Date Published: February 3, 2017
Publisher: Public Library of Science
Author(s): Marlien Pieters, Sunelle A. Barnard, Du Toit Loots, Dingeman C. Rijken, Pablo Garcia de Frutos.
Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma.
Plasminogen activator inhibitor type-1 (PAI-1) is a serine protease inhibitor (serpin) [1, 2], which acts as a main inhibitor of fibrinolysis . Elevated plasma PAI-1 levels have been associated with a risk for developing atherothrombosis [4–6] due to its antifibrinolytic properties, by reducing the clearance of fibrin in plaques , and also via its influence on cellular migration, matrix remodelling and activation of growth factors [7, 8]. Plasma PAI-1 exists either in an active or latent form, or in complex with tissue plasminogen activator (tPA) [9–11]. The active form of PAI-1 is unstable, with a half-life of approximately two to three hours, after which it will spontaneously convert to the inactive, latent form [9, 12]. PAI-1 is produced by various cells such as endothelial cells, hepatocytes, smooth muscle cells, adipocytes, and platelets [11, 13]. In platelets, PAI-1 is stored in the alpha granules and is released during platelet activation and aggregation [11, 14, 15].
This study investigated the effect of residual platelets present in plasma, on plasma PAI-1 and PAI-1-related assay results. The presence of platelets in plasma significantly influenced plasma PAI-1ag levels in a concentration dependent manner, likely due to an increase in mainly plasma latent PAI-1. Only in the presence of large amounts of platelets such as in PRP, functional effects in terms of plasma fibrinolytic potential are seen, suggesting the presence of a comparatively lower concentration of active PAI-1 in platelets. It was furthermore demonstrated that platelets present in plasma, do not initially release all of their PAI-1 content and that further release of PAI-1 can occur upon further / complete in vitro platelet degradation.