A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, β-thromboglobulin and platelet factor 4 in healthy volunteers

Abstract

A radioimmunoassay was developed for the platelet α-granule protein thrombospondin; concentrations of thrombospondin as low as 3 ng ml −1 could be measured. There was no interference from other components of human biological fluids and no crossreactivity with β-thromboglobulin (β-TG) or platelet factor 4 (PF4). Plasma samples were stable when stored at −20°C. Normal human plasma contained 105.0 ± 31.0 ng thrombospondin ml −1 compared with β-TG concentrations of 37.2 ± 10.9 ng ml −1 and PF4 concentrations of 14.7 ± 10.1 ng ml −1 when samples were carefully taken into a platelet inhibitor cocktail and processed at 0–4°C. Release of thrombospondin during clotting of blood occurred at the same time as that of β-TG and PF4 and resulted in a serum concentration of 17.5 ± 5.5 μg ml −1. Assay of whole blood gave a platelet thrombospondin content of 89.1 ± 28.3 ng/ 10 6 platelets. The concentration in normal urine fluctuated widely from 3 to 22.5 ng ml −1, and was unrelated to urine flow. The half-life of thrombospondin in vivo was about 9 h, much longer than that of either β-TG or PF4. Unlike PF4, it was not released into the blood following an intravenous heparin injection. Bovine, ovine, canine and porcine sera contained thrombospondin which crossreacted immunologically with the human molecule; these species would be suitable animal models for the study of thrombospondin and its value as a platelet release marker.