Preferential binding of heparin to granulocytes of various species

To address the problem of heparin binding to leukocytes of various animal species.

Leukocytes of the various species were incubated with fluorescent-labeled, low molecular mass heparin (LMMH). Fluorescence intensity on granulocytes, lymphocytes, and monocytes was analyzed by flow cytometry analysis.

Leukocytes were prepared from EDTA-anticoagulated blood of human subjects, rats, rabbits, dogs, pigs, and sheep 3 times.

The leukocyte populations were identified by their light scatter properties. In addition, phycoerythrin-labeled CD4, CD13, and CD14 antibodies were used to identify human lymphocytes, granulocytes, and monocytes; CD4, GR-1, and CD11b antibodies were used for mouse, and CD45, RP 3, and ED 9 antibodies were used for identification of rat leukocyte subpopulations.

Granulocytes, monocytes, and lymphocytes of all species bound LMMH in dose-dependent manner. Binding of LMMH-tyramine (tyr)-fluorescein-5-isothiocyanate (FITC) to granulocytes was higher in human subjects, rats, rabbits, dogs, and pigs, compared with binding to monocytes and lymphocytes. Mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. Binding of LMMH-tyr-FITC was reversible in the presence of unlabeled heparin of LMMH. More then 99% of human, rat, rabbit, dog, and sheep granulocyte populations were distinguished from monocytes and lymphocytes by means of their fluorescence intensity owing to LMMH-tyr-FITC. This separation was not obtained for mouse and pig granulocytes.

Evidence of specific heparin binding to granulocytes of many species indicates the relevance of fluorescent-labeled LMMH for biological investigations.

Binding of heparin and LMMH to granulocytes, lymphocytes, and monocytes may have a substantial role in atherosclerosis, inflammation, malignancy, and immunologic diseases.