Brogren Helén, Karlsson Lena, Andersson Maria, Wang Lingwei, Erlinge David, Jern Sverker
Clinical Experimental Research Laboratory, Department of Medicine, Sahlfrenska University Hospital/Ostra, Cardiovascular Institute, Göteborg, Sweden.
Blood. 2004 Dec 15;104(13):3943-8. doi: 10.1182/blood-2004-04-1439. Epub 2004 Aug 17.
Previous studies have suggested that plasminogen activator inhibitor 1 (PAI-1) released from platelets convey resistance of platelet-rich blood clots to thrombolysis. However, the majority of PAI-1 in platelets is inactive and therefore its role in clot stabilization is unclear. Because platelets retain mRNA and capacity for synthesis of some proteins, we investigated if platelets can de novo synthesize PAI-1 with an active configuration. PAI-1 mRNA was quantified with real-time polymerase chain reaction and considerable amounts of PAI-1 mRNA were detected in all platelet samples. Over 24 hours, the amount of PAI-1 protein as determined by an enzyme-linked immunosorbent assay increased by 25% (P = .001). Metabolic radiolabeling with (35)S-methionine followed by immunoprecipitation confirmed an ongoing PAI-1 synthesis, which could be further stimulated by thrombin and inhibited by puromycin. The activity of the newly formed PAI-1 was investigated by incubating platelets in the presence of tissue-type plasminogen activator (tPA). This functional assay showed that the majority of the new protein was in an active configuration and could complex-bind tPA. Thus, there is a continuous production of large amounts of active PAI-1 in platelets, which could be a mechanism by which platelets contribute to stabilization of blood clots.
先前的研究表明，血小板释放的纤溶酶原激活物抑制剂1（PAI-1）使富含血小板的血凝块对溶栓产生抵抗。然而，血小板中的大多数PAI-1是无活性的，因此其在血凝块稳定中的作用尚不清楚。由于血小板保留mRNA并具有合成某些蛋白质的能力，我们研究了血小板是否能够从头合成具有活性构型的PAI-1。用实时聚合酶链反应对PAI-1 mRNA进行定量，在所有血小板样品中均检测到相当数量的PAI-1 mRNA。在24小时内，通过酶联免疫吸附测定法测定的PAI-1蛋白量增加了25%（P = .001）。用（35）S-甲硫氨酸进行代谢性放射性标记，然后进行免疫沉淀，证实了PAI-1的持续合成，凝血酶可进一步刺激其合成，而嘌呤霉素可抑制其合成。通过在组织型纤溶酶原激活物（tPA）存在的情况下孵育血小板，研究新形成的PAI-1的活性。该功能测定表明，大多数新蛋白处于活性构型，并且可以与tPA形成复合物结合。因此，血小板中持续产生大量有活性的PAI-1，这可能是血小板促进血凝块稳定的一种机制。
