Nature of the high molecular weight fraction of fibrinolytic digests of human fibrinogen

96

BIOCHIMICAET BIOPHYSICAA(;TA

BBA 35172 N A T U R E OF T H E H I G H MOLECULAR W E I G H T FRACTION OF F I B R I N O L Y T I C DIGESTS OF HUMAN F I B R I N O G E N

G. A. J A M I E S O N AND 1). J. G A F F N E Y , JR.*

Blood Program Research Laboratory, The American National Red Cross, Washington, D.C., and Department of Biochemistry, Schools of ~VIedicine and Dentistr% Georgetown University, Washington, D.C. (U.S..4.) (Received J u l y 25th, ~967)

SUMMARY

I. The high molecular weight fraction (Fraction D) of complete plasmin digests of human fibrinogen gave a regular pattern of six clearly marked bands on starch or acrylamide gel electrophoresis. Comparable, but different, patterns were found at earlier stages of the digestion. No new antigenic determinants were revealed by this separation. 2. An equal or greater number of bands was found by starch gel electrophoresis in 6 M urea both before and after reduction and alkylation. 3- Gel filtration experiments suggested that the weight average molecular size of Fraction D (about 83 ooo) was not reduced in the presence of 6 M urea either before or after reduction and alkylation and was identical with that of each of the individual components. 4- These results indicate that Fraction D, which contains one of the antigenic determinants of fibrinogen as well as the 'fibrin polymerization inhibitor' of fibrinolytic digests, is not a single species but a mixture of molecules of closely related structure.

INTRODUCTION

Extensive studies in several laboratories have shown that fibrinolytic digests of fibrinogen and fibrin possess anticoagulant activity 1-4 due to the presence of a component which interferes in the conversion of fibrin monomer to fibrin by incorporation into the fibrin clot 5. Separately, NUSSENZWEIGet al. showed that complete digestion of native fibrinogen resulted in the separation of two antigenic determinants 8 and that these fibrinolytic digests could be resolved into five components by chromatography on DEAE-cellulose 7. The major component from this fractionation (Fraction D) was apparently identical with the fibrin polymerization inhibitor of ALKJAERSlG, FLETCHER AND SHERRY5. It comprised approx. 50% of the digestion products and * P r e s e n t a d d r e s s : Medical R e s e a r c h Council, A b n o r m a l H a e m o g l o b i n R e s e a r c h U n i t , D e p a r t m e n t of B i o c h e m i s t r y , Cambridge, G r e a t Britain.

Bioehim. Biophys. dcta, 154 (1968) 9 6 - i o 9

FIBRINOLYTIC DIGEST OF FIBRINOGEN

97

contained one of the antigenic determinants of fibrinogen. It behaved as a single component on agar gel electrophoresis and immunoelectrophoresis and in the ultracentrifuge. Since biophysical studies indicated a mol. wt. of 83 ooo (s,0,w 5.2 S) it appeared that only two such fragments could be obtained from intact fibrinogen (mol.wt. 320 000) (ref. 7). The present work demonstrates that the high molecular weight fragment obtained after complete plasmin digestion of either human fibrinogen or fibrin is, in fact, a heterogeneous mixture of at least six components separable by electrophoresis on starch and acrylamide gels. This paper is concerned with electrophoretic and other changes during the course of plasmin digestion and with the characterization of the multiple components of this high molecular weight fraction which has been variously termed 'Fraction D '7, 'fibrin polymerization inhibitor 's or 'anticoagulant fraction of incubated fibrinogen '3.. Preliminary accounts of some of these results have already appeared 8& MATERIALS AND METHODS

H u m a n fibrinogen was prepared from eight unit pools of fresh plasma by a modification (J. H. PERT AND A. J. JOHNSON, unpublished results) of the ethanolglycine procedure 1° in the presence of e-aminocaproic acid 11. Traces of e-aminocaproic acid remaining in the preparation were removed b y dialysis against two changes of a solution containing o.85 ~o sodium chloride o.4% sodium citrate (pH 7.4) for 48 h at 3-4 ° prior to use. Fibrinogen prepared in this way was over 96% clottable and contained negligible amounts of plasmin and plasminogen as determined by clot stability b o t h in the absence and presence of streptokinase. H u m a n plasmin was produced by the Division of Laboratories of the Michigan Department of Health for the American Red Cross 12. It was activated in 6% glycerol and contained 71 caseinolytic units per ml or 12 units per mg of protein. Vertical gel electrophoresis was performed in borate-starch geP 3 or in acrylamide slab la or disc ~5with T r i s - E D T A - b o r a t e ~6or Tris-glycine 15buffer systems. Disc electrophoresis was routinely carried out at 5 mA/tube for 3 tl. Elimination of the spacer gel did not alter the pattern of banding. Formate or formate-urea buffers were used in the second dimension for two-dimensional gel electrophoresislL Plasmin digests were routinely prepared using a pH-stat (Radiometer, Ltd., Copenhagen) at pH 7.5. Fibrinogen samples were adjusted to approx. 1% protein concentration in o.85 % saline solution-o.4 ~o sodium citrate buffer (pH 7.4) and inoculated with plasmin at a level of o. 14 caseinolytic units per mg offibrinogen. The solutions were maintained at constant pH by the addition of o.o5 M N a O H and no special precautions were taken to exclude atmospheric carbon dioxide. In certain experiments " A rigorous n o m e n c l a t u r e for the p r o d u c t s at the various stages of the fibrinolytic digestion process p r e s e n t s difficulties in the absence of clear criteria of h o m o g e n e i t y and, in appropriate cases, correlation with physiological activity. I n the p r e s e n t work the completed digest is defined as t h a t which exhibits no f u r t h e r changes in migration p a t t e r n on b o r a t e - s t a r c h gel electrophoresis following the f u r t h e r addition of h u m a n plasmin; this is a s u b s e q u e n t stage to t h a t characterized b y the constancy of the agar gel i m m u n o e l e c t r o p h o r e t i c patterns. The components of this digest are designated Fraction A, B, etc. according to the description of NUSSENZWEIG et al.L The individual c o m p o n e n t s of F r a c t i o n D identified in the present w o r k are designated b y a p p r o p r i a t e subscripts, D1, D 2, D3, etc. with D 1 being the b a n d with the highest anionic mobility w h e n examined in the b o r a t e - s t a r c h gel electrophoretic system. B i o c h i m . B i o p h y s . Acta, 154 (1968) 96 1o9

98

G.A. JAMIESON, P. J. GAFFNEY, JR.

the reaction was s t o p p e d at various points during the course of digestion b y the a d d i t i o n of s o y b e a n t r y p s i n i n h i b i t o r ( W o r t h i n g t o n Biochemical Corp., Freehold, N. J.) at a level of o.2 m g / m i to aliquots of the mixture. Small a m o u n t s of toluene or alcohol were a d d e d to i n h i b i t b a c t e r i a l g r o w t h when the digestion was allowed to proceed overnight. F r a c t i o n D was o b t a i n e d b y c h r o m a t o g r a p h y on D E A E - c e l l u l o s e (Brown a n d Co., Berlin, New H a m p s h i r e ) using the m e t h o d of NUSSENZWEIG et ald b u t modified in certain cases b y the use of a linear g r a d i e n t from o . o i M b i c a r b o n a t e buffer (pH 9.I, 5oo ml) to the same buffer m a d e o.I M with respect to sodium chloride (5oo ml). Virtually i d e n t i c a l elution profiles were o b t a i n e d with b o t h linear and concave gradients. Gel filtration was carried out on S e p h a d e x G - I o o ( P h a r m a c i a C o m p a n y , U p p sala, Sweden ; column size, 80 cm ~< 2.5 cm) which h a d been swollen previously in I M sodium chloride and then e q u i l i b r a t e d with w a t e r a d j u s t e d to p H 8 with a m m o n i u m h y d r o x i d e , or with b i c a r b o n a t e buffer (pH 9.I). p H values below 6 resulted in precipit a t i o n of various c o m p o n e n t s of the digests. T h r o m b i n clotting times were d e t e r m i n e d b y the m e t h o d of LATALLO et al. is using 2oo/~g of fibrinogen digestion p r o d u c t s t o g e t h e r with 6 o o / , g of fibrinogen in a t o t a l v o l u m e of o.2 ml. U n d e r these conditions the clotting t i m e after zero rain of digestion was 8 sec. Bovine t h r o m b i n was o b t a i n e d from Parke, Davis a n d Co. as a freeze-dried p o w d e r containing IOOO units/vial. I t was dissolved in I ml of 2 5 % glycerol saline a n d stored in the frozen state. This solution was d i l u t e d I :IO prior to use. P r o t e i n c o n c e n t r a t i o n s were d e t e r m i n e d using the b i u r e t ~° or Folin phenol 21 methods. RESULTS

Digestion process. U n d e r the conditions of digestion outlined the solution b e c a m e u n c l o t t a b l e b y t h r o m b i n after 8 rain a n d the u p t a k e of base in the p H - s t a t h a d v i r t u a l l y s t o p p e d after I h. However, changes in the a b s o r b a n c e (280 m#) of the s u p e r n a t a n t

Fig. i. Sedimentation patterns of complete fibrinogen digest. Photographs were taken after 9o, lO6, and 122 min at 59 57° rev./min. Phase-plate angle 45 °. Protein concentration 0.9% in o.i M phosphate (pH 8.0). Sedimentation is from left to right. solution, following a d d i t i o n of trichloracetic acid to 5 % concentration, i n d i c a t e d t h a t digestion was still progressing up to 4 h. W h e n aliquots o b t a i n e d at various stages of t h e digestion process were a d d e d to i n t a c t fibrinogen the clotting t i m e of the m i x t u r e increased from 8 sec at zero t i m e to a m a x i m u m of 95 sec after 15 min digestion ('peak point'), t h e n decreased to a c o n s t a n t Biochim. Biophys. Acta, 154 (I968) 96-I,~ 9

FIBRINOLYTIC DIGEST OF FIBRINOGEN

99

value of 24 sec after 3o rain digestion ('elbow point'). These striking changes in the clotting time during digestion are similar to those previously observed in other laboratories21, 22. No significant differences were observed in any of these parameters by making sequential additions of plasmin or by adding massive excesses (5o-fold increase) at the completion of base uptake or after incubation overnight. In most

Fig. 2. (a) B o r a t e - s t a r c h gel electrophoresis of fibrinogen digests. (1) P e a k digest (I 5 min), (II) complete digest (5 h), (III) 'elbow' (3 ° min) digest• Conditions: 25 mA, time 15 h. The anode is a t t h e bottom• (b) Disc acrylamide gel electrophoresis. (I) Fibrinogen, ( I I ) ' p e a k ' (12 min), ( [ l I ) ' e l b o w ' (3 ° min) a n d (IV) 'completed' (15 h). Conditions: Tris-glycine buffer la, 5 mA/tube, time 3 h.

preparative experiments the digestion mixture was removed from the pH-stat at the the completion of base uptake and set aside at room temperature overnight ('completion'). When examined in the ultracentrifuge the sedimentation pattern indicated multiple components with the major one sedimenting at a rate of 5.2 S (Fig. I). Two intersecting bands were obtained by agar gel immunoelectrophoresis against antihuman fibrinogen, identical with previous observations 6. Gel electrophoresis. Striking differences in the effect of fibrinogen digestion products on the clotting time of fibrinogen were obtained at 'peak', 'elbow' and 'completion' points. A complex pattern of bands was observed in each case on starch and acrylamide-disc gel electrophoresis indicating that multiple fragments of fibrinogen were obtained within the first 15 min of digestion and that further digestion resulted in increasing concentrations of the bands of higher mobility rather than changes in the number of individual components (Figs. 2a, b). Two electrophoretically distinct components were apparent in the 15 min ('peak') digest which were even more clearly Biochim. Biophys. ,4cl~, I54 (I968) 96 to9

IOO

G . A . JAMIESON, P. J. (;AFFNEY, JR.

marked in the 30 min ('elbow') sample and six bands of almost equal intensity were obtained in the complete digest. Following gel filtration on Sephadex G-IOO the major portion of the digest was eluted at close to the colunm volume for all three points although increasing amounts of lower molecular weight material appeared during the course of digestion (Fig. 3). These results are consistent with a tool. wt. of at least 8o-Ioo ooo for the major portion of the digest and indicate that the heterogeneity of the later digests is not due to an aggregation of smaller fragments which might arise in the early stages of proteolysis. The elution pattern from DEAE-cellulose for the digest obtained at 'completion' was virtually identical with that obtained by NUSSENZWEIG et alfl under slightly

8.C

6.C 4.C

2.0

o

c

(M 8.C

6.G

4.G

2.G

6 . oc

4.0 2.0

0

2:5 FRACTION

50

7'5

NUMBER

Fig. 3. Sephadex G-Ioo elution p a t t e r n s of digests: (upper) 15 min digest, (middle) 3 ° rain digest, (lower) complete (15 h) digest. Eluant, dilute NHaOH (pH 8.0).

different conditions, being characterized b y a single large peak (Fraction D) with two minor peaks (Fractions A and E) which were eluted at lower and higher ionic strengths. In the completed digest the Fraction B/C corresponded to a relatively small amount of the total protein. However, the elution patterns at the 'peak' and 'elbow' points were markedly different although a large component corresponding to Fraction D was eluted in each case (Fig. 4). Fractions B and C appeared to comprise a significant portion of the digest at the 'elbow point' while Fraction A appeared only in the later Biochim. Biophys. Acta, 1.54 (1968) 96-1o9

FIBRINOLYTIC DIGEST OF FIBRINOGEN

I0I

1.0 05 ~ o o co N IG

0

IO0 FRACTION

200

300

NUMBER

Fig. 4- DEAE-cellulose c h r o m a t o g r a p h y of fibrinogen digests. The elution scheme was a modification of t h a t of NOSSENZWEIG et alY and utilized a linear gradient f r o m o.oi to o.i M NaC1 in o.oi M c a r b o n a t e buffer at p H 9.I. Peak E was eluted with 0.2 M NaC1. Fractions (A, B/C, D and E) are designated according to the scheme of NUSSENZWEIG. Upper: Peak (I 5 min) digest; middle: elbow (3 ° min) digest; lower: complete (15 h) digest.

stages of digestion. It is of interest that hexose and sialic acid appeared only in Fractions A and E of the complete digest and that the mobilities of the individual bands of Fraction D were unaffected b y neuraminidase. In other experiments the time course of plasmin digestion was followed by acrylamide disc electrophoresis for a larger series of points (Fig. 5)- Under these conditions fibrinogen migrated only a short distance into the gel. However, after 2 min digestion the fibrinogen band had completely disappeared and was replaced by a component which migrated relatively rapidly although the digestion mixture still remained clottable with thrombin. Further digestion showed little change in the mobility of this band but it slowly disappeared with the production of several bands of higher mobility and gradually increasing intensity. Completed digest. Since it was realized that incomplete and continuing digestion could lead to a multiplicity of products on gel electrophoresis, attention was focused on the final product resulting from long term digestion. Stable and reproducible electrophoretic patterns were obtained using the digestion procedure outlined in M A T E R I A L S AND METHODS and these patterns were not altered by the addition of a 5e-fold excess of plasmin at the completion of the normal digestion process. Starch gel electrophoresis of these ,completed' digests showed a fast-moving diffuse spot, which Biochim. Biophys. Acta, 154 (1968) 96-1o9

I()2

G . A . JAMIESON, P. J. GAFFNES", JR.

t, r 0 Z

16 ~o

6o tg@ t.f'@

Fig. 5. Acrylamide disc electrophoresis of fibrinogen digest. The digestion was s t o p p e d w i t h so 3, bean t r y p s i n inhibitor (SBTI) at various times of digestion as indicated in minutes. Since the p h o t o g r a p h is a composite of two separate runs the gel origins are n o t exactly colinear. I n t e r n a l controls were r u n in each case. Conditions of electrophoretic r u n : Tris glycine buffer, 5 m A / t u b e , time 3 h.

stained weakly with amido black and which could be washed out of the gel during destaining procedures, followed by six regularly spaced bands of approximately equal intensity. However, progressive dilution of the digest prior to electrophoresis showed that bands D 3 and D 4 were considerably more intense than the other four. When these digests were examined by acrylamide disc or slab electrophoresis it was necessary to select conditions such that the high molecular weight component migrated into the gel sufficiently to display its heterogeneity (Fig. 6). Although the number of components separated by these three electrophoretic methods was identical further attempts were made to obtain improved resolution by the starch and disc acrylamide methods. An excellent resolution was obtained on starch using the discontinuous buffer system 24 but the bands themselves were not as widely separated as in the normal borate system. Similar, but not improved, separations were obtained in borate and phosphate buffers Bh)chim. Biophys. dcla, 154 (i968) 96 lO 9

F I B R I N O L Y T I C D I G E S T OF F I B R I N O G E N

lO3

Fig. 6. A c r y l a m i d e disc electrophoresis. T h e h i g h molecular w e i g h t fraction (Fraction D) r u n u n d e r s t a n d a r d conditions 1~ for t, 2 a n d 3 h, respectively.

at pH 8.0 in both electrophoretic systems. Identical patterns were obtained using fibrin as substrate both before and after washing the clot to remove fibrinopeptides. Electrophoretic exarnit, ation of purified fraction, s. Since multiple electrophoretic bands, presumably of high molecular weight, were obtained from fractions eluted at the column volume by gel filtration of the completed digest on Sephadex G-ioo, further experiments were carried out using purified fractions. Extensive studies have been carried out by TRIANTAPHYLLOPOULOS AND TRIANTAPHYLLOPOULOS~4 on the properties of the 'anticoagulant fraction of incubated fibrinogen' isolated from fibrinogen digests by precipitation at 25-5o% ammonium sulfate concentration. When re-examined by the various forms of gel electrophoresis this fraction was found to give the pattern of a fast diffuse band and six slower, regularly spaced bands characteristic of the whole digest. When Fraction D was prepared b y the method of NUSSENZWEIGet al. 7 it migrated as a single peak in the ultracentrifuge and gave a single sharp precipitin line on agar gel imnmnoelectrophoresis. However, on starch or acrylamide gel eleetrophoresis a pattern was obtained identical with that of the whole digest or the anticoagulant fraction of incubated fibrinogen except that the fast-moving diffuse band was absent (Fig. 7). Comparison with other components of the fractionation showed that this fast-moving band was identical with Fraction E, while Fractions A and B/C did not appear on gels run under these conditions, either because they migrated into the buffer compartments or did not stain effectively. The separation of the six clearly marked electrophoretic bands of Fraction D did not reveal any new antigenic determinants since a series of continuous precipitin Biochim. Biophys. Acta, 154 (1968) 96 lO9

Zo 4

(;. A. JAMIESON, P. J. GAFFNEY, JR.

a

b

c

d

Fig. 7. E l e c t r o p h o r e t i c p a t t e r n s of F r a c t i o n l) u n d e r v a r i o u s c o n d i t i o n s : (a) disc a c r y l a m i d e , T r i s - - E D T A b o r a t e buffer, 5 m A / t u b e , t i m e 3 h; (b) disc acrylamide, Tris glycine buffer, 5 m A / tube, t i m e 3 h; ( c ) s l a b (3 nlm) acrylamide, Tris E D T A borate, 12o m A , t i m e 4 b, 2 - 5 ° ; (d) s t a r c h - b o r a t e , 2 5 m A , t i m e 15 11. All were r u n at room t e m p e r a t u r e unless otherwise indicated.

arcs was obtained on starch gel immunoelectrophoresis against anti-human fibrinogen (Fig. 8a). Similar reactions of identity were obtained when the individual components were examined b y the Ouchterlony technique after being recovered from appropriate gel sections b y freezing and thawing (Fig. 8b). Tzvo-di.mensional electrophoresis. When Fraction D was separated into six components b y b o r a t e - s t a r c h gel electrophoresis in one dimension and then allowed to migrate in a second dimension under the same conditions, or in the discontinuous buffer system, no extra bands were produced indicating t h a t the heterogeneity under alkaline conditions was not due to a system of metastable components. Studies on the separation of hydrogen and disulfide bonded subunits of proteins have been facilitated b y the use of starch gels containing urea or u r e a - m e r c a p t o e t h a n o l run under acid conditions 25. In the present case the bands resolved in the borate gel were not further resolved when run in a second dimension into an acid gel prepared in formate buffer (pH 3.1). However, the addition of 6 M urea or or 6 M urea and fl-mercaptoethanol to the formate gel produced several new bands in the second dimension (Fig. 9). Although quantitative experiments relating the reduction of disulfide bridges Biochim. Biophys. Acta, 154 (1968) 96-1o9

FIBRINOLYTIC DIGEST OF FIBRINOGEN

I05

to the gel electrophoretic patterns were not carried out these results suggested a considerable increase in the complexity of the electrophoretic patterns in the presence of both 6 M urea and 6 M urea-o.o7 M/5-mercaptoethanol. Gel filtration. Since the two-dimensional gel electrophoresis of "fraction D" in

I

J a

|

l

I

D 1

D~

D~

D4

I

I

l

!

D2

D3

D4

J D5

! D6

i D1

J D5

! D6

Fig. 8. Gel-precipitin r e a c t i o n s of F r a c t i o n D. (a) S t a r c h gel i m m u n o e l e c t r o p h o r e s i s . T h e letters r e p r e s e n t t h e p e a k s of t h e i n d i v i d u a l precipitin arcs o b t a i n e d a g a i n s t a n t i - h u m a n fibrinogen. (b) P a t t e r n o b t a i n e d on e x a m i n a t i o n b y t h e O u c h t e r l o n y t e c h n i q u e of t h e e x u d a t e s squeezed f r o m t h e excised b a n d s after freezing a n d t h a w i n g .

Biochim. Biophys. Acta, 154 (1968) 96-1o9

lO6

c,. A. JAMIESON, P. J. GAFFNEY, JR.

Fig. 9. S t a r c h gel e l e c t r o p h o r e s i s of F r a c t i o n D. (a) V e r t i c a l b o r a t e s t a r c h gel e l e c t r o p h o r e s i s of F r a c t i o n D. The anode is a t t h e b o t t o m . The gel s t r i p was r u n in a second d i m e n s i o n i n t o (b) s t a r c h - f o r m a t e (pH 3.~), (c) s t a r c h f o r m a t e - 6 M urea, (d) s t a r c h - f o r n l a t e - 6 M u r e a - o . o 7 M flm e r c a p t o e t h a n o l . T h e c a t h o d e is to t h e r i g h t in t h e acid gels.

urea-formate suggested the presence of hydrogen or disulfide bonded components, it was chromatographed on Sephadex G-Ioo in phosphate buffer (o.o5 M, p H 8.o) and in phosphate buffer containing 6 M urea, both before and after reduction and alkylation. The elution volumes of the fractions were identical to the void volumes of the column under the various conditions of ionic strength and protein modification (Table I). TABLEI ELUTION

CHARACTERISTICS

OF HIGH MOLECULAR

WEIGHT

FRACTIONS

C o l u m n s (72 cm × 1.5 cm) of S e p h a d e x G - i o o were p r e p a r e d in s o l u t i o n s as i n d i c a t e d . The v o i d v o l u m e (Vo) of each c o l u m n was d e t e r m i n e d u s i n g Blue D e x t r a n 2o00 (mol. wt. 2-IOS). The e l u t i o n v o l u m e s (Ve) were d e t e r m i n e d in p h o s p h a t e buffer, in 6 M u r e a a n d in 6 M u r e a a f t e r r e d u c t i o n a n d a l k y l a t i o n , as i n d i c a t e d .

Solvent

o.05 M p h o s p h a t e buffer, p H 8.o 6 M u r e a in o.o 5 M p h o s p h a t e buffer, p H 8.o

Column void volume (Vo)

Elution volume of ' Fraction D" ( V e )

32.4

3z.4

39.8

--

Biochim. Biophys. Acta, 154 (1968) 96-1o9

Ve[ Vo

...............................................

(a) Phosphate buffer

(b) 6 M urea

38.8

(c) 6 M urea after reduction and alkylation

--

i

38.9

o.97

FIBRINOLYTIC DIGEST OF FIBRINOGEN

10 7

DISCUSSION

Since the original observations of SEEGERS, NIEFT AND VANDENBELT26 that spontaneously lysed fibrinolytic digests of fibrinogen gave rise to two electrophoretica]ly distinct components, studies on the nature of these digests have been characterized by the recognition of increasing complexity both in the number of components and in the range of their physiological activities 27. The best resolution has been effected in the studies of NUSSENZWEIG,SELIGMANNAND GRABAR6 who separated five components by chromatography on DEAE-cellulose and determined their ultracentrifugal and immunological characteristics. The properties of the digests used in the present studies are identical with those reported for the complete digests obtained under slightly different conditions; namely, by digestion of fibrinogen with plasmin itself '~, as against the use of added plasnfinogen activator such as streptokinase 7, or spontaneous lysis by endogenous plasmin a. Although the 'Fraction D' isolated in the present study meets the previous criteria for homogeneity, recognition of a high degree of heterogeneity in this fraction has been achieved by the use of gel electrophoretic techniques, specifically with starch and acrylamide under closely controlled conditions of buffer composition and time. The observation that the multiple electrophoretic bands obtained in one dimensional electrophoresis were not further resolved by electrophoresis in a second dimension, in either alkaline or acid gels, indicated that these were not metastable components. The further resolution of these bands, in the presence and absence of added/5-mercaptoethanol, in gels made 6 M with respect to urea, suggested that the dismutation of hydrogen or disulfide-bonded subunits might be responsible for the electrophoretic heterogeneity in the first dimension. However, elution from Sephadex G-Ioo at a constant void volume indicated that no major change in molecular size was effected by reduction and alkylation in the presence of buffers containing 6 M urea. These results suggest that the extra bands produced in the second dimension m a y be due to an increased resolution effected by changes in the physical properties of the gel and not by the actual separation of subunits. It m a y be noted that HENSCHEN2s has found that the individual chains of fibrinogen are not disaggregated in 6 M urea following reduction and alkylation. Since six separate bands of identical molecular weight are present in the high molecular weight fraction arising by fibrinolytic digestion it appears that at least six distinct populations of fibrinogen molecules are present in the original substrate and in the fibrin derived therefrom. Preliminary results on these individual components, which have been partially purified by preparative gel electrophoresis, indicate differences in their amino acid composition, particularly with respect to lysine, as well as in their end group analyses as determined by the dinitrophenol method. One possibility is that fibrinogen itself exists in several genetically determined forms in the original plasma pool each of which gives rise to a distinct and homogeneous "Fraction D". However, patterns obtained on samples of fibrinogen isolated from eight individual donors were identical with those obtained on plasma pools but the effect of possible individual microheterogenelty in human fibrinogen is not excluded 29. Other possibilities concern the nature of the digestion process itself. Transpeptidation cannot be ruled out in modifying the structure of the digested fragments although the remarkable reproducibility of the patterns obtained under varying Biochim. Biophys. Acta, 154 (1968) 96-1o9

lO8

(;. A. J A M I E S O N , P. J . G A F F N E Y , J R .

conditions suggests that this is not the case. Secondly, there is the possibility that certain areas of the fibrinogen molecule are particularly susceptible to denaturation, in the sense of the loss of their helical structure, and that it is in these areas that proteolysis occurs. It has been observed that the best resolution of the various bands is obtained on digests carefully prepared from fresh fibrinogen. Prolonged storage or excessive warming of the digest results in a general smearing out of the electrophoretic pattern. Thirdly, there is the possibility that these nmltiple bands arise by partial proteolysis of the parent protein. That is, that while several bonds may be equally accessible to the proteolytic enzyme in the native state their subsequent susceptibility depends oil which of the original bonds is first cleaved. These different conformations of the digested protein might be expected to lead to electrophoretic differences. Experiments on human transferrin have shown a similar pattern of banding on gel electrophoresis of the high molecular weight fraction of various proteolytic digests 3° suggesting that this m a y be a relatively common phenomenon. Structural changes in these fibrinolytic digests are reflected in the changes in clotting time observed during the course of plasma in digestion. Although different electrophoretic patterns have been observed for the 'peak', 'elbow' and 'complete' digests it has not been possible to correlate these structural changes with the differences in anticoagulant activity. However, these differences, and particularly the time course of digestion obtained by disc acrylanfide electrophoresis indicate that the intact fibrinogen structure is rapidly destroyed although the digest still contains clottable protein and that further digestion is associated with an increase in the concentration of the components of higher electrophoretic mobility. However, at all these points the digests are characterized by a major cationic component corresponding to "Fraction D" in the completed digests and by high molecular weight fractions, as shown by the gel filtration experiments, which extfibit nmltiple banding on gel electrophoresis. Taken together, these results indicate an unexpected degree of structural heterogeneity in the high molecular weight fraction of complete fibrinogen digests, which contains one of the antigenic determinants of human fibrinogen as well as 'fibrin polymerization inhibitor' activity, and show that these properties cannot be considered to be associated with a single molecular species.

ACKNOWLEDGEMENTS

The authors are indebted to Mrs. JOAN CRISWELLZANFAGNAand to Mrs. CAROL WU for timir assistance during various phases of the work. This project was supported by U.S. Public Health Service Grant H E o94~8. REFERENCES i 2 3 4 5 6 7

S. D. D. A. •' V. V.

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