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- Krisinger, Michael, et al.
(författare)
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Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma.
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 276, s. 6586-6602
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Tidskriftsartikel (refereegranskat)abstract
- Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P(10)S(12)D(23)Q(32)N(33)) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca(2+) titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca(2+) requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity.
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| 2. |
- Oslakovic, Cecilia, et al.
(författare)
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Anionic Phospholipids Lose their Procoagulant Properties when Incorporated into High-Density Lipoproteins.
- 2009
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 284:9, s. 5896-5904
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Tidskriftsartikel (refereegranskat)abstract
- Blood coagulation involves a series of enzymatic protein complexes that assemble on the surface of anionic phospholipid. To investigate whether apolipoproteins affect coagulation reactions, they where included during the preparation of anionic phospholipid vesicles using a detergent solubilization-dialysis method. Apolipoprotein components of high-density lipoproteins, especially apolipoprotein A-I, had pronounced anticoagulant effect. The anionic phospholipids lost their procoagulant effect when the vesicle preparation method was performed in the presence of apolipoprotein A-I. The anionic phospholipid-apolipoprotein A-I particles were 8-10 nm in diameter and contained around 60-80 phospholipid molecules, depending on the phospholipid composition. The phospholipids of these particles were unable to support the activation of prothrombin by factor Xa in the presence of factor Va, and unable to support binding of factor Va, while binding of prothrombin and factor Xa were efficient. Phospholipid transfer protein was shown to mediate transfer of phospholipids from liposomes to apolipoprotein A-I containing reconstituted high-density lipoprotein. In addition, serum was also shown to neutralize the procoagulant effect of anionic liposomes and to efficiently mediate transfer of phospholipids from liposomes to either apolipoprotein A-I or apolipoprotein B containing particles. In conclusion, apolipoprotein A-I was found to neutralize the procoagulant properties of anionic phospholipids by arranging the phospholipids in surface areas that are too small to accommodate the prothrombinase complex. This anionic phospholipid scavenger function may be an important mechanism to control the exposure of such phospholipids to circulating blood and thereby prevent inappropriate stimulation of blood coagulation.
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