Molecular Determinants of Phospholipid Synergy in Blood Clotting

Abstract

Many regulatory processes in biology involve reversible association of proteins with membranes. Clotting proteins bind to phosphatidylserine (PS) on cell surfaces, but a clear picture of this interaction has yet to emerge. We present a novel explanation for membrane binding by GLA domains of clotting proteins, supported by biochemical studies, solid-state NMR analyses, and molecular dynamics simulations. The model invokes a single “phospho-l-serine-specific” interaction and multiple “phosphate-specific” interactions. In the latter, the phosphates in phospholipids interact with tightly bound Ca2+ in GLA domains. We show that phospholipids with any headgroup other than choline strongly synergize with PS to enhance factor X activation. We propose that phosphatidylcholine and sphingomyelin (the major external phospholipids of healthy cells) are anticoagulant primarily because their bulky choline headgroups sterically hinder access to their phosphates. Following cell damage or activation, exposed PS and phosphatidylethanolamine collaborate to bind GLA domains by providing phospho-l-serine-specific and phosphate-specific interactions, respectively.

Characterization of platelet aminophospholipid externalization reveals fatty acids as molecular determinants that regulate coagulation

“…5). In agreement, lyso-PE (which contains no sn2 fatty acid) was previously shown to not support activity of the tenase complex in PS liposomes (27). Our finding that short chain PE poorly supports coagulation whereas short chain PS supports coagulation normally, suggests that these molecules interact differentially with coagulation factors through their Gla domains.…”

“…Each Gla domain is thought to have one specific interaction with the carboxylate on the headgroup of PS and approximately six interactions with phosphates on any PL except PC. This idea means that PS is required for coagulation to take place but PE can enhance the rate of these reactions (27). To date, the interaction between APL and coagulation factors was (C and D) Platelets were activated with 10 μM rotenone for 180 min, or with 1 μM ABT-737 for 60 min, after preincubation with inhibitors (Q-VD-OPH, caspase inhibitor, 10 μM), EGTA (1 mM), BAPTA-AM (10 μM), then surface APL analyzed as described in Materials and Methods.…”

“…Specifically, tissue factor/factor VIIa, factor VIIIa/IXa (tenase complex), and factor Va/Xa (prothrombinase complex) interact with surface APL in a Ca 2+ -dependent manner, enhancing enzymatic activity (27,33,34). This interaction occurs through multiple γ-carboxylated glutamic acids (Gla domains), and the mechanisms by which these interactions occur have been described at an atomic level (35).…”

“…The role of APL headgroups in facilitating coagulation at the plasma membrane of activated platelets is well known; however, whether the fatty acids attached to APL can also regulate this phenomenon is unknown (27). To address this idea, liposomes were generated that contain the amount of APLs found to be externalized, at a physiological platelet concentration.…”

The Charge Properties of Phospholipid Nanodiscs

“…Although good charge measurement data are available for proteins and nucleic acids (8,9), obtaining charge measurements in lipids has posed experimental difficulties. This lack of solid charge information is unfortunate because the membrane composition, including charge, is of fundamental importance for a variety of cellular and physiological functions (10).…”