α-Granule proteins derive from both synthetic and endocytic pathways ²² . Synthetic pathways traffic translated proteins from ER to α-granules. The endocytic pathway enables megakaryocytes and mature platelets to acquire plasma proteins by the process of endocytosis at the plasma membrane ²³ . Multiple individual proteins and protein complexes mediate trafficking of these separate pathways ( Figure 1A). Such proteins include coat proteins such as clathrin, adaptor proteins AP1 and AP2, and proteins required for vesicle trafficking, including soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins, SNARE regulators, particularly Sec1/Munc18 proteins, and small GTPases such as Rabs. As a first step, soluble clathrin molecules recruited to either trans-Golgi network (TGN) or plasma membrane self-assemble into a lattice structure and interact with APs to form clathrin-coated pits. Platelets contain clathrin-associated adaptor proteins AP1, AP2, and AP3 ²⁴ . Since AP2 localizes only to plasma membrane where it functions in the endocytotic pathway and AP3 is critical for lysosomal and LRO trafficking, the deficiency of which leads only to dense granule deficiency as in HPS subtype 2, AP1 is assumed to be employed by the synthetic pathway in α-granule biogenesis, although there is no direct evidence for this or for the role of other coat proteins such as COPI in α-granule biogenesis ²⁵ . Vesicles carrying α-granule cargo budding off from either TGN or plasma membrane are subsequently directed to multivesicular bodies (MVBs) via endosomes ²⁶ .

MVBs are transient late endosomal structures that contain internal vesicles formed from inward budding of the limiting membrane of the endosome ²⁷ . Initially assumed to only direct proteins to be degraded in the lysosomes, these structures are now known to have multiple other functions, including granule trafficking in various cell types. MVBs serve as an intermediate stage of granulogenesis in megakaryocytes ²⁶ . α-Granule cargoes from both synthetic and endocytic pathways can be identified in MVBs ²⁶ . Both α- and dense granules mature from MVBs but use distinct machinery ²⁶ . For example, defects in VPS33B and NBEAL2 lead only to α-granule deficiency but not to dense granule defects. VPS33B, a Sec1/Munc18 protein deficient in arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome, was the first protein involved in α-granule biogenesis to be identified ^{28, 29} . VPS16B, its partner, works in association ³⁰ . Although many platelet-specific details remain poorly understood, the SNARE binding function of VPS33 and VPS16 in vesicular trafficking as a component of two large protein complexes—class C core vacuole/endosome/tethering (CORVET), containing isoforms VPS33B and VPS16B, and homotypic fusion and protein sorting (HOPS), containing isoforms VPS33A and VPS16A—has been characterized in yeast ³¹ . The other proteins of these complexes have membrane-, AP-, and Rab-binding properties, thus bringing together the basic machinery required for endosomal maturation. NBEAL2 deficiency as a cause of GPS was first described in 2011 ^{14, 32, 33} . Nbeal2 ^-/- mice exhibit a phenotype similar to that of patients with GPS, including macrothrombocytopenia, splenomegaly, and myelofibrosis, but the exact molecular function of NBEAL2 is not known ³⁴ . It acts at a later state of α-granule development, independently of VPS33B, as Nbeal2 ^-/- platelets express some P-selectin that externalizes upon platelet activation. NBEAL2 is under direct transcriptional control of GATA1, a mutation in which results in a syndrome similar to GPS, in addition to myelodysplasia ³⁵ . It is one of the nine BEACH (beige and Chediak-Higashi) domain-containing proteins, hypothesized to be scaffolds for fission and fusion membrane events ³⁶ . Genetic defects in Chediak-Higashi syndrome 1 ( CHS1), another member of this family, lead to platelet dysfunction secondary to dense granule deficiency in addition to immunodeficiency and other manifestations ³⁶ .

Protein sorting and packaging into the developing α-granule occur via varying mechanisms dependent on the protein type. Many membrane proteins, such as P-selectin, contain signal peptides that direct them to the developing granule ³⁷ . Notably, P-selectin uses distinct signal peptides for trafficking to the α-granules in platelets and Weibel–Palade bodies in endothelial cells ³⁸ . Another mechanism is protein aggregation, which is employed by large soluble proteins such as multimerin and VWF ^{39, 40} . VWF self-assembles into large homoaggregates that ultimately form tubular structures occupying a distinct sub-compartment within α-granules ⁴¹ . Sorting sequences contribute to trafficking of many smaller soluble proteins to α-granules. PF4 is one such protein that has a four-amino acid sequence within its hydrophilic loop that directs it to the maturing α-granule ⁴² . Other examples of small proteins that employ sorting sequences are RANTES and NAP2. Cationic glycosaminoglycans within α-granules may also serve to retain these small chemokines ⁴³ . Exogenous proteins are trafficked through an endocytic pathway into α-granules via either receptor-mediated endocytosis or pinocytosis. Fibrinogen, which is internalized via integrin αIIbβ ₃, is a classic example of this route, which subsequently uses adaptor protein Disabled-2 for formation of clathrin-coated vesicles ^{44, 45} . Proteins that are incorporated into platelets via pinocytosis include immunoglobulins as well as angiogenesis regulators such as VEGF, endostatin, and FGF ^{23, 46} . Vesicle-associated membrane protein 3 (VAMP-3), a v-SNARE (discussed below), regulates platelet endocytosis. VAMP-3 ^-/- platelets show impaired αIIbβ ₃-mediated fibrinogen uptake ⁴⁷ . In addition, loss of VAMP-3 impairs trafficking of both endocytosed and pinocytosed cargo between Rab4 (early endosomes) and Rab11 (recycling endosomes) positive compartments, although its mechanism remains unclear. Endocytosis of plasma proteins starts in megakaryocytes but continues in mature circulating platelets. For example, platelets from patients with complete factor V deficiency endocytose and release factor V supplemented in transfused plasma for prolonged periods greater than the half-life of factor V ⁴⁸ .

Dense granules are platelet-specific LROs ⁷ . These granules are distinct from classic secretory granules in that they are derived from the endosomal system instead of directly from TGN ( Figure 1B). They also share some characteristics with lysosomes as their intra-granular pH is acidic and they possess lysosome-resident proteins, such as the tetraspanin CD63. However, CD63 is not restricted to dense granules in platelets, and the lack of other specific cargoes that can be followed biosynthetically has made evaluation of dense granule biogenesis challenging. There is evidence that early endosomes contribute to dense granule biogenesis ⁴⁹ . In addition, like α-granules, dense granules are believed to be sorted in MVBs, although the only direct evidence of this is the accumulation of CD63 and serotonin in MVBs in megakaryocytes ⁵⁰ . HPS and related disorders together with their murine counterparts have served as a great source of understanding of biogenesis of LROs, and melanosomes are the prototype organelle that has been studied.

In total, at least 10 different HPS genes encode subunits of four distinct ubiquitously present protein complexes: adaptor protein-3 (AP3) and biogenesis of lysosome-related organelles complex (BLOC) 1, 2, and 3 ^{51– 54} . These complexes localize mainly to the endosomal compartment and are essential for biogenesis of LROs. Deficiency or alteration in these proteins results in two common manifestations: albinism due to abnormal melanogenesis and a bleeding disorder due to dense granule deficiency. Some HPS subtypes display other manifestations, such as pulmonary fibrosis, inflammatory bowel disease, and immunodeficiency ⁵¹ . Functions of these individual proteins and protein complexes are being understood with increasing detail. In melanosomes, BLOC1 (complex of HPS7, HPS8, HPS9, Muted, Cappuccino, Snapin, BLOS2, and BLOS3) is required for the exit of melanosome cargoes from endosomes into tubular transport carriers ⁵⁵ . BLOC2 (complex of HPS3, HPS5, and HPS6) directs these carriers specifically to the melanosomes. Alternatively, cargoes can be directed into developing melanosomes in an AP3-dependent pathway, which in turn can be BLOC1-independent or -dependent ^{55– 58} . BLOC3 (complex of HPS1 and 4) functions after cargo delivery in pathways out of melanosomes, specifically in retrieval and recycling of the BLOC1-dependent v-SNARE VAMP-7 ⁵⁹ . Owing to concurrence of albinism and dense granule deficiency in HPS, pathways similar to those described above are thought to function in dense granule biogenesis in megakaryocytes, although there is no direct evidence. The exact molecular functions of many of the HPS and related proteins are also being characterized, mainly in melanosomes. HPS9, or Pallidin, a component of BLOC1, is known to interact with syntaxin 13, a SNARE protein involved in vesicle membrane fusion during trafficking ⁶⁰ . BLOC2 constituents HPS3 and HPS6 have been described to bind clathrin and dynactin p150Glued, respectively ^{61, 62} . BLOC3 functions as a guanine nucleotide exchange factor for cell type-specific Rab GTPases, such as Rab32 and Rab38 in melanocytes ^{63, 64} . A direct role of Rab32 and Rab38 in dense granule biogenesis in megakaryocytes has also been implicated ^{13, 64} . RUNX1 mutations lead to dense granule but not α-granule deficiency due to dysregulation of Pallidin (HPS9) transcription ⁶⁵ .

Dense granule contents, such as bioactive amines and adenine nucleotides, are transported into the maturing dense granules via specific membrane pumps, such as vesicular nucleotide transporter (VNUT), which has been proposed as a candidate for ADP and ATP accumulation in dense granules, and multidrug resistance-associated protein 4 (MRP4), which uptakes cAMP into dense granules ^{66– 68} . MRP4 ^-/- mice show significant platelet dysfunction due to cytosolic accumulation of cAMP and lack of cAMP in dense granules, as do inhibitors of MRP4, such as probenecid ^{67, 69} . York platelet syndrome is characterized by thrombocytopenia and striking giant electron-opaque organelles. It is caused by a calcium-selective release-activated calcium (CRAC) channelopathy, which results in defective calcium storage ⁷⁰ .

Membrane fusion is facilitated by SNARE proteins, a family of highly conserved eukaryotic proteins essential for vesicle fusion ⁷⁸ . SNARE proteins are classified into two groups on the basis of their location: v-SNAREs, located on the vesicle/granule membrane, and t-SNAREs, located on the target membrane (for example, plasma membrane). Related v- and t-SNAREs interact through SNARE domains, which are α-helices of about 60 amino acids, assembled into amphipathic, heptad repeats. SNAREs can also be classified as R-SNAREs (typically v-SNAREs) or Q-SNAREs (typically t-SNAREs), depending on the presence of an arginine or glutamine residue, respectively, in the central position of the SNARE domain ⁷⁹ . Four SNARE domains—one each from the v-SNARE plus three t-SNAREs—form a coiled-coil structure that brings the two opposing membranes together (for example, granule and plasma membrane) against repulsive electrostatic forces of the two lipid membranes in an aqueous environment ( Figure 2) ⁸⁰ .

VAMPs constitute the largest group of v-SNAREs. Platelets contain VAMP-2, -3, -7, and -8; VAMP-8 is the most abundant and functionally important in human platelets, followed by VAMP-7 ⁸¹ . Loss of VAMP-8 in mice causes defective α- and dense granule exocytosis, and platelet thrombus formation in vivo, without excessive bleeding ⁸² . On the other hand, loss of VAMP-7 in mice leads to defective platelet spreading, and altered α- and dense granule exocytosis, without impacting platelet thrombus formation or bleeding ⁷³ . Moreover, VAMP-7 is located peripherally in the spreading platelet whereas VAMP-8 concentrates in the central granulomere ⁷² . These observations suggest a distinct role for these v-SNAREs in platelet function. Interestingly, VAMP-8 has also been linked to early-onset myocardial infarction in genome-wide association studies, suggesting a syndrome of platelet hyper-responsiveness ⁸³ . VAMP-3, which is important in the endocytotic pathway of α-granule biogenesis, has minimal function in platelet exocytosis ⁴⁷ . The role of platelet-specific VAMPs has not been well established in granulogenesis.

Of the t-SNAREs, proteomic studies suggest that platelet contains syntaxin 2, 4, 6, 7, 8, 11, 12, 16, 17, and 18 and soluble NSF attachment proteins (SNAPs) 23, 25, and 29 ⁸⁴ . Of these, syntaxin 11 and SNAP 23 are the only t-SNAREs found to be essential for platelet granule exocytosis. As with v-SNAREs, most data come from mouse models lacking one or more specific t-SNAREs. Loss of syntaxin 11, which forms complexes with SNAP 23 and VAMP-8, is associated with abnormal exocytosis of all three of the major platelet granules ⁸⁵ . In humans, familial hemophagocytic lymphohistiocytosis type 4 is caused by lack of syntaxin 11 ⁸⁶ . Loss of syntaxin 8 has been associated with minor defects in dense granule exocytosis ⁸⁷ .

To prevent indiscriminate release of granular content, fusion of vesicle and target membranes is tightly regulated by SNARE regulators. Some SNARE regulators are chaperones (for example, Munc18b), while others promote formation of membrane-fusion complexes and direct where the fusion occurs (for example, Munc13-4, Munc18 isoforms, Rabs, STXBP5/Tomosyn 1, and exocyst complex).

Munc18b is the most important syntaxin chaperone belonging to the Sec/Munc family of proteins present in the platelet, forming specific complexes with t-SNAREs. Munc18b deficiency leads to decreases in platelet levels of syntaxin 11, consistent with its role as a chaperone, resulting in impaired granule exocytosis ⁸⁸ . Homozygous deficiency, as seen in familial hemophagocytic lymphohistiocytosis type 5, leads to severe defects in all platelet granule exocytosis, whereas heterozygous deficiency leads to intermediate defects ⁸⁹ . VPS33B, discussed in α-granule biogenesis above, also belongs to the Sec/Munc family of proteins ²⁹ .

Syntaxin binding protein 5 (STXBP5), or tomosyn 1, binds to the cytoskeleton and to t-SNARE heterodimers (syntaxin 11 and SNAP-23) through the presence of a v-SNARE-like domain at its C-terminal. Its deficiency causes defective granule exocytosis, and mice lacking STXBP5 show excessive bleeding ⁹⁰ . Interestingly, STXBP5 negatively regulates VWF release from the endothelial cells, and polymorphisms in STXBP5 gene are associated with increased plasma VWF levels and cardiovascular disease ⁹¹ .

Rab proteins that belong to the Ras superfamily of GTPases function as master regulators of the complex network of intracellular membrane trafficking pathways ⁹² . Rabs perform this regulatory function by binding to effector proteins in the GTP-bound, or “on”, state ⁹³ . Some of these Rab effectors are SNARE regulators. Multiple Rabs, including Rab3b, 6c, and 8, are phosphorylated upon platelet activation, and their inhibition decreases platelet exocytosis ⁹⁴ . Among these, Rab4 is crucial for α-granule exocytosis whereas Rab27b is a key regulator of dense granule biogenesis and exocytosis ^{95, 96} . Munc13-4 is a Rab27b effector protein, essential for dense granule function. Munc13-4 forms calcium-dependent bridges between the dense granule and plasma/OCS membrane, facilitating membrane fusion ^{97, 98} . Rab27 ^-/- and Munc13-4 ^-/- platelets have defective dense granule exocytosis and a bleeding diathesis. These platelets also display defective exocytosis of α-granules and lysosomes, which can be overcome by the addition of ADP, a key dense granule component ⁹⁹ . This reversal by ADP, as also occurs in HPS platelets, demonstrates the critical role of autocrine signaling from released dense granule cargo for complete platelet activation ^{15, 100} .

Synatotagmin-like proteins (SLPs), particularly SLP1 and SLP4, that bind calcium/lipids are also known to regulate dense granule exocytosis and may act as calcium sensors. SLP1—which forms a complex with Rap1, a Ras-like GTPase, and RAP1GEF2, its guanine nucleotide exchange factor—is a negative regulator of dense granule release ¹⁰¹ . SLP4, a Rab27 effector, on the other hand, is a positive regulator of dense granule release ¹⁰² .

Tethering complexes, particularly the exocyst complex, which is known to play a role in polarized secretion, may also be involved in the regulation of dense granule exocytosis ¹⁰³ . Exocyst complex is targeted to the plasma membrane by Ral, a Ras-like GTPase, which is expressed in platelets and activated upon platelet stimulation. Blocking of Ral-GTP binding to exocyst complex impairs dense granule exocytosis.

NSF and soluble NSF attachment proteins (SNAPs) are also important regulators of platelet exocytosis ¹⁰⁴ . These proteins disassemble SNARE complexes to allow recycling of v-SNAREs and t-SNAREs for the next round of membrane fusion ¹⁰⁵ . The inhibitory effect of nitric oxide on platelet exocytosis is at least partly due to its reversible inhibition of NSF ¹⁰⁶ .

Many SNAREs and their regulators, such as SNAP23 and Munc18, are known to be protein kinase C substrates, linking platelet activation and ensuing signaling cascades to the exocytosis machinery. Platelet signaling and protein phosphorylation and their role in regulated platelet exocytosis are beyond the scope of this review. The reader is referred to excellent reviews on this topic ^{107– 109} .