Ca 2+ switches the effect of PS-containing membranes on Factor Xa from activating to inhibiting: implications for initiation of blood coagulation

Calcium (Ca2+) plays a pivotal role in cellular and organismal physiology. The Ca2+ ion has an intermediate protein-binding affinity, thus it can serve as an on/off switch in regulation of different biochemical processes. The serum level of ionized Ca2+ is regulated with normal ionized Ca2+ being in the range from 1.10 to 1.29 mM. Hypocalcaemia (free Ca2+ < 1.1mM) in critically ill patients is commonly accompanied by hemostatic abnormalities, ranging from isolated thrombocytopenia to complex defects such as disseminated intravascular coagulation, commonly thought to be due to insufficient functioning of anticoagulation pathways. A small amount of Factor Xa (fXa) produced by Factor VIIa and exposed tissue factor is key to initiating blood coagulation by producing enough thrombin to induce later stages of coagulation. FXa must bind to phosphatidylserine (PS)-containing membranes to produce thrombin at a physiologically significant rate. In this work, we show that overall fXa activity on PS-containing membranes is sharply regulated by a “Ca2+ switch” centered at 1.16 mM, below which fXa is active and above which fXa forms inactive dimers on PS-exposing membranes. Our data lead to a mathematical model that predicts the variation of fXa activity as a function of both calcium and membrane concentrations. Because the critical Ca2+ concentration is at the lower end of the normal plasma ionized Ca2+ concentration range, we propose a new regulatory mechanism by which local Ca2+ concentration switches fXa from an intrinsically active form to a form requiring its cofactor (fVa) to achieve significant activity

Phosphatidylserine-Induced Factor Xa Dimerization and Binding to Factor Va Are Competing Processes in Solution

A soluble, short chain phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), binds to discrete sites on FXa, FVa, and prothrombin to alter their conformations, to promote FXa dimerization (Kd ~ 14 nM), and to enhance both the catalytic activity of FXa and the cofactor activity of FVa. In the presence of calcium, C6PS binds to two sites on FXa, one in the epidermal growth factor like (EGF) domain and one in the catalytic domain; the latter interaction is sensitive to Na+ binding and probably represents a protein recognition site. Here we ask whether dimerization of FXa and its binding to FVa in the presence of C6PS are competitive processes. We monitored FXa activity at 5, 20 and 50 nM FXa while titrating with FVa in the presence of 400 µM C6PS and 3 or 5 mM Ca2+ to show that the apparent Kd of FVa-FXa interaction increased with increasing FXa concentration at 5 mM Ca2+, but the Kd was only slightly affected at 3 mM Ca2+. A mixture of 50 nM FXa and 50 nM FVa in the presence of 400 µM C6PS yielded both Xa homodimers and Xa ·Va heterodimers but no FXa dimers bound to FVa. A mutant FXa (R165A) that has reduced prothrombinase activity showed both reduced dimerization (Kd~147 nM) and reduced FVa binding (apparent Kd, = 58, 92 and 128 nM, respectively for 5, 20 and 50 nM R165A FXa). Native gel electrophoresis showed that the GLA-EGFNC fragment of FXa (lacking the catalytic domain) neither dimerized nor formed a complex with FVa in the presence of 400 µM C6PS and 5 mM Ca2+. Our results demonstrate that the dimerization site and FVa binding site are both located in the catalytic domain of FXa and that these sites are linked thermodynamically

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (Kd = ∼10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60–70% functional prothrombinase complex at saturating factor Va concentrations. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va2 (Kd = ∼6.5 μm) and to factor Xa (Kd = ∼91 μm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (Kdapp = ∼40 nm) of a partially active prothrombinase complex between factor Xa and factor Va2, compared with Kdapp for C6PS ∼2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting

Phosphatidylserine and FVa regulate FXa structure

Human coagulation factor Xa (FXa) plays a key role in blood coagulation by activating prothrombin to thrombin on “stimulated” platelet membranes in the presence of its cofactor factor Va (FVa). Phosphatidylserine (PS) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used fluorescence resonance energy transfer (FRET) to monitor changes in FXa length in response 1] to soluble PS (dicaproyl-phosphatidylserine; C6PS), 2] to PS membranes, and 3] to FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa fluor 555) at FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites, a regulatory site (Reg site) at the N-terminus (previously identified as involving the Gla and EGFN domains) and a presumptive protein-recognition site in the catalytic domain (Prot site). Binding of C6PS to the regulatory site increased the inter-probe distance by ~ 3 Å, while saturation of both sites further increased the distance by ~ 6.4 Å. FXa binding to a membrane produced a smaller length increase (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane than when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS or PS-containing membranes bound to FXa, the overall change in length was comparable (~ 5.6–5.8 Å), indicating that C6PS and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analyzing structure-function relationships in FXa and in the FXa.FVa2 complex

Effects of Water Soluble Phosphotidylserine on Bovine Factor X(a): Functional and Structural Changes Plus Dimerization

Previous work has shown that two molecules of a soluble form of phosphatidylserine, C6PS, bind to human and bovine factor X(a). Activity measurements along with the fluorescence of active-site-labeled human factor X(a) showed that two linked sites specifically regulate the active site conformation and proteolytic activity of the human enzyme. These results imply, but cannot demonstrate, a C6PS-induced factor X(a) conformational change. The purpose of this paper is to extend these observations to bovine factor X(a) and to demonstrate that they do reflect conformational changes. We report that the fluorescence of active-site-labeled bovine factor X(a) also varied with C6PS concentration in a sigmoidal manner, whereas amidolytic activity of unlabeled enzyme varied in a simple hyperbolic fashion, also as seen for human factor X(a). C6PS induced a 70-fold increase in bovine factor X(a)'s autolytic activity, consistent with the 60-fold increase in proteolytic activity reported for human factor X(a). In addition, circular dichroism spectroscopy clearly demonstrated that C6PS binding to bovine factor X(a) induces secondary structural changes. In addition, C6PS binding to the tighter of the two sites triggered structural changes that lead to Ca(2+)-dependent dimer formation, as demonstrated by changes in intrinsic fluorescence and quantitative native gel electrophoresis. Dimerization produced further change in secondary structure, either inter- or intramolecularly. These results, along with results presented previously, support a model in which C6PS binds in a roughly sequential fashion to two linked sites whose occupancy in both human and bovine factor X(a) elicits different structural and functional responses

Pancreatic Cancer and Venous Thromboembolism

Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of all pancreatic cancers and is the most fatal of all cancers. The treatment response from combination chemotherapies is far from satisfactory and surgery remains the mainstay of curative strategies. These challenges warrant identifying effective treatments for combating this deadly cancer. PDAC tumor progression is associated with the robust activation of the coagulation system. Notably, cancer-associated thrombosis (CAT) is a significant risk factor in PDAC. CAT is a concept whereby cancer cells promote thromboembolism, primarily venous thromboembolism (VTE). Of all cancer types, PDAC is associated with the highest risk of developing VTE. Hypoxia in a PDAC tumor microenvironment also elevates thrombotic risk. Direct oral anticoagulants (DOACs) or low-molecular-weight heparin (LMWH) are used only as thromboprophylaxis in PDAC. However, a precision medicine approach is recommended to determine the precise dose and duration of thromboprophylaxis in clinical setting

A Current of Anti-Japanese Sentiments : Anti-Japanese sentiment of Liberal Criticism

It was not only the intellectuals who had congregated in All-China Federation of National Salvation Association that called for the realization of resistance to Japan and democracy before "7・7" (i.e. "The Marco Polo Bridge Incident"). In the past studies, however, the public anti-Japanese movements, which had gathered into All-China Federation of National Salvation Association, have been mainly discussed. In the present work I would like to analize the anti-Japanese sentiments of Lo Lung-chi (羅隆基) and his fellows, who had close relation to The National Socialist Party (中国国家社会党), using Liberal Criticism (『自由評論』) as the basic material. The period investigated is that from the winter of 1935 to the autumn of 1936. The aim of this thesis is as follows: (1) to make clear the community between the anti-Japanese sentiments of All-China Federa tion of National Salvation Association and those of the intellectuals of the Liberal Criticism faction, and to demonstrate that this community brought about the gradual consolidation of several anti-Japanese movements into one after "9・18" (i.e. "The Manchurian Incident"); (2) to clarify the difference in opinion between All-China Federation of National Salvation Association and the intellectuals of the Liberal Criticism faction, and to point out that this explains the essential difference in their views as to the Chinese Revolution after the anti-Japanese resistance was over