Small GTPase Rab4 Regulates Ca2+-induced α-Granule Secretion in Platelets

Abstract

Upon activation, platelets release many active substances stored in ␣-and dense-core granules. However, the molecular mechanisms governing regulated exocytosis are not yet fully understood. Here, we have established an assay system using permeabilized platelets to analyze the Ca 2؉ -induced exocytosis of both types of granules, focusing on RabGTPases. Incubation with Rab GDP dissociation inhibitor, an inhibitory regulator of RabGTPases, reduced membrane-bound RabGTPases extensively, and caused strong inhibition of the Ca 2؉ -induced secretion of von Willebrand factor (vWF) stored in ␣-granules, but not that of [ 3 H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 cofractionated with vWF and P-selectin (an ␣-granule marker) upon separation of platelet organelles by density gradient centrifugation. Incubation of the permeabilized platelets with cell extracts expressing the dominant negative mutant of His-tagged Rab4S22N, but not with those of similar mutant His-Rab3BT36N, inhibited the vWF secretion, whereas neither of the cell extracts affected the [ 3 H]5-HT secretion. Importantly, the inhibition of vWF secretion was rescued by depleting the cell extracts of the His-Rab4S22N with nickel beads. Thus, in platelets, the regulatory mechanisms governing ␣-and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca 2؉ -induced exocytosis of ␣-granules.Certain types of cells contain specialized granules, which are released in response to extracellular stimuli. The process is known as regulated exocytosis, which is often triggered by the increased concentration of intracellular calcium ions, followed by docking and fusion of granule membrane with plasma membrane (1). Platelets contain three types of such granules, densecore granules, ␣-granules, and lysosomes, which are secreted upon activation of platelets (2, 3). Dense-core granules contain small molecules such as 5-hydroxytryptamine (5-HT) 1 and ␣-granules contain newly synthesized proteins such as von Willebrand factors (vWF). Despite the biological significance of these secretions in platelet function (2), the molecular mechanism remains unclear. Recent advances have shed light on the mechanism of intracellular membrane docking/fusion. An ATPase, N-ethylmaleimide-sensitive factor (NSF), and its binding protein, soluble NSF attachment protein (␣-SNAP), are required for priming SNAP -receptors (SNARE) (4) on vesicular (v-) and target (t-) membranes. Then, the cognate v-and t-SNARE (trans) complex on both sides of the membranes are formed, resulting in their docking/fusion (5, 6). Many v-and t-SNARE homologues have been identified from mammals to yeast, and the SNARE complex-mediated mechanism has been demonstrated to play essential roles in many pathways of intracellular vesicle transport (7). In the case of platelets, NSF (8) and ␣-SNAP (8, 9) have recently been shown to be required for the granule secretion. Furthermore, syntaxin2 and synaptosome-associated protein of 23 kDa (SNAP23), which are t-SNARE proteins...

Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

“…Rab4 also regulates exocytic events from other post-Golgi compartments in cells with specialized functions (34). In adipocytes, Rab4 controls recycling of the insulin-regulated glucose transporter GLUT4 (37), and recombinant Rab4 stimulates ␣-granule secretion in platelets in vitro (38). All Rab proteins contain highly conserved domains required for guanine nucleotide binding, GTP/GDP exchange, and GTP hydrolysis that are essential for their proper targeting and function (31).…”

Direct Demonstration of Involvement of Protein Kinase Cα in the Ca2+-induced Platelet Aggregation

“…Washed human platelets from healthy donors were prepared (24), resuspended in ice-cold Buffer A (50 mM Hepes/KOH, pH 7.2, 78 mM KCl, 4 mM MgCl 2 , 0.2 mM CaCl 2 , 2 mM EGTA, 1 mM dithiothreitol, and the calculated free [Ca 2ϩ ] was ϳ20 nM (25)) containing 4 mg/ml bovine serum albumin and 20 ng/ml prostaglandin E 1 and kept at 4°C. The platelets were incubated with 0.6 g/ml SLO at 4°C for 10 min and washed once to remove unbound SLO (19,20,26,27), The treated platelets were resuspended in ice-cold Buffer A containing 4 mg/ml bovine serum albumin at a density of 5 ϫ 10 8 /ml, quantified with a Coulter Counter, and incubated at 30°C for 5 min to make holes in their plasma membrane (19,20,26,27). The permeabilized platelets were kept on ice for 15-30 min with 2 mg of proteins/ml human platelet cytosol (or rat brain cytosol), an ATP-regenerating system (19,20,26,27), and tested substances.…”

“…For the granule secretion, we have recently demonstrated the direct involvement of PKC (19). We have established a semi-intact secretion assay (19,20) where the secretion does not occur upon stimulation without adding exogenous cytosol, indicating the existence of cytosolic essential factors. We purified an essential factor for the secretion and identified it to be PKC␣ (19).…”

“…In this method, we permeabilized only the plasma membrane judging from observations that the intracellular membrane structures of the platelets appeared to be intact morphologically (30) and that vWF stored in ␣-granules did not leak out (19,20). Because the condition used here with 0.6 g/ml SLO induced leakage of 80% cytosolic lactate dehydrogenase from platelets (19,20), it was presumed that most of ATP and cytosol were also lost by diffusion through the pores in the plasma membrane. Therefore, we exogenously added ATP and cytosol in the assay to reconstitute the aggregation.…”

“…The platelets were incubated with 0.6 g/ml SLO at 4°C for 10 min and washed once to remove unbound SLO (19,20,26,27), The treated platelets were resuspended in ice-cold Buffer A containing 4 mg/ml bovine serum albumin at a density of 5 ϫ 10 8 /ml, quantified with a Coulter Counter, and incubated at 30°C for 5 min to make holes in their plasma membrane (19,20,26,27). The permeabilized platelets were kept on ice for 15-30 min with 2 mg of proteins/ml human platelet cytosol (or rat brain cytosol), an ATP-regenerating system (19,20,26,27), and tested substances. Because fibrinogen has been added in previously established aggregation assays using washed platelets at 0.4 mg/ml by and 0.38 mg/ml by Santos et al (29), we also added fibrinogen (Sigma) in our assay at the concentration of 0.4 mg/ml.…”