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Mechanism of human platelet activation by endotoxic glycolipid-bearing
mutant Re595 of Salmonella minnesota
S Timmons, , J Grabarek, M Kloczewiak and J Hawiger
The mechanism through which human blood platelets interact with gram-
negative bacteria with well-defined structural variations in endotoxic
lipopolysaccharide was studied. Secretion of 14C-serotonin and aggregation
of platelets separated from plasma proteins were observed on challenge with
rough mutant Re595 of Salmonella minnesota possessing a glycolipid outer
layer composed of Lipid A and 2-keto-3-deoxyoctonate (KDO) but lacking
heptose phosphate in the core and O-polysaccharide in its outer portion.
Both 14C-serotonin secretion and platelet aggregation were
concentration-dependent, with a half-maximum response at the ratio of one
bacterial colony-forming unit (CFU) to two platelets. The aggregation of
human platelets induced by mutant Re595 was divalent cation-dependent and
required secretion of ADP and fibrinogen from platelet storage granules
because it was inhibited by chelators, by the ADP-splitting enzyme apyrase,
and by monospecific antifibrinogen Fab fragments. The synthetic peptide
analog of the platelet receptor recognition site on the gamma chain of
fibrinogen, gamma 400-411, inhibited platelet aggregation induced by mutant
Re595 (IC50 160 mumol/L), whereas serotonin secretion was unaffected.
Tetrapeptide, RGDS, analogous to human fibrinogen alpha chain (alpha
572-575) and to the cell adhesion site of fibronectin, also inhibited
aggregation induced by mutant Re595 (IC50 60 mumol/L). Secretion of 14C-
serotonin was preceded by a very rapid phosphorylation of a platelet
protein of mol wt 47,000, which is associated with protein kinase C
activation. Myosin light chain (mol wt 20,000) was also phosphorylated.
Both phosphoproteins were dephosphorylated while secretion was reaching
maximum. Furthermore, release of 3H-arachidonic acid from platelet
phospholipids and generation of thromboxane B2 via the cyclooxygenase
pathway were observed. Inhibition of this pathway with acetylsalicylic acid
(10(-4) mol/L) or indomethacin (5 X 10(-4) mol/L) reduced 14C- serotonin
secretion and platelet aggregation. The role of Lipid A in the interaction
of mutant Re595 with human platelets was deduced from the inhibitory effect
of the Lipid A-binding protein present in Limulus amebocyte lysate.
Likewise, polymyxin B, known to complex with Lipid A, was inhibitory. The
reactivity of mutant Re595 toward platelets was attenuated by mild acid
hydrolysis, during which KDO was dissociated from the glycolipid, and by
alkaline hydrolysis, which breaks ester- linked fatty acids in Lipid A. In
contrast to mutant Re595, strain S218 of S minnesota bearing "complete"
endotoxic lipopolysaccharide did not induce secretion and aggregation of
human platelets.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 68,
Issue 5,
pp. 1015-1023,
11/01/1986
Copyright © 1986 by The American Society of Hematology
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