A major role for Scar/WAVE-1 downstream of GPVI in platelets

BACKGROUND: The small GTPase Rac1 plays a critical role in lamellipodia assembly in platelets on matrix proteins in the absence or presence of G protein-coupled receptor (GPCR) agonists. Rac mediates actin assembly via Scar/WAVE, a family of scaffolding proteins that direct actin reorganization by relaying signals from Rac to the Arp2/3 complex. OBJECTIVE: To evaluate the role of Scar/WAVE-1 in mediating platelet activation and cytoskeletal reorganization. METHODS AND RESULTS: Using specific antibodies, we demonstrate that murine platelets, like human platelets, express Scar/WAVE-1 and Scar/WAVE-2. Lamellipodia formation in Scar/WAVE-1(-/-) platelets is markedly inhibited on immobilized collagen-related peptide (CRP) and on laminin, both of which signal through the collagen receptor GPVI. In contrast, lamellipodia formation on collagen, which requires release of the GPCR agonists ADP and thromboxane A(2), is not altered. Immobilized fibrinogen supports limited formation of lamellipodia in murine platelets, which is not altered in Scar/WAVE-1(-/-) platelets. As with Rac1(-/-) platelets, Scar/WAVE-1(-/-) platelets exhibit a marked inhibition of aggregation in response to CRP, whereas the response to the GPCR agonist thrombin is not altered. Platelet aggregation on immobilized collagen under shear, which is dependent on signaling by matrix and GPCR agonists, was unaltered in the absence of Scar/WAVE-1. CONCLUSION: This study demonstrates a major role for Scar/WAVE-1 in mediating platelet cytoskeletal reorganization and aggregate formation downstream of activation by GPVI but not by GPCR agonists.     

中性粒细胞对洗涤血小板聚集功能的影响

目的:观察非激活或激活的中性粒细胞(PMN)对洗涤血小板聚集功能的影响。方法:采用Born方法测定血小板聚集功能。结果:非激活的PMN(5×10⁹cells/L)上清液对ADP和花生四烯酸(AA)诱导的血小板聚集具有显著抑制作用,阿司匹林可增强这种抑制作用;肉豆寇佛波醇(fMLP)及血小板活化因子(PAF)激活的PMN悬液或其上清液均能活化血小板聚集,且PMN悬液的诱聚作用比其上清液更强;阿司匹林对fMLP或PAF激活的PMN悬液或其上清液均无明显抑制作用。结论:不同状态(非激活或激活状态)的PMN对正常血小板的反应性表现出完全相反的作用,即非激活的PMN抑制血小板反应性,而激活的PMN则具有促血小板活化聚集作用。     

Molecular and cellular basis of restenosis after percutaneous coronary intervention: the intertwining roles of platelets, leukocytes, and the coagulation-fibrinolysis system

The major limitation of percutaneous coronary intervention (PCI) is restenosis. Restenosis is considered to be an overreaction of the natural healing process after traumatic balloon dilatation. An elaborate web of cellular and molecular responses, including the interaction of platelets, leukocytes, and the coagulation-fibrinolysis system, as well as the secretion of various growth factors and pro-inflammatory cytokines, contributes to neointimal hyperplasia and the development of restenosis. Moreover, platelet and neutrophil activation after stenting appears to be different from that after balloon angioplasty alone. Pharmacotherapy targeting the cell-to-cell interaction between platelets and neutrophils may potentially offer an effective treatment strategy against restenosis after PCI.     

Emerging Biomarkers Beyond Leukotrienes for the Management of Nonsteroidal Anti-inflammatory Drug (NSAID)-Exacerbated Respiratory Disease

Nonsteroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is a unique condition characterized by aspirin/NSAID hypersensitivity, adult-onset asthma, and/or chronic rhinosinusitis with nasal polyps. Arachidonic acid metabolism dysregulation and intense eosinophilic/type 2 inflammation are central mechanisms in NERD. Studies have been conducted on various biomarkers, and urinary leukotriene E4 is considered the most available biomarker of NERD. However, the pathophysiology of NERD is heterogeneous and complex. Epithelial cells and platelets can interact with immune cells in NERD, and novel biomarkers related to these interactions have recently been investigated. We summarize emerging novel biomarkers of NERD and discuss their roles in the management of NERD.     

Platelet counts and outcome in the pediatric intensive care unit

Objectives:

Thrombocytopenia is commonly observed in critically ill patients. This study was undertaken to evaluate the variation in platelet counts and the risk factors associated with thrombocytopenia and mortality in pediatric intensive care patients. In addition, prognostic value of platelet counts for outcome in pediatric intensive care unit was studied.

Study Design:

Prospective, observational cohort analysis.

Setting:

8- bedded pediatric intensive care unit of a tertiary care teaching hospital.

Patients:

All consecutively admitted patients (n=138) staying in the pediatric intensive care unit (PICU) for at least 48h over a 7 months period were studied.

Measurements and Main Results:

Thrombocytopenia was defined as platelet counts <150.0/nL. Median 1^st day Pediatric Risk of Mortality Score (PRISM) was 5 (range 0-30) and median ICU stay was 4 days (range 2-98 days). Twenty five percent patients had at least one episode of thrombocytopenia during the stay. Twenty percent of these patients had thrombocytopenia on admission and rest (80%) developed it during the PICU stay. Seventy one percent (19) of the patients developed thrombocytopenia by fourth day of admission. Patients with PICU acquired thrombocytopenia had statistically significant lower baseline, nadir and 4th day platelet counts and a significantly higher drop in platelet counts (56% vs. 6% P<0.001) as compared to non thrombocytopenic patients. PRISM score, long PICU stay, sepsis, coagulopathy, and creatinine levels were significantly associated with occurrence of thrombocytopenia. Patients with thrombocytopenia had higher probability of bleeding (34% vs. 15%, P=0.01). Higher platelet counts on admission were associated with significantly reduced risk of thrombocytopenia (P=0.00) Baseline, nadir and day-4 platelet counts, presence of thrombocytopenia on admission, sepsis, coagulopathy and a higher mean PRISM score on univariate analysis were significantly associated with mortality. Leucopenia or leucocytosis, thrombocytopenia and coagulopathy were found to significantly affect outcome. Drop in platelet counts was found to have slightly higher discriminative value for mortality prediction than PRISM on the ROC curve. The survivors had higher platelet counts throughout the PICU stay and after an initial fall in platelet counts in the PICU showed a significantly higher rise in the platelet counts in the following days than the non-survivors.

Conclusions:

Thrombocytopenia is common in PICU. Patients requiring cardiopulmonary resuscitation or with circulatory shock, coagulopathy, sepsis and with more severe disease have higher risk of developing thrombocytopenia. Thrombocytopenic patients have a higher risk of bleeding. Drop in platelet counts >27% and thrombocytopenia were independently related to mortality. Serial measurements of platelet counts are better predictors of pediatric intensive care outcome than one-time values. Any drop in platelet counts even without thrombocytopenia needs an urgent and extensive evaluation.     

Human platelets express Toll-like receptor 3 and respond to poly I:C

Platelets functions in hemostasis have been widely studied. Currently, growing evidence shows that platelets have also a role in the immune innate response. Recently, protein expression of Toll-like receptors (TLR’s) 2, 4, 7, 8, and 9, and the presence of TLRs 1 and 6 mRNA in human platelets was described. Up to now the functionality of TLR-2, 4 and 9 in human platelets has been demonstrated. Due to the relevance of TLRs functions to PAMPS (pathogen-associated molecular patterns) recognizing, we evaluated the presence of TLR3 in human platelets founding low percentages of platelets expressing surface or intracellular TLR3 protein. The activation with thrombin induced an increase in the percentage of platelets expressing surface TLR3 and higher levels of TLR3 expression in the whole population. Human platelets responded to poly I:C by increasing [Ca²⁺]_i, the percentages of cells expressing TLR4 and CD62P, and by releasing CXCL4 and IL-1β in comparison to unstimulated platelets. These results demonstrate that human platelets express TLR3 and are capable of responding to poly I:C, suggesting that these cells might influence the immune innate response when detecting viral dsRNA.     

血小板膜外周型苯二氮卓受体在初发脑梗死后抑郁中的作用

 目的：观察卒中后抑郁（PSD）患者血小板膜外周型苯二氮卓受体（PBRs）的变化,探讨PBRs在PSD中的作用。方法：卒中后抑郁组为初发脑梗死后PSD患者43例、脑梗死组为初发脑梗死患者59例、对照组为健康献血者46名。采用Hamilton抑郁量表（HAMD）评定患者抑郁程度。提取外周血血小板膜,应用放射性配基［3H］PK11195结合实验测定PBRs特异结合活性。结果：［3H］PK11195结合活性3组之间有显著差异（P<0.01）。与对照组［（298.2±25.1) pmol/（g protein）］比较,脑梗死组［3H］PK11195结合活性［（1 410.8±41.4） pmol/(g protein)］显著升高（P<0.01）。与脑梗死组比较,PSD组［3H］PK11195结合活性［（361.7±30.6） pmol/g protein］显著降低（P<0.01）。PSD组男性、女性患者之间比较,［3H］PK11195结合活性差异不显著。PSD组［3H］PK11195结合活性与患者病程无显著相关性（r=0.27,P＞0.05）,与HAMD评分呈显著负相关（r=-0.44,P＜0.01）。结论:PSD患者血小板膜PBRs结合活性下降, PBRs影响抑郁程度。     

Sialylation of ICAM-2 on Platelets Impairs Adhesion of Leukocytes via LFA-1 and DC-SIGN

Intercellular adhesion molecule (ICAM)-2 is highly expressed on platelets and endothelium and is a counter-receptor for the leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1) and for the dendritic cell-specific, ICAM-grabbing non-integrin (DC-SIGN) protein. In this study, we investigated structural and functional differences between ICAM-2 from platelets and that from endothelial cells. The isoelectric point (pI) of ICAM-2 from HUVEC was pH 3.5-4.3, whereas that of platelet ICAM-2 was more acidic at pH 3.0-3.7. This charge difference was abolished by treatment with N-glycanase or neuraminidase, thus it was due to cell-specific N-linked glycosylation. Purified, immobilized platelet ICAM-2 supported 50% less adhesion of LFA-1-bearing T cells than did purified HUVEC ICAM-2 and no adhesion was observed of monocyte-derived immature dendritic cells via DC-SIGN to platelet ICAM-2. Treatment of platelet ICAM-2 with neuraminidase abolished these functional differences. These findings demonstrated that physiologic sialylation of platelet ICAM-2 renders it less able than endothelial ICAM-2 to support adherence of leukocytes.