JOURNAL OF IMMUNOLOGICAL METHODS zyxwvutsrqponmlk ELSEVIER Journal of Immunological Methods 191 (1996) 55-63 Microtiter plate immunoassay for the evaluation of platelet adhesion to fibronectin ’ Masaru Chiba, Syed W. Malik, Ulrich Specks * zyxwvutsrqponmlkjihgfedcbaZYXWV Thoracic Diseases Research Unit. Mayo Clinic and Foundation, Rochester. MN, USA Received 13 April 1995; revised I 1 December 1995; accepted 6 January 1996 Abstract Investigations of platelet adhesion to adhesive proteins have been pursued to understand the basic mechanisms of hemostasis and thrombosis. Most assays used to determine platelet adhesion under stasis conditions rely on radiolabeled platelets. We describe a new microtiter immunoassay to study platelet adhesion to adhesive proteins under stasis conditions. Direct comparison of platelet adhesion to fibronectin using a standard platelet adhesion assay based on “Cr-labeled platelets and the new immunoassay showed that the optical density values obtained with the immunoassay are directly proportional to the number of platelets bound. The choice of platelet suspension buffer appears crucial for the design of such experiments, because the adhesion of resting platelets to fibronectin was significantly higher when platelets were suspended in Tyrode’s buffer rather than Tris buffer. Platelet adhesion to fibronectin is increased in response to thrombin stimulation. This increase can be inhibited by synthetic RGD peptides. The thrombin-induced increase of platelet adhesion to fibronectin could be detected with antibodies against actin and glycoprotein IIb-IIIa, but not against the o-granule constituent platelet factor 4 (PF4). This assay is very versatile, because it avoids the use of radioactivity, and allows the parallel processing of a large number of samples. In addition, the parallel use of antibodies against different platelet antigens allows the screening for platelet activation events associated with the measured platelet adhesion. Keywords: Platelet; Adhesion; Microtiter immunoassay; Fibronectin; Thrombin; Platelet factor 4 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR 1. Introduction Abbreviations: BSA, bovine serum albumin; PBS, phosphate buffered saline. * Corresponding author. At: Thoracic Diseases Research Unit, Guggenheim Bldg. 642 A, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA. Tel.: (507J2842301; Fax: (507)2844521; e-mail: specks.uhich@mayo.edu. ’ Supported by a grant-in-aid from the Minnesota Affiliate of the American Heart Association (to U.S.) and funds from the Mayo Foundation. This work was done during the tenure of a Clinician-Scientist Award from the American Heart Association to U.S. Elsevier Science B.V. SSDI 0022- 1759(96)0002 1 -X Platelet adherence to vessel wall matrix proteins such as collagens, as well as plasma adhesive pro- teins such as fibronectin, vitronectin, von Willebrand factor or fibrinogen has been investigated exten- sively in order to understand the basic mechanisms of the initial events involved in hemostasis and thrombosis (Roth, 1992). Several different assay systems have been devel- oped to study the interaction of platelets with immo- bilized adhesive proteins under stasis and high shear