THE JOURNAL OF Bro~ocrc~~ CHEMISTRY Vol. 254, No. 2, Issue of January 25, pp. 508-517, 1979 Printed in U.S.A.

Isolation and Characterization of Single Chain Bovine Factor V*

(Received for publication, June 28, 1978, and in revised form, August 17, 1978)

Michael E. Nesheim,$ Kurt H. Myrmel,§ Lyndon Hibbard, and Kenneth G. Mann7

From the Hematology Research Section, Mayo Clinic, Rochester. Minnesota 55901

A procedure for the isolation of bovine Factor V has been developed. The Factor V is isolated from bovine plasma by a series of steps including barium citrate adsorption, polyethylene glycol precipitation, QAE-cel- lulose adsorption, hydrophobic chromatography on oc- tyl Sepharose, ammonium sulfate fractionation, pre- parative electrophoresis on acrylamide gels, and ii- nally, phenyl Sepharose chromatography. During iso- lation, judicious use of inhibitors including benzami- dine hydrochloride, soybean trypsin inhibitor, and di- isopropylphosphorofluoridate has been applied to pre- vent activation of the Factor V to Factor V,. The activ- ity of the isolated protein increases by a factor of 80 when stimulated by catalytic amounts of thrombin. The specific activity of the material after thrombin activa- tion is 1250 units/mg of protein when evaluated versus a bovine Factor V standard in human factor V-deficient plasma. The isolated protein is a single component when analyzed by a variety of electrophoretic tech- niques and has been characterized in terms of its gross physical and chemical properties. Bovine Factor V is a single chain glycoprotein which has a molecular weight of 330,000. The single chain nature of the molecule has been established by sedimentation equilibrium studies of the native molecule and on the molecule in 6 M

guanidinium chloride with and without disulfide bond reduction. In addition to these mass measurements, the single chain nature of the molecule has been estab- lished by hydrodynamic estimation of the random coil volume by sedimentation velocity studies of the re- duced carboxyamidomethylated protein in 6 M guani- dinium chloride. Native Factor V has a sedimentation coefficient ~$0 ,,,, of 9.19 S, which indicates the molecule is highly asymmetric. The frictional ratio, f/&in for the molecule is estimated to be 2.01, and the axial ratio of the equivalent prolate ellipsoid is 25:l. Thus, present data suggest that Factor V is a rod-like molecule com- posed of a single chain.

Great progress in understanding the molecular details of the blood coagulation process at the level of the “prothrom- binase” complex was made with the isolation to homogeneity (2-7) and subsequent study (8-12) of prothrombin and Factor

* This research was supported by Grant HL-17430D. A preliminary report of this study was presented to the American Society of Biolog- ical Chemists Meeting, June, 1978, Atlanta, GA, (1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “acluer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Blood Banking and Hemostasis Training Grant HL-07069.

5 Present address, Ortho Diagnostics, Raritan, NJ 08869. 1 American Heart Established Investigator, to whom correspond-

ence concerning this paper should be addressed.

X,. Further understanding, however, has been impeded some- what by the lack of a procedure for isolating Factor V to homogeneity.

Although there have been numerous reports of the purifi- cation of Factor V (13-22), no preparation has satisfied the criteria normally accepted and traditionally emplqyed in pro- tein chemistry as evidence for homogeneity (such as a single component on gel electrophoresis). Nonhomogeneity in nu- merous preparations of Factor V can be inferred from reports of amino acid composition which vary widely (13, 17), from reported molecular weight values ranging from 30,000 to 1,200,OOO (20, 22), and from conflicting reports of a variety of subunit structures (17, 19, 22).

Nonetheless, valuable insights into the cofactor function of Factor V have been obtained (23). In studies using partially purified Factor V, and aimed at characterizing prothrombin and its activation products it was inferred that the prothrom- bin Fragment 2 region interacts with Factor V (24). Partici- pation of Factor V in prothrombin conversion has been shown to be obligately dependent upon Ca2+ and facilitated by phos- pholipid (25). Alterations in the esterase activity of Factor X, by partially purified Factor V have also been observed (26).

It was recognized in early studies (15, 27), and shown more conclusively later on (22), that Factor V issubject to activation by thrombin. In the present work the explicit assumption was made at the outset that the activation of Factor V is a consequence of the proteolytic conversion of an inactive or minimally active pro-cofactor to active cofactor. The goal of the present work was to isolate and study the putative pro- cofactor. Thus, all samples were assayed for activity not only before but also after deliberate activation by thrombin. Activ- ity measured prior to deliberate activation by thrombin was assumed to be the result of endogeneous activated cofactor, while activity observed after deliberate thrombin activation was assumed to be a measure of total potential activity in the form of a pro-cofactor.

This paper contains a report on the isolation of homogene- ous bovine Factor V and on its gross physical properties.

EXPERIMENTAL PROCEDURES

Materials

Tris base was obtained from either Sigma (TRIZMA) or Schwarz/Mann. Soybean trypsin inhibitor was from Sigma. Boric acid and CaCl, were obtained from Fisher Scientific. Quaternary aminoethyl cellulose was obtained from Schleicher and Schuell, and washed by the procedure of Sophianopoulous and Vestling (28). Sepharose and phenyl Sepharose were obtained from Pharmacia. Ascorbic acid was purchased from Eastman. Diisopropylphosphoro- fluoridate was obtained from Sigma and benzamidine hydrochloride from Aldrich. Guanidinium chloride was obtained from Heico; con- centrations were determined by refractometry (29). Ammonium sul- fate (ultrapure) was obtained from Schwarz/Mann. Sodium dodecyl sulfate was purchased from Pierce. Rabbit brain thromboplastin was prepared according to the method of Bowie et al. (30). Bovine blood was obtained either by venipuncture from the Institute Hills Farm of

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Bovine Factor V Isolation 509

the Mayo Clinic, or occasionally by postmortem exsanguination at Hormel in Austin, MN, or Erdman’s in Kasson, MN. Factor V- deficient plasma was prepared by a modification of the procedure of Lewis and Ware (31). Human blood collected in 0.1 M sodium oxalate (9:l (v/v)) was obtained from the Mayo Clinic Blood Bank. Following centrifugation to remove cells, the resulting plasma was transferred to sterile bottles and incubated under 5% CO2 at 37°C for 12 days. The incubated plasma had a pH of 7.0 to 7.4 and a one-stage prothrombin time greater than 150 s which could be corrected to approximately 22 s by the addition of bovine plasma. Rabbit brain cephalin was obtained from Sigma. Prior to use in prethrombin 1 activation studies, it was dispersed in 0.02 M Tris-HCl, pH 7.4, 0.15 M NaCl at a concentration of 0.65 mM. Human prethrombin 1 was prepared as described by Downing et al. (32). A procedure described by the same authors was used to prepare bovine Factor X,. Factor X, activity was assayed as described by Bajaj and Mann (5) and had a specific activity of 1115 units/mg. All other reagents were of the highest purity available.

Assay of Factor V and Factor V,,

A 50-~1 aliquot of sample, appropriately diluted in buffer (0.02 M imidazole-HCl, 0.15 M NaCl, pH 7.4), was added to a culture tube (13 x 75 mm) followed by 50 ~1 of Factor V-deficient plasma plus 50 ~1 of thromboplastin. After a brief incubation at 37°C 50 ~1 of 0.025 M CaC12 was added to initiate the assay and the time required to form a clot at 37’C was recorded. Factor V, activity of the sample was determined by comparison to a standard curve prepared from a serial dilution of bovine plasma (not activated). Fig. 1 is a typical standard curve for the assay. One unit of Factor V, activity was defined as that amount found in 1 ml of bovine plasma prepared from blood freshly drawn into N volume of 2.85% trisodium citrate. To measure the Factor V, present after thrombin activation, the sample was treated with thrombin (final concentration, 1 NIH unit/ml) for 60 s at 37°C. It was then diluted 20- to 50-fold and assayed as described above. The small amount of thrombin added was not sufficient to interfere with the measurement. The activity observed by assay after thrombin treatment was presumed to be a measure of total pro-cofactor content, i.e. Factor V. Unless otherwise stated all assay results are given in terms of total activity observed after thrombin treatment, plus an activation quotient defined as the ratio of the activity observed after thrombin treatment to the activity observed before thrombin treat- ment.

Human prethrombin 1 activations with varying concentrations of Factor V were carried out in plastic tubes containing 0.25 ml of 0.02 M Tris-HCl, pH 7.4, 0.15 M NaCl, 2 miw CaC12, 0.13 mM cephalin, and prethrombin 1 (0.071 mg/ml). The solution was incubated at 37°C for 5 min after which an appropriate aliquot of Factor V stock solution (260 units/ml) was added to a final concentration of 0 to 10 units/ml. Factor X, was then immediately added to a final concentration of 1.0 unit/ml. Incubation was continued at 37°C and aliquots were with- drawn at intervals for thrombin assay by the NIH procedure (33) as modified by Mann et al. (34).

Factor V Isolation

Blood Collection-For most isolation experiments, 9 parts of bo- vine blood were drawn by venipuncture into 1 part anticoagulant consisting of 2.85% trisodium citrate, 0.02% soybean trypsin inhibitor, 10 mM benzamidine hydrochloride, and 10m4 M DFP.’ The red cells were removed by centrifugation at 4000 x g, 4”C, for 30 min. The plasma was carefully removed by siphon, and subsequent steps were carried out as soon after blood collection as possible.

Barium Citrate Adsorption-To each liter of plasma from the previous step, 80 ml, 1 M BaClz was added dropwise over a period of approximately 20 min, and stirring was vigorously maintained at 4°C. After stirring for an additional 30 min the barium citrate was removed by centrifugation for 30 min at 4°C and 5000 X g.

PEG-6000 Fractionation-Sufficient 50% of PEG-6000 was added dropwise at 4’C to render the supernatant from the previous step 4% in PEG-6000. Vigorous stirring was maintained throughout the addi- tion and for 30 min thereafter. The precipitate was then removed by centrifugation for 30 min at 4°C and 5000 x g.

QAE-cellulose Adsorption-To the vigorously stirred supernatant from the PEG-6000 step was added QAE-cellulose (chloride form, 100

’ The abbreviations used are DFP, diisopropylphosphorofluoridate; QAE-cellulose, diethyl-(2.hydroxypropyl)aminoethyl cellulose; DodSO,, sodium dodecyl sulfate; PEG, polyethylene glycol.

FACTOR P ASSAY STANDARD CURVE

100,

0.001 0.005 0.025 0.100 0.0025 0.010 0.050

Factor a, U / ml

FIG. 1. Factor V assay standard curve. The logarithm of clotting time is plotted against the logarithm of added Factor V. The standard unit was defined as the amount of Factor V (prior to activation by thrombin) found in 1 ml of fresh bovine plasma prepared from blood drawn into 2.85% trisodium citrate (N volume). In the absence of added Factor V the clot time exceeded 150 s. Assays were performed in human Factor V-deficient plasma as described under “Experimen- tal Procedures.”

ml of packed resin prepared as described above per liter of PEG-6000 supernatant). After stirring 5 min at 4°C an equal volume of cold buffer (0.025 M Tris-HCl, 5 mM CaCl?, 1 mM benzamidine-HCl, pH 7.4) plus an equal volume of cold water were added. Stirring at 4°C was continued for an additional 30 min. The QAE-cellulose was then removed by vacuum filtration through a coarse, sintered glass funnel. Partial vacuum was used to enhance flow rates but care was taken to avoid excessive foaming at the lower surface of the filter. The filtered resin was washed with buffer followed by buffer in 0.1 M NaCl. The volume of each wash was 5 to 10 times the packed bed volume of the resin. Factor V was then eluted with a minimum volume of buffer containing 0.5 M NaCl. The protein was concentrated by adding solid ammonium sulfate to 60% saturation (0.36 g/ml) followed, after 20 min of stirring at 4’C by centrifugation for 20 min at 4°C and 10,000 x g.

Octyl Sepharose Chromatography-The ammonium sulfate pellet from the previous step was dissolved in approximately 100 ml of buffer (0.02 M Tris/borate, 1 InM CaClz, 0.8 M NaCl, pH 8.3) at 22°C and loaded over a 30-min period onto a column (2.4 x 20 cm) of octyl Sepharose equilibrated with the same buffer. The column was then washed with buffer at a rate of 4 ml/min until the absorbance at 280 nm of the eluate was less than 0.05. A 500.ml linear gradient buffered with 0.02 M Tris/borate, 1 mivr CaC12, pH 8.3, that started with 0.1 M NaCl and ended with buffer only was used to elute the Factor V. Peak fractions were pooled, chilled to ice temperature, and concentrated by adding solid ammonium sulfate at 70% saturation (0.44 g/ml). After 30 min on ice the protein was pelleted by centrifugation for 20 min at 4°C and 13,000 x g.

Preparative Electrophoresis-The pellet from the preceding step was dissolved in 5 ml of 0.01 M Tris/borate, 1 mM CaC12, 10% glycerol, pH 7.4, and applied to the preparative electrophoresis apparatus described by Nesheim (35). The apparatus was a custom-made device (similar to those available commercially) in which electrophoresis is performed in a gel cast between two cooled, concentric cylinders of 20- and 42-mm diameter, respectively. The gel was 30 mm in length, prepared from 5%) acrylamide and 0.14% bisacrylamide. Gel formation was accomplished by adding 0.25 ml of 28% ammonium persulfate and 0.15 ml of N,N,N’,N’-tetramethylethylenediamine/lOO-ml gel. Contin- uous elution to a fraction collector was maintained via the buffer through a space between the bottom of the gel and a dialysis mem- brane. The buffer used throughout was 0.05 M Tris/borate, 5 mM CaC12, pH 8.3. Prior to sample application, the gel was pre-electro- phoresed in two stages. In the first stage, voltage was applied until a dye marker (bromphenol blue) had traversed the gel. In the second stage, 2.0 ml of 0.05 M ascorbic acid plus 0.025 M NaOH in 10% glycerol/H20 with a trace of bromphenol blue was subjected to electrophoresis at 70 V until dye had migrated through the gel. Five

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Bovine Factor V Isolation

milliliters of protein sample was then added and electrophoresis was some low molecular weight material was present and plots of log AY performed at 4’C, 100 V (about 80 mA). Fractions of 15 ml were versus r2 were curved. Under these circumstances, weight-average collected at 12-min intervals. molecular weight (&<,.) values were obtained using the expression:

Phenyl Sepharose-To the pooled fractions from the previous step was added an equal volume of cold 2 M NaCl. The resulting solution was then applied at a rate not exceeding 1 to 2 ml/min to a 5-ml column of phenyl Sepharose (1.5~cm diameter) at 4’C equilibrated in 0.01 M Tris/borate, pH 8.3, 1 InM CaC12, 1 M NaCl. The column was washed with about 30 ml of equilibrating buffer. Factor V was eluted in a minimal volume of equilibrating buffer minus NaCl. The eluted material was dialyzed overnight against 10 to 20 volumes of 50% glycerol/water, 0.5 mrvr benzamidine hydrochloride. The protein, con- centrated by a factor of 3 by dialysis, was subsequently stored at -20°C.

(2) RT cb

” =

W2w$‘P) J b crdr

a

The curved plots obtained from studies of native Factor V in the native state were also analyzed in terms of point weight averages (&fur) and the whole cell z-average molecular weight (&) was ob- tained by extrapolating the M,,, values to the bottom of the cell. In any event, the difference between I@~ and ar was only 7.2%.

Analytical Gel Electrophoresis

Native gel electrophoresis was performed using the discontinuous systems described by Davis (36) or Williams and Riesfeld (37), or with a continuous system employing 0.1 M Tris/borate, 1 mM CaClz, pH 8.3. Gel electrophoresis in DodSOl was carried out in acrylamide ‘according to the method of Weber and Osborn (38) as modified by Mann et al. (34). or in agarose according to the method of Fass et aE. (39). Protein bands were detected with Coomassie blue. Gels were destained electrophoretically.

Amino Acid Composition and Carbohydrate Analysis

Tryptophan was determined by the method of Liu and Chang (40). Cysteine was determined as cysteic acid by the method of Moore (41). The remaining amino acids were determined from 24-, 48., and 72-h 6 N HCl hydrolysates (110°C). Analyses were performed with a Beckman 119 amino acid analyzer utilizing Beckman AA15 resins. Amino sugars were determined on the amino acid analyzer following hydrolysis in 4 N HCl for 6 h at 110°C. Simple sugars were determined by the phenolsulfuric acid method described by Ashwell (42) using glucose as a reference standard.

The partial specific volume (6) used for calculations of molecular weight was computed from the amino acid composition of Factor V using the method of Cohn and Edsall (45).’ A value of 0.625 ml/g for V was used for the weight fraction of carbohydrate (46, 47). For sedimentation equilibrium studies conducted under native conditions, the value of U calculated from compositions, 0.712 ml/g was used for 4’; since in dilute salt solutions, preferential solvent interactions are minimal and +’ is essentially equal to U. However, in concentrated guanidinium chloride solutions, preferential guanidination of protein may occur (48) and 9’ may be decreased when compared to U by as much as 0.01 ml/g. Two possibilities were considered in calculating molecular weights for sedimentation equilibrium studies performed in 6 M guanidine chloride: 1) preferential solvent interaction is mini- mal and 4’ is identical with U with (0.712 ml/g); 2) maximal interaction of a protein with solvent had occurred resulting in a decrease in 4’ by 0.01 ml/g (0.702 ml/g).

Extinction Coefficient

Protein concentration was determined by ultracentrifugation using the synthetic boundary procedure of Babul and Stellwagen (43). The absorbance at 280 nm used in the calculation of the extinction coefficient was corrected for scatter by subtraction of 1.7 times the absorbance observed at 320 nm.

Sedimentation velocity experiments were carried out in double sector synthetic boundary cells at rotor speeds between 56,000 and 60,000 rpm and temperatures between 21 and 23’C. Schlieren optics were used for all sedimentation velocity analyses. Studies on native Factor V were conducted in 0.025 M Tris-HCl, 1 mM benzamidine hydrochloride, 5 mM calcium chloride, 0.1 M sodium chloride, pH 7.4. Sedimentation studies on the reduced carboxamidomethylated pro- tein in the denatured state were carried out in 6 M guanidine hydro- chloride, pH 6.0 (49). In both cases, limiting sedimentation values were obtained by extrapolation of data to zero protein concentration. In the native studies, the normal standard state of water at 20°C (s& J was used, whereas for the protein in 6 M guanidine chloride, the standard state used was 6 M guanidine chloride at 25°C (s&c).

Ultracentrifuge Studies

Sedimentation equilibrium studies were performed using the short column, high speed technique of Yphantis (44). Molecular weight analyses were conducted with Factor V samples under three sets of conditions: 1) studies of the native molecular weight of the Factor V molecule were conducted with the protein in 0.025 M Tris-HCl, 1 mM benzamidine hydrochloride, 5 mM CaC12, 0.1 M NaCl, pH 7.4; 2) for studies of the subunit structure of Factor V in its denatured state with disulfide bonds intact, analyses were performed with protein which had been dialyzed against 6 M guanidinium chloride, pH 6.0; 3) for studies of the subunit structure with disulfide bonds reduced, Factor V was reduced in 6 M guanidinium chloride, 0.1 M 2-mercap- toethanol, pH 8.6, and carboxamidomethylated with an excess of iodoacetamide. Sedimentation equilibrium studies were performed at constant temperatures between 20°C and 23°C using 3-mm solution columns in double sector cells equipped with sapphire windows.

The frictional coefficient for Factor V in the native state was calculated from the velocity and sedimentation equilibrium data and compared to the minimum frictional coefficient obtainable for an equivalent anhydrous sphere (50). The calculated f/f”,,,) value was analyzed in terms of frictional ratio using the data of Oncley (51) assuming 0.2 g of water/g of protein.

The number of amino acid residue units in the (presumed) ran- domly coiled, reduced, carboxyamidomethylated Factor V in 6 M

guanidinium chloride was calculated using the expression derived empirically by Tanford and co-workers (49) relating polymer chain length and sedimentation coefficient:

(3) s;sG

= 0.286 no.473 (l-+‘p)

For most analyses, plots of log AY versus r2 were linear and a single molecular weight was obtained throughout the cell. Under these circumstances, molecular weights were calculated using the expres- sion:

In Equation 3, n is the number of monomer units in the molecule. Multiplication of the number of monomer units by the mean residue weight obtained from amino acid composition results in a molecular weight based upon polymer hydrodynamic volume.

RESULTS

(1)

2 RT dlnc

M=

w2( I-$‘p) dr2

Isolation

All isolation steps were performed without interruption in

in which 4’ is the effective partial specific volume of the protein, w is the angular velocity, and p is the solvent density. Values for dlnc/dr’ were obtained from least squares analyses of plots of long fringe displacement (AY) uersas the square of the radial distance (?) for points above 100 pm fringe displacement.

Gel electrophoretic analysis of protein samples were conducted before and after each sedimentation equilibrium analysis. In studies of the native molecule, it was clear that some degradation had occurred during the periods of analysis. As a result of this degradation,

A The partial specific volumes used in the calculation of sedimen- tation equilibrium molecular weights were calculated from the amino acid (and carbohydrate) composition and were reported to three significant figures. However, the 14 or so partial specific volumes of each amino acid which are added to achieve this value have been determined to only two significant figures. Although partial specific volumes calculated in this manner are almost always reported to three significant figures, as was the case in the present work, it is apparent that no partial specific volume calculated from the amino acid composition is valid to more than two significant figures.

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Bovine Factor V Isolation 511

order to minimize the likelihood of activation or degradation of the protein. The individual steps were arranged such that the material obtained from one step was suitable in both pH and ionic strength for direct application to the subsequent step, thus obviating the need for lengthy sample preparation by dialysis. Although intermediate steps of precipitation with ammonium sulfate were included, they were used primarily for convenience and as a means of obtaining a slight increase in specific activity with good yields. The ordering of steps in the indicated fashion permitted the completion of the isolation in about 35 h from the time of blood collection.

Purification

A summary of the steps of purification, including yields, specific activities and activation quotients is presented in Table I.

Step 1. Reparation of Plasma--In most cases blood was drawn by venipuncture and collected into trisodium citrate plus protease inhibitors. The inhibitors presumably sup- pressed activation and degradation of Factor V during blood collection and subsequently through Step 4.

Step 2. Barium Citrate Adsorption of PEasma-Although no net purification was obtained, yields were nearly quanti- tative and potential activator(s) were removed.

Step 3. Precipitation with Polyethylene Glycol WOO-This step, in which Factor V remained in the supernatant, gave upwards of a 2-fold purification over the previous step with nearly quantitative yields.

Step 4. QAE-cellulose Chromatography-A batch proce- dure using a filter provided about a 30-fold purification over the previous step with a 70% recovery of added activity. The results of the QAE-cellulose procedure are depicted in the histogram of Fig. 2 in which total protein and Factor V recovered after each elution step is represented. Two washes were used: the first wash consisted of initial buffer; the second was with initial buffer in 0.1 M NaCl. Factor V was eluted with initial buffer in 0.5 M NaCl. The Factor V, which was approx- imately 2% pure at this point, was concentrated by precipita- tion with 60% ammonium sulfate.

Step 5. Octyl Sepharose Chromatography-This procedure typically resulted in a lo- to 20-fold purification over the previous step with yields in excess of 80%. The elution profile of Factor V from octyl Sepharose is shown in Fig. 3. Both absorbance at 280 nm and Factor V activity recovered are plotted against fraction number. The first peak which extends from about Fraction 5 to Fraction 25 was observed upon sample loading and the initial wash with buffer in 0.8 M NaCl. The next peak, beginning with Fraction 35, was eluted about midway through a linear gradient starting with buffered 0.1 M NaCl and ending in buffer only. Factor V activity indicated by the dashed line was eluted in the second peak. Chroma- tography on octyl Sepharose typically proceeded as indicated

in Fig. 3. Occasionally, however, for unknown reasons, when the linear gradient of decreasing ionic strength was applied, Factor V activity began to elute as a broad peak resulting in the protein being inconveniently dispersed in large volumes. Thus, once the gradient was started, column fractions were continuously monitored for activity, and, if the resulting elu- tion profile indicated the onset of a broad peak, the gradient was discontinued and elution was continued with low ionic strength buffer only (10 mM Tris/borate, 1 mM CaCla, pH 8.3). The activity then was eluted sharply and in high yield. Al- though the specific activity of this material was about 30% lower than that which would have been realized with the gradient, this material was suitable for the next step of puri- fication. Pooled fractions following gradient elution (Fractions 36 to 42 in Fig. 3) typically contained Factor V at about 40% purity. This material was concentrated by precipitation with 70% ammonium sulfate.

Step 6. Preparative Polyacrylamide Gel Electro- phoresis-The results of a typical preparation are shown in Fig. 4. Both the absorbance at 280 nm and total Factor V activity are plotted. The peak preceding Factor V contained

fa Protein q Factor V

Filtrate 0.1 M NaCl 0.5M NaC plus wash eiuate eluate

FIG. 2. Results of batch chromatography on QAE-cellulose. Total protein, estimated by absorbance at 280 nm, and total Factor V (after thrombin activation), are indicated by vertical bars according to the units on the left and right vertical axis, respectively. Elution condi- tions are given below the three sets of bars. The set of bars on the left indicate protein and Factor V found in the filtrate plus a low ionic strength wash. The set of bars in the middle resulted from subsequent washing with buffer containing 0.1 M NaCl. The bars on the right indicate the result of elution with buffer in 0.5 M NaCl.

TABLE I Summary of the isolation of bovine Factor V

step Volume Total protein Total Factor V Activation &d activity auotient Specific activity Purification Yield

units/Am -fold % 0.50 1.00 100 0.45 0.90 81.3 0.60 1.20 75.0

19.5 39.0 55.0 27.0 54.0 44.0

475 950 37.0 573 1146 36.0

1090 2178 26.7 1250 2500 21.4

Plasma Barium citrate supernatant 4% PEG-6000 supernatant QAE-cellulose eluate 60% (NH&SO4 pellet Octyl Sepharose pool 70% (NH4)2S04 pellet Preparative electrophoresis pool Phenyl Sepharose pool in 50%

glycerol

1,840 92,000 46,000 51.0 1,930 90,000 40,500 52.5 2,000 57,400 34,400 46.5

240 1,300 25,300 34.0 60 756 20,400 33.3

104 36.2 17,200 34.4 5 28.8 16,500 66.0

78 11.3 12,300 79.9 4 7.87 9,840 80.0

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Bovine Factor V Isolation

1.0

0.8

0.6

04

0.2

0 10 20 30 40 50 60

Fraction number FIG. 3. Chromatography on octyl Sepharose. The units on the left

and right vertical axes are absorbance at 280 nm (0) and Factor V activity (0), respectively. Fraction numbers are on the horizontal axis. The column (2.4 x 20 cm) was equilibrated at 20°C with 0.02 M Tris/borate, 0.8 M NaCl, 5 mM CaC12, pH 8.3; the same buffer with which the sample was applied and washing was accomplished. Follow- ing the wash, Factor V was eluted upon application of 500-ml linear gradient beginning with buffer plus 0.1 M NaCl and ending in buffer only. The gradient was started at Fraction 25. The flow rate was about 4 ml/min. Fraction volumes were 20 ml.

0.3 0 z 0.2

T

0.1 1 , *JJL, , roni 10 20 30 40 50 60

FIG. 4. Preparative electrophoresis. The units on the left and right vertical axis are absorbance at 280 nm (0) and Factor V activity (O), respectively. The buffer was 0.05 M Tris/borate, 5 mM CaC12, pH 8.3. Prior to sample application, the gel was pre-electrophoresed with ascorbic acid. Electrophoresis was performed at 4°C at 100 V, 80 mA. Fractions of 15 ml were collected every 12 min. In Panel A, the activation quotient of Factor V as a function of fraction number is plotted. In Panel B, Factor V specific activity values (total activity per absorbance unit following activation with thrombin) are plotted against fraction number.

multiple protein components. Factor V activity, indicated by the dashed line in Fig. 4, was eluted exclusively in the second peak. Also plotted in the insets of Fig. 4 are specific activity of total Factor V and activation quotients (by thrombin) in fractions across the peak. The Factor V specific activity is constant over much of the protein peak but decreases on the trailing edge. Concomitant with the decrease in Factor V specific activity on the trailing edge is a decrease in activati- bility by thrombin. The activation quotient values range from about go-fold at the peak to less than 20-fold on the trailing edge of the peak. For typical preparations the yields of Factor V were between 70 and 80%.

Step 7. Phenyl Sepharose Chromatography-This proce- dure was used primarily as a means of concentrating the Factor V, although a slight increase in specific activity was accomplished. The material was concentrated by phenyl Sepharose to the extent that final solutions, when dialyzed

against glycerol/water/benzamidine solutions as indicated un- der “Experimental Procedures,” resulted in a solution con- taining Factor V at about 2 mg/ml.

The isolated Factor V, stored as described under “Experi- mental Procedures” was stable for at least 4 months, losing neither total activity nor activatibility and showing no evi- dence of proteolysis when analyzed by DodS04 gel electro- phoresis.

Analysis of Purity by Gel Electrophoresis

The relative homogeneity of the isolated Factor V was assessed by polyacrylamide gel electrophoretic analysis. The results are shown in Fig. 5. Gel A represents analysis in the Tris/borate/CaClz system, Gel B the Davis system (36), and Gel C that of Williams and Reisfeld (37). The material in all three cases appears to give a single band. Gel D of Fig. 5

ABC DEF FIG. 5. Electrophoretic analysis of isolated Factor V. The support

medium of Gels A through D was 5% polyacrylamide. Gel A was buffered with 0.1 M Tris/borate, 5 mM CaC12, pH 8.3. Gels B and C were buffered as described by Davis (35), and Williams and Reisfeld (36), respectively. Gel D was 0.1% in DodSOl as described by Weber and Osborn (37). Electrophoretic analysis in DodSOl and 2.0% aga- rose, as described by Fass et al. (38) are depicted in Gels E and F; both before E and after F reduction with 2-mercaptoethanol.

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Bovine Factor V Isolation 513

represents analysis in DodSOd and 5% polyacrylamide. A single band is observed with no evidence of noncovalently associated subunits of differing molecular weight. Because of the limited mobility of Factor V in DodS04 and polyacryl- amide, gels containing the detergent were prepared in agarose. The results are shown in Gels E and F; before and after reduction with 2-mercaptoethanol, respectively. It is to be noted that no change in the apparent molecular weight was observed upon reduction, indicating an absence of multiple disulfide linked chains. In Gel F (reduced), some faster moving minor components are evident but these were unique to this particular sample and were not seen with other preparations. It is surmised that in the preparation analyzed on Gel F some minor intrachain proteolysis had occurred in a small fraction (5%) of the sample which became evident upon reduction.

The results shown in Fig. 5 indicate that the Factor V isolated by the procedure summarized in Table I is homoge- neous by the criteria of gel electrophoresis, and consists of a single chain, high molecular weight polypeptide exhibiting no evidence of noncovalent structure or disulfide linkages be- tween chains.

Sedimentation Equilibrium Analysis

Because of the lability of Factor V over long periods of time at room temperature, the best sedimentation equilibrium data were obtained on protein solutions in 6 M guanidinium chlo- ride. Under these conditions, no proteolytic degradation of the protein occurred, and Factor V solutions could be analyzed without fear of proteolytic cleavage. Fig. 6 presents data obtained with Factor V in 6 M guanidinium chloride at 14,000 and 16,000 rpm. Under these conditions, linear plots of log A Y versus r” were obtained indicating effective homogeneity in each experiment. In addition, the similar molecular weights

7.3

6.8

6.3 1

In (ny)

5.8 !

5.3 -

4.8 - 4.35

1000

500

dY

250

100

49.9 50.4 50.9

r2 (cm2)

FIG. 6. Sedimentation equilibrium in 6 M guanidine hydrochloride: plots of the logarithm of fringe displacement AY uersus square of the distance from the center of rotation (r’). Factor V was dissolved in 6 M guanidine hydrochloride (pH 6.0) at 0.2 mg/ml. Respective rotor speeds for the two depicted experiments were 14,000 (0) and 16,000 (0). The lines are drawn from least squares analyses which include all fringe displacements greater than 100 pm. Cell bottoms are indi- cated by (rb’).

determined at different rotor speeds (using U: 325,000 at 16,000 rpm and 331,000 at 14,000 rpm) attest to the homogeneity of the sample.

Sedimentation equilibrium analysis of reduced carboxami- domethylated Factor V in 6 M guanidinium chloride were not as simply analyzed as those for the protein with disulfide bonds intact. Upon electrophoretic analysis in DodS04 under reducing conditions some preparations of Factor V showed small amounts of highly heterogeneous low molecular weight material, apparently representing internal cleavages around disulfide bonds in a subpopulation of Factor V molecules. An example of this is shown in one of the electrophoretograms (F) of Fig. 5. In order to avoid unnecessarily complicating subsequent calculations, only those preparations which dis- played minimal degradation (by DodS04 analysis) were sub- ject to sedimentation equilibrium analysis under reducing conditions. Sedimentation equilibrium analysis of reduced, carboxamidomethylated Factor V in 6 M guanidinium chloride on the best preparations gave a molecular weight of 326,000.

Sedimentation equilibrium analyses were also conducted with Factor V under native conditions. These analyses were complicated to some degree, however, by the fact that some degradation of the sample occurred during the 24 to 30 h required for the attainment of equilibrium, as observed by electrophoretic analysis of samples taken after the conclusion of the experiments. Because of this degradation, sedimenta- tion equilibrium analysis of Factor V preparations in the native state gave evidence of some heterogeneity and data were interpreted in terms of weight, M,., and z, &f,, averages for the entire cell. In the native state, Factor V preparations had an a<,. of 311,000 and an &f, of 335,000. Because of the minor degradation which occurred during the analysis, it is felt that k, is probably more representative of the molecular weight of intact Factor V in the native state. The molecular weight data obtained by sedimentation equilibrium analysis on Factor V are presented in Table II.

Sedimentation Velocity Studies

In all sedimentation velocity studies in the native and reduced denatured state, only a single sharp sedimenting boundary was observed. The limiting sedimentation coeffi- cient, s:‘,),.., obtained for native Factor V is 9.19 S. This is a relatively low sedimentation coefficient considering the phys- ical size of the molecule, and indicates that the Factor V molecule is relatively asymmetric. The frictional coefficient

TABLE II

Molecular weight of bovine Factor V

Native: iI%<, = 310,700 &I, = 334,900

Denatured: M(+‘)” = 311,600 M(@ = 328,400

Denatured: (--s--s- bonds re- duced) M(+‘)” = 308,700 M(v)’ = 325,600

“Best” value M’ = 329,600

(1 Molecular weight computed presuming maximal interaction with solvent or +’ = 0.702.

” Molecular weight computed assuming no preferential interaction with solvent, or $I’ = 0.712.

’ Average value for M using Ii4, (native) and the denatured protein analyses computed based upon no preferential interaction (i.e. +’ = 0.712).

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

514 Bovine Factor V Isolation

calculated for Factor V is 1.722 x lo-’ g/s and the frictional ratio (f/f,,,,,) is 2.01. Extrapolation of the Oncley (51) nomo- gram relating axial ratios of molecules from hydrodynamic and mass data suggest that the Factor V molecule may be represented in solution by a prolate ellipsoid with an axial ratio of 25:l.

Sedimentation velocity studies were also conducted on re- duced, carboxamidomethylated Factor V in 6 M guanidinium chloride. These studies were initiated because of the indication from sedimentation equilibrium and DodS04 electrophoretic data, that the Factor V molecule is not composed of subunits but rather appears to be a single chain of molecular weight near 330,000. It was decided to assess the molecular weight of Factor V in terms of its hydrodynamic volume as a random coil. Conventionally, either intrinsic viscosity (52) measure- ments or gel filtration (53) are used to deduce the polymer chain length and hydrodynamic volume of reduced proteins in 6 M guanidinium chloride. In this instance, however, the available quantities of protein and its large apparent physical size precluded the use of viscosity measurements and gel filtration, respectively. Thus, the effective polymer chain length of Factor V was evaluated using sedimentation velocity as described by Tanford and co-workers (49). The limiting sedimentation coefficient s&c and computed polymer chain length obtained for Factor V with disulfide bonds reduced and carboxamidomethylated in 6 M guanidinium chloride are pre- sented in Table III. The polypeptide chain length of Factor V based upon direct analysis of mass and composition is 2,541 residues. The 11.9% carbohydrate present on the Factor V polypeptide chain could be expected to contribute to the hydrodynamic volume of the molecule, but in a magnitude difficult to predict because of the unknown location and distribution of the carbohydrate chains. The value of 2,600 to 3,000 residues in the polypeptide chain of Factor V determined from s&, (; is in good agreement with the value obtained from composition, especially in view of the fact that one would expect the experimental value to be slightly larger than that for polypeptide alone because of the contribution of the car- bohydrate side chain(s). The computed mass, based upon sedimentation analysis of the presumed random coil, including the contribution of the 11.9% carbohydrate, results in a mo- lecular weight by sedimentation velocity (presuming prefer- ential solvent interaction) of 336,000 for the Factor V mole- cule. These data obtained by sedimentation velocity studies of the reduced random coil thus support the concept that Factor V is composed of a single chain of molecular weight near 330,000.

Amino Acid Composition, Carbohydrate Content, and Extinction Coefficient

The amino acid composition and carbohydrate content of Factor V are tabulated in Table IV. Of note is the abundance of combined aspartic acid (+ asparagine) and glutamic acid (+ glutamine) (592 mol/mol of Factor V) compared to a total of 258 mol of lysine plus arginine and the presence of a fairly high proline content (8%). Glucosamine and neutral sugars are also present. The amino acid composition of Table IV differs markedly from those reported by Esnouf and Jobin (13) and Dombrose and Seegers (17).

Protein concentrations used in the calculation of the extinc- tion coefficient of Factor V were determined in the ultracen- trifuge by the method of Babul and Stellwagen (45). The absorbance of a 1% solution of Factor V at 280 nm, l-cm pathlength, was calculated to be 9.6.

Factor V-dependent Activation of Human Prethrombin 1

Previous studies have shown that partially purified Factor

TABLE III Estimation of random coil (R.C.) chainlength

s% ~:,,“~‘I = 2.36 S n,$.‘< = 2,630 n ” = 2,980 n, onl,l = 2,541 Mean amino residue = 113 g

weight” MI< c +,I’ = 297,000 Ml< (‘ i( = 337,000 MKC ’ = 290,000

fl Number of residues and polypeptide molecular weight (exclusive of carbohydrate) computed based upon presumption of maximal interaction with solvent, I$’ = 0.702.

“Number of residues and polypeptide chain molecular weight (exclusive of carbohydrate) computed presuming minimal interaction with solvent, I$’ = 0.712.

’ Number of residues and mass of the polypeptide chain (exclusive of carbohydrate) computed based upon the molecular weight and composition of the protein.

” Computed exclusive of carbohydrate side chain.

TABLE IV

Amino acid composition of bovine Factor V

Moles amino acid per mol of Factor V (molecular weight, 329,600).

Aspartic acid 299 Threonine 121 Serine 204 Glutamic acid 293 Proline 199 Cystine” 24 Glycine 146 Alanine 131 Valine 108 Methionine 43 Isoleucine 124 Leucine 240 Tyrosine 94 Phenylalanine 88 Lysine 148 Histidine 71 Arginine 110 Tryptophanh 21 Glucosamine 65 Neutral sugars’ 11.9%

” Determined as cysteic acid by the method of Moore (41). ’ Determined by the method of Liu and Chang (40). ’ Determined by the phenolsulfuric procedure described by Ashwell

(42).

V markedly accelerates the rate of thrombin production when added to an in vitro system containing prothrombin, Factor X,, phospholipid, and calcium ions. The Factor V effect is even more dramatic with prethrombin 1 (24), since neither calcium ions nor phospholipid alone increase the rate of its Factor X,-catalyzed conversion to thrombin. The ability of Factor V to accelerate thrombin production is consistent with its accepted cofactor role in the clotting mechanism. Thus, while the ability of the material isolated as described in this paper to correct the prolonged prothrombin time of Factor V- deficient plasma indicates that the material is, by definition, Factor V, its identity was further tested by its ability to accelerate the Factor X,-catalyzed activation of human pre- thrombin 1 in a well defined system of purified components.

The rates of thrombin production were determined at fixed prethrombin 1, calcium, and Factor X, concentrations, while the Factor V concentration in the system was varied. For each concentration of Factor V, the initial rate of thrombin pro- duction was measured (following an initial “lag”). The results are presented in Fig. 7. The values of the ordinates and abscissas of the plotted points are in NIH units of thrombin per ml per min and units of added Factor V activity per ml,

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Bovine Factor V Isolation 515

160 -

20

0 1 2 3 4 5 6 7 8 9 10

Factor V, U/ml FIG. 7. Initial rates of human prethrombin 1 activation with vary-

ing amounts of Factor V. The initial velocity of thrombin formation following an initial lag are indicated by the vertical axis in NIH units of thrombin/ml/min. The units of Factor V activity on the horizontal axis are those realizable after activation with thrombin. Measured amounts of Factor V, (prior to activation with thrombin) initially added were about one-eightieth the values indicated on the horizontal axis. The activation was carried out as described under “Experimental Procudures.” The observed rate of thrombin formation in the absence of added Factor V was 0.69 NIH unit/ml/min.

respectively. The maximum initial velocity achieved at 37°C with prethrombin 1 at 71 pg/ml, Factor X, at 1 unit/ml, and Factor V activity in excess of 5 units/ml (after activation by thrombin) was 145 NIH units of thrombin/ml/min. In the absence of Factor V the rate was 0.69 unit/ml/min. Thus, after the lag, added unactivated Factor V caused a 210-fold increase in the reaction rate. From these results the isolated material is identified as Factor V not only by assays in Factor V-deficient plasma, but also by its behavior in a system of components characterized on a molecular level.

DISCUSSION

The isolation procedure described in this paper yields bo- vine Factor V which appears to be homogeneous when ana- lyzed by a variety of techniques including gel electrophoresis, sedimentation equilibrium, and sedimentation velocity. The procedure provides overall yields of about 20%, or 4 to 5 mg of Factor V per liter of starting plasma, and has proven to be very reproducible, in that material of the same purity, specific activity, and activatibility by thrombin is obtained from prep- aration to preparation.

One of the goals of the work described in this paper was the isolation of unactivated and thus presumably uncleaved Fac- tor V. Successful isolation of the nonactivated cofactor re- quired means for both measuring the extent of activation (if any) and preventing its occurrence during both blood collec- tion and subsequent purification procedures. The extent of activation was measured by assaying all samples both before and after thrombin treatment, thereby providing measures of both Factor V, and Factor V content. Prevention of activation was accomplished by incorporation of a spectrum of protease inhibitors at each step of isolation from blood collection by venipuncture through QAE-cellulose chromatography.

Although the specific isolations of Factor V detailed in this paper were performed from beginning to end without stopping, it was subsequently observed that material isolated after QAE-cellulose or octyl Sepharose could be stored at 4°C as ammonium sulfate-precipitated pellets without loss of activity for at least brief periods (days) prior to continuation of puri- fication. In contrast to the results of Saraswathi et al. (22) we

have observed that whether blood was drawn by venipuncture or collected by postmortem exsanguination at the slaughter- house made no detectable difference in the quality of the final product, The total amount of Factor V recoverable from a

given volume of slaughterhouse blood was somewhat dimin- ished, however, compared to that obtainable by venipuncture

In addition to the use of inhibitors and rapid isolation steps to minimize activation, the procedure developed for the iso- lation of Factor V relies heavily upon the use of the two relatively new techniques, hydrophobic chromatography and preparative electrophoresis on polyacrylamide gels. The ra- tionale for the use of hydrophobic chromatography followed from the putative lipid-binding properties of Factor V (54). One of the primary values of hydrophobic chromatography on octyl Sepharose is that it renders the material sufficiently pure to make preparative electrophoresis, with its great flexi- bility and high resolution, but limited capacity, useful in the final stages of purification.

Comparison of the relative purity of the Factor V described in this paper to that isolated previously is complicated by variability in both assay conditions and the defined unit of Factor V activity. Without exception, previous authors (13-22) have reported assay results in terms of Factor V, activity only, whereas the results in the current paper are expressed in terms of total Factor V, or that activity observed after acti- vation by thrombin. In addition, the most commonly used unit of Factor V activity is the amount contained in 1 ml of human plasma. In the present work, the chosen standard unit was that amount found in 1 ml of bovine plasma, which by assay contains about 5 times the activity measurable in human plasma. Nonetheless, the Factor V specific activity (following thrombin activation) of material isolated previously by others can be estimated by multiplying activation quotients (when reported) by reported Factor V, specific activities. These values, when normalized to a bovine plasma standard, can then be used semiquantitatively as a basis for comparison of the relative purity of various preparations of Factor V. When the indicated calculations are performed, specific activities ranging from 30 units/mg (14) to 339 units/mg (18) are found. The specific activity of the preparation reported here is about 1250 units/mg. These values indicate that the previously published preparations (13-22) most likely contained consid- erable amounts of inactive, or non-Factor V protein.

As with apparent specific activities, the lability of Factor V coupled with its ability to be activated and differences in assay conditions create problems in using the “purification factor” from plasma as a criterion for assessing the relative purity of Factor V isolated in different laboratories. Purification factors from plasma varying from 2,000 (22) to 16,000 (17) have been published, the latter of which greatly exceeds the value of 2,500 found in the present work. However, previously reported values were based on Factor V, measurements only without accounting for the possible activation of Factor V during isolation. It can be noted that in the present work, if only Factor V. had been assayed, and if all results had been the same except that a final product having an activation quotient of 1, rather than 80, had been obtained, the apparent purifi- cation factor from plasma would have been 127,500, rather than 2,500 as reported. The latter hypothetical value was obtained by multiplying the observed value of 2,500 by the activation quotient of the starting plasma.

A summary of the physical properties of Factor V is pre- sented in Table V. All studies conducted using sedimentation equilibrium analysis are consistent with the Factor V molecule being composed of a single carbohydrate-bearing polypeptide chain of molecular weight near 330,000. Sedimentation equi- librium analysis of the molecular weight of the protein in the

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

516 Bovine Factor V Isolation

TABLE V

Factor V gross properties

M = 330,000 sli,,. ((. = 9.19 s

U = 0.712 ml/gm f/fm = ‘2.01

a/b - 0.04 (25) E&, , urn = 9.6

native state versus the denatured state in 6 M guanidinium chloride with disulfide bonds reduced, is ordinarily taken as a measure of the subunit structure. In the present work, sedi- mentation under native conditions and under denaturing con- ditions in 6 M guanidinium chloride with disulfide bonds either intact or reduced, yields the same molecular weight, indicating the apparent absence of both complex, noncovalent subunit structure and interchain disulfide bond-linked subunits. In view of the very large single chain molecular weight assessed for Factor V using measurements of mass by sedimentation equilibrium, and reports of complex subunit structure in the literature (17, 19, 22), it was deemed useful to assess the molecular weight of Factor V by estimation of the random coil chain length. Using this approach, the sedimentation coeffi- cient obtained for Factor V was appropriate for a molecule containing 2,600 to 2,900 residues and a molecular weight of 337,000 to 382,000 including carbohydrate, thus precluding the possibility that the Factor V molecule is composed of multiple polypeptide chains that are not denatured in 6 M guanidinium chloride, or possesses residual cross-links not susceptible to 2- mercaptoethanol redu-tion. Not precluded by this analysis is the possibility that the Factor V molecule is composed of multiple subunits joined by mercaptoethanol-resistant cross- links which are at, or near, the termini of each potential chain. The likelihood of this eventuality, however, is small. It can therefore be concluded with reasonable certainty that the Factor V molecule has a native molecular weight of 330,000 and is composed of a single chain of this dimension. Prelimi- nary NHz-terminal sequence analysis conducted with Factor V is also consistent with a single chain nature of this molecule. Using the automated degradation procedure of Edman and Begg (56) 0.85 mol of alanine/mol of Factor V and NHz- terminal sequence Ala-Lys-Leu-Met are observed.

Although considerable experimentation was directed to- ward a search for Factor V subunit structure, no evidence of subunits was found. This is inconsistent with the various complex subunit structures reported by Dombrose and See- gers (17) and Kandall et al. (19). The previous studies, how- ever, may have been complicated by {xtraneous protein in Factor V preparations.

The sedimentation coefficient observed for Factor V is unusually low (9.19 S), suggesting that the molecule is not globular. Rather, the sedimentation coefficient of Factor V is reminiscent of that obtained for fibrinogen, a highly extended rod-like molecule (50). The sedimentation coefficient of Factor V, when compared with the molecular weight, permits the calculation of an f/fmln value of 2.01, similar to the value of 2.34 observed with fibrinogen (50). The frictional ratio com- puted for Factor V suggests that the shape of the molecule may be represented by an equivalent prolate ellipsoid with an axial ratio of 25:l. Evidence that isolated Factor V is hydro- dynamically similar to that in plasma comes from the obser- vation that Factor V activity in porcine plasma sediments at 9.1 S in sucrose density gradients (57). Some of the molecular weight values reported for Factor V were determined by gel filtration (14, 18, 20-22) and sedimentation velocity measure- ments (13). In view of the results of the hydrodynamic studies reported in this paper, the use of gel filtration under native

conditions to estimate the molecular weight of Factor V would, because of the grossly nonspherical shape of the pro- tein, lead to erroneous molecular weight estimates, especially when using globular proteins as standards. Sedimentation velocity experiments used alone for determining the molecular weight of Factor V would also be of limited value because of the rod-like shape of the Factor V molecule.

Both fibrinogen and Factor V are substrates for thrombin, as is the elusive Factor VIII molecule (58) which, up to the time of writing this paper, has resisted isolation. Factor VII1 appears to have a cofactor function with Factor IX, similar to that of Factor V with Factor X,. It is intriguing to note the sedimentation coefficient observed by Brockway and Fass (59) for Factor VIII procoagulant activity dissociated from von Willebrand’s factor is 9.2 S, very similar to the value obtained for isolated Factor V. There is, however, no Factor VIII procoagulant activity associated with the final product re- ported in this paper.

The isolation and physical characterization of homogeneous Factor V will significantly expedite determination of the cor- relation between its structure and function, and advance our understanding, on a molecular level, of the role of this vital and intriguing cofactor in the process of blood coagulation. In addition, studies of the cofactor nature of Factor V may well advance our understanding of the participation of protein cofactors in biological processes in general.

AchnoLuZedgrnents-We gratefully acknowledge the valuable ad- vice of Dr. David Fass of the Mayo Clinic, and the excellent technica assistance of Gary Clark, James Taswell, and Lisa Thorvig in purifying and analyzing Factor V. In addition, we express gratitude for the patience and expertise of Ruth Kendall in typing the manuscript.

1.

2. 3. 4. 5. 6.

7.

8.

9.

IO.

11. 12.

13. 14.

15. 16.

17.

18. 19.

20.

21.

22.

23.

24.

REFERENCES

Nesheim, M. E., Myrmel, K. H., Hibbard, L., and Mann, K. G. (1978) Fed. Proc. 37(6), 1750

Esnouf, M. P., and Williams, W. J. (1962) Biochem. J. 84, 62 Jackson, C. M., and Hanahan, D. J. (1968) Biochemistry 7,4506 Jesty, J., and Esnouf, M. P. (1973) Biochem. J. 131, 791-799 Bajaj, S. P., and Mann, K. G. (1973) J. Biol. Chem. 248,7729-7741 Cox, A. C., and Hanahan, D. J. (1970) Biochim. Biophys. Acta

207,49-64 Ingwall, J. S., and Scheraga, H. A. (1969) Biochemistry 8,

1860-1869 Fujikawa, K., Legaz, M. E., and Davie, E. W. (1974) Biochemistry

13,4508 Fujikawa, K., Titani, K., and Davie, E. W. (1975) Proc. N&Z.

Acad. Sci. U. S. A. 72, 3359 Jesty, J., Spencer, A. K., Nakashima, Y., Nemerson, Y., and

Konigsberg, W. (1975) J. Biol. Chem. 250,4497-4504 Mann, K. G. (1976) Methods Enzymol. 45B, 123-156 Magnusson, S., Petersen, T. E., Sottrup-Jensen, L., and Claeys,

H. (1975) Cold Spring Harbor Con& Cell Proliferation 2, 123-149

Esnouf, M. P., and Jobin, F. (1967) Biochem. J. 102, 660-665 Barton, P. G., and Hanahan, D. J. (1967) Biochim. Biophys. Acta

133, 506-518 Colman, R. W. (1969) Biochemistry 8, 1438-1445 Philip, G., Moran, J., and Colman, R. W. (1970) Biochemistry 9,

2212-2218 Dombrose, F. A., and Seegers, W. H. (1974) Thromb. Diath.

Huemorrh. 57 (suppl), 241-254 Day, W. C. (1975) Biochim. Biophys. Acta 386,352-361 Kandall, C. L., Rosenberg, R., and Colman, R. W. (1975) Eur. J.

Biochem. 58,203-211 Chulkova, T., and Hernandez, G. (1975) Clin. Chem. Acta 62,

21-28 Smith, C. M., and Hanahan, D. J. (1976) Biochemistry 15,

1830-1838 Saraswathi, S., Rawala, R., and Colman, R. W. (1978) J. Biol.

Chem. 253, 1024-1029 Colman, R. W. (1976) in Progress in Hemostasis and Thrombosis

(Spaet, T. H., ed) Vol. 3, Grune and Stratton, NY Bajaj, S. P., Butkowski, R. J., and Mann, K. G. (1975) J. Biol.

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Bovine Factor V Isolation 517

Chem. 250,2150-2156 25. Jobin, F., and Esnouf, M. P. (1967) Biochem. J. 102,666-674 26. Colman, R. W. (1970) Br. J. Hematol. 19, 675 27. Ware, A. G., Murphy, R. C., and Seegers, W. H. (1947) Science

106,618 28. Sophianopoulous, A. J., and Vestling, C. S. (1962) Biochem. Prep.

9, 102-107 29. Nozaki, Y. (1972) Methods Enzymol. 26, C, 43-50 30. Bowie, E. J. W., Thompson, J. H., Jr., Didisheim, P., and Owen,

C. A., Jr. (1971) Mayo Clinic Laboratory Manual of Hemosta- sis, W. B. Saunders, Philadelphia

31. Lewis, M. L., and Ware, A. G. (1953) Proc. Sot. Exp. Biol. Med. 84,640

32. Downing, M. R., Butkowski, R. J., Clark, M. M., and Mann, K. G. (1975)5. Biol. Chem. 250,8897-8906

33. Minimum Reouirements for Dried Thrombin (1946) 2nd Revi- sion, National Institutes of Health, Bethesda, MD

34. Mann, K. G., Heldebrant, C. M., and Fass, D. N. (1971) J. Biol. Chem. 246, 5994-6001

35. Nesheim, M. E. (1978) Ph. D. thesis, Department of Biochemistry, University of Minnesota, Minneapolis, MN

36. Davis. B. J. (1964) Ann. N. Y. Acad. Sci. 121. 404-427 37. Williams, D. E., and Reisfeld, R. A. (1964) Ann. N. Y. Acad. Sci.

121,373-381 38. Weber, K, and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 39. Fass, D. N., Knutson, G. J., and Bowie, E. J. W. (1978) J. Lab.

Clin.Med. 91, 307-320 40. Liu, T.-Y., and Chang, Y. H. (1971) J. Biol. Chem. 246,2842-2848 41. Moore, S. (1963) J. Biol. Chem. 238, 235-237 42. Ashwell, G. (1966) Methods Enzymol. 8,93-95

43. Babul, J., and Stellwagen, E. (1969) Anal. Biochem. 28,216-221 44. Yphantis, D. A. (1964) Biochemistry 3,297-317 45. Cohn, E. J., and Edsall, J. T. (1943) Proteins, Amino Acids, and

Peptides as Ions and Dipolar Ions, pp. 370-381, Reinhold Publishing Co., New York, NY

46. Muir, H., and Hardingham, T. E. (1975) MTP International Review of Sciences, Biochemistry Series One, Vol. 5, pp. 153-222, University Park Press, Baltimore, MD

47. Gibbons, R. A. (1966) Glycoproteins, Vol. 5, BBA Library, pp. 29-95. Elsevier Publishing Co.. New York. NY

48. Hade, K., and Tanford, C. (i967) J. Am. Chem. Sot. 89,5034 49. Tanford, C., Kawahara, K., and Lapanje, S. (1967) J. Am. Chem.

sot. 89, 729 50. Tanford, C. (1961) Physical Chemistry of Macromolecules, pp.

356-361, John Wiley and Sons, New York, NY 51. Oncley, J. L. (1951) Ann. N. Y. Acad. Sci. 41, 121 52. Mann, K. G., Fish, W. W., Cox, A. C., and Tanford, C. (1970)

Biochemistry 9, 1348-1354 53. Mann, K. G.. and Fish. W. W. (1972) Methods Enzvmol. 26.28 54. Colman, R. W., and Weinberg,’ R. ik (1976) Methbds Enz~mol.

45B, 107-122 55. Maurer, H. R. (1971) Disc Electrophoresis, p. 59, Walter de

Gruyter, Berlin 56. Edman, P., and Begg, G. (1967) Eur. J. Biochem. 1,80-91 57. Myrmel, K. H. (1978) Ph. D. thesis, Department of Biochemistry,

University of Minnesota, St. Paul, MN 58. Legaz, M. E., and Davie, E. W. (1976) Methods Enzymol. 45B,

83-89 59. Brockway, W. J., and Fass, D. N. (1977) J. Lab. Clin. Med. 89,

1295

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from

M E Nesheim, K H Myrmel, L Hibbard and K G MannIsolation and characterization of single chain bovine factor V.

1979, 254:508-517.J. Biol. Chem. 

  http://www.jbc.org/content/254/2/508Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/254/2/508.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on April 10, 2020

http://ww

w.jbc.org/

Dow

nloaded from