Targeting cargo to an unconventional secretory system within megakaryocytes allows the release of transgenic proteins from platelets.

BACKGROUND: Platelets are essential for hemostasis, and thrombosis, and play vital roles during metastatic cancer progression and infection. Hallmarks of platelet function are activation, cytoskeletal rearrangements, and the degranulation of their cellular contents upon stimulation. While alpha and dense granules are the most studied platelet secretory granules, the dense tubular system (DTS) also functions as a secretory system for vascular thiol isomerases. However, how DTS cargo is packaged and transported from megakaryocytes (MKs) to platelets is poorly understood. OBJECTIVES: To underpin the mechanisms responsible for DTS cargo transport and leverage those for therapeutic protein packaging into platelets. METHODS: A retroviral expression system combined with immunofluorescence confocal microscopy was employed to track protein DTS cargo protein disulfide isomerase (PDI) fused to eGFP (eGFP-PDI) during platelet production. Murine bone marrow transplantation models were used to determine the release of therapeutic proteins from platelets. RESULTS AND CONCLUSIONS: We demonstrated that the endoplasmic reticulum retrieval motif Lys-Asp-Glu-Leu (KDEL) located at the C-terminus of PDI was essential for the regular transport of eGFP-PDI-containing granules. eGFP-PDIΔKDEL, in which the retrieval signal was deleted, was aberrantly packaged and its expression was upregulated within clathrin-coated endosomes. Finally, we found that ectopic transgenic proteins, such as tissue factor pathway inhibitor and interleukin 2, can be packaged into MKs and proplatelets by adding a KDEL retrieval sequence. Our data corroborate the DTS as a non-canonical secretory system in platelets and demonstrate that in vitro-generated MKs and platelets may be used as a delivery system for transgenic proteins during cellular therapy.

The bone marrow is the primary site of thrombopoiesis.

ABSTRACT: Megakaryocytes (MKs) generate thousands of platelets over their lifespan. The roles of platelets in infection and inflammation has guided an interest to the study of extramedullary thrombopoiesis and therefore MKs have been increasingly reported within the spleen and lung. However, the relative abundance of MKs in these organs compared to the bone marrow and the scale of their contribution to the platelet pool in a steady state remain controversial. We investigated the relative abundance of MKs in the adult murine bone marrow, spleen, and lung using whole-mount light-sheet and quantitative histological imaging, flow cytometry, intravital imaging, and an assessment of single-cell RNA sequencing (scRNA-seq) repositories. Flow cytometry revealed significantly higher numbers of hematopoietic stem and progenitor cells and MKs in the murine bone marrow than in spleens or perfused lungs. Two-photon intravital and light-sheet microscopy, as well as quantitative histological imaging, confirmed these findings. Moreover, ex vivo cultured MKs from the bone marrow subjected to static or microfluidic platelet production assays had a higher capacity for proplatelet formation than MKs from other organs. Analysis of previously published murine and human scRNA-seq data sets revealed that only a marginal fraction of MK-like cells can be found within the lung and most likely only marginally contribute to platelet production in the steady state.

Fibrin protofibril packing and clot stability are enhanced by extended knob-hole interactions and catch-slip bonds.

Fibrin polymerization involves thrombin-mediated exposure of knobs on one monomer that bind to holes available on another, leading to the formation of fibers. In silico evidence has suggested that the classical A:a knob-hole interaction is enhanced by surrounding residues not directly involved in the binding pocket of hole a, via noncovalent interactions with knob A. We assessed the importance of extended knob-hole interactions by performing biochemical, biophysical, and in silico modeling studies on recombinant human fibrinogen variants with mutations at residues responsible for the extended interactions. Three single fibrinogen variants, γD297N, γE323Q, and γK356Q, and a triple variant γDEK (γD297N/γE323Q/γK356Q) were produced in a CHO (Chinese Hamster Ovary) cell expression system. Longitudinal protofibril growth probed by atomic force microscopy was disrupted for γD297N and enhanced for the γK356Q mutation. Initial polymerization rates were reduced for all variants in turbidimetric studies. Laser scanning confocal microscopy showed that γDEK and γE323Q produced denser clots, whereas γD297N and γK356Q were similar to wild type. Scanning electron microscopy and light scattering studies showed that fiber thickness and protofibril packing of the fibers were reduced for all variants. Clot viscoelastic analysis showed that only γDEK was more readily deformable. In silico modeling suggested that most variants displayed only slip-bond dissociation kinetics compared with biphasic catch-slip kinetics characteristics of wild type. These data provide new evidence for the role of extended interactions in supporting the classical knob-hole bonds involving catch-slip behavior in fibrin formation, clot structure, and clot mechanics.

Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability, and resistance to fibrinolysis.

Fibrinogen is essential for blood coagulation. The C-terminus of the fibrinogen α-chain (αC-region) is composed of an αC-domain and αC-connector. Two recombinant fibrinogen variants (α390 and α220) were produced to investigate the role of subregions in modulating clot stability and resistance to lysis. The α390 variant, truncated before the αC-domain, produced clots with a denser structure and thinner fibres. In contrast, the α220 variant, truncated at the start of the αC-connector, produced clots that were porous with short, stunted fibres and visible fibre ends. These clots were mechanically weak and susceptible to lysis. Our data demonstrate differential effects for the αC-subregions in fibrin polymerisation, clot mechanical strength, and fibrinolytic susceptibility. Furthermore, we demonstrate that the αC-subregions are key for promoting longitudinal fibre growth. Together, these findings highlight critical functions of the αC-subregions in relation to clot structure and stability, with future implications for development of novel therapeutics for thrombosis.