Differential effects of novel anticoagulants: FXa versus FIIa inhibition on coagulation and inflammation

S of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Differential effects of novel anticoagulants: FXa versus FIIa inhibition on coagulation and inflammation S. Nazir, K. Shahzad, I. Gadi, M. Al-Dabet, S. Kohli, S. Ranjan, F. Bock, B. Isermann (Magdeburg, Germany, Nashville, United States) Basic Science 16.02.2017, 11:00 12:00 Objective: Thrombin is the key protease in regard to thrombus formation; the same is not true in regard to protease dependent signaling. Hence, we postulate that inhibition of either fXa or fIIa may have comparable effects in regard to coagulation, but convey different effects in regard to inflammation and receptor dependent regulation of cellular effects. Methods: WT mice were either treated with low and high dose of dabigatran or Rivaroxaban for 1 week and then were analyzed for tail bleeding assay or for arterial injury induced thrombosis formation and LAD ligation (ischemic/reperfusion) induced myocardial infarction. Results: Rivaroxaban and dabigatran have comparable dose-dependent effects in regard to bleeding and thrombosis in in vivo models. However, while fIIa inhibition abrogates the anti-inflammatory effect of fIIa already at low dosages, e.g. at dosages at which bleeding time is already prolonged, this is not the case when using the fXa inhibitor Rivaroxaban. Although Infarcted heart areas were similar in both groups, fxa inhibition abolished proinflammatory cytokines, IL-6, TNF-alpha and macrophages infiltration in infarcted heart tissue. In addition we observed higher levels of blood plasma endogenous activated protein C (aPC) in fXa inhibition group, as compared to in fIIa inhibition group. Western Blot analysis revealed NF-Kβ levels significantly less in fxa inhibited group as compared to fIIa inhibition. Conclusion: Taken together, these results strongly support that inhibition of fIIa and fXa have similar profiles in regard to the regulation of hemostasis, but differ in their ability to modulate the inflammatory response, with fXa inhibition being superior. Congress President GTH 2017 Prof. Dr. med. Johanna A. Kremer Hovinga, Inselspital, 3010 Bern [email protected] Congress Organisation: Meister ConCept GmbH, Bahnhofstrasse 55, CH-5001 Aarau T +41 62 836 20 90, F +41 62 836 20 97, [email protected] ABSTRACTS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Annexin A7 (ANX7) regulates collagen-dependent platelet activation and Ca2+ signalingS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Annexin A7 (ANX7) regulates collagen-dependent platelet activation and Ca2+ signaling S. Geue, B. Walker-Allgaier, D. Eißler, P. Münzer, F. Lang, M. Gawaz, O. Borst (Tübingen, Germany) Basic Science 16.02.2017, 11:00 12:00 Objective: Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca2+ concentration ([Ca2+]i) and is followed by platelet secretion, aggregation and thrombus formation with consecutive vascular occlusion. This study aimed to determine the role of ANX7 in collagen-dependent platelet Ca2+ signaling and function in platelets from ANX7 knockout (anx7-/-) mice. Methods: Fura-2-AM spectrofluorometric Ca2+ measurements, flowchamber and light transmission aggregometry were performed with platelets from ANX7 knockout mice. Results: Stimulation of the collagen receptor glycoprotein VI (GPVI) by collagen or collagen-related peptide (CRP) leads to platelet activation due to increased intracellular calcium concentration ([Ca2+]i). Fura-2-AM spectrofluorometric Ca2+ measurements revealed that ANX7 deficiency strongly blunted Ca2+ mobilisation from intracellular stores and impaired extracellular Ca2+ influx in response to CRP and collagen. As platelet activation is linked with integrin αIIbβ3 activation and subsequent aggregation, light transmission aggregometry was performed uncovering a significant impaired platelet aggregation in anx7-/platelets compared to anx7+/+ platelets after stimulation with CRP and collagen. Furthermore, GPIV-dependent platelet dense granule secretion (reflected by ATP release) as well as arterial thrombus formation under high shear rates (1700 s-1) on collagen were significantly diminished in anx7-/platelets as compared to anx7+/+ platelets Conclusion: In conclusion, ANX7 plays a pivotal role upon collagen-triggered platelet activation and thrombus formation at least due to impaired activation-dependent increase of [Ca2+]i. Congress President GTH 2017 Prof. Dr. med. Johanna A. Kremer Hovinga, Inselspital, 3010 Bern [email protected] Congress Organisation: Meister ConCept GmbH, Bahnhofstrasse 55, CH-5001 Aarau T +41 62 836 20 90, F +41 62 836 20 97, [email protected] ABSTRACTS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland P2Y6 depletion enhances metabolic activity and ameliorates outcome of high fat diet induced obesity in miceS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland P2Y6 depletion enhances metabolic activity and ameliorates outcome of high fat diet induced obesity in mice J. Merz, P. Stachon, P. Albrecht, F. Ünal, S. von Garlen, A. Heidenreich, N. Hoppe, B. Duffner, C. Bode, A. Zirlik (Freiburg, Deutschland) Basic Science 16.02.2017, 11:00 12:00 Objective: With 17.3 million deaths per year cardiovascular diseases are the major cause of death worldwide. A prolonged obese body composition is strongly related with a higher risk of cardiovascular disease (CVD) incidence and mortality. Extracellular nucleotides are known to modulate metabolic pathways such as glucose homeostasis, cholesterol homeostasis, and adipocyte differentiation. These extracellular nucleotides signal through purinergic receptors, e.g. P2Y6, which fine-tune metabolic processes providing a potential target in the treatment of obesity induced metabolic syndrome. We hypothesized a role of P2Y6 in the outcome of highfat diet induced obesity. Methods: P2Y6 deficient (ko) 8 week old male mice and C57Bl6/N wildtype (wt) mice were fed for 20 weeks a high-fat or normal diet. Metabolic activity was assessed by metabolic caging at week 17. After 20 weeks mice were euthanized and whole body composition analyzed by necropsy. Blood plasma was analyzed by enzymelinked immunosorbent assay. Results: We observed a decelerated gain of body weight in P2Y6 deficient animals (after 12 weeks wt: 45.4 ± 0.9g (n=13) and P2Y6 ko: 40.6 ± 1.0g (n=13), p=0.0022). After necropsy P2Y6 ko mice revealed reduced fat depositions (e.g. pericardial adipose tissue of P2Y6 ko mice: 58.3 ± 5.4mg (n=13) versus wt animals: 75.2 ± 4.8mg (n=9), p < 0.05). Brown adipose tissue was elevated in P2Y6 ko animals under HFD (P2Y6 ko: 380 ± 27.9mg (n=8) versus wt: 303.3 ± 23.2mg (n=10), p < 0.05). P2Y6 deficient mice have a higher O2 consumption (P2Y6 ko: 2666 ± 79ml/h/kg (n=6) versus wt: 2315 ± 62ml/h/kg (n=6), p=0.0059), increased CO2 exhalation (P2Y6 ko: 2006 ± 66ml/h/kg (n=6) versus wt: 1724 ± 44ml/h/kg (n=6), p=0.0051) and increased heat (P2Y6 ko: 12.64 ± 0.38kcal/h/kg (n=6) versus wt: 11.16 ± 0.42kcal/h/kg (n=6), p<0.05) but no difference in movement or food intake. Plasma cholesterol levels were decreased in P2Y6 ko animals (100.2 ± 28.6mg/dl (n=5)) compared to wt animals under HFD (219.5 ± 41.4mg/dl (n=3), p<0.05). Conclusion: In conclusion our data showed a reduction of HFD induced fat deposition in P2Y6 ko mice due to an increased metabolic activity. These effects make P2Y6 a promising target for the treatment of obesity induced metabolic syndrome and prevention of CVDs. Congress President GTH 2017 Prof. Dr. med. Johanna A. Kremer Hovinga, Inselspital, 3010 Bern [email protected] Congress Organisation: Meister ConCept GmbH, Bahnhofstrasse 55, CH-5001 Aarau T +41 62 836 20 90, F +41 62 836 20 97, [email protected] ABSTRACTS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Introduction of a guideline for the treatment of synovitis in patients with hemophilia B. Habermann, A. Seuser, T. Hilberg (Mainz, Germany, Bonn, Germany, Wuppertal, Germany)S of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Introduction of a guideline for the treatment of synovitis in patients with hemophilia B. Habermann, A. Seuser, T. Hilberg (Mainz, Germany, Bonn, Germany, Wuppertal, Germany) Basic Science 16.02.2017, 11:00 12:00 Objective: Treatment of patients with hemophilia (PwH) has changed through the last decades. Especially the implementation of a prophylactic treatment with factor concentrates in young patients has reduced the incidence of severe bleedings. Nevertheless, many patients suffer from synovitis due to previous bleedings and there it still a lack of knowledge of how and when to treat a synovitis in those patients. Since a synovitis after a bleeding event is the start point of joint degeneration, it is crucially important to start an adequate treatment at the right moment. The aim of our work was, to develop a guideline based on the evidence in literature to support physicians, medical staff and patients in the treatment of synovitis in PwH. Methods: All member societies of the AWMF were invited to take part in the work of our guideline group. The group consisted of experts in the fields of hemophilia and hematology, nuclear medicine, pediatrics, physiotherapy, orthopedics, radiology, and sports medicine. Based on the regulations of the AWMF, the present and past literature on synovitis and hemophilia was acquired, systematically reviewed and thereafter assessed by the Oxford rating system. All papers and abstracts were scanned and finally reviewed in detail by the working group. The content was evaluated and summarized to a comprehensive paper. Results: We developed the underlying work for a guideline that may help physicians, medical staff and patients to treat a synovitis according to the available evidence in literature. The result is subdivided in diagnostics, treatment and secondary prophylaxis of synovitis. Besides a comprehensive paper we plan to develop short versions for physicians and patients that offer a quick guide in the daily routine work. Conclusion: The guideline may be a valuable tool for physicians, medical staff and patients for the treatment of early and late onset synovitis. With it, treatment of the PwH will be based on what has been published in literature in the near future. However, the review of the literature has shown that many established treatment options can’t be substantiated with sufficient evidence in literature and are only recommended by experts’ opinion. Nevertheless, the content of our guideline can be also a central basis for necessary further research. Congress President GTH 2017 Prof. Dr. med. Johanna A. Kremer Hovinga, Inselspital, 3010 Bern [email protected] Congress Organisation: Meister ConCept GmbH, Bahnhofstrasse 55, CH-5001 Aarau T +41 62 836 20 90, F +41 62 836 20 97, [email protected] ABSTRACTS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland The coagulation Factor XIII (FXIII) A subunit activation peptide directs the evolution of FXIIIA as a proteolytically activated dimeric moleculeS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland The coagulation Factor XIII (FXIII) A subunit activation peptide directs the evolution of FXIIIA as a proteolytically activated dimeric molecule A. Biswas, S. Singh, V. Ivaskevicius , J. Oldenburg (Bonn, Germany) Basic Science 16.02.2017, 11:00 12:00 Objective: To trace the evolutionary origins of Factor XIII (FXIIIA) subunit activation peptide (FXIII-AP) and its importance in the speciation events leading to generation of FXIIIA subunit dimeric molecule. Methods: Nucleotide and amino acid sequences of coagulation FXIIIA subunit, the activation peptide itself and of other Transglutaminase paralogues were downloaded from pubmed. Sequences were manually curated to eliminate redundancy and reduce error rates arising from partial/incorrect sequences. After generating high quality multiple alignments phylogenetic analysis was performed for both types of sequences on the GUI platform of MEGAversion7. On a structural level all zymogenic and activated forms of FXIIIA and other known TGs were downloaded from protein structure database. The TGs for whom no structures were available, threading based structures were generated on the ITASSER server. Structure alignment and visual comparative analysis was performed on the YASARA platform. Results: The FXIII-AP has evolved from the membrane anchorage region of TGM1 as a result of a loss of function deletion. The evolution of the FXIII-AP has directed the evolution of FXIIIA subunit as a functionally similar but structurally distinct (dimer) class of enzyme within the transglutaminases. The N terminal region of the FXIII-AP shows a higher substitution rate than the C-terminal part (which is highly conserved) indicating that while the C-terminal part is structural in function, the N-terminal part is meant for protein-protein interaction. Cross conservation analysis suggests that part of the activation peptide originates from a sequence on the beta sandwich domain of TGM5 and Erythrocyte membrane protein band 4.2 and moves through TGM1 to FXIIIA through a series of gain of function and loss of function mutations. Conclusion: FXIII-AP is not only an important region for the activation of FXIIIA subunit; it has also played a very important role in the specialized evolution of FXIIIA. Congress President GTH 2017 Prof. Dr. med. Johanna A. Kremer Hovinga, Inselspital, 3010 Bern [email protected] Congress Organisation: Meister ConCept GmbH, Bahnhofstrasse 55, CH-5001 Aarau T +41 62 836 20 90, F +41 62 836 20 97, [email protected] ABSTRACTS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland CLIC1 supports mechanisms related to thrombosis and vascular repairS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland CLIC1 supports mechanisms related to thrombosis and vascular repair L. M. Knowles, P. Niewald, E. Ampofo, A. Drawz, I. Müller, H. Eichler, J. Pilch (Homburg, Germany) Basic Science 16.02.2017, 11:00 12:00 Objective: Chloride intracellular channel 1 (CLIC1) has been shown to be involved in thrombus formation and angiogenesis but the functional context of CLIC1 action remains largely unexplored. The objective of this study is to determine if CLIC1 supports cell adhesive processes that are relevant for endothelial and platelet function. Methods: Human umbilical venous endothelial cells (HUVEC) were treated with CLIC1 siRNA or the CLIC1 small molecule inhibitor IAA94 and probed for cell proliferation on plastic (2D) as well as cell invasion/survival after embedding in fibrin (3D). Platelets were treated with IAA94 to assess the effect of CLIC1 on aggregation. Flow cytometry was used to determine integrin alphaIIbbeta3 activation as well as CLIC1 cell surface expression on platelets. The subcellular localization of CLIC1 in HUVEC/platelets was analyzed with fluorescence or confocal microscopy. The effect of CLIC1 on thrombus formation in vivo was assessed by intravital fluorescence microscopy in a mouse dorsal skin fold chamber model. Results: Treatment of HUVEC with CLIC1 siRNA or IAA94 had a strong anti-proliferative effect in 2D and caused significant reduction of invasion and survival in 3D. At the same time, we detected relocation of CLIC1 into lamellipodia (2D) and invadopodia (3D) in untreated HUVEC. This particular distribution of CLIC1 is functionally significant as we found reduced membrane ruffling in combination with increased stress fiber formation in CLIC1 siRNA-treated HUVEC. Relocation of CLIC1 into the cell periphery was also detectable in platelets, which expressed CLIC1 on the cell surface in an RGD-dependent manner. CLIC1 relocation to the platelet membrane was inhibited after treatment with IAA94, which also reduced integrin alphaIIbbeta3 activation. This in turn led to impaired platelet aggregation in vitro and prolonged vaso-occlusion in a mouse model of photo-chemical thrombus formation in vivo. Conclusion: Our results show that CLIC1 is regulated by adhesive interactions with integrin ligands that cause CLIC1 to relocate to the cell surface. This process in turn appears to be relevant for integrin-mediated functions involved in platelet thrombus formation, angiogenesis and vascular repair. Congress President GTH 2017 Prof. Dr. med. Johanna A. Kremer Hovinga, Inselspital, 3010 Bern [email protected] Congress Organisation: Meister ConCept GmbH, Bahnhofstrasse 55, CH-5001 Aarau T +41 62 836 20 90, F +41 62 836 20 97, [email protected] ABSTRACTS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Collagen V alpha 2 as a direct target of miR-143 in arteriogenesis T. Hammerschick, K. Troidl, K. T. Preissner, S. Fischer (Giessen, Germany, Bad Nauheim, Germany)S of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Collagen V alpha 2 as a direct target of miR-143 in arteriogenesis T. Hammerschick, K. Troidl, K. T. Preissner, S. Fischer (Giessen, Germany, Bad Nauheim, Germany) Basic Science 16.02.2017, 11:00 12:00 Objective: Arteriogenesis describes the formation of collateral arteries from preexisting small vessels, that involves the proliferation of endothelial and smooth muscle cells as well as the recruitment of leukocytes. This process is induced by physical forces, most importantly fluid shear stress (FSS). It was shown that FSS leads to a change in the expression pattern of several microRNAs (miRs) in growing collateral arteries; especially miR-143 is highly expressed. Accordingly, a miR-143 knockdown resulted in the inhibition of the growth of collateral arteries in mice. Collagen V alpha 2 (COLVA2) was identified by us as a candidate target gene of miR-143 carrying the predicted target sequence and being downregulated in growing collaterals. Methods: To confirm the direct interaction between miR-143 and COLVA2 mRNA, a Dual Luciferase Reporter Assay was performed by cloning the COLVA2-3’UTR-sequence into the miR-specific plasmid vector psiCHECK^TM-2. Results: Co-transfection of this plasmid for 48 h together with an artificial miR-143 construct (but not with the scrambled miRNA-construct) into endothelial cells (Ea.hy926; express only low amounts of miR-143) resulted in increased miR-143 expression while luciferase activity decreased. These data indicate that miR-143 modulates COLVA2 expression by directly targeting the 3'UTR-region of the COLVA2 mRNA. Accordingly, 48 h co-transfection of the plasmid with anti-miR-143 (but not with the scrambled anti-miRNA) in murine vascular smooth muscle cells or NIH/3T3-fibroblasts respectively, which both express high amounts of miR-143, induced the knockdown of miR 143 and upregulated the luciferase expression. Conclusion: These findings demonstrate that miR-143 directly regulates COLVA2 expression, which may play a decisive role in vessel and tissue remodeling during arteriogenesis. Congress President GTH 2017 Prof. Dr. med. Johanna A. Kremer Hovinga, Inselspital, 3010 Bern [email protected] Congress Organisation: Meister ConCept GmbH, Bahnhofstrasse 55, CH-5001 Aarau T +41 62 836 20 90, F +41 62 836 20 97, [email protected] ABSTRACTS of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Phosphorylation of the protein phosphatase 2A inhibitor alpha-endosulfine (ENSA) at two distinct sites, serine 67 and serine 109, in human platelets E. Walter, O. Pagel, R. Zahedi, A. Smolenski, S. Gambaryan, U. Walter, K. Jurk (Mainz, Germany, Dortmund, Germany, Dublin, Ireland, St. Petersburg, Russian Federation)S of the 61 Annual Meeting of the Society of Thrombosis and Hemostasis Research 15-18 February 2017 Basel, Switzerland Phosphorylation of the protein phosphatase 2A inhibitor alpha-endosulfine (ENSA) at two distinct sites, serine 67 and serine 109, in human platelets E. Walter, O. Pagel, R. Zahedi, A. Smolenski, S. Gambaryan, U. Walter, K. Jurk (Mainz, Germany, Dortmund, Germany, Dublin, Ireland, St. Petersburg, Russian Federation) Basic Science 16.02.2017, 11:00 12:00 Objective: The 13 kDa protein alpha-endosulfine (ENSA; or ARPP-19e), originally identified as sulfonylurea receptor ligand, belongs to the highly conserved cAMP-regulated phosphoprotein (ARPP) family with a conserved PKA phosphorylation site (serine 109). In Drosophila and Xenopus, Greatwall kinase phosphorylated ENSA (at serine 67), is a powerful inhibitor of the protein phosphatase 2A (PP2A). In Xenopus, PKA-evoked ENSA S109 phosphorylation keeps the oocyte in prophase whereas S67 phosphorylation is necessary for M-phase entry (1). Very little is known about ENSA in human platelets. Methods: Proteomic and phosphoproteomic studies with human platelets and platelet proteins were carried out as described (2). ENSA was cloned from human platelets and expressed in HEK293 cells and E.coli BL21. ENSA phosphorylation was studied in washed human platelets and with recombinant proteins. Results: In our proteomic studies (2) ENSA was detected in human platelets at significant levels (7800 copies/platelet). By our quantitative platelet phosphoproteomic studies, ENSA S109 was found to be strongly phosphorylated (> 10 fold stimulation) in response to cAMP-(Iloprost) and cGMP-elevating (NO-donors, Riociguat) platelet inhibitors (3, unpublished). ENSA S67 phosphorylation was also detected by phosphoproteomics but down-regulated by these platelet inhibitors. Human platelets have two major ENSA transcripts which allowed its cloning and expression in HEK293 cells and E.coli BL21. Wildtype ENSA as well as various ENSA mutants (at S109 and S67) were then generated, purified and tested as PKA or PKG substrates. Further studies with a phosphosite-specific antibody demonstrated an increase of ENSA S67 phosphorylation in platelets in response to the phosphatase inhibitor okadaic acid. Conclusion: The PP2A phosphatase inhibitor ENSA of human platelets is an excellent PKA and PKG substrate in intact cells and as purified protein. Platelet ENSA S67 phosphorylation was detected for the first time. Ongoing studies aim to identify the ENSA S67 protein kinase and the functional role of this phosphorylation.