Homocysteine Inhibits Inactivation of Factor Va by Activated Protein C*

Anetta UndasDagger , E. Brady Williams§, Saulius Butenas, Thomas Orfeo, and Kenneth G. Mann

From the Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405-0068, § Department of Chemistry, College of St. Catherine, St. Paul, Minnesota 55105-1730, and Dagger  Department of Medicine, Jagiellonian University School of Medicine, Cracow 31-066, Poland

Received for publication, May 15, 2000, and in revised form, November 15, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by alpha -thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min-1 at 0, 0.03, 0.1, 1 mM homocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mM beta -mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [35S]homocysteine (10-450 µM) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507-709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with beta -mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s).



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homocysteine is a nonprotein-forming sulfhydryl amino acid derived from the metabolism of methionine. Homocysteine is metabolized in human cells through two pathways of remethylation to methionine and one pathway of transsulfuration to cysteine with cystathionine as an intermediate (1). Congenital defects in the enzymes involved in methionine metabolism, 5,10-methylenetetrahydrofolate reductase, N5-methyltetrahydrofolate methyltransferase, and cystathionine beta -synthase, along with deficiencies in the essential cofactors folic acid and vitamins B6 or B12 can result in elevated levels of plasma homocysteine. In healthy individuals, plasma total homocysteine (the sum of homocysteine and the homocysteinyl derivatives homocystine and cysteine-homocysteine, whether free or bound to proteins) was between 5 and 15 µM (2, 3). A number of case-control and prospective studies have shown that hyperhomocysteinemia, present in 5-7% of the population, is an independent risk factor for atherosclerotic vascular disease in the coronary (4-6), cerebrovascular (7), and peripheral vessels (8, 9) and also for venous thromboembolism (10-13).

The relationship(s) between elevated levels of homocysteine in the blood and thrombosis has been a subject of considerable investigative effort; however, the mechanisms by which hyperhomocysteinemia leads to increased risk of thrombosis are not clear (14-17). In vitro studies provide evidence that supraphysiologic homocysteine levels have a direct toxic effect on the endothelium, probably through free radical generation during oxidation of the homocysteine sulfhydryl group (18, 19). Homocysteine has also been reported to impair the binding of tissue-type plasminogen activator to its endothelial cell receptor, annexin II (20), to induce tissue factor expression (21) and to suppress heparan sulfate expression (22). Several studies suggest that homocysteine may interfere with the protein C pathway (23-26). The most important function of this pathway appears to be the inactivation of factor Va, the essential cofactor for the prothrombinase complex which converts prothrombin to thrombin.

Human factor V (Mr = 330,000) is activated by alpha -thrombin cleavages at Arg-709, Arg-1018, and Arg-1545 (27-28). The product, factor Va, is composed of a heavy chain, residues 1-709 (Mr = 105,000), derived from the NH2 terminus of factor V (A1-A2 domains) and a noncovalently associated light chain, residues 1546-2196 (Mr = 74,000), derived from the COOH terminus (A3-C1-C2 domains) (27-28). Factor Va is proteolytically inactivated by activated protein C (APC)1 by three cleavages of the heavy chain at Arg-506, Arg-306, and Arg-679 (29, 30). The cleavage of factor Va at Arg-506 is enhanced in the presence of a phospholipid surface, whereas cleavage at Arg-306 is lipid-dependent (30). After cleavage at Arg-306, the A2 domain (residues 307-709) dissociates as two fragments (residues 307-506 and 507-709) from the factor Va molecule, leading to the complete inactivation of factor Va (31, 32).

The regulation of factor Va is a key process for maintaining hemostasis, as evidenced by two congenital thrombotic disorders, i.e. the G1691A mutation (Arg-506 to Gln) in factor V Leiden and protein C deficiency (33, 34). There is also evidence for impaired factor Va inactivation by APC in the absence of the factor V Leiden mutation, which is associated with increased risk of venous thrombosis (35).

The present study was undertaken to evaluate the ability of homocysteine at concentrations of 1 mM or less to modulate activation of human factor V and inactivation of human factor Va. We examined the effect of homocysteine on these processes using clotting assays and Western blot analyses along with radiolabeling of factor V by [35S]homocysteine. Our data suggest a potential contributor to the prothrombotic tendency observed in hyperhomocysteinemia.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

DL-Homocysteine, L-homocystine, DL-homocysteine thiolactone, and L-cysteine as well as HEPES were purchased from Sigma. 1,2-Dioleoyl-sn-glycero-3-[phospho-L-serine] (sodium salt) (PS) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (PC) were purchased from Avanti (Alabaster, AL). Acrylamide, bisacrylamide, TEMED, ammonium persulfate, and nitrocellulose were purchased from Bio-Rad. Pooled normal plasma (number of donors >30) was purchased from George King Bio-Medical (Overland Park, KS). Factor V was purified from human plasma by immunoaffinity chromatography as previously described (36). Factor V concentrations were estimated spectrophotometrically. Human APC and alpha -thrombin were gifts from Hematologic Technologies Inc. (Essex Junction, VT). Hirudin was obtained from Genentech (South San Francisco, CA). Phospholipid vesicles (PCPS) composed of 75% PC and 25% PS were prepared according to Barenholz et al. (37), with the concentrations determined by phosphorous assay. Monoclonal antibodies against the heavy chain of human factor Va (alpha FVaHC 17) were from the Biochemistry Antibody Core Laboratory (University of Vermont). Goat anti-mouse IgG peroxidase was purchased from Southern Biotech (Birmingham, AL). Molecular weight standards were purchased from Life Technologies, Inc. The chemiluminescent substrate, luminol, was purchased from PerkinElmer Life Sciences as was the fluorography reagent, Entensify. L-[35S]Methionine and L-[35S]cysteine were purchased from Amersham Pharmacia Biotech.

Methods

Homocysteine Modification and Functional Assays-- Factor V was dialyzed against 20 mM HEPES, 0.15 M NaCl, 5 mM CaCl2, pH 7.4 (HBS-Ca2+) with or without the addition of homocysteine or other thiols (solubilized in 0.15 M HCl) at varying concentrations at 4 °C for 2 h followed by dialysis against HBS-Ca2+ for 2 h. Homocystine was solubilized in 0.15 M HCl. Factor V was activated by treatment with 1 nM alpha -thrombin for 10-13 min at 37 °C. alpha -Thrombin was inhibited by the addition of hirudin at a 4-5-fold molar excess. APC at various concentrations (see legends to figures) was added to factor Va in the presence or absence of phospholipids. At selected time intervals, samples were removed and diluted into HBS-Ca2+ for assay. Factor Va activity was immediately measured in a clotting assay at 37 °C using factor V-deficient plasma (38). In a typical assay, 50 µl of factor V or Va diluted in HBS-Ca2+ was added to an equal volume of factor V-deficient human plasma followed by the addition of 100 µl of PT (prothrombin time) reagent (Simplastin, Organon Teknika, Durham, NC). All experiments were repeated three times on separate days with comparable results. Time course data for the APC inactivation of factor Va were fit to a single exponential equation using software IGOR Pro Version 3.1, WaveMetrics Inc. (Lake Oswego, OR). Initial slopes from the fit curves were used to calculate the pseudo first order rate constants. Pseudo first order rate constants are all normalized to constant APC concentration (2.5 nM).

Gel Electrophoresis and Western Blotting-- Aliquots from the activation/inactivation experiments were withdrawn and quenched in 2% beta -mercaptoethanol, 2% SDS for electrophoretic analysis. Samples were separated on a 5 to 15% linear gradient gel (SDS-PAGE) under reducing conditions according to Laemmli (39). Transfer from the gel to nitrocellulose (Bio-Rad) was performed as previously described (40). Immunoreactive fragments were detected using the monoclonal antibody alpha FVaHC 17, which recognizes an epitope located between residues 307 and 506 in the heavy chain of factor Va (41). Blots were developed using the Renaissance chemiluminescent reagent (Dupont). Film was developed in a Kodak X-Omat on Reflection film (PerkinElmer Life Sciences). Densitometry of immunoblots was performed on a Hewlett-Packard ScanJet 4C/T equipped with a transparency adapter for backlighting the x-ray film (Hewlett-Packard, Palo Alto, CA) and analyzed using NIH Image 1.6 (Bethesda, MD).

Activated Protein C Resistance Assay-- Factor V was dialyzed against HBS-Ca2+ containing 1 mM beta -mercaptoethanol (BME) or 2 mM homocysteine (HCY) for 2 h at 4 °C and then for 2 h at 4 °C against HBS-Ca2+. Dialysis was performed in filled, capped bottles in buffer that had been boiled in a reflux apparatus and purged extensively with nitrogen. The resulting stocks, FVBME (2.2 µM) and FVHCY (2.0 µM) were used immediately. FVHCY could be flash-frozen in methanol/dry ice and successfully recovered, whereas FVBME proved sensitive to freezing, showing a decrease in APC sensitivity after thawing. Mixtures of FVBME and FVHCY with a total factor V concentration of 400 nM in which the amount of FVHCY varied from 0 to 100% were constructed by appropriate dilution in HBS-Ca2+, and their relative susceptibility to APC proteolysis assessed by a modification of the method of Dahlback (42). Five µl of each factor V mixture was added to 95 µl of factor V-deficient (immunodepleted) plasma followed by the addition of 100 µl of aPTT (activated partial thromboplastin time) reagent (Organon Teknika). This reformulated plasma ([factor V] = 20 nM) was incubated for 5 min at 37 °C. One hundred µl of 3 nM APC in HBS-30 mM Ca2+ or 100 µl of HBS-30 mM Ca2+ was then added, the mixture rocked at 37 °C, and clotting time was recorded. Clotting times were plotted against the percentage of FVBME present. Best-fit lines were determined by linear regression analysis. The slopes of these lines from 3 separate sets of FVBME/FVHCY mixtures varied by less than 10%. To assay plasma, 100 µl of patient or normal plasma was combined directly with 100 µl of aPTT reagent and then processed as above.

Plasma samples from individuals with elevated homocysteine levels were a generous gift from Dr. Andrzej Szczeklik, Jagiellonian University School of Medicine, Cracow, Poland. Plasmas were obtained from four asymptomatic men with genetically determined hyperhomocysteinemia stemming from a mutation in the 5,10-methylenetetrahydrofolate reductase gene (C677T). The presence of factor V Leiden was excluded on the basis of polymerase chain reaction analysis. Blood was drawn after a 16-h overnight fast, and plasma homocysteine concentration was determined according to Mansoor et al. (43).

Radiolabeling of Factor V with [35S]Homocysteine-- L-[35S]Methionine (201 mCi/mmol) was converted to L-[35S]homocysteine thiolactone and subsequently to L-[35S]homocysteine using the method of Baernstein (44). The product was identical with authentic homocysteine by thin layer and paper chromatography and contained a small amount (<10%) of homocystine. Reported concentrations of L-[35S]homocysteine were determined by liquid scintillation counting. Radiolabeled L-homocysteine was added to factor V that had been dialyzed against HBS-Ca2+ at room temperature for 2 h. Subsequently, factor V was activated with alpha -thrombin, and then, upon addition of hirudin and PCPS vesicles, factor Va was inactivated by APC. At designated time points, aliquots were removed from the mixture and subjected to SDS-PAGE. Protein bands were visualized by staining the gel with Coomassie Blue and destaining by diffusion. Gels were then soaked in the fluorography reagent as per the manufacturer's instructions and dried, and radiolabeled protein was detected using Fuji Medical x-ray film (Fuji Medical Systems USA, Inc., Stamford CT). Densitometry was performed on the Coomassie-stained gel before drying and on the resulting autoradiographs as described for immunoblots. Details are given in the legends to figures.

Kinetic analyses of [35S]homocysteine modification of factor V were performed in HBS-Ca2+ at room temperature in which times of treatment were varied at a fixed [35S]homocysteine concentration, or concentrations of [35S]homocysteine were varied and the reaction allowed to proceed for a fixed time. Reactions were quenched with iodoacetic acid (Sigma) that had been recrystallized from heptane. Aliquots from reacting mixtures of factor V and [35S]homocysteine were combined with an equal volume of 100 mM iodoacetic acid, 0.5 M Tris, pH 8.7, before the addition of SDS-PAGE sample buffer. The radiolabeled factor V was resolved by electrophoresis on 4-12% linear gradient SDS-polyacrylamide gels under nonreducing conditions, and the gels were processed as described above. Densitometric data from resulting autoradiographs were normalized to correct for variations in protein loading and then converted to the percentage of maximum values using the highest densitometric value within a data set. These transformed data were then fit to a double exponential equation using software IGOR Pro Version 3.1, WaveMetrics Inc.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of Factor V by alpha -Thrombin-- There was no significant alteration in alpha -thrombin activation of factor V after dialysis against 1 mM homocysteine in HBS-Ca2+ for 2 h. Homocystine, homocysteine thiolactone, and cysteine incubated with factor V under the same conditions did not affect activation of factor V by alpha -thrombin (data not shown).

Inactivation of Factor Va by APC in the Presence of Phospholipids-- Preincubation of factor V with 1 mM homocysteine for 2 h inhibited the APC anticoagulant effect toward factor Va (Fig. 1). Factor V was treated with homocysteine at 1 mM, 100 µM, or 30 µM at 4 °C for 2 h, dialyzed against HBS-Ca2+ for an additional 2 h, and then activated by alpha -thrombin. A progressive decrease in the initial rates of APC inactivation of factor Va, generated from the homocysteine-treated factor V, is associated with increasing homocysteine concentrations (Fig. 1). The rate constant for the APC-catalyzed inactivation of factor Va, generated from factor V treated with 1 mM homocysteine, is about one-third the rate constant for control experiments (Fig. 1).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of increasing concentrations of homocysteine. Factor V was dialyzed against HBS-Ca2+ containing homocysteine at the following concentrations: 0 (), 30 µM (triangle ), 100 µM (), 1 mM (open circle ) as described under "Methods." Subsequently, factor V (100 nM) was activated by alpha -thrombin (1 nM) at 37 °C for 10 min followed by the addition of hirudin (5 nM). Upon the addition of PCPS vesicles (20 µM) and then APC (2.5 nM), the activity of factor Va was evaluated at 1, 3, 5, and 10 min using a one-stage clotting assay (see "Methods"). The results were expressed as the percentage of initial activity of factor Va as a function of time. The inset presents the rate constants for APC inactivation. Data represent one of three experiments.

To test whether the interference was related to a decrease in APC activity after incubation with homocysteine, APC was preincubated with homocysteine at 1 mM for 30, 60, and 120 min at 4 °C. No differences were observed in the rate of inactivation of 100 nM factor Va by 2.5 nM APC whether treated with homocysteine or untreated (data not shown). Contrary to previous reports (23, 25), we did not observe a direct effect of homocysteine on APC activity per se.

Inactivation of Factor Va by APC in the Absence of Phospholipids-- The cleavage at Arg-306 is phospholipid-dependent, whereas the cleavage at Arg-506 is enhanced in the presence of phospholipids (30). In the absence of phospholipids, the rate of APC inactivation of factor Va derived from factor V treated with 1 mM homocysteine (above) was significantly delayed compared with controls (Fig. 2). The ~3-fold decrease in the rate constant induced by homocysteine is similar to that observed in the presence of phospholipids. Since the lipid-independent inactivation proceeds only through cleavage at Arg-506, the significant impairment of APC inactivation of factor Va without phospholipids indicates that homocysteine must slow cleavage at Arg-506.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of homocysteine in the absence of phospholipids. Factor V was dialyzed against HBS-Ca2+ with 1 mM homocysteine (open circle ) or without (), as described under "Methods." Subsequently, factor V (100 nM) was activated by alpha -thrombin (1 nM) at 37 °C for 10 min followed by the addition of hirudin (5 nM). After the addition of APC (10 nM), the activity of factor Va was evaluated at 1, 3, 5, and 10 min using a one-stage clotting assay (see "Methods"). The results were expressed as the percentage of initial activity of factor Va as a function of time. The inset presents the rate constants for APC inactivation. Data represent one of three experiments.

Immunoblot Analysis of Factor Va Inactivation by APC-- Factor V (100 nM) was treated with either 1 mM homocysteine, 1 mM homocystine, or HBS-Ca2+ at 4 °C for 2 h. After activation by alpha -thrombin (see "Methods"), the factor Va preparations were cleaved by 2.5 nM APC at 37 °C, and the time course of each reaction was followed by Western blot analysis using the monoclonal antibody alpha FVaHC 17, which recognizes an epitope in the fragment 307-506 (36), the ultimate APC cleavage product of factor Va heavy chain. Data for this experiment over 60 min are presented in Fig. 3. Lanes 1-6 correspond to the control; lanes 13-18 correspond to the homocystine-treated factor V. The samples from treatment with homocysteine are presented in lanes 7-12. In both the control and in the homocystine-treated samples, the 60-min reaction sample (lane 6 and lane 18) show almost complete cleavage of the heavy chain and the appearance of fragments corresponding to the final cleavage product of factor Va. In contrast, some heavy chain remains intact in the homocysteine-treated samples at 60 min (lane 12). The data show that homocysteine inhibits cleavage of factor Va heavy chain by APC and that the reaction requires the reduced form of the amino acid. This effect on inactivation is homocysteine-specific, since for factor V preincubated with 1 mM L-cysteine for 2 h, the time course of the inactivation of factor Va by APC was identical to those in control and homocystine experiments (data not shown).



View larger version (83K):
[in this window]
[in a new window]
 
Fig. 3.   Immunoblots of factor Va after treatment. Human factor V was dialyzed in HBS-Ca2+ alone, with homocysteine (1 mM), or with homocystine (1 mM), as described under "Methods." Factor V (100 nM) was then incubated with alpha -thrombin (0.5 nM) at 37 °C for 13 min, followed by the addition of hirudin (2 nM). In the presence of PCPS vesicles (40 µM), factor Va was inactivated by APC (2.5 nM) at 37 °C. At six time points (see below), aliquots taken from the mixture were added to 2% beta -mercaptoethanol, 2% SDS (see "Methods") After transfer to nitrocellulose, immunoreactive fragments were detected with the monoclonal antibody alpha FVaHC 17. Lanes 1-18 depict aliquots withdrawn immediately before (0 min) and at 1, 5, 10, 20, and 60 min after the addition of APC to factor Va untreated (1-6), dialyzed against 1 mM homocysteine (7-12), and against 1 mM homocystine (13-18). Approximately 100 ng of protein was applied per lane. The position of the molecular weight markers is indicated on the left. HC, heavy chain.

Factor V Purified from Plasma Treated with Homocysteine-- Human plasma contains a wide range of the sulfhydryl compounds, including cysteine, cysteinylglycine, homocysteine, and glutathione, which are in the reduced (<5%) or oxidized form or are protein-bound (43). Total plasma concentration of cysteine, the most abundant aminothiol, is around 250 µM, with levels of reduced cysteine amounting to 10 µM (43). Moreover, it is known that homocysteine binds to numerous plasma proteins, mainly albumin (plasma concentration 0.5-0.7 mM) at an equimolar ratio. To determine whether the addition of homocysteine to normal human plasma would alter the APC sensitivity of factor V subsequently purified from such plasma, human plasma was incubated with homocysteine (10 mM) at 4 °C for 2 h, and then factor V was purified, as described under "Methods." Homocysteine added to plasma effectively retarded the inactivation of subsequently purified and alpha -thrombin-activated factor V by APC in the presence of PCPS vesicles (Fig. 4). The ~3-fold decrease in the rate constant is the same as observed when purified factor V was treated with 1 mM homocysteine (Fig. 1). Slower inactivation of factor Va, derived from factor V purified from plasma treated with homocysteine, was also observed in the absence of phospholipids (data not shown).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of homocysteine on factor V in normal plasma. Homocysteine (10 mM) (open circle ) or buffer control () was added to plasma for 2 h (see "Methods"). Factor V was purified using immunoaffinity chromatography according to Katzmann et al. (36). Factor V (100 nM) was treated with 1 nM alpha -thrombin, after which hirudin (5 nM), PCPS vesicles (40 µM), and finally APC (2.5 nM) were added to the mixture. At 1, 3, 5, 7, and 10 min after the addition of APC, samples taken from the mixture were assayed for factor Va activity. The inset presents the rate constants for APC inactivation. Data represent one of three experiments.

Radiolabeling of Human Factor V with [35S]Homocysteine-- To determine whether homocysteine can be incorporated into human factor V, factor V was incubated with 450 µM [35S]homocysteine at room temperature for 2 h. It was then activated by alpha -thrombin, and the resulting factor Va was treated with 6 nM APC in the presence of phospholipids. At selected time points aliquots were removed and subjected to analysis by SDS-PAGE. Coomassie Blue staining of the gel with nonreduced samples (Fig. 5A) displays [35S]homocysteine-treated factor V (lane 1), the derived factor Va (lane 2), and the time course of APC degradation of factor Va (lanes 3-8). Degradation of the heavy chain of factor Va, derived from [35S]homocysteine-treated factor V, is not complete at 180 min (lane 8). In contrast, APC degradation of factor Va, derived from untreated factor V, is complete under these conditions in 60 min (30). The APC cleavage of factor Va, derived from [35S]homocysteine-treated factor V, yielded products of Mr = 45,000 (), Mr = 30,000 (), and Mr = 28/26,000 (). Bands corresponding to the light chain of factor Va are stable throughout the 180-min incubation with APC. This pattern is identical to that seen with untreated factor V (30). Comparing the Coomassie Blue-stained (Fig. 5A) gel with the corresponding autoradiograph (Fig. 5B) revealed that only the protein fragments that contain free sulfhydryl groups (45, 46) (intact factor V (lane 1), the heavy and light chains of factor Va (lane 2), the B region (lanes 2-8), and the doublet of Mr = 28/26,000 (lanes 3-8)) incorporate [35S]homocysteine. Consistent with the pattern observed by Coomassie Blue staining, the intensity of the radiolabeled heavy chain band decreased with time, whereas the intensity of the light chain bands remained unchanged. The fragments of Mr = 45,000 () and 30,000 (), which do not contain any free cysteines, are not visualized on the autoradiograph (Fig. 5B).



View larger version (89K):
[in this window]
[in a new window]
 
Fig. 5.   Modification of human factor V with [35S]homocysteine. Factor V (600 nM) was incubated with 450 µM [35S]homocysteine in HBS-Ca2+ at 25 °C for 2 h (lane 1) and then treated with alpha -thrombin (12 nM) for 10 min (lane 2). After the addition of hirudin (60 nM) followed by PCPS vesicles (40 µM), factor Va was subjected to APC (6 nM) proteolysis. At selected time intervals (5, 10, 30, 60, 120, and 180 min in lanes 3-8, respectively), 110-µl aliquots of the mixture were withdrawn and analyzed by a 5-15% linear gradient SDS-PAGE under nonreducing conditions. Duplicate aliquots were also removed before and 10, 30, and 120 min after the addition of APC, mixed with 2% beta -mercaptoethanol, 2% SDS, and analyzed by a 5-15% linear gradient SDS-PAGE. Coomassie Blue-stained gel run under nonreducing conditions (panel A) and its autoradiograph (panel B) are shown. Coomassie Blue-stained gel run under reducing conditions (panel C) and the corresponding autoradiograph (panel D) are shown. The positions of the molecular weight markers are indicated. HC, heavy chain; LC, light chain.

Samples obtained before and 10, 30, and 120 min after the addition of APC were removed, mixed with 2% beta -mercaptoethanol/SDS, and analyzed by SDS-PAGE. When samples were reduced, none of the bands seen on the gel stained with Coomassie Blue (Fig. 5C) could be detected on the autoradiograph of this gel (Fig. 5D) even with periods of autoradiography 20 times longer than those required to detect bands on the gels with the nonreduced samples. Thus the radiolabel in factor V/Va was totally removed by the disulfide-reducing agent.

To test whether factor V in plasma can incorporate homocysteine, human plasma was incubated with 450 µM [35S]homocysteine at room temperature for 2 h. SDS-PAGE analysis of nonreduced samples was performed. The Coomassie Blue-stained gel and the corresponding autoradiograph showed the presence of a band with the same mobility as purified human factor V (data not shown).

Time course analyses of the incorporation of homocysteine into factor V were performed across a range of concentrations using [35S]homocysteine. At 450 µM [35S]homocysteine, radiolabeling of factor V was complete in less than 90 s (data not shown). At concentrations of homocysteine between 10 and 450 µM, a 5-min reaction period at room temperature was sufficient to achieve levels of radiolabeling within 15% of those observed after 60 min. Fig. 6A illustrates the time course for the reaction with 300 nM factor V and 25 µM [35S]homocysteine. When iodoacetic acid-quenched time points from reactions of factor V and [35S]homocysteine were subjected to proteolysis by alpha -thrombin and analyzed by SDS-PAGE, no differences in the rates of homocysteine incorporation into the heavy and light chains of factor Va were observed (data not shown). At elevated homocysteine concentrations, e.g. 450 µM, periods of incubation with factor V greater than 60 min resulted in an additional, slower increase in the level of factor V radiolabeling. alpha -Thrombin digestion indicated that this radiolabeling reflected adduct formation in the B region of factor V, presumably with Cys-1085 (data not shown).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Kinetics of factor V modification by [35S]homocysteine. Factor V (300 nM) was incubated with [35S]homocysteine at concentrations ranging from 10 to 450 µM in HBS-Ca2+ at 25 °C for times ranging from 15 s to 120 min. Reactions were quenched with iodoacetic acid, and the radiolabeled factor V was resolved by SDS-PAGE under nonreducing conditions. Relative levels of radiolabeled factor V were determined by densitometric analysis of the resulting autoradiographs. Panel A shows a time course analysis of the reaction of 25 µM [35S]homocysteine with factor V. The curve fit results in a percentage of maximum density value at infinite time of 93.5%. Panel B shows the result of a 5-min treatment of factor V at the following [35S]homocysteine concentrations: 10, 20, 40, 80, 100, 200, and 450 µM. The curve fit results in a percentage of maximum density at infinite time of 105.1%.

To estimate the equilibrium constant describing the reaction of factor V with homocysteine, factor V (300 nM) was incubated at room temperature for 5 min with increasing concentrations of [35S]homocysteine, the radiolabeled product resolved by SDS-PAGE, and the relative levels of factor V radiolabeling determined by densitometry. The resulting binding curve is shown in Fig. 6B. Half-maximal modification of factor V was achieved at ~80 µM homocysteine.

Radiolabeling experiments carried out with L-[35S]cysteine (124 mCi/mmol) indicate that this thiol also readily forms disulfide adducts with factor V when reacted under conditions and concentrations used with [35S]homocysteine (data not shown). As observed in the case of [35S]homocysteine, disulfide formation between factor V and L-[35S]cysteine was apparent after alpha -thrombin proteolysis in the heavy chain, the light chain, and the B region; proteolysis by APC located the heavy chain radiolabeling only in the 28/26,000 doublet (residues 507-709).

Effects of BME-- Since our data suggest that homocysteine is incorporated into factor V through the formation of disulfide bonds, we hypothesized that every plasma-derived factor V preparation would reflect some level of derivatization with homocysteine and, thus, some impairment with respect to APC inactivation. To test this hypothesis, we dialyzed factor V against 1 mM beta -mercaptoethanol for 2 h. Factor Va, derived from the thiol-reduced factor V, was inactivated by APC about 50% more rapidly than the control (Fig. 7).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of BME. Factor V was dialyzed against HBS-Ca2+with 1 mM BME (open circle ) or without (), as described under "Methods." Factor V (100 nM) was activated with alpha -thrombin (0.5 nM), followed by the addition of hirudin (2 nM). In the presence of PCPS vesicles (40 µM), factor Va was inactivated by APC (2.5 nM) at 37 °C. The activity of factor Va was evaluated at 1, 3, 5, and 10 min after the addition of APC (see "Methods"). The results were expressed as the percentage of initial activity of factor Va versus time. The inset presents the rate constants for APC inactivation of factor Va.

APC Resistance Assay Performed in Plasmas Containing Various Mixtures of Reduced (FVBME) and Homocysteinylated (FVHCY) Factor V-- A series of reconstituted plasmas were generated with varying proportions of partially reduced factor V (FVBME) and homocysteinylated factor V (FVHCY) (see "Methods") and tested in an APC resistance assay format. Clotting times for each factor V mixture were measured after incubation in the presence or absence of 1 nM APC. The clotting time for each factor V mixture was then plotted against the percentage of reduced factor V (FVBME) present in that mixture. Fig. 8 shows the results of such an experiment. The composition of the factor V mixture had no effect on the rate of clot formation in the absence of APC (closed circles). However with APC, clotting times in those same mixtures increased as a function of the percentage of FVBME present: from 56 s with 0% FVBME,100% FVHCY to 89 s with 100% FVBME, 0% FVHCY (open circles). A pooled plasma obtained from normal individuals gave clotting times of 46 s in the absence of APC and 85 s in the presence of APC, corresponding to a FVBME/FVHCY composition of 88%/12% (Fig. 8).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8.   APC resistance in plasmas containing various mixtures of reduced (FVBME) and homocysteinylated (FVHCY) factor V. Mixtures of FVBME and FVHCY with a total factor V concentration of 400 nM were constructed, diluted 20-fold in factor V-deficient plasma, and tested for reactivity with APC. Each factor V mixture was assessed in the presence (open circle ) or absence () of 1 nM added APC. Clotting time is plotted against the percentage of FVBME present in each mixture. Data are from a representative experiment. Best-fit lines were calculated by linear regression analysis. Clotting times measured in the presence of 1 nM APC for plasmas from patients (see "Methods") with defined levels of plasma homocysteine (40, 44, 76, and 80 µM) are indicated with arrows as is the clotting time from pooled plasma obtained from normal donors.

Plasmas from four individuals (see "Methods") with homocysteine levels of 40, 44, 76, and 80 µM were then tested in this assay format (Fig. 8). In the absence of APC, clotting times for patient plasmas always fell between 40-45 s, the same range as observed with the mixtures constructed in factor V-deficient plasma (Fig. 8). In the presence of APC, clotting times for the pair with 40 µM levels of homocysteine averaged 79 s, corresponding to a FVBME/FVHCY distribution of 68%/32%; clotting times for the plasmas with ~80 µM homocysteine averaged 71 s, corresponding to a FVBME/FVHCY composition of 44%/56%. Thus plasmas with higher homocysteine levels behave in the APC resistance assay as mixtures with a higher proportion of FVHCY.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We present evidence that in vitro homocysteine treatment of factor V can protect alpha -thrombin-derived factor Va from inactivation by APC. This effect is observed at concentrations of homocysteine as low as 30 µM, similar to those found in the blood of patients with "moderate hyperhomocysteinemia." As the homocysteine concentration used to treat factor V increases, impairment of APC-mediated proteolysis of human factor Va is more evident (Fig. 1). Our data indicate that the rate of APC cleavage at Arg-506 of homocysteinylated factor Va is diminished about 2.5-fold. (Fig. 2). The magnitude of this inactivation impairment is similar to that observed when APC inactivation of factor V Leiden is assessed in a function-based assay (47).

The most probable mechanism by which homocysteine inhibits APC inactivation of factor Va is by forming heterologous disulfide bonds. The heavy chain of bovine factor Va contains four cysteine residues in the A1 domain and six in the A2 domain (45). All cysteines in the A1 domain are associated with two disulfide bridges, whereas in the A2 domain, two free cysteines are located at positions 538 and 589 (45). On the basis of homology, the A2 domain of the heavy chain of human factor Va is likely to contain two free cysteines, Cys-539 and Cys-585 (Fig. 9). Since the light chain of bovine factor Va contains two free cysteines (Cys-1953 and Cys-2100), two free cysteine residues are anticipated in human factor Va light chain (46). The autoradiograph depicted in Fig. 5B provides evidence that both the heavy and light chains of factor Va are radiolabeled with [35S]homocysteine. Of the three fragments resulting from the APC cleavage of the heavy chain of factor Va, only the 28/26,000 doublet (residues 507-709), which contains Cys-539 and Cys-585, incorporated [35S]homocysteine (Fig. 5B). Under reducing conditions (2% beta -mercaptoethanol) [35S]homocysteine was released from factor V, since none of the bands seen on the Coomassie Blue-stained gel (Fig. 5C) were visualized on the corresponding autoradiograph (Fig. 5D). These results are consistent with the conclusion that incubation of human factor V with [35S]homocysteine results in the formation of protein-S-[35S]homocysteine disulfide adducts in a pattern consistent with the involvement of the five free cysteines of factor V. 



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 9.   Factor V and Va with cleavage sites by thrombin and APC. Positions of all free cysteines and disulfide bridges are depicted.

It has been reported that reduction of disulfide bonds by homocysteine or other reducing agents can result in the impaired function of such proteins as protein C or thrombomodulin (25). No evidence of disruption of intrachain disulfides of factor V was seen in our study either by direct or functional criteria. The APC-generated heavy chain fragments containing only disulfides (Mr = 30,000 and Mr = 45,000, see Fig. 9) were not radiolabeled when factor V were treated for time periods up to 2 h either with 450 µM [35S]homocysteine (Fig. 5B) or 450 µM L-[35S]cysteine (data not shown). Factor V subjected to our treatment regimens with 1 mM thiol, whether DL-homocysteine, L-cysteine, or beta -mercaptoethanol, was indistinguishable from untreated factor V in the kinetics of its activation by alpha -thrombin and in the activity of its product, factor Va. In addition, 1 mM L-cysteine exerted no effect on APC-mediated proteolysis of factor Va, despite incorporation of L-[35S]cysteine into the heavy and light chains of the protein. Taken together, these observations do not support a mechanism in which homocysteine efficacy derives from disulfide exchange reactions between homocysteine and the structural disulfides of the factor V molecule.

Our data offer some grounds for speculation concerning the mechanism of adduct formation between factor V and aminothiol. Blood constitutes a dynamic system where the thermodynamic and kinetic constants defining the chemistries of disulfide formation through oxidation and subsequent disulfide exchange reactions compete with the biological processes maintaining the specific circulating half-lives of the individual proteins, oxidized aminothiols, and reduced aminothiols involved in these reactions. In normal individuals, the total concentration of aminothiols (including homocysteine, cysteine, cysteinylglycine, and glutathione) is in the range of 300 µM, with cysteine levels constituting about 80% of the total (43, 48). Aminothiols in the reduced form constitute about 7% of the total. Greater than 50% of the oxidized pool for each major aminothiol occurs as a protein-S-S-X adduct (43). The ratio of reduced to total aminothiol for each thiol appears specific for that thiol: homocysteine, 1% (48); cysteine, 5% (43); cysteinylglycine, 10% (43); and glutathione, 60% (43). Our data detailing the modification of factor V by homocysteine combined with our preliminary observations indicating a similar reactivity with cysteine can be extrapolated to predict that factor V derived from plasma with 10-20 µM reduced aminothiol will have about 20% of its free cysteines in the form of heterologous disulfides. Thus in our reactions between homocysteine or cysteine and purified factor V, the majority of the initial bond-forming events would be oxidation reactions, presumably involving molecular oxygen as the electron acceptor. Factor V shows significant sequence homology with the copper-binding protein ceruloplasmin (49) and has been demonstrated to contain 1 mol of copper/mol of factor V (50). The spectral properties of the factor V copper complex are consistent with those of other copper-binding proteins expressing oxidase activity (50). We have observed that factor V fully homocysteinylated with unlabeled homocysteine and then incubated with [35S]homocysteine rapidly radiolabels in all regions of factor V containing free cysteines (data not shown). This indicates that disulfide exchange between free aminothiol and factor V homocysteine disulfide adducts will also be an ongoing process.

Results of the experiments with factor V treated with 1 mM beta -mercaptoethanol suggest that reduction of heterologous disulfide bonds between factor V free cysteines and homocysteine leads to a markedly faster APC inactivation of factor Va. Since plasma concentrations of homocysteine differ among even healthy subjects, the degree of impairment of APC inactivation of factor Va could vary significantly. Our observations may offer an explanation for the reported differences in APC activity toward factor Va derived from different preparations of factor V.

Kinetic studies using [35S]homocysteine indicate that the equilibrium between free homocysteine and homocysteinylated factor V is reached in less than 5 min. This equilibrium is characterized by an apparent Kd of 80 µM. Since the circulating half-life of factor V is about 720 min, it can be estimated that a fairly complete spectrum of factor V modification (10-85%) could occur within the physiologically relevant range of elevated homocysteine concentrations (10-450 µM). No difference in the rates of modification between the free cysteines in the heavy chain and those of the light chain was observed, suggesting an equivalence in the reactivity of these sites. Combined with the observation that modification of factor V by homocysteine does not alter its rate of activation by alpha -thrombin, we hypothesized that a two component model for factor V populations might be sufficient to characterize the relationship between homocysteine level and the APC sensitivity of derived factor Va. One component is unmodified, virgin factor V, produced in vitro by treating purified factor V from pooled normal plasma with beta -mercaptoethanol (FVBME) and representing the source of factor Va most rapidly inactivated by APC (Fig. 7). The other component is homocysteine-modified factor V, generated in vitro by treating factor V with 2 mM homocysteine (FVHCY), representing the source of "impaired" factor Va. The rate constant for the latter inactivation by APC is ~25% that for unmodified factor Va. Plasma factor V may then be conceived of as a mixture of these species, the amounts of each being a function of plasma homocysteine and factor V concentrations and the equilibrium constant. When such mixed populations were systematically constructed from FVBME and FVHCY stocks and tested in an APC resistance assay format, clotting times increased in a linear manner with increasing representation by FVBME in the factor V mixture (Fig. 8). This curve was used as a standard curve for translating plasma homocysteine levels into estimates of their factor Va sensitivity to APC degradation. Preliminary results with plasmas from four individuals with elevated homocysteine levels support the potential for this approach. Plasma homocysteine levels of 40, 44, 76, and 80 µM yield calculated values for percent unmodified factor V of 67, 65, 51, and 50%, respectively, and predict clotting times of 79, 78, 73.4, and 73 s using the standard curve shown in Fig. 8. The measured clotting times for these plasmas were 80, 78, 72, and 70 s. The clotting time of 85 s observed with the normal pool plasma translates into a plasma homocysteine concentration of 10.9 µM, a value within the range of normal homocysteine levels (2, 3, 43).

In vivo, increased thrombin formation would be the consequence of the reduced rate of APC-mediated proteolysis of factor Va, because this process leads to accumulation of active cofactor for the prothrombinase complex (30). Decreased efficiency of the APC-mediated factor Va inactivation may thus contribute to the prothrombotic activity of homocysteine. Our study may explain why the coexistence of factor V Leiden and hyperhomocysteinemia has a synergistic effect and confers a markedly higher risk of thrombotic disorder, as reported in a large epidemiological study by Ridker et al. (51).

Since platelets contain factor V that is secreted upon platelet activation, the procoagulant activity of homocysteine through modification of platelet factor V may be important in regulating coagulation reactions at the site of vascular injury. If this is the case, the property of homocysteine described here could also contribute to an increased risk of arterial thrombosis reported in patients with hyperhomocysteinemia. In summary, our results indicate that homocysteine markedly impairs the APC-mediated inactivation of factor Va. This effect is likely to be the consequence of modification of factor V through attachment of homocysteine to free cysteine(s). Our study strongly suggests that at the least the action of homocysteine on factor Va inactivation is specific, involves the entire molecule, and takes place at submillimolar homocysteine concentrations. The novel pathophysiological mechanism reported here may partly explain the thrombotic tendency observed in patients with hyperhomocysteinemia. Although the relationship between the pathology of hyperhomocysteinemia and APC resistance is by no means established by the present work, our data do indicate that homocysteine modification of factor V is a potential confounder of current APC resistance assays.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the excellent technical assistance of Jed Pauls and thank Ted Foss for writing the slope-finding software. We thank Dr. Andrzej Szczeklik and Dr. Teresa B. Domagala for providing plasmas from individuals with a genetically determined hyperhomocysteinemia and Dr. Richard Jenny for providing human APC and alpha -thrombin. We thank the United States Information Agency Fulbright Scholar Program for its support.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Merit Award HL-34575 (to K. G. M.) and the United States Information Agency Fulbright Scholar Program (to A. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Given Bldg., Health Science Complex, University of Vermont, College of Medicine, Burlington, VT 05405-0068. Tel.: 802-656-0335; Fax: 802-862-8229; E-mail: kmann@protein.med.uvm.edu.

Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M004124200


    ABBREVIATIONS

The abbreviations used are: APC, activated protein C; BME, beta -mercaptoethanol; HCY, homocysteine; TEMED, N,N,N',N'-tetramethylethylenediamine; PS, 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (sodium salt); PC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; PCPS, phospholipid vesicles; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Selhub, J., and Miller, J. W. (1991) Am. J. Clin. Nutr. 55, 131-138[Abstract]
2. Kang, S. S., Wong, P. W. K., and Malinow, M. R. (1992) Annu. Rev. Nutr. 12, 279-298[CrossRef][Medline] [Order article via Infotrieve]
3. Malinow, M. R., Bostom, A. G., and Krauss, R. M. (1999) Circulation 99, 178-182[Free Full Text]
4. Stampfer, M. J., Malinow, M. R., Willet, W. C., Newcomer, L. M., Upson, B., Ullman, D., Tischler, P. V., and Hennekens, C. H. (1992) J. Am. Med. Assoc. 268, 877-881[Abstract]
5. Nygard, O., Nordrehaug, J. E., Refsum, H., Ueland, P. M., Farstad, M. F., and Vollset, S. E. (1997) N. Engl. J. Med. 337, 230-236[Abstract/Free Full Text]
6. Schwartz, S. M., Siscovick, D. S., Malinow, M. R., Rosendaal, F. R., Beverly, R. K., Hess, D. L., Psaty, B. M., Longstreth, W. T., Koepsell, T. D., Raghunathan, T. E., and Reitsma, P. H. (1997) Circulation 96, 412-417[Abstract/Free Full Text]
7. Perry, I. J., Morris, R. W., Ebrahim, S. B., Ueland, P. M., and Shaper, A. G. (1995) Lancet 346, 1395-1398[Medline] [Order article via Infotrieve]
8. Van den Berg, M., Stehouwer, C. D. A., Bierdrager, E., and Rauwerda, J. A. (1996) Arterioscler. Thromb. Vasc. Biol. 16, 165-171[Abstract/Free Full Text]
9. Boushey, C. J., Beresford, S. A. A., Omenn, G. S., and Motulsky, A. G. (1995) J. Am. Med. Assoc. 274, 1049-1057[Abstract]
10. den Heijer, M., Koster, T., Blom, H. J., Bos, G. M. J., Briet, E., Reitsma, P. H., van den Brouke, J. P., and Rosendaal, F. R. (1996) N. Engl. J. Med. 334, 759-762[Abstract/Free Full Text]
11. Falcon, C. R., Cattaneo, M., Panzeri, R., Martinelli, I., and Mannucci, P. M. (1994) Arterioscler. Thromb. 14, 1080-1083[Abstract]
12. Fermo, I., D'Angelo, S. V., Paroni, R. P., Mazzola, G., Caloni, G., and D'Angelo, A. (1995) Ann. Intern. Med. 123, 747-753[Abstract/Free Full Text]
13. Cattaneo, A. (1999) Thromb. Haemostasis 81, 165-176[Medline] [Order article via Infotrieve]
14. Rees, M. M., and Rodgers, G. M. (1993) Thromb. Res. 71, 337-359[Medline] [Order article via Infotrieve]
15. Welch, M. F., and Loscalzo, J. (1998) N. Engl. J. Med. 338, 1042-1050[Free Full Text]
16. Domagala, T. B., Undas, A., Libura, M., and Szczeklik, A. (1998) J. Cardiovasc. Risk 5, 239-247[Medline] [Order article via Infotrieve]
17. D'Angelo, A., and Selhub, J. (1997) Blood 90, 1-11[Free Full Text]
18. Loscalzo, J. (1996) J. Clin. Invest. 98, 5-7[Free Full Text]
19. Stamler, J. S., Osborne, J. A., Jaraki, O., Rabbani, L. E., Mullins, M., Singel, D., and Loscalzo, J. (1993) J. Clin. Invest. 91, 308-318[Medline] [Order article via Infotrieve]
20. Hajjer, K. A. (1993) J. Clin. Invest. 91, 2873-2879[Medline] [Order article via Infotrieve]
21. Fryer, R. H., Wilson, B. D., Gubler, D. B., Fitzgerald, L. A., and Rodgers, G. M. (1993) Arterioscler. Thromb. 13, 1327-1233[Abstract]
22. Nishinaga, M., Ozawa, T., and Shimada, K. (1993) J. Clin. Invest. 92, 1381-1386[Medline] [Order article via Infotrieve]
23. Rodgers, G. M., and Kane, W. H. (1986) J. Clin. Invest. 77, 1909-1916[Medline] [Order article via Infotrieve]
24. Rodgers, G. M, and Conn, M. T. (1990) Blood 75, 895-901[Abstract]
25. Lentz, S. R., and Sadler, J. E. (1991) J. Clin. Invest. 88, 1906-1914[Medline] [Order article via Infotrieve]
26. Hayashi, T., Honda, G., and Suzuki, K. (1992) Blood 79, 2930-2936[Abstract]
27. Nesheim, M. E., and Mann, K. G. (1979) J. Biol. Chem. 254, 1326-1334[Abstract]
28. Jenny, R. J., Pittman, D. D., Toole, J. J., Kriz, R. W., Aldape, R. A., Hewick, R. M., Kaufman, R. J., and Mann, K. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4846-4850[Abstract]
29. Kalafatis, M., and Mann, K. G. (1993) J. Biol. Chem. 268, 27246-27257[Abstract/Free Full Text]
30. Kalafatis, M., Rand, M. D., and Mann, K. G. (1994) J. Biol. Chem. 269, 31869-31880[Abstract/Free Full Text]
31. Mann, K. G., Hockin, M. F., Begin, K. J., and Kalafatis, M. (1997) J. Biol. Chem. 272, 20678-20683[Abstract/Free Full Text]
32. Hockin, M. F., Cawthern, K. M., Kalafatis, M., and Mann, K. G. (1999) Biochemistry 38, 6918-6934[CrossRef][Medline] [Order article via Infotrieve]
33. Bertina, R. M., Koeleman, B. P. C., Koster, T., Rosendaal, F. R., Dirven, R. J., de Ronde, H., van der Velden, P. A., and Reitsma, P. H. (1994) Nature 369, 64-67[CrossRef][Medline] [Order article via Infotrieve]
34. Lane, D. A., Mannucci, P. M., Bauer, K. A., Bertina, R. M., Bochkov, N. P., Boulyienko, V., Chandy, M., Dahlback, B., Ginter, E. K., Miletich, J. P., Rosendaal, F. R., and Seligsohn, U. (1996) Thromb. Haemostasis 76, 651-670[Medline] [Order article via Infotrieve]
35. de Visser, M. C., Rosendaal, F. R., and Bertina, R. M. (1999) Blood 93, 1271-1276[Abstract/Free Full Text]
36. 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]
37. Barenholz, Y., Gibbes, D., Litman, B. J., Goll, J., Thompson, T. E., and Carlson, F. D. (1977) Biochemistry 16, 2806-2810[Medline] [Order article via Infotrieve]
38. Nesheim, M. E., Katzmann, J. A., Tracy, P. B., and Mann, K. G. (1981) in Methods in Enzymology, Proteolytic Enzymes Part C (Lorand, L., ed) , pp. 249-285, Academic Press, Inc., New York
39. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
40. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract]
41. Kalafatis, M., Bertina, R. M., Rand, M. D., and Mann, K. G. (1995) J. Biol. Chem. 270, 4053-4057[Abstract/Free Full Text]
42. Dahlback, B., Carlsson, M., and Svensson, P. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1004-1008[Abstract]
43. Mansoor, M. A., Svardal, A. M., and Ueland, P. (1992) Anal. Biochem. 200, 218-229[Medline] [Order article via Infotrieve]
44. Baernstein, H. D. (1934) J. Biol. Chem. 106, 451-456
45. Xue, J., Kalafatis, M., Silveira, J. R., Kung, C., and Mann, K. G. (1994) Biochemistry 33, 13109-13116[Medline] [Order article via Infotrieve]
46. Xue, J., Kalafatis, M., Silveira, J. R., Kung, C., and Mann, K. G. (1993) Biochemistry 32, 5917-5923[Medline] [Order article via Infotrieve]
47. Egan, J. O., Kalafatis, M., and Mann, K. G. (1997) Protein Sci. 6, 2016-2027[Abstract/Free Full Text]
48. Mansoor, M. A., Svardal, A. M., Schneede, J., and Ueland, P. M. (1992) Clin. Chem. 38, 1316-1321[Abstract/Free Full Text]
49. Church, W. R., Jernigan, R. L., Toole, J., Hewick, R. M., Knopp, J., Knutson, G. J., Nesheim, M. E., Mann, K. G., and Fass, D. N. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6934-6937[Abstract]
50. Mann, K. G., Lawler, C. M., Vehar, G. A., and Church, W. R. (1984) J. Biol. Chem. 259, 12949-12951[Abstract/Free Full Text]
51. Ridker, P. M., Hennekens, C. H., Selhub, J., Miletich, J. P., and Malinow, M. R. (1997) Circulation 95, 1777-1782[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.