A Colorimetric Method for the Measurement of Platelet Adhesion in Microtiter Plates

Abstract

A procedure for the determination of the adhesion of human platelets to protein-coated culture microwells was developed. The number of platelets was quantitated by measuring the activity of acid phosphatase, a platelet enzyme whose activity is stable independently of platelet stimulation and is not released. Isolated and washed platelets were incubated in 96-well microtiter plates with flat-bottom wells that had been precoated with various compounds, including collagen, fibrinogen, human plasma, and human albumin. At the end of incubation (optimal time: 40-60 min), nonadherent platelets were washed out, adherent platelets were solubilized with Triton X-100, and the acid phosphatase activity was measured by using the substrate p-nitrophenyl phosphate. The p-nitrophenol produced was measured with a microplate reader at 405 nm and the percentage of adhesion was calculated with reference to known platelet standards. ADP and thrombin stimulated platelet adhesion in a dose-dependent manner to fibrinogen and human plasma, but not to human albumin. Platelets adhered to collagen even in the absence of stimulants. Simultaneous evaluation of adhesion and aggregation demonstrated that with ADP as stimulus, but not with thrombin, the two platelet responses were dissociated. Microscopic examination of culture wells showed that most of platelets adhered as single cells and not as aggregates. The sensitivity of this method allowed the assay of platelet adhesion by using only 2.5 × 105 platelets/well.