Abstract
Anti-platelet integrin GPIIIa49-66 Ab obtained from HIV-ITP patients (or raised in rabbits) induces complement-independent platelet oxidative fragmentation and death by activating platelet 12-lipoxygenase (generation of 12(S)-HETE) and NADPH oxidase (
JCI, 113:973, 2004
). Platelet oxidative fragmentation is measured by flow cytometry of generated microparticles as well as intracellular DCFH oxidation. We now report that oxidative fragmentation in human platelets is preceded by Ca++ flux and P-selectin activation, n=6. However, the activation mechanism is different from classic platelet activation in that it is not inhibited by PGE1 or dibutryl cyclic AMP and is operative with Gαq−/− mouse platelets, whereas under these conditions, thrombin-induced platelet activation is completely inhibited, n=5–6. We chose to identify putative physiologic ligands that behave similarly to the GPIIIa49-66 Ab, and are therefore capable of regulating platelet reactive oxygen species (ROS) as well as arterial thrombus formation. The GPIIIa49-66 platelet peptide was used as bait to screen a 7-mer peptide phage display library. A peptide was found with 70% homology at the C-terminal position of ADAMTS-18, an ‘orphan’ disintegrin and metalloproteinase with thrombospondin (TSR)-like motifs, with unknown substrate. We have found it present in HUVEC as well as human pulmonary artery endothelial cells, on fixed sections of pathology specimens employing immunohistochemistry with a specific rabbit Ab raised against a C-terminal 18 mer peptide ADAMTS-18 (no staining with preimmune Ab). Recombinant ADAMTS-18 was produced in HEK 293 T cells and shown to induce ROS and oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. HUVEC ADAMTS-18 activity could be inhibited by a human scFv Ab raised against its C-terminal 18 mer peptide, as well as the ADAMTS-18 peptide itself, but not by a rabbit Ab against the N-terminal domain or an irrelevant peptide. Endothelial cell secretion and activation of ADAMTS-18 was optimally induced with 0.5 u/ml thrombin at 2 – 4 hrs, n=3–4. The truncated 385 amino acid C-terminal rADAMTS-18 fragment containing the 4 TSR motifs (produced in E.coli) had full activity at (<0.3 uM) whereas the C-terminal 66 amino acid fragment not containing the 18-mer binding site was inactive at 65 fold higher concentration, n=4. The physiologic significance of ADAMTS-18 was supported by demonstrating its secretion into plasma following iv injection of 4–16 u/ml thrombin into mice. Wild type mice have no detectable ADAMTS-18 in their plasma, with a sensitive ELISA assay (1 ng detectability). Thrombin stimulated mice secrete ADAMTS-18 in a concentration dependent manner. Platelet aggregates produced ex vivo with ADP and fibrinogen were destroyed with ADAMTS-18 as documented by LDH release at 1, 2 and 4 hrs of 83, 241 and 260 fold respectively, of PBS buffer control. In vivo tail vein bleeding time was shortened 4.5 fold with 1 hr prior infusion of 25 ug of a polyclonal rabbit IgG against ADAMTS-18, but not with preimmune IgG, n=10. Thus, a new mechanism is proposed for platelet activation, ROS release, death and platelet thrombus regulation, via platelet membrane oxidative fragmentation induced by thrombin-induced secretion and activation of ADAMTS-18.Author notes
Disclosure: No relevant conflicts of interest to declare.
2007, The American Society of Hematology
2007
