Rinku Majumder

Soluble Phosphatidylserine Triggers Assembly in Solution of a Prothrombin-activating Complex in the Absence of a Membrane Surface

Factor Xa (FXa) binding to factor Va (FVa) on platelet-derived membranes containing surface-exposed phosphatidylserine (PS) forms the “prothrombinase complex” that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FVa, FVa1 and FVa2. These two isoforms differ by a 3-kDa polysaccharide chain (at Asn2181 in human FVa1 (Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., Lentz, B. R., and Kane, W. H. (1999)Biochemistry 38, 11448–11454)) and have different coagulant activities. We examined the interaction of the two bovine isoforms with active site-labeled FXa, finding no significant difference. A soluble form of PS (C6PS) bound to FVa1 and FVa2 with comparable affinities (K d = 11–12 μm) and changes in FVa intrinsic fluorescence. At concentrations well below its critical micelle concentration, C6PS binding to bovine FVa2 enhanced its affinity for FXa in solution by nearly 3 orders of magnitude (K d eff = 40–2 nm over a C6PS range of 30–400 μm) but had no effect on the affinity of FVa1for FXa (K d = 1 μm). This results in a soluble complex between FXa and FVa2, whose expected molecular weight was confirmed by calibrated native gel electrophoresis. This complex behaved as a normal Michaelis-Menten enzyme in its ability to produce thrombin from meizothrombin (apparentk cat/K m ≅ 109 m −1 s−1). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.

Effects of Water Soluble Phosphotidylserine on Bovine Factor Xa: 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.

Inhibition of Intrinsic Xase by Protein S: A Novel Regulatory Role of Protein S Independent of Activated Protein C

Objective—Protein S is a vitamin K–dependent plasma protein that functions in the feedback regulation of thrombin generation. Our goal was to determine how protein S regulates the intrinsic pathway of blood coagulation. Methods and Results—We used plasma, including platelet-rich plasma, and in vitro methods to determine how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: (1) activated partial thromboplastin time assays with protein S–supplemented plasma confirmed that protein S prolongs clotting time; (2) a modified activated partial thromboplastin time assay with factor IX (fIX)–deficient plasma confirmed that protein S affects fIX-initiated clotting; (3) a fIXa/factor VIIIa (fVIIIa)–mediated thrombin generation assay with either platelet-rich plasma or factor-deficient plasma, initiated with a limiting amount of tissue factor, was regulated by protein S; (4) in the presence of phosphatidylserine vesicles, protein S inhibited fIXa in the absence and presence of fVIIIa; and (5) protein S altered only the KM for factor X activation by fIXa in the absence of fVIIIa and both kcat and KM in the presence of fVIIIa. Conclusion—From our findings, it can be concluded that protein S inhibits fIXa in the presence or absence of fVIIIa in an activated protein C–independent way.

Factor Xa Binding to Phosphatidylserine-Containing Membranes Produces an Inactive Membrane-Bound Dimer

Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXas active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.

Functional and Structural Characterization of Factor Xa Dimer in Solution

Previous studies showed that binding of water-soluble phosphatidylserine (C6PS) to bovine factor Xa (FXa) leads to Ca2+-dependent dimerization in solution. We report the effects of Ca2+, C6PS, and dimerization on the activity and structure of human and bovine FXa. Both human and bovine dimers are 10(6)- to 10(7)-fold less active toward prothrombin than the monomer, with the decrease being attributed mainly to a substantial decrease in k(cat). Dimerization appears not to block the active site, since amidolytic activity toward a synthetic substrate is largely unaffected. Circular dichroism reveals a substantial change in tertiary or quaternary structure with a concomitant decrease in alpha-helix upon dimerization. Mass spectrometry identifies a lysine (K(270)) in the catalytic domain that appears to be buried at the dimer interface and is part of a synthetic peptide sequence reported to interfere with factor Va (FVa) binding. C6PS binding exposes K(351) (part of a reported FVa binding region), K(242) (adjacent to the catalytic triad), and K(420) (part of a substrate exosite). We interpret our results to mean that C6PS-induced dimerization produces substantial conformational changes or domain rearrangements such that structural data on PS-activated FXa is required to understand the structure of the FXa dimer or the FXa-FVa complex.

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 (K(d) ~ 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 Ca(2+) to show that the apparent K(d) of FVa-FXa interaction increased with an increase in FXa concentration at 5 mM Ca(2+), but the K(d) was only slightly affected at 3 mM Ca(2+). 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 weakened dimerization (K(d) ~ 147 nM) and weakened FVa binding (apparent K(d) values of 58, 92, and 128 nM for 5, 20, and 50 nM R165A FXa, respectively). Native gel electrophoresis showed that the GLA-EGF(NC) 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 Ca(2+). 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.

Factor Xa dimerization competes with prothrombinase complex formation on platelet-like membrane surfaces

Exposure of phosphatidylserine (PS) molecules on activated platelet membrane surface is a crucial event in blood coagulation. Binding of PS to specific sites on factor Xa (fXa) and factor Va (fVa) promotes their assembly into a complex that enhances proteolysis of prothrombin by approximately 10⁵. Recent studies demonstrate that both soluble PS and PS-containing model membranes promote formation of inactive fXa dimers at 5 mM Ca²⁺. In the present study, we show how competition between fXa dimerization and prothrombinase formation depends on Ca²⁺ and lipid membrane concentrations. We used homo-FRET measurements between fluorescein-E-G-R-chloromethylketone (CK)-Xa [fXa irreversibly inactivated by alkylation of the active site histidine residue with FEGR (FEGR-fXa)] and prothrombinase activity measurements to reveal the balance between fXa dimer formation and fXa-fVa complex formation. Changes in FEGR-fXa dimer homo-FRET with addition of fVa to model-membrane-bound FEGR-fXa unambiguously demonstrated that formation of the FEGR-fXa-fVa complex dissociated the dimer. Quantitative global analysis according to a model for protein interaction equilibria on a surface provided an estimate of a surface constant for fXa dimer dissociation (K(fXa×fXa)(d, σ)) approximately 10-fold lower than K(fXa×fVa)(d,σ) for fXa-fVa complex. Experiments performed using activated platelet-derived microparticles (MPs) showed that competition between fXa dimerization and fXa-fVa complex formation was even more prominent on MPs. In summary, at Ca²⁺ concentrations found in the maturing platelet plug (2-5 mM), fVa can compete fXa off of inactive fXa dimers to significantly amplify thrombin production, both because it releases dimer inhibition and because of its well-known cofactor activity. This suggests a hitherto unanticipated mechanism by which PS-exposing platelet membranes can regulate amplification and propagation of blood coagulation.

Soluble phosphatidylserine binds to two sites on human factor IXa in a Ca2+ dependent fashion to specifically regulate structure and activity.

Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.

Anticoagulant Protein S Targets the Factor IXa Heparin-Binding Exosite to Prevent Thrombosis

Objective— PS (protein S) is a plasma protein that directly inhibits the coagulation FIXa (factor IXa) in vitro. Because elevated FIXa is associated with increased risk of venous thromboembolism, it is important to establish how PS inhibits FIXa function in vivo. The goal of this study is to confirm direct binding of PS with FIXa in vivo, identify FIXa amino acid residues required for binding PS in vivo, and use an enzymatically active FIXa mutant that is unable to bind PS to measure the significance of PS–FIXa interaction in hemostasis. Approach and Results— We demonstrate that PS inhibits FIXa in vivo by associating with the FIXa heparin-binding exosite. We used fluorescence tagging, immunohistochemistry, and protein–protein crosslinking to show in vivo interaction between FIXa and PS. Importantly, platelet colocalization required a direct interaction between the 2 proteins. FIXa and PS also coimmunoprecipitated from plasma, substantiating their interaction in a physiological milieu. PS binding to FIXa and PS inhibition of the intrinsic Xase complex required residues K132, K126, and R170 in the FIXa heparin-binding exosite. A double mutant, K132A/R170A, retained full activity but could not bind to PS. Crucially, Hemophilia B mice infused with FIXa K132A/R170A displayed an accelerated rate of fibrin clot formation compared with wild-type FIXa. Conclusions— Our findings establish PS as an important in vivo inhibitor of FIXa. Disruption of the interaction between PS and FIXa causes an increased rate of thrombus formation in mice. This newly discovered function of PS implies an unexploited target for antithrombotic therapeutics.

Mini-review on "A novel one-step purification of mouse factor IX".

Factor IX (FIX) is a 70-kDa, single-chain, vitamin K-dependent glycoprotein present in trace amounts (~95 nM) in blood plasma1,2. FIX is synthesized as a zymogen that is converted to a serine protease, FIXa, by the activated form of factor XI3,4. FIXa has a central function in the intrinsic pathway of blood coagulation, in that it acts as an activator of factor X (FX), directly upstream of the common pathway1,5. Upon activation, FXa concomitantly converts prothrombin to thrombin to initiate the formation of the fibrin lattice6,7.