Impaired hemostatic activity of healthy transfused platelets in inherited and acquired platelet disorders: Mechanisms and implications.

Platelet transfusions can fail to prevent bleeding in patients with inherited platelet function disorders (IPDs), such as Glanzmann's thrombasthenia (GT; integrin αIIbß3 dysfunction), Bernard-Soulier syndrome [BSS; glycoprotein (GP) Ib/V/IX dysfunction], and the more recently identified nonsyndromic RASGRP2 variants. Here, we used IPD mouse models and real-time imaging of hemostatic plug formation to investigate whether dysfunctional platelets impair the hemostatic function of healthy donor [wild-type (WT)] platelets. In Rasgrp2-/- mice or mice with platelet-specific deficiency in the integrin adaptor protein TALIN1 ("GT-like"), WT platelet transfusion was ineffective unless the ratio between mutant and WT platelets was ~2:1. In contrast, thrombocytopenic mice or mice lacking the extracellular domain of GPIbα ("BSS-like") required very few transfused WT platelets to normalize hemostasis. Both Rasgrp2-/- and GT-like, but not BSS-like, platelets effectively localized to the injury site. Mechanistic studies identified at least two mechanisms of interference by dysfunctional platelets in IPDs: (i) delayed adhesion of WT donor platelets due to reduced access to GPIbα ligands exposed at sites of vascular injury and (ii) impaired consolidation of the hemostatic plug. We also investigated the hemostatic activity of transfused platelets in the setting of dual antiplatelet therapy (DAPT), an acquired platelet function disorder (APD). "DAPT" platelets did not prolong the time to initial hemostasis, but plugs were unstable and frequent rebleeding was observed. Thus, we propose that the endogenous platelet count and the ratio of transfused versus endogenous platelets should be considered when treating select IPD and APD patients with platelet transfusions.

New insights into cytoskeletal remodeling during platelet production.

The past decade has brought unprecedented advances in our understanding of megakaryocyte (MK) biology and platelet production, processes that are strongly dependent on the cytoskeleton. Facilitated by technological innovations, such as new high-resolution imaging techniques (in vitro and in vivo) and lineage-specific gene knockout and reporter mouse strains, we are now able to visualize and characterize the molecular machinery required for MK development and proplatelet formation in live mice. Whole genome and RNA sequencing analysis of patients with rare platelet disorders, combined with targeted genetic interventions in mice, has led to the identification and characterization of numerous new genes important for MK development. Many of the genes important for proplatelet formation code for proteins that control cytoskeletal dynamics in cells, such as Rho GTPases and their downstream targets. In this review, we discuss how the final stages of MK development are controlled by the cellular cytoskeletons, and we compare changes in MK biology observed in patients and mice with mutations in cytoskeleton regulatory genes.

Regulation of differentiation of MEG01 to megakaryocytes and platelet-like particles by Valproic acid through Notch3 mediated actin polymerization.

Valproic acid (VPA) is one of the HDAC inhibitors used for the treatment of neurological disorders and hematological malignancies. Its role in self-renewal and proliferation of hematopoietic stem cells (HSCs) is well studied, but little is known about its involvement in regulating megakaryopoiesis and thrombopoiesis. In this study, we evaluated the role of VPA in megakaryopoiesis by using MEG-01, a megakaryoblast cell line. Our results show that VPA treatment differentiates MEG-01 cells to megakaryocytes (MK) and platelet-like particles. It was confirmed by augmented expression of MK and PLT-specific markers, higher ploidy, and PLT functionality. We assessed the molecular events underlying megakaryopoiesis. In the present study, we found an upregulation of Notch3 and its downstream target PDGFR-ß upon VPA treatment. The direct role of Notch3 in megakaryopoiesis has not yet been studied. PDGFR-ß is known to control actin organization during vascular smooth muscle cell differentiation. The actin cytoskeleton plays important role during proplatelet and PLT formation. We found an upregulation of Rac/Cdc42 GTPase and its downstream effectors that are the key players during actin polymerization events. We speculate that VPA induces PLT formation through Notch-3 signaling that in turn modulates actin polymerization that is one of the crucial steps necessary for thrombopoiesis. These studies were also confirmed with knockdown of Notch3 in MEG01 by using ShRNA approach as well as with apheresis-derived CD34+ cells. Altogether, these findings provide an evidence for a novel role of Notch3 in regulating platelet formation.

Data on the effect of oral feeding of Arachidonic acid or Docosahexanoic acid on haematopoiesis in mice.

Stem cells have peculiar property to self-renew and differentiate. It is important to control their fate in safe and effective ways for their therapeutic use. The mediators of essential polyunsaturated fatty acids (PUFAs) namely Arachidonic acid (AA) and Docosahexanoic acid (DHA) are known to play a role in haematopoiesis via various metabolic pathways [1]. However the direct effect of purified AA or DHA on haematopoiesis has not been well investigated yet. We have reported that oral administration of PUFAs enhanced haematopoiesis in mice [2]. Signaling Leukocyte Antigen Molecule (SLAM) (CD48-CD150+) phenotype consists of pure population of haematopoietic stem cells (HSCs). Herein we observed higher percentage of SLAM (CD48-CD150+) phenotype in the bone marrow (BM) cells of mice fed with AA or DHA compared to PBS fed control mice. Data from engraftment study depicts that BM from AA/DHA-fed mice showed higher absolute number of donor cells in recipient mice compared to control. The enhanced hematopoiesis observed in AA/DHA-fed mice was returned to normal when the mice were kept on normal diet for six weeks (after ten days of oral feeding). We confirmed GCMS (Gas Chromatography-Mass Spectroscopy) retention times of AA and DHA by co-injecting fatty acid extract from AA or DHA fed mice with purified AA or DHA standards respectively. Representative flow cytometry profile of Lin-Sca-1+c-kit+(LSK) cells showed higher expression of CXCR4 protein and ligands of Wnt, Notch1 signaling in BM of AA/DHA-fed mice.

Oral feeding with polyunsaturated fatty acids fosters hematopoiesis and thrombopoiesis in healthy and bone marrow-transplanted mice.

Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1+ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure.

Arachidonic acid and Docosahexanoic acid enhance platelet formation from human apheresis-derived CD34+ cells.

An Aberration in megakaryopoiesis and thrombopoiesis, 2 important processes that maintain hemostasis, leads to thrombocytopenia. Though platelet transfusions are used to treat this condition, blood banks frequently face a shortage of platelets. Therefore, methods to generate platelets on a large scale are strongly desirable. However, to generate megakaryocytes (MKs) and platelets (PLTs) in numbers sufficient for clinical application, it is essential to understand the mechanism of platelet production and explore efficient strategies accordingly. We have earlier reported that the N-6 and N-3 poly-unsaturated fatty acids (PUFAs), Arachidonic acid (AA)/Docosahexanoic acid (DHA) have beneficial effect on the generation of MKs and PLTs from umbilical cord blood derived CD34+ cells. Here we tested if a similar effect is observed with peripheral blood derived CD34+ cells, which are more commonly used in transplantation settings. We found a significant enhancement in cell numbers, surface marker expression, cellular ploidy and expression of cytoskeletal components during PLT biogenesis in cultures exposed to media containing AA/DHA than control cultures that were not exposed to these PUFAs. The test cells engrafted more efficiently in NOD/SCID mice than control cells. AA/DHA appears to have enhanced MK/PLT generation through upregulation of the NOTCH and AKT pathways. Our data show that PUFAs could be valuable additives in the culture system for large scale production of platelets for clinical applications.