Contribution of Different Types of Phospholipids in Blood Coagulation

In case of an injury platelets become activated and proper interaction of blood clotting proteins with membranes of activated platelets is essential for maintaining a balanced hemostasis. Hemophilia and thrombosis are the two extremes of an imbalanced hemostasis where patients with hemophilia suffer from severe bleeding and those with thrombosis from unnecessary blood clots that block normal flow of blood. There are several therapeutics available for treating patients suffering from different types of hemophilia and thrombosis, but lack of specific and efficient medications requires scientist to look for new and more specific activators or inhibitors of the blood clotting cascade. This will not be possible unless we have a detailed knowledge of the nature of the interactions between blood clotting proteins and phospholipid membranes. In this study, we benefitted from combining the biochemical and biophysical techniques with Nanodisc technology (Sligar lab), molecular dynamics (MD) simulations (Tajkhorshid lab) and solid state-NMR (SSNMR) (Rienstra lab), as a part of a collaborative project at university of Illinois. This approach enabled us to look into the protein-phospholipid interactions in blood clotting at a molecular scale. The first part of my thesis focuses mainly on γ-carboxyglutamate-rich (GLA) domains of factor X (fX) and prothrombin as representative GLA domains of clotting factors, which have been the subject of several protein-phospholipid interaction studies. Based on the results of this project, we proposed a new mechanism for the binding of GLA domains of clotting proteins to phospholipid membranes. The blood clotting reactions take place on membranes with exposed phosphatidylserine (PS). GLA domains are the most common PS-binding motifs in blood coagulation. A further intriguing aspect of the membrane’s contributions to clotting is that phosphatidylethanolamine (PE) supports little or no clotting activity in the absence of PS, but strongly synergizes with small amounts of PS to enhance many clotting reactions. The mechanism of PE/PS synergy is poorly understood, although several hypotheses have been proposed which rely on specific properties of PE’s phosphoethanolamine headgroup. We proposed a novel, general model for GLA domain binding to membranes; the ABC (Anything But Choline) hypothesis, supported by biochemical studies, solid-state NMR analyses and molecular dynamics simulations. This hypothesis invokes two types of protein-phospholipid interactions: “phospho-Lserine-specific” and “phosphate-specific.” In the latter, the accessible phosphate groups in phospholipids interact with tightly bound Ca in GLA domains. We proposed that based on this model, phospholipids that can satisfy the phosphate-specific interactions should be able to synergize with PS to support fX activation. We showed that almost any glycerophospholipid other than phosphatidylcholine (PC) synergizes strongly with PS to enhance factor X activation by factor VIIa/tissue factor. We proposed that PC and sphingomyelin (the major external phospholipids of healthy cells) provide an anticoagulant surface to healthy cells because their bulky, highly hydrated choline headgroups sterically hinder access to their phosphates. Following cell activation, lysis, or damage, PE and PS are exposed on the outer leaflet where they can collaborate to create binding sites for GLA domains, by providing phosphate-specific and phospho-Lserine-specific interactions, respectively.