Microtiter plate immunoassay for the evaluation of platelet adhesion to fibronectin

Masaru Chiba, Syed W. Malik, Ulrich Specks

Research output: Contribution to journalArticle

7 Scopus citations

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 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 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 α-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.

Original languageEnglish (US)
Pages (from-to)55-63
Number of pages9
JournalJournal of Immunological Methods
Volume191
Issue number1
DOIs
StatePublished - May 10 1996

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Keywords

  • adhesion
  • fibronectin
  • microtiter immunoassay
  • platelet
  • platelet factor 4
  • thrombin

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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