Open Access Rambam Maimonides Medical Journal CLINICAL IMPLICATIONS OF BASIC RESEARCH Heparanase—A Link between Coagulation, Angiogenesis, and Cancer Yona Nadir, M.D., Ph.D.,* and Benjamin Brenner, M.D. Thrombosis and Hemostasis Unit, Department of Hematology, Rambam Health Care Campus and Technion–Israel Institute of Technology, Haifa, Israel ABSTRACT Heparanase that was cloned from and is abundant in the placenta is implicated in cell invasion, tumor metastasis, and angiogenesis. Recently we have demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. Heparanase was shown to up-regulate tissue factor (TF) expression and interact with tissue factor pathway inhibitor (TFPI) on the cell surface, leading to dissociation of TFPI from the cell membrane of endothelial and tumor cells, resulting in increased cell surface coagulation activity. More recently, we have shown that heparanase directly enhances TF activity, resulting in increased factor Xa production and activation of the coagulation system. Data indicate increased levels and possible involvement of heparanase in vascular complications in pregnancy. Taking into account the prometastatic and proangiogenic functions of heparanase, overexpression in human malignancies, and abundance in platelets and placenta, its involvement in the coagulation machinery is an intriguing novel arena for further research. KEY WORDS: Heparanase, tissue factor, tissue factor pathway inhibitor, cancer INTRODUCTION Cancer patients have a prothrombotic state between procoagulants and anticoagulants.1 because of the ability of cancer cells to activate the Overexpression of tissue factor (TF), cancer coagulation system and to interact with procoagulant—a cysteine protease that activates hematopoietic cells, thus tilting the balance factor X—and acquired activated protein C Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ECM, extracellular matrix; HS, heparan sulfate; HUVEC, human umbilical vein endothelial cell; IUGR, intrauterine growth-restricted; LMWH, low- molecular-weight heparin; MM, multiple myeloma; SNPs, single nucleotide polymorphism; TF, tissue factor; TFPI, tissue factor pathway inhibitor; VEGF, vascular endothelial growth factor. Citation: Nadir Y, Brenner B. Heparanase—A Link between Coagulation, Angiogenesis, and Cancer. RMMJ 2012;3 (1):e0002. doi:10.5041/RMMJ.10069 Copyright: © 2012 Nadir and Brenner. This is an open-access article. All its content, except where otherwise noted, is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conflict of interest: No potential conflict of interest relevant to this article was reported. * To whom correspondence should be addressed. E-mail: [email protected] RMMJ|www.rmmj.org.il 1 January 2012  Volume 3  Issue 1  e0002 Heparanase—A Link between Coagulation, Angiogenesis, and Cancer resistance,2 are thought to be the main factors for PROMETASTATIC PROPERTIES OF coagulopathy in malignant disorders. TF is a trans- HEPARANASE membrane receptor that is constitutively The clinical significance of the enzyme in tumor expressed in tumors, i.e. human leukemias, progression emerges from a systematic evaluation lymphomas, adenocarcinomas, and sarcomas.3 TF of heparanase expression in primary human also plays a role in cellular signaling, contributing tumors. Immunohistochemistry, in-situ hybridiza- to tumor growth and metastasis.3,4 The only tion, RT-PCR, and real-time PCR analyses known endogenous modulator of blood coagula- revealed that heparanase is up-regulated in tion initiated by TF is tissue factor pathway inhib- essentially all human tumors examined. These itor (TFPI)—a plasma Kunitz-type serine protease include carcinomas of the colon,17,18 thyroid,19 inhibitor.5,6 Growing evidence suggests the liver,20 pancreas,21,22 bladder,23,24 cervix,25 breast,26 involvement of tumor-derived proteins, including gastric,27,28 prostate,29 head and neck,30,31 as well heparanase, in activation of the coagulation as multiple myeloma,32 leukemia, and lymph- system. oma.33 In most cases, elevated levels of heparanase were detected in about 50% of the HEPARANASE tumor specimens, with a higher incidence in Heparanase is an endo-β-D-glucuronidase capable pancreatic (78%) and gastric (80%) carcinomas, of cleaving heparan sulfate (HS) side chains at a and in multiple myeloma (86%). In all cases, limited number of sites, yielding HS fragments of normal tissue adjacent to the malignant lesion still appreciable size (~5–7 kDa).7,8 Heparanase expressed little or no detectable levels of activity has long been detected in a number of cell heparanase, suggesting that epithelial cells do not types and tissues. Importantly, heparanase activity normally express the enzyme. In several correlated with the metastatic potential of tumor- carcinomas, most intense heparanase staining was derived cells, attributed to enhanced cell localized to the invasive front of the tumor,23,28,30 dissemination as a consequence of HS cleavage supporting a role for heparanase in cell invasion. and remodeling of the extracellular matrix (ECM) Furthermore, patients that were diagnosed as barrier.9,10 Similarly, heparanase activity was heparanase-positive exhibited a significantly implicated in neovascularization, inflammation, higher rate of local and distant metastasis as well and autoimmunity, involving migration of as reduced postoperative survival, compared with vascular endothelial cells and activated cells of the patients that were diagnosed as heparanase- immune system.9–11 A single human heparanase negative.18,22,23,28,32 Collectively, these studies cDNA sequence was independently reported by provide strong clinical support for the promet- several groups.12–15 Thus, unlike the large number astatic function of heparanase. Interestingly, of proteases that can degrade polypeptides in the patient survival was noted to correlate not only ECM, one major heparanase appears to be used by with heparanase levels, but also with its cells to degrade the HS side chains of HS localization. In addition to its presence in the proteoglycans. Expression of heparanase is cytoplasm, heparanase was also noted to assume restricted primarily to the placenta, keratinocytes, nuclear localization, demonstrated by cell platelets, and activated cells of the immune fractionation,34 and by immunostaining of system, with little or no expression in connective cultured cells34 and tumor biopsies.27,35 Inter- tissue cells and most normal epithelia.9,10 Up- estingly, nuclear localization was correlated with regulated expression of heparanase was noted in maintained cellular differentiation35 and favorable essentially all human tumors examined, as well as outcome of patients with gastric27,35 and head and in inflammation, wound healing, and diabetic neck carcinomas,36 suggesting that heparanase is nephropathy.9–11 During embryogenesis, the intimately involved in gene regulation. Whether enzyme is preferentially expressed in cells of the gene transcription and maintained cellular developing vascular and nervous systems.16 differentiation is due to direct interaction of Rambam Maimonides Medical Journal 2 January 2012  Volume 3  Issue 1  e0002 Heparanase—A Link between Coagulation, Angiogenesis, and Cancer heparanase with the DNA or is a consequence of and subsequent complex formation.41–43 More- heparanase-mediated nuclear-HS degradation is over, cell surface HS can facilitate the catabolism yet to be demonstrated. In addition, heparanase of coagulation factors such as factor VIII.44 Other up-regulation in primary human tumors coagulation inhibitors such as TFPI also associate correlated in some cases with larger tumors,20,26,28 with the luminal face of the endothelial cell and with enhanced microvessel density,18,20,24,32 plasma membrane via HS.45 HS is also important providing clinical support for the proangiogenic constituents of the subendothelial basement function of the enzyme. membrane, where they cross-link various com- ponents, e.g. laminin and collagens, thereby HEPARANASE POLYMORPHISMS contributing to the integrity of the blood vessel wall.46 HS, unfractionated heparin, and other Heparanase gene single nucleotide poly- heparin derivatives have been investigated as morphisms (SNPs) were characterized in Jewish heparanase inhibitors, and some of them exerted populations of Israel.37 Four Israeli Jewish pop- antimetastatic activity in animal models.47 Both ulations (Ashkenazi, North African, Mediter- the type of the polysaccharide backbone and the ranean, and Near Eastern) were examined for degree of sulfation seem to affect the heparanase- seven heparanase gene SNPs. Four out of seven inhibiting activity of sulfated polysaccharides.48,49 SNPs were found to be polymorphic. Population However, different heparin preparations display comparisons revealed significant differences in significantly different antiheparanase activity,48,49 SNPs allele frequency between Near Eastern and indicating that this activity is also dependent on each of the other three populations. Genotype and more subtle structural features. Recently, allele frequencies in Jewish populations were heparanase’s strong affinity to heparins was utilized different from non-Jewish populations, except for in vitro to reverse heparin’s effect. Heparanase was a certain similarity to Caucasians.37 Ostrovsky et shown to reverse the anticoagulant activity of al. found an association of heparanase gene SNPs unfractionated heparin on the coagulation with hematological malignancies.38 Genotype pathway as well as on thrombin activity. In frequency comparisons revealed a significant addition, heparanase abrogated the factor Xa association of specific SNPs with multiple inhibitory activity of low-molecular-weight myeloma (MM), acute myeloid leukemia (AML), heparin (LMWH). The procoagulant effects of and acute lymphoblastic leukemia (ALL) patients. heparanase were also exerted by its major Examination of heparanase gene mRNA functional heparin-binding peptide.50 expression by real-time RT-PCR indicated a significantly lower heparanase expression level in ALL patients and a higher expression level in MM HEPARANASE AS A CO-FACTOR TO TF and AML patients, compared to healthy controls.38 ACTIVITY The findings were not verified in ALL patients from Northern Ireland.39 Ralph et al. reported on Tissue factor is constitutively expressed in various an association between a specific heparanase SNP cell types, including pericytes adjacent to the and stage of ovarian cancer disease, while the vessel wall, but absent from the blood cell and association was not found in vascular endothelial endothelial cell surface. This localization is crucial growth factor (VEGF) SNPs.40 Further research is for hemostasis since it prevents a direct contact needed to explore the clinical relevance of between TF and the circulating blood. Immuno- heparanase polymorphism detection. histochemical studies revealed that many tumors express high levels of TF, including leukemia cells,51 raising the possibility of a TF role in the INTERACTION OF HEPARANASE WITH pathogenesis of cancer.1 We have demonstrated HEPARINS that heparanase overexpression in human Anticoagulant activities of cell surfaces have been leukemia, glioma, and breast carcinoma cells predominantly attributed to HS,41,42 which is results in a marked increase in TF levels verified by composed of repeating hexuronic and D- immunoblot and real-time PCR analyses.52 glucosamine sulfated disaccharide units. HS has Likewise, TF was induced by exogenous addition of been shown to exert anticoagulant activities on recombinant heparanase to tumor cells and cells, on ECM, and in tissues due to its catalyzing primary endothelial cells, induction that was function for protease inhibition by antithrombin mediated by p38 phosphorylation and correlated Rambam Maimonides Medical Journal 3 January 2012  Volume 3  Issue 1  e0002 Heparanase—A Link between Coagulation, Angiogenesis, and Cancer with enhanced procoagulant activity. TF induction HEPARANASE AND TFPI was further confirmed in heparanase- overexpressing transgenic mice and, moreover, TFPI is a plasma Kunitz-type serine protease correlated with heparanase expression levels in inhibitor and the only known endogenous leukemia patients.52 Lately, heparanase was found modulator of blood coagulation initiated by TF.5,6 to exert also non-enzymatic activities, inde- TFPI concentration in plasma is increased in pendent of its involvement in ECM degradation patients with acute myocardial infarction.57,58 and alterations in the extracellular micro- There are also reports on the plasma levels of TFPI environment.53 For example, inactive heparanase in relation to disseminated intravascular coag- enhances Akt signaling and stimulates PI3K- and ulation59 and to other diseases, such as diabetes p38-dependent endothelial cell migration and mellitus,60 renal diseases,61 and cancer.62,63 invasion.54 It also promotes VEGF expression via Recently we demonstrated that exogenous addi- the Src pathway.55 Up-regulation of TF adds tion or overexpression of heparanase by trans- another example of the multiple non-enzymatic fected cells resulted in release of TFPI from the functions of heparanase. Recently, we have cell surface and its accumulation in the cell culture demonstrated that heparanase may serve as a co- medium.64 Importantly, the in-vitro studies were factor of TF, suggesting that heparanase is directly supported by elevation of TFPI levels in the involved in activation of the coagulation cascade.56 plasma of transgenic mice overexpressing The findings were supported by experiments heparanase. Moreover, increased levels of TFPI indicating that heparanase increases the level of have been noted in the plasma of cancer factor Xa in the presence of TF/VIIa and the effect patients,62,63 reflecting, possibly, induction of is enzymatically independent. The newly heparanase expression and elevation of its plasma generated Xa had the same molecular weight as levels revealed by a newly developed ELISA Xa cleaved by TF/VIIa and was active as depicted assay.65 In human umbilical vein endothelial cell by increased conversion of prothrombin to (HUVEC) and tumor-derived cell lines, release of thrombin. Increased Xa generation in the TFPI from the cell surface correlated with presence of heparanase was shown to be relevant enhanced TF-mediated coagulation. This effect in the clinical setting. Thus, apart from the ability was evident already 30 min following heparanase of heparanase to increase Xa levels in normal addition and prior to the induction of TF52 or TFPI human plasma, a statistically significant positive expression. Thus, heparanase enhances local correlation was found in patients with acute coagulation activity by two independent leukemia and healthy donors between the plasma mechanisms: induction of TF expression52 and levels of heparanase and Xa.56 TFPI dissociation from the cell surface. Both Figure 1. A model of the interaction between heparanase (Hepa), TF, and TFPI. Heparanase interacts with TF resulting in increased generation of factor Xa and enhancement of the coagulation system. Heparanase also up-regulates TF expression and releases TFPI from the cell surface, rendering the cell surface highly procoagulant. TFPI and heparanase may circulate as a complex in the plasma. Rambam Maimonides Medical Journal 4 January 2012  Volume 3  Issue 1  e0002 Heparanase—A Link between Coagulation, Angiogenesis, and Cancer functions require secretion of heparanase, but not from placenta tissue. Additionally, estrogen was its enzymatic activity. The underlying mechanism found to up-regulate heparanase gene expression is apparently release of TFPI due to its physical in human endometrium71 and breast cancer.72 interaction with the secreted heparanase, as Recently, we investigated the role of heparanase in clearly evident by co-immunoprecipitation the placenta, focusing on its effect on TF, TFPI, experiments,64 reflecting a functional interaction TFPI-2, and VEGF-A.73,74 In these two studies between heparanase and a membrane protein. placenta samples of women with recurrent abortions and thrombophilia (weeks 6–10) were Elevated levels of heparanase may be compared to control cases of pregnancy generated locally upon degranulation of terminations and placentas of normal vaginal neutrophils, mast cells, and platelets,66 further deliveries, and intrauterine growth-restricted facilitating blood coagulation at the site of platelet (IUGR) babies were compared to control cases of activation. The hemostatic function of heparanase, elective cesarean sections, applying real-time RT- executed by inducing TF expression and releasing PCR and immunostaining. Sections obtained from TFPI from the endothelial cell surface, provides a miscarriages and vaginal and IUGR deliveries mechanism by which heparanase contributes to revealed increased (2–3-fold) levels of tumor complication, in addition to its established heparanase, VEGF-A, and TFPI-2 compared to proangiogenic and prometastatic activities.67,68 placentas from controls in maternal as well as in fetal placenta elements. A possible common A MODEL FOR INTERACTION BETWEEN denominator of the cases is vascular insufficiency: HEPARANASE, TF, AND TFPI in vaginal deliveries lasting intermittently for a few hours; in miscarriages and IUGR babies it Platelets and tumor cells have abundant amounts may represent a prolonged state. As heparanase of heparanase.53 Activation of the coagulation directly activates the coagulation system,56 system, including platelet activation, occurs in increased heparanase found in the placentas may malignant and angiogenic processes.69 contribute to placental vascular complications as Heparanase is directly involved in activation of the summarized in Figure 2. coagulation system by enhancing factor Xa production in the presence of the TF/VIIa complex. Additionally, heparanase released from activated platelets and tumor cells induce up- regulation of TF in the cells. Heparanase- mediated release of TFPI from the cell surface, together with its induction of TF, renders the cell surface highly procoagulant. Heparanase may also form complexes with TFPI and circulate in the plasma, possibly binding to endothelial cells and other intravascular components, i.e. platelets and microparticles. These aspects are depicted in Figure 1. Pregnancy causes an acquired hypercoagulable state, and women with a prior tendency to thrombosis may present with clinical symptoms of placental vascular complications. Maternal thrombophilia can be associated with placental Figure 2. Heparanase, TFPI-2, and VEGF-A are vascular events, although 30%–50% of vascular elevated in placentas with vascular insufficiency. gestational pathologies cannot be accounted for by A schematic summary of two studies73,74 implying the currently available tests for thrombophilia.70 elevated levels of heparanase, TFPI-2, and VEGF-A in Thus, an understanding of the hemostasis in the placentas of women with recurrent fetal losses, IUGR placenta, especially the dominant factors that deliveries, and normal vaginal deliveries. In these regulate the delicate hemostatic balance three conditions vascular insufficiency occurs. As throughout pregnancy, is essential. Heparanase is heparanase has a procoagulant role, it potentially can abundant in the placenta and was originally cloned contribute to thrombosis in these placentas. Rambam Maimonides Medical Journal 5 January 2012  Volume 3  Issue 1  e0002 Heparanase—A Link between Coagulation, Angiogenesis, and Cancer CONCLUSIONS 10. Vlodavsky I, Friedmann Y. Molecular properties and involvement of heparanase in cancer metasta- Heparanase was recently revealed as an important sis and angiogenesis. J Clin Invest 2001;108:341–7. modulator of blood coagulation. The elevation of 11. Dempsey LA, Brunn GJ, Platt JL. Heparanase, a heparanase levels in human tumors, together with potential regulator of cell-matrix interactions. the prothrombotic state of most neoplasms, Trends Biochem Sci 2000;25:349–51. Full Text suggests possible clinical relevance of the procoagulant function of heparanase. In addition 12. Hulett MD, Freeman C, Hamdorf BJ, Baker RT, Harris MJ, Parish CR. Cloning of mammalian hep- its increased levels in pregnancy vascular compli- aranase, an important enzyme in tumor invasion cations accentuate heparanase significance in and metastasis. Nat Med 1999;5:803–9. Full Text other proangiogenic states. In order to augment the understanding of heparanase we lately 13. Kussie PH, Hulmes JD, Ludwig DL, et al. Cloning developed an assay to evaluate heparanase pro- and functional expression of a human heparanase gene. Biochem Biophys Res Commun 1999;261: coagulant activity in the plasma,75 enabling further 183–7. Full Text extensive research in the field. Targeting domains of heparanase that mediate its enzymatic activity- 14. Toyoshima M, Nakajima M. Human heparanase. dependent and independent functions may prove Purification, characterization, cloning, and expres- beneficial for patients with proangiogenic and sion. J Biol Chem 1999;274:24153–60. Full Text prothrombotic conditions. 15. Vlodavsky I, Friedmann Y, Elkin M, et al. 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