Monocyte chemoattractant activity of Ser195-->Ala active site mutant recombinant alpha-thrombin.
alpha-Thrombin is chemotactic for human monocytes with optimal activity between 10-100 nM. The mechanism by which this response is mediated remains a point of controversy. The purpose of this study was to compare the chemotactic activity of proteolytically inactive thrombin (active site Ser195-->Ala mutant or Phe-Pro-Arg-chloromethyl ketone-inactivated thrombin) to thrombin and the "tethered ligand" thrombin receptor agonist peptide SFLLRN (single-letter amino acid code). Monocyte chemotaxis was compared to an optimal concentration (10 nM, considered to be 100%) of formyl-Met-Leu-Phe (fMLP). Proteolytically inactive thrombin (38% of fMLP) had similar chemotactic activity to active thrombin (46% of fMLP) at a concentration of 100 nM. Chemotaxis to SFLLRN was comparable to that of a control hexapeptide (FSLNLR) which is not an agonist for the tethered ligand thrombin receptor. Cross-desensitization experiments showed that pretreatment of monocytes with either mutant or active thrombin reduced subsequent chemotaxis to both thrombin chemotaxins. Pretreatment with SFLLRN did not decrease subsequent chemotaxis to either form of thrombin. Calcium flux measurements showed that both active thrombin and SFLLRN induced a rapid increase in monocyte and platelet intracellular calcium concentration. However, there was no intracellular calcium change in response to mutant thrombin or FSLNLR. Likewise, active thrombin and SFLLRN induced a rapid net increase in polymerized actin, but mutant thrombin and FSLNLR did not. By contrast, both active and mutant thrombin induced a polarization of monocyte morphology and actin distribution. This polarization has been associated with directed migration in many cell types. SFLLRN, however, induced a symmetrical increase in polymerized actin. These results suggest that measurements of intracellular calcium and polymerized actin are not perfect surrogate tests for true chemotactic activity. These results show that thrombin proteolysis is not required for monocyte chemotaxis and may be mediated by interaction with a binding site other than the tethered ligand thrombin receptor.
Crago, AM; Wu, HF; Hoffman, M; Church, FC
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