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+ .

Curcumin is a polyphenolic phytonutrient that has antineurodegenerative properties. In this study, we investigated the anti‐amyloidogenic properties of curcumin. Following incubation with curcumin, intrinsic tryptophan fluorescence emission of apolipoprotein (apo) A‐I was strongly quenched. At the same time, curcumin fluorescence emission was enhanced. The fluorescence emission spectra of curcumin in the presence of amyloid‐like aggregates formed by methionine‐oxidized (ox) apoA‐I varied, depending on whether curcumin was added before, or after, aggregate formation. The impact of curcumin on the structure of the aggregating material was revealed by the lower amount of β‐structure in ox‐apoA‐I amyloid‐like aggregates formed in the presence of curcumin, compared to aggregates formed without curcumin. However, the kinetics of ox‐apoA‐I amyloid‐like aggregate formation was not altered by the presence of curcumin. Moreover, electron microscopy analysis detected no discernable differences in amyloid morphology when ox‐apoA‐I amyloid‐like aggregates were formed in the presence or absence of curcumin. In conclusion, curcumin interacts with apoA‐I and alters the structure of ox‐apoA‐I amyloid‐like aggregates yet does not diminish the propensity of ox‐apoA‐I to form aggregates.

Phospholipid membranes are an important part of the blood clotting cascade and can alter rates of clotting reactions depending on their phospholipid composition. Seven proteins in blood clotting bind reversibly to phospholipid membranes through γ‐carboxyglutamate‐rich (GLA) domains. Although the GLA domains of these proteins are very similar structurally, their membrane binding affinities vary by almost three orders of magnitude. We employed surface plasmon resonance binding studies and enzymatic assays to systematically investigate the phospholipid specificity of these seven GLA domains. It has long been thought that GLA domains bind preferentially to bilayers containing phosphatidylserine (PS), but we found, surprisingly, that two of the GLA domain‐containing blood clotting proteins (factor VII and protein C) bound preferentially to membranes containing phosphatidic acid (PA) or phosphatidylinositol phosphate (PIP), compared to PS. Furthermore, PA and PIP strongly enhanced the enzymatic activities of factor VIIa and activated protein C. Incidentally, of the seven blood clotting proteins with GLA domains, factor VII and protein C are known to have the lowest binding affinities for PS‐ membranes. Our findings provide new insights into the membrane binding mechanism for these two medically important GLA domain‐containing clotting proteins, through PA‐ and PIP‐specific binding interactions.

1175 Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Previous studies from our lab and others have shown that phosphatidylethanolamine (PE) poorly supports clotting reactions by itself, but strongly synergizes with PS to promote several membrane-dependent steps in the blood clotting cascade, although the mechanism for PE-PS synergy has been unclear. We recently put forward a new mechanistic explanation – which we termed the ABC or Anything But Choline hypothesis – for how PS and PE synergize to enhance factor X (fX) activation by the factor VIIa-tissue factor complex (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). The membrane contribution to this reaction is dominated by the affinity of fX for the membrane surface; since fX binds to membranes via its gamma-carboxyglutamate-rich (GLA) domain, the ABC hypothesis therefore focuses on the mechanisms by which GLA domains engage the phospholipid bilayer. We identified two main types of GLA domain-phospholipid interactions: a single phospho-L-serine-specific binding site in each GLA domain; and multiple ”phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains. In the current study, we test the ABC hypothesis in the context of the prothrombinase complex – i.e., activation of prothrombin by the membrane-bound complex of fXa and factor Va (fVa). Using a variety of approaches including surface plasmon resonance analyses, we measured the contributions of varying phospholipid compositions to the membrane binding affinities of fXa, fVa and prothrombin, as well as to the enzymatic activity of prothrombinase. Our results suggest that phospholipid synergy in prothrombinase activity differs in certain respects from that observed for the factor VIIa-tissue factor complex. Not only did PS synergize with PE for enhancing the activity of prothrombinase, but phosphatidylglycerol (PG) and phosphatidylacid (PA) also synergized with PE, albeit more weakly than with PS (i.e., significantly higher levels of PG or PA in the presence of PE were required to achieve prothrombinase activities comparable to mixtures of PS and PE). In contrast, PE failed to synergize with either PG or PA to support fX activation by the factor VIIa-tissue factor complex. These differences primarily arise from differential membrane binding of the substrates for these two complexes (fX for factor VIIa-tissue factor and prothrombin for prothrombinase). The data suggest that the phospho-L-serine-specific binding site in the GLA domain of prothrombin may not be as stringent as that of fX, as high levels of PG or PA can substitute for PS in membrane binding of prothrombin but not for fX. This study provides further insights into the membrane's role in regulating blood clotting reactions, specifically the binding interactions between GLA domains and membrane surfaces. Disclosures: No relevant conflicts of interest to declare.