Platelet adhesion to photodynamic therapy-treated extracellular matrix proteins

Photochemistry and Photobiology, Apr 2002 by Fungaloi, Patrick, van Eps, Randolph Statius, Wu, Ya-Ping, Blankensteijn, Jan, Et al

Platelet Adhesion to Photodynamic Therapy-treated Extracellular Matrix Proteins(para)

ABSTRACT

Photodynamic therapy (PDT) produces reactive species that alter vascular wall biology and vessel wall proteins. In this study, we examined platelet adhesion to PDT-- treated (photosensitizer = Photofrin; fluence 100 J/cm^sup 2^; lambda = 630 nm) extracellular matrix (ECM), fibrinogen, von Willebrand factor (vWF) and collagen Types I and III, under flow conditions in a recirculating perfusion chamber. Platelet adhesion was quantified by image analysis. The effect of PDT on vWF was assessed by measuring the binding of domain-specific antibodies to treated vWF. PDT significantly decreased platelet adhesion to the ECM, fibrinogen and vWF. However, PDT of collagen resulted in significantly increased platelet adhesion, with large aggregate formation. PDT affected mostly the A1 (glycoprotein [GP]-Ib-binding site), A2 and A3 (collagen-- binding site) domains of vWF but not the D'-D3 (factor VIII-binding site) and B-C1 (GP-IIb/IIIa-binding site) domains. In conclusion, PDT can alter the ECM, resulting in decreased platelet adhesion. However, vessels with high collagen content, such as veins and small arteries, may become increasingly prone to thrombosis. The results of this study may thus play a role in understanding the thrombogenic properties and mechanisms of vascular PDT.

Abbreviations: ECM, extracellular matrix; GP, glycoprotein; HA, human albumin; HUVEC, human umbilical vein endothelial cells; OPD, o-phenylenediamine; PDT, photodynamic therapy; PBS, phosphate-buffered saline; PS, photosensitizer; vWF, von Willebrand factor.

INTRODUCTION

One of the first steps occurring after vascular injury is the adherence of platelets to the subendothelium and the exposed media (1). This is a complex mechanism that involves several steps (2-4). Briefly, after denudation of endothelial cells, there is exposure of the extracellular matrix (ECM), of which the major adhesive proteins are fibrinogen, von Willebrand factor (vWF) and the fibrillar collagen Types I and III (5). Platelets can bind directly to fibrinogen and vWF already present in the ECM. The role of collagen is more complex, as collagens are not only important adhesive proteins but also potent platelet agonists leading to platelet aggregation (6-8). Platelet adhesion to collagen can occur directly by interaction of collagen with platelet membrane proteins, of which glycoprotein (GP)-Ia/IIa (integrin alpha2beta1) is the most important. Platelet adhesion to collagen can also occur indirectly by binding with circulating vWF to create a bridge between both the platelet GP-lb receptor and collagen. This process activates the platelets and causes a conformational change, thus exposing the GP-IIb/IIIa receptor on the platelet membrane. The GP-IIb/IIIa receptor, in turn, binds to adhesive proteins like vWF, fibronectin, fibrinogen and vitronectrin to form a firm bridge between platelets, thus causing platelet aggregation.

Photodynamic therapy (PDT) utilizes light of a specific wavelength and energy to activate a photosensitive dye, thereby creating highly reactive species to produce free radicals, which, in turn, have biological effects. Recently, the use of PDT has expanded from its initial ontological application to other fields (9). In tumor treatment, one important mechanism of PDT is based on its thrombogenic properties by causing thrombosis in the tumor microvasculature and, thus, ischemia (10,11). This thrombogenic effect has also been used as an effective means of treating age-related macular degeneration caused by choroidal neovascularization (12). Whereas some studies show PDT to be highly effective in inducing thrombosis in the microvasculature with up to 90% blood stasis rate (13), in cardiovascular application (14,15) using larger blood vessels, there are no reports of such occurrence (16). This study was thus undertaken to further investigate this difference.

Recent studies have demonstrated that PDT alters the ECM profoundly (17). In this study we examine how PDT affects ECM, vWF, fibrinogen and collagen thrombogenicity by measuring platelet adhesion. Considering the multifactorial roles of vWF and collagen in mediating platelet adhesion, further studies were carried out to examine how PDT affects these specific proteins.

METHODS AND MATERIALS

Preparation of ECM. Human umbilical vein endothelial cells (HUVEC)-derived ECM, a well-known model for the subendothelial layer, was used (18,19). Briefly, HUVEC were isolated from the umbilical cord, as described (20). Endothelial cells from the second passage were cultured on glass coverslips (18 x 18mm; Menzel Glaser, Braunschweig, Germany) and grown to confluence in 5-7 days. ECM was isolated by exposing the endothelial cells to 0.1 M NH40H in phosphate-buffered saline (PBS). The isolated matrix was stored in PBS at 4 deg C for up to 2 weeks before assay.

Coating of cover-slips. Plasma vWF was purified from human cryoprecipitate, using a Bio-Gel A-15m (BioRad Laboratories, Veenendaal, The Netherlands) column as described (21) and stored at -20 deg C. Coverslips were cleaned overnight in a chromium trioxide solution, rinsed with distilled water and coated by incubating each coverslip with 100 (mu)L of purified vWF in PBS (20 (mu)g/mL) for I h, followed by incubation in 1% human albumin (HA). For fibrinogen and collagen coating, coverslips were cleaned with ethanol, rinsed and air-dried. Coverslips were incubated with 100 (mu)g/mL fibrinogen (Enzyme Research Labs, South Bend, IN) for 1 h, followed by incubation in 1% HA. Human placenta collagen Types I and III (Sigma Chemical Co.. St. Louis, MO) were solubilized in 50 mM acetic acid and sprayed at a density of 30 (mu)g/cm^sup 2^ on ethanol-cleaned coverslips with a retouching airbrush (Badger model 100, Badger Brush Co.. Franklin Park, IL). After the spraying procedure, the coverslips were incubated for I h with 1% HA in PBS (10 mM phosphate buffer, pH 7.4; 0.15 M NaCl). For certain experiments, coverslips coated with collagen Type III were blocked with 1% HA for 1 h, followed by incubation with 20 (mu)g/mL purified vWF. All coated coverslips were stored in PBS at 4 deg C for up to 2 days before perfusion.