Heparin, urokinase, and ancrod alter neutrophil function

Abstract

Neutrophils (polymorphonuclear neutrophils [PMNs]) have been implicated as mediators of reperfusion injury. Heparin, urokinase, and ancrod are agents used routinely to prevent and treat thrombosis, yet their effects on PMN function are unknown. Therefore human PMNs were obtained and incubated for 30 minutes with either saline solution or one of the following pharmacologic agents, each tested at three different concentrations: group 1, saline solution (control, n = 14); groups 2 through 4, heparin (5000 units/ml, n = 8; 2500 units/ml, n = 6; and 1250 units/ml, n = 6, respectively); groups 5 through 7, urokinase (50,000 units/ml, n = 8; 25,000 units/ml, n = 6; and 12,500 units/ml, n = 6, respectively), and groups 8 through 10, ancrod (70 units/ml, n = 8; 35 units/ml, n = 6; and 17.5 units/ml, n = 6). Superoxide anion production was measured by the reduction of cytochrome c in a spectrophotometric assay. Chemotaxis was evaluated by the number of PMNs migrating across a filter with a Neuro Probe chamber (Neuro Probe, Cabin John, Md.). Phagocytosis was determined by the ingestion of opsonized zymosan particles by PMNs. Serum obtained from each PMN donor was used both to opsonize the zymosan and as a chemoattractant in the chemotaxis assay. Statistical comparison was evaluated by analysis of variance, and post hoc comparisons for each agent with control were performed with the unpaired Student t test. No agent, at any dose, significantly changed superoxide anion production compared with control cells. All three agents significantly inhibited PMN chemotaxis (p < 0.01). In the control group the number of PMNs counted was 27.6 ± 4.9. In groups 2 through 4 (heparin) the numbers were 15.2 ± 1.2, 18.3 ± 1.6, and 19.8 ± 2.8, respectively; in groups 5 through 7 (urokinase) they were 17.1 ± 2.0, 18.5 ± 2.7, and 22.2 ± 3.1, respectively; and in groups 8 through 10 (ancrod) they were 16.7 ± 3.1, 18.2 ± 3.2, and 20.3 ± 4.3, respectively. In the phagocytotic assay, heparin, urokinase, and ancrod reduced phagocytic activity in the high-dose (p < 0.01) and intermediate-dose groups (heparin, group 3, and urokinase, group 6, p < 0.01; ancrod, group 9, p < 0.05). However, in the low-dose groups only heparin produced a significant inhibition (p < 0.05). In the control group the percentage of PMNs that had ingested the opsonized zymosan was 19.8% ± 4.5%. In groups 2 through 4 (heparin) the numbers were 11.0 ± 1.9, 13.2 ± 2.6, and 16.0 ± 3.3, respectively; in groups 5 through 7 (urokinase) they were 12.2 ± 1.5, 14.5 ± 4.0, and 21.7 ± 6.9, respectively; and in groups 8 through 10 (ancrod) they were 13.0 ± 1.8, 15.5 ± 2.6, and 17.7 ± 5.7, respectively. We conclude that (1) heparin, urokinase, and ancrod inhibit PMN chemotaxis and phagocytosis in vitro and (2) superoxide anion production, which reflects PMN stimulation, was not affected by these agents in vitro.