Intracellular Trafficking, Localization, and Mobilization of Platelet-Borne Thiol Isomerases

Abstract

Objective: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation.

Approach and results: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice).

Conclusions: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.

Keywords: blood platelets; megakaryocytes; membrane fusion; protein disulfide isomerases; secretory vesicles.

Figure 1. Intracellular distribution of PDI and ERp57 during MK development

IFM images (mid-cell Z-sections with YZ and XY profiles or extended focus renders) are shown for representative cultured mouse MK stained for ERp57 (magenta), PDI (green), CD41 (red) and DNA (blue; bars = 5μm except where noted). A) In early endomitotic cells ERp57 and PDI show a similar extranuclear distribution (see Video 1). B) In larger cells undergoing maximal protein synthesis PDI remains widely distributed, and in some cells ERp57 is highly concentrated in ribbon-like structures that may represent ER (inset detail bar = 1μm; see Video 2). C) As late-stage MKs flatten out, both thiol isomerases are present throughout the extranuclear region (see Video 3) and D) in proplatelet extensions (main cell body at right out of field), where they are present throughout the shafts and in terminal buds (see Video 4).

Figure 2. Thiol isomerases are not associated with MK trans-Golgi network or recycling endosomal compartments, or with platelet α-granules

A) IFM imaging of a representative mature human MK shows ERp57 (green) distributed throughout the cell having little overlap with trans-Golgi defined by TGN46 (red) or recycling endosomes containing TFR (magenta; mid-cell Z-section, bar = 10 μm). B) In a human platelet, PDI (green) and ERp57 (red) overlap with each other but not with P-selectin (magenta; mid-cell Z-sections, bars = 1μm). C) Platelets stained for CD41/integrin αIIb (green), ERp57 (red) and P-selectin (magenta) from WT and Nbeal2^-/- mice (the latter lacking α-granule cargo) show similar intracellular distributions of thiol isomerase (mid-cell Z-sections, bars = 1 μM). D) Immunoblot analysis shows similar levels of PDI and ERp57 in WT and Nbeal2^-/- mouse platelet lysates, and E) similar expression of PDI in lysates from normal human (NH) and Gray platelet syndrome (GPS) patient platelets (actin used as loading control).

Figure 3. Platelet thiol isomerases associate with cell-surface membrane structures and overlap with SERCA3 on the inner surface of the cell outer membrane

A) Human platelet lysates were fractionated by sucrose gradient and fractions (1-13) analyzed by immunoblotting for PDI, ERp57, β3 integrin, RabGDI b, TSP-1 and calreticulin (marker sizes in kilodaltons at left). Peak concentrations of PDI and ERp57 overlapped with β3 integrin corresponding to the cell surface membrane fraction, with cytosolic RabGDI b, and the DTS marker calreticulin and showed little overlap with α-granule borne TSP-1 (Figure representative of 3 experiments). B) IFM imaging of fixed resting human platelets stained for ERp57 (green) and SERCA3 (red) shows overlap in a compartment defined by the latter on the inner surface of the cell outer membrane, delineated by the colocalization channel (yellow; mid-cell Z-sections, bars = 1 μM).

Figure 4. Mobilization of thiol isomerases in activated platelets requires actin polymerization

IFM imaging of human platelets activated with 4 μM U46619 in the presence (+LAT) or absence (-LAT) of latrunculin A, fixed and stained for tubulin (magenta), P-selectin (red) and either: A) CD42B/GP1b (light blue) and VWF (green), or B) PDI (light blue) and ERp57 (green). Reorganization of cytoskeletal tubulin and surface mobilization of P-selectin, VWF and ERp57 is evident in platelets not treated with latrunculin, while treated cells show intracellular protein distribution patterns similar to unactivated platelets (Fig.2B). Maximum intensity renders, bars = 1 μM. C) The effect of latrunculin treatment on the surface mobilization of thiol isomerases, as compared to that of P-selectin, was quantified by flow cytometry. Platelets were activated with 1U/ml of thrombin, the binding of the primary antibody for thiol isomerases was detected using a FITC-conjugated species-specific secondary antibody, the PE/Cy5 anti-human CD62P was used to detect P-selectin. The percentage of inhibition of the Median Fluorescence Intensity was calculated (**P<0.01, one-way ANOVA, followed by Dunnett's test, n=3).

Figure 5. Surface mobilization of platelet thiol isomerase is independent of Munc13-4

A) Immunoblot analysis of platelet lysates from wild type (WT) and Unc13d^Jinx mice shows lack of Munc13-4 in the latter and similar levels of PDI and ERp57 (GAPDH loading control). B) Flow cytometry measurement of surface P-selectin (CD62P) and PDI in platelets under resting conditions or after stimulation with PAR4-AP in the presence/absence of ADP shows that P-selectin mobilization is significantly impaired in Unc13d^Jinx platelets but PDI mobilization is greater than WT. ADP significantly increased PDI mobilization as compared to WT or resting Unc13d^Jinx platelets (*P<0.05, *** P<0.001, one-way ANOVA, followed by Dunnett's test or two-way ANOVA, followed by Bonferroni's test, n=4).

Figure 6. Model comparing synthesis, trafficking, packaging and mobilization of platelet thiol isomerases and α-granule cargo

A) In the maturing MK, proteins destined for α-granules (e.g. VWF, TSP-1) are trafficked from the ER via a pathway that leads through the trans-Golgi network to multivesicular bodies. B) Thiol isomerases associate with membranes destined for the platelet DTS and cell surface. C) As proplatelet extensions form secretory granule cargo is packaged, while thiol isomerases move with membranes into forming platelets. D) In mature platelets, thiol isomerases are concentrated near the surface membrane and in the near-surface DTS. E) In activated platelets actin polymerization and membrane fusion facilitates the surface mobilization and secretion of granule cargo, while thiol isomerases mobilized without the need for membrane fusion.