![]() ![]() Trizol was from Gibco BRL, Life Technology. 35S-labeled methionine was from Amersham Biosciences (Buckinghamshire, United Kingdom). Human aortic endothelial cells (HAECs) were purchased from Clonetics, BioWhittaker (Walkersville, MD). All PCR consumables were purchased from Applied Biosystems (Foster City, CA). The RiboGreen RNA Quantitation Kit was from Molecular Probes (Leiden, The Netherlands). ![]() Two different types of medium 199 were used, M199 without phenol red from Gibco BRL, Life Technology (Paisley, United Kingdom) and M199 without phenol red and methionine purchased from JHR Biosciences (Lenexa, KS). Because the major part of the newly synthesized PAI-1 was found to be active, this could provide a mechanism by which platelet-rich clots maintain resistance to fibrinolysis. 23 In the present work, we provide direct experimental evidence that platelets contain large amounts of PAI-1 mRNA that is translationally active. ![]() 20-22 We have recently shown that mRNA extracted from platelets can be accurately quantified by real-time polymerase chain reaction (PCR). 14-16 Although platelets lack nuclear DNA they retain mRNA from the megakaryocyte, 17-19 and it has been shown that platelets retain the ability for protein synthesis and can synthesize at least some proteins. Previous studies have shown that only 5% to 10% of the PAI-1 present in the platelets is in an active configuration that could complex-bind and thereby inhibit tPA. On the other hand, the pathophysiologic importance of the platelet PAI-1 pool for inhibition of the fibrinolytic system has been difficult to reconcile with the fact that the majority of PAI-1 in platelets exists in a predominantly inactive or latent form. 12 Also, studies in transgenic mice have suggested that PAI-1 not only influences the resistance to thrombolysis but also the rate of progression of thrombus formation following vascular injury. ![]() 10 The hypothesis that PAI-1 released from platelets is an important determinant of thrombolysis resistance is supported by in vitro clot lysis studies on platelets from a patient with complete loss of PAI-1 expression 11 as well as studies on thrombi generated in the Chandler loop. 9 It is likely that the major part of the PAI-1 in thrombi has been released from activated platelets because platelets contain large amounts of the inhibitor in their α-granules. Arterial clots contain 2- to 3-fold more PAI-1 than venous clots, 7, 8 and the relative PAI-1 content determines their resistance to thrombolysis. 6 Its activity in plasma is regulated by specific inhibitors, the most important of which is plasminogen activator inhibitor 1 (PAI-1). 2004 104:3943-3948)Ĭlot lysis in vivo results primarily from the enzymatic activation of the fibrinolytic system, and stimulated secretion of tissuetype plasminogen activator (tPA) from the vascular endothelium is the key event in this activation. 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. This functional assay showed that the majority of the new protein was in an active configuration and could complexbind tPA. The activity of the newly formed PAI-1 was investigated by incubating platelets in the presence of tissue-type plasminogen activator (tPA). Metabolic radiolabeling with 35S-methionine followed by immunoprecipitation confirmed an ongoing PAI-1 synthesis, which could be further stimulated by thrombin and inhibited by puromycin. Over 24 hours, the amount of PAI-1 protein as determined by an enzyme-linked immunosorbent assay increased by 25% ( P =. PAI-1 mRNA was quantified with real-time polymerase chain reaction and considerable amounts of PAI-1 mRNA were detected in all platelet samples. 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. However, the majority of PAI-1 in platelets is inactive and therefore its role in clot stabilization is unclear. Previous studies have suggested that plasminogen activator inhibitor 1 (PAI-1) released from platelets convey resistance of platelet-rich blood clots to thrombolysis. ![]()
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