Proteins and disulfide groups in the aggregation of dissociated cells of sea sponges.

A sponge extract that produced specific aggregation of dissociated cells was treated with various enzyme preparations to determine which enzymes would destroy its aggregating properties. The results indicate that proteins play a key role in the aggregating effect of the extract on dissociated, glutaraldehyde-fixed sponge cells. Further studies confirm the necessity of calcium for the aggregation and indicate the necessity of intact disulfide groups.

Inhibition of lung tumor colonization by leech salivary gland extracts from Haementeria ghilianii.

Salivary gland extract from the South American leech Haementeria ghilianii, administered i.v. on the same day as the i.v. inoculation of T241 sarcoma cells, completely suppresses colonization of the mediastinal lymph nodes and markedly reduces the number and size of lung tumor colonies produced by this tumor. Additional studies indicate that the extract contains various types of proteinase inhibitors and has the capacity to inhibit clotting and platelet aggregation by tumor material and collagen. Although not yet proved by direct evidence, these activities may be involved in the inhibitory effect of lung tumor colonization by the leech extract.

Aggregation of platelets and cell membrane vesiculation by rat cells transformed in vitro by Rous sarcoma virus.

Primary rat embryo cells and established normal rat kidney cells transformed in vitro by Rous sarcoma virus induced the aggregation of rat platelets in vitro. The aggregating activity was shown to be specific for the transformed cells and was absent in the normal parent cells. The aggregation reaction is accompanied by the release of serotonin from the platelets. Further analysis and purification of this activity from the transformed cells demonstrated that the activity is shed from the cells growing in culture and is associated with membrane vesicles of heterogenous size. The normal cells also produced vesicles in culture; however, the level of vesicle productio was less than that from transformed cells, and the platelet aggregation and serotonin release activities were greatly reduced or absent in these vesicles.

Thrombospondin, a potentiator of tumor cell metastasis.

The platelet-secreted protein thrombospondin (TSP) potentiates tumor cell metastasis. Human TSP injected i.v. into mice 5 min prior to i.v. injection of T241 sarcoma cells potentiates lung tumor colony formation. Several lines of evidence suggest that the TSP-enhancing effect involves both TSP-mediated tumor cell adhesion and the host's hemostatic system: TSP potentiates the initial, rapid sequestering of tumor cells in the lung; TSP promotes the adhesion of tumor cells in vitro; the effect of TSP on tumor cell metastasis is dependent on the presence of platelets and a normal plasma clotting system, since TSP does not potentiate lung tumor colony formation in either thrombocytopenic mice or mice anticoagulated with Coumadin. Our results suggest a central role for TSP in the metastatic process.

Leech salivary gland extract from Haementeria officinalis, a potent inhibitor of cyclophosphamide- and radiation-induced artificial metastasis enhancement.

Studies were designed to determine whether salivary gland extract (SGE) from the leech Haementeria officinalis could inhibit enhancement of lung tumor colonization induced by pretreatment of mice with cyclophosphamide (CY) or local thoracic irradiation (LTI). Tumor nodules in the lung were generated by i.v. injections of T241 sarcoma and FSA fibrosarcoma cells into syngeneic C57BL/6 and C3Hf/Kam mice, respectively. CY (200 mg/kg) was given i.p. 1 or 4 days prior to i.v. injection of tumor cells. In other mice, a single dose of 1000 rads of LTI was given 1 day before tumor cells. Three i.v. or i.p. injections of SGE at doses of 600 to 800 micrograms of protein per injection given at 2-hr intervals between 2 hr before and 4 hr after CY, LTI, or tumor cell injection strongly inhibited and, in some cases, abolished the artificial metastasis enhancing effect of CY and LTI. SGE was similarly effective in inhibiting the enhancement of lung colonization when given before or after cytotoxic agents. Using [125I]iododeoxyuridine-labeled tumor cells, it was observed that SGE did not affect the initial lodgement of tumor cells in the lung, but it greatly facilitated their subsequent release from the lung. In normal mice, the SGE was active when given on the day or 1 day before but not when given 4 days before tumor cells. The antimetastatic effect of SGE was ascribed to its anti-platelet-aggregating, anticoagulant, and antiproteolytic enzyme activities.

Cloning and expression of cDNA encoding antistasin, a leech-derived protein having anti-coagulant and anti-metastatic properties.

As a factor Xa inhibitor, antistasin is a potent anti-coagulant and anti-metastatic agent that is found in the salivary gland of the Mexican leech Haementaria officinalis. cDNA clones that encode antistasin have been isolated. Subsequent sequence analysis and comparison with the amino acid sequence of the mature protein indicates that antistasin is produced as a pre-protein containing a 17-amino acid signal peptide. Antistasin exists as at least two variants. By sequence analysis of multiple cDNA clones, we found two additional sites for amino acid substitutions, confirming variants that differ from each other by amino acid changes at a minimum of four residues. These sequence variations appear to be the result of allelic variation rather than gene duplication as deduced from DNA blot analyses. Sequence data suggest that antistasin may have evolved from a smaller ancestral gene by a duplication event giving rise to a two-fold structural homology between the N- and C-terminal halves of the molecule. Insect cells transfected with a recombinant baculovirus expressed antistasin which was biologically active and had an electrophoretic mobility identical to that of the native molecule.

Promotion of lung tumor colonization in mice by the synthetic thrombin inhibitor (no. 805) and its reversal by leech salivary gland extracts.

The role of anticoagulation per se in the reduction of experimental or spontaneous metastasis still remains to be determined, as shown by the conflicting results reported by the literature using different conventional anticoagulants. A new compound has been synthesized (compound no. 805) which prolongs or suppresses coagulation via specific inhibition of thrombin and its possible use in a model of experimental metastasis to clarify the role of anticoagulants in tumor spread was investigated. Contrary to our expectations, this compound increased rather than decreased the number of lung colonies induced by intravenous injections of a variety of murine neoplasias. Studies of the mechanism of this effect indicated that the compound increases retention of tumor cells by the lung without apparent impairment of the natural cell immune system, suggesting that the synthetic thrombin inhibitor may enhance vascular attachment of tumor cells. The promoting effect of compound no. 805 on metastasis was totally reversed by the administration of leech salivary gland extracts, which appear to protect capillaries from damage produced by cyclophosphamide, as revealed by other studies.

Involvement of a cathepsin B-like cysteine proteinase in platelet aggregation induced by tumor cells and their shed membrane vesicles.

Murine 15091A mammary adenocarcinoma cells and membrane vesicles spontaneously shed from these tumor cells in culture can induce aggregation of washed human platelets. A spectrum of proteinase inhibitors was tested for their ability to inhibit 15091A induced platelet aggregation. Of the inhibitors tested the most effective were those selective for cysteine proteinases. The effect of the spectrum of proteinase inhibitors on 15091A induced platelet aggregation was compared to the effect on cathepsin B-like cysteine proteinase activity in homogenates of 15091A tumor cells and their spontaneously shed vesicles. The results suggest that there is a correlation between activity of a cathepsin B-like proteinase in 15091A cells and vesicles and the ability of these cells and vesicles to induce aggregation of washed human platelets.

Protease inhibitors in Haementeria leech species.

Inhibitors, of trypsin, plasmin, alpha-chymotrypsin and granulocyte elastase were demonstrated in salivary gland extracts from two species of leeches. Haementeria ghilianii and Haementeria officinalis. Preliminary fractionation of salivary gland extracts from Haementeria ghilianii allowed separation of protease inhibitors from hementin a fibrinogenolytic blood anticoagulant. It was found that the anticoagulant activity resided only in hementin-containing fractions and did not parallel protease inhibitory activity.

Role of plasma, platelets, and endothelial cells in tumor metastasis.

This review studies interactions of tumor cells with a particular host system which is normally responsible for hemostasis and the physiological integrity of the blood vessel luminal surface. With malignancy components of this system are frequently activated, producing abnormalities of blood coagulation, increased platelet responses, and conditions favoring tumor growth and metastasis. Activation of the clotting cascade is mediated by tumor and macrophage procoagulants, acting via Factor X or VII. Thrombin and fibrin are formed. Thrombin also interacts with platelets and the endothelium, potentiating or decreasing coagulation. Generation of thrombin or other tumor mechanisms activate platelets, leading to direct aggregation or secretion of ADP, serotonin, and/or intermediates of the arachidonate metabolism. Vascular lesions caused by tumor attack, platelet secretion, or exogenous agents promoting metastasis may also activate the hemostatic system. It is not yet fully understood how activation of the clotting system, including platelets, contributes to metastasis. Secretion of platelet products appears, however, to be heavily involved. Based on putative mechanisms of action, anticoagulants, platelet inhibitors, thrombocytopenic or vascular repairing agents have been used to control tumor spread. Results depended on the agent and experimental model of metastasis used. Except for coumarin, which was beneficial even against spontaneous metastases, other anticoagulants and platelet inhibitors, excluding perhaps Nafazatrom, gave equivocal results. Thrombocytopenic agents, however, were effective in every tumor system and with any experimental model of metastasis, indicating that platelets play a role in this process. Also consistent were the inhibitory effects of leech salivary gland extract (probably a vascular repairing agent) against lung tumor colonization promoted by ionizing radiation, cyclophosphamide, and cortisone.

Total suppression of pregnancy in mice by post-coital administration of neuraminidase.

Because of certain analogies between the spread of tumors and different steps of pregnancy culminating in egg implantation and invasive growth, pregnant mice were treated at different intervals post coitum with two thrombocytopenic agents which can reduce metastases by interfering with vascular lodgement and growth of tumor cells. It was found that both neuraminidase and antiplatelet serum were active against pregnancy, the enzyme being by far the more effective of the two. The mechanism of this effect involves both egg implantation and egg development. The effect cannot be explained solely by the thrombocytopenia produced by both agents.

Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation.

The purpose of this study was to purify and characterize the agent responsible for the antimetastatic activity of an extract of the salivary glands (SGE) of the Mexican leech Haementeria officinalis. When administered intravenously in mice on the same day as the intravenous inoculation of T241 sarcoma cells, SGE markedly reduces the number and size of lung tumor colonies. In designing a purification protocol for the antimetastatic agent, we postulated that the antimetastatic agent would also display anticoagulant activity. Thus, we discovered that heparin affinity chromatography followed by anion-exchange chromatography results in a fraction highly enriched in both potent anticoagulant activity and potent antimetastatic activity. Approximately, 200-300 micrograms of purified protein is isolated from 150 mg of SGE. As little as 15 micrograms of this material inhibits tumor cell metastasis to the same extent as 1.0 mg of the unfractionated SGE. When analyzed on sodium dodecyl sulfate gels the active fraction consists mainly of one polypeptide band having an apparent molecular weight of approximately 17,000 under either reducing or nonreducing conditions. The protein has a pI of approximately 9.5 and a molecular weight of approximately 17,000 under nondenaturing conditions. A specific antiserum prepared against the 17,000-dalton protein indicated that this protein is the major anticoagulant and antimetastatic agent of leech salivary gland extract. We have termed this anticoagulant, antimetastatic agent "antistasin." We hypothesize that antistatin inhibits coagulation via factor Xa, and not thrombin, since factor Xa, but not thrombin, is rapidly inactivated upon addition of antistasin. The mechanism of antistasin's antimetastatic activity is currently under investigation.

Platelet aggregating material in mouse tumor cells. Removal and regeneration.

The platelet aggregating principle of two mouse ascites tumors and of their cell-free supernatants released spontaneously has been studied. It was found that the principle disappeared from the cells after trypsin digestion and that part of it was recovered in the cell-free trypsinate. Digested cells regenerated the principle during subsequent incubation by a process requiring protein synthesis. The principle was found to be spontaneously released by intact cells into the medium and sensitive to proteolytic attack. The principle was not present in five varieties of nonneoplastic cells. Since previous work by the authors indicates that the principle is present in numerous other tumor cell lines, its study might reveal it to be an indicator of malignant transformation or malignant progression.

Abortifacient effects of Vibrio cholerae exo-enterotoxin and endotoxin in mice.

To study antifertility properties of microbial toxins, exoenterotoxin and endotoxin from Vibrio cholerae were injected intravenously into mice at different times during pregnancy. The two substances induced termination of pregnancy, but the patterns of abortifacient activity were different. Exotoxin terminated pregnancy in mice when administered between Days 4 and 10 of gestation, but abortifacient activity was reduced in animals more than 10 days pregnant; exogenous progesterone did not protect the pregnancies. Endotoxin was most effective in terminating pregnancy when injected after mid-gestation and the active principle was heat-stable; exogenous progesterone was not able to prevent the effects of endotoxin. Animals treated with endotoxin on Day 17 often gave birth to live young prematurely; indomethacin reduced the incidence of premature littering. The results demonstrate that exo- and endotoxins have antifertility properties and both appear to act on intrauterine targets rather than inducing progestin deficiency.

PIP: Examination of the effects of exo-enterotoxin and endotoxin from Vibrio cholerae following intravenous injection into mice at different stages of gestation. Although the patterns of abortificient activity were different, both substances induced termination of pregnancy. When administered between gestation days 4 and 10, exotoxin terminated pregnancy. However, animals more than 10 days pregnant exhibited reducted abortificient activity. Endotoxin, when injected after mid-gestation and when the active principle was heat-stable, was the most effective pregnancy terminator. Exogenous progesterone did not protect the pregnancies nor was it able to prevent the endotoxic effects. Premature parturition was induced in 1/2 to 2/3 of the mice receiving endotoxin on gestation Day 17 while indomethacin reduced the incidence of premature littering. The study indicates that exoand endotoxins exhibit antifertility properties. Rather than inducing progestin deficiency, both appear to act on intrauterine targets.

Thrombogenic activity of mouse and human tumors: effects on platelets, coagulation, and fibrinolysis, and possible significance for metastases.

Twelve mouse tumors and 29 human malignancies were assayed in vitro for their capacity to aggregate platelets and induce release of radiolabelled serotonin, and for their ability to coagulate blood plasma and digest the fibrin clot. It was discovered that many human and mouse tumors can induce release of radiolabelled serotonin but that the quantitative relationships between this activity of tumors and their capacity to aggregate platelets was variable, permitting tumors to be classified into 3 different types. The procoagulant and fibrinolytic activity was also quite variable. Since no correlation was found between the 4 assayed tumor activities they appear to be independent, separate thrombogenic properties of tumors. Although the information gathered by this study is still fragmentary, some speculations can be made about the role of these activities in treatment of malignant tumors and in determing patterns of body distribution and control of metastases.

Antistasin, an inhibitor of coagulation and metastasis, binds to sulfatide (Gal(3-SO4) beta 1-1Cer) and has a sequence homology with other proteins that bind sulfated glycoconjugates.

Antistasin, a 15-kDa salivary protein from the Mexican leech Haementeria officinalis, inhibits both blood coagulation and the metastasis of tumors (Tuszynski, G. P., Gasic, T. B., and Gasic, G. J. (1987) J. Biol. Chem. 262, 9718-9723). Antistasin binds to heparin-agarose, suggesting the protein interacts with sulfated glycoconjugates. The specificity of the interaction between antistasin and heparin was tested by measuring the binding of antistasin to various lipids and by comparing the ability of several charged glycoconjugates to inhibit binding. Of the lipids tested, antistasin binds with high affinity only to sulfatide (Gal(3-SO4)beta 1-1Cer) and does not bind to comparable levels of phospholipids, neutral glycosphingolipids, gangliosides, or cholesterol-3-SO4. The binding of antistasin to sulfatide is inhibited by dextran sulfate, fucoidan, and heparin, with I50 values of 1.5, 9.2, and 16 micrograms/ml, respectively. Comparable levels of chondroitin sulfates A, B, C, keratan sulfate, or hyaluronic acid do not inhibit binding. Comparisons of the amino acid sequences of antistasin and other sulfatide or heparin-binding proteins revealed a region of homology, based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg, which may be a sulfated glycoconjugate binding domain. In addition, homologies were found with the alternate complement pathway protein properdin and coat proteins from malaria circumsporozoites and Herpes simplex I.

Coagulation factors X, Xa, and protein S as potent mitogens of cultured aortic smooth muscle cells.

Smooth muscle cells (SMCs) in the rat carotid artery leave the quiescent state and proliferate after balloon catheter injury. The precise signals responsible for this SMC mitogenesis need to be elucidated. Although platelet-derived growth factor (PDGF), a potent SMC mitogen, is released from activated platelets, damaged endothelium, and macrophages, it cannot be solely responsible for this proliferation. In search of other SMC growth factors, we have examined several proteins of the coagulation cascade. At nanomolar concentrations, factors X, Xa, and protein S promote cultured rat aortic SMC mitosis. In contrast, factor IX is only weakly mitogenic, whereas factor VII and protein C fail to stimulate SMC division. Protein S, the most mitogenic of these coagulation cascade factors, stimulates DNA synthesis in cultured SMCs with a time course similar to that of PDGF-AA and without the delay observed for transforming growth factor beta. Antistasin and tick anticoagulant peptide, two specific factor Xa inhibitors, inhibit SMC mitogenesis due to Xa and protein S. Coagulation factors that possess mitogenic activity may contribute to intimal SMC proliferation after vascular injury as a result of angioplasty or vascular compromise during atherogenesis.

Comparison of basement membrane matrix degradation by purified proteases and by metastatic tumor cells.

We have examined the nature of biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) using different methodologies. The first strategy was quantitation of the release of surface-bound 125I and a second the documentation by SDS-PAGE of the appearance of putative cleavage products and the loss of high-molecular-weight components from the matrix. Basement membrane matrix bands resolved on SDS-PAGE were identified by their protease sensitivities as well as by Western immunoblots using monoclonal antibodies developed for this study. Radioiodinated components were found predominantly at positions on the gel equivalent to 160-200 kd and 400 kd proteins. Since these labeled moieties were sensitive to bacterial collagenase digestion and stained with anticollagen type IV antibodies, they were determined to represent various configurations of collagen type IV. Several other lower-molecular-weight bands also stained with the anticollagen IV antibodies. Monoclonal antibodies reactive with laminin exhibited a complex staining pattern on the gels, which included the expected 200 and 400 kd components. We confirmed that lens capsule basement membrane contained only a single heparan sulfate glycosaminoglycan species, and tumor cell-induced glycosaminoglycan degradation within the basement membrane matrix was detected using cellulose acetate electrophoresis. Distinctive putative cleavage products were resolved on SDS-PAGE gels from matrices subjected to digestion by a variety of purified proteases as well as by metastatic tumor cells or their conditioned media. Tumor cells of different histiotypes produced different characteristic cleavage patterns, suggestive of the existence of several pathways of matrix degradation. Overall, primary tumor cells exhibited a greater degradative activity towards the basement membrane matrix than did long-term tissue culture-passaged cells. The same tumor cell line could exhibit considerably different patterns of both protein and glycosaminoglycan degradation depending on recent culture history. The relevance of these biochemical studies to the pathogenesis of malignant neoplasms is shown by: 1) the evaluation of degradative activities of B16 tumor cell populations exhibiting enhanced lung-colonizing phenotypes, and 2) the ability of a known antimetastatic moiety with antiprotease activity (Haementeria leech species salivary gland extract) to protect matrix components from degradation by tumor cell-conditioned medium.

The amino acid sequence of antistasin. A potent inhibitor of factor Xa reveals a repeated internal structure.

Antistasin is a 15-kDa protein from the salivary glands of the Mexican leech, Haementeria officinalis, which manifests anticoagulant activity by inhibiting factor Xa. Previous work demonstrating the presence of this activity in salivary gland extracts and its partial purification has been reported (Tuszynski, G. P., Gasic, T. B, and Gasic, G.J. (1987) J. Biol. Chem. 262, 9718-9723). The present study includes further purification to homogeneity of antistasin and its subsequent fragmentation and complete amino acid sequence determination. The protein, which possesses 119 amino acid residues, is blocked at its amino terminus by the presence of a pyroglutamic acid residue and has an unusually high cysteine content, with 20 cysteine residues. The primary structure of antistasin shows no homology to hirudin, a 65-residue anticoagulant protein from the medicinal leech, Hirudo medicinalis. Of great interest is the finding of significant internal homology within antistasin where a 2-fold internal repeated structure is observed. At least four isoforms of antistasin have been identified in leech salivary gland extracts by high performance liquid chromatography analysis, and partial amino acid sequence analysis of these isoforms indicates they differ by 1 or 2 amino acid residues.