Receptor-mediated chemotactic response of a macrophage cell line (J774) to thrombin

Human alpha-thrombin, the procoagulant activation product of prothrombin, elicits chemotaxis in several macrophage-like continuous cell lines, most notably J774. Effective alpha-thrombin concentrations eliciting cell movement range from 10(-10) to 10(-6) M, with the optimal response occurring at about 10(-8) M. At the latter concentration, the response is equivalent, on a molar basis, to that observed with f-Met-Leu-Phe-OH, the positive control used in these experiments. Blockage of alpha-thrombin's active center with diisopropylfluorophosphate or by tryptic proteolysis of the procoagulant exosite (i.e., gamma-thrombin) does not decrease chemotactic activity. However, formation of enzymatically inactive complexes with AT3 or hirudin eliminates chemotactic potency. Competition chemotaxis assays, carried out by comparing the abilities of varying concentrations of test substances placed in the upper compartment of Boyden chambers to inhibit gradient-oriented movement of cells toward a fixed concentration of thrombin in the lower compartment, demonstrate that, although the modified forms of thrombin cross-compete, formylated peptide will not inhibit thrombin's chemotactic effects. Binding studies carried out using 125I-alpha-thrombin on paraformaldehyde-fixed J774 cells show a Kd of approximately 7.5 nM with an estimated 14,100-binding sites/cell. This Kd is in agreement with the optimal chemotactic thrombin dose (approximately 10 nM). On the basis of the competition chemotaxis assays and the radiolabeled thrombin-binding data, it is proposed that unique thrombin-specific chemotactic receptors exist on J774 cell membranes, and that these receptors are distinct from those mediating chemotaxis by agents such as formylated peptides.