Defective Zn2+ homeostasis in mouse and human platelets with \u03b1- and \u03b4-storage pool diseases

Sanjeev Kiran Gotru,Paquita Nurden,Stefan Eber,Georgi Manukjan,Harald Schulze,Elmina Mammadova‐Bach ,Leonard Wagner,Andreas Greinacher,Sanne L N Brouns ,Heike M Hermanns,Joachim Volz ,Johan W M Heemskerk,Carsten Deppermann,C M Schambeck ,Magdolna Nagy,Ulrich J Sachs ,Bernhard Nieswandt,Johanna P Van Geffen,Julia Eilenberger,Attila Braun

doi:10.1038/s41598-019-44751-w

Abstract

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ 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 Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ 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 Zn2+ 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 Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.

Highlights

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation
We show that the free ionic form of Zn2+ has a granular localization in both human and murine platelets, which is rapidly lost upon platelet activation
Given the unclearness of the location of intra-platelet ionic Zn2+ and the role of platelet Zn2+ release in clot formation, we re-evaluated these topics in human and mouse platelets

Summary

Results and Discussion

Given the unclearness of the location of intra-platelet ionic Zn2+ and the role of platelet Zn2+ release in clot formation, we re-evaluated these topics in human and mouse platelets. TPEN treatment reduced the Zn2+ level in a similar manner in WT and Unc13d−/− platelets, but not in Nbeal2−/− platelets (Fig. 1C); suggesting that the free ionic Zn2+ store in Nbeal2−/− platelets is limited These alterations may point to an impaired Zn2+ transport into the (low numbers of) available secretory granules. No significant change was found in platelet releasate from Unc13d−/− mice neither in the presence nor in the absence of Zn2+, likely due to the abolished δ-granule and reduced α-granule secretion which strongly reduced fibrin clot formation in this experimental condition (Fig. 2B). Our data indicate that several genetic dysorders impairing platelet granular content or release, especially in patients with GPS, could negatively influence platelet Zn2+-dependent fibrin formation and this defect could be partially rescued by Zn2+ supplementation. Our results suggest that determination of the platelet Zn2+ content with FluoZin[3] could be a novel prognostic biomarker for patients related storage pool disease and other bleeding disorders

Material and Methods

Author Contributions

Additional Information