:Ag), VWF activity by latex immunoassay (VWF:Ltx), VWF multimer analysis and VWF multimer densitometry ratio of large vs medium to low molecular weight multimers (>15 mers/2–15 mers).

Results
The 6 dysfunctional prosthetic valve patients included patient-prosthesis mismatch (2) and (one each), thrombosed mechanical aortic prosthesis, torn biological aortic prosthesis, severe tissue mitral prosthetic regurgitation, and post-mitral valve repair severe mitral regurgitation. Four of six patients had clinically significant bleeding. Most aortic stenosis and all 6 prosthetic dysfunction patients had abnormal VWF multimers which is rare in normally functioning prostheses and completely absent in normal donors. VWF:Ltx/Ag was lower in AS and MVR than normal. VWF:Ltx/Ag ratio, VWF:multimer ratio and PFA-CADP were all significantly abnormal in patients with dysfunctional prosthetic valve. VWF:multimer ratios in normally functioning prostheses tended to be intermediate between normals and than those of patients with severe AS or prosthetic valve dysfunction (Table).

Conclusions
VWF testing including quantitative von Willebrand multimer analysis and PFA-CADP reliably separate dysfunctional from normally functioning cardiac prostheses, and should be considered in the evaluation of patients with cardiac prostheses, especially those who have clinically significant bleeding.

Disclosures:
No relevant conflicts of interest to declare.