Platelet leukocyte interactions in vascular disease

描述(申请人的目标清单)具体目标1.使用活体显微镜在小鼠肠系膜和肌腱循环血栓形成模型中评估血小板-白细胞相互作用的动力学。具体目的2.评估血管损伤后血小板上的白细胞沉积是否有助于血管钝化，即恢复到血小板不再被招募的状态。将在野生型小鼠、β3整合素-/-小鼠、P-选择素-/-小鼠、α-Mβ-/-小鼠和PECAM-1-/-小鼠身上测量股动脉损伤后6小时内选定的30分钟内放射性标记血小板的沉积。具体目的3.探讨血小板-白细胞相互作用在血管损伤后白细胞迁移和血管内膜增生中的作用。使用我们的小鼠模型，在野生型小鼠和缺乏β3整合素、P-选择素、αMbeta2和PECAM-1的小鼠中，将评估前6小时内白细胞向血管中层的迁移，并将其与4周后发生的内膜增生相关。具体目的4.体外评估正常人全血与不锈钢支架接触过程中血小板-白细胞相互作用对以下方面的影响：1)血栓形成；2)组织因子；凝血酶；激活的血小板(P-选择素)；激活的中性粒细胞(α-Mbeta2)；血小板-中性粒细胞、血小板-单核细胞和血小板-血小板聚集体；血小板微粒；血管内皮生长因子；细胞因子1-309；和“可溶性”CD40配体；3)支架上血小板沉积的钝化，以及4)支架血栓的形态。将使用针对GPIIb/IIIa和/或αVbeta3、P-选择素、αMbeta2和PECAM-1的封闭抗体进行研究，以评估这些受体的作用。具体目的5.评估具有血栓形成危险因素的患者和对照组在采取干预措施改变危险因素前后，体内循环中是否存在血小板-白细胞的相互作用，并将这些发现与全身炎症状态的标记物相关联(见项目1)。将对血液进行血小板-中性粒细胞聚集物、血小板-单核细胞聚集物、血小板-血小板聚集物、白细胞αMbeta2表达、血小板P-选择素表达和血小板微粒的检测。这些数据将与在流室研究中获得的循环组织因子、C反应蛋白、组织因子途径抑制、凝血酶原片段F1.2和血栓形成的数据相关联。

DESCRIPTION (Applicant's list of Aims) Specific Aim 1. To assess the dynamics of platelet-leukocyte interactions using intravital microscopy in mouse models of thrombosis in the mesenteric and cremasteric circulations. Specific Aim 2. To assess whether leukocyte deposition on platelets after vascular injury contributes to blood vessel passivation, that is, a return to a state in which platelets are no longer recruited. Deposition of radiolabeled platelets over select 30 min periods for 6 hours after femoral artery injury will be measured in wild-type mice, beta3 integrin -/- mice, P-selectin -/- mice, alphaMbeta -/- mice, and PECAM-1 -/- mice. Specific Aim 3. To assess the role of platelet-leukocyte interactions in leukocyte transmigration and the development of intimal hyperplasia after vascular injury. Using our murine model, leukocyte transmigration into the blood vessel media during the first 6 hours will be assessed and correlated with the intimal hyperplasia that develops after 4 weeks in wild-type mice and mice deficient in beta3 integrins, P-selectin, alphaMbeta2, and PECAM-1. Specific Aim 4. To assess ex vivo the effects of platelet-leukocyte interactions during contact of normal whole human blood with a stainless steel stent on: 1) thrombus formation; 2) release into the downstream circulation of tissue factor; thrombin; activated platelets (P- selectin); activated neutrophils (alphaMbeta2); platelet-neutrophil, platelet- monocyte, and platelet-platelet aggregates; platelet microparticles; VEGF; the cytokine 1-309; and "soluble" CD40 ligand; 3) passivation of platelet deposition on the stent, and 4) stent thrombus morphology. Studies using blocking antibodies to GPIIb/IIIa and/or alphaVbeta3, P-selectin, alphaMbeta2, and PECAM-1 will be conducted to assess the contributions of these receptors. Specific Aim 5. To assess the presence of platelet- leukocyte interactions in the systemic circulation of patients with risk factors for thrombosis and controls, before and after interventions to modify the risk factors, and correlate these findings with markers of a systemic inflammatory state (see Project 1). Blood will be tested for platelet- neutrophil aggregates, platelet-monocyte aggregates, platelet-platelet aggregates, leukocyte alphaMbeta2 expression, platelet P-selectin expression, and platelet microparticles. These data will be correlated with data obtained on circulating tissue factor, C-reactive protein, tissue factor pathway inhibitory, prothrombin fragment F1.2, and thrombus formation in flow chamber studies.