Microtiter plate immunoassay for the evaluation of platelet adhesion to fibronectin.
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 51Cr-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 crucial for the design of such experiments, because the adhesion of resting platelets to fibronectin is increased in response to thrombin stimulation. This increase 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 alpha-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.
Chiba
M
M
Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905 , USA.
Malik
S W
SW
Specks
U
U
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism; D002021:Buffers; D015550:Cell Degranulation / Q000187:drug effects / Q000276:immunology; D005353:Fibronectins / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010973:Platelet Adhesiveness / Q000187:drug effects / Q000276:immunology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D002021:Buffers; D005353:Fibronectins; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; C047981:arginyl-glycyl-aspartic acid; D013917:Thrombin