Histidine-Rich Glycoprotein Modulates the Toxic Effects of High-Dose Polyphosphate in Mice.

BACKGROUND: Polyphosphate (polyP), a procoagulant released from platelets, activates coagulation via the contact system and modulates cardiomyocyte viability. High-dose intravenous polyP is lethal in mice, presumably because of thrombosis. Previously, we showed that HRG (histidine-rich glycoprotein) binds polyP and attenuates its procoagulant effects. In this study, we investigated the mechanisms responsible for the lethality of intravenous polyP in mice and the impact of HRG on this process. METHODS: The survival of wild-type or HRG-deficient mice given intravenous synthetic or platelet-derived polyP in doses up to 50 mg/kg or saline was compared. To determine the contribution of thrombosis, the effect of FXII (factor XII) knockdown or enoxaparin on polyP-induced fibrin deposition in the lungs was examined. To assess cardiotoxicity, the ECG was continuously monitored, the levels of troponin I and the myocardial band of creatine kinase were quantified, and the viability of a cultured murine cardiomyocyte cell line exposed to polyP in the absence or presence of HRG was determined. RESULTS: In HRG-deficient mice, polyP was lethal at 30 mg/kg, whereas it was lethal in wild-type mice at 50 mg/kg. Although FXII knockdown or enoxaparin administration attenuated polyP-induced fibrin deposition in the lungs, neither affected mortality. PolyP induced dose-dependent ECG abnormalities, including heart block and ST-segment changes, and increased the levels of troponin and myocardial band of creatine kinase, effects that were more pronounced in HRG-deficient mice than in wild-type mice and were attenuated when HRG-deficient mice were given supplemental HRG. Consistent with its cardiotoxicity, polyP reduced the viability of cultured cardiomyocytes in a dose-dependent manner, an effect attenuated with supplemental HRG. CONCLUSIONS: High-dose intravenous polyP is cardiotoxic in mice, and HRG modulates this effect.

Simultaneous inhibition of endocytic recycling and lysosomal fusion sensitizes cells and tissues to oligonucleotide therapeutics.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

Specific targeting of the KRAS mutational landscape in myeloma as a tool to unveil the elicited antitumor activity.

Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.

Prekallikrein inhibits innate immune signaling in the lung and impairs host defense during pneumosepsis in mice.

Prekallikrein (PKK, also known as Fletcher factor and encoded by the gene KLKB1 in humans) is a component of the contact system. Activation of the contact system has been implicated in lethality in fulminant sepsis models. Pneumonia is the most frequent cause of sepsis. We sought to determine the role of PKK in host defense during pneumosepsis. To this end, mice were infected with the common human pathogen Klebsiella pneumoniae via the airways, causing an initially localized infection of the lungs with subsequent bacterial dissemination and sepsis. Mice were treated with a selective PKK-directed antisense oligonucleotide (ASO) or a scrambled control ASO for 3 weeks prior to infection. Host response readouts were determined at 12 or 36 h post-infection, including genome-wide messenger RNA profiling of lungs, or mice were followed for survival. PKK ASO treatment inhibited constitutive hepatic Klkb1 mRNA expression by >80% and almost completely abolished plasma PKK activity. Klkb1 mRNA could not be detected in lungs. Pneumonia was associated with a progressive decline in PKK expression in mice treated with control ASO. PKK ASO administration was associated with a delayed mortality, reduced bacterial burdens, and diminished distant organ injury. While PKK depletion did not influence lung pathology or neutrophil recruitment, it was associated with an upregulation of multiple innate immune signaling pathways in the lungs already prior to infection. Activation of the contact system could not be detected, either during infection in vivo or at the surface of Klebsiella in vitro. These data suggest that circulating PKK confines pro-inflammatory signaling in the lung by a mechanism that does not involve contact system activation, which in the case of respiratory tract infection may impede early protective innate immunity. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines.

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.

Blood-derived plasminogen drives brain inflammation and plaque deposition in a mouse model of Alzheimer's disease.

Two of the most predominant features of the Alzheimer's disease (AD) brain are deposition of ß-amyloid (Aß) plaques and inflammation. The mechanism behind these pathologies remains unknown, but there is evidence to suggest that inflammation may predate the deposition of Aß. Furthermore, immune activation is increasingly being recognized as a major contributor to the pathogenesis of the disease, and disorders involving systemic inflammation, such as infection, aging, obesity, atherosclerosis, diabetes, and depression are risk factors for the development of AD. Plasminogen (PLG) is primarily a blood protein synthesized in the liver, which when cleaved into its active form, plasmin (PL), plays roles in fibrinolysis, wound healing, cell signaling, and inflammatory regulation. Here we show that PL in the blood is a regulator of brain inflammatory action and AD pathology. Depletion of PLG in the plasma of an AD mouse model through antisense oligonucleotide technology dramatically improved AD pathology and decreased glial cell activation in the brain, whereas an increase in PL activity through α-2-antiplasmin (A2AP) antisense oligonucleotide treatment exacerbated the brain's immune response and plaque deposition. These studies suggest a crucial role for peripheral PL in mediating neuroimmune cell activation and AD progression and could provide a link to systemic inflammatory risk factors that are known to be associated with AD development.

The contact system proteases play disparate roles in streptococcal sepsis.

Sepsis causes an activation of the human contact system, an inflammatory response mechanism against foreign surfaces, proteins and pathogens. The serine proteases of the contact system, factor XII and plasma kallikrein, are decreased in plasma of septic patients, which was previously associated with an unfavorable outcome. However, the precise mechanisms and roles of contact system factors in bacterial sepsis are poorly understood. We, therefore, studied the physiological relevance of factor XII and plasma kallikrein in a mouse model of experimental sepsis. We show that decreased plasma kallikrein concentration in septic mice is a result of reduced mRNA expression plasma prekallikrein gene, indicating that plasma kallikrein belong to negative acute phase proteins. Investigations regarding the pathophysiological function of contact system proteases during sepsis revealed different roles for factor XII and plasma kallikrein. In vitro, factor XII decelerated bacteria induced fibrinolysis, whereas plasma kallikrein supported it. Remarkably, depletion of plasma kallikrein (but not factor XII) by treatment with antisense-oligonucleotides, dampens bacterial dissemination and growth in multiple organs in the mouse sepsis model. These findings identify plasma kallikrein as a novel host pathogenicity factor in Streptococcus pyogenes sepsis.

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides.

Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs.

Knockdown of circulating C1 inhibitor induces neurovascular impairment, glial cell activation, neuroinflammation, and behavioral deficits.

The cross-talk between blood proteins, immune cells, and brain function involves complex mechanisms. Plasma protein C1 inhibitor (C1INH) is an inhibitor of vascular inflammation that is induced by activation of the kallikrein-kinin system (KKS) and the complement system. Knockout of C1INH was previously correlated with peripheral vascular permeability via the bradykinin pathway, yet there was no evidence of its correlation with blood-brain barrier (BBB) integrity and brain function. In order to understand the effect of plasma C1INH on brain pathology via the vascular system, we knocked down circulating C1INH in wild-type (WT) mice using an antisense oligonucleotide (ASO), without affecting C1INH expression in peripheral immune cells or the brain, and examined brain pathology. Long-term elimination of endogenous C1INH in the plasma induced the activation of the KKS and peritoneal macrophages but did not activate the complement system. Bradykinin pathway proteins were elevated in the periphery and the brain, resulting in hypotension. BBB permeability, extravasation of plasma proteins into the brain parenchyma, activation of glial cells, and elevation of pro-inflammatory response mediators were detected. Furthermore, infiltrating innate immune cells were observed entering the brain through the lateral ventricle walls and the neurovascular unit. Mice showed normal locomotion function, yet cognition was impaired and depressive-like behavior was evident. In conclusion, our results highlight the important role of regulated plasma C1INH as it acts as a gatekeeper to the brain via the neurovascular system. Thus, manipulation of C1INH in neurovascular disorders might be therapeutically beneficial.

Kininogen deficiency or depletion reduces enhanced pause independent of pulmonary inflammation in a house dust mite-induced murine asthma model.

High-molecular-weight kininogen is an important substrate of the kallikrein-kinin system. Activation of this system has been associated with aggravation of hallmark features in asthma. We aimed to determine the role of kininogen in enhanced pause (Penh) measurements and lung inflammation in a house dust mite (HDM)-induced murine asthma model. Normal wild-type mice and mice with a genetic deficiency of kininogen were subjected to repeated HDM exposure (sensitization on days 0, 1, and 2; challenge on days 14, 15, 18, and 19) via the airways to induce allergic lung inflammation. Alternatively, kininogen was depleted after HDM sensitization by twice-weekly injections of a specific antisense oligonucleotide (kininogen ASO) starting at day 3. In kininogen-deficient mice HDM induced in Penh was completely prevented. Remarkably, kininogen deficiency did not modify HDM-induced eosinophil/neutrophil influx, T helper 2 responses, mucus production, or lung pathology. kininogen ASO treatment started after HDM sensitization reduced plasma kininogen levels by 75% and reproduced the phenotype of kininogen deficiency: kininogen ASO administration prevented the HDM-induced increase in Penh without influencing leukocyte influx, Th2 responses, mucus production, or lung pathology. This study suggests that kininogen could contribute to HDM-induced rise in Penh independently of allergic lung inflammation. Further research is warranted to confirm these data using invasive measurements of airway responsiveness.

Depletion of coagulation factor XII ameliorates brain pathology and cognitive impairment in Alzheimer disease mice.

Vascular abnormalities and inflammation are found in many Alzheimer disease (AD) patients, but whether these changes play a causative role in AD is not clear. The factor XII (FXII) -initiated contact system can trigger both vascular pathology and inflammation and is activated in AD patients and AD mice. We have investigated the role of the contact system in AD pathogenesis. Cleavage of high-molecular-weight kininogen (HK), a marker for activation of the inflammatory arm of the contact system, is increased in a mouse model of AD, and this cleavage is temporally correlated with the onset of brain inflammation. Depletion of FXII in AD mice inhibited HK cleavage in plasma and reduced neuroinflammation, fibrinogen deposition, and neurodegeneration in the brain. Moreover, FXII-depleted AD mice showed better cognitive function than untreated AD mice. These results indicate that FXII-mediated contact system activation contributes to AD pathogenesis, and therefore this system may offer novel targets for AD treatment.

Limited role of kininogen in the host response during gram-negative pneumonia-derived sepsis.

High-molecular-weight kininogen (HK), together with factor XI, factor XII and prekallikrein, is part of the contact system that has proinflammatory, prothrombotic, and vasoactive properties. We hypothesized that HK plays a role in the host response during pneumonia-derived sepsis. To this end mice were depleted of kininogen (KNG) to plasma HK levels of 28% of normal by repeated treatment with a specific antisense oligonucleotide (KNG ASO) for 3 wk before infection with the common human sepsis pathogen Klebsiella pneumoniae via the airways. Whereas plasma HK levels increased during infection in mice treated with a scrambled control ASO (Ctrl ASO), HK level in the KNG ASO-treated group remained reduced to 25-30% of that in the corresponding Ctrl ASO group both before and after infection. KNG depletion did not influence bacterial growth in lungs or dissemination to distant body sites. KNG depletion was associated with lower lung CXC chemokine and myeloperoxidase levels but did not impact neutrophil influx, lung pathology, activation of the vascular endothelium, activation of the coagulation system, or the extent of distant organ injury. These results were corroborated by studies in mice with a genetic deficiency of KNG, which were indistinguishable from wild-type mice during Klebsiella-induced sepsis. Both KNG depletion and KNG deficiency were associated with strongly reduced plasma prekallikrein levels, indicating the carrier function of HK for this zymogen. This study suggests that KNG does not significantly contribute to the host defense during gram-negative pneumonia-derived sepsis.

Inhibition of the extrinsic or intrinsic coagulation pathway during pneumonia-derived sepsis.

Pneumonia is the most frequent cause of sepsis, and Klebsiella pneumoniae is a common pathogen in pneumonia and sepsis. Infection is associated with activation of the coagulation system. Coagulation can be activated by the extrinsic and intrinsic routes, mediated by factor VII (FVII) and factor XII (FXII), respectively. To determine the role of FVII and FXII in the host response during pneumonia-derived sepsis, mice were treated with specific antisense oligonucleotide (ASO) directed at FVII or FXII for 3 wk before infection with K. pneumoniae via the airways. FVII ASO treatment strongly inhibited hepatic FVII mRNA expression, reduced plasma FVII to ~25% of control, and selectively prolonged the prothrombin time. FXII ASO treatment strongly suppressed hepatic FXII mRNA expression, reduced plasma FXII to ~20% of control, and selectively prolonged the activated partial thromboplastin time. Lungs also expressed FVII mRNA, which was not altered by FVII ASO administration. Very low FXII mRNA levels were detected in lungs, which were not modified by FXII ASO treatment. FVII ASO attenuated systemic activation of coagulation but did not influence fibrin deposition in lung tissue. FVII ASO enhanced bacterial loads in lungs and mitigated sepsis-induced distant organ injury. FXII inhibition did not affect any of the host response parameters measured. These results suggest that partial inhibition of FVII, but not of FXII, modifies the host response to gram-negative pneumonia-derived sepsis.

Selective depletion of factor XI or factor XII with antisense oligonucleotides attenuates catheter thrombosis in rabbits.

Central venous catheter thrombosis can cause venous obstruction and pulmonary embolism. To determine the extent to which catheter thrombosis is triggered by the contact or extrinsic pathway of coagulation, we used antisense oligonucleotides (ASOs) to selectively knock down factor (f)XII, fXI, or high-molecular-weight kininogen (HK), key components of the contact pathway, or fVII, which is essential for the extrinsic pathway. Knockdown of contact pathway components prolonged the activated partial thromboplastin time and decreased target protein activity levels by over 90%, whereas fVII knockdown prolonged the prothrombin time and reduced fVII activity to a similar extent. Using a rabbit model of catheter thrombosis, catheters implanted in the jugular vein were assessed daily until they occluded, up to a maximum of 35 days. Compared with control, fXII and fXI ASO treatment prolonged the time to catheter occlusion by 2.2- and 2.3-fold, respectively. In contrast, both HK and fVII knockdown did not significantly prolong the time to occlusion, and dual treatment with fVII- and fXI-directed ASOs produced a time to occlusion similar to that with the fXI ASO alone. These findings suggest that catheter thrombosis is triggered via the contact pathway and identify fXII and fXI as potential targets to attenuate this complication.

Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates.

OBJECTIVE: During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. APPROACH AND RESULTS: We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. CONCLUSIONS: These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.

STAT6-targeting antisense oligonucleotides against solitary fibrous tumor.

Solitary fibrous tumor (SFT) is a rare, non-hereditary soft tissue sarcoma thought to originate from fibroblastic mesenchymal stem cells. The etiology of SFT is thought to be due to an environmental intrachromosomal gene fusion between NGFI-A-binding protein 2 (NAB2) and signal transducer and activator protein 6 (STAT6) genes on chromosome 12, wherein the activation domain of STAT6 is fused with the DNA-binding domain of NAB2 resulting in the oncogenesis of SFT. All NAB2-STAT6 fusion variations discovered in SFTs contain the C-terminal of STAT6 transcript, and thus can serve as target site for antisense oligonucleotides (ASOs)-based therapies. Indeed, our in vitro studies show the STAT6 3' untranslated region (UTR)-targeting ASO (ASO 993523) was able to reduce expression of NAB2-STAT6 fusion transcripts in multiple SFT cell models with high efficiency (half-maximal inhibitory concentration: 116-300 nM). Encouragingly, in vivo treatment of SFT patient-derived xenograft mouse models with ASO 993523 resulted in acceptable tolerability profiles, reduced expression of NAB2-STAT6 fusion transcripts in xenograft tissues (21.9%), and, importantly, reduced tumor growth (32.4% decrease in tumor volume compared with the untreated control). Taken together, our study established ASO 993523 as a potential agent for the treatment of SFTs.

Plasminogen deficiency suppresses pancreatic ductal adenocarcinoma disease progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal metastatic disease associated with robust activation of the coagulation and fibrinolytic systems. However, the potential contribution of the primary fibrinolytic protease plasminogen to PDAC disease progression has remained largely undefined. Mice bearing C57Bl/6-derived KPC (KRasG12D , TRP53R172H ) tumors displayed evidence of plasmin activity in the form of high plasmin-antiplasmin complexes and high plasmin generation potential relative to mice without tumors. Notably, plasminogen-deficient mice (Plg- ) had significantly diminished KPC tumor growth in subcutaneous and orthotopic implantation models. Moreover, the metastatic potential of KPC cells was significantly diminished in Plg- mice, which was linked to reduced early adhesion and/or survival of KPC tumor cells. The reduction in primary orthotopic KPC tumor growth in Plg- mice was associated with increased apoptosis, reduced accumulation of pro-tumor immune cells, and increased local proinflammatory cytokine production. Elimination of fibrin(ogen), the primary proteolytic target of plasmin, did not alter KPC primary tumor growth and resulted in only a modest reduction in metastatic potential. In contrast, deficiencies in the plasminogen receptors Plg-RKT or S100A10 in tumor cells significantly reduced tumor growth. Plg-RKT reduction in tumor cells, but not reduced S100A10, suppressed metastatic potential in a manner that mimicked plasminogen deficiency. Finally, tumor growth was also reduced in NSG mice subcutaneously or orthotopically implanted with patient-derived PDAC tumor cells in which circulating plasminogen was pharmacologically reduced. Collectively, these studies suggest that plasminogen promotes PDAC tumor growth and metastatic potential, in part through engaging plasminogen receptors on tumor cells.

Selective depletion of plasma prekallikrein or coagulation factor XII inhibits thrombosis in mice without increased risk of bleeding.

Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulation factor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had ∼ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases.

Knockdown of coagulation factor VII has potential anticoagulant and anti-inflammatory effects in a mouse model of group A streptococcal sepsis.

Infections originating from subcutaneous tissues are among the most common invasive infections caused by group A streptococcus (GAS) and associated with systemic coagulation activation. The role of intrinsic coagulation factors on GAS virulence has recently been determined, but the role of the extrinsic coagulation factor VII is unknown. Using a mouse model, in which GAS-sepsis emerges from a subcutaneous infection, we show that FVII is a negative acute phase protein. F7 knockdown using antisense oligonucleotides resulted in an attenuated systemic coagulation activation and inflammatory response in septic animals. The findings indicate FVII's ability to modify the host response.

Histidine-rich glycoprotein attenuates catheter thrombosis.

Factor XII (FXII) knockdown attenuates catheter thrombosis in rabbits. Because histidine-rich glycoprotein (HRG) modulates FXIIa activity, we hypothesized that HRG depletion would promote catheter thrombosis. To test this, rabbits were given either antisense oligonucleotides (ASOs) against HRG or FXII, a control ASO, or saline. The activated partial thromboplastin time (aPTT), prothrombin time (PT), and catheter-induced thrombin generation were determined in blood collected before and after treatment. Compared with the controls, the HRG- and FXII-directed ASOs reduced hepatic messenger RNA and plasma levels of HRG and FXII, respectively, by >90%. Although HRG knockdown shortened the aPTT by 2.5 fold, FXII knockdown prolonged it by fourfold; neither of the ASOs affected the PT. Catheter segments shortened the lag time and increased peak thrombin in the plasma from control rabbits; effects were significantly enhanced and attenuated in the plasma from rabbits given the HRG- and FXII-directed ASOs, respectively. Catheters were then inserted into the right external jugular vein of the rabbits, and the time for catheter occlusion was determined. The catheter occlusion times with the control ASO or saline were 62 ± 8 minutes and 60 ± 11 minutes, respectively. The occlusion time was significantly reduced to 34 ± 9 minutes, with HRG knockdown and significantly prolonged to 128 ± 19 minutes with FXII knockdown. HRG levels are decreased with sepsis or cancer, and such patients are prone to catheter thrombosis. Because HRG modulates catheter thrombosis, our findings suggest that HRG supplementation may prevent this problem.