Biosynthesis of major platelet proteins in human blood platelets

Abstract

We studied de novo protein biosynthesis in platelets of normal adult donors and in newly formed platelets isolated from splenectomized patients with idiopathic thrombocytopenic purpura (ITP). After metabolic labelling of platelet proteins, performed with different radiolabelled amino acids or carbohydrates, a tenfold increase in incorporation of radioactivity into trichloroacetic-acid-precipitable material was obtained with ITP platelets compared to control platelets. Electron microscopic studies of ITP platelets revealed the presence of rough endoplasmic reticulum and polyribosomes, providing morphological evidence for protein synthesis. SDS-PAGE of radiolabelled ITP platelet proteins followed by autoradiography showed that [35S]methionine and [3H]leucine were incorporated into almost all Coomassie-blue-stained proteins whereas [3H]cdrbohydrates only labelled a few bands. Using crossed-immunoelectrophoresis we identified some of the labelled platelet compounds and demonstrated that major membrane glycoproteins (GPIb, IIb, ZIIa) and alpha-granule proteins, such as fibrinogen, thrombospondin, albumin and von Willebrand factor, were synthesized in newly formed circulating platelets.Human platelets play an essential role in hemostasis and thrombosis. Following vascular injury, platelets adhere to exposed subendothelial tissue, become activated and secrete the content of their storage organelles. Released alpha-granule proteins, such as fibrinogen, von Willebrand factor, thrombospondin and factor V, interact with specific platelet membrane receptors to induce aggregation, thrombin conversion and fibrin formation for eventual clot retraction [I], whereas released platelet-derived growth factors promote tissue repair processes [2].Since human platelets are anucleated cytoplasmic fragments of megakaryocytes with a short lifespan in the peripheral blood, synthesis of platelet proteins is thought to occur almost solely in the precursor cell, the megdkaryocyte. Platelet-specific membrane glycoproteins have indeed been identified in megakaryocytes [3 -61, which are also known to contain platelet-related proteins such as von Willebrand factor [7] [12]. However, the site of biosynthesis of some platelet alpha-granule proteins, such as thrombospondin, albumin or immunoglobulins [ 131, remains uncertain since several of these proteins are not specific to platelets and are synthesized by other cell types, e.g. hepatocytes, endothelial cells, fibroblasts or lymphocytes. Biosynthesis therefore could occur either in megakaryocytes, where the proteins are packaged into alpha granules for subsequent release from circulating platelets, or in other cell types followed by an active uptake of the preformed proteins by the megakaryocyte, or possibly in both megakaryocytes and other cells. stable messenger RNA derived from nucleated megakaryocytes is present in circulating platelets [14-251. This biosynthetic activity has, however, been considered to be rather vestigial. Recently Belloc et al. reported increased protein synthesis and larg...

Expression of Cholesterol Sulfotransferase (SULT2B1b) in Human Platelets

“…Although platelets lack a nucleus, they do contain a rough endoplasmic reticulum and polysomes and are known to engage in protein synthesis. 26 The presence of SULT2B1b protein in platelet preparations was confirmed by Western analysis as well as by measuring catalytic activity and the content of cholesterol sulfate produced. The finding of 566 pmol of cholesterol sulfate per 10 9 untreated platelets is in keeping with a previous report where the content of cholesterol sulfate in human platelets was found to range from 164 to 512 pmol/10 9 platelets.…”

Human Platelets Express Organic Anion-Transporting Peptide 2B1, an Uptake Transporter for Atorvastatin

“…We also demonstrated expression of OATP2B1 mRNA in human platelets and megakaryocytes, using real-time RT-PCR. Protein synthesis by anucleated platelets was reported two decades ago (Kieffer et al, 1987), but only recently was de novo synthesis of several polypeptides by human platelets convincingly demonstrated (Lindemann et al, 2001). Denis et al (2005) found constitutive pre-mRNA in human platelets, and critical factors were able to splice it upon activation, generating mature mRNA and new protein.…”

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

“…Besides uptake from plasma, platelets are capable of synthesizing fibrinogen 49 and other proteins through an intact translation machinery that lacks DNA but consists of mRNA, ribosomes, and amino acids. 50 Interestingly, fibrinogen stored in the platelet ␣-granules completely consists of ␥A/␥A fibrinogen.…”