| Summary: | 碩士 === 高雄醫學院 === 醫學研究所 === 85 === Escherichia coli is one of the most common infectious Gram-
negative bacteria clinically. The incidence of Gram-negative
bacteremia has increased in the past few decades and the overall
mortality remains high (20~40%) in spite of major advancements
in clinical medicine. Sepsis and its sequelae (sepsis syndrome,
septic shock and multiple organ failure) are increasing common
in hospitalized patients . During the late stage of sepsis,
especially Gram negative septicemia, it is often associated with
platelet-related vascular injury and disseminated intravascular
coagulation (DIC). In general, the release of several mediators
from leukocytes or endothelial cells is considered to activate
the coagulopathy. Few reports have been mentioned about the
effect of cytosolic fraction of organism, even though the
membranous fraction (endotoxin) has been well documented.
This study was designed to investigate whether the heat-treated
pathogenic E.coli cytosolic proteins influence the aggregability
of platelets in vitro, and the possible mechanism was
investigated. The contents of this study are composed of：1.
The effect of heated and non-heated E.coli cytosolic extraction
proteins on platelet aggregation in human.2. The dose-dependent
effect of heated E.coli extraction protein on platelet
aggregation3. The effect of BSA and E.coli DnaK on platelet
aggregation in human4. The effect of anti-HSP 72/73 on
heated E.coli extraction protein- induced platelet
aggregation5. Analysis of heated and non-heated E.coli cytosolic
extraction proteins by two-dimensional electrophoresis E.
coli was obtained from infectious appendix from patient suffered
from appendititis and appendectomy. They were cultured in eosin-
methylene blue(EMB) for identification. E.coli was cultured by
T.S.B. broth media in 30℃ . Heat shock treatment was performed
in a 42℃ thermocontrolled water bath. Cytosolic protein
fraction was extracted by sonication and centrifugation. The
supernatant of crude extraction was used. Platelet rich plasma(
PRP) and platelet-poor plasma (PPP) were prepared from citrated
cord whole blood after normal delivery who had not ingested
aspirin or other drug for at leasttwo weeks prior to donation.
Platelet aggregation test was evaluated by PACKS-4 aggregometer
within 4 hours. The results showed that the extraction from
heated-E.coli significantly increased the platelet aggregation
(Wilcoxon test , p < 0.05,n=13 ).Furthermore, the effect showed
a dose-dependent manner. Same amount of BSA did not influence
platelet aggregation. Constitutive hsp of E.coli (DnaK) did
not influence the platelet aggregation. However, the
hyperaggregability induced by heated E.coli cytosolic extraction
disappeared by adding anti-hsp 72 antibody. Two-dimensional gel
electrophoretic analysis showed that a protein spot with
molecular weight 27kDa and pI 6.03 significantly increased in
the heated E.coli cytosolic protein . It was concluded that
1) the whole cytosolic protein of heated E.coli promote the
ability of platelet aggregation by a dose dependent manner. 2)
the accelerative factor is not the DnaK. 3) However , anti-human
hsp72/73 Antibody can neutralize the hyperaggregability
phenomenon. 4) A protein with MW27 kDa and pI 6.03, which was
present in heated E.coli cytosolic extraction, may play a
critical role in the effect .Through the present study, we
believe that the result canafford a new consideration in
understanding the pathogenesis of DIC during gram-negative
septicemia, and contribute to make a more effective therapeutic
plan clinically.Keywords：sepsis, disseminated intravascular
coagulation, E. coli, platelet aggregation,
heat shock proteins
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