Phosphatidylserine and Phosphatidylethanolamine Bind to Protein Z Cooperatively and with Equal Affinity

Abstract

Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa) by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS) and phosphatidylethanolamine (PE) with equal affinity (Kd~48 μM); further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process.

Fig 1. Binding of C6PS and C6PE to PZ measured by intrinsic fluorescence.

To detect binding, intrinsic tryptophan fluorescence intensities of 150 nM PZ in 50 mM Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM CaCl₂, 0.6% PEG were measured at 25° C as a function of added C6PS (●) or C6PE (○). Solid lines show fits of the data to a simple single-site binding model, which predicted apparent K_d values for binding of C6PS and C6PE as 47.7 ± 10.7 μM and 47.6 ±8.2 μM, respectively. Fluorescence titrations were also performed under two more conditions: in presence of 5 mM EDTA with C6PS (■) and C6PE (□); and in presence of 1 mM Ca²⁺ with C6PS (▲) and C6PE (Δ).

Fig 2. Binding of different six-carbon chain soluble lipids to PZ.

Intrinsic tryptophan fluorescence intensities of 150 nM PZ in 50 mM Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM CaCl₂, 0.6% PEG were measured as a function of added A. C6PA (○), B. C6PC (●), C. C6PG (Δ) and D. C6(D)PS (▼) to obtain binding constants. The apparent K_d values for binding are 165 ± 25 μM, 129 ± 32, 131± 39, for PA, PC and PG respectively. (D)-PS shows extremely weak binding with K_d~900 μM.

Fig 3. Binding of human PZ to phospholipid vesicles.

Fluorescence measurements were performed in 50 mM Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM CaCl₂, 0.6% PEG by adding increasing concentrations of PZ to 1 μM-labeled DOPC: DOPS: DOPE: Dansyl-PE vesicles of varying composition: A. 69:1:27.5:2.5 (●) 96.5:1: 0: 2.5 (○), 97.5: 0:0:2.5 (Δ); B. 60:10: 27.5: 2.5 (●) and 87.5:10: 0: 2.5 (○).Details of the experimental procedure are described in Methods.

Fig 4. Binding of PZ to DEGR-Xa [(5-(Dimethylamino)-1-naphthalenesulfonyl]-Glutamylycylarginyl)-Xa] on phospholipid membranes.

DEGR-Xa (30 nM) fluorescence emission intensities were measured with increasing concentrations of PZ in the presence of 50 μM membrane composed of DOPS: DOPC 30:70 (●), DOPE: DOPC 30:70 (○), 100% DOPC (▼) and in the absence of any membrane (□). Dissociation constants in the presence of PC: PS and PC: PE were 8.6 ± 2.2 nM and 20.6 ± 3 nM, respectively. There was no association of fXa and PZ observed in presence of 100% PC or in absence of any membrane.

Fig 5. FXa-PZ interaction on phospholipid membranes as measured by AT-mediated inhibition of fXa activity.

The amidolytic activity of 5 nM fXa in a buffer containing 50 mM Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM Ca²⁺ and 0.6% PEG was measured in the presence of AT, with or without PZ in the absence and presence of membranes (DOPC: DOPS 70: 30; DOPC: DOPE 70: 30, 100% DOPC) using substrate S2765.

Fig 6. Cartoon diagram describing the major findings of the study.

The diagram shows that PZ does not bind to 100% PC membrane and does not form a complex with fXa (A); PZ binds to PS or PE containing membranes with comparable affinity and form a stable PZ-fXa complex (B & C); PS and PE synergistically act to enhance the binding affinity of PZ towards membrane (D).