Summary
Thrombin-activated human platelets release substance(s) of a prote-ic nature which
induce an increase in the intracellular calcium concentration in polymorphonuclear
leukocytes (PMN). Aim of this study was to characterize the platelet released product(s)
responsible for PMN stimulation.
PMN-stimulating activity was isolated from platelet supernatant by FPLC and HPLC.
The N-terminal sequence analysis revealed that the purified fractions consisted in
90% of a peptide of 73 amino acids and in 10% of a peptide of 74 amino acids; both
are truncated forms of the connective tissue-activating peptide III (CTAP-III), a
platelet a-granule product, and have 3 and 4 additional amino acids at the N-terminus
compared with the neutrophil-activating peptide 2 (NAP-2): Asp-Leu-Tyr and Ser-Asp-Leu-Tyr,
respectively. Treatment of platelet supernatant (previously depleted of PMN-activating
nucleotides) with Affi-gel heparin resulted in the disappearance of PMN-stimulating
effects, suggesting that NAP-2 variants, which are heparin-binding proteins, account
for ATP-independent PMN-stimulating activity of the supernatant. Cross-desensitization
between rNAP-2 and the platelet supernatant and inhibition by the anti-NAP-2 antibody
are in agreement with this conclusion. Although NAP-2 and its variants are reportedly
generated from the inactive precursors, CTAP-III and platelet basic protein, through
a proteolytic cleavage, NAP-2 variants were not generated in our system by proteases
deriving from platelets or contaminating leukocytes. Indeed, treatment of intact platelet
suspensions with different protease inhibitors failed to modify the calcium stimulating
activity of the resulting supernatants. In conclusion, thrombin-activated platelets
release NAP-2 variants which are not generated outside the platelets by proteolytic
processing but are released in an active form. This finding enhances our understanding
of platelet-PMN interaction in thrombosis and inflammation.
