Preferential binding of heparin to granulocytes of various species

Job Harenberg From the Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.

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Reinhard Malsch From the Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.

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Lukas Piazolo From the Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.

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Günther Huhle From the Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.

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Dieter L. Heene From the Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.

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Abstract

Objectives

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

Design

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.

Sample Population

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

Procedure

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.

Results

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 or LMMH. More than 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.

Conclusion

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

Clinical Relevance

Binding of heparin and LMMH to granulocytes, lymphocytes, and monocytes may have a substantial role in atherosclerosis, inflammation, malignancy, and immunologic diseases. (Am J Vet Res 1996;57:1016–1020)

Abstract

Objectives

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

Design

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.

Sample Population

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

Procedure

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.

Results

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 or LMMH. More than 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.

Conclusion

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

Clinical Relevance

Binding of heparin and LMMH to granulocytes, lymphocytes, and monocytes may have a substantial role in atherosclerosis, inflammation, malignancy, and immunologic diseases. (Am J Vet Res 1996;57:1016–1020)

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