Blood clotting reactions on nanoscale phospholipid bilayers

Abstract

Blood clotting reactions, such as those catalyzed by the tissue factor/factor VIIa complex (TF:VIIa), assemble on membrane surfaces containing anionic phospholipids such as phosphatidylserine (PS). In fact, membrane binding is critical for the function of most of the steps in the blood clotting cascade. In spite of this, our understanding of how the membrane contributes to catalysis, or even how these proteins interact with phospholipids, is incomplete. Making matters more complicated, membranes containing mixtures of PS and neutral phospholipids are known to spontaneously form PS-rich membrane microdomains in the presence of plasma concentrations of calcium ions, and it is likely that blood clotting proteases such as TF:VIIa partition into these PS-rich microdomains. Unfortunately, little is known about how membrane microdomain composition influences the activity of blood clotting proteases, which is typically not under experimental control even in "simple" model membranes. Our laboratories have developed and applied new technologies for studying membrane proteins to gain insights into how blood clotting protease-cofactor pairs assemble and function on membrane surfaces. This includes using a novel, nanoscale bilayer system (Nanodiscs) that permits assembling blood clotting protease-cofactor pairs on stable bilayers containing from 65 to 250 phospholipid molecules per leaflet. We have used this system to investigate how local (nanometerscale) changes in phospholipid bilayer composition modulate TF:VIIa activity. We have also used detailed molecular dynamics simulations of nanoscale bilayers to provide atomic-scale predictions of how the membrane-binding domain of factor VIIa interacts with PS in membranes.Most steps in the blood clotting cascade require the assembly of a serine protease together with its cognate regulatory protein on a membrane surface [1]. Both the coagulation serine protease (for example, factors VIIa, IXa or Xa) and its protein cofactor (tissue factor, factor VIIIa or factor Va, respectively) contain specific membrane-interactive domains that direct these proteins to assemble and function on membrane surfaces. Furthermore, the protein substrates (factors IX, X and prothrombin) of these membrane-bound enzymes also bind reversibly to membranes. Membrane surfaces can only support the binding and assembly of clotting factors if they contain exposed anionic phospholipids, with phosphatidylserine (PS) being the most active. But membrane binding does more than just localize these enzymes and substrates to regions of tissue trauma; the membrane also plays a critical role in catalysis, as blood clotting enzymes are thousands of times less active when released from the membrane surface. In spite of the critical importance of membrane binding, we still have a very incomplete understanding of how the membrane surface enhances blood clotting reactions. Furthermore, we lack a detailed understanding of how the membrane binding domains of blood clotting proteins actually interact with anionic phosph...

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