Chemically modified heparins inhibit fibrinogen-bridged indirect adhesion between tumor cells and platelets

Abstract

The interaction between platelets and tumor cells is critical for the hematogenous metastasis of tumor cells. We recently reported that fibrinogen was capable of bridging and enhancing the interaction of platelets and tumor cells under conditions of physical shear force. In the present study, we aimed to detect the effects of 8 chemically modified heparins on the binding of fibrinogen to platelets or tumor cells using flow cytometry assays, as well as the fibrinogen-bridged adhesion of platelets and tumor cells using flow chamber assays. The results showed that fibrinogen binds to platelets and tumor cells in a β3 integrin-dependent manner and bridges the adhesion between platelets and tumor cells. Heparin and certain chemically modified heparins, including borohydride-reduced (RO)-, carboxyl-reduced (CR)- and 2-O, 3-O-desulfated (2/3ODS)-heparins, inhibited the β3 integrin-dependent adhesion of fibrinogen to platelets or tumor cells, and consequently blocked the fibrinogen-bridged indirect adhesion of platelets to tumor cells. These data indicate that chemically modified heparins should be potential inhibitors for the fibrinogen-bridged indirect adhesion of platelets and tumor cells, which provides a novel explanation of the anti-adhesion property of heparin and proposes a new anti-metastatic target for cancer treatment.

Figure 1

Fibrinogen binds to tumor cells and platelets in a β3 integrin- dependent manner. (A) A total of 100 μl of 2 mg/ml fibrinogen (Alexa Fluor488 conjugated) were incubated with 100 μl of 2×10⁶/ml A375 or B16F10 cells in phosphate-buffered saline (PBS) containing Ca²⁺ (PBS samples) or EDTA (Ctr samples) for 30 min. Tumor cells adhered with fibrinogen were assessed by flow cytometry after being washed three times with PBS. Preincubation of melanoma cells with integrin antibodies was performed prior to the adhesion of fibrinogen to tumor cells (β1 or β3 samples). (B) Adhesion of murine or human platelets to fibrinogen layers were analyzed by static adhesion assays. A total of 100 μl of 2×10⁹/ml platelets (Calcein AM dyed) in PBS containing Ca²⁺ (PBS samples) or EDTA (Ctr samples) were added to the wells that were precoated with 100 μl of 2 mg/ml fibrinogen, and adhesion was carried out for 30 min. Platelets adhered to fibrinogen were assessed using a plate reader after washing three times with PBS. Preincubation of platelets with integrin antibodies was performed prior to the adhesion of platelets to fibrinogen (β1 or β3 samples). Ctr, control; EDTA, ethylenediaminetetraacetic acid.

Figure 2

Fibrinogen enhances the adhesion of tumor cells and platelets in a concentration-dependent manner. Platelet monolayers on glass slides were washed with PBS/0.1% bovine serum albumin for 2 min. A375 cells were preincubated with or without (Ctr) various concentrations of fibrinogen for 30 min, the treated cells were then perfused over the layers under 1.2 dyn/cm². The adhered tumor cells on platelet monolayers were counted in 10 randomly selected fields after 3 min. PBS, phosphate-buffered saline; Ctr, control.

Figure 3

β3 integrins expressed in tumor cells and platelets are involved in indirect adhesion. The effects of β3 integrin-blocking antibodies on the indirect adhesions were assessed using flow chamber assays. (A) B16F10 and (B) A375, tumor cells (β3-T or β1-T) or platelets (β3-P) were incubated with mAbs or PBS (PBS). Ctr were the control samples, which were the direct adhesions of platelets and tumor cells, performed without fibrinogen. mAbs, monoclonal antibodies; PBS, phosphate-buffered saline; Ctr, control.

Figure 4

Chemically modified heparins inhibit the binding of fibrinogen to tumor cells or platelets. The binding of fibrinogen (2 mg/ml) to melanoma cells (A375 and B16F10) or platelets was assessed by flow cytometry or static adhesion assays (platelets). Prior to the assays being performed, cells were incubated with 1 mg/ml of 8 chemically modified heparins or PBS for 30 min, and the inhibitory rates of adhesions were calculated using the results from these assays. PBS, phosphate-buffered saline; NDS, N-desulfated/N-acetylated; CRS, carboxyl-reduced sulfated; CR, carboxyl-reduced; RO, borohydride-reduced.

Figure 5

Chemically modified heparins inhibit the fibrinogen-mediated indirect adhesion of tumor cells and platelets. The indirect adhesion of A375 cells to platelet monolayers mediated by fibrinogen were assessed using flow chamber assays. Prior to incubation with fibrinogen, tumor cells were preincubated with PBS or varying concentrations of heparin, LMWH or chemically modified heparins. The adhesions of tumor cells to platelet monolayers were carried out following incubations, and the number of adhered tumor cells was quantified. Ctr were the control samples of direct adhesion of tumor cells and platelets, without the addition of fibrinogen or heparins. LMWH, low molecular weight heparin; Ctr, control; PBS, phosphate-buffered saline; NDS, N-desulfated/N-acetylated; CRS, carboxyl-reduced sulfated; CR, carboxyl-reduced; RO, borohydride-reduced.