Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M511419200 on January 23, 2006

J. Biol. Chem., Vol. 281, Issue 13, 8773-8779, March 31, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/13/8773    most recent
M511419200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Toso, R.
Right arrow Articles by Camire, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Toso, R.
Right arrow Articles by Camire, R. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Role of Hirudin-like Factor Va Heavy Chain Sequences in Prothrombinase Function*

Raffaella Toso{ddagger} and Rodney M. Camire{ddagger}§1

From the {ddagger}Department of Pediatrics, Division of Hematology, The Children's Hospital of Philadelphia and §University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, October 20, 2005 , and in revised form, December 9, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659–698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa709 (des710–1545), FVa699 (des700–1545), FVa692 (des693–1545), FVa678 (des679–1545), and FVa658 (des659–1545). We found that FVa709, FVa699, FVa692, and FVa678 exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the Km and kcat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa709, FVa699, FVa692, and FVa678 bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa658 had an ~1% clotting activity and reduced affinity for FXa membranes (~20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa658 exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Blood coagulation factor Va (FVa)2 is a heterodimeric protein composed of a heavy chain (residues 1–709; 105 kDa) and a light chain (residues 1546–2196; 74 kDa) which arises from limited proteolysis of the pro-cofactor factor V (FV) (1). Factor Va reversibly associates with the serine protease factor Xa (FXa) on an appropriate membrane surface in the presence of calcium ions to form prothrombinase. This enzyme complex cleaves two peptide bonds in prothrombin, resulting in the generation of {alpha}-thrombin (IIa) (2). This reaction is also catalyzed by membrane-bound FXa; however, incorporation of FVa within prothrombinase has a profound effect on the rate of IIa generation. Additionally, prothrombinase has remarkable specificity, as its only known biological substrate is prothrombin (2).

Although the molecular basis underlying the rate-enhancing effect of FVa remains largely unknown, recent contributions have provided insight into how prothrombinase recognizes its protein substrate (3–5). These studies support a model in which binding specificity is determined by two resolvable steps involving an interaction at an exosite followed by active site docking. These exosites have yet to be fully defined, but there is some evidence that at least part of the enzyme exosite lies on the catalytic domain of FXa (6–8). Because it is well established that prothrombin binds the cofactor (9–16), this interaction may also play an important role in substrate recognition.

There is increasing evidence that catalytic domain structures on prothrombin provide an exosite docking surface for prothrombinase (3, 17, 18). A series of observations suggest that the proexosite I region, the precursor site to IIa exosite I (19), contributes either directly or indirectly to this substrate binding site. For example, reagents that block proexosite I such as hirugen and bothrojaracin inhibit macromolecular substrate cleavage by prothrombinase or the FXa-FVa complex (3, 20, 21). Additionally, proexosite I prethrombin-1 mutants and a naturally occurring prothrombin variant (Arg67 to His, chymotrypsin numbering system (22)) are poor substrates for prothrombinase and FXa-FVa (23, 24). Interestingly, these probes or mutations reduced the rate of IIa generation only in the presence of the cofactor, suggesting a link between FVa and proexosite I. Consistent with this, IIa exosite I mutants and exosite I probes inhibit FV activation (25–27). Additionally, detailed studies by Bock and co-workers have provided compelling evidence that the FVa heavy chain binds to exosite I of IIa (26, 28). Taken together, these data are consistent with the idea that proexosite I plays an important role in macromolecular substrate recognition by facilitating productive interactions between FVa and prothrombin.

Based on these findings it is reasonable to hypothesize that the corresponding binding site on FVa for proexosite I is found within the hirudin-like C-terminal region of the FVa heavy chain (659–698; Fig. 1). Evidence to support this derives from recent experiments employing synthetic FVa peptides. One of these peptides (DYDYQ; 695–699, see Fig. 1), like hirugen, was found to bind specifically to IIa and inhibit prothrombin activation, suggesting this region of FVa provides a substrate binding site within prothrombinase (29, 30). In support of this, changing DYDY to KFKF (FVa2K2F) significantly reduced FVa cofactor activity within prothrombinase (30). However, it is unlikely that these data provide a complete explanation since proteolysis within the C-terminal FVa heavy chain region by a variety of enzymes only modestly reduces cofactor function in certain assay systems (29, 31–34).

To evaluate the importance of this region of FVa to prothrombinase function in more detail, we have employed a novel strategy to express recombinant FVa derivatives with specific truncations within the C terminus of the FVa heavy chain and used a series of physical and kinetic measurements to assess the contribution of this region to substrate binding within prothrombinase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Bovine serum albumin (BSA) and 5,5'-dithiobis(2-nitrobenzoic acid) were purchased from Sigma. Hippuryl-D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide (S2238) was purchased from Diapharma Group, Inc. (West Chester, OH), and its concentration was verified (in water) using E342 = 8270 M-1 cm-1 (35). Oregon Green488 maleimide and succinimidyl acetothioacetate were from Molecular Probes (Eugene, OR). D-Phenylalanyl-L-prolyl-L-arginine (FPR) chloromethyl ketone was from Calbiochem. SulfoLink coupling gel was purchased from Pierce. Dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide was purchased from Hematologic Technologies (Essex Junction, VT). All tissue culture reagents were from Invitrogen except insulin-transferrin-sodium selenite, which was from Roche Applied Science. L-{alpha}-Phosphatidylserine (brain, sodium salt) and L-{alpha}-phosphatidylcholine (egg yolk) were purchased from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL). Small unilamellar phospholipid vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine (PCPS) were prepared as described previously (36). The concentration of the phospholipid vesicles was determined by phosphorous assay (37). Unless otherwise noted, all functional assays were performed at 25 °C in 20 mM Hepes, 0.15 M NaCl, 2 mM CaCl2, 0.1% polyethylene glycol 8000, pH 7.5 (assay buffer).

Proteins—Human prothrombin, FX, and FV were isolated from plasma as described previously (6, 38, 39). Prethrombin-1, prethrombin-2, and IIa were prepared from human prothrombin and purified using established procedures (40). A recombinant B-domainless form of FV (rFV-DT) used to prepare rFVa was expressed and purified as previously described (41). Human plasma derived FV (PD-FV) and rFV-DT were proteolytically processed with IIa to generate PD-FVa and rFVa, and both were subsequently purified on a Poros HS/20 (0.46 x 10 cm) column as described (41, 42). Oregon Green488-human FXa (OG488-FXa) was prepared and characterized as described (6).

Protein concentrations were determined using the following molecular weights and extinction coefficients (Formula): prothrombin, 72,000 and 1.47 (40); prethrombin-1 49,900 and 1.78 (40); prethrombin-2, 37,500 and 1.95 (40); IIa, 37,500 and 1.94 (43); FXa, 45,300 and 1.16 (44); PD-FVa, 173,000 and 1.78; rFVa, 175,000 and 1.78 (41). The calculated molecular weights for the rFVa derivatives were taken as: rFVa709, 175,000; rFVa699, 174,060; rFVa692, 173,190; rFVa678, 171,478; rFVa658, 171,165; an extinction coefficient of 1.78 was used for each derivative. N-terminal sequence analysis was performed in the laboratory of Dr. Alex Kurosky and Steven Smith at the University of Texas Medical Branch at Galveston.

Construction of rFVa Derivatives—The human FV cDNA was subcloned into the pED expression plasmid as previously described (41). Splicing by overlap extension was used to generate a series of FV derivatives in which the entire B-domain and portions of the C terminus of the heavy chain were deleted. To facilitate intracellular proteolytic processing, a DNA sequence encoding a PACE/furin recognition site was inserted between the new C terminus on the heavy chain and the light chain. The following five constructs were prepared (RKRRKR was inserted before amino acid 1546 for all constructs, see Fig. 1): rFVa709 (des710–1545), rFVa699 (des700–1545), rFVa692 (des693–1545), rFVa678 (des679–1545), and rFVa658 (des659–1545). Specific oligonucleotides used for rFVa709 were as follows: primer A, 5'-GAAGAGGTGGGAATACTT-3' corresponds to the cDNA sequence coding for amino acid residues 319–325; primer B, 5'-GCGCTTTCTACGCTTTCTCCTAATTCCTAATGCTGC-3' in which the first 18 bases correspond to cDNA sequence coding for a PACE/furin recognition sequence (RKRRKR), and the last 18 bases correspond to cDNA sequence coding for residues 709–704; primer C, 5'-AGAAAGCGTAGAAAGCGCAGCAACAATGGAAACAGAAGA-3' in which the first 18 bases correspond to cDNA sequence coding for RKRRKR and the last 21 bases correspond to cDNA sequence coding for residues 1546–1552; primer D, 5'-TCTGTCCATGATAAGAAATGG-3', which corresponds to the FV cDNA sequence coding for residues 1877–1871. The resulting DNA fragment was digested with Bsu36I and SnaBI, gel-purified, and subcloned into pED-FV digested with the same enzymes. To ensure the absence of polymerase-induced errors, the entire modified cDNA was sequenced. The remaining constructs were prepared in the same way, except primer B was appropriately changed.

Expression and Purification of rFVa Derivatives—Plasmids encoding each of the rFVa constructs were transfected into baby hamster kidney cells, and stable clones were established essentially as described (41). Protein expression levels varied from 1 to 4 µg/106cells/24 h. Each of the rFVa derivatives was expanded into triple flasks, and approximately 1 liter of conditioned media was harvested daily for 4 days and immediately loaded onto a SP-Sepharose (Amersham Biosciences) column equilibrated in 20 mM Hepes, 0.18 M NaCl, 5 mM CaCl2, pH 7.4. The column was washed with the same buffer and then eluted with 0.65 M NaCl. Fractions containing FVa activity were pooled and concentrated by ultrafiltration (Millipore), and the buffer was exchanged to reduce the NaCl concentration. The protein was then loaded onto a Poros HS/20 (0.46 x 10 cm) column equilibrated with 20 mM Hepes, 0.15 M NaCl, 5 mM CaCl2, pH 7.4. Bound protein was eluted with a gradient of increasing NaCl (0.15–1.0 M). The final yield was ~0.5–2.0 mg of rFVa/liter of conditioned media, and the purified protein was stored at -80 °C. Protein purity was assessed by SDS-PAGE using pre-cast 4–12% gradient gels (Invitrogen) under reducing conditions using the MOPS buffer system followed by staining with Coomassie Brilliant Blue R-250.

FV-specific PT-based Clotting Assay—Factor Va (200 nM) derivatives were prepared in assay buffer. Samples were then diluted to less than 1 nM in assay buffer with 0.1% albumin and specific clotting activity using FV-deficient plasma (George King Bio-medical Inc., Overland Park, KS) was performed as described (33).

Fluorescence Intensity Measurements—Samples (2.5 ml) in assay buffer were maintained at 25 °C in 1 x 1-cm2 stirred quartz cuvettes, and steady state fluorescence intensity was measured using {lambda}ex = 480 and {lambda}em = 520 nm with a long pass filter (KV500, Schott, Duryea, PA) in the emission beam. Measurements, including controls, were performed essentially as described (3, 6).

Kinetics of Protein Substrate Cleavage—Steady state initial velocities of macromolecular substrate cleavage by prothrombinase were determined discontinuously at 25 °C as described (17, 45). The kinetic parameters of prothrombinase-catalyzed prothrombin, prethrombin-2/fragment 1.2, or prethrombin-1 activation (Km and Vmax) were determined in assay buffer by measuring the initial rate of IIa formation at increasing concentrations of macromolecular substrate. Assay mixtures contained PCPS (20 or 50 µM), FVa (20 nM), and various concentrations of prothrombin (0–1.5 µM), prethrombin-2/fragment 1.2 (0–1.5 µM; 1.5 molar excess of fragment 1.2), or prethrombin-1 (0–12 µM). The reaction was initiated with 0.1 nM FXa for prothrombin and prethrombin-2/fragment 1.2 or 0.5 nM FXa for prethrombin-1.

Initial rates of prothrombin activation in the absence of membranes were determined essentially as described (20). Reaction mixtures in assay buffer contained FVa (5 nM; 200 nM for rFVa658) and various concentrations of prothrombin (0–15 µM). The reaction was initiated with 10 nM FXa, and the rate of thrombin generation was measured as described (17, 45).


Figure 1
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1.
Construction of recombinant FVa variants. Schematic representation of the likely processing steps of rFVa variants, which harbor a PACE/furin recognition sequence (RKRRKR) between the heavy (1–709) and light (1546–2196) chains. After synthesis, we speculate that intracellular proprotein convertases (i.e. PACE/furin) remove the RKRRKR sequence from the light chain, and the newly exposed basic residues are removed by an Arg/Lys carboxypeptidase from the heavy chain. The resulting two-chain protein is then secreted into the media. This model is consistent with current understanding regarding the processing events of proteins that harbor internal pairs of basic amino acids (53–55). The hirudin-like C-terminal region of the heavy chain is shown with acidic regions 1 and 2 indicated. SPCs, serine proprotein convertases.

 
Chromatography of FVa on IIa-agarose—N{alpha}-[(acetylthio)acetyl]-FPR-chloromethyl ketone was prepared and characterized as previously reported (46, 47). N{alpha}-[(acetylthio)acetyl]-FPR-IIa and N{alpha}-[(acetylthio)-acetyl]-FPR-IIa coupled to agarose were prepared using previously established procedures (26). The IIa-agarose matrix contained 3.7 mg of IIa coupled/ml of gel. Free binding sites were blocked with BSA. The BSA-agarose matrix used as the control for nonspecific binding contained 10 mg of BSA coupled/ml of gel. Columns were equilibrated in 50 mM Hepes, 110 mM NaCl, 5 mM CaCl2, 0.1% polyethylene glycol 8000, pH 7.4. Factor Va (~200 µg) was loaded onto each column at a flow rate of 8 ml/h at 22 °C and washed with the same buffer. Protein was eluted with 50 mM Hepes, 2 M NaCl, 5 mM CaCl2, 0.1% polyethylene glycol 8000, pH 7.4. Fractions (0.5 ml) were collected, and the absorbance at 280 nm and clotting activity were measured.

Data Analysis—Data were analyzed according to the referenced equations by nonlinear least squares regression analysis using the Marquardt algorithm (48). The qualities of the fits were assessed by the criteria described (49). Fitted parameters are reported ±95% confidence limits. Dissociation constants (Kd) and stoichiometries (n) for the interaction between FXa and membrane-bound FVa were obtained from the dependence of the fluorescence intensity on the concentrations of cofactor (50). Initial velocity measurements of prothrombin cleavage by prothrombinase were analyzed by fitting the data to the Henri-Michaelis-Menten equation (51), to yield fitted values for Km and Vmax.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Construction of rFVa Variants—To directly assess the contribution of hirudin-like FVa sequences (659–698) to cofactor function, the entire B-domain and specific heavy chain sequences were removed, and a PACE-furin cleavage site was introduced between these deleted sequences and the light chain generating rFVa658 (des659–1545), rFVa678 (des679–1545), rFVa692 (des693–1545), rFVa699 (des700–1545), and rFVa709 (des710–1545; see Fig. 1). Recombinant FVa709 served as a control as it should be equivalent to PD-FVa and rFVa. This system takes advantage of the endogenous intracellular proteolytic processing machinery allowing for internally cleaved two-chain FVa variants to be secreted into the cell culture media. We used this strategy to overcome potential problems in the activation of the deletion variants by IIa or RVV-V (factor V, cleaving protease isolated from Russell's viper venom) because of the elimination of possible enzyme binding sites contributed by heavy chain and B-domain sequences.


Figure 2
View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 2.
SDS-PAGE analysis of purified proteins. Purified proteins (3 µg/lane) were subjected to SDS-PAGE with disulfide bond reduction (50 mM dithiothreitol) and visualized by staining with Coomassie Blue R250. Lane 1, human PD-FVa; lane 2, rFVa; lane 3, rFVa709; lane 4, rFVa699; lane 5, rFVa692; lane 6, rFVa678; lane 7, rFVa658. The apparent molecular weights of the standards are indicated on the left. The gel was run for an extended period of time to maximize separation of the heavy chains.

 
Characterization of rFVa Variants—Western blot analysis of conditioned media derived from stable cell lines revealed that each variant was intracellularly processed and secreted in the two chain form (data not shown). These derivatives were subsequently purified to homogeneity by ion exchange chromatography. SDS-PAGE analyses (Fig. 2) indicated that the proteins migrated according to the expected molecular weight. N-terminal sequence analysis of the light chains for each derivative showed that the PACE/furin cleavage site was completely removed, as the N terminus was found to be SNNGNRRN (Table 1). Because it has been previously shown that baby hamster kidney cells process C-terminal basic amino acids from the light chain of protein C (52) and rFVa709 appears functionally equivalent to plasma-derived and rFVa (see Table 1), we speculate that the RKRRKR sequence is removed form the C terminus of the heavy chain, most likely by an Arg/Lys carboxypeptidase (53–55).


View this table:
[in this window]
[in a new window]
  		TABLE 1
Functional and physical characterization of FVa variants

 
Assessment of Specific Activity—Initial characterization of the FVa derivatives using a one-stage PT-based clotting assay with FV-deficient plasma indicated that rFVa709, rFVa699, rFVa692, and rFVa678 had specific clotting activities that were comparable with wild-type FVa (Table 1). These data show that removal of amino acids 679–709 has little if any impact on cofactor function in this assay system. These results deviate somewhat from previous reports which found a ~50–60% reduction in clotting activity using FVa cleaved by proteases within the C-terminal region of the heavy chain (29, 33). Interestingly, further truncation to position 658 resulted in a substantial reduction in activity. To verify the results of the clotting assay, a prothrombinase assay employing purified reagents was established. Progress curves of the conversion of prothrombin to IIa using limiting amounts of cofactor showed that rFVa658 had reduced cofactor activity, whereas the other variants had activity profiles similar to wild-type FVa (Table 1). These data indicate that 1) acidic region 2 (679–709) does not influence in an obvious way FVa cofactor function within prothrombinase, and 2) deletion of residues 659–709 impairs assembly of FVa in prothrombinase or otherwise alters cofactor activity.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3.
Chromatography of FVa variants on IIa-agarose. Elution profile of FVa variants (200 µg) on a 3-ml IIa-agarose column (3.7 mg IIa/ml of gel). Protein concentration was monitored by absorbance at 280 nm. Elution with buffer containing 2 M NaCl was started at the point indicated by the arrow. The symbols are as follows: {blacktriangleup}, PD-FVa; {triangleup}, rFVa; {blacksquare}, rFVa709; {square}, rFVa699; {diamondsuit}, rFVa692; •, rFVa678; {circ}, rFVa658. For clarity, rFVa658 is shown on the bottom panel. The data are representative of two similar experiments.

 
FVa Binding to IIa-agarose—Studies with immobilized IIa and kinetic inhibition experiments with (pro)exosite I probes indicate that IIa and prothrombin likely share a common mode of binding to FVa (20, 28). Affinity chromatography studies were used to qualitatively assess whether the truncated FVa variants bind IIa. As shown in Fig. 3, with the exception of rFVa658, each derivative was retained by the IIa-agarose column and eluted after the addition of 2 M NaCl. Electrophoretic analysis of the samples after chromatography did not reveal any significant proteolytic degradation (data not shown). None of the FVa constructs bound to a BSA-agarose column used to test nonspecific binding (data not shown). The data demonstrate that acidic region 2 (679–709) does not provide a binding site for IIa, a surprising contrast to conclusions obtained from peptide inhibition studies (29, 30). Although the results with rFVa658 indicate that acidic region 1 (659–678) may provide a binding site for IIa, we cannot rule out the possibility that this derivative is altered in some other fashion precluding its binding to IIa-agarose. Overall the chromatography data correlate well with the functional measurements (Table 1) in terms of which variant is altered. However, close inspection of the functional data reveals that there is a discrepancy in the magnitude of the reduction between the two assay systems for rFVa658. This discrepancy suggests that the underlying nature of the reduced cofactor activity of this variant is complex and likely reflects alterations to multiple aspects of cofactor function.

Binding of the Cofactors to FXa Membranes—To examine this further we evaluated the ability of each of the FVa variants to bind FXa membranes. Equilibrium fluorescence measurements were used to establish binding parameters describing the interaction between the cofactor and membrane-bound FXa. Using a fixed concentration of OG488-FXa, subsequent titration with incremental additions of PD-FVa, rFVa, rFVa709, rFVa699, rFVa692, or rFVa678 (Fig. 4) yielded a saturable increase in fluorescence intensity with comparable dissociation constants (Kd) and stoichiometries (n) (Table 2). These data indicate that complete removal of acidic region 2 (see Fig. 1) does not detectably influence binding interactions with FXa membranes. Similar results were obtained from kinetic assessment of cofactor binding to FXa membranes (data not shown). In contrast, rFVa658 bound with a reduced affinity (~20-fold) to FXa membranes. This finding implies that amino acids within acidic region 1 (659–678) somehow play a role in the high affinity interaction with FXa membranes. The simplest interpretation is that these residues provide a primary binding site. An alternative explanation is that structural changes that accompany deletion of these residues adversely impact FXa binding. It is also possible that these same structural changes influence IIa binding (Fig. 3). The decreased affinity of rFVa658 for FXa membranes explains at least in part the decreased cofactor activity observed in the functional measurements (Table 1), since both of these assays use limiting amounts of cofactor.


View this table:
[in this window]
[in a new window]
  		TABLE 2
Equilibrium binding constants for prothrombinase assembly Increasing concentrations of the different cofactor species were incubated with 10 nM OG488-FXa and 50 µM PCPS, and fluorescence intensity was recorded as described under "Experimental Procedures" (see Fig. 4). The errors in the fitted constants represent 95% confidence limits. Data are representative of two to three similar experiments.

 


Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4.
Fluorescence (F) measurements of the assembly of prothrombinase. Reaction mixtures containing 10 nM OG488-FXa and 50 µM PCPS in assay buffer were titrated with increasing concentrations of PD-FVa ({blacktriangleup}), rFVa ({triangleup}), rFVa709 ({blacksquare}), rFVa699 ({square}), rFVa692 ({diamondsuit}), rFVa678 (•), or rFVa658 ({circ}). Fluorescence intensity was measured at 25 °C. The lines are drawn after analysis to independent, noninteracting sites, and the fitted values are given in Table 2.

 
Kinetics of Macromolecular Substrate Cleavage—To further evaluate the ability of these modified cofactors to function within prothrombinase, initial velocity studies using prothrombin as the protein substrate with saturating amounts of cofactor were established (Fig. 5). In this system the steady state kinetic constants for each of the cofactors were essentially equivalent (Table 3). Surprisingly, even though rFVa658 had altered IIa binding, it exhibited essentially normal kinetic parameters for prothrombin upon full assembly in prothrombinase with the overall second-order rate constant (kcat/Km), differing by only ~1.5-fold compared with wild type. This decrease arose from a reduction in the maximal catalytic rate (kcat) with no obvious change in the Km. When the concentration of rFVa658 was increased, there was no significant difference in the kcat value (data not shown), suggesting a modest decrease in intrinsic cofactor function. These data indicate that removal of both acidic regions from the FVa heavy chain does not significantly impair the interaction of the prothrombinase complex with its physiological substrate prothrombin.


View this table:
[in this window]
[in a new window]
  		TABLE 3
Kinetic constants for macromolecular substrate cleavage Kinetic constants were derived from initial velocity studies conducted with increasing concentrations of substrate. Concentrations of reaction components can be found under "Experimental Procedures." The errors in the fitted constants represent 95% confidence limits. The data are representative of two to three independent measurements.

 


Figure 5
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 5.
Determination of kinetic constants for cleavage of prothrombin. The initial velocity of IIa generation was determined at increasing concentrations of prothrombin with 0.1 nM FXa, 20 µM PCPS, and 20 nM PD-FVa ({blacktriangleup}), rFVa ({triangleup}), rFVa709 ({blacksquare}), rFVa699 ({square}), rFVa692 ({diamondsuit}), rFVa678 (•), or rFVa658 ({circ}). The lines were drawn after analysis of all data sets to a rectangular hyperbola, and the fitted constants can be found in Table 3. The data are representative of two similar experiments.

 
It has been previously demonstrated that hirugen inhibits IIa generation only when using FXa-FVa or when using a non-membrane-bound protein substrate such as prethrombin-1 (3, 20). Because eliminating hirudin-like sequences on the FVa heavy chain did not significantly influence the kinetics of prothrombin activation, initial velocity studies were conducted with prothrombin in the absence of membranes (FXa-FVa) or with prethrombin-1 and prothrombinase. Consistent with the results presented above, in the absence of anionic membranes each of the recombinant variants, including rFVa658, had a Km for prothrombin that was comparable with wild-type FVa (Table 3). Because the conditions chosen for this experiment do not permit reliable calculation of the kcat, the Vmax is presented. With the exception of rFVa658, each of the cofactors had a similar Vmax for the reaction, suggesting that the fractional saturation of FXa with each of the FVa species was similar. The reduced Vmax obtained with rFVa658 likely reflects its reduced affinity for FXa (Fig. 4). Similar to results obtained with prothrombinase (Table 3), the data indicate that removal of hirudin-like sequences from the C terminus of the FVa heavy chain does not alter prothrombin binding to the FXa-FVa complex. In contrast to these results, differences in the initial rates of IIa generation were observed with each of the truncated FVa derivatives when using prethrombin-1. Measurements of the conversion of varying concentrations of prethrombin-1 to IIa were conducted using fixed, saturating amounts of membranes and cofactor. The deviations in rate principally arose from an increased Km value (~5–10-fold) for rFVa699, rFVa692, and rFVa678 (Table 3). Factor Va658 had a 15-fold increase in Km and a ~4.5-fold reduced kcat. Similar results were obtained with prethrombin-2 (data not shown). Interestingly, saturation of prethrombin-2 with fragment 1.2 completely restored the Km values to that seen with wild-type FVa (data not shown). Thus, elimination of the fragment 1 or fragment 1.2 domains revealed differences in cofactor function after removal of even a small portion of the heavy chain (rFVa699). These differences in the Km, however, are relatively small considering that sequences within the C terminus of the FVa heavy chain were completely removed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is now well established that exosite interactions play a principal role in macromolecular substrate recognition by prothrombinase (5). Although extended surfaces on FXa are at least partly involved (6–8, 56), there is increasing evidence that productive binding interactions between prothrombin and FVa also play a role. Recent work suggests that the proexosite I region on the zymogen contributes to this recognition surface for FVa within prothrombinase (25–28). Furthermore, peptide inhibition studies have shown that sequences (695–699) within the hirudin-like C-terminal region of the FVa heavy chain may provide a binding site for prothrombin (29, 30).

Drawing on these ideas, we directly examined the importance of these cofactor sequences by engineering a series of variants lacking portions of the C terminus of the heavy chain. The current work provides strong evidence that any possible binding interaction between these cofactor sequences and prothrombin do not contribute in a detectable way to the enhanced function of prothrombinase. This is not to say that removal of amino acids 659–709 had no influence on cofactor function, since rFVa658 had reduced affinity for FXa membranes. This finding likely explains the reduction in cofactor activity for rFVa658 noted in Table 1 since both assays employed limiting amounts of FVa and were very sensitive to changes in the FXa-FVa interaction (1). Additionally, we were also able to show that whereas removal of residues 679–709 had no obvious influence on the FVa-IIa interaction, further truncation to position 658 abrogated binding to IIa-agarose. At present we cannot discern whether this was caused by global changes in protein structure or if this region of FVa directly binds IIa. Regardless of the explanation, rFVa658 had essentially normal kinetic parameters for prothrombin when assembled into prothrombinase. This surprising finding indicates that whereas this region of FVa may be important for IIa binding, it has little to do with prothrombin recognition within prothrombinase.

The influence of the C-terminal heavy chain to FVa function has been the subject of several investigations with somewhat conflicting results. Multiple studies have shown that removal of FVa sequences within the 679–697 heavy chain region using a variety of proteases has little if any effect on cofactor function (29, 32–34). The relatively modest changes that were noted were only observed when using a one-stage PT-based clotting assay (29, 33). A more pronounced effect was found when using a protease purified from the venom of Naja naja oxiana (cleavage at His682). However, the reduction in cofactor activity was primarily attributed to altered FXa binding with only a minor change in the Km for prothrombin (a 4-fold increase) (31).

In contrast to these observations, recent mutagenesis and peptide inhibition studies have suggested that a binding site for prothrombin is located within 695DYDYQ699 of the FVa heavy chain (see Fig. 1) (29, 30). For example, the quadruple mutant (Asp695 -> Lys, Tyr 696-> Phe, Asp 697-> Lys, Tyr 698-> Phe) rFVa2K2F had markedly reduced activity (>20-fold) in conditioned media compared with wild-type FVa (30). It should be pointed out, however, that the molecular basis underlying this reduction was not determined. In support of these data, the hirudin-like peptide DYDYQ was found to inhibit prothrombin activation when using prothrombinase as the enzyme. This observation, however, deviates from results obtained with bothrojaracin and hirugen. These proexosite I probes were only effective inhibitors if substrate-membrane binding was eliminated or if negatively charged phospholipids were omitted from the reaction (20, 21). It was suggested that assembly of FXa-FVa-prothrombin on phospholipid membranes somehow counteracted the effectiveness of the inhibitors. This implies that data obtained with DYDYQ cannot be interpreted in a way that is equivalent to these proexosite I probes. These observations together with the results of the current study cast doubt on the importance of FVa residues 659–709 in providing a productive binding site for prothrombin within prothrombinase.

Although removal of the FVa hirudin-like heavy chain region had little influence on prothrombin activation, this was not the case when using prethrombin-1. A significant difference in the rate of IIa generation was found with each of the truncated FVa variants fully assembled into prothrombinase. Surprisingly, the effect on the Km (4–14-fold) was similar for each variant with no obvious relationship between the magnitude of the change and the length of the heavy chain remaining. The significance and nature of this change in substrate recognition and how it relates to the mechanism of prothrombin recognition by prothrombinase is not clear. The data are consistent with the conclusion that the C terminus of the FVa heavy chain somehow plays a role in prethrombin-1 substrate recognition. These deviations in the Km, however, were not related to the absence of substrate membrane binding as indistinguishable Km values for prothrombin were obtained with each of the variants using the FXa-FVa complex. This result was somewhat surprising considering that hirugen is known to effectively inhibit prethrombin-1 activation using prothrombinase and prothrombin activation in the absence of anionic membranes (20). Interestingly, recent data indicate that removal of fragment 1 or fragment 1.2 from prothrombin alters the environment of the protease domain, in particular the (pro)exosite I region (57, 58). It was suggested that modulation of (pro)exosite I affinity by the fragment 1 domain may affect interactions of prothrombin activation species with FVa (58). Whether our observations with the truncated FVa variants and prethrombin-1 are related to this is not clear, and more studies are needed to help better understand these observations.

In summary, the results of the current study challenge existing conclusions that hirudin-like sequences within the C-terminal heavy chain region of FVa provide a primary binding site for prothrombin. The deletion approach employed offered a direct assessment of FVa cofactor function and IIa binding. Analyzing loss of function data using either mutant proteins or peptides can be problematic because accounting for indirect effects is often difficult. On the other hand, maintenance of cofactor function has obvious interpretative advantages. We cannot rule out the possibility that removal of these heavy chain sequences was accompanied by favorable compensating effects. This scenario, however, requires invoking a secondary mechanism by which prothrombinase recognizes its macromolecular substrate. Our findings do not exclude a dominant role for proexosite I in macromolecular substrate recognition mediated by FVa within prothrombinase. Rather, the data show that this productive binding site on FVa cannot be attributed to the C terminus of the FVa heavy chain.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants P01 HL-74124-01, Project 2 (to R. M. C.), and National Research Service Award T32 HL-07439-26 (to R. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Div. of Hematology, 302E Abramson Research Center, The Children's Hospital of Philadelphia, 3615 Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-9968; Fax: 215-590-3660; E-mail: rcamire{at}mail.med.upenn.edu.

2 The abbreviations used are: FVa, activated factor V; FV, factor V; FXa, factor Xa; IIa, {alpha}-thrombin; BSA, bovine serum albumin; FPR, D-phenylalanyl-L-prolyl-L-arginine; PCPS, small unilamellar phospholipid vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine; rFV-DT, recombinant partial B-domainless (des811–1491) factor V; PD-FV, plasma-derived factor V; OG488-FXa, factor Xa modified with Oregon Green488; rFVa709, recombinant FV with amino acids 710–1545 deleted; rFVa699, amino acids 700–1545 deleted; rFVa692, amino acids 693–1545 deleted; rFVa678, amino acids 679–1545 deleted; rFVa658, amino acids 659–1545 deleted; MOPS, 4-morpholinepropanesulfonic acid; PACE, paired basic amino acid cleaving enzyme; PT, prothrombin time. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Sriram Krishnaswamy for useful suggestions and critical reading of the manuscript. We also acknowledge Hua Zhu for technical assistance and Dr. Alex Kurosky and Steven Smith at the University of Texas Medical Branch at Galveston for N-terminal sequence analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mann, K. G., and Kalafatis, M. (2002) Blood 101, 20-30[CrossRef][Medline] [Order article via Infotrieve]
  2. Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P. E., and Krishnaswamy, S. (1990) Blood 76, 1-16[Abstract/Free Full Text]
  3. Betz, A., and Krishnaswamy, S. (1998) J. Biol. Chem. 273, 10709-10718[Abstract/Free Full Text]
  4. Boskovic, D. S., and Krishnaswamy, S. (2000) J. Biol. Chem. 275, 38561-38570[Abstract/Free Full Text]
  5. Krishnaswamy, S. (2005) J. Thromb. Haemost. 3, 54-67[CrossRef][Medline] [Order article via Infotrieve]
  6. Buddai, S. K., Toulokhonova, L., Bergum, P. W., Vlasuk, G. P., and Krishnaswamy, S. (2002) J. Biol. Chem. 277, 26689-26698[Abstract/Free Full Text]
  7. Church, W. R., Messier, T. L., Tucker, M. M., and Mann, K. G. (1988) Blood 72, 1911-1921[Abstract/Free Full Text]
  8. Wilkens, M., and Krishnaswamy, S. (2002) J. Biol. Chem. 277, 9366-9374[Abstract/Free Full Text]
  9. Esmon, C. T., Owen, W. G., Duiguid, D., and Jackson, C. M. (1973) Biochim. Biophys. Acta 310, 289-294[Medline] [Order article via Infotrieve]
  10. van de Waart, P., Hemker, H. C., and Lindhout, T. (1984) Biochemistry 23, 2838-2842[CrossRef][Medline] [Order article via Infotrieve]
  11. Guinto, E. R., and Esmon, C. T. (1984) J. Biol. Chem. 259, 13986-13992[Abstract/Free Full Text]
  12. Luckow, E. A., Lyons, D. A., Ridgeway, T. M., Esmon, C. T., and Laue, T. M. (1989) Biochemistry 28, 2348-2354[CrossRef][Medline] [Order article via Infotrieve]
  13. Kotkow, K. J., Deitcher, S. R., Furie, B., and Furie, B. C. (1995) J. Biol. Chem. 270, 4551-4557[Abstract/Free Full Text]
  14. Deguchi, H., Takeya, H., Gabazza, E. C., Nishioka, J., and Suzuki, K. (1997) Biochem. J. 321, 729-735[Medline] [Order article via Infotrieve]
  15. Blostein, M. D., Rigby, A. C., Jacobs, M., Furie, B., and Furie, B. C. (2000) J. Biol. Chem. 275, 38120-38126[Abstract/Free Full Text]
  16. Boskovic, D. S., Giles, A. R., and Nesheim, M. D. (1990) J. Biol. Chem. 265, 10497-10505[Abstract/Free Full Text]
  17. Krishnaswamy, S., and Walker, R. K. (1997) Biochemistry 36, 3319-3330[CrossRef][Medline] [Order article via Infotrieve]
  18. Boskovic, D. S., Troxler, T., and Krishnaswamy, S. (2004) J. Biol. Chem. 279, 20786-20793[Abstract/Free Full Text]
  19. Anderson, P. J., Nesset, A., Dharmawardana, K. R., and Bock, P. E. (2000) J. Biol. Chem. 275, 16428-16434[Abstract/Free Full Text]
  20. Anderson, P. J., Nesset, A., Dharmawardana, K. R., and Bock, P. E. (2000) J. Biol. Chem. 275, 16435-16442[Abstract/Free Full Text]
  21. Monteiro, R. Q., and Zingali, R. B. (2002) Thromb. Haemost. 87, 288-293[Medline] [Order article via Infotrieve]
  22. Bode, W., Mayr, I., Bauman, Y., Huber, R., Stone, S. R., and Hofsteenge, J. (1989) EMBO J. 8, 3467-3475[Medline] [Order article via Infotrieve]
  23. Chen, L., Yang, L., and Rezaie, A. R. (2003) J. Biol. Chem. 278, 27564-27569[Abstract/Free Full Text]
  24. Akhavan, S., De Cristofaro, R., Peyvandi, F., Lavoretano, S., Landolfi, R., and Mannucci, P. M. (2002) Blood 100, 1347-1353[Abstract/Free Full Text]
  25. Esmon, C. T., and Lollar, P. (1996) J. Biol. Chem. 271, 13882-13887[Abstract/Free Full Text]
  26. Dharmawardana, K. R., and Bock, P. E. (1998) Biochemistry 37, 13143-13152[CrossRef][Medline] [Order article via Infotrieve]
  27. Myles, T., Yun, T. H., Hall, S. W., and Leung, L. L. (2001) J. Biol. Chem. 276, 25143-25149[Abstract/Free Full Text]
  28. Dharmawardana, K. R., Olson, S. T., and Bock, P. E. (1999) J. Biol. Chem. 274, 18635-18643[Abstract/Free Full Text]
  29. Kalafatis, M., Beck, D. O., and Mann, K. G. (2004) J. Biol. Chem. 278, 33550-33561[CrossRef]
  30. Beck, D. O., Bukys, M. A., Singh, L. S., Szabo, K., and Kalafatis, M. (2004) J. Biol. Chem. 279, 3084-3095[Abstract/Free Full Text]
  31. Bakker, H. M., Tans, G., Thomassen, M. C., Yukelson, L. Y., Ebberink, R., Hemker, H. C., and Rosing, J. (1994) J. Biol. Chem. 269, 20662-20667[Abstract/Free Full Text]
  32. Egan, J. O., Kalafatis, M., and Mann, K. G. (1997) Protein Sci. 6, 2016-2027[Medline] [Order article via Infotrieve]
  33. Camire, R. M., Kalafatis, M., and Tracy, P. B. (1998) Biochemistry 37, 11896-11906[CrossRef][Medline] [Order article via Infotrieve]
  34. Safa, O., Morrissey, J. H., Esmon, C. T., and Esmon, N. L. (1999) Biochemistry 38, 1829-1837[CrossRef][Medline] [Order article via Infotrieve]
  35. Lottenberg, R., and Jackson, C. M. (1983) Biochim. Biophys. Acta 742, 558-564[CrossRef][Medline] [Order article via Infotrieve]
  36. Higgins, D. L., and Mann, K. G. (1983) J. Biol. Chem. 258, 6503-6508[Abstract/Free Full Text]
  37. Gomori, G. (1942) J. Lab. Clin. Med. 27, 955-960
  38. Baugh, R., and Krishnaswamy, S. (1996) J. Biol. Chem. 271, 16126-16134[Abstract/Free Full Text]
  39. Katzmann, J. A., Nesheim, M. E., Hibbard, L. S., and Mann, K. G. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 162-166[Abstract/Free Full Text]
  40. Mann, K. G. (1976) Methods Enzymol. 45, 123-156[Medline] [Order article via Infotrieve]
  41. Toso, R., and Camire, R. M. (2004) J. Biol. Chem. 279, 21643-21650[Abstract/Free Full Text]
  42. Kalafatis, M., Krishnaswamy, S., Rand, M. D., and Mann, K. G. (1993) Methods Enzymol. 222, 224-236[Medline] [Order article via Infotrieve]
  43. Lundblad, R. L., Kingdon, H. S., and Mann, K. G. (1976) Methods Enzymol. 45, 156-176[Medline] [Order article via Infotrieve]
  44. Di Scipio, R. G., Hermodson, M. A., Yates, S. G., and Davie, E. W. (1977) Biochemistry 16, 698-706[CrossRef][Medline] [Order article via Infotrieve]
  45. Camire, R. M. (2002) J. Biol. Chem. 277, 37863-37870[Abstract/Free Full Text]
  46. Bock, P. E. (1988) Biochemistry 27, 6633-6639[CrossRef][Medline] [Order article via Infotrieve]
  47. Bock, P. E. (1992) J. Biol. Chem. 267, 14974-14981[Abstract/Free Full Text]
  48. Bevington, P. R., and Robinson, K. D. (1992) Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill Inc., New York
  49. Straume, M., and Johnson, M. L. (1992) Methods Enzymol. 210, 87-105[Medline] [Order article via Infotrieve]
  50. Krishnaswamy, S. (1990) J. Biol. Chem. 265, 3708-3718[Abstract/Free Full Text]
  51. Segal, I. H. (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems., John Wiley & Sons, Inc., New York
  52. Foster, D. C., Sprecher, C. A., Holly, R. D., Gambee, J. E., Walker, K. M., and Kumar, A. A. (1990) Biochemistry 29, 347-354[Medline] [Order article via Infotrieve]
  53. Rouille, Y., Duguay, S. J., Lund, K., Furuta, M., Gong, Q., Lipkind, G., Oliva, A. A., Jr., Chan, S. J., and Steiner, D. F. (1995) Front. Neuroendocrinol. 16, 322-361[CrossRef][Medline] [Order article via Infotrieve]
  54. Zhou, A., Webb, G., Zhu, X., and Steiner, D. F. (1999) J. Biol. Chem. 274, 20745-20748[Free Full Text]
  55. Rockwell, N. C., Krysan, D. J., Komiyama, T., and Fuller, R. S. (2002) Chem. Rev. 102, 4525-4548[CrossRef][Medline] [Order article via Infotrieve]
  56. Betz, A., Vlasuk, G. P., Bergum, P. W., and Krishnaswamy, S. (1997) Biochemistry 36, 181-191[CrossRef][Medline] [Order article via Infotrieve]
  57. Anderson, P. J., Nesset, A., and Bock, P. E. (2003) J. Biol. Chem. 278, 44482-44488[Abstract/Free Full Text]
  58. Anderson, P. J., and Bock, P. E. (2003) J. Biol. Chem. 278, 44489-44495[Abstract/Free Full Text]
  59. Pryzdial, E. L. G., and Mann, K. G. (1991) J. Biol. Chem. 266, 8969-8977[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 BloodHome page
M. H. A. Bos, M. Boltz, L. St. Pierre, P. P. Masci, J. de Jersey, M. F. Lavin, and R. M. Camire
Venom factor V from the common brown snake escapes hemostatic regulation through procoagulant adaptations
Blood, July 16, 2009; 114(3): 686 - 692.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Toso, H. Zhu, and R. M. Camire
The Conformational Switch from the Factor X Zymogen to Protease State Mediates Exosite Expression and Prothrombinase Assembly
J. Biol. Chem., July 4, 2008; 283(27): 18627 - 18635.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Y. Kim and M. E. Nesheim
Further Evidence for Two Functional Forms of Prothrombinase Each Specific for Either of the Two Prothrombin Activation Cleavages
J. Biol. Chem., November 9, 2007; 282(45): 32568 - 32581.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. J. Gale, S. Yegneswaran, X. Xu, J.-L. Pellequer, and J. H. Griffin
Characterization of a Factor Xa Binding Site on Factor Va near the Arg-506 Activated Protein C Cleavage Site
J. Biol. Chem., July 27, 2007; 282(30): 21848 - 21855.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Zhu, R. Toso, and R. M. Camire
Inhibitory Sequences within the B-domain Stabilize Circulating Factor V in an Inactive State
J. Biol. Chem., May 18, 2007; 282(20): 15033 - 15039.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/13/8773    most recent
M511419200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Toso, R.
Right arrow Articles by Camire, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Toso, R.
Right arrow Articles by Camire, R. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement