||The role of Rho kinase in activated platelets and pathogenesis of platelet-monocyte aggregates in atherothrombosis
||Institute of Basic Medical Sciences
動脈血栓形成起因為粥狀動脈硬化斑塊損傷時會釋放血栓前物質和促進血小板聚集。血小板是參與血栓形成的主要角色，並與血管中的免疫細胞共同作用。在本篇研究中，我們研究在動脈血栓中活化後的血小板如何與單核細胞相互作用。我們藉由經皮冠狀動脈介入治療收集患者的冠狀動脈血液，並測量血小板活性。急性冠心症患者冠狀動脈中的血小板具有較高的偽足和活性。另外核醣體基因圖譜表明冠狀動脈中的血小板有大量的Rho GTPases及其下游表現因子。RhoA活化下游ROCK，並且ROCK會增加冠狀動脈血小板中的表面P-selectin蛋白。在體外實驗和急性冠心症患者的血斑塊中觀察到血小板和單核細胞之間的聚集。此外，我們發現活化後的血小板促進了單核細胞的遷移，而此ROCK抑制劑可以抑制此遷移。凝血酶誘導的血小板表面P-selectin蛋白和MIF蛋白分泌增加，並且透過ROCK訊號傳遞路徑增加CCR2的表現。CCR2在單核細胞-血小板聚集物中表現高於沒有血小板作用的單核細胞。最後，我們使用亞洲篩查陣列微珠芯片(Asian Screening Array BeadChip)，辨別與心血管事件有關的SNP。值得注意的是，患者基因帶有RHOA SNP rs11706370主要等位基因在心血管事件中的風險較高。在動脈血栓中，血小板透過ROCK重塑細胞骨架和P-selectin蛋白表現，並且聚集單核細胞並且與CCR2高表現的單核細胞協同誘發血栓炎症。
Arterial thrombosis is initiated by atherosclerotic plaque damage, prothrombotic material release and platelet aggregation. Platelets are primary mediators involved in thrombosis and cooperate with vascular and immune cells. Herein, we investigated how activated platelets interacted with monocytes in atherothrombosis. We collected patients’ blood from coronary arteries during percutaneous coronary intervention (PCI), and measured platelet activity. Platelets from coronary arteries had higher pseudopodia expression and activity in patients with acute coronary syndrome (ACS). Ribosome profiling of platelets from coronary blood mapped a vigorous upregulation of Rho GTPases and its downstream effectors. RhoA activates downstream Rho associated coiled-coil containing protein kinase (ROCK), and ROCK increased surface P-selectin in coronary blood platelets. The interaction between platelets and monocytes was observed in vitro, and monocyte-platelet aggregates (MPAs) was found in ruptured coronary plaques of ACS. Further we found that activated platelets promoted monocyte transmigration, which could be suppressed in the presence of ROCK inhibitor. The increased surface P-selectin and macrophage migration inhibitory factor (MIF) secretion on thrombin-induced platelets interacted with monocytes to upregulate monocyte chemokine receptor 2 (CCR2) expression via the ROCK pathway. The expression of CCR2 was higher in MPAs than that of monocytes without platelets. Finally, using the Asian Screening Array BeadChip, we identified patients containing homozygous major alleles of the RHOA SNP rs11706370 represented with higher risks in cardiovascular events. Through ROCK activated cytoskeleton remodeling and P-selectin expression, platelets recruited and interacted synergistically with high CCR2 expressing monocytes to induce thrombo-inflammation in atherothrombosis.
Chinese abstract II
Figure list VIII
Table list XI
Chapter 1. Introduction 1
1.1 Coronary artery disease and therapy 1
1.2 Platelets in atherothrombosis 2
1.3 GPCR in platelets 5
1.4 The translatome of platelets activation 7
1.5 Monocytes-platelets aggregates in atherothrombosis 8
Chapter 2. Objective and Specific Aims 10
Chapter 3. Materials and Methods 12
3.1 Clinic study design 12
3.1.1 Patients’ enrollment 12
3.1.2 Study protocol 13
3.2 Platelets isolation 13
3.2.1 Patients’ and heathy donors’ platelets 13
3.2.2 Washed platelets for ex vivo 14
3.3 Platelets activity analysis 14
3.4 Translatome analysis 14
3.4.1 RNA sequencing libraries. 14
3.4.2 Bioinformatic analysis of ribosome profiling data. 15
3.4.3 Gene ontology (GO) analysis 16
3.5 Immunofluorescence (IF) staining 16
3.5.1 Morphology observation of platelets 16
3.5.2 Platelets-monocytes/macrophages aggregates in coronary thrombus 17
3.5.3 Immunofluorescence staining of platelets-THP-1 aggregates 17
3.6 RhoA pull-down activation analysis 18
3.7 Western blotting 18
3.8 Clot formation assay 19
3.9 Fluorescence-activated cell sorting (FACS) analysis 19
3.9.1 Measurement of P-selectin and CCR2 level under thrombin-induced platelets and monocytes 19
3.9.2 Measurement of P-selectin and CCR2 level from patients’ platelets and monocytes 20
3.10 Soluble P-selectin and MIF level from coronary culprit cite of CAD patients 20
3.11 Cell culture 21
3.12 Transmigration assays 21
3.13 SNP Genotyping Assays 21
3.13.1 Sample preparation 21
3.13.2 DNA extraction 22
3.13.3 Human Genome-Wide Arrays 22
3.13.4 TaqMan qPCR Assay 23
3.14 Statistical analysis 23
Chapter 4. Results 25
4.1 Patient Characteristics 25
4.2 Platelets activity was higher within the coronary blood circulation than the peripheral blood among ACS population 25
4.3 The Rho family was enriched in platelets from coronary blood of ACS. 26
4.4 The RhoA–ROCK signaling pathway is activated in platelets from coronary blood of ACS. 27
4.5 P-selectin on platelet surface: the interaction with ROCKs in thrombin-induced platelet aggregation 28
4.6 The interaction with ROCKs in thrombin-induced platelets and monocytes 29
4.6.1 Platelets drove monocyte migration and promoted CCR2 expression of monocytes 29
4.6.2 Platelet-cultured medium drove monocyte migration and promoted CCR2 expression of monocytes 30
4.6.3 Compared THP-1 counts, CCR2 and P-selectin surface expression of THP-1 interacted with platelets or platelet-cultured medium 31
4.6.4 Low concentration of ROCK inhibitor did not reduce the transmigration of THP-1 cells 31
4.6.5 Block P-selectin in cultured medium reduce the transmigration of THP-1 cells 32
4.6.6 Inflammatory cytokine, IL6, TNFα, ICAM1 increased in MPAs 32
4.6.7 The correlation of platelets and monocytes via ROCKs in CAD patients 33
4.7 Association of SNPs with RhoA-ROCK-P-selectin pathway and patients’ cardiovascular events 33
4.8 MIF, a chemokine of platelets: the interaction with ROCKs in thrombin-induced platelet aggregation 34
Chapter 5. Discussion 36
5.1 WBC counts and smoking of patients linked to the thrombo-inflammation 36
5.2 Platelet activity between coronary and peripheral blood 36
5.3 GPCR and Rho GTPase activated platelets 38
5.4 ROCK1 and ROCK2 have different roles in platelets 39
5.5 Platelet P-selectin played a dominant role in thrombo-inflammation 40
5.6 Platelet MIF played a dominant role in thrombo-inflammation 41
5.7 The interactions of MPAs increased in athero-thrombosis 42
5.8 CCR2 on monocytes leads to inflammation 43
5.9 SNPs are related CVD 43
Chapter 6. Conclusion 45
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