腦中風是全世界主要死因的第三位，並且是導致成年人長期嚴重生理殘障的主要的原因。血小板活化與血小板－白血球的交互作用在缺血性腦中風的病理學上扮演重要的角色。目前抗血小板藥物被廣泛地用於預防缺血性腦中風，而不同的抗血小板藥物對血小板的抑制有其不同的藥理作用。血液中的許多物質都會刺激血小板細胞質內的鈣離子濃度升高，引起血小板內的二級訊號傳遞，進而導致血小板活化。急性腦中風所造成主要的腦損傷並非發生在中風的當時，而是在中風後的數小時內逐漸發展所致。一般認為腦中風後，白血球在缺血的大腦區域會釋放出發炎的細胞激素和神經毒素，並且透過
血小板－白血球－血管內皮細胞的交互作用促使微血管進一步阻塞。在本論文中，我們探討急性腦中風後病患血小板細胞內鈣離子的連續性變化。研究血小板的活性、白血球活性、以及血小板－白血球交互作用在急性缺血性腦中風病人的改變程度，並加以分析這些生物標記和臨床預後之間的相關性。再者我們比較急性缺血性腦中風病患使用阿斯匹靈（aspirin）和保栓通（clopidogrel）治療對血小板活性的影響。

急性腦中風病人之血小板內鈣離子流動調節異常

本實驗針對31位急性腦中風病人與27位對照組病患之血液，以花生四烯酸(arachidonic acid)、二磷酸腺甘酸(ADP)、血小板活動原素(PAF)、以及thrombin刺激血小板的活化，利用對鈣離子敏感的螢光的染料fura-2來測試病患血小板細胞內鈣離子的連續變化。研究發現：血小板內的基礎鈣離子濃度不論在細胞培養液有無鈣離子的情況下，急性腦中風病人組皆高於對照組。在含鈣離子培養液的狀態下，使用PAF和ADP刺激下血小板內鈣離子濃度上升的程度在急性腦中風組較對照組有顯著增加。不過，在培養液無鈣離子的狀態下，只有PAF刺激後血小板內鈣離子濃度上升的程度在急性腦中風組比較對照組有顯著增加。血小板內的基礎鈣離子濃度在腦中風病人中風一個月後仍然明顯高於對照組。

血小板活化因子在急性腦中風不同子類型的表現和價值

本實驗收錄32位微小血管阻塞與22位大血管阻塞之急性腦中風病患。以流式細胞儀測量急性腦中風後不同時間點之血小板活化因子（包括CD62P，CD63 和CD40L）以及血小板－白血球交互作用，與28位腦中風恢復期病人(中風之後的3到9個月)、以及28位對照組作比較。研究顯示：血液中CD62P、CD63、血小板－單核球、以及血小板－淋巴球交互作用，在急性腦中風病患的表現量相較於腦中風恢復期患者以及對照組有顯著增加。CD62P和CD63的表現量在大血管阻塞組都比微小血管阻塞組有顯著增加，而CD62P在兩組的表現量到腦中風後一個月仍有顯著的差異。而住院時CD40L的表現量在預後不佳病人比預後良好病人有顯著的升高。

白血球黏著因子對急性缺血性腦中風病患的臨床價值

本實驗中我們測量急性腦中風後不同時間點之白血球黏著因子（包括PSGL-1、Mac-1和 LFA-1）的表現量。急性腦中風病人PSGL-1的表現量在嗜中性白血球和單核性白血球上與對照組相比有明顯升高，其表現量從急性期到中風後90天都呈現顯著差異。單核性白血球上Mac-1、LFA-1與嗜中性白血球上Mac-1的表現量，在急性腦中風後七天內相較於對照組呈現統計上顯著的升高。而且，嗜中性白血球上PSGL-1的表現量與腦中風後三個月預後呈現負相關。

阿斯匹靈與保栓通對缺血性中風病人血小板活化的影響

本實驗中針對70位急性缺血性腦中風病人，分成使用阿斯匹靈與保栓通(clopidogrel)兩組，連續性的評估其血小板活化因子。研究顯示：血液中CD62P、CD63、以及CD40L的表現量，在急性腦中風病患相較於對照組呈現顯著增加。使用保栓通治療的病人在腦中風之後第一週CD62P、CD63和CD40L的表現量比使用阿斯匹靈治療組呈現顯著的降低。而且， CD62P和CD63表現量在腦中風後的一個月兩組仍有顯著的差異。

結論

急性缺血性腦中風病患其血小板活性、以及血小板－白血球交互作用，相較於腦中風恢復期病人或對照組呈現顯著的增加。而大血管阻塞性腦中風比微小血管阻塞性腦中風表現出更高的血小板活化程度與更活躍地血小板－白血球交互作用。缺血性腦中風後血小板出現的鈣離子流動異常可能是透過PAF與ADP為媒介所造成，而保栓通是一種抑制ADP接受體的抗血小板藥物，在急性腦中風後一個月內呈現出比阿斯匹靈更強的抑制血小板活化作用，原因可能是經由抑制血小板表面ADP接受體引發的鈣離子上升所造成。另外，藉由白血球表面黏著因子的變化，我們知道白血球、血小板與血管內皮細胞的交互作用在缺血性腦中風後仍持續進行，而其產生的發炎反應可能是造成腦梗塞進一步惡化的原因。研究也顯示血小板表面CD40L的表現量可以作為預測缺血性腦中風後三個月預後的指標。

Stroke is the third most common leading cause of death worldwide and is a major cause of serious long-term disability among adults. Platelet activation and it’s interaction with leukocytes plays an important role in the pathophysiology of ischemic stroke. Anti-platelet drugs are widely used for secondary prevention after cerebral ischemia of non-cardioembolic origin and different anti-platelet drugs exert different pharmacologic effects on platelets. Several stimulations cause elevation of cytosolic Ca2+ level ([Ca2+]i), activating the secondary messenger in the platelet, and then leading to platelet activation. The majority of damage following acute stroke does not occur immediately, but rather develops gradually over the course of the following hours. Leukocytes are believed to liberate inflammatory cytokines and other neurotoxins in the ischemic brain and to promote microvascular occlusion through platelet-leukocyte-endothelium interactions in the ischemic penumbra.
In this thesis, we tested the serial changes of platelet [Ca2+]i movement in patients after acute ischemic stroke. We evaluated platelet activation markers, leukocyte adhesion molecules and platelet-leukocyte interaction in patients with acute ischemic stroke. We also analyzed the relationship between these biomarkers and the clinical outcome. Furthermore, we compared the antiplatelet effect of aspirin and clopidogrel in patients after acute stroke

Dysregulation of Ca2+ Movement in Platelets from Patients with Acute Ischemic Stroke

Thirty-one patients with acute ischemic stroke and 27 at-risk controls were enrolled in this study. The platelet [Ca2+]i was measured using a fluorescent dye fura-2 after stimulation with arachidonic acid (AA), adenosine diphosphate (ADP), platelet-activation factor (PAF), and thrombin. The basal [Ca2+]i was higher in the stroke group than in the at-risk controls irrespective of the presence or absence of extracellular Ca2+. In the Ca2+-containing medium, both PAF and ADP, but not AA and thrombin, significantly increased the platelet [Ca2+]i in the stroke patients than in the at-risk controls. However, in the Ca2+-free medium, only PAF significantly increased the platelet [Ca2+]i in the stroke patients than in the at-risk controls. The basal [Ca2+]i and PAF-induced platelet [Ca2+]i rises were still higher in the stroke patients at the subacute stage than in the at-risk controls.

Levels and Value of Platelet Activation Markers in Different Subtypes of Acute Non-Cardio-Embolic Ischemic Stroke

Platelet activation markers (CD62P, CD63, and CD40L) and platelet-leukocyte interaction were measured by flow cytometry at different time points in 54 (32 small-vessel and 22 large-vessel diseases) acute ischemic stroke patients, 28 convalescent stroke patients (3 to 9 months after acute stroke), and 28 control subjects. Patients with ischemic stroke had significantly increased circulating CD62P, CD63, platelet-monocyte interaction, and platelet-lymphocyte interaction in the acute stage compared with the convalescent stage and control groups. Levels of CD62P and CD63 were significantly higher in the large-vessel disease group than in the small-vessel disease group, and differences in CD62P were significant even at one month. The CD40L level in the poor outcome group was significantly higher than that in the good outcome group.

The Value of Leukocyte Adhesion Molecules in Patients after Ischemic Stroke

We examined serially the change of PSGL-1, Mac-1, and LFA-1 expression on leukocytes by using flow cytometry at various time points in 65 acute ischemic stroke patients and 60 controls. PSGL-1 expression on neutrophils and monocytes was significantly higher from Day 1 to Day 90 after stroke as compared with control subjects. The expression of monocyte Mac-1, LFA-1, and neutrophil Mac-1 were more significantly increased on Day 1 and 7 after stroke as compared with the control subjects. Furthermore, the neutrophil PSGL-1 expression on admission was independently associated with early neurologic deterioration.

Serial Changes in Platelet Activation Markers with Aspirin and Clopidogrel after Acute Ischemic Stroke

We designed a prospectively randomized case-control study and 70 patients with non-cardioembolic stroke who were treated with either aspirin (100 mg/d) or clopidogrel (75 mg/d) after acute ischemic stroke were evaluated. Ischemic stroke patients had significantly increased circulating CD62P, CD63, and CD40L in the acute stage as compared to the control group. Levels of CD62P, CD63 and CD40L were more significantly reduced in the clopidogrel group than in the aspirin group in the first week after stroke. Furthermore, differences in CD62P and CD63 levels were significant even at one-month post-stroke.

Conclusion

Stroke patients have enhanced platelet activity and platelet-leukocyte interaction in acute and convalescent phase of ischemic stroke compared with controls. Large-vessel cerebral infarction elicits higher platelets activation than small-vessel infarction in the acute phase of stroke. The dysregulation of Ca2+ movement, through the PAF- and ADP- receptor mediated pathway, in platelets may persist up to the subacute stage of ischemic stroke. It is possible that clopidogrel, an ADP-receptor inhibitor, elicit stronger antiplatelet effect than aspirin in the acute and convalescent phases after ischemic stroke. Sustained leukocyte-endothelium interaction, as reflected by expression of Mac-1 and LFA-1 on circulating leukocytes, may cause substantial inflammatory reaction and lead to secondary injury of potentially salvageable neurons after cerebral infarction. Ischemic stroke patients presenting with a higher CD40L level on admission were associated with a 3-month poor outcome.

中文摘要 I
ABSTRACT V
TABLE OF CONTENTS X
ABBREVIATIONS XIII
CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW 1
1-1 STROKE 2
1-1-1 Classification of stroke 2
1-1-2 Pathophysiology of ischemic stroke 4
1-2 PLATELETS AND ISCHEMIC STROKE 5
1-2-1 The platelet activation in acute ischemic stroke 5
1-2-2 Platelet-leukocyte interaction in acute ischemic stroke 7
1-2-3 Adhesion of platelet to endothelium in acute ischemic stroke 8
1-3 CALCIUM REGULATES PLATELET ACTIVATION 9
1-4 LEUKOCYTE-ENDOTHELIUM INTERACTION IN ISCHEMIC STROKE 10
1-4-1 The leukocyte activation in acute ischemic stroke 10
1-4-2 Leukocyte-endothelium adhesion 10
1-4-3 The role of leukocyte apoptosis 12
CHAPTER 2 MOTIVES AND RESEARCH OBJECTIVES 13
2-1 MOTIVES 14
2-2 RESEARCH OBJECTIVES 17
CHAPTER 3 MATERIALS AND METHODS 19
3-1 PARTICIPANTS 20
3-2 CLINICAL ASSESSMENT AND OUTCOME EVALUATION 21
3-3 TREATMENT 21
3-4 SAMPLE COLLECTION 22
3-5 ASSESSMENT OF PLATELETS CYTOSOLIC [CA2+]I 23
3-6 FLOW CYTOMETRY 24
3-6-1 Assessment of platelet activity 24
3-6-2 Assessment of platelet-leukocyte interaction 25
3-6-3 Assessment of leukocytes activity 26
3-6-4 Quantification of leukocyte apoptosis 27
3-7 ENZYME-LINKED IMMUNOSORBENT ASSAYS 27
3-7-1 Assessment of serum leukocyte and endothelium adhesion markers 27
3-7-2 Histone-associated DNA fragment ELISA 28
3-8 STATISTICAL ANALYSIS 29
CHAPTER 4 DYSREGULATION OF CA2+ MOVEMENT IN PLATELETS FROM PATIENTS WITH ACUTE ISCHEMIC STROKE 31
4-1 INTRODUCTION 32
4-2 MATERIALS AND METHODS 34
4-3 RESULTS 39
4-4 DISCUSSION 45
CHAPTER 5 LEVELS AND VALUE OF PLATELET ACTIVATION MARKERS IN DIFFERENT SUBTYPES OF ACUTE NON-CARDIO-EMBOLIC ISCHEMIC STROKE 49
5-1 INTRODUCTION 50
5-2 MATERIALS AND METHODS 52
5-3 RESULTS 57
5-4 DISCUSSION 67
CHAPTER 6 THE VALUE OF LEUKOCYTE ADHESION MOLECULES IN PATIENTS AFTER ISCHEMIC STROKE 73
6-1 INTRODUCTION 74
6-2 MATERIALS AND METHODS 76
6-3 RESULTS 80
6-4 DISCUSSION 88
CHAPTER 7 SERIAL CHANGES IN PLATELET ACTIVATION MARKERS WITH ASPIRIN AND CLOPIDOGREL AFTER ACUTE ISCHEMIC STROKE 91
7-1 INTRODUCTION 92
7-2 MATERIALS AND METHODS 94
7-3 RESULTS 98
7-4 DISCUSSION 105
CHAPTER 8 CONCLUSIONS AND FUTURE WORKS 109
8-1 CONCLUSIONS 110
8-2 CLINICAL APPLICATION OF THE THESIS 111
8-3 LIMITATIONS OF THE RESEARCH 112
8-4 FUTURE WORKS 113
REFERENCES 115
APPENDIX 133

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