Defibrinating enzyme from timber rattlesnake (Crotalus H. Horridus ) venom: A potential agent for therapeutic defibrination I. Purification and properties

THROMBOSIS RESEARCH Printed in the United

Vol. States

6, pp. 151-169, Pergamon Press,

1975 Inc.

DEFIBBINATING ENZYME FROM TIMBER RATTLESNAKE (CHOTALUS HA HORRIDUS) VENOM: A POTENTIAL AGENT FOR THERAPEUTIC DEFIBRINATION I. PURIFICATION AND PHCPEHTIES Carlos A. Bonilla Department of Physiology and Biophysics, School of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523 (Received 5.12.1974; in revised form 9.1.1975. Accepted by Editor W.H. Seegers)

ABSTRACT

A defibrinating (thrombin-like) enzyme, isolated from timber rattlesnake (C. -_ h. horridus) venom by gel filtration, ion exchange and adsorption chromatography was found to be homogeneous by (i) analytical ultracentrifugation, (ii) electrophoresis in-highly cross-linked (12.8%), acidic, 6M urea polyacrylamide gels. The molecular weight was 19,610 based on amino acid composition, 19,500 by sedimentation velocity and 19,500 by sedimentation equilibrium. The enzyme is specific for the fibrinogen-fibrin conversion and does not affect other blood clotting factors; it exhibits an unusually high stability to acid pH and high temperature and it is partially inhibited by heparin only in very high concentrations. The ease of isolation of this enzyme and its apparent lack of side effects "in viva" warrant further investigation into its mechanism of action and its potential use as an agent for therapeutic defibrination.

INTRODUCTION The high incidence of thromboembolic disease and the role which abnormal polymerization of fibrinogen to fibrin plays in disseminated intravascular coagulation (11, hyaline membrane disease (21, and marantic endocarditis (3) has prompted the investigation of enzymes from snake venoms which may have potential use for therapeutic defibrination. Such enzymes have been isolated and purified to a high degree of homogeneity from the venoms of the Malayan pit viper (Agkistrodon rhodastoma) (4), the Eastern diamondback rattlesnake (Crotalus adamanteus) (51, the Southern copperhead snake (Agkistrodon contortrix contortrix) (61, the American Lance headed pit viper (Bothrops atrox) (7), and Asiatic snakes (Agkistrodon acutus) (8). Of these enzymes, only two are available commercially for investwnal use: Agkistrodon rhodostoma

Supported by grants from The Colorado Heart Association and a National Institutes of Health Special Research Fellowship (lFO3 DE45781-03).

151

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R (AncrodR: Twyford Laboratories,and Bothrops atrox (Peptilase:

Pentapharm Limited) and only AncrodR has been subjected t=ensive in viva and in vitro -investigationsin preparation for clinical use (9,101. The present stzyy ports the isolation and purification in homogeneous form of a defibrinating enzyme from the venom of the timber rattlesnake (C. h. horridus). The potential use of this enzyme for therapeuticdefibrinazonhas been described (11) and the hemodynamiceffects of slow and rapid defibrinationin experimental animals using the -C. h. horridus enzyme have been investigated (12). MATERIALS Venom Timber rattlesnake (C. -- h. horridus) venom was collected from healthy specimenskept in our laboratoryunder controlled conditionsof temperature, humidity and lighting. Immediatelyfollowing collectionthe venom was cleared by centrifugation,lyophilized,and stored at -40°C as described in earlier publications (13,14).

Chemicals All chemical reagents were the best cozunercially available analytical grade; hydrochloricacid and other reagents for amino acid analysis were seguanal grade.from Pierce Chemical Company, Sephadex gel filtrationmedia from Pharmacia; cellulose ion exchanges (DE-52 and CM-521 from Reeve-Angel:TBABcellulose (cellex-T)and hydroxylapatite(Bio-gelBT) from Bio-Bad Laboratories. Synthetic substrates (BAEE and TAME) and enzyme inhibitorswere from KM-Nutritional Biochemicals.

Diagnostic reagents Control human plasma, thromboplastin,thromboplastingenerationkits and prothrombin free plasma were obtained from Bio-Quest Laboratories. Other factor-deficientplasmas and Russell's viper venom in cephalin were Sigma products: thrombin, topical was purchased from Parke-Davis and Human Fibrinogen (Grade L) was a gift from Dr. J. Jouhar, Kabi Pharmaceuticals (Sweden). Fibrinogen and fibrinogen degradation product (FDP) detection kits were obtained from Burroughs Wellcome.

METHODS Gel filtrationon Sephadex G-100 Venom (5-10 g) was dissolved in 50-100 mls of 0.12 M TRIS-HCl-0.2 M NaCl, pH 8.6, centrifugedat 49,500 for 30 minutes in a refrigeratedcentrifuge

(4O), sorval S-34 rotor. The supernatantfractionwas then applied to a 5.0 x 100 cm column of Sephadex G-100 using upward flow adaptors (actualbed height: 90-cm) and elution carried out with the same buffer. When larger amounts of venom (11-20g) were gel filtered, recycling chromatographyunder similar conditions was used (13).

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Ion exchange chromatography 1.

DEAE+Cellulose*

The active fractions from the Sephadex G-100 column were pooled, dialyzed for 24 hours against distilled, deionized water at 4" and lyophilized. of 0.005 prepared Stepwise 8.0, and 2.

The lyophilized fraction was dissolved in a minimal volume (5-1Omls) M TRIS-HCl, pH 8.0, applied to a column of DEAE-cellulose (DE-52) according to the method of Sober (15) and eluted with the same buffer. elution was then carried out with 0.005 M TRIS-HCl-0.2 M NaCl, pH 1.0 M NaCl. CM-Cellulose

The pooled, active fraction from the DEAE-cellulose column was dialyzed (24 h, 4O, against 4000 vol. of HzO), lyophilized, re-dissolved in a small volume of 0.04 M sodium citrate (pH 3.7), dialyzed (6 hrs) against the same buffer and applied to a CM-cellulose (15) column (2.5 x 45 cm) equilibrated with the same starting buffer. Stepwise elution was carried out as follows: 0.04 M sodium citrate (pH 3.7), 0.40 M sodium citrate (pH 3.7) and 1.0 M NaCl. The fractions containing the active, defibrinating enzyme were pooled, dialyzed against distilled Hz0 (24 hrs) in the cold (4') and lyophilized. 3.

TEA+Cellulose

When required, final purification of the defibrinating enzyme was accomplished in TEAE-cellulose or hydroxylapatite columns. The ion exchange cellulose is more practical due to higher flow rates but the apatite columns are just as efficient at removing contaminant proteins in this final stage of purification. The defibrinating enzyme applied to TEAR-cellulose columns is eluted in a frontal peak with 0.005 M TRIS-citrate (pH 7.4) or with 0.10 M phosphate buffer (pH 7.4) from the hydroxylapatite columns.

Gel electrophoresis Electrophoresis in 12.8% polyacrylamide acidic gels containing 6 M urea was performed as described earlier (14), without modification.

Amino acid analysis Amino acid analyses in duplicate were carried out on homogeneous samples of the defibrinating enzyme according to the method originally described by Spa&man, Stein, and Moore (16) using a Durrum analyzer model D-500 with built in computer. Salt free samples were prepared by exhaustive dialysis against double-distilled deionized water. Aliquots were hydrolyzed in duplicate for 24 hours at llO°C, and the results averaged. No attempt was made to determine the presence or absence of carbohydrate in the purified enzyme.

*Abbreviations used in this work: Defibrizyme, for defibrinating enzyme from C. h. horridus venom; DEAR, diethylaminoethyl; CM, carboxymethyl; TEAE, -triethylaminoethyl.

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Cysteine determination Half-cystine was determined by performic acid oxidation and acid hydrolysis followed by amino acid analysis. Values reported are total cysteic acid obtained from cystine plus cysteine content (17).

Tryptophane determination Tryptophane content was obtained from analysis of 94 hour alkaline hydrolysis as described by Drsze (18) and by the calorimetric procedure of Spies and Chambers (19).

Sedimentation studies Analytical ultracentrifugation was carried out with a Beckman-Spinco Model E instrument, equipped with Schlieren and Baleigh interference optics. A standard 12 mm double-sector cell was used in all the sedimentation studies. The sedimentation coefficient, S, was calculated as described by Schachman (20) and the diffusion coefficient determined in the ultracentrifuge using a capillary synthetic boundary cell and interference optics. The rotor speed was 7,447 rpm. The diffusion coefficient, D, was calculated from the boundary spreading as described by Schachman (20) and Longsworth (21). For low-speed sedimentation equilibrium runs a rotor speed of 12,590 rpm was used with interference optics. The initial protein concentration, Co, of the sample was determined from the number of fringes observed in a synthetic boundary run as described by Richards and Schachman (22). In our sedimentation studies, the value of partial specific-volume, c, was taken as 0.730 ml/mg in order to calculate the molecular weight of the defibrinating enzyme. The Svedberg equation was used for the calculation of molecular weight from S and D values (23).

Enzyme analysis Pooled fractions from the Sephadex G-100 column were analyzed for L-amino acid oxidase (24), 5'-nucleotidase (25), phosphodiesterase (25).,and phospholipase-A (26).

Coagulation methodology One stage prothrombin times were calculated as described by Quick (27). Clot formation was measured electronically with a fibrometer (Baltimore Biological Laboratories, #60415). The effect of Heparin on defibrinating enzyme activity was tested by incubating the enzyme (0.1 ml) with Heparin (10-1000 u/ml) for 30 minutes at 37" at which time control plasma (0.1) was added and the clotting times determined. To test the effect of the enzyme on fibrinogen, known amounts of the pure defibrinating enzyme were added to a solution of Grade L-human fibrinogen, 96% clottable at a concentration of 0.35-0.40% prepared as described by Baughman (28). To 0.2 ml of the fibrinogen solution either thrombin (0.1 ml) or defibrinating enzyme (0.1 ml) was added and the clotting times recorded.

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RESULTS Purification procedure A summary of the purification procedure is shown in Fig. 1. The sequential steps are designed to remove all traces of the highly toxic basic protein neurotoxin present in the crude venom of the timber rattlesnake (29-311, and to obtain other biologically active fractions, in addition to defibrizyme, in highly purified form. The same scheme can be used for analytical and preparative fractionation and as much as 20 grams of crude venom can be separated at a given time provided that recycling chromatography or two large (5 x 100 cm) columns in tandem be used in the initial (Sephadex G-100) gel filtration step. The best resolution is achieved when the ratio of crude venom (mg) to buffer (ml.)does not exceed 1OO:l. The use of Sephadex G-150 or G-200 does not significantly improve resolution and the G-100 is chosen because of its higher flow rates.

CRUDE VENOM (C.h. horrldus) IO-2oq c&yRlTRlgAT~o~ TRiS-HCI

, pH fi.6°~50-100ml)

CLEARED VENOM SEPHADEX-G-100 0.12M TRIS-HCI-0.2M SII PL-A DEAE-CELLULOSE

SIA SIB ? LAO,PDE, 5’NTD

NaCI.PH8.8 Sill TOXIN Dill

Jy--ijq

CM’

w

DEFl8RQYME

CMIV TII FIG. 1

Outline of procedure for purification of defibrinating enzyme from Crotalus h. horridus venom. LAO, L-amino acid oxidase; PDE, phosphodiesterase; S'NTD, 5'nucleotidase; PL-A, phospholipase-A.

Gel filtration As shown in Fig. 2, gel filtration of crude -C. h. horridus venom on Sephadex G-100 separates four different components designated as follows:

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IA, a large molecular weight fraction excluded from the column, its pharmacological actions are unknown at this time; IB, contains the L-amino acid oxidase, phosphodiesteraseand 5'-nucleotidaseactivitiesof the venom; all of the defibrinatingenzyme, phospholipase-Aand some proteolyticactivity reside in fraction II; fraction III contains most of the small molecular weight basic protein toxin (30) and some proteolytic and hemorrhagicactivity.

SEPHADEXG-100 (5.0x90.0 cm) 24.0

z20.0 s x $ a 5:12.0 3 3 16.0 : 8.0

FRACTKlN NUMEFt(9.3 mls/llJBO FIG. 2 Sephadex G-100 gel filtrationof cleared crude venom from -C. h. horridus M NaCl, pH 8.6. The venom. Load: 7 grams in 50 ml of 0.12 M TBIS-I-Xl-O.2 pooled fractions are indicatedby the thick horizontalbar. Other details are described in the text. Ion exchange chromatography 1. DEAE-Cellulose Chromatographyof the pooled, dialyzed, lyophilizedfraction II on DE-52 yields three peaks (Fig. 3) of which the unretarded fraction (DI) contains most of the defibrinatingenzyme activity. Some activity is normally found in fraction DII and the ratio for the two peaks varies drasticallyfor different lots of crude venom. The removal of most of the phospholipase-A activity as fraction DII makes the DE-S2 step an absolute necessity in the purificationscheme.

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15x

-t MAE-CELLULOSE (2.5x45.0cm)

0.005M TRIS-Hcl dpH8.0

OOO5M

TRIS-HcCO.2MNoCI l.OMNoCl &+H3.0

,I

IO

20

30

40 SO 60 70 SO FRACTION [email protected]/TU8E)

90

150

FIG. 3 DEAE-cellulose chromatography of fraction II obtained from Sephadex G-100 gel filtration of C. h. horridus venom. The breakthrough peak, DI, containing --. the bulk of the defibrinating enzyme was pooled, dialyzed, lyophilized and chromatographed on CM-cellulose to remove contaminant proteins.

2.

CM-Cellulose

The elution profile obtained from ion exchange chromatography of fraction D-II on CM-cellulose is shown in Fig. 4. All of the defibrinating enzyme activity is found in the slightly retarded fraction (CM-II). Fractions CM-III and CM-IV are the most toxic components of -C. h. horridus venom (30). The reasons for instituting this purification step are described in the discussion section. 3.

TEAJ+Cellulose

Final purification of the defibrinating enzyme was achieved by chromatography of fraction CM-II on cellex T (Fig. 5) or hydroxylapatite (not shown).

Gel electrophoresis The electrophoretic patterns obtained with crude venom and the purified defibrinating enzyme are shown in Fig. 6 and indicate that, using the procedure herein described, the enzyme can be recovered in homogeneous or nearly homogeneous form. In approximately 65% of our preparations a contaminant protein which does not penetrate very far into the gel (Slot 7, Fig. 6) can be observed. This source of heterogeneity, however, is not detectable in the ultracentrifuge and, to this point, we have been unable to identify the nature of this component.

158

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T

CM- CELLULOSE ( 2.5 x 45.0 cm)

12.0 0.4OMNO pH 3.7 .-_

1.00 M NaCl

.

f P

6.0/%!I+

a

CMTl

CMm

c

4.0 -

.

\

.

/ 20-

. J_.,:

Idc’*.,*

.** IO

20

30 Fl?A::ON

,

,

00

so

Q

NUEER C&l&&

FIG. 4 profile of fraction DI (Fig. 3) on CM-cellulose. The fractions indicated by the thick horizontal bar were pooled, dialyzed and lyophilized and further purified on a TF,AE-cellulosecolumn.

Elution

TEAE- CELLULOSE (2.Sx45.Ocml 12.0

.

FRACTION NUMBER FIG.

5

Several batches of venom were purified through the CM-cellulose step and the resulting fractions (CM-II) pooled, and chromatographed on a TEAS-cellulose column. Fraction TI is the highly purified defibrinating enzyme.

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FIG. 6 Polyacrylamide gel electrophoresis of crude -C. h. horridus venom and purified defibrinating enzyme. Conditions for electrophoresis: voltage, 250 V (constant); current, 100 MA; pre-run, 3.5 h; electrolyte, 0.37 M glycine-citric acid buffer, pH 2.9; load, lo-40 Hl (28 mg/ml); length of separation, 4 h (4'); gel concentration, 14% cyanogum-41. 1 = lysozyme marker; 2-4 = -C. h. horridus crude venom, 10, 20, and 40 ~1; 5-7 = defibrinating enzyme, 10, 20, and 40 ~1; 8 = lysozyme marker.

Amino acid analysis The averaged results of duplicate 24-hour, 6 N HCl hydrolyzates are shown in Table I. The results indicate that the amino acid composition of the defibrinating enzyme from C. h. horridus venom differs from thrombin and the thrombin-like enzymes isolated from other snake venoms. The molecular weight was estimated from amino acid analysis by calculating the composition in the following manner: molar ratios of each amino acid (Table I, Column 1) to alanine which was taken as 1.00 (Column 2). The minimum residue weight (Column 3) was then obtained from the product of the averaged ratio (Column 2) times the residue weight. The minimum residue weights

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were summed and divided by 19,500, the molecular weight of the homogeneous enzyme determined from analytical centrifugation. The resulting factor (7.9) was then multiplied by the averaged ratio (Column 2) to obtain the number of residues for each amino acid (Column 4), which was subsequently rounded off to determine the nearest integral residue (Column 5). The molecular weight obtained from amino acid composition is 19,610 which is in close agreement with a molecular weight of 19,500 determined by physical methods. Although no attempt was made in this investigation to detect the presence of carbohydrate in the purified defibrinating enzyme, two factors: a) recovery of the amino acid nitrogen and ammonia on a weight basis accounted for about 96-97 per cent of the weight of the material , and b) the very close correlation between the molecular weights determined from sedimentation analysis and amino acid analysis; suggest, that, this defibrinating enzyme contains

TABLE1 Amino Acid Composition of befibrinating Enzyme From -C. h. horridus Venom

Amino acid

24 hr average

Patio of alanine

Minimum residue weight

Residues

Nearest integer

Molecular weight

p mole Alanine

0.0088

1.00

89.1

Arginine

0.0046

0.52

Aspartic acid

0.0241

Half-cystine

7.9

8

713

109.6

4.1

4

843

2.74

364.7

21.6

22

2,928

0.0055

0.63

76.4

4.9

5

606

Glutamic acid

0.0162

1.84

337.8

14.5

15

2,754

Glycine

0.0116

1.32

99.1

10.4

10

751

Histidine

0.0039

0.44

92.2

3.5

4

838

Isoleucine

0.0059

0.67

87.9

5.3

5

656

Leucine

0.0092

1.05

137.8

8.3

8

1,050

Lysine

0.0073

0.84

153.5

6.6

7

1,279

Methionine

0.0016

0.18

26.8

1.4

1

149

Phenylalanine

0.0065

0.74

122.2

5.8

6

991

Proline

0.0060

0.68

78.3

5.4

5

575

Serine

0.0129

1.47

154.5

11.6

12

1,261

Threonine

0.0072

0.82

97.7

6.5

7

834

Tryptophan

0.0051

0.58

118.4

4.6

5

1,021

Tyrosine

0.0070

0.80

174.2

6.3

6

1,306

Valine

0.0103

1.17

137.1

9.2

9

1,055

139

19,610

2,457.3

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lower concentrations of carbohydrate than those reported for Arvin (20%) and the thrombin-like enzyme from C. adamanteus venom (5.4%). Further characterization of the pure enzyme shozd clarify this point.

Sedimentation analysis The chromatographically purified defibrinating enzyme behaved as a single component in sedimentation velocity experiments in the analytical ultracentrifuge (Fig. 7). Its sedimentation coefficient at 59,780 rpm and 20" was 2.56 S (Fig. 8); this value appeared to be independent of its protein concentration at 60,000 rpm, 20" from 1.0 to 3.5 mg per ml. The molecular weight calculated from sediment&ion velocity was 19,500 and from sedimentation equilibrium 19,400. Calculation of the diffusion coefficient from boundary spreading gave a value of D20, = 11.42 x lo-' cm'sec-'(Fig. 9). w

FIG. 7 Sedimentation patterns of purified defibrinating enzyme in 70 nM potassium sodium phosphate buffer pH 7.1. Photographs were taken at the times indicated in the figure after attaining a speed of 59,780 r-pm. Sedimentation is from left to right. Other details are described in the text.

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= .0415

l166-0160

IO060

41.17, .lO-7cm UC“

I.790 -

IO-

177oi 0

a

’ ’ ’ . . * ’ ’ . 16 32466460%llZ126144160,76(1O241224 TIME (MINJ

’

’

8

0

0

4

6

I2

16

20

24

26

32

TlME lM1N.I

FIG. 8

FIG. 9

Determination of the sedimentation coefficient of defibrisyme using the method of Schachman (20). The operating speed was 59,780 rpm, and traces recorded at I-minute intervals. The buffer was 0.1 M phosphate at pH 7.1.

Determination of the diffusion coefficient (D) of defibrinating enzyme from C. h. horridus venom according to thrmzhod of boundary spreading (20,211. A capillary synthetic boundary cell and interference optics were used. The rotor speed was 7,447 r-pm. Other details in the text.

Plots of log C versus X2 (c = protein concentration; X = distance from axis of rotation to the center of the fringe pattern in centimeters) gave straight lines, a good indication that the preparation is a homogeneous protein.

Stability of the purified enzyme The effects of heparin, pH, and temperature on the clotting activity of defibrizyme are shown in Table II. Heparin does not significantly inhibit the clotting activity of the purified enzyme in concentrations ranging from 10 to 1000 units/ml. Long term exposure of the enzyme to acidic pH (4.01) at 37' has little or no effect, while exposure to extremely alkaline pH (12.40) for a

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similar length of time (48 hours) induced a significant decrease in clotting activity. Further evidence for the stability of the enzyme to acid pH is shown by the absence of loss of activity during the purification procedure on carboxymethyl cellulose using sodium citrate buffer at a pH of 3.70. The enzyme remains fully stable following storage for over 24 months at -20" and for over 6 months at 4' in either lyophilized form or in solution at pH 8.0 (0.005 M TRIS-HCl buffer). Temperature effects indicate no loss of activity upon incubation for 15 minutes at 65“ but a decrease in activity following 15 minutes incubation at 96'. From preliminary data collected, but not herein described, it may be safely assumed that the enzyme is a small molecular weight serine esterase highly specific for fibrinogen. These results will be presented in a followup communication on the mechanism of action and the nature of the fibrinopeptides released by defibrizyme upon incubation with fibrinogen.1

TABLE 2 Effects Of Heparin, Temperature And pH On -In Vitro Clotting Activity Of Defibrinating Enzyme Clotting times (seconds) Defibrinating enzyme concentration 10 mg/ml

1 mg/ml

Heparin (u/ml) 1000

11.47 + 0.38

100

6.03 + 0.38

10

3.85 f 0.10

0

3.46 + 0.06

Temperature ('C)

1

37

5.95 + 0.05

60

5.90 r 0.00

95

8.00 + 0.07

PH 4.01

9.65 + 0.11

8.00

5.40 + 0.16

12.40

102.18 + 1.98

C. A. Bonilla.

Manuscript in preparation.

30.20 + 0.60

240.95 It: 5.41

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Ultraviolet absorption Defibrizyme exhibits a typical protein absorption profile with a maximum at 280 run,a minimum at 250 nm and no absorption in the visible spectrum.

Coagulation methodology A. One stage prothrombin times. As shown in Table 3, the defibrinating enzyme resembles thrombin in its ability to clot control human plasma in the absence of thromboplastin. In general, in the assay system described in this work, 1.0 mg of defibrinating enzyme has the approximate clotting activity of 10 NIH units of Parke-Davis thrombin.

TABLE 3 Thrombin-Like Activity Of Defibrinating Enzyme Thrombin concentration clotting time* (units/O.1 ml)

Defibrizyme concentration clotting time* @g/o.1 ml)

10.0

14.9

1.25

16.1

5.0

22.7

0.625

20.8

2.5

36.2

0.313

31.8

1.25

57.7

0.156

50.8

0.625

97.7

0.078

228.3

0.010

291.8

*Each value represents the mean of seven determinations obtained with an automatic clotting detector (Fibrometer). A reaction mixture consisting of water (0.1 ml) and control plasma (0.1 ml) was incubated for 3 minutes (37') at which time thrombin (0.1 ml) or defibrinating enzyme (0.1 ml) was added and the clotting times recorded.

B. Human fibrinogen (79% clottable) was converted to fibrin by the defibrinating enzyme in a dose dependent manner (Table 4). Based on this fibrinogen assay it again appears as though 1 mg/ml of the purified enzyme has an equivalent clotting activity to 10 NIH B/ml of purified, Parke-Davis thrombin. It should be pointed out at this time that because defibrizyme does not activate factor XIII, the nature of the fibrin clot differs from that of thrombin-induced clots (11). This point will be described in greater detail in a subsequent communication to this journa1.l

1

C. A. Bonilla.

Manuscript in preparation.

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TABLE 4 Effect Of Defibrinating Enzyme On Purified Fibrinogen Thrombin concentration W/ml) 100

Defibrizyme (mg/ml)

Clotting Time* 11.4 f 0.52**

Clotting Time*

10

15.8 f

0.91**

50

17.9 f 1.74

5

19.0 f

1.19

25

28.5 f 1.08

2.5

56.6 f

3.18

12.5

31.3 + 1.88

1.25

78.2 f

6.62

0.625

131.0 + 12.88

*Each value represents the mean of 11 to 17 determinations. To 0.2 ml of the fibrinogen solution either thrombin (0.1 ml) or defibrinating enzyme (0.1 ml) was added and the clotting times (seconds) recorded with a Fibrometer. **Mean 2 standard error.

FIG. 10 Comparative gel electrophoresis of (from left to right): C. adamanteus thrombin-like enzyme isolated by the procedure herein descxbed. C. adamane thrombin-like enzyme isolated by the method of Markland and DGs (Ref. 5). C. h. horridus basic protein toxin; Defibrizyme, 40, 20 and 10 ~1; C. h. horriduscrude venom, 40, 20 and 10 1.11; and lysozyme marker. Other detaxsas in Fig. 6.

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DISCUSSION The hypofibrinogenimic state induced by the administration of purified thrombin-like enzyme from C. h. horridus venom has been investigated (11). Following intravenous admixstration of the enzyme to dogs or cats at a close of 1.0 mg/kg body weight, fibrinogen levels drop to below 30 mg per 100 ml of blood and consequently an incoagulable state is achieved due to low fibrinogen levels, that is, due to hypofibrinogenimia. Post mortem examination in experimental animals indicates a complete absence of intravascular clotting ana, thus, it can be assumed that the fibrin formed by the action of the defibrinating enzyme on fibrinogen differs from that of thrombin. This has been recently confirmed in our laboratory (11) since it can be shown that, unlike thrombin, tiefibrizymedoes not activate the fibrin stabilizing factor (factor XIII). The lack of deleterious effects observed upon defibrination with this enzyme and its very low antigenicity has prompted our detailed investigation of its potential use for therapeutic defibrination in clinical medicine. The present report outlines a simple, reproducible ma relatively inexpensive Procedure for the purification of the defibrinating enzyme from -C. h. horridus. Isolation of defibrinating enzyme requires an initial gel filtration step designed to remove the bulk of the small molecular weight toxin, followed by DEAE-cellulose chromatography which is used t9 remove phospholipase-A present in the original fraction. An additional ion exchange chromatographic step on CM-cellulose is then carried out in order to remove remnant contaminating neurotoxin, and final purification is then achieved on either TEAE-cellulose or hydroxyapatite. The final preparation of the enzyme is homogeneous about 35 to 40% of the time, but this depends strictly on the batch of venom used as the starting material. In approximately 65% of our preparations a contaminant protein is present in trace amounts but its presence aOf not, in any way , modify the activity of the defibrinating enzyme. The nature of this contaminant has not been elucidated. As described in this report, the purification Procedure outlined is adaptable to large scale production of the enzyme, a prerequisite for wide scale animal experimentation in preparation for clinical trials. It is hopea that the ease of extraction of the defibrinating enzyme in homogeneous or nearly homogeneous form will enable a large number of coagulation research centers to undertake the required detailed investigation into potential teratogenic effects, bioavailability in the presence of plasma proteins and its interaction with drugs widely used in the patient with hemorrhagic and thrombotic disease. This is particularly important specially in view of the lack of general availability and high cost of defibrinating enzymes, i.e.: AncrodR, now being marketed in the United States. The timber rattlesnake is widely distributed in the eastern and midcentral states of the united States, it is one of the three largest of the North American rattlesnakes, ana it adapts quite well to captivity so, should the need arise, the snake could be farmed for the purpose of venom collection. To this point, in our experimental animals (cats, dogs), gross organ pathology and serum enzymology have been negative (11); this, however, needs confirmation in other animal species. Although the enzyme has some of the biological characteristics accrued to AncrodR (Arvin), the thrombin-like enzyme from the Malayan pit viper.& rhoaostoma) venom, it differs in having a much smaller molecular weight, 1.e.; 19,600 compared to approximately 30,000, in its amino acid composition and in its stability to acid pH and high temperature. It is very interesting to note that when the venom of the Eastern diamondback rattlesnake, C. adamadeus, was fractionated under similar conditions to those described G this paper for C. h. horridus, a defibrinating

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enzyme identical to the latter in amino acid composition and molecular weight as determined by sedimentation analysis was obtained. The molecular weight reported (5) for the thrombin-like enzyme from C. adamanteus is 32,700 compared to approximately 20,000 obtained in this study. The amino acid composition is also at variance with our findings , and from the extreme stability of the enzyme at acid pH, it must be assumed that in the C. adamanteus venom there exist two proteins with thrombin-like activity which can be obtained in homogeneous for-u-bydifferent fractionation procedures. As has been shown in Fig. 10, the enzymes from -C. h. horridus and C. adamanteus isolated by the procedure herein described have identical electrophoretic mobilities. Up to this point we have not been able to explain the reasons for the aforementioned differences and/or similarities in the two enzyme preparations from C. adamanteus venom (Dr. Markland, personal communication). The venom sourcZ, i.e.; commercial preparations versus in situ collected and treated venom, however, do play a very important rolFithe isolation of specific proteins and peptides from rattlesnake venoms, specially in view of their very high protease content. There is little reason not to suspect, that, the contaminant protein present in a large percentage of our final preparations of the defibrinating enzyme is, in fact, a product of proteolytic degradation since it is always present in the pooled, crude -C. h. horridus venom which we obtain from commercial sources (see Fig. 10, Slots 7-91, but which is absent in most of the venom collected in our own serpentarium. Finally, it is obvious from the above results that the defibrinating enzyme from timber rattlesnake venom offers a great deal of promise as a potential agent for therapeutic defibrination in the United States.

ACKNOWLEDGEMENT I would like to express my gratitude to Dr. Diwan Singh, Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, for his expert help and advice on the sedimentation analysis of the pure defibrinating enzymes from C. h. horridus and C. adamanteus venoms.

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