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Platelet factor 4 is shown to be a chemotactic protein for human polymorphonuclear leukocytes and monocytes at concentrations found in human serum and reached locally in injured tissue. The maximum chemotactic response to platelet factor 4 nearly equals that achieved with saturating concentrations ofthe chemotactic activity derived from the fifth component of human complement, C5. Cells desensitized to C5 chemotactic activity retain chemotactic responsiveness to platelet factor 4. Serum contains inhibitory capacity against the chemotactic activity associated with platelet factor 4. Our results suggest that the local release of platelet factor 4 may be an important stimulus attracting inflammatory cells to sites of blood vessel injury. Chemotaxis assays were performed in modified Boyden chambers (Ahlco, Southington, CT) by a double-filter method by using a 5-pum (monocyte) or 2-;im (neutrophil) (Nucleopore, Pleasanton, CA) filter overlaying a 0.45-pum filter (Millipore) (10). The upper compartments ofthe chambers were filled with 0.7 ml of cell suspension. The lower compartments contained 1.7 ml of medium only or medium with either platelet factor 4 or chemotactic activity derived from the fifth component of complement (C5f) that was obtained from whole serum by incubation with zymosan and e-aminocaproic acid (1.0 M) at 370C, followed by gel filtration over Sephadex G-100 (11). Twice the C5f activity necessary for 50% maximal chemotactic response (EDW) (in 25 jil) was used as a positive control in each experiment. To distinguish chemotaxis from chemokinesis, platelet factor 4 was simultaneously incubated with the cells in the upper compartment and platelet factor 4, CMf, or buffer alone in the lower compartment. In other experiments, test cells were desensitized to C5f by preincubation with C5ffor 15 min at room temperature (12). After gentle washing three times, the cells were tested promptly for chemotactic activity.For chemotaxis measurements, the chambers were incubated for 60 min (neutrophils) or 90 min, (mononuclear cells) at 37°C and then disassembled. The filters were stained with hematoxylin; after coding to conceal the identity from the reader, the filters were quantified for cell movement by recording the number of cells per high power (x400) grid migrating to the bottom of the upper filter. Five fields were averaged on each filter. Cell migration was corrected for blanks, in which the lower compartment contained only medium. Blank values for monocyte migration averaged 47 (+2.7) and for neutrophil migration averaged 62 (±7.7). All assays were done in triplicate. RESULTS Fig. 1 illustrates the relationship between chemotactic activity and increasing platelet factor 4 concentrations. Cell migration toward platelet factor 4 occurred at platelet factor 4 concentrations of 1 pug/ml and increasing concentrations ofplatelet factor 4 to 5 ,ug/ml resulted in increased monocyte and neutrophil migration. At 5 pug/ml, the migration response of test cells to platelet factor 4 nearly equaled (or fully equa...

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New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues with the use of appropriately designed coenzyme analogs. The resultant semisynthetic enzymes can have catalytic activities very different from those of the corresponding native enzymes. For example, papain has been converted into a highly effective oxidoreductase by covalent modification of the sulfhydryl group of the active site cysteine residue (Cys25) with flavins such as 8-bromoacetyl-10-methylisoalloxazine. Thus, it is now possible to enhance the catalytic versatility of existing enzymes through the process of "chemical mutation" of the active site.

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