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Neutrophil attractant/activation protein-1 (NAP-1) has sequence similarity to platelet factor-4 (PF-4) and to NAP-2 (a truncated from of connective tissue activating protein-III [CTAP-III(des 1-15)]. We compared chemotactic activity for neutrophils of these related proteins. We also included for comparison CTAP-III, CTAP-III(des 1-13), the C-terminal dodecapeptide of PF-4 [PF-4(59-70)], and C5a. Chemotactic potency (EC50) was highest for NAP-1 and C5a. Although chemotactic efficacy (peak percentage of neutrophils migrating) was comparable for C5a, NAP-1, and NAP-2, the NAP-2 response occurred only at concentrations 100-fold higher than the NAP-1 EC50 of 10(8) M. Data for the CTAP-III proteins confirmed that CTAP-III is not an attractant and that chemotactic activity appears as a result of cleavage of residues at the N-terminus to make CTAP-III(des 1-13) or NAP-2 [CTAP-III(des 1-15)]. Chemotactic activity of PF-4 was low and variable, with no significant response by neutrophils from six of nine subjects. In contrast, PF-4(59-70) regularly induced high chemotactic responses, although the EC50 of 1.6 x 10(5)M was 1,000-fold greater than that of NAP-1. The binding of fluoresceinated NAP-1 to neutrophils was inhibited by unlabeled NAP-1 or NAP-2 but not by PF-4 or PF-4 (59-70). This suggests that NAP-2 interacts with the neutrophil NAP-1 receptor. Despite the low chemotactic potency of NAP-2, it is a potential attractant at sites of injury because of the relatively large amounts of the parent CTAP-III released from platelets, as indicated by a serum concentration of approximately 10(-6) M.

Although analytic measurements of connective tissue components in biological fluids would frequently be useful in diagnosis and/or management of many inflammatory, neoplastic and metabolic diseases, such information is often unavailable due to the methodologic problems posed by these natural products. This report outlines relatively simple procedures leading to the colorimetric measurement of glycosaminoglycans in urine and other body fluids and to measurement of hydroxyproline in urine. Normal values and potential applications are presented.

Connective tissue-activating peptides (CTAPs) stimulate human synovial cells to exhibit a higher rate of DNA synthesis, glycolysis, and glycosaminoglycan formation. These bioactive peptides have been isolated from human platelets (CTAP-III), lymphocytes, tumor cells, and neutrophilic leukocytes. Several other growth factors, such as somatomedins A and C and nonsuppressible insulin-like activity (soluble), have been shown to be dependent on the circulating levels of pituitary GH. In this study, we examined the human GH (hGH) dependence of CTAP-III. Platelets from children with reduced or absent hGH were examined for the presence of CTAP-III. The peptide was detected qualitatively by polyacrylamide gel electrophoresis and Ouchterlony double diffusion. CTAP-III antigen, measured by RIA, was found in normal amounts in platelet lysates from normal persons and GH-deficient patients. Biological activity of the peptide was suggested by the ability of platelet lysates to stimulate the formation of glycosaminoglycans and increase sulfate incorporation into glycosaminoglycans formed in cell cultures. In addition, normal and hGH-deficient platelet lysates contained potent mitogenic activity which increased thymidine incorporation into DNA. Platelets from GH-deficient patients also released CTAP-III normally on exposure to thrombin.