Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice (original) (raw)
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2022
Background: Heparin anticoagulation increases the bleeding risk during extracorporeal life support (ECLS) and thus must be limited and monitored carefully. This study determined whether factor XII (FXII) silencing using short interfering RNA (siRNA) can provide ECLS circuit anticoagulation, maintain normal tissue coagulation, and reduce the need for careful anticoagulation monitoring.Methods: Adult male, Sprague-Dawley rats were randomized to four groups (n = 3 each) based on anticoagulant: (1) no anticoagulant, (2) heparin, (3) FXII siRNA, or (4) non-targeting siRNA. In group 1, no anticoagulant was administered. In group 2, heparin was administered intravenously before and during ECLS at an activated clotting time of 180–250 seconds. In groups 3 and 4, siRNA was administered intravenously three days before the initiation of ECLS via lipidoid nanoparticles. The rats were placed on pumped, arteriovenous ECLS with a small 3D-printed mock-oxygenator, and blood flow was maintained at 2...
Journal of Experimental Medicine, 2012
Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation...
Platelet Protease Nexin-1, a Serpin That Strongly Influences Fibrinolysis and Thrombolysis
Circulation, 2011
Background-Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma, but we have shown recently that PN-1 is present within the ␣-granules of platelets. Methods and Results-In this study, the role of platelet PN-1 in fibrinolysis was investigated with the use of human platelets incubated with a blocking antibody and platelets from PN-1-deficient mice. We showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tissue plasminogen activator and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. Protease nexin-1 is thus a major determinant of the lysis resistance of platelet-rich clots. Moreover, in an original murine model in which thrombolysis induced by tissue plasminogen activator can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1-deficient mice. Surprisingly, general physical health, after tissue plasminogen activator-induced thrombolysis, was much better in PN-1-deficient than in wild-type mice.
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.
Thrombin Activates Factor XI on Activated Platelets in the Absence of Factor XII
—Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor–initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo. (Arterioscler Thromb Vasc Biol. 1999;19:170-177.) Key Words: factor XIa factor XIIa factor IXa human blood coagulation contact system C oagulation factor XI circulates as a homodimer consisting of 2 identical disulfide-linked polypeptide chains (M r 80 000 each). 1 The zymogen protein is activated by a single cleavage of each polypeptide chain to give rise to the activated form (factor XIa). 2 Factor XIa activates zymogen factor IX to factor IXa. 3–5 Unlike classic hemophilia A or B (factor VIII or factor IX deficiency), one cannot predict whether patients with factor XI deficiency will exhibit a bleeding diathesis based on their level of factor XI activity alone. Some patients who are homozygous for factor XI deficiency show a severe bleeding tendency while others are asymptomatic. 6,7 Although the bleeding tendency of patients who are heterozygous for factor XI is controversial, 1 recent study showed that up to half of these patients have a bleeding tendency. 8 Attempts have been made to correlate bleeding in factor XI– deficient patients with low levels of von Wille-brand factor, 8 blood group, 8 and lack of an alternatively spliced form of platelet factor XI. 9,10 Activated factor XII can activate factor XI both in plasma 11,12 and on activated platelets. 13,14 However, deficiency of factor XII is not associated with a bleeding tendency. 15 This led investigators to propose an alternate mechanism for activation of factor XI based on the observation that thrombin can activate factor XI in a purified system 16,17 or in plasma. 18,19 It remains unclear whether or not thrombin is a physiologically relevant activator of factor XI. In most studies, factor XI activation by thrombin has required the presence of dextran sulfate (DS) or nonphysiological concentrations of sulfatide cofactors. It has also been suggested that high-molecular-weight kininogen, which strongly associates with factor XI, and fibrinogen, which is the preferred sub-strate for thrombin, would block thrombin activation of factor XI in plasma. 20 Another study showed that factor XI was not activated by thrombin in plasma in the presence of kaolin. 12 However, 1 group suggested that thrombin activation of minute amounts of factor XI in plasma protected fibrin clots from fibrinolysis, presumably by increasing the amount of thrombin generated within the clot. 21 If thrombin is a physiological activator of factor XI, it is not known what the in vivo cofactor for this activity might be. It has been hypothesized that endogenous glycosaminogly-cans 22 or platelets 23 might promote activation of factor XI by thrombin in vivo. One study found that a physiological glycosaminoglycan, heparan sulfate, could support activation of factor XI in plasma, but only when unphysiologically high levels of thrombin were added. 19 Platelets can bind factor XI and provide a site for activation of factor IX by factor XIa. 5 We hypothesized that platelets could serve as a cofactor for activation of small but physiologically important amounts of factor XI by thrombin. We have tested this hypothesis by using a defined, tissue factor–initiated, cell-based model of coagulation to examine factor XI activation by mechanisms
Mouse antithrombotic assay. Inhibition of platelet thromboembolism by disintegrins
Thrombosis Research, 1993
The mouse antithrombotic assay represents a model of fatal pulmonary thromboembolism induced by intravenous injection of collagen and epinephrine. Mice were protected by low doses of two disintegrins, albolabrin (10 l.@/mouse) and eristostatin (0.6 pg/mouse), whereas high doses of a thrombin inhibitor and an inhibitor of von Willebrand Factor binding to glycoprotein Ib were not effective. Injection of collagen and epinephrine resulted in the drop of platelet count and accumulation of platelet aggregates in 'the lung that appears to be the immediate cause of death. Albolabrin or eristostatin administration did not prevent the decrease of platelet count. Injection of albolabrin resulted in the formation of smaller and reversible platelet aggregates in the lungs and decreased accumulation of SlCr-labeled platelets in the lung suggesting that this disintegrin decreases formation of platelet aggregates in viva We compared the effects of allbolabrin and eristostatin on platelet aggregation, tail bleeding time, and survival of challenged animals. Eristostatin was about 5 times more potent in inhibiting platelet aggregation in vitro than albolabrin and 38 times more potent than albolabrin in protecting animals from sudden death. Both disintegrins, at the same doses (0.6-5 ltglmouse), caused similar dose-dependent prolongation of the bleeding time; however, only eristostatin exerted a protective effect. In conclusion, a) the mouse antithrombotic assay is a suitable model to screen and to evaluate the potency of platelet fibrinogen receptor antagonists in vivo; b) the results of the antithrombotic assay correlate better with the inhibition of platelet aggregation in vitro than with the prolongation of bleeding time.
Monitoring platelet dependent thrombin generation in mice
Thrombosis Research, 2010
Calibrated automated thrombin generation assay was adapted to measure thrombin generation in platelet rich plasma from mice. Vena cava phlebotomy appeared the best technique for blood sampling. The concentration-effect curves of tissue factor and platelet count have been determined. Corn trypsin inhibitor 2 μM inhibits contact activation effectively. Recombinant human thrombomodulin does not inhibit thrombin generation in mouse plasma but activated protein C (20 nM) does. Thrombin generation was dose dependently diminished by low molecular weight heparin and increased by high concentrations of exogenous factor VIII i.e. the assay can detect both hypo-and hypercoagulability.
Defective thrombus formation in mice lacking coagulation factor XII
Journal of Experimental Medicine, 2005
Blood coagulation is thought to be initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. In contrast, coagulation factor XII (FXII)–mediated fibrin formation is not believed to play an important role for coagulation in vivo. We used FXII-deficient mice to study the contributions of FXII to thrombus formation in vivo. Intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in the formation and stabilization of platelet-rich occlusive thrombi. Although FXII-deficient mice do not experience spontaneous or excessive injury-related bleeding, they are protected against collagen- and epinephrine-induced thromboembolism. Infusion of human FXII into FXII-null mice restored injury-induced thrombus formation. These unexpected findings change the long-standing concept that the FXII-induced intrinsic coagulation pathway is not important for clotting in vivo. The results establish FXII as essent...
Thrombosis and Haemostasis
Adhesion of platelets to immobilized collagen induces the expression of anionic phospholipids, e. g. phosphatidylserine (PS), in the outer leaflet of the plasma membrane of these platelets. In contrast, of the platelets that adhere to immobilized fibrinogen only a small sub-population representing 10 ± 3% of the total population of the fibrinogenadherent platelets has exposed PS as probed by annexin V binding. Although the presence of PS is thought to be critical for thrombin generation at the platelet surface, no information is available about the effect of this differential PS exposure on the ability of adherent platelets to support thrombin generation. Perfusion of the fibrinogen-or collagen-adherent platelets with solutions containing factor Xa and prothrombin resulted in thrombin generation that i) increased linear during the first perfusion minutes, ii) was about two-fold faster at collagenadherent than at fibrinogen-adherent platelets and iii) was for more than 98% restricted to the surface of the adherent platelets. It appeared that the lower thrombin generating capacity of fibrinogen-adherent platelets is not due to a lower overall surface density of PS, but is caused by lower amounts of platelet-bound factor Va. Firstly, in both cases thrombin generation could be completely attenuated with antibodies against human factor Va, and secondly, in the presence of an excess of exogenous plasma-derived factor Va similar initial rates of thrombin formation were measured for collagen-and fibrinogen-adherent platelets. Our findings suggest a unique role for immobilized collagen in maintaining haemostasis.