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Keywords:

  • low-molecular-weight heparin;
  • metastasis;
  • thrombosis;
  • tissue factor pathway inhibitor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Summary.  The importance of coagulation activation in cancer patients is suggested by the clinical finding of hypercoagulability, experimental enhancement of metastasis and angiogenesis by coagulation factors such as tissue factor (TF) and thrombin and the possible antitumor effects of anticoagulant agents. Tinzaparin is a low-molecular-weight heparin (LMWH) with a relatively high molecular weight distribution and high sulfate to carboxylate ratio. In addition to its ability to inhibit thrombin and factor Xa, tinzaparin is particularly effective at releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of both procoagulant and non-coagulant effects of TF. The present study was undertaken to investigate the effect of tinzaparin on lung metastasis using a B16 melanoma model in experimental mice. Tinzaparin's anticoagulant effect in mice and its ability to release TFPI from human endothelial cells at various time points were demonstrated. Subcutaneous (s.c.) injection of tinzaparin (10 mg kg−1) 4 h before intravenous administration of melanoma cells (2.0 × 105) markedly (89%) reduced lung tumor formation (3 ± 2) compared with controls (31 ± 23; P < 0.001). In a second group of animals, tinzaparin (10 mg kg−1, s.c.) administered daily for 14 days following the initial (pretumor cell) dose, before assessment of lung seeding, reduced tumor formation by 96% (P < 0.001). No bleeding problems were observed in any of the tinzaparin-treated animals, despite a 4-fold prolongation of the whole blood clotting time after a single s.c. dose of tinzaparin (10 mg kg−1). Administration of tumor cells (2 × 106) caused a rapid and significant fall in platelet count 15 min after injection (a sensitive marker of intravascular coagulation) in controls (939 ± 37 vs. 498 ± 94 × 106 mL−1, P < 0.01), but this was prevented by tinzaparin treatment (921 ± 104 × 106 mL−1). These data provide further experimental evidence to support the potential for LMWH as antimetastatic agents.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The link between coagulation and malignancy has been recognized for over a century and is now well accepted [1,2]. Hypercoagulability in cancer patients is extremely common and is usually associated with a chronic low-grade intravascular coagulation [2,3]. Thrombosis is a common cause of death in cancer patients and pulmonary embolism (PE) is more commonly present in cancer patients studied at autopsy than in those without malignancy [4]. It may also be the presenting symptom of an underlying malignancy [5].

It is now evident that coagulation and platelet activation in malignancy may also have significance in the biology of tumor growth and dissemination. Thus, there is mounting evidence that components of tumor-induced hypercoagulability may not only lead to thrombosis, but may also enhance tumor angiogenesis and metastasis [6–8]. A number of factors that positively or negatively regulate angiogenesis are related to the coagulation pathway [9,10]. These include platelet-derived products such as vascular endothelial growth factor (VEGF), platelet factor 4, and thrombospondin, and terminal products of coagulation including fibrin and its fibrin degradation products. There has also been considerable research interest in a possible role of tissue factor (TF) in tumor biology. This protein, which normally acts as a cellular receptor for factor (F)VIIa and initiates the coagulation pathway [11], is present on the surface of many tumor cell types and is largely responsible for tumor cell procoagulant activity (PCA) [12]. Tumor cell-induced thrombin generation via TF/FVIIa and subsequent platelet activation has been shown to enhance hematogenous metastasis in experimental animal models [8,12]. TF has also been shown to promote metastasis via non-procoagulant functions [13].

The effects of unfractionated heparin have been studied both as a single agent and in combination with other drugs in animal models of malignancy. While some studies showed that heparin delayed the growth of implanted primary tumors [14], most demonstrated an inhibitory effect against metastasis [15–18]. Furthermore, co-operative effects of heparin with other molecules such as α2-glycoprotein, interferon and tumor necrosis factor against tumor growth and metastasis have been reported [19,20]. We previously demonstrated that the significant fall in peripheral blood platelet count that follows intravenous (i.v. tail vein) injection of procoagulant fibrosarcoma cells in experimental animals corresponds to the accumulation of platelets in the lungs (the organ of first encounter) [8]. Both tumor cell-induced thrombocytopenia and experimental metastasis can be significantly inhibited by warfarin as well as unfractionated heparin [8,21].

Tinzaparin (Innohep®; Pharmion Corporation, Boulder, CO, USA) is the most recent low-molecular-weight heparin (LMWH) to be approved by the US Food and Drug Administration. It is produced by controlled heparinase depolymerization of unfractionated porcine heparin and has an average molecular weight of 6500 Da (Fig. 1). Tinzaparin is indicated for the treatment of acute symptomatic deep vein thrombosis (DVT) with or without PE when administered in conjunction with warfarin [22]. Typically, tinzaparin is given subcutaneously once daily without the need for laboratory monitoring [23]. Similar to the other LMWHs, tinzaparin inhibits factors (F)Xa and FIIa by binding to the plasma protease inhibitor antithrombin. Furthermore, tinzaparin is particularly effective at releasing tissue factor inhibitor (TFPI) from endothelial cells [24] and we have previously shown that TFPI exerts significant antitumor effects in experimental models of hematogenous metastasis [25]. The aim of the present study therefore was to determine the effect of subcutaneous (s.c.) tinzaparin on tumor cell-induced thrombocytopenia and experimental metastasis.

image

Figure 1. Tinzaparin is produced by heparinase depolymerization of heparin.

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Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Tinzaparin is made by the controlled enzymatic depolymerization of heparin isolated from porcine intestinal mucosa using heparinase from Flavobacterium heparinum. A 2-O-sulfo-4-enepyranosuronic acid comprises the majority of the components at the non-reducing end and a 2-N, 6-O-disulfo-D-glucosamine structure at the reducing end of their chain (Fig. 1).

Cell culture

Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics (Walkersville, MD, USA). HUVEC was grown to 80–90% confluence in endothelial growth medium (EGM) containing hEGF (10 ng mL−1), hydrocortisone (1 mg mL−1), gentamicin (50 mg mL−1), amphotercin-B (50 µg mL−1), blood brain extract (0.012 mg mL−1) and 2% fetal bovine serum (FBS) equilibrated with 95% air/5% CO2 at 37 °C. HUVEC cells were serially passaged and maintained in endothelial growth medium in cell culture flasks coated with 0.2% gelatin (Sigma Chemical Co., St Louis, MO, USA). Confluent cultures of endothelial cells between the third and sixth passages were washed with Hank's balanced salt solution and harvested with a solution con/taining 0.025% trypsin and 0.01% EDTA and counted.

Release of endothelial TFPI

The effect of tinzaparin (1 µg) on endothelial cell TFPI release into culture media over time was examined. TFPI was measured in culture medium supernatants by enzyme-linked immunoassay following incubation in microtiter wells precoated with a rabbit antihuman TFPI polyclonal antibody. Bound TFPI was detected using a biotinylated monoclonal antibody specific for the Kunitz domain 1 of TFPI [26]. Subsequent binding of the streptavidin-conjugated horseradish peroxidase (HRP) was detected colorimetrically after addition of tetramethylbenzidine substrate. TFPI levels were calculated from a standard calibration curve generated using purified TFPI.

Tumor cells

B16 murine malignant melanoma cells (ATCC, Rockville, MD, USA) were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS, penicillin and streptomycin (Sigma). They were cultured to 70% confluence and harvested with trypsin–EDTA (Sigma) and washed twice with PBS. Cells were resuspended in PBS at a concentration of either 1 × 106 cells mL−1 for experimental metastasis or at 1 × 107 cells mL−1 for tumor cell-induced thrombocytopenia experiments.

Animals

Female C57/Bl6 mice (Harlan, Indianopolis, IN, USA) weighing 18–21 g were used for this study. All procedures were in accordance with Institutional Animal Care and Use Committee and institutional guidelines.

Anticoagulant effect of tinzaparin

The anticoagulant effect of tinzaparin was measured 4 h after s.c. or 15 min after i.v. (tail vein) injection of tinzaparin (10 mg kg−1) in experimental mice. Blood samples were collected by cardiac puncture (0.5–1.0 mL) into plastic tubes containing 3.2% citrate (9 : 1 v/v). Platelet counts were determined electronically using a Coulter MD electronic counter (Coulter Electronics, Miami, FL, USA). Whole blood clotting times were measured in a Sonoclot™ Coagulation Analyzer after recalcifying whole blood (400 µL) with 0.1 m CaCl2 (40 µL). Activated partial thromboplastin times (APTT) were measured on citrated plasma using the AMAX coagulation analyzer (Sigma).

Effect of tinzaparin on tumor cell-induced thrombocytopenia

Tinzaparin (10 mg kg−1) was administered subcutaneously as described above. This relatively high level of tinzaparin was chosen to ensure adequate anticoagulation according to our observations that clotting occurs faster in mice than in humans, as well as considering the fact that coagulation factors and platelet numbers are higher in mice compared with humans [27]

Control animals were injected subcutaneously with PBS. Tumor cells (2 × 106) were injected intravenously via the tail vein 4 h after tinzaparin administration. Platelet counts were measured in both control and tinzaparin-treated animals, before and 15 min after i.v. injection of tumor cells.

Effect of tinzaparin on experimental metastasis

Tinzaparin was administered by s.c. injection (10 mg kg−1 in 0.2 mL), followed 4 h later by the i.v. injection of tumor cells (2.0 × 105 in 0.2 mL PBS) via the tail vein. In a second experimental group, in addition to the pretumor cell dose, tinzaparin (10 mg kg−1) was also administered subcutaneously daily for 14 days. Control animals received PBS. No bleeding problems were observed in any of the tinzaparin-treated animals. On day 15, animals were killed by an overdose of Halothane anesthetic and the lungs were removed from the thoracic cage en bloc, washed in PBS and fixed in Bouin's solution. Pulmonary tumor nodules on the surface of the lungs were counted macroscopically.

Statistical analysis

All data were normally distributed as assessed by normal probability plots (StatisticaTM for Windows). Summary statistics were therefore presented as means and standard deviation and differences between groups were analyzed using Student's t-test. Throughout the work statistical significance was assumed when P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Anticoagulant and TFPI-releasing effects of tinzaparin

Four hours after s.c. injection of tinzaparin (10 mg kg−1), the whole blood recalcification time was prolonged 4-fold (P < 0.001). APTT results were > 200 s, also reflecting the presence of a significant circulating anticoagulant effect in the tinzaparin-treated animals. No changes in platelet count were observed in tinzaparin-treated animals compared with control (data not shown). At the dose employed (10 mg kg−1) tinzaparin was well tolerated and no bleeding or any other apparent side-effects were observed during the study. The ability of tinzaparin to release TFPI from endothelial cells was demonstrated in vitro. Treatment of HUVECs with tinzaparin (1 µg) resulted in a marked and time-dependent release of TFPI, rising to > 50 ng/2 × 106 cells mL−1 after 8 h (Table 1).

Table 1.  Effect of tinzaparin (1 µg) on the release of tissue factor pathway inhibitor (TFPI) from human endothelial cells (HUVEC)
Time (h) post tinzaparinMean (± SD) TFPI (ng/2 × 106 cells mL−1)
00.8 ± 0.3
12.2 ± 0.5
212.5 ± 2.4
438.0 ± 3.5
852.5 ± 5.4

The effect of tinzaparin on tumor cell-induced thrombocytopenia

In control animals, the peripheral blood platelet count (initially 939 ± 37 × 106) fell rapidly (498 ± 94 × 106) following i.v. injection of tumor cells (P < 0.01; Fig. 2). In tinzaparin-treated animals, in contrast, no such effect on platelet count was observed (921 ± 104 × 106 following tumor cell injection). Thus, pretreatment with tinzaparin effectively abolished tumor cell-induced thrombocytopenia.

image

Figure 2. The effect of tinzaparin on tumor cell-induced thrombocytopenia. Melanoma cells were injected 4 h after subcutaneous administration (10 mg kg−1) of tinzaparin. Blood (n = 4) was collected for platelet count 15–30 min after tumor cell injection. Tinzaparin completely abolished the thrombocytopenia caused by intravenous tumor cell injection.

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Experimental metastasis

A single s.c. injection of tinzaparin (10 mg kg−1) 4 h before i.v. administration of melanoma cells (2.0 × 105) markedly (89%) reduced mean (± SD) lung tumor formation (3 ± 2) compared with controls (31 ± 23; P < 0.001). In a second experimental group, extending anticoagulant therapy by administering s.c. tinzaparin (10 mg kg−1) daily for 14 days following the pretumor cell dose, reduced tumor formation by 96% (P < 0.001; Fig. 3).

image

Figure 3. The effect of single (pretumor cell) dose (10 mg kg−1; n = 8) and multiple doses (pretumor cell and daily for 14 days; n = 8) of tinzaparin on experimental lung metastasis. Control animals (n = 8) were injected with PBS.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The etiology of thrombosis in malignancy is multifactorial. Hypercoagulability in cancer patients may be mediated by chemotherapy and radiotherapy [28,29] but the increased level of coagulation activation is at least partly due to the presence of TF and possibly other types of procoagulant on the surface of tumor cells such as cancer procoagulant (CP), a cysteine proteinase procoagulant from fetal and malignant tissues, which directly activates FX in the absence of FVII [30–32].

In the present study we have shown that s.c. tinzaparin strongly inhibits tumor cell-induced thrombocytopenia and experimental metastasis when administered before i.v. administration of tumor cells. This effect was slightly, but not significantly increased when additional tinzaparin was given after tumor cell injection. Our previous studies using both coumadin and unfractionated heparin have shown that delaying anticoagulation (i.e. administering the agents some hours after tumor cell injection) significantly reduced their antimetastatic effect [8]. These observations confirm that in the injectable model of metastasis used in this study, maximal antitumor effect is achieved if the host animal is adequately anticoagulated at the time of tumor cell injection. Our data also provide further evidence to show that inhibiting the host thrombin generating capacity greatly suppresses experimental metastasis. Injected tumor cells rapidly cause intravascular coagulation in lung capillaries. Platelet activation and fibrin deposition occur either directly on the surface or in close vicinity of the trapped tumor cells. Tumor cells are retained in the pulmonary circulation for a longer period in control animals compared with anticoagulated or fibrinogen-deficient animals [8,33]. The prolonged microvascular presence of tumor cells may increase the possibility of extravasation and successful metastasis.

Activation of coagulation, with subsequent generation of thrombin, is believed to occur primarily as a result of TF expression by the tumor cells. We have previously shown that inhibiting TF activity with a monoclonal antibody effectively prevented melanoma-induced thrombocytopenia and subsequent lung seeding [34]. Also, we have specifically demonstrated (by flow cytometry) that TF is present on the surface of B16 melanoma cells [25]. The thrombocytopenia that is observed following i.v. tumor cell injection is secondary to thrombin generation since B16 cells did not cause any significant activation of platelets directly as determined by aggregometry using washed platelets, platelet-rich plasma or whole blood (data not shown). In addition, the observations that anti-TF antibodies, TFPI and heparins all inhibit tumor cell-induced platelet aggregation in vivo provide strong evidence to support the role of thrombin in that process.

Expression of αIIβ3 on cancer cells and platelets is stimulated by thrombin which is generated by tumor cell procoagulant activity or due to vascular damage. Thrombin generation can promote tumor cell adhesion to the vascular endothelium [35–37]. The tumor cell-induced thrombin production and subsequent platelet aggregation are positively correlated with cancer progression and metastatic potential [38–40]. Thus it is likely that in the injectable model of metastasis used in the present study, tinzaparin is interfering with the initial adhesion and arrest of blood-borne melanoma cells by inactivation of thrombin and inhibition of platelet aggregation. Heparin treatment has also been shown to reduce the formation of ‘spontaneous’ lung metastasis in mice from subcutaneously implanted mammary carcinoma and improve survival of the animals [41].

Using several different approaches, we also demonstrated that TFPI is effective in this experimental model. First we showed that injection (i.v.) of recombinant TFPI significantly reduced the metastasis of B16 melanoma. Second, we found that stably transfected TFPI (+) B16 melanoma cells produced significantly fewer lung metastases compared with wild type and vector control cells. Finally, we demonstrated that mice receiving i.v. somatic gene transfer of TFPI expression vector developed significantly fewer lung nodules than controls [25]. Heparin is capable of releasing TFPI from vascular endothelial cells [42]. Among the LMWHs, tinzaparin is particularly effective at releasing TFPI [43] and this may therefore represent an additional mechanism by which this LMWH exerts an antimetastatic effect. In the present study, we confirmed the ability of tinzaparin to release endothelial TFPI, showing that release from HUVECs was time-dependent and continued for at least 8 h after tinzaparin exposure. This is broadly compatible with the observation that tinzaparin [single s.c. dose of 175 IU kg−1 (DVT treatment dose) to healthy volunteers] rapidly causes an increase in plasma TFPI, which is sustained for up to 2–5 h after injection, and only returns to basal levels after 16 h [43,44]. Of potential importance in cancer therapy is the observation that tinzaparin and its relatively higher molecular weight and sulfated fractions are more potent stimulants of endothelial TFPI release compared with other LMWHs [43].

In summary, s.c. administration of tinzaparin effectively prevented tumor-associated coagulopathy and lung seeding in the B16 melanoma model of experimental metastasis. The favorable pharmacokinetic attributes of this agent compared with unfractionated heparin, together with its superior ability to release TFPI for relatively long periods from vascular endothelial cells compared with other LMWH, provide a rationale for its use in oncology as an antimetastatic as well as an antiangiogenic agent.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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