Materials
Dulbeco Modified Eargle’s Medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from Hyclone^â (Utah, USA). Hirudin was obtained from Feinchemie GmbH (Germany). Apyrase, indomethacin, EDTA and enzyme-free cell dissociation solution (c5914) were purchased from Sigma^â (USA).

Subjects
Thirty healthy volunteers age 25-55 years were enrolled into the study. All of them did not ingest aspirin (ASA), any other NSAIDs, or antiplatelet drugs for at least for 2 weeks and they fasted overnight before the day of the study.

Written informed consent was obtained from all subjects. The study protocol was approved by The Ethics Committee for Human Research, Faculty of Medicine Siriraj Hospital.

HuCCA cell culture
Primary HuCCA cells were established in our laboratory. Cells were cultured in T-75 flasks with DMEM containing 15 % FBS, 100 unit/ml penicillin G, 100 mg/ml streptomycin and grown to confluence until used at 37°C in a humidified atmosphere of 5 % CO₂ in air¹⁸. Cells were harvested with enzyme-free cell dissociation solution (c5914) and resuspended in DMEM to yield a concentration of 10⁷ cells/ml for platelet aggregation experiments. Viability, as determined by tryptan blue exclusion, was >80%.

Platelet aggregation study
Blood samples were obtained by vein puncture and gently mixed with 8 U/ml heparin as an anticoagulant. Platelet rich plasma (PRP) containing 4.5±1.0 x 10⁸ platelets/ml. was prepared to test with each of DMEM (150 ml; control) or HuCCA cell suspensions alone (1.0x107 cells/150 ml, final concentration = 1.5x10⁶ cells/ml) or HuCCA cell suspensions plus hirudin (10, 20 and 40 unit) or plus apyrase (5, 10 and 20 unit) or plus EDTA (2, 3 and 4 mM) or plus indomethacin (0.1, 1 and 2 mM). Platelet free plasma (PFP) was also prepared for baseline calibration. Platelet aggregatability was determined turbidly according to the method of Born¹⁹ within 2 - 3 hours of blood collection.  

Using aggregation & a shape change monitor (BORN-MICHAL, Pharmacological Research, England) the percent maximum aggregation within 7 minutes was evaluated by comparing the amplitudes of the aggregation curves of PRP after being co-incubated with each tested sample to the amplitudes of the aggregation curves of PFP multiplied by 100.

Statistical analysis
Results are shown as mean ± SE from duplicate determinations (samples) of three subjects on separate experimental days (n=3). Student’s unpaired t-tests were used to determine the significance of differences between means and p-values of less than 0.05 were accepted as statistically significant.

1. Effects of HuCCA cells on platelet aggregation

HuCCA cells can induce platelet aggregation while conditioned medium 24, 48 and 72 hours (serum free medium from HuCCA cells) cannot (data not shown). The potency of induction activity related to the number of HuCCA cells. Figure 1A demonstrates that HuCCA cells induced platelets to aggregate in a concentration-dependent manner within the range of HuCCA cell 3.25 x 10⁵ to 1.5 x 10⁶ cells/ml. Figure 1B demonstrates that all three batches of HuCCA cells used in this study reveal the similar effects. Three batches of HuCCA cells were CL-2, CL-6 and CL-19 HuCCA cell lines, derived from HuCCA cells of cholangiocarcinoma patients (CL) number 2, number 6 and number 19, respectively.

2. Effects of hirudin, apyrase, EDTA and indomethacin on HuCCA cells induced platelet aggregation
Platelet aggregation induced by HuCCA cells was inhibited by hirudin, EDTA and indomethacin but not by apyrase.

2.1 Effects of hirudin on HuCCA cell induced platelet aggregation.
Hirudin inhibited HuCCA cell induced platelet aggregation in a dose dependent manner (Figure 2A). The maximum aggregation of HuCCA cell treated platelets plus hirudin (10, 20 and 40 units) were 77.27 ± 13.68%, 60.53 ± 11.14% and 34.42 ± 8.13%, respectively (n=3). The significant inhibition was found at all doses of hirudin (p < 0.05; Figure 2B).

2.2 Effects of apyrase on HuCCA cell induced platelet aggregation.
Apyrase did not inhibit HuCCA cell induced platelet aggregation (Figure 3A). There was no  difference between the maximum aggregation of HuCCA cell treated platelets plus apyrase and of  the control (HuCCA cells alone). The maximum aggregation of HuCCA cell treated platelets plus apyrase (5, 10 and 20 units) were 97.66 ± 0.38%, 97.66 ± 0.50% and 97.66 ± 0.83%, respectively.(n=3, Figure 3B).

2.3 Effects of EDTA on HuCCA cell induced platelet aggregation.
EDTA inhibited HuCCA cell induced platelet aggregation in a dose dependent manner (Figure 4A). The maximum of HuCCA cell treated platelets plus EDTA (2, 3 and 4 mM) were 65.70 ± 8.47%, 36.12 ± 10.65% and 4.48 ± 2.20%, respectively (n=3). The significant inhibition was found at all doses of EDTA. (p < 0.05; Figure 4B).

2.4 Effects of indomethacin on HuCCA induced platelet aggregation.
Indomethacin inhibited HuCCA cell induced platelet aggregation in a dose dependent manner (Figure 5A). The maximum aggregation of HuCCA cell treated platelets plus indomethacin (0.1, 1 and 2 mM) were 77.15 ± 10.58%, 50.92 ± 2.80% and 31.35 ± 4.87%, respectively (n=3). The significant inhibition was found at all doses of indomethacin (p < 0.05; Figure 5B).

The study showed that HuCCA cells can induce platelet aggregation while their condition medium cannot (Figure 1A). The induction potency was related to the number of HuCCA cells (Figure 1A). This finding is similar to many other investigations that various tumor cells such as pancreatic cancer cell, fibrosarcoma, adenocarcinoma, lung cancer cell and brain tumors^8,10,20 can induce platelet aggregation in vitro. Platelets may be activated by mediators released from tumor cells, or by the direct contact with the molecules on the surface membrane of tumor cells.^21-24 Our results also revealed that the condition medium (serum free medium from HuCCA cells) did not induce platelet aggregation (data not shown). It might imply that the fresh mediators released from tumor cells are needed in the initiation of TCIPA. However, the contact of tumor cells with platelets may be another important factor because this contact may even be necessary to TCIPA in some cell systems. It may mediate via adhesion receptors on tumor cells (integrin a₂b₁, integrin aIIb/b₃-like receptors, Lewis antigens, thrombospondin receptor) and on platelets (integrina₂b₁, integrin aIIb/b₃, thrombospondin receptor, P-selectin).^25-29 However, some studies reported that direct contact of tumor cells with platelets may not be necessary in TCIPA. Heinmoller et al (1995) also reported that monoclonal antibodies against adhesion receptors have no effects on pancreatic cancer cell induced platelet aggregation.⁸

The mechanisms of TCIPA have been severally described. These depend on the different origins and organs of tumor cells. During TCIPA by adenocarcinoma, melanoma and neuroblastoma cell lines, ADP was released, platelet receptors were, then, stimulated and platelets were, finally, induced to aggregate.³⁰ Some tumors can induce platelet aggregation and subsequently activate the coagulation cascade through the generation of thrombin.^9,30 Furthermore, TCIPA is shown to associate with the production of eicosanoids that amplifies platelet aggregation such as thromboxane A₂.^31,32 Along with the production of eicosanoids, Steinert et al (1993) have shown that platelet surface glycoprotein aIIbb₃, the receptor for fibrinogen, plays an important role in platelet aggregation by tumor cells.³³ Moreover, elevation in plasma vWF levels, the common part of an acute-phase reaction, or during endothelial cell perturbation in cancer patients, may contribute to platelet hyperreactivity.^34-35

Hirudin, apyrase, EDTA and indomethacin are some signaling molecules that are used as pharmacological tools to understand the mechanisms underlying TCIPA. In this study, HuCCA cell induced platelet aggregation was inhibited by hirudin, EDTA and indomethacin but not by apyrase. Hirudin is a specific inhibitor of thrombin and hence, the inhibition of platelet aggregation by hirudin may indicate the important pathway of thrombin in the mechanisms of HuCCA cell induced platelet aggregation. Here, we showed that hirudin inhibited HuCCA cell induced platelet aggregation in a dose dependent manner (Figure 2). This result indicated that the signalling mechanism of HuCCA cell induced platelet aggregation is thrombin-dependent reactions.

Apyrase, is an ADP scavenger molecule. HuCCA cell induced platelet aggregation was not inhibited by apyrase which possibly indicated that the ADP pathway was not involved in this TICPA (Figure 3). This result is in contrast to the previous works of Oleksowicz et al (1997) and Mohanty et al (1984) who reported that breast cancer and fibrosarcoma induced platelet aggregation possibly via the ADP pathway.^35,36 However, our result is similar to the study of Heinmoller et al (1995) and Katagiri et al (1991) who demonstrated that apyrase did not effect platelet aggregation induced by pancreatic cancer cells and human melanoma cells.^8,37 The different results found in these studies may be due to the various types of tumor cells used.

EDTA is the chelation of extracellular calcium. EDTA can inhibit HuCCA cell induced platelet aggregation (Figure 4) which indicated that extracellular calcium ions are an important factor required for this TCIPA. Many studies have accordingly shown that tumor cell lines are able to induced heparinized platelets to aggregate.^3,8,10,20,38

Indomethacin is non specific COX inhibitor. Indomethacin was used to determine whether HuCCA cell induced platelet aggregation through the cyclooxygenase pathway. The results showed that indomethacin can inhibit HuCCA cell induced platelet aggregation (Figure 5) which indicated that this TCIPA was possibly mediated by TXA₂ formation through the cyclooxygenase pathway. The result is similar to the works of Honn et al and Bastida et al (1987) who reported that platelet aggregation in response to tumor cells is dependent upon both cyclooxygenase and lipoxygenase pathways.^31,39 However, some investigators have reported that platelet aggregation is independent of the cyclooxygenase pathway.⁴⁰

The results obtained from this study may provide an explanation for the high incidence of thrombosis in cholangiocarcinoma patients. However, thrombin and TXA₂ have been accused of exerting various different effects during tumor invasion and metastasis.^41-42 It remains to be determined whether the mediators released during platelet aggregation by tumor cells affect tumor invasion and metastasis.