Impact of allele-selective silencing of von Willebrand factor in mice based on a single nucleotide allelic difference in von Willebrand factor.

INTRODUCTION: Von Willebrand factor (VWF) plays a pathophysiological role in hemostatic disorders. Partial inhibition of the VWF gene through small interfering RNA (siRNA)-mediated allele-selective silencing could be a promising therapeutic strategy. For von Willebrand disease, allele-selectively inhibiting dominant-negative VWF-alleles might ameliorate the phenotype. For thrombotic disorders, partial VWF reduction can lower thrombotic risk, while avoiding bleeding. Previously, we demonstrated the feasibility of Vwf-silencing in homozygous C57BL/6J (B6) or 129S1/SvImJ (129S) mice. The present study investigated allele-selective Vwf-silencing in a complex heterozygous setting of crossed B6 and 129S mice and its subsequent hemostatic impact. MATERIALS AND METHODS: Heterozygous B6.129S mice were treated with siRNAs targeting Vwf expressed from either B6- (siVwf.B6) or 129S-alleles (siVwf.129S). Plasma VWF and lung Vwf mRNA were determined. siVwf.B6-treated B6.129S mice were subjected to ferric chloride-induced mesenteric vessel thrombosis and tail-bleeding. RESULTS: In B6.129S mice, siVwf.B6 reduced Vwf mRNA of the targeted B6-allele by 72% vs. only 12% of the non-targeted 129S-allele (41% total mRNA reduction), lowering plasma VWF by 46%. Oppositely, siVwf.129S reduced Vwf mRNA by 45%, now selectively inhibiting the 129S-allele over the B6-allele (58% vs. 9%), decreasing plasma VWF by 43%. The allele-selective VWF reduction by siVwf.B6 coincided with decreased thrombus formation in mesenteric arterioles, without prolonging tail-bleeding times. CONCLUSIONS: This study demonstrates the feasibility of allele-selective Vwf-silencing in a heterozygous setting, achieving a controlled close to 50% reduction of plasma VWF. The observed thromboprotection and absence of prolonged bleeding times underline the potential of allele-selective Vwf-silencing as a therapeutic strategy in hemostatic disorders.

Small interfering RNA-mediated allele-selective silencing of von Willebrand factor in vitro and in vivo.

An imbalance in von Willebrand factor (VWF) may either lead to bleeding (von Willebrand disease, VWD) or thrombosis. Both disorders have shortcomings in the currently available treatments. VWF itself could be a potential therapeutic target because of its role in both bleeding and thrombosis. Inhibiting VWF gene expression through allele-selective silencing of VWF with small interfering RNAs (siRNAs) could be a personalized approach to specifically inhibit mutant VWF in VWD or to normalize increased VWF levels in thrombotic disorders without complete VWF knockdown. Therefore, we investigated a method to allele-selectively silence the VWF gene in mice as a therapeutic strategy. Fourteen candidate siRNAs targeting murine Vwf of either the C57BL/6J (B6) or the 129S1/SvImJ (129S) strain were tested in vitro in cells expressing B6- and 129S-Vwf for inhibitory effect and allele-selective potential. Together with a nonselective siVwf, 2 lead candidate siRNAs, siVwf.B6 and siVwf.129S, were further tested in vivo in B6 and 129S mice. Efficient endothelial siRNA delivery was achieved by siRNA encapsulation into 7C1 oligomeric lipid nanoparticles. Treatment with the nonselective siVwf resulted in dose-dependent inhibition of up to 80% of both lung messenger RNA and plasma VWF protein in both mouse strains. In contrast, the allele-selective siVwf.B6 and siVwf.129S were shown to be effective in and selective solely for their corresponding mouse strain. To conclude, we showed efficient endothelial delivery of siRNAs that are highly effective in allele-selective inhibition of Vwf in mice, which constitutes an in vivo proof of principle of allele-selective VWF silencing as a therapeutic approach.

Influence of rosuvastatin on apolipoproteins and coagulation factor levels: Results from the STAtin Reduce Thrombophilia trial.

Background: The STAtins Reduce Thrombophilia trial showed that, in patients with prior venous thrombosis, rosuvastatin decreased various coagulation factor levels. Objectives: Here, we investigated the hypothesis that statins decrease coagulation factor levels through shared mechanisms of synthesis or regulatory pathways with apolipoproteins. Methods: We measured the levels of apolipoprotein (Apo)A-I, A-II, A-IV, (a), B-100, B-total, C-I, C-II, C-III, and E in patients (n = 126) randomized to 28 days of rosuvastatin use. We assessed the association between apolipoproteins and coagulation factors at baseline using linear regression. The mean difference in apolipoprotein levels between baseline and after 28 days of rosuvastatin use was determined through linear regression, adjusting for age, sex, and body mass index. Coagulation factors were added to this model to determine if the lowering of apolipoproteins by rosuvastatin was linked with coagulation factor levels. Results: At baseline, levels of all apolipoproteins, except Apo(a), were positively associated with FVII, FIX, and FXI. Apolipoproteins levels, except for ApoA-I, A-IV, and Apo(a), were decreased after 28 days of rosuvastatin. ApoB-100 showed the largest mean decrease of -0.43 g/L (95% CI = -0.46 to -0.40). The decrease in ApoC-I and C-III levels was associated with a decrease in FVII, whereas the decrease in apoA-II, B-100, and B-total was associated with a decrease in FXI. The decrease in apolipoproteins was neither associated with FVIII or vWF decrease nor with endogenous thrombin potential changes. Conclusions: Rosuvastatin decreases the level of several apolipoproteins, but this decrease was associated only with a decrease in FVII and XI and not with FVIII/vWF.

Tumor-expressed factor VII is associated with survival and regulates tumor progression in breast cancer.

Cancer enhances the risk of venous thromboembolism, but a hypercoagulant microenvironment also promotes cancer progression. Although anticoagulants have been suggested as a potential anticancer treatment, clinical studies on the effect of such modalities on cancer progression have not yet been successful for unknown reasons. In normal physiology, complex formation between the subendothelial-expressed tissue factor (TF) and the blood-borne liver-derived factor VII (FVII) results in induction of the extrinsic coagulation cascade and intracellular signaling via protease-activated receptors (PARs). In cancer, TF is overexpressed and linked to poor prognosis. Here, we report that increased levels of FVII are also observed in breast cancer specimens and are associated with tumor progression and metastasis to the liver. In breast cancer cell lines, tumor-expressed FVII drives changes reminiscent of epithelial-to-mesenchymal transition (EMT), tumor cell invasion, and expression of the prometastatic genes, SNAI2 and SOX9. In vivo, tumor-expressed FVII enhanced tumor growth and liver metastasis. Surprisingly, liver-derived FVII appeared to inhibit metastasis. Finally, tumor-expressed FVII-induced prometastatic gene expression independent of TF but required a functional endothelial protein C receptor, whereas recombinant activated FVII acting via the canonical TF:PAR2 pathway inhibited prometastatic gene expression. Here, we propose that tumor-expressed FVII and liver-derived FVII have opposing effects on EMT and metastasis.

Absence of COVID-19-associated changes in plasma coagulation proteins and pulmonary thrombosis in the ferret model.

BACKGROUND: Many patients who are diagnosed with coronavirus disease 2019 (COVID-19) suffer from venous thromboembolic complications despite the use of stringent anticoagulant prophylaxis. Studies on the exact mechanism(s) underlying thrombosis in COVID-19 are limited as animal models commonly used to study venous thrombosis pathophysiology (i.e. rats and mice) are naturally not susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ferrets are susceptible to SARS-CoV-2 infection, successfully used to study virus transmission, and have been previously used to study activation of coagulation and thrombosis during influenza virus infection. OBJECTIVES: This study aimed to explore the use of (heat-inactivated) plasma and lung material from SARS-CoV-2-inoculated ferrets studying COVID-19-associated changes in coagulation and thrombosis. MATERIAL AND METHODS: Histology and longitudinal plasma profiling using mass spectrometry-based proteomics approach was performed. RESULTS: Lungs of ferrets inoculated intranasally with SARS-CoV-2 demonstrated alveolar septa that were mildly expanded by macrophages, and diffuse interstitial histiocytic pneumonia. However, no macroscopical or microscopical evidence of vascular thrombosis in the lungs of SARS-CoV-2-inoculated ferrets was found. Longitudinal plasma profiling revealed minor differences in plasma protein profiles in SARS-CoV-2-inoculated ferrets up to 2 weeks post-infection. The majority of plasma coagulation factors were stable and demonstrated a low coefficient of variation. CONCLUSIONS: We conclude that while ferrets are an essential and well-suited animal model to study SARS-CoV-2 transmission, their use to study SARS-CoV-2-related changes relevant to thrombotic disease is limited.

Atherothrombosis model by silencing of protein C in APOE*3-Leiden.CETP transgenic mice.

Murine atherosclerosis models are key for investigation of atherosclerosis pathophysiology and drug development. However, they do not feature spontaneous atherothrombosis as a final stage of atherosclerosis. Transgenic mice expressing both the human mutant apolipoprotein E form APOE*3-Leiden and human cholesteryl ester transfer protein (CETP), i.e. APOE*3-Leiden.CETP mice, feature a moderate hyperlipoproteinemia and atherosclerosis phenotype. In contrast to apolipoprotein E deficient (Apoe-/-) mice, APOE*3-Leiden.CETP mice respond well to lipid-lowering and anti-atherosclerotic drugs. The aim of the study was to investigate whether silencing of anticoagulant Protein C (Proc) allows APOE*3-Leiden.CETP mice to feature thrombosis as a final stage of atherosclerosis. Female APOE*3-Leiden.CETP mice were fed a Western-type diet to induce advanced atherosclerosis, followed by an injection with a small interfering RNA targeting Proc (siProc). Presence of atherosclerosis and atherothrombosis was determined by histologic analysis of the aortic root. Atherosclerosis severity in the aortic root area of APOE*3-Leiden.CETP mice varied from type "0" (no lesions) to type "V" lesions (advanced and complex lesions). Atherothrombosis following siProc injection was observed for 4 out of 21 APOE*3-Leiden.CETP mice (19% incidence). The atherothrombosis presented as large, organized, fibrin- and leukocyte-rich thrombi on top of advanced (type "V") atherosclerotic plaques in the aortic root. This atherothrombosis was comparable in appearance and incidence as previously reported for Apoe-/- mice with a more severe atherosclerosis (19% incidence). APOE*3-Leiden.CETP mice with modest hyperlipidemia and atherosclerosis can develop atherothrombosis upon transient Proc-silencing. This further extends the use of these mice as a test model for lipid-lowering and anti-atherosclerotic drugs.

Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rates.

Genome-wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for von Willebrand factor (VWF). To explain the link between Slc44a2 and VT, we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100 s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100 s-1. This adhesion could occur independently of ß2 integrin and was enhanced when neutrophils were preactivated with lipopolysaccharide. Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit," inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. The fact that neutrophils expressing Slc44a2/HNA-3b have a different response on VWF in the conditions tested could thus explain the association between HNA-3b and a reduced risk for VT in humans.

Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequent VWF single nucleotide polymorphisms to inhibit mutant VWF. The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes.

SLC44A2 deficient mice have a reduced response in stenosis but not in hypercoagulability driven venous thrombosis.

BACKGROUND: Genome wide association studies (GWAS) identified SLC44A2 as a novel susceptibility gene for venous thrombosis (VT) and previous work established that SLC44A2 contributed to clot formation upon vascular injury. OBJECTIVE: To further investigate the role of SLC44A2 in VT by utilizing SLC44A2 deficient mice (Slc44a2-/- ) in two representative disease models. METHODS: Mice were included in a hypercoagulability model driven by siRNA-mediated hepatic gene silencing of anticoagulants Serpinc1 (antithrombin) and Proc (protein C) and a flow restriction (stenosis) model induced by partial ligation of the inferior vena cava. RESULTS: In the hypercoagulability model, no effect in onset was observed in Slc44a2-/- animals; however, a drop in plasma fibrinogen and von Willebrand factor coinciding with an increase in blood neutrophils was recorded. In the neutrophil dependent stenosis model after 48 hours, Slc44a2-/- mice had significantly smaller thrombi both in length and weight with less platelet accumulation as a percentage of the total thrombus area. During the initiation of thrombosis at 6 hours post-stenosis, Slc44a2-/- mice also had smaller thrombi both in length and weight, with circulating platelets remaining elevated in Slc44a2-/- animals. Platelet activation and aggregation under both static- and venous and arterial shear conditions were normal for blood from Slc44a2-/- mice. CONCLUSIONS: These studies corroborate the original GWAS findings and establish a contributing role for SLC44A2 during the initiation of VT, with indications that this may be related to platelet-neutrophil interaction. The precise mechanism however remains elusive and warrants further investigation.

Plasma Protein Signatures of a Murine Venous Thrombosis Model and Slc44a2 Knockout Mice Using Quantitative-Targeted Proteomics.

The plasma compartment of the blood holds important information on the risk to develop cardiovascular diseases such as venous thrombosis (VT). Mass spectrometry-based targeted proteomics with internal standards quantifies proteins in multiplex allowing generation of signatures associated with a disease or a condition. Here, to demonstrate the method, we investigate the plasma protein signatures in mice following the onset of VT, which was induced by RNA interference targeting the natural anticoagulants antithrombin and protein C. We then study mice lacking Slc44a2, which was recently characterized as a VT-susceptibility gene in human genome-wide association studies. We use a recently developed panel of 375 multiplexed mouse protein assays measured by mass spectrometry. A strong plasma protein siganture was observed when VT was induced. Discriminators included acute phase response proteins, and proteins related to erythrocyte function. In mice lacking Slc44a2, protein signature was primarily overruled by the difference between sexes and not by the absent gene. Upon separate analyses for males and females, we were able to establish a signature for Slc44a2 deficiency, in which glycosylation-dependent cell adhesion molecule-1 and thrombospondin-1 were shared by both sexes. The minimal impact of Slc44a2 deficiency on the measured plasma proteins suggests that the main effect of Slc44a2 on VT does not lay ultimately in the plasma compartment. This suggests further investigation into the role of this VT-susceptibility gene should perhaps also question the possible involvement in cellular mechanisms.

Low human and murine Mcl-1 expression leads to a pro-apoptotic plaque phenotype enriched in giant-cells.

The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) plays an important role in survival and differentiation of leukocytes, more specifically of neutrophils. Here, we investigated the impact of myeloid Mcl-1 deletion in atherosclerosis. Western type diet fed LDL receptor-deficient mice were transplanted with either wild-type (WT) or LysMCre Mcl-1fl/fl (Mcl-1-/-) bone marrow. Mcl-1 myeloid deletion resulted in enhanced apoptosis and lipid accumulation in atherosclerotic plaques. In vitro, Mcl-1 deficient macrophages also showed increased lipid accumulation, resulting in increased sensitivity to lipid-induced cell death. However, plaque size, necrotic core and macrophage content were similar in Mcl-1-/- compared to WT mice, most likely due to decreased circulating and plaque-residing neutrophils. Interestingly, Mcl-1-/- peritoneal foam cells formed up to 45% more multinucleated giant cells (MGCs) in vitro compared to WT, which concurred with an increased MGC presence in atherosclerotic lesions of Mcl-1-/- mice. Moreover, analysis of human unstable atherosclerotic lesions also revealed a significant inverse correlation between MGC lesion content and Mcl-1 gene expression, coinciding with the mouse data. Taken together, these findings suggest that myeloid Mcl-1 deletion leads to a more apoptotic, lipid and MGC-enriched phenotype. These potentially pro-atherogenic effects are however counteracted by neutropenia in circulation and plaque.

Use of "C9/11 Mismatch" Control siRNA Reveals Sequence-Related Off-Target Effect on Coagulation of an siRNA Targeting Mouse Coagulation Factor XII.

Recently, our group reported that a small interfering RNA (siRNA) targeting coagulation factor XII (siF12) leads to an unexpected prothrombotic response in a mouse model where venous thrombosis follows inhibition of endogenous anticoagulants. In this study, we aimed to clarify this unexpected response by evaluating the effects of this siF12 (here, siF12-A) on plasma coagulation through thrombin generation (TG). Besides a routine negative control siRNA (siNEG), we included extra siRNA controls: one siRNA similar to siF12-A except for positions 9-11 of the siRNA that are replaced with its complementary base pairs (siF12-AC9/11), and a second siRNA against F12 (siF12-B). Three days after injection, a significant increase in TG peak height was observed solely for animals injected with siF12-A and siF12-AC9/11, which is considered prothrombotic. As this change in coagulation was unrelated to FXII we conclude that it was off-target. For siRNA studies we now recommend to include mismatch siRNA controls, such as the C9/11 mismatch control used in this study, and to consider plasma coagulation in off-target analysis.

Mouse venous thrombosis upon silencing of anticoagulants depends on tissue factor and platelets, not FXII or neutrophils.

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus.

Characterization of hemostasis in mice lacking the novel thrombosis susceptibility gene Slc44a2.

INTRODUCTION: Recent genome wide association studies (GWAS) identified a novel susceptibility locus for thrombosis, harbouring the SLC44A2 gene which encodes the Solute Carrier Family 44 Member 2 protein (SLC44A2). Thus far, SLC44A2 has not been studied in the context of thrombosis, and may be a unique contributor to thrombotic disease. Here we utilize mice lacking SLC44A2 (Slc44a2-/-) to evaluate a possible role of SLC44A2 in hemostasis. METHODS: Slc44a2-/- mice were evaluated in key aspects of normal hemostasis including a challenge of vascular damage by applying laser induced injury to the cremaster muscle arteriole. RESULTS: Slc44a2-/- mice had comparable levels of thrombin generation and gene expression of coagulation related genes, as compared to littermate wild type controls. Lower levels of circulating plasma Von Willebrand factor (VWF) were measured in Slc44a2-/- mice, while no difference in VWF multimerization or vascular localization was detected. Upon in vivo laser injury of the cremaster arterioles, we detected an impairment of clot formation for Slc44a2-/- mice. CONCLUSIONS: Although mice lacking SLC44A2 are normal for several hemostasis parameters, we do observe a reduction of plasma VWF levels and an altered response upon vascular damage, which suggests that SLC44A2 contributes to hemostasis upon injury. These findings are in line with the reported GWAS data and support further research on SLC44A2 in thrombosis.

Predilection of Low Protein C-induced Spontaneous Atherothrombosis for the Right Coronary Sinus in Apolipoprotein E deficient mice.

Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe-/-) mice. The aims of the current study were (1) to analyze if plaque characteristics or circulating factors could be linked to atherothrombosis susceptibility, (2) to increase the incidence of atherothrombosis by transiently increasing blood pressure, and (3) to direct atherothrombosis to an additional predefined vascular site by applying a semi-constrictive collar around the carotid artery. siProc-driven spontaneous atherothrombosis in the aortic root of Apoe-/- mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe-/- mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, serum amyloid A, and IL-6 were not different amongst siProc treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe-/- mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.

Leukocyte Bim deficiency does not impact atherogenesis in ldlr -/- mice, despite a pronounced induction of autoimmune inflammation.

Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim -/- or wild type bone marrow transplanted ldlr -/- mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim -/- transplanted mice displayed splenomegaly and overt lymphocytosis. CD4+ and CD8+ T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim -/- mice. Bim -/- mice displayed massive intraplaque accumulation of Ig complexes and of lesional T cells, although this did not translate in changes in plaque size or stability features (apoptotic cell and macrophage content). The surprising lack in plaque phenotype despite the profound pro-atherogenic immune effects may be attributable to the sharp reduction of serum cholesterol levels in WTD fed bim -/- mice.

Silencing of Anticoagulant Protein C Evokes Low-Incident but Spontaneous Atherothrombosis in Apolipoprotein E-Deficient Mice-Brief Report.

OBJECTIVE: Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to progress toward an atherothrombotic phenotype. APPROACH AND RESULTS: On lowering of plasma protein C levels with small interfering RNA (siProc) in 8-week Western-type diet-fed atherosclerotic apolipoprotein E-deficient mice, 1 out of 4 mice displayed a large, organized, and fibrin- and leukocyte-rich thrombus on top of an advanced atherosclerotic plaque located in the aortic root. Although again at low incidence (3 in 25), comparable thrombi at the same location were observed during a second independent experiment in 9-week Western-type diet-fed apolipoprotein E-deficient mice. Mice with thrombi on their atherosclerotic plaques did not show other abnormalities and had equally lowered plasma protein C levels as siProc-treated apolipoprotein E-deficient mice without thrombi. Fibrinogen and thrombin-antithrombin concentrations and blood platelet numbers were also comparable, and plaques in siProc mice with thrombi had a similar composition and size as plaques in siProc mice without thrombi. Seven out of 25 siProc mice featured clots in the left atrium of the heart. CONCLUSIONS: Our findings indicate that small interfering RNA-mediated silencing of protein C in apolipoprotein E-deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications.

Oligonucleotides targeting coagulation factor mRNAs: use in thrombosis and hemophilia research and therapy.

Small interfering (si) RNAs and antisense oligonucleotides (ASOs; here for simplicity reasons, both referred to as oligonucleotides) are small synthetic RNA or DNA molecules with a sequence complementary to a (pre)mRNA. Although the basic mechanisms of action between siRNAs and ASO are distinct, a sequence-specific interaction of the both oligonucleotides with the target (pre)mRNA alters the target's fate, which includes highly effective sequence-specific blockade of translation and consequently depletion of the corresponding protein. For a number of years, these oligonucleotides have been used as a tool in biological research to study gene function in vitro. More recently, safe and specific delivery of these oligonucleotides to the liver of mammals has been achieved and optimized. This not only allowed their use for in vivo gene studies in physiology and disease, but also opened the opportunity for the development of a new generation of RNA-specific drugs for therapeutic purposes. In 2013, the first oligonucleotide product targeting RNA from the hepatic cholesterol pathway was approved. For blood coagulation, a large portion of key proteins are produced in the liver, and thereby siRNAs and ASOs can also be used as appropriate tools to target these proteins in vivo. In this review, we describe the first use of oligonucleotides for this purpose from zebrafish to primates. As the use of oligonucleotides allows avoidance of early lethality associated with full deficiency of several coagulation factors, it has proved to be of value for studying these proteins in physiology and disease. Currently, oligonucleotides are tested as therapeutics, with the ultimate goal to beneficially modulate the hemostatic balance in thrombosis and hemophilia patients. We discuss both the preclinical and clinical studies of a number of siRNAs and ASOs with the potential to be introduced as drugs for prophylactic and/or treatment of thrombosis or hemophilia. We conclude that for the coagulation field, oligonucleotides are of value for research purposes, and now the moment has come to fulfill their promise as therapeutics.

Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice.

Recently, platelets, neutrophils, and factor XII (FXII) have been implicated as important players in the pathophysiology of venous thrombosis. Their role became evident in mouse models in which surgical handling was used to provoke thrombosis. Inhibiting anticoagulation in mice by using small interfering RNA (siRNA) targeting Serpinc1 and Proc also results in a thrombotic phenotype, which is spontaneous (no additional triggers) and reproducibly results in clots in the large veins of the head and fibrin deposition in the liver. This thrombotic phenotype is fatal but can be fully rescued by thrombin inhibition. The mouse model was used in this study to investigate the role of platelets, neutrophils, and FXII. After administration of siRNAs targeting Serpinc1 and Proc, antibody-mediated depletion of platelets fully abrogated the clinical features as well as microscopic aspects in the head. This was corroborated by strongly reduced fibrin deposition in the liver. Whereas neutrophils were abundant in siRNA-triggered thrombotic lesions, antibody-mediated depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, or thrombus morphology. In addition, absence of circulating neutrophils did not affect quantitative liver fibrin deposition. Remarkably, siRNA-mediated depletion of plasma FXII accelerated the onset of the clinical phenotype; mice were affected with more severe thrombotic lesions. To summarize, in this study, onset and severity of the thrombotic phenotype are dependent on the presence of platelets but not circulating neutrophils. Unexpectedly, FXII has a protective effect. This study challenges the proposed roles of neutrophils and FXII in venous thrombosis pathophysiology.