Recently, we showed that platelet alpha-granule fibrinogen is derived entirely from endocytic uptake and not from megakaryocyte synthesis, as previously thought. In this present report, we identify the receptor that mediates endocytosis. We have found that barbourin, a unique disintegrin that is a specific antagonist of alpha IIb beta 3, inhibits the endocytic uptake of fibrinogen into alpha-granules. Continuous intravenous infusion of barbourin (200 micrograms/h) into guinea pigs blocked collagen-induced platelet aggregation as well as endocytosis of biotinylated fibrinogen into megakaryocytes; however, endocytosis of biotinylated albumin by megakaryocytes was not affected. Thus, we have shown that endocytosis of fibrinogen into megakaryocyte and platelet alpha-granules is receptor-mediated, and that alpha IIb beta 3 is the primary receptor.
In a previous study we provided evidence for a circuitous pathway by which circulating plasma proteins enter megakaryocyte granules by an endocytic mechanism and are returned to the circulation in platelets (1987. Proc. NatL. Acad. Sci. USA. 84:861-865). Horseradish peroxidase (40,000 mol wt) was injected into guinea pigs and its uptake into megakaryocyte organelles examined by electron microscopy and cytochemistry.In the present study we tested the ability of guinea pig megakaryocytes to take up intravenously injected albumin, IgG, and fibrinogen. We used two types of proteins to study the endocytic pathway: (a) heterologous human proteins, which were detected immunohistochemically using antibodies that do not crossreact with the native guinea pig counterparts; and (b) human and guinea pig proteins labeled with the small (250 mol wt), inert molecule, biotin, which were detected using an antibody against biotin. We detected all three of the injected proteins in bone marrow megakaryocytes in patterns identical to those of native counterparts. The injected protein consistently appeared in platelets 24 h later and was secreted in response to thrombin. We conclude that there are at least two mechanisms by which guinea pig megakaryocyte granules acquire proteins (a) endogenous synthesis, as demonstrated by others, and (b) endocytosis of plasma proteins synthesized by other types of cells.
To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of a granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into a granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some a granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of a granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.One of the most striking characteristics of platelets is the large variety of proteins that are secreted during the release reaction. Platelets contain counterparts of several plasma proteins (albumin, fibrinogen, and others) in addition to platelet-specific proteins (platelet factor 4 and f3-thromboglobulin). These proteins are localized in a granules, the primary storage organelle for platelet secretory proteins. Predictably, many of these proteins have been shown to be synthesized in megakaryocytes, the bone marrow precursor cell from which platelets are derived. Platelet-specific proteins appear to be synthesized solely by megakaryocytes, whereas proteins such as fibrinogen, coagulation factor V, and von Willebrand factor are synthesized by other cell types in addition to megakaryocytes (1,2).Recently, we discovered that immunoglobulin G is located in platelet a granules and is secreted after platelet activation (3 In Vivo Studies. Male Hartley guinea pigs (300-400 g) were anesthetized with ether, and 75 mg of HRP was dissolved in 1 ml of physiologic saline and injected into the femoral vein. The animals were sacrificed with ether at short (15, 45, and 75 min) and long (7 hr, 24 hr, 48 hr, And 4 days) intervals after the injection. Blood was collected into acid/citrate/dextrose by heart puncture immediately before the guinea pigs were killed. Platelets were counted by use of an automated analyzer (Coulter S + IV). Platelet-rich plasma was obtained by centrifugation of the blood at 100 x g for 10 min at 22°C. Megakaryocytes were isolated from bone marrow according to a described technique (4), e...
It has been assumed that endogenous synthesis by the platelet precursor cell, the bone marrow megakaryocyte, is the major source of platelet a-granule protein. To test this hypothesis, we used mRNA phenotyping to detect in megakaryocytes the presence of mRNA transcripts specific for various proteins. Our results indicate that megakaryocytes synthesize platelet factor 4, a protein relatively specific for platelets, but do not express mRNA transcripts for the fibrinogen, albumin, or IgG found in a-granules. We have previously shown that megakaryocytes endocytose circulating proteins, including fibrinogen, albumin, and IgG, and incorporate them into a-granules. Thus, platelets appear to contain a unique type of secretory granule whose contents originate by both endogenous synthesis and endocytosis from plasma. Under basal conditions, the source of a-granule fibrinogen is plasma. (J. Clin. Invest. 1990Invest. . 86:1364Invest. -1368.
Improper handling of specimens results in artifactually high Mean Platelet Volume (MPV) measurements limiting their usefulness as a clinical tool. MPV measurement and Scanning Electron Microscopy (SEM) were performed on split specimens collected from normal dogs using two anticoagulants and two temperatures over a period of 4 hours. Platelets exposed to EDTA and maintained at 4 degrees C (39.2 degrees F) exhibited the highest artifactual increase in MPV, while those exposed to citrate and maintained at 37 degrees C (98.6 degrees F) exhibited minimal change. The increase in MPV was accompanied by platelet shape change from a smooth disc to an irregular sphere with filopodia. It is recommended that citrated specimens maintained at 37 degrees C be used in all MPV measurements.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.