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, 286 (41), 35535-42

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

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Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

Rinku Majumder et al. J Biol Chem.

Abstract

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (K(d) = ∼10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60-70% functional prothrombinase complex at saturating factor Va concentrations. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va(2) (K(d) = ∼6.5 μm) and to factor Xa (K(d) = ∼91 μm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (K(d)(app) = ∼40 nm) of a partially active prothrombinase complex between factor Xa and factor Va(2), compared with K(d)(app) for C6PS ∼2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.

Figures

FIGURE 1.
FIGURE 1.
Prothrombinase activity on DOPC:DOPE:DOPS membranes. A, rates of thrombin generation were determined at 37 °C in reaction mixtures containing 25 mm Tris, pH 7.4, 150 mm NaCl, 2.7 mm KCl, 0.6% PEG 8000, 3 mm CaCl2, 0.2 nm bovine factor Xa, a fixed concentration of prothrombin (1.4 μm), varying factor Va concentrations, and 5 μm of DOPC:DOPE vesicles of varying compositions: 70:30 (○), 80:20 (♢), and 90:10 (▿). Three independent experiments are shown for each phospholipid vesicle composition. The line represents global fitting (GraphPad Prism) of the data while varying parameters Kdapp and Vsat, as described under “Experimental Procedures.” The inset shows the rates of thrombin formation in the presence of 30% egg PE:70% egg PC membranes. B, binding of DEGR-Xa to factor Va2 in the presence of 30:70 DOPE:DOPC membranes was determined in the presence of increasing concentration of factor Va2 and at a fixed concentration of DEGR-Xa (10 nm). The buffer was same as in A. The line represents the best fit of the data to a simple single-site binding model as obtained using Sigma Plot. C, rates of thrombin generation were determined at 37 °C in reaction mixtures containing 3 mm CaCl2, 0.2 nm bovine factor Xa, 5 nm bovine factor Va, 5 μm of vesicles of varying compositions: 100% DOPC (●), 80:20 DOPC:DOPE (▴), 69:30:1, and DOPC:DOPE:DOPS (■). The lines show the fit of these data using Sigma Plot to a standard Michaelis-Menten model to determine the kcat and Km of prothrombin activation with membranes of different lipid compositions, as described in the text.
FIGURE 2.
FIGURE 2.
DOPE enhances prothrombinase activity on membranes containing low concentrations of DOPS. Rates of thrombin generation were determined at 37 °C in buffer containing 25 mm Tris, pH 7.4, 150 mm NaCl, 2.7 mm KCl, 10 mg/ml BSA, 3 mm CaCl2, 0.2 nm bovine factor Xa, 1.4 μm bovine prothrombin, bovine factor Va, and 5 μm DOPC:DOPS:DOPE vesicles of varying compositions: 69:1:30 and 99:1:0 (A), 65:5:30 and 95:5:0 (B), and 60:10:30 and 90:10:0 (C). In A, four independent experiments are shown. Global fitting of the data for Vsat and Kdapp was performed using GraphPad Prism version 4.00. In B and C, the error bars represent one standard error of the parameter for triplicate measurements in a representative experiment.
FIGURE 3.
FIGURE 3.
Binding of factor Va to phospholipid vesicles. Samples contained 150 mm NaCl, 20 mm Tris, pH 7.4, and 0.6 μm labeled DOPC:DOPS:DOPE:dansyl-PE vesicles of varying compositions: 70:0:27.5:2.5 (A), 69:1:27.5:2.5 and 96.5:1:0:2.5 (B), 65:5:27.5:2.5 and 92.5:5:0:2.5 (C), and 60:10:27.5:2.5 and 87.5:10:0:2.5 (D). Fluorescence measurements (F) were made as described under “Experimental Procedures” after incremental addition of bovine factor Va at 25 °C. The lines represent the best fit of data in one representative experiment. The inset in A shows the binding of bovine Va to egg PE:egg PC (20:80) membranes.
FIGURE 4.
FIGURE 4.
Binding of rHFVa and Factor Xa to soluble C6PE. The intrinsic fluorescence intensity of 0.1 μm rHFVa (A) or 0.1 μm human factor Xa (B) in 20 mm Tris, 150 mm NaCl, and 5 mm CaCl2, pH 7.5, was measured as a function of C6PE concentration at 23 °C. The data were analyzed according to a simple single-binding site model to yield a Kd of 6.5 ± 0.5 μm for rHFVa and 91 ± 1.4 μm for factor Xa. The insets to A and B show the binding of bovine factors Va/Xa with C6PE (Kd values are 8 ± 1.2 and 90 ± 14 μm for factors Va and Xa, respectively). C presents fluorescence of DEGR-Xa with the addition of rHVa2 in the presence of 400 μm C6PE. The fit of a single-binding site model to the data provided the Kd (40 ± 3 nm) for Xa-DEGR-Va2 complex formation.

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