Factor X-activating activity in normal and malignant colorectal tissue

THROMBOSIS RESEARCH 52; 207-217, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

FACTOR X-ACTIVATING

ACTIVIN

J.L. Francis,

IN NORMAL AND MALIGNANT

COLORECTAL

TISSUE

K. El-Daruni, O.S. Roath, and I. Taylor*

University Department of Haematology and University Surgical Unit*, Level F, SoutharrQton General Hospital, Southampton so9 4XY, United Kingdom.

(Received 4.7.1988; accepted in original form 22.8.1988 by Editor J.R. O'Brien)

ABSTRACT The factor X-activating activity (FXAA) of homogenates from human colorectal tumours and corresponding normal colonic mucosa from the same patients was assessed with a specific chromogenic substrate technique. FXAA was detected in all normal and tumour tissue tested, but was significantly higher in tumour tissue. The procoagulant activity was inhibited by DFP, but was unaffected by iodoacetamide and mercuric chloride. FXAA was largely abolished by prior incubation of both normal and turrour tissue hmgenates with a rabbit anti-human factor VII serum, but was greatly enhanced by the addition of purified factor VII. FXAA was partially adsorbed on to aluminium hydroxide and almost completely abolished by treatment with barium citrate. It is concluded that the FXM of both normal and malignant colorectal tissue is the result of tissue factor-factor VII interaction.

INTFKKLCTICN The clinical association of cancer and thrombosis (11, and the histological relationship of fibrin and tumour deposits (2) are well recognised and there are numerous reports of thrcmboe&olic complications and intravascular coagulation in patients with malignant disease (3). The observation that malignant tissue shortened the plasma recalcification time significantly mot-e than most normal tissues (4) suggested that tumour cells secrete a procoagulant activity which might account for these findings. The finding of tissue factor-like activity in a variety of human malignant tissues is well documented (5-81, although this is not the only mechanism by which malignant cells can activate the blood coagulation system.

Key words: Blood coagulation, colorectal cancer

factor VII, factor X, tissue factor,

207

208

CLOTTING FACTORS IN RECTAL TISSUE

Vol. 52, No. 3

Pineo et al (9) showed that a glycoprotein isolated from a variety of m~lcus extracts directly activated factor X, raising the possibility that this procoagulant might be responsible for the intravascular coagulation associated with mucus-producing adenocarcinomas. Subsequently, Cordon and coworkers (10) showed that a number of malignant tumours contained a factor X-activating procoagulant. This activity, termed “Cancer Procoagulant A” (CPA), has since been reported in a number of experimental (11,121 and human (13,14) malignancies, and purified to homogeneity (15). CPA initiates coagulation in both normal and factor VII-deficient plasma, has a molecular weight of 68 KD and has the properties of a cysteine protease (15). Recently, we described a specific factor X-activating activity in both normal and malignant tissue from 50 patients with colorectal carcinoma which we then assumed to be identical to CPA (16). We now report on further experiments which provide evidence that this activity is in fact, distinct from Cancer Procoagulant A.

MATERIALS AND MEMOOS

Tissue

sanples

Tissue satrples were obtained from 63 patients undergoing surgery for resection of colorectal carci ncma. The resected tissue was collected before addition of formalin or any other solution. Samples of tumour were cut from areas of viable malignant tissue, avoiding the necrotic tumour centre. Portions of macroscopically uninvolved mucosa were then stripped from the proximal end of the bowel sqles, keeping well clear of the resected tumour margins. Tissue

hanwenization

The tissue smles were weighed, snap frozen in liquid nitrogen and horrogenised by cryofragmentation in a Braun Mikrodismembrator (F.T. Scientific The powderedtissue was thoroughly mixed Instrmnts Ltd, Tewkesbury, U.K.). with a ten-fold (w/v) volume of Tris-HCl buffer (O.O5M, pH 7.8). The resultant homogenates were then centrifuged at 10 000 X g for 1 minute and the clear supernatant used in al 1 assays. Procoaqu 1ant

assay

Factor X activating activity was determined in a specific chromogenic assay. Into the wells of a flat-bottomed microtitre plate were pipetted 20 ~1 of normal or tun-our tissue extract, 20 ~1 calcium chloride (O.O25M), 20 ~11 (2 units/ml) purified bovine factor X (Sim Chemical many) and 40 ~1 buffer The plate was incubated in a Dynatech shaker(0.05M Tris-HCl, pH 7.8). Generation of factor Xa was then stopped by incubator at 37oC for 2 hours. addition of 100 ~1 assay buffer containing 7.5 rrlvl EDTA. 40 t.11 of the chromogenic substrate CBS 31.39 (3 rrM, Diagnostic Stag0 Ltd) was added, and the This reaction was then incubation continued for a further 30 minutes. terminated by addition of 50 l.11 glacial acetic acid and thorough mixing. The optical density of each well was then read at 410 nm in a Dynatech MR700 plate To reader against a blank containing assay buffer in place of factor X. correct for non-calcimdependent protease activity, the absorbance of a well

CLOTTING FACTORS IN RECTAL TISSUE

Vol. 52, No. 3

209

containing buffer in place of calcium chloride was subtracted from the total Procoagulant activity was expressed in absorbance units per gram of activity. tissue extracted, and as the ratio of results obtained in turnout- and Separate experiments (data not shown) ensured corresponding normal mLlcosa. that no factor X activation occurred in the absence of tissue homogenate during the incubation period used in the assay, and that the reaction rate was linear over the range of procoagulant activities studied. Controls The controls used in 42.5 r_1g/ml, Diagnostic activating enzyme, and was prepared from rabbit (Sigma Chemical many) Jennings (17). In some additional control. Effect

of

factor

frost experiments consisted of Russell Viper venom (RVV, Stag0 Ltd), as a known serine protease factor Xtissue factor-factor VII complex (TF-VII). The latter brain thrcmboplastin and purified bovine factor VII according to the method described by Hubbard and experiments, rabbit brain thromboplastin served as an

VII

To determine the effect of exogenous factor VII on the generation of factor Xa, procoagulant assays were performed as detailed above, but with the inclusion of an extra well containing 20 ~1 purified bovine factor VII (40 units/ml, Sigma Chemical corr(sany). Reactions performed in the presence of exogenous factor VII were stopped 3 mins after addition of chromogenic substrate because of the greater speed of this reaction. For comparison, the results of these experiments were expressed as AAA/hr, standardised for a constant tissue weight. Effect

of

anti-factor

VII

3 t-11 of a rabbit anti-human factor VII antibody (100 inhibitory units/ml, Diagnostic Stago Ltd) were added to 90 ~1 of tissue hmgenate or control and incubated at 37oC for 30 minutes. Aliquots of the mixture were then assayed for residual factor X-activating activity as detailed above. Effect

of

inhibitors

Tissue homogenates, pooled f ran colorectal tours and corresponding normal mLlcosa, as well as RW and TF-VII controls were incubated with diisopropylfluorophosphate (DFP, 5 IT+I f.c), iodoacetamide (1 r&l f.c.) or mercuric chloride (0.1 r&l, f.c) for 30 minutes at 37oC. Aliquots of the mixtures were then assayed for procoagulant activity as detailed above. Hmgenates incubated with buffer instead of inhibitors served as non-inhibited controls.

Effect of adsorption Aluminium hydroxide: 0.9 ml mnate or control was incubated with 0.1 ml aluminium hydroxide gel (25%) for 3 mins at 37oC. The mixture was than centrifuged at 10 000 X g for 1 minute and the supernatant assayed for residual procoagulant activity. Barium citrate: 25 ~1 25% sodium citrate was added to 0.9 ml hcmogenate or control. 25 ~1 1.5M bariun chloride was then added and mixed continuously for 2 minutes. The mixture was then centrifuged to rmve the insoluble barium

210

CLOTTING FACTORS IN RECTAL TISSUE

citrate activity.

precipitate,

Statistical

and the

supernatant

assayed

for

residual

Vol. 52, No. 3

procoagulant

analysis

All statistical analyses were performed using the STATGRAPHICS statistical software package running on an Amstrad PC1640. The results of procoagulant assays were not normally distributed and results are therefore presented as medians and interquartile ranges. Differences between groups were determined with the Mann-Whitney U-test or Wilcoxon test for paired samples.

RESULTS Factor

X-activating

activity

Statistical analysis of factor X-activating activity (FXAA) in normal and malignant tissue is shown in Table 1. The median FXAA level in turrour tissue was significantly higher (p
TABLE 1 Factor X-activating activity in homogenates of tumours and corresponding adjacent, non-involved mucosa from patients with colorectal carcinoma (n=63). extracted and as the ratio of tumour Results are expressed as A41’J/gm of tissue and normal values. Median

Normal (non-ma1 i gnant ) mLlcosa Tumour tissue Ratio ( turnour/normal )

Effect

of

factor

103 176 2.15

Interquarti

le

range

55 - 182 65 - 499 1.11 - 4.17

VII

The addition of purified factor VII markedly increased the generation of factor Xa by all normal and malignant tissue homogenates tested (p
of

anti-factor

VII

Incubation of the tissue homogenates with a rabbit anti-human factor antibody resulted in almost complete abolition of procoagulant activity normal and malignant sqles tested (p
VII in all in

Vol. 52, No. 3

211

CLOTTING FACTORS IN RECTAL TISSUE

prccoagulant activity was significantly greater (p
TABLE 2 Effect of adding purified factor VII on factor Xa generation (AA/hr) colonic mccosa, colorectal tumurs and a thrcmtmplastin control.

Normal colonic rrmosa Without FVII With FVII Color-e&al turmurs Without FVII With FVII Thrmboplastin control Without FVII With FVII

Inter-quartile

by nor-ml

n

Median

range

14 14

0.33 23.72

0.21 - 0.57 16.0 - 40.0

14 14

0.56 28.16

0.45 - 1.25 ii.86 - 40.0

4 4

1.22 23.04

0.98 - 1.41 22.7 - 23.8

TABLE 3 Effect of adding a rabbit anti-human factor VII antibody to normal and malignant tissue hmgenates. Results of tissue -1es are expressed as A410/gm tissue and as percentage inhibition, while control values are given in absorbance units. Median Normal (n=12): Pm-treatment Post-treatment Inhibition (%) Tumur (n=12): Pm-treatment Post-treatment Inhibition (%) Russell Viper Venom (n=5): Pre-treatment Past-treatmnt Inhibition (%) Tissue factor-N11 cmplex (n=4): Pre-treatmnt Post-treatment Inhibition (%)

Interquartile

94.0 10.5 93.2

61.0 - 196.5 3.5 - 18.5 86.7 - 95.2

110.5 5.5 95.5

102.5 - 471.5 3.5 - 11.5 94.1 - 98.0

1.12 1.06 3.91

1.10 - 1.13 1.05 - 1.08 3.34 - 4.34

1.71 0.43 75.5

1.65 - 1.78 0.28 - 0.51 69.2 - 83.8

range

212

CLOTTING FACTORS IN RECTAL TISSUE

Vol. 52, No. 3

TABLE 4 Effect of inhibitors on the factor X activating activity of pooled normal and malignant tissue ho-rogenates and controls. Results for tissue samples are given as A410/gm tissue extracted, and the percentage inhibition compared to the buffer control is shown in parentheses. Results for RVV and TF-VII controls are expressed in absorbance units. Inhibitor

Buffer DFP Iodoacetamide HgClz

Normal

57.0 17.5 49.5 97.5

(0) (69) (13) (0)

Tumour

96.0 22.5 128.0 150.5

(0) (67) (0) (0)

RVV

1.22 0.09 1.20 1.24

TF-FVII

(0) (92) (2) (0)

1.34 0.05 1.30 1.40

(0) (96) (3) (0)

TABLE 5 Effect of Al(CH)3 and barium citrate absorption on the procoagulant activity normal and malignant tissue harogenates. Tissue results are expressed as A41°/gram tissue while controls are given as absorbance units. Median

Interquartile

range

Al(CHj3 Normal: Pre-treatment (n=l6) Post-treatment Reduction (%) Tumour: Pre-treatment (n=l6) Post-treatment Reduction (%) TF-VII: Pre-treatment (n=2) Post-treatment Reduction (X1 RVV: Pretreatment (n=2) Post-treatment Reduction (%>

182.7 102.0 41.8 444.0 127.2 51.4 1.43 0.028 98.0 1.5 1.4 8.6

73.0 25.7 25.1 113.0 31.7 34.5 1.20 0.022 97.9 1.49 1.38 7.60

-

437.2 245.5 66.3 802.7 426.7 89.2 1.66 0.035 98.2 1.59 1.43 9.70

Barium citrate Normal: Pre-treatment (n=l8) Post-treatment Reduction (%) Turnour: Pre-treatment (n=l8) Post-treatment Reduction (%) TF-VII: Pretreatment Post-treamnt (n=2) Reduction (%) Pre-treatment RVV: Post-treatment (n=2) Reduction (%)

125.5 19.5 77.6 254.7 38.0 86.9 1.43 0.026 98.0 1.54 1.58 0

69.0 7.5 63.5 175.0 9.0 83.3 1.20 0.021 97.4 1.49 1.50

-

174.0 45.0 89.4 735.5 44.0 94.9 1.66 0.031 98.7 1.59 1.67

of

Vol. 52, No. 3

CLOTTING

FACTORS

IN RECTAL TISSUE

213

Effect of inhibitors Incubation of tissue hmgenates with DFP inhibited the generation of factor Xa. Slight inhibition was observed with iodoacetamide in the pooled normal colon hmgenate, but not in the tumur satrple. However, this represented only a small change in absorbance value and may have been within experimental error. No inhibition was seen with mercuric chloride which rather resulted in an increase in procoagulant activity. Both RW and TF-VII controls were markedly or mercuric chloride. The data is inhibited by DFP, but not by iodoacetamide sumnarised in Table 4. Effect of adsorption Treatmsnt of both normal and turfour hoirogenates with Al(CH)3 resulted in the loss of scme 40-50% of prccoagulant activity (p
DISCUSSION The relationship between blood coagulation and the spread of malignant disease is supported by an increasing amount of clinical and experimental evidence. The possibility that turfour cells might secrete procoagulant activities has been extensively explored, although any relationship of procoagulant type and activity to the course of the malignant process remains unclear. The presence of a specific factor X-activating protease in a variety of human and experimental tumrs is well docurrented (10-14, 18-20). This procoagulant is primarily characterised by its ability to activate factor X in the absence of factor VII, thus distinguishing it frcm tissue factor which requires factor VII to initiate coagulation. Applying a different tissue hmgenization procedure and a modified, microtitre plate chrmgenic assay to a further 63 patients, this study has confirmed our previous report (16) of a prccoagulant, capable of directly activating factor X, in both normal colonic mucosa and colorectal tumours. The activity was significantly greater in tumour tissue than in normal mucosa, and the ratio of turrour:normal procoagulant activity was elevated by 20% or trore in 73% of the patients tested. In our previous study (16) we had assumed that the use of a chromogenic substrate assay, in which purified factor X was the only clotting factor present, ensured the detection of a specific, factor VII-independent, factor X-activating activity. Thus, the assay was thought to detect the presence of "Cancer Procoagulant A" (CPA) as extensively described by Gordon and coworkers (10,13-15,18-20). However, it now appears that the factor X-activating activity detected by this chromogenic assay may not in fact be identical with this cysteine protease. In the first place, CPA has only been described in malignant tissue, whereas our data indicates that an apparently identical procoagulant activity is present in both normal and tmour samples. The possibility that the macroscopically non-malignant tissue

214

CLOTTING FACTORS IN RECTAL TISSUE

Vol. 52, No. 3

designated 'normal' in this study may contain some malignant cells cannot be discounted. However, we have previously demnstrated factor X-activating activity in a limited number of histologically-proven non-malignant sarrples (16). Secondly, as an enzyrfe with a cysteine active site, CPA is inhibited by iodoacetamide and mercuric chloride (14,151. In contrast, the procoagulant activity presently described was not significantly blocked by these reagents, but was inhibited by the serine protease inhibitor DFP. Two new observations shed further light on the nature of this factor Xactivating procoagulant. Firstly, it is almzst corrpletely (>90%) inhibited by addition of an anti-factor VII antibody, suggesting an identity with plasma factor VII, or the presence of a factor VII-like material. Secondly, the generation of factor Xa was greatly enhanced by the addition of purified factor VII. As the factor VII had no factor X-activating activity of its own, the increase in factor Xa production can only be explained by the presence of free tissue factor. Taken together, these findings are corrpatible with the hypothesis that hmgenates of both normal colonic n-ucosa and color-e&al tours contain both free tissue factor and activated factor VII, presumably complexed to tissue factor. It would appear from these data that tissue factor is present in excess, while the higher level of factor X-activating activity in malignant tissue may reflect an increased concentration of the factor VII -t-tent (and hence a higher content of the TF-VII ccrrplex). The possibility that the specific factor X-activating activity of colorectal tissue might be due to tissue factor-factor VII interaction is also indirectly Firstly, it has been supported by several observations by other investigators. suggested that FXAA might represent a new type of ganma-carboxylated protein dependent on vitamin K for its synthesis (21). This hypothesis is based on the effect of oral anticoagulants and experimental vitamin K deficiency on the growth and spread of various experimental turnours (22,231 and reduction in cellular procoagulant activity (23,241. Whether the procoagulant described in the present study is vitamin K-dependent is not yet known. However, the fact that this activity is dependent on calcium ions and binds to aluminium hydroxide and barium salts is corrpatible with the presence of gamma-carboxyglutamic acid residues (25), and the apparent identity of the factor X activator to plasma factor VII would also support this hypothesis. Secondly, recent evidence suggests that the procoagulant associated with Lewis Lung Carcinoma (3LL) cells (12) is not a single protein, but is composed of a cellular cmnt, and a vitamin K-dependent serum factor, possibly factor VII (26). Thirdly, the factor X-activating activity found in arouse exudate macrophages appears to be a serine protease, and apparently results from the interaction and corrplexing of membrane-bound tissue factor and a factor VIIlike substance (27). However, in the latter two studies, the identity of factor VII was not confirmed immunologically. Finally, the procoagulant activity of miotic fluid, which directly activates factor X, has properties compatible with those of a stable thromboplastin-factor VII complex (28). Interestingly, in a separate report (291, the amniotic fluid procoagulant was found to be inmunologically identical to "Cancer Procoagulant" derived from rabbit V2 carcinoma. In conclusion, the procoagulant described in the present study, although capable of activating factor X directly, does not appear to be identical to the cysteine protease previously described in several experimental and hunan tumxlt-s, including colorectal adenocarcincina. Our data suggest that factor X-

activating colorectal

215

CLOTTING FACTORS IN RECTAL TISSUE

Vol. 52, No. 3

activity turmurs,

is a property of and is the result

both normal colonic mucosa and malignant of tissue factor-factor VII interaction.

ACKN(WLED(MENTS We are grateful patients under

to Mr McGinn, their care.

Mr Royle

and Mr Steer

for

allowing

us to study

Medicale

de l’Hote1

and Therapeutics, London, 1878.

translated

REFERENCES

Phlegmasia alba dolens. In: Vol 3, 1865, pp. 654-656,

Clinioue

de

1.

TROUSSEAU, A. Paris Balliere, __?

2.

BILLROTH, T. frm the 8th

3.

RICKLES, F.R. and EDWARDS, R.L. Trousseau’s Syndrome revisited.

4

O’MEARA, R.A.Q. 479, 1958.

5.

SAKURAGAWA, N., TAKAHASHI, K., HCSHIYAMA, M., JIME?O, C., ASHIZAMA, K., MATSATUOAKA, M. and OHNISHI, Y. The extract from the tissue of gastric cancer as procoagulant in disseminated intravascular coagulation syndrome. Thrombosis Res. 10, 457-463, 1977.

6.

SZCZEPANSKI, M., LUCER, C., and fibrinolytic activities Cancer 30, 329-333, 1982.

7.

MUSSONI, L., CONFORTI, G., GAMBACCRTI-PASSERINI, C., ALESSIO, G., PEPE, S., VAGHI, M., ERBA, E., AMATO, G., LANDINI, F., MANGICNI,C., MXASCA, L., SEMERARO, N. and DCNATI, M.B. Procoagulant and fibrinolytic activity of human ovarian carcinoma cells in culture. Eur. J. Cancer Clin. Oncol. 22, 373-380, 1986.

8.

MEMOLI, V.A. and ROUSSEAU, S.M. Coagulation-cancer ZACHARSKI, L.R., Blood 68, 394-399, interaction in situ in renal cell carcinoma.

Lectures edition,

on Surgical Pathology New Sydenham Society,

Coagulative

Activation Blood 62,

properties

of blood coagulation 14-31, 1983.

of cancers.

Irish.

J.

in cancer:

Med. 394,

474-

ZAWADZKI, J. and TOLLOCZKA, T. Procoagulant of gastric and colorectal cancer. Int. J.

9.

PINEO, G.F., coagulation coagulation 1973.

10

GORDON, S.G., FRANKS, J.J. and LEWIS, activating procoagulant from malignant 137, 1975.

1986.

REGOECZI, E., HATTON, M.W.C. and BRAIN, M.C. The activation by extracts of mucus: A possible pathway of intravascular J. Lab. clin Med. 82, 255-266, accompanying adenocarcinomas.

B. Cancer tissue.

procoagulant A: A factor Thrombosis Res. 6, 127-

of

X-

CLOTTING FACTORS IN RECTAL TISSUE

216

Vol. 52, No. 3

11.

CURALOLO, L., COLUCCI, M., CAMBINI, A.L., -1, M.B. and SEMERARO, N. Evidence that cells from activate factor X. Br. J. Cancer 40, 228-233,

12.

HILGARD, P. and WHUR, P. Factor X-activating carcinoma. Br. J. Cancer 41, 642-643, 1980.

13.

FRANKS, J.J. and LEWIS, B. Comparison of procoagulant GoRm, S.G., activities in extracts of normal and malignant human tissue. J. Natl. Cancer Inst. 62, 773-776, 1979.

14.

WNATI, M.B., GAMBACCRTI-PASSERINI, C., CASALI, B., FALANGA, A., VANNCTTI, P . , FOSSATI , G. , SEMERARC, N. and GORDON, S.G. Cancer procoagulant in human tumor cells: Evidence from melanoma patients. Cancer Res. 46, 6471-6474, 1986.

15.

FALANGA, A. and GORDON, S.G. Isolation and characterization procoagulant: A cysteine proteinase from malignant tissue. 34, 5558-5567, 1985.

16.

DOVER, R., GOETING, N.L.M., TAYLOR, I., Factor X-activating activity in patients J. Surg. 74, 1122-1124, 1987.

17.

HUBBARD, A.R. and JENNINGS, C.A. Inhibition of the complex: involvewnt of factor Xa and lipoproteins. 527-537, 1987.

18.

GORDON, S.G. and LEWIS, B.J. Comparison of culture medium from normal and transformed 2467-2472, 1979.

19.

A factor X-activating GORDON, S.G. and CROSS, B.A. J. Clin. Invest. 67, 1665-1671, malignant tissue.

20.

GILBERT, activity Res. 43,

21.

DELAINI, F., CCLUCCI, M., De BELLIS VITTI, G., LOCATI, D., -1, SEMERARC, N. and DCNATI, M.B. Cancer cell procoagulant: A novel dependent activity. Thrombosis Res. 24, 263-266, ,198l.

L.C. and GORDON, S.G. and metastatic capacity 536-540, 1983.

A., MORASCO, L., DCNATI, experimental tumours can 1979. activity

from

Lewis

Lung

of cancer Biochemistry

ROATH, O.S. and FRANCIS, J.L. with colorectal carcinoma. Brit A

tissue factor-factor Thrombosis Res. 46,

procoagulant f i broblasts.

activity Cancer

cysteine 1981.

VII

in tissue Res. 38,

protease

from

Relationship between cellular procoagulant Cancer of B16 mouse melanoma variants.

A., vitamin

K-

22.

Cancer ccl 1 procoagulants and their DCNATI, M.B. and SEMERARC, N. Haemostasis 14, 422-429, 1984. pharmacological modulation.

23.

COLUCCI, M., DELAINI, F., de BELLIS VITTI, G., LCCATI, D., FCGGI, A., Warfarin inhibits both procoagulant activity SEMERARO, N. and DCNATI, M.B. Biochem. Pharmacol and metastatic capacity of Lewis Lung carcinoma ccl 1s. 32, 1689-1691, 1983.

24.

POGGI, A., CCLUCCI, M., DELAINI, F., SEMERARC, N. and CONATI, procoagulant activity of Lewis Lung carcinoma cells from mice 1980. warfarin. Eur. J. Cancer 16, 1641-1642,

M.B. Reduced treated with

.

Vol. 52, No. 3

25.

26.

217

CLOTTING FACTORS IN RECTAL TISSUE

Synthesis and secretion of vitamin K-dependent clotting SUTTIE, J.W. factors. In: Plasma Protein Secretion by the Liver H. Glaumann, T. and C. Redman (eds), Academic Press, London, 1983, pp. 375-403.

Peters

Evidence for a FASCC, M.J., WILSON, A.C., LINCOLN, D. and GIERTHY, J. warfarin-sensitive serum factor that participates in factor X activation Lewis Lung turm- cells. Int. J. Cancer 39, 631-637, 1987. Macrophage factor X activator for synthesis of ccmponents.

27.

SHANDS, J.W. requirements

28.

PUSEY, M.L. and MENDE, T.J. amniotic fluid II. The role 1985.

29.

GCi?DoN, S.G., HASIBA, U., CROSS, B.A., POOLE, M.A. and FALANGA, A. Cysteine proteinase procoagulant frm arrmion-chorion. Blood 66, 1261-1265, 1985.

Studies on the of factor VII.

formation: Blood 65,

by

Metabolic 169-175, 1985.

procoagulant Thrombosis

activity Res. 39,

of human 571-585,