Binding of fluorescent-labeled low molecular mass heparin to latex microspheres and to rat and human leukocytes.

Abstract

Fluorescent-labeled LMMH may be used to investigate the pharmacokinetics of heparins. FITC has been specifically bound by endpoint attachment to LMMH-tyramine. In the present article two in vitro methods are described with the intention of developing new ex vivo test systems. To determine the concentration of heparin, protamine has been bound to the surface of magnetic latex particles. LMMH-tyramine-FITC bound dose dependently in a linear range from 0.1 to 30 micrograms/mL to the protamine-coated latex particles. The fluorescence intensity of the latex particles was analyzed using flow cytometry. No differences in the dilution curves were observed between buffer system, rat and human plasma. This indicated that no interference of heparin binding took place with plasma proteins with respect to the binding to protamine. In the second method the binding of LMMH-tyramine-FITC on rat and human leukocytes was analyzed using flow cytometry. LMMH-tryamine-FITC bound dose dependently to rat and human granulocyes, monocytes, and lymphocytes. A linear relationship of binding was obtained for all three cell lines; the sensitivity was 0.1 microgram/mL for monocytes and lymphocytes and 0.03 microgram/mL for granulocytes without differences between rat and human cells. The data demonstrate a dose-dependent binding of LMMH-tyramine-FITC to protamine coated latex beads and to rat and human leukocytes.