Defective Zn2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases

Abstract

Zinc (Zn 2+ ) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn 2+ storage and release. To visualize Zn 2+ storage in human and mouse platelets, the Zn 2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d −/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn 2+ release upon activation. Platelets from Nbeal2 −/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn 2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn 2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2 −/− and Unc13d −/− mice, and the impairment could be partially restored by extracellular Zn 2+ . Altogether, we conclude that the release of ionic Zn 2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn 2+ in platelet-dependent fibrin formation.

The Yin and Yang of ACE/ACE2 Pathways: The Rationale for the Use of Renin-Angiotensin System Inhibitors in COVID-19 Patients

“…As already mentioned, free Zn 2+ promotes clot stability by binding to fibrinogen (see Box 4 and [91]) and chloroquine has been shown to have an anti-thrombotic activity by inhibiting platelet activation [125]. To this regard, upon platelet activation the release of Zn 2+ store from their secretory granules has been shown to participate to the pro-coagulant activity in platelet-dependent fibrin formation [126], suggesting that an elevated free Zn 2+ concentration might occur and contribute to thrombotic predisposition in COVID-19 patients, a phenomenon possibly countered by chloroquine.…”

Zinc Homeostasis in Platelet-Related Diseases

“…The phospholipid membrane of eukaryotic cells is impermeable for Zn 2+ , therefore transport and storage of Zn 2+ is ensured by Zn 2+ transporters [2,8]. Resting platelets can uptake Zn 2+ from the blood plasma and store it, indicating the existence of active Zn 2+ transport and storage mechanisms [15,25,26]. Zn 2+ concentration in blood serum was found to be higher than in blood plasma [27,28], suggesting that activated platelets could release a significant amount of Zn 2+ during blood clotting.…”

“…Hence, approximatively 50–80 pieces of α-granule fulfill the body of a single platelet [30,31,32], which possibly stores a significant amount of Zn 2+ . Therefore, it was proposed that the largest Zn 2+ store could be located in platelet α-granules [26,29]. In addition, Zn 2+ has a high affinity to bind fibrinogen, albumin, histidine-rich glycoprotein (HRG) and factor XIII, which are also accumulated in α-granules of platelets, suggesting that Zn 2+ store exists as a protein-bound form in this type of granules [33].…”

“…Using inductively coupled plasma mass spectrometry (ICP-MS), Gorodetsky et al, reported that approximately 40% of Zn 2+ is stored in granules, while 60% of Zn 2+ is stored in other intracellular organelles and also in the cytoplasm [33]. Recently, the labile Zn 2+ pool was visualized by our group and quantified in human and mouse platelets using a Zn 2+ specific fluorescence dye FluoZin3 [26]. The specificity of the dye was characterized with Zn 2+ supplementation or chelation of Zn 2+ with ZnCl 2 or TPEN, respectively.…”

“…The specificity of the dye was characterized with Zn 2+ supplementation or chelation of Zn 2+ with ZnCl 2 or TPEN, respectively. Using confocal microscopy, FluoZin3 staining showed several stained foci in the platelet cytoplasm, which became markedly reduced when platelets were spread on a fibrinogen-coated surface [26]. In addition, the rapid loss of intracellular FluoZin3 staining measured by flow cytometry, correlated to Zn 2+ efflux in activated platelets [26].…”