Dataset Information

Enhancing functional platelet release in vivo from in vitro-grown megakaryocytes using small molecule inhibitors.

ABSTRACT: In vitro-grown megakaryocytes for generating platelets may have value in meeting the increasing demand for platelet transfusions. Remaining challenges have included the poor yield and quality of in vitro-generated platelets. We have shown that infusing megakaryocytes leads to intrapulmonary release of functional platelets. A Src kinase inhibitor (SU6656), a Rho-associated kinase inhibitor (Y27632), and an aurora B kinase inhibitor (AZD1152) have been shown to increase megakaryocyte ploidy and in vitro proplatelet release. We now tested whether megakaryocytes generated from CD34+ hematopoietic cells in the presence of these inhibitors could enhance functional platelet yield following megakaryocyte infusion. As expected, all inhibitors increased megakaryocyte ploidy, size, and granularity, but these inhibitors differed in whether they injured terminal megakaryocytes: SU6656 was protective, whereas Y27632 and AZD1152 increased injury. Upon infusion, inhibitor-treated megakaryocytes released threefold to ninefold more platelets per initial noninjured megakaryocyte relative to control, but only SU6656-treated megakaryocytes had a significant increase in platelet yield when calculated based on the number of initial CD34+ cells; this was fourfold over nontreated megakaryocytes. The released platelets from drug-treated, but healthy, megakaryocytes contained similar percentages of young, uninjured platelets that robustly responded to agonists and were well incorporated into a growing thrombus in vivo as controls. These studies suggest that drug screens that select megakaryocytes with enhanced ploidy, cell size, and granularity may include a subset of drugs that can enhance the yield and function of platelets, and may have clinical application for ex vivo-generated megakaryocytes and platelet transfusion.

SUBMITTER: Jarocha D

PROVIDER: S-EPMC5873227 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Enhancing functional platelet release in vivo from in vitro-grown megakaryocytes using small molecule inhibitors.

Jarocha Danuta D Vo Karen K KK Lyde Randolph B RB Hayes Vincent V Camire Rodney M RM Poncz Mortimer M

Blood advances 20180301 6

In vitro-grown megakaryocytes for generating platelets may have value in meeting the increasing demand for platelet transfusions. Remaining challenges have included the poor yield and quality of in vitro-generated platelets. We have shown that infusing megakaryocytes leads to intrapulmonary release of functional platelets. A Src kinase inhibitor (SU6656), a Rho-associated kinase inhibitor (Y27632), and an aurora B kinase inhibitor (AZD1152) have been shown to increase megakaryocyte ploidy and in ...[more]

PMID: 29545255

Similar Datasets

Project description:PurposeType 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet β-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems.ResultsHere, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 β-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM.ConclusionGiven the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.

Project description:AbstractFibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.

Dataset Information

Enhancing functional platelet release in vivo from in vitro-grown megakaryocytes using small molecule inhibitors.

Publications

Enhancing functional platelet release in vivo from in vitro-grown megakaryocytes using small molecule inhibitors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets