Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

Koedam, J. A.; Meijers, J. C. M.; Sixma, Jan J.; Bouma, Bonno N.

doi:10.1172/jci113721

Cited by 224 publications

(143 citation statements)

References 48 publications

(27 reference statements)

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“…38 VWF protects FVIII from proteolytic degradation in the circulation and also interferes with the binding of FVIII to its clearance receptors. 39,40 Recently, VWF has also been shown to inhibit the uptake of FVIII by antigen presenting cells thereby providing a possible modulating effect on the development of an immune response to infused FVIII in patients with hemophilia A. 41 In addition, studies in megakaryocytes have suggested that co-release of VWF and FVIII may protect FVIII from inhibitory antibodies.…”

Section: Discussionmentioning

confidence: 99%

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Biggelaar¹,

Bouwens²,

Kootstra³

et al. 2009

Haematologica

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BackgroundGene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion. Design and MethodsHuman blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human Bdomain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy. ResultsBlood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6±1.0 pmol/1×10 6 cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6-to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15-22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies. ConclusionsWe conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A.

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Section: Discussionmentioning

confidence: 99%

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Biggelaar¹,

Bouwens²,

Kootstra³

et al. 2009

Haematologica

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“…FX was purified from fresh-frozen plasma by immunoaffinity chromatography, followed by Q-Sepharose chromatography as described previously (24). Protein C was purified from fresh-frozen plasma by barium sulphate precipitation, followed by immunoaffinity chromatography and activated as published (25,26). Citrated FXI-deficient plasma was purchased from George King Biomedical Inc. (Kansas, KS).…”

Section: Methodsmentioning

confidence: 99%

Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma

Maas

Meijers

Marquart

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.coagulation | coagulation factor V | coagulation factor XI | feedback activation T he mechanism behind the activation of the intrinsic pathway of coagulation in vivo has been subject to discussion since the introduction of the model of coagulation (1, 2). The importance of the intrinsic pathway for physiological hemostasis is apparent from the bleeding phenotypes that are seen in factor VIII (FVIII) deficiency, factor IX (FIX) deficiency and, to a lesser extent, in factor XI (FXI) deficiency. In vitro, activation of the intrinsic pathway can be initiated by factor XII (FXII) on negatively charged surfaces, which results in clotting. However, the role of FXII-dependent coagulation in physiological hemostasis is controversial. FXII deficiency, for instance, protects against thrombosis in murine models (3), which conflicts with the protective role of high FXII antigen levels in epidemiological studies on thrombosis in humans (4), as well as with the absence of bleeding symptoms in FXII-deficient individuals. Additionally, FXII hyperactivity leads to angioedema, rather than to thrombosis (5), which corresponds with evidence that activation of the kallikrein-kinin system by FXII can take place independent of coagulation (6).As FXI deficiency, unlike FXII deficiency, is associated with mild bleeding phenomena, a FXI activator other than FXII is likely. Thrombin, the final and central enzyme of the coagulation cascade, can directly activate FXI, which leads to downstream formation of more thrombin (7,8). This feedback activation mechanism offered an alternative explanation for the existence of the intrinsic pathway: The amplification of minute amounts of thrombin, ...

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“…Variant Gln506-FV, normal FV, and prothrombin were purified from plasma (30,31), and variant and normal FV were converted by thrombin to FVa as described (30,32). The thrombin amidolytic substrate, CBS 34-47, was obtained from American Bioproducts (Parsippany, New Jersey, USA); Innovin (recombinant human tissue factor reagent) from Baxter-Dade, (Miami, Florida, USA); bee venom and Naja mossambica mossambica phospholipase A 2 from Sigma Chemical Co. (St. Louis, Missouri, USA); and FV-deficient plasma from George King Biomedical (Overland Park, Kansas, USA).…”

Section: Methodsmentioning

confidence: 99%

High-density lipoprotein enhancement of anticoagulant activities of plasma protein S and activated protein C

Griffin¹,

Kojima²,

Banka³

et al. 1999

J. Clin. Invest.

208

144

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Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels are associated, respectively, with either increased risk or apparent protective effects for atherothrombosis. The ability of purified LDL and HDL to downregulate thrombin formation, a contributor to atherothrombotic processes, was assessed. Purified HDL, but not LDL, significantly enhanced inactivation of coagulation factor Va by activated protein C (APC) and protein S, and HDL stimulated protein S-dependent proteolytic inactivation of Va by APC, apparently due to cleavage at Arg306 in Va. In normal plasma, added HDL enhanced APC/protein S anticoagulant activity in modified prothrombin-time clotting assays. When the anticoagulant potency of HDL was compared with phospholipid (PL) vesicles of well-defined composition using this assay, HDL appeared qualitatively different from PL vesicles because HDL showed only good anticoagulant activity, whereas PL vesicles were rather procoagulant. When 20 normal plasmas were tested using this clotting assay, apoA-I levels correlated with anticoagulant response to APC/protein S (r = 0.47, P = 0.035), but not with activated partial thromboplastin time-based APC resistance ratios. Because HDL enhances the anticoagulant protein C pathway in vitro, we speculate that HDL may help downregulate thrombin generation in vivo and that this anticoagulant action is one of HDL's beneficial activities.

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Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor.

Cited by 224 publications

References 48 publications

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma

High-density lipoprotein enhancement of anticoagulant activities of plasma protein S and activated protein C

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