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.

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by three orders of magnitude. Here we employed Nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained L-serine versus D-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins—factor VIIa and activated protein C—than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared to the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Receptor tyrosine kinases (RTKs) are a group of enzymes involved in a variety of physiological and pathological processes. The human Ror1 is a member of the RTK family with unknown ligand and biological function. Overexpression of Ror1 has recently been reported in B-cell chronic lymphocytic leukemia. The aim of this study was to explore the expression profile of Ror1 in acute lymphoblastic leukemia (ALL) cells. Therefore, leukemic cells were isolated from the bone marrow and/or peripheral blood (PB) of 57 ALL patients. Immunophenotyping was performed by flow cytometry and mRNA expression was detected by RT-PCR. Overexpression of Ror1 mRNA was detected in 23 of 57 (40%) ALL patients. A similar expression pattern was observed in ALL cell lines, with 4 of 12 (33%) being positive. Stimulation of normal PB mononuclear cells with pokeweed mitogen and phorbol myristate acetate induced substantially higher Ror1 mRNA expression compared to unstimulated cultured cells. There has been neither a significant association between Ror1 expression and the immunophenotypic profile of the leukemic cells, nor with other clinical or hematological features of the patients. In conclusion, our findings propose Ror1 as a new tumor-associated antigen and a potential tool for targeted immunotherapy and monitoring of minimal residual disease in ALL.

Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations. These studies are now providing structural insights at atomic resolution into clotting protein-membrane interactions.

Dear Sirs, A number of studies have shown that the γ-carboxyglutamate-rich (GLA) domains of vitamin K-dependent clotting proteins require Ca 2+ to fold properly and bind to membranes (1, 2). Although plasma contains about 1.25 mM free Ca 2+ and 0.5 mM Mg 2+ (3), in vitro assays of clotting factor function often employ supraphysiologic Ca 2+ concentrations (2.5-5 mM Ca 2+), and no Mg 2+. Sekiya et al. showed that Mg 2+ enhances factor IX (fIX) structure and function in combination with physiologic Ca 2+ concentrations (4, 5). Subsequent reports showed that, in the presence of plasma concentrations of Ca 2+ , Mg 2+ enhances the activity of factor VIIa (fVIIa) bound to tissue factor (TF) (6-10). The concept is that GLA domains typically bind seven or eight Ca 2+ when it is the only divalent metal ion present (at supraphysiologic Ca 2+ concentrations), but at plasma concentrations of Ca 2+ and Mg 2+ , two or three of these "calcium" binding sites are actually occupied by Mg 2+ , with functional consequences (11). Mg 2+ does not support clotting reactions in the absence of Ca 2+ (12), also consistent with the notion that only a subset of the metal ion binding sites in GLA domains can be productively occupied by Mg 2+ .