HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY A Critical Role for N-ethylmaleimide–Sensitive Fusion Protein (NSF) in Platelet Granule Secretion

The molecular mechanisms that regulate membrane targeting/fusion during platelet granule secretion are not yet understood. N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements of a conserved molecular machinery for membrane targeting/fusion that have been detected in platelets. We examined whether NSF, an ATPase that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule secretion. Peptides that mimic NSF sequence motifs inhibited both a-granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar amino acids in a scrambled sequence inhibited platelet secretion. The peptides that inhibited platelet granule secretion also inhibited the human recombinant a-SNAP–stimulated ATPase activity of recombinant NSF. It was also found that anti-NSF antibodies, which inhibited recombinant a-SNAP–stimulated ATPase activity of NSF, inhibited platelet granule secretion in permeabilized cells. The inhibition by anti-NSF antibodies was abolished by the addition of recombinant NSF. These data provide the first functional evidence that NSF plays an important role in platelet granule secretion. r 1999 by The American Society of Hematology.