Saumet Anne, de Jesus Nando, Legrand Chantal, Dubernard Véronique
INSERM Unité 553, Institut d'Hématologie Université Paris 7, Hôpital Saint-Louis, 75010 Paris, France.
Biochem J. 2002 May 1;363(Pt 3):473-82. doi: 10.1042/0264-6021:3630473.
Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from alpha-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted alpha-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation, and was inhibited by cytochalasin D, an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors, as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. alphaIIbbeta3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking alphaIIbbeta3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes, as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1, in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of alphaIIbbeta3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin microfilament system which may promote platelet aggregate cohesion.
血小板反应蛋白-1（TSP-1）是一种黏附性糖蛋白，当它从活化血小板的α颗粒分泌出来时，能够与细胞表面结合并参与血小板聚集体的形成。在本研究中，我们发现凝血酶激活会导致大量分泌的α颗粒TSP-1与肌动蛋白细胞骨架迅速且特异性地结合。TSP-1与细胞骨架的这种结合与血小板分泌相关，但与聚集无关，并且会被肌动蛋白聚合抑制剂细胞松弛素D所抑制。TSP-1与肌动蛋白细胞骨架的结合是由膜受体介导的，使用MAII（一种TSP-1特异性单克隆抗体）可证明这一点，它既能抑制TSP-1与活化血小板的表面结合，也能抑制其与细胞骨架的结合。TSP-1及其潜在的膜受体，如αIIbβ3整合素、CD36和CD47，会同时与肌动蛋白细胞骨架结合。然而，对一名缺乏αIIbβ3的I型Glanzmann血小板无力症患者以及另一名几乎检测不到CD36的患者的血小板研究表明，TSP-1的表面表达及与肌动蛋白细胞骨架的结合均正常。同样，使用功能阻断性抗CD47抗体B6H12对对照血小板进行实验，无法推断出CD47参与TSP-1与肌动蛋白细胞骨架的结合。最后，通过酪氨酸磷酸化蛋白和激酶向肌动蛋白细胞骨架的转位观察到的信号复合物组装，在对照血小板以及血小板无力症和CD36缺陷血小板中，均与TSP-1的细胞骨架结合同时发生。我们的结果表明肌动蛋白细胞骨架在TSP-1分子的膜表面表达过程中发挥作用，并提示TSP-1受体可能与独立于αIIbβ3或CD36发生的信号事件偶联。这些结果为表面结合的TSP-1与收缩性肌动蛋白微丝系统之间的联系提供了新的见解，这可能促进血小板聚集体的黏附。
