Selective sorting of alpha-granule proteins

Summary

One of the main functions of blood platelets is to secrete a variety of substances that can modify a developing thrombus, regulate the growth of the vasculature, promote wound repair, and contribute to cell-adhesive events. The majority of this vast array of secreted proteins is stored in alpha-granules. Until recently, it was assumed that platelets contained one homogeneous population of alpha-granules that undergo complete de-granulation during platelet activation. This review focuses on the mechanisms of alpha-granule biogenesis and secretion, with a particular emphasis on recent findings that clearly demonstrate that platelets contain distinct subpopulations of alpha-granules that undergo differential release during activation. We consider the implications of this new paradigm of platelet secretion, discuss mechanisms of alpha-granule biogenesis, and review the molecular basis of transport and delivery of alpha-granules to assembling platelets.

Introduction

Platelets play important roles in many aspects of vascular biology over and above their well-characterized function in hemostasis. A body of experimental and clinical data has linked platelets to inflammation, wound healing, atherosclerosis and recent studies have pointed to a key regulatory role for platelets in angiogenesis. One of the more interesting characteristics of platelets is the large number of biologically active molecules carried in their granules. Their localization in platelet alpha-granules allows them to achieve high local concentrations when they are released from platelets at the site of vascular injury. A recent proteomic analysis of molecules released during platelet activation has identified over 300 proteins [1]. This vast array of proteins includes adhesive proteins, chemokines, cytokines, coagulation factors, mitogenic factors, angiogenesis regulatory proteins, and fibrinolytic agents. The majority of these proteins are housed in alpha-granules, the main storage granule within platelets. Platelets contain from 40 to 80 alpha-granules, each measuring between 200 to 500 nm and enclosed by a membrane. This review focuses on the biology of alpha-granules, with a particular emphasis on recent findings demonstrating that platelets contain distinct subpopulations of alpha-granules. These distinct subpopulations undergo differential release during platelet activation, and provide a new mechanism to regulate secretion. We review the mechanisms of alpha-granule biogenesis as well as the molecular basis by which alpha granules are transported and delivered to platelets during thrombopoiesis. Future investigations into the mechanics of alpha-granule heterogeneity and the regulatory mechanisms of differential release may yield strategies to manipulate platelet secretion for therapeutic benefit.

A new model of platelet alpha-granule biology: Distinct subpopulations of alpha-granules

The classic textbook view assumes that alpha-granule proteins are indiscriminately packaged into one homogeneous population of alpha-granules, and that these granules are all secreted together during platelet activation. However, recent observations suggest that platelets contain a heterogeneous population of alpha-granules and that this organization may facilitate differential release of specific subpopulations of alpha-granules. While studying the role of platelets in regulating new blood cell development, we discovered that platelet alpha-granule proteins were organized into separate and distinct granules [2]. An association between angiogenesis and platelets has long been recognized, but the cause and effect relationship linking the two has been unclear [3,4]. If platelets contained both stimulators and inhibitors of angiogenesis packaged into a homogeneous population of alpha-granules, the question becomes how can you attain a pro-angiogenic or anti-angiogenic effect? The simultaneous release of a mixture of both pro- and anti-angiogenic regulatory proteins randomly packaged into a homogeneous population of alpha-granules should cancel the effect of each other. We have recently discovered that angiogenesis regulatory proteins are in fact segregated among distinct sets of alpha-granules in platelets: The major pro-angiogenic regulatory protein VEGF is housed in one set of alpha-granules, whereas the major anti-angiogenic regulatory protein endostatin is packaged into another set of alpha-granules. Double immunofluorescence labeling of VEGF (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic FGF, confirms the segregation of stimulators and inhibitors into separate and distinct alpha-granules in human platelets. Heterogeneous populations of alpha-granules were also observed in mouse megakaryocytes. Similar granule heterogeneity was reported by Seghal and Storrie, who demonstrated using confocal microscopy that the adhesive proteins von Willebrand factor and fibrinogen are as well located in separate and distinct alpha-granules in human platelets [5,6]. Both of these studies identify a novel property of platelet biology that has important implications and raises many new questions. While each of these studies suggests the presence of two classes of alpha-granules, they raise the possibility that platelets may contain multiple subpopulations of alpha-granules.

How are distinct subpopulations of alpha-granules established in platelets?

The alpha-granule is a unique secretory organelle that acquires its protein content by both biosynthesis predominantly at the megakaryocyte level and endocytosis at both the megakaryocyte and circulating platelet levels. To understand how platelets obtain a heterogeneous population of alpha-granules, we need understand the basic mechanisms of alpha-granule development. While the cell biological pathways that regulate alpha-granule assembly are not fully understood, several studies suggest multivesicular bodies play a crucial intermediary role in alpha-granule biogenesis [7,8]. Alpha-granules initially develop from budding vesicles in the Golgi complex within megakaryocytes, from where they mature into multivesicular bodies, which can interact with the endocytic vesicles. MVBs, which are very abundant in immature megakaryocytes and decline with cellular maturation, are thought to be common precursors to both alpha- and dense granules. MVBs, which appear as a membranous sac containing numerous small vesicles, seem to provide a common sorting compartment for both alpha- and dense-granules, and we hypothesize that MVBs also function as a sorting hub to rout proteins into distinct classes of alpha-granules. Two types of MVBs have been identified in megakaryocytes. Type I MVBs contain only internal vesicles, and Type II contain internal vesicles and an electron-dense matrix [9]. While the molecular basis by which these granules develop into distinct units is unclear, one possibility is that heterogeneity among the internal membranes plays an important role. Insight into the sorting of granules that occurs in the MVB is based on research on two disorders that are associated with complete deficiency in a particular granule component. The Hermansky-Pudlak Syndrome (HPS) proteins interact through Biogenesis of Lysosome-related Organelle Complexes (BLOCs). In patients with subtype BLOC-2 and BLOC-3 delta granules were not present while alpha granules were intact showing that these granule types are indeed independently sorted [10]. Another syndrome that has provided insight into granule sorting is arthrogryposis, renal dysfunction, and cholestasis or ARC syndrome. This syndrome is characterized by mutations in the novel Sec1/Munc18 protein termed VPS33B that is essential for intracellular vesicle trafficking. Patients with ARC syndrome have recently been shown to have intact dense granules but absent alpha-granules [11]. The details regarding how these two populations of granules are released is also not completely understood. Platelet secretion has been proposed to be regulated by SNARE proteins through signaling cascades such as protein kinase C activation and elevation of [Ca2+]_i [12]. Ren and colleagues have further revealed that Vesicle Associated Membrane Protein 8 (VAMP8) is required for granular release from the platelet [13,14].

Transport and packaging of alpha granules into assembling platelets

Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions, which serve as assembly lines for platelet production. One of the key steps in platelet production is the transport and delivery of alpha-granules into nascent platelets. Platelet maturation appears to finalize at proplatelet tips where a microtubule coil is formed [15]. To complete its construction of mature platelets, once the fundamental cytoskeletal components have been delivered to and assembled in the platelet ends, the tips must fill with their granule content. The microtubule bundles in the shaft serve as the tracks on which alpha-granules are sent to the maturing platelet buds. Distinct subpopulations of alpha-granules are clearly visible along proplatelets, indicating that the segregation of proteins into distinct classes of alpha-granules occurs before proplatelet production initiates [2]. Alpha-granule movement along proplatelets has been observed in living cells by loading megakaryocytes with green-labeled fibrinogen, which is taken up by megakaryocytes and packaged into the alpha-granules [16]. The filling process occurs sequentially as α-granules traffic in single file to the platelet buds. Alpha-granule traffic is observed to be bidirectional along the proplatelets, as predicted from the mixed polarity of proplatelet microtubules, although traffic directed outward initially dominates. Cargo is eventually trapped at the proplatelet end within the nascent platelet buds [16]. Both the bidirectional nature of granule movement and the general slowness of traffic suggest that the underlying goal of this transport process is not to drive the cargo to the ends of proplatelets but instead to disperse the cargo throughout proplatelets as well as to mix the various granules/organelles with the proplatelet. Moreover, these findings indicate that if proplatelets are shed into blood as suggested by Benhke and observed in the sinusoids of living mice that they can use the granules dispersed throughout to mature multiple platelets[17] [18]. It should be noted that to date, the movement and delivery of only one subpopulation of alpha-granules, those loaded with fluorescently-labeled fibrinogen, has been visualized [16]. Thus, it is unclear if other subpopulations of alpha-granules move in the same manner along proplatelets and operate under similar mechanisms. Differential regulation of the transport and delivery of distinct subpopulations of alpha-granules into nascent platelets could provide an elegant mechanism to establish what type of alpha-granules and how many are packaged into a maturing platelet.

Selective release of alpha-granules

By adhering to the endothelium of injured organs and tissues and then secreting their granular contents, platelets deposit high concentrations of active molecules in a regulated and localized manner. While the general assumption in the field has been that platelets undergo total degranulation during activation, the presence of distinct subpopulations of alpha granules immediately suggested that platelets may regulate the differential release of distinct classes of alpha-granules. We tested this hypothesis and demonstrated that treatment of human platelets with the selective Protease-Activated-Receptor (PAR)-4 agonist resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR-1 agonist liberated VEGF, but not endostatin-containing granules. These observations demonstrated that separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of alpha-granules in platelets provides a mechanism by which platelets can locally stimulate or inhibit angiogenesis. In an elegant separate study, Seghal and Storrie also demonstrated selective release of distinct subpopulations of alpha-granules [6] [5]. The authors demonstrated that glass activation of platelets induced the release of fibrinogen-containing granules, but not vWf-containing granules. These findings raise a number of issues. While selective release has been observed with glass and PAR peptides, do physiological agonists release specific granules? And if so, does the strength of the agonist regulate the secretion of one exclusive class of granules? Do platelets contain distinct signal transduction pathways that lead to the secretion of one select class of granules?

Can we use new platelet alpha-granule biology to make “designer platelets?”

Circulating in an adult human, there are nearly a trillion platelets, each with the capacity to release over 300 proteins. It is clear that this secretion event can influence many physiological and pathological processes, and thus the recent finding of distinct subpopulations of alpha-granules that undergo differential release provides a new opportunity to manipulate platelet secretion for therapeutic benefit. The ability to selectively modulate the content, differential packaging, and selective release of distinct classes of alpha-granules may assume considerable importance. For example, the development of drugs designed to stimulate or inhibit the exocytosis of an individual class of alpha-granules would provide a new class of therapeutic agents. In addition to providing a transport and delivery vehicle for physiological proteins housed in alpha-granules, platelets may also function as drug delivery systems for targeted agents. For example, recently Verheul and colleagues were able to show that the monoclonal antibody, bevacizumab, which specifically targets VEGF can be taken up by the platelet and will suppress VEGF expression within the platelet [19]. The therapeutic benefits of these “drug loaded” platelets have yet to be explored. However, given that bevacizumab is being used to treat many angiogenesis-related malignancies the implications of this work could be far reaching. This marks the beginning of a new era for platelet biology in which platelets are not only seen as important for transporting physiological factors but may also be manipulated for therapeutic benefit.

Final thoughts

This review should make it clear that platelets contain distinct subpopulations of alpha-granules that can undergo differential release. This new platelet property undoubtedly requires precise packaging of alpha-granules and a highly orchestrated release reaction. A better understanding of the cell biological pathways by which platelets develop distinct alpha-granule populations as well as the signal transduction mechanisms that orchestrate selective release should yield strategies to manipulate platelet secretion for therapeutic benefit.