Platelet aggregation in flowing blood in vitro. I. Production by controlled ADP convective diffusion and quantification by videodensitometry

Abstract

The overall objective of this work has been to quantify and to control the effects of flow and diffusion of adenosine diphosphate (ADP) on platelet adhesion and aggregation in whole blood. Flow conditions were chosen such that the study has maximal relevance to arterial thrombosis, where platelets are believed to play a prominent role. Toward this end, a blood flow chamber was designed and constructed wtihin which platelet aggregates form at and adhere to a specified, microscopic site on Cuprophan (PT 150, Enka Glanzstoff) membrane. Important features include controlled surface shear rate, controlled ADP diffusion (assessed by separate sutdies with [ 3H]ADP) through the membrane and into the flowing blood at the microscopic site, and a design permitting continuous in situ observation and quantification (e.g., aggregate mean thicknesses and thickness growth rates) using videomicroscopy and dual window densitometry of video recordings. Video calibration is carried out using differing, but known thicknesses of blood film between the light source and microscope objective. Although the wavelength of the incident light is 800 ± 90 nm (mean ± half-bandwidth for 50% peak intensity), the Lambert-Beer law is applicable to the present system, as shown by separate determination of the relative constancy of the absorptivities of both whole blood and packed platelets over the range from 700 to 900 nm. The chamber, videomicroscope, and videodensitometer constitute a whole blood platelet aggregometer, one with which shear rate (99 to 986 sec −1 or more) and entry rate of ADP into citrated or heparinized blood (leading to membrane-blood interface concentrations of ADP of micromolal to millimolal in present experiments) may be varied independently. The calibrated output of the device is a recording of the mean thickness of adherent aggregates within a standardized electronic “window” as a function of time. From such a recording, which may be correlated with simultaneous morphologic observations by videomicroscopy, estimates may be made of time to embolization, maximum mean aggregate thickness, and rate of increase of mean aggregate thickness.