Factor XII silencing using siRNA prevents thrombus formation in a rat model of extracorporeal life support (original) (raw)
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Biomaterials
Blood contact with high surface area medical devices, such as dialysis and extracorporeal life support (ECLS), induces rapid surface coagulation. Systemic anticoagulation, such as heparin, is thus necessary to slow clot formation, but some patients suffer from bleeding complications. Both problems might be reduced by 1) replacing heparin anticoagulation with artificial surface inhibition of the protein adsorption that initiates coagulation and 2) selective inhibition of the intrinsic branch of the coagulation cascade. This approach was evaluated by comparing clot formation and bleeding times during short-term ECLS using zwitterionic polycarboxybetaine (PCB) surface coatings combined with either a potent, selective, bicyclic peptide inhibitor of activated Factor XII (FXII900) or standard heparin anticoagulation. Rabbits underwent venovenous ECLS with small sham oxygenators for 60 min using three means of anticoagulation (n = 4 ea): (1) PCB coating + FXII900 infusion, (2) PCB coating + heparin infusion with an activated clotting time of 220-300s, and (3) heparin infusion alone. Sham oxygenator blood clot weights in the PCB + FXII900 and PCB + heparin groups were 4% and 25% of that in the heparin group (p < 10 − 6 and p < 10 − 5), respectively. At the same time, the bleeding time remained normal in the PCB + FXII900 group (2.4 ± 0.2 min) but increased to 4.8 ± 0.5 and 5.1 ± 0.7 min in the PCB + heparin and heparin alone groups (p < 10 − 4 and 0.01). Sham oxygenator blood flow resistance was significantly lower in the PCB + FXII900 and PCB + heparin groups than in the heparin only group (p < 10 − 6 and 10 − 5). These results were confirmed by gross and scanning electron microscopy (SEM) images and fibrinopeptide A (FPA) concentrations. Thus, the combined use of PCB coating and FXII900 markedly reduced sham oxygenator coagulation and tissue bleeding times versus the clinical standard of heparin anticoagulation and is a promising anticoagulation method for clinical ECLS.
Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation
Proceedings of the National Academy of Sciences, 2007
Upon vascular injury, locally controlled haemostasis prevents lifethreatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural ''foreign surface'' and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies.
European Journal of Cardio-Thoracic Surgery, 2015
OBJECTIVES: Heparin and protamine are standard for anticoagulation and reversal for cardiopulmonary bypass (CPB). The REGADO biosciences protocol 1 (REG1) anticoagulant system, consisting of the Factor IXa (FIXa)-inhibitor pegnivacogin and its reversal agent (anivamersen), has been studied in patients undergoing coronary catheterization and in CPB in sheep and pigs. Prior to first human use in CPB, we wanted to test the safety and efficacy of REG1 in a primate model. METHODS: Fourteen baboons undergoing 2 h of CPB followed by 1 h of reperfusion were studied. Three received heparin/protamine and 11 received 1 of 2 doses of pegnivacogin followed by anivamersen. Thrombin-generating capacity was tested in additional in vitro experiments. RESULTS: Targeted drug levels and near-complete FIXa inhibition were achieved. Bypass was run uneventfully in all animals without any clotting in the circuit and bleeding was minimal in the two groups. However, in contrast to heparin-treated baboons, those receiving pegnivacogin/anivamersen displayed thrombi in the bypass cannulae upon cannulation and kidney cortical infarcts. Inter-species comparisons revealed that in the presence of high levels of FIXa inhibition, tissue factor-mediated thrombin generation in baboons was much higher than that in other species. CONCLUSIONS: These data highlight the limitations of the baboon model for assessing factor-specific coagulation inhibitors during CPB. The justification for Phase 1 human studies using REG1 for CPB is unclear.
Antithrombotic Effect of Tissue Factor Inhibition by Inactivated Factor VIIa: An Ex Vivo Human Study
Arteriosclerosis, Thrombosis, and Vascular Biology, 2002
FFR-rFVIIa is an inactivated recombinant factor VIIa (rFVIIa) that inhibits the binding of factor VIIa to tissue factor (TF). It has been shown to prevent TF-induced thrombosis in animals. The present study is a substudy of the Active Site Inhibited Seven (ASIS) trial and examines the antithrombotic effect of 3 doses of FFR-rFVIIa in 24 patients undergoing percutaneous coronary intervention (PCI). Group 1 (nϭ9) received 400 g/kg FFR-rFVIIa and 40 to 50 U/kg heparin, group 2 (nϭ7) received 200 g/kg FFR-rFVIIa and 100 U/kg heparin, and group 3 (nϭ8) received 50 g/kg FFR-rFVIIa and 100 U/kg heparin. Blood thrombogenicity was assessed as total thrombus area and fibrin deposition on the perfusion chamber at shear rate conditions typical of mild-moderate coronary stenosis. Baseline blood thrombogenicity was evaluated a day before PCI, after heparin administration. A second perfusion chamber study was performed just before PCI, 15 minutes after the administration of heparin and FFR-rFVIIa. Thrombus formation at a high shear rate was markedly reduced in groups 1 and 2 after drug administration, by 79% to 84% and 76% to 87%, respectively (PϽ0.004 [group 1], PϽ0.04 [group 2]). In group 3, moderate thrombus reduction of 46% to 48% was achieved (PϽ0.04). Fibrin deposition in all 3 groups was nearly eliminated after drug administration. Our data demonstrate that FFR-rFVIIa has a potent antithrombotic effect at different shear rates and severe arterial injury conditions. (Arterioscler Thromb Vasc Biol. 2002;22:1036-1041.)
Trials, 2022
Background: Although life-saving in selected patients, ECMO treatment still has high mortality which for a large part is due to treatment-related complications. A feared complication is ischemic stroke for which heparin is routinely administered for which the dosage is usually guided by activated partial thromboplastin time (aPTT). However, there is no relation between aPTT and the rare occurrence of ischemic stroke (1.2%), but there is a relation with the much more frequent occurrence of bleeding complications (55%) and blood transfusion. Both are strongly related to outcome. Methods: We will conduct a three-arm non-inferiority randomized controlled trial, in adult patients treated with ECMO. Participants will be randomized between heparin administration with a target of 2-2.5 times baseline aPTT, 1.5-2 times baseline aPTT, or low molecular weight heparin guided by weight and renal function. Apart from anticoagulation targets, treatment will be according to standard care. The primary outcome parameter is a combined endpoint consisting of major bleeding including hemorrhagic stroke, severe thromboembolic complications including ischemic stroke, and mortality at 6 months. Discussion: We hypothesize that with lower anticoagulation targets or anticoagulation with LMWH during ECMO therapy, patients will have fewer hemorrhagic complications without an increase in thromboembolic complication or a negative effect on their outcome. If our hypothesis is confirmed, this study could lead to a change in anticoagulation protocols and a better outcome for patients treated with ECMO.
Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice
Blood, 2016
Recently, platelets, neutrophils, and factor XII have been implicated as important players in the pathophysiology of venous thrombosis. Their role became evident in mouse models where surgical handlings were used to provoke thrombosis. Inhibition of anticoagulation in mice using small interfering (si) RNA 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. In the present study, this model was used to investigate the role of platelets, neutrophils, and factor XII. 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 wer...
Journal of Thrombosis and Haemostasis, 2008
Background: Heparin and low molecular weight heparin (LMWH) are widely used for prevention and treatment of thromboemobolic events, but may occasionally cause uncontrollable bleeding. Heparin can readily be antagonized with protamine, but this is less effective against LMWH. Objectives: To test the effects of rFVIIa or an analogue of rFVIIa, NN1731, on heparin-and LMWH-induced bleeding in rats. Methods: Initially the doses of heparin and tinzaparin (a LMWH) were determined by dose-titration. Following pretreatment with heparin or tinzaparin in rats, tail-transection was performed, and the effect of rFVIIa and NN1731 on the bleeding was observed. Results: rFVIIa (5, 10 and 20 mg kg)1) reduced bleeding time and blood loss caused by heparin-and tinzaparin-induced bleeding, using doses of 200 IU kg)1 (n = 8) and 500 IU kg)1 (n = 9), respectively. Similarly, 10 mg kg)1 NN1731 significantly reduced both heparin-and tinzaparin-induced bleeding to the normal level. Following severe anticoagulation with 1800 IU kg)1 tinzaparin, 10 mg kg)1 NN1731 reduced and normalized the bleeding, while the effect of 20 mg kg)1 rFVIIa failed to reach statistical significance. These data are consistent with the hypothesis that rFVIIa/NN1731 are capable of generating sufficient thrombin locally on the surface of activated platelets to induce hemostasis in the presence of heparin/LMWH. Conclusions: This study suggests that rFVIIa and NN1731 may have the potential to control bleedings caused by heparin or LMWH.
Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor
Nucleic Acids Research, 2002
Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequencespecific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3′-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3′ mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.
Anti-thrombotic and haemorrhagic effects of active site-inhibited factor VIIa in rats
British Journal of Haematology, 2001
Active site-inhibited factor VIIa (FFR-rFVIIa) competes with factor VIIa (FVIIa) for binding to tissue factor (TF) and exerts an anti-thrombotic effect. We report an evaluation of the anti-thrombotic properties of FFR-rFVIIa in a model of thrombosis involving two thrombogenic surfaces. Uncoated glass capillaries or glass capillaries coated with TF were incorporated into an arterioarterial shunt in the rat and the occlusion time (OT) of the shunt was determined. An anti-thrombotic activity of FFR-rFVIIa was shown only on the TF-coated surface: the OT of the shunt was significantly prolonged, from 167^34 s in control animals to 312^42 s after i.v. bolus administration of 4 mg/kg FFR-rFVIIa. This OT was similar to those observed with the uncoated shunts in untreated animals (353^84 s). In vitro preincubation of the TF-coated shunt with FFR-rFVIIa significantly prolonged the OT to 245^45 s in the absence of detectable amounts of FFR-rFVIIa in the plasma. rFFR-rFVIIa weakly prolonged the tail template bleeding time by a factor of 1´5. This effect was more pronounced in animals pretreated with heparin. The anti-thrombotic and prohaemorrhagic effects of FFR-rFVIIa were totally reversed by administration of an equidose of rFVIIa. These results provide new information on the pharmacological properties of FFR-rFVIIa that will be useful for its clinical development.