Ins and outs of platelet α-granule proteins

Abstract

Although platelets are small anucleate cell fragments, they are highly organized cells rich in different types of organelles. Platelets contain three secretory granules, dense granules, α-granules and lysosomes which are released upon platelet activation at sites of injury. Most of these α-granule proteins are synthesized by the platelet precursor cells, the megakaryocytes (MKs). Platelets and MKs are, however, unique in that they also take up plasma proteins. The mechanisms behind the endocytosis of plasma proteins as well as the exocytosis machinery are, however, still poorly understood. In our work, we employ protein-protein interaction studies and cell-based internalization studies to get more insight into the mechanisms underlying the endocytosis of plasma proteins in MKs. In addition, we utilize mass spectrometry-based approaches to assess the role of the platelet integrins in α-granule cargo enrichment as well as the mechanism of the release thereof. α-granule coagulation factor V (FV) derives entirely from receptor-mediated endocytosis by megakaryocytes. In our work we provided evidences of a novel role for the lectin galectin8 (Gal8) as mediator of FV uptake. Surface plasmon resonance (SPR) analysis showed a reversible and dose-dependent binding of Gal8 and FV. By inhibiting Gal8 functions and/or targeting its expression in the megakaryocyte-like cells (DAMI), we demonstrated that Gal8-FV complex formation is a prerequisite for FV uptake. Further we explored the potential role of other interaction partners of Gal8, i.e. GPIIbIIIa and GPIb-GPIX-GPV, in FV endocytosis. Mass spectrometry (MS) analysis of platelet lysates from leukocyte adhesion deficiency type III (LAD-III) and Glanzmann's thrombasthenia (GT) patients show that the level of GPV and not of GPIIbIIIa correlates with the amount of FV. Biochemical approaches show that Gal8 can act as a cross-linker between FV and GPV, suggesting a potential role for GPV as Gal8 counter-receptor. Fibrinogen, the most abundant α-granule protein, can be taken up from plasma by both circulating platelets and megakaryocytes. Here, by mass spectrometry analysis of the platelet content from GT and LAD-III patients we explored the role of integrins on platelets cargo enrichment.The results show that GPIIbIIIa only contributes to the uptake of fibrinogen, and that activation of this integrin is not required to facilitate fibrinogen endocytosis. Novel differentially expressed proteins that may be associated with the investigated diseases are identified as well. Moreover MS analysis further revealed the genetic defect of a previously uncharacterized GT patient. In a different line of research, we explored the platelet secretory efficiency of α-granulesin patients with Familial Hemophagocytic Lymphohistiocytosis-5 (FHL-5). Disease-causing mutations have been mapped onto the gene encoding syntaxin-binding-protein2 (Munc18-2). We use MS, flow cytometry and electron microscopy to identify the protein product of a Munc18-2 splice-site variant (1247-1G>C) and the effect thereof on platelet degranulation. This Munc18-2 variant has been established to cause impaired degranulation of neutrophils. In spite of previous suggestions, flow cytometry, electron microscopy and MS studies reveal that the splice-site variant does not significantly affect platelet. Moreover MS analysis revealed the still unknown protein product resulting from the 1247-1G>C mutation.