行政院國家科學委員會補助專題研究計畫成果報告
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※ 幽門螺旋桿菌誘發的血小板凝集作用之機轉 ※
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計畫類別：;個別型計畫 整合型計畫
計畫編號：NSC96－2320－B－110－008－
執行期間： 96 年 8 月 1 日至 97 年 7 月 31 日
計畫主持人：陳和瑟 （國立中山大學 生物醫學研究所）
共同主持人：吳政毅 （高雄醫學大學 醫學系內科）
計畫參與人員： 吳登強 （高雄醫學大學 醫學系內科）
劉大智 （高雄醫學大學 醫學研究所）
蔡秀貞 （高雄市立小港醫院 檢驗科）
葉正忠 （國立中山大學 生物醫學研究所）
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：國立中山大學 生物醫學研究所
中 華 民 國 97 年 9 月 9 日
行政院國家科學委員會專題研究計畫成果報告
幽門螺旋桿菌誘發的血小板凝集作用之機轉
The Mechanisms of Platelets Aggregation induced by Helicobacter pylori
計畫編號：NSC 96-2320-B-110-008
執行期限：96 年 8 月 1 日至 97 年 7 月 31 日
主持人：陳和瑟（國立中山大學 生物醫學研究所）
共同主持人：吳政毅（高雄醫學大學 醫學系內科）
計畫參與人員：葉正忠、蔡秀貞、吳登強、劉大智
Abstract
Background: Helicobacter pylori had a
tendency toward myocardial infarction, coronary heart disease and idiopathic thrombocytopenic purpura through triggering platelet aggregation.
Objectives: To elucidate the roles of Fcγ
receptor IIA genotypes and lipopolysaccharides in Helicobacter pylori induced aggregation and to explore the functions of P-selectin in it.
Methods: Fifteen healthy blood donors were
enrolled in this study. Platelets aggregation was assayed by Platelet Aggregometer and Fcγ receptor IIA genotype was determined by allelic specific nested polymerase chain reaction. The presence of Helicobacter pylori in aggregates was screened by scanning electron microscopy and Helicobacter pylori specific polymerase chain reaction. The expression levels of P-selectin were analyzed by flow cytometry.
Results: Helicobacter pylori induced
dependent and the concentration of 2 × 107 CFU/mL bacteria was the threshold for inducing platelet aggregation. Lipopolysaccharides alone, even extracted from the pro-aggregatory strain, was unable to induce aggregation. Different Fcγ receptor IIA genotypes had no effect on aggregation; whereas the freshness of
Helicobacter pylori as well as platelets was
crucial in aggregation. It was found that
Helicobacter pylori were contained in the
platelet aggregates and failed to induce aggregation in the P-selectin depleted plasma.
Conclusions: Helicobacter pylori trigger
platelet aggregation regardless of Fcγ receptor IIA genotypes. Anti-Helicobacter pylori antibodies and P-selectin are essential factors in
Helicobacter pylori induced platelet
aggregation.
Keywords: Fcγ receptor IIA; Helicobacter
pylori; Lipopolysaccharides; Platelet
aggregation; P-selectin
pylori, Helicobacter pylori; PBS, phosphate
buffered saline; PRP, platelet rich plasma; PPP, platelet poor plasma; LPS, lipopolysaccharides; AA, arachidonic acid; ADP, adenosine diphosphate; Col, collagen; Epi, epinephrine
Results and Discussion
No control of FcγRIIA genotypes on H.
pylori-induced aggregation
The necessity of anti-H. pylori antibody was examined on H. pylori -induced platelets aggregation using the pro-aggregatory strain of
H. pylori (49503). Results showed that H. pylori
(49503) induced platelets aggregation only occurred in the anti-H. pylori antibody positive PRP (Fig 1).
Fig 1
The allelic specific nested PCR analyses for three representative individuals are shown in Fig 2.
Fig 2
A 278-bp specific PCR product amplified with primer P4A, but not with primer P5G was shown in donor 1. In contrast, this product could be amplified only with primer P5G in donor 3. Both primers, P5G and P4A, amplified the 278-bp product from donor 2. In all reactions, the 440-bp internal control fragment of the CRP gene was present. Donors 1, 2, and 3 represented homozygous FcγRIIA-H/H131, heterozygous FcγRIIA-R/H131, and homozygous
FcγRIIA-H/H131, respectively. All three FcγRIIA genotypes appeared in aggregation (+) groups with H. pylori antibody (+) suggests that FcγRIIA genotypes have no control on H.
pylori-induced platelet aggregation. In another
word, platelet aggregation could be induced by the pro-aggregatory bacteria as long as the PRP contains its antibodies.
H. pylori LPS fail to induce aggregation
Different H. pylori strains: ATCC 49503, (CagA+
/VacA+
), ATCC 43504 (CagA+
/VacA+
)
and ATCC 51932 (CagA-/VacA-) were
examined for platelets aggregation. Results were shown in Fig 3.
Fig 3
Platelet aggregation was occurred during infected with H. pylori (49503). However, it did occur in a few occasions with H. pylori (51932), but it never happened with the H. pylori (43504). Results confirmed the previous description that the aggregation was independent on CagA and
VagA virulence factors. The other virulence
factors-LPS extracted from these strains were examined for their ability to induce aggregation. Figure 3 demonstrated that the LPS extracted from these bacteria failed to induce aggregation, even the LPS of pro-aggregatory H. pylori (49503). Human platelet aggregation was associated with bacterial LPS. The LPS from
Escherichia coli could bind to platelets and the
LPS of some specific H. pylori strain carried the sialyl Lewis x antigen and the sialy Lewis x structure could be recognized by three members of the selectin receptor family have been reported; therefore, platelet aggregation were investigated by the direct stimulation of H.
pylori LPS in this study. Results clarified that
LPS alone, even it was extracted from pro-aggregatory strains of H. pylori (49503), were unable to induce aggregation. It suggests that the whole bacteria organism was involved in aggregation induction. Except the platelet aggregation induction, H. pylori could also induce apoptosis on platelets as well as on the aggregates and the dead bacteria or of which have lost their activity during storage were also unable to induce aggregation. These evidences
may explain why to eradicate H. pylori from the gastric mucosa was associated with improvement of immune thrombocytopenic purpura (ITP).
Anti-P-selectin antibody inhibited H. pylori -induced aggregation
In order to investigate the role of P-selectin in H. pylori induced aggregation, the expression of P-selectin was measured by flow cytometry. Results demonstrated that the increased expression of P-selectin was only observed in the pro-aggregatory H. pylori strain (49503) and was concentration dependent. With the addition of anti-P-selection antibodies (CD62P) prior to the aggregation analysis, the pro-aggregatory bacteria failed to induce aggregation (as shown in Fig 4). These were not seen on other activator induced platelet aggregation, such as arachidonic acid (AA), adenosine diphosphate (ADP) and collagen (Col), except for epinephrine (Epi). The H. pylori induced-platelet aggregation was also failed on washed platelets. It was due to the washed platelets lack of H.
pylori IgG. However, the levels of aggregation
did not further decrease in the washed platelets when anti-P-selection antibodies (CD62P) was added (Fig 4) suggests that some platelets were activated during platelets wash.
Fig 4
The pro-aggregatory strain bacteria induced the expression of P-seletin, that was not observed in the non-aggregatory strain. Although significant influence of anti-P-selectin IgG in epinephrine induced-aggregation; it was completely inhibited H. pylori induced-platelet aggregation. Results suggests that H. pylori (43504) was unable to induce platelet aggregation might due to its lacking the ability to induce the expression of P-selectin. It is concluded that anti-H. pylori IgG, and freshly released P-selectin are essential factors in H
