Leven R M, Tablin F
Department of Anatomy, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612.
Exp Hematol. 1992 Dec;20(11):1316-22.
We have used an in vitro culture system to study the role of extracellular matrix (ECM) in the fragmentation of guinea pig bone marrow megakaryocytes (MK) and the formation of proplatelet fragments from these cells. Proplatelet formation is stimulated by culturing the cells on a hydrated type I collagen gel in the presence of serum. MK express integrin proteins alpha 5, alpha 6, beta 1, and the alpha v beta 3 complex as demonstrated by immunofluorescence. A monoclonal antibody, LM 609, to the alpha v beta 3 vitronectin receptor blocked proplatelet formation, whereas the monoclonal antibodies to the beta 1, alpha 5, and alpha 6 integrin proteins did not inhibit proplatelet formation. The tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits proplatelet formation; however, there was no inhibition by the fibronectin receptor-specific peptide GRGDdSP. The fibrinogen gamma chain peptide HHLGGAKQAGDV, which binds to the platelet membrane glycoprotein complex IIb-IIIa but not to the vitronectin receptor (VnR), did not inhibit proplatelet formation, nor did two different laminin peptides. In the absence of serum, 5.7% of MK spontaneously formed processes or fragmented. The addition of 50 micrograms/ml of vitronectin to serum-free cultures increased proplatelet formation to 21.5% of the MK, equal to cultures with 10% serum. Stimulation of proplatelet formation by vitronectin in serum-free cultures was inhibited by LM 609. Antibody staining with anti-bovine vitronectin antibody showed that MK contain intracellular vitronectin. These data show that guinea pig MK express alpha 5, alpha 6, beta 1, and alpha v beta 3 integrins. However, only the MK vitronectin receptor and its interaction with vitronectin plays an essential role in proplatelet formation in vitro.
我们利用体外培养系统研究细胞外基质（ECM）在豚鼠骨髓巨核细胞（MK）碎片化以及这些细胞形成前血小板片段过程中的作用。通过在含有血清的水合I型胶原凝胶上培养细胞来刺激前血小板的形成。免疫荧光显示，MK表达整合素蛋白α5、α6、β1和αvβ3复合物。针对αvβ3玻连蛋白受体的单克隆抗体LM 609可阻断前血小板的形成，而针对β1、α5和α6整合素蛋白的单克隆抗体则不抑制前血小板的形成。四肽精氨酸-甘氨酸-天冬氨酸-丝氨酸（RGDS）可抑制前血小板的形成；然而，纤连蛋白受体特异性肽GRGDdSP却没有抑制作用。与血小板膜糖蛋白复合物IIb-IIIa结合但不与玻连蛋白受体（VnR）结合的纤维蛋白原γ链肽HHLGGAKQAGDV以及两种不同的层粘连蛋白肽均未抑制前血小板的形成。在无血清条件下，5.7%的MK会自发形成突起或碎片化。向无血清培养物中添加50微克/毫升的玻连蛋白可使前血小板的形成增加至MK的21.5%，与含有10%血清的培养物相当。在无血清培养物中，玻连蛋白对前血小板形成的刺激作用被LM 609抑制。用抗牛玻连蛋白抗体进行抗体染色显示，MK含有细胞内玻连蛋白。这些数据表明，豚鼠MK表达α5、α6、β1和αvβ3整合素。然而，只有MK玻连蛋白受体及其与玻连蛋白的相互作用在体外前血小板形成过程中起关键作用。
