Transport Rate Limited Catalysis on Macroscopic Surfaces: The Activation of Factor X in a Continuous Flow Enzyme Reactor

Andree, Harry A. M.; Contino, Paul; Repke, Doris I.; Gentry, Rodney D.; Nemerson, Yale

doi:10.1021/bi00180a034

Cited by 35 publications

(27 citation statements)

References 30 publications

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“…This agrees well with the maximal estimate of ϳ10 fX molecules/leaflet based solely on the surface area of a Gla domain. Using 0.7 nm 2 /phospholipid, this corresponds to a surface coverage of 29 pmol fX/cm 2 , which is significantly higher than reported in the literature in experiments using fX binding to liposomes (33,36). We attribute this apparently higher coverage to the fact that Nanodiscs have boundaries beyond which the globular portions of these proteins can project (as demonstrated graphically by the TF⅐VIIa complex in Fig.…”

Section: Resultsmentioning

confidence: 51%

The Local Phospholipid Environment Modulates the Activation of Blood Clotting

Shaw¹,

Pureza

Sligar

et al. 2007

Journal of Biological Chemistry

141

180

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Examples abound of membrane-bound enzymes for which the local membrane environment plays an important role, including the ectoenzyme that triggers blood clotting, the plasma serine protease, factor VIIa, bound to the integral membrane protein, tissue factor. The activity of this enzyme complex is markedly influenced by lipid bilayer composition and further by tissue factor partitioning into membrane microdomains on some cell surfaces. Unfortunately, little is known about how membrane microdomain composition controls factor VIIa-tissue factor activity, as reactions catalyzed by membrane-tethered enzymes are typically studied under conditions in which the experimenter cannot control the composition of the membrane in the immediate vicinity of the enzyme. To overcome this problem, we used a nanoscale approach that afforded complete control over the membrane environment surrounding tissue factor by assembling the factor VIIa⅐tissue factor complex on stable bilayers containing 67 ؎ 1 phospholipid molecules/leaflet (Nanodiscs). We investigated how local changes in phospholipid bilayer composition modulate the activity of the factor VIIa⅐tissue factor complex. We also addressed whether this enzyme requires a pool of membrane-bound protein substrate (factor X) for efficient catalysis, or alternatively if it could efficiently activate factor X, which binds directly to the membrane nanodomain adjacent to tissue factor. We have shown that full proteolytic activity of the factor VIIa⅐tissue factor complex requires extremely high local concentrations of anionic phospholipids and further that a large pool of membrane-bound factor X is not required to support sustained catalysis.In both normal hemostasis and many life-threatening thrombotic diseases, blood clotting is triggered when tissue factor (TF), 5 an integral membrane protein, binds the plasma serine protease factor VIIa (fVIIa) (1). The resulting two-subunit membrane-bound enzyme TF⅐VIIa activates the plasma zymogen factors IX (fIX) and X (fX) by limited proteolysis. Erwin Chargaff (of nucleic acids fame) demonstrated in the 1940s that TF procoagulant activity requires it to be associated with phospholipids, and research over the ensuing decades has demonstrated that TF is a membrane-spanning protein that must be incorporated into bilayers containing anionic phospholipids for optimal activity (reviewed by Bach (2)). Despite this extensive history, we still have an indistinct picture of how anionic phospholipids contribute so profoundly to the enzymatic activity of membrane-bound protease complexes involved in blood clotting (3).Membranes composed of mixed phospholipids (and other lipids) can form membrane microdomains with locally different surface properties. A noted example is the formation of cholesterol-and sphingolipid-rich lipid rafts and caveolae on cell surfaces (4, 5). Experiments using giant unilamellar vesicles have shown that even liposomes containing simple binary mixtures of neutral and anionic phospholipids spontaneously form anionic phospholipid-rich me...

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Section: Resultsmentioning

confidence: 51%

The Local Phospholipid Environment Modulates the Activation of Blood Clotting

Shaw¹,

Pureza

Sligar

et al. 2007

Journal of Biological Chemistry

141

180

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“…This is because binding simply delays the steady state, but not its value, a phenomenon our group demonstrated in a tubular flow reactor. 18 Substitution of the 27-m platelet layer with an equal layer of 1.5-m in diameter, polystyrene microspheres did not significantly reduce the appearance of FX a , and adding 5 times the thickness of microspheres only marginally decreased its appearance. Thus the physical impedance imposed by platelets likely results from their asymmetric shape and their ability to spread over surfaces when activated.…”

Section: Discussionmentioning

confidence: 99%

Platelet deposition inhibits tissue factor activity: in vitro clots are impermeable to factor Xa

Hathcock¹,

Nemerson²

2004

Blood

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Upon plaque rupture or vascular injury, tissue factor (TF) protein in the vessel wall becomes exposed to flowing blood, initiating a cascade of reactions resulting in the deposition of fibrin and platelets on the injured site. Paradoxically, the growing thrombus may act as a barrier, restricting the convective and diffusive exchange of substrates and coagulation products between the blood and reactive vessel wall, thus limiting the role TF plays in thrombus growth. In this study, various in vitro, platelet-fibrin clots were prepared on TF:VIIa-coated surfaces and the rate at which factor (F) X in the well-mixed clot supernatant permeates the clot and is converted to X a was monitored over several hours. The apparent diffusion coefficients of FX (a) in fibrin and plateletfibrin clots at 37°C was 2.3 ؋ 10 ؊7 and 5.3 ؋ 10 ؊10 cm 2 /second, respectively, indicating that the mean time required for FX (a) , and likely FIX (a) , to diffuse 1 mm in a fibrin clot is 4 hours, and in the presence of platelets, 3.6 months. As complete human thrombotic occlusion has been observed within 10 minutes, an alternative source of procoagulant activity that can localize to the outer surface of growing thrombi, such as platelet factor XI or blood-borne TF, appears essential for rapid thrombus growth. IntroductionIn the generally accepted view of thrombosis, rupture or erosion of an atherosclerotic plaque exposes procoagulant regions on the vascular wall that bind platelets and initiate thrombus formation. The exposed material thought responsible for initiating coagulation is tissue factor (TF), which complexes with VII (a) to activate factor (F) IX and FX. Immediately following injury, FIX and FX are rapidly delivered to the vessel wall by flowing blood resulting in localized thrombin generation. However, as fibrin and platelets accrue to the injured surface, flow to the underlying TF region becomes restricted and substrates must percolate through an overlying platelet-fibrin mesh in order to become activated ( Figure 1). Furthermore, to achieve effective prothrombinase activity (X a :V a ) near the outer surface of a growing thrombus, the products (IX a or X a ) must diffuse a substantial distance back through the overlying mass. Through its interaction with VIIIa, factor IX (a) plays a crucial role in amplifying the amount of X a generated; However, since the only accepted source of Ixa in most coagulation models is its generation by TF:VII a , IX a would have to percolate a substantial distance through the overlying platelets and fibrin in order to generate tenase activity near the surface of the growing thrombus. (Factor XI-mediated activation of IX through the intrinsic pathway is generally not regarded as an essential step in thrombosis and is addressed later.) Hence, to obtain occlusive thrombi, the substrates (IX and X) and their respective enzymes, either individually or working in concert, must travel several millimeters through a growing thrombus without the aid of convection. As diffusion of proteins the size of fact...

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“…Furthermore, a macroscopic phospholipid membrane (with embedded TF) was used rather than unilamellar vesicles because this model mimics the plasma membrane of TF-bearing cells better than small unilamellar vesicles. Moreover, several studies have shown that the kinetics of activation and inactivation of blood coagulation enzyme complexes are dependent on the characteristics of the phospholipid surface like the radius of the phospholipid vesicle (35,38), microscopic homogeneity (39), and ratio of reactant-bearing vesicles over non-bearing vesicles (25). Additionally, with macroscopic surfaces it is easier to separate physically the surface-bound and fluid-phase reactions.…”

Section: Discussionmentioning

confidence: 99%

“…1 reveals that, in contrast to the TF⅐PC surface, it takes for the TF⅐PSPC surface about 1 min before FXa activity starts to increase linearly. This delayed rise in solution FXa activity most likely reflects binding of FXa to the TF⅐PSPC surface (35). Therefore, the initial rate of FX activation is defined as the linear increase in solution phase FXa 1.5 min after the start of the reaction.…”

Section: Initial Rate Of Fx Activation At the Surface Of A Rotating Dmentioning

confidence: 99%

Inhibition of Tissue Factor-Factor VIIa-catalyzed Factor X Activation by Factor Xa-Tissue Factor Pathway Inhibitor

Salemink¹,

Franssen²,

Willems³

et al. 1999

Journal of Biological Chemistry

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The physiological inhibitor of tissue factor (TF)⅐factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPI FL ) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TF⅐FVIIa-catalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF⅐PC) or 25% phosphatidylserine and 75% phosphatidylcholine (TF⅐PSPC) was measured in the presence of preformed complexes of FXa⅐TFPI FL Blood coagulation in vivo is initiated when circulating factor VII(a) binds in a calcium-dependent way to its cofactor, tissue factor (TF) 1 (see Refs. 1 and 2 for a review). This complex formation results in enhanced catalytic activity of factor VIIa (FVIIa), which via limited proteolysis, activates factors X (FX) and IX (FIX) (3). TF is a transmembrane glycoprotein, which under normal conditions is expressed only in extravascular tissues (4, 5).The main physiological regulator of TF-induced blood coagulation is tissue factor pathway inhibitor (TFPI) (6, 7), a single chain glycoprotein of 42 kDa and a member of the Kunitz family of serine protease inhibitors. TFPI contains an acidic N terminus, followed by three tandemly repeated Kunitz-type inhibition domains, and a basic C-terminal tail (8). Site-directed mutagenesis has revealed that the first Kunitz domain binds to FVIIa and that the second Kunitz domain interacts with the active site of FXa (9). No such functions could be attributed to the third Kunitz domain (10). Yet, various interactions have been ascribed to this domain, e.g. with lipoproteins and heparin, but their importance for the inhibitory function of TFPI is not clear (11,12). On the other hand, the basic C-terminal region of TFPI (residues 240 -276) has been shown to play a crucial role in the anticoagulant activity of this inhibitor (13,14). Despite numerous studies, it remains unclear how this basic C terminus modulates the anticoagulant activity of TFPI (15)(16)(17)(18)(19). TFPI inhibits the generation of FXa and FIXa by the TF⅐FVIIa complex in a unique, two-step reaction (20). First, TFPI binds Ca 2ϩ independently to FXa, thereby inhibiting the FXa catalytic activity (9). In a second step, the FXa⅐TFPI complex binds in a Ca 2ϩ -dependent way to TF⅐FVIIa. This results in the formation of the quaternary complex TF⅐FVIIa⅐FXa⅐TFPI, in which the proteolytic activity of the TF⅐FVIIa complex is fully neutralized. The effect of TFPI on TF⅐FVIIa activity in the absence of FXa is negligible (21,22), implying that the true inhibitor of TF⅐FVIIa activity is the FXa⅐TFPI complex. The rate of complex formation of FXa and TFPI is enhanced by negatively charged phospholipids for full-length TFPI (TFPI FL ) but not for TFPI 1-161 , a truncated variant lacking the third Kunitz domain and the potential phospholipid binding C-terminal tail (16,23).Recently (24), we demonstrated that TFPI FL in complex with FXa has a much higher affinity for anionic ...

show abstract

Transport Rate Limited Catalysis on Macroscopic Surfaces: The Activation of Factor X in a Continuous Flow Enzyme Reactor

Cited by 35 publications

References 30 publications

The Local Phospholipid Environment Modulates the Activation of Blood Clotting

The Local Phospholipid Environment Modulates the Activation of Blood Clotting

Platelet deposition inhibits tissue factor activity: in vitro clots are impermeable to factor Xa

Inhibition of Tissue Factor-Factor VIIa-catalyzed Factor X Activation by Factor Xa-Tissue Factor Pathway Inhibitor

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