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.

Hepatitis C Virus p7 Induces Membrane Permeabilization by Interacting with Phosphatidylserine

“…Because of its significance in cellular processes, the identification of interactions between proteins and PS is essential for understanding its regulatory roles [21]. Several PS-binding proteins have been reported [22]; however, only a few studies have reported the interaction between viral proteins and lipids despite its essential function on viral entry and budding [23]. In this study, we showed that HCV p7 prefers PS over other negatively charged phospholipids (PA, PG, PI, and CL).…”

Sensitivity of peripheral membrane proteins to the membrane context: A case study of phosphatidylserine and the TIM proteins

“…Since the Hill coefficients are above 1, the binding of the three TIM proteins are cooperative and thus sensitive to PS surface density [53]. Unlike the cooperativity measured for Annexin V, the PKC family, or Protein Z, the cooperativity of the TIM proteins cannot be explained by multiple PS or Ca binding sites [25,26,28]. Alternative mechanisms can underly cooperative binding such as the protein’s non-specific electrostatic interactions with neighboring PS.…”

“…Annexin V, a PS-receptor suspected to inhibit blood coagulation by outcompeting coagulation factors with C2 and Gla PS-binding domains, binds cooperatively to PS and calcium, indicating a preference for membranes with high PS surface density [25,26]. Similarly, Protein Kinase C (PKC) family members also bind PS cooperatively [27] and the coagulation factor Protein Z binds both PS and phosphatidylethanolamine (PE), another lipid normally enriched on the inner leaflet that flips out during apoptosis [28]. Considering that PS receptors are each specialized for particular functions, such as the recognition of apoptosis, signal transduction, exocytosis, phosphorylation of phosphoinositide, and coagulation [10], it is likely that PS-binding proteins recognize both PS and the membrane context of PS presentation.…”

Minor plasma lipids modulate clotting factor activities and may affect thrombosis risk

“…C6PS also appears to promote dimer formation of factor Xa and such dimerization may prevent factor Xa binding to factor Va due competition . Furthermore, this short chain lipid binds to factor IXa and protein Z, possibly affecting functions of these molecules. Short chain phospholipids might be produced by oxidation, but this has not been reported to our knowledge; so the pathophysiologic relevance of short chain PLs like C6PS remains unknown.…”