Soluble Platelet Release Factors as Biomarkers for Cardiovascular Disease

Platelets are the main players in thrombotic diseases, where activated platelets not only mediate thrombus formation but also are involved in multiple interactions with vascular cells, inflammatory components, and the coagulation system. Although in vitro reactivity of platelets provides information on the function of circulating platelets, it is not a full reflection of the in vivo activation state, which may be relevant for thrombotic risk assessment in various disease conditions. Therefore, studying release markers of activated platelets in plasma is of interest. While this type of study has been done for decades, there are several new discoveries that highlight the need for a critical assessment of the available tests and indications for platelet release products. First, new insights have shown that platelets are not only prominent players in arterial vascular disease, but also in venous thromboembolism and atrial fibrillation. Second, knowledge of the platelet proteome has dramatically expanded over the past years, which contributed to an increasing array of tests for proteins released and shed from platelets upon activation. Identification of changes in the level of plasma biomarkers associated with upcoming thromboembolic events allows timely and individualized adjustment of the treatment strategy to prevent disease aggravation. Therefore, biomarkers of platelet activation may become a valuable instrument for acute event prognosis. In this narrative review based on a systematic search of the literature, we summarize the process of platelet activation and release products, discuss the clinical context in which platelet release products have been measured as well as the potential clinical relevance.

CXCL12-Abundant Reticular (CAR) Cells Direct Megakaryocyte Protrusions Across the Bone Marrow Sinusoid Wall

Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.

PAK1 Regulates MEC-17 Acetyltransferase Activity and Microtubule Acetylation during Proplatelet Extension

Mature megakaryocytes extend long processes called proplatelets from which platelets are released in the blood stream. The Rho GTPases Cdc42 and Rac as well as their downstream target, p21-activated kinase 2 (PAK2), have been demonstrated to be important for platelet formation. Here we address the role, during platelet formation, of PAK1, another target of the Rho GTPases. PAK1 decorates the bundled microtubules (MTs) of megakaryocyte proplatelets. Using a validated cell model which recapitulates proplatelet formation, elongation and platelet release, we show that lack of PAK1 activity increases the number of proplatelets but restrains their elongation. Moreover, in the absence of PAK1 activity, cells have hyperacetylated MTs and lose their MT network integrity. Using inhibitors of the tubulin deacetylase HDAC6, we demonstrate that abnormally high levels of MT acetylation are not sufficient to increase the number of proplatelets but cause loss of MT integrity. Taken together with our previous demonstration that MT acetylation is required for proplatelet formation, our data reveal that MT acetylation levels need to be tightly regulated during proplatelet formation. We identify PAK1 as a direct regulator of the MT acetylation levels during this process as we found that PAK1 phosphorylates the MT acetyltransferase MEC-17 and inhibits its activity.

Harnessing a Novel Dyrk1a-Ablim2-MKL1 Regulatory Module in Megakaryocyte Morphogenesis to Enable Scalable Platelet "Pharming"

Growing clinical demands for platelet transfusions combined with supply limitations have created shortages which are trending toward a global crisis. Major efforts have been taken to address key issues of platelet sources, storage, and utilization. Recent progress in ex vivo culture-based production of megakaryocytes (Mk) and platelets, "pharming," has highlighted the potential for novel, donor-independent sources amenable to antigenic editing and cryo-stockpiling. Such cultures can be easily initiated from umbilical cord blood (CB) progenitors, induced pluripotent stem cells (iPSC), or directly re-programmed somatic cells. The major roadblock associated with these Mk sources consists of their fetal ontogenic status, which is beneficial for expansion but severely limits platelet production. The ability to elicit in pre-expanded Mk an adult program of morphogenesis (polyploidization, enlargement, and proplatelet formation) would enable circumvention of this scalability barrier. A master regulator of adult Mk morphogenesis consists of the transcriptional coactivator MKL1 which undergoes nuclear translocation in response to RhoA-mediated actin polymerization, stimulated by thrombopoietin and environmental mechano-sensing. Nuclear MKL1 associates with the transcription factor SRF1 to upregulate cytoskeletal remodeling factors, including filamin A and Hic-5, that act as morphogenesis effectors. Our previous studies identified in infantile CB Mk a failure in MKL1 upregulation resulting from repression by the oncofetal RNA-binding factor IGF2BP3. Pharmacologic suppression of IGF2BP3 with BET inhibitors rescued MKL1 expression and improved platelet production but caused cycle arrest preventing polyploidization. As an alternative approach to abrogate the fetal blockade in Mk morphogenesis, we sought to promote MKL1 activity by targeting a kinase, Dyrk1a, which had been shown to restrain MKL1 from nuclear translocation. Treatment of infantile CB Mk with a variety of Dyrk1-selective inhibitors including harmine and EHT 1610 strongly enhanced polyploidization (p = 0.015 and 0.009 respectively), enlargement (p < 0.005) , and in vitro platelet release (2 fold each, p = 0.001 and 0.007 respectively), attaining levels seen with adult Mk. When xenotransplanted into NSG mice, harmine-treated CB Mk demonstrated enhanced capability for in vivo platelet release (about 5 fold, p = 0.016). CB stem cells expanded with the AHR antagonist SR1 and an iPSC-Mk cell line also responded to Dyrk1 inhibition with robustly increased morphogenesis. Several findings implicated MKL1 in this response: 1) induction of nuclear translocation by the inhibitors, 2) induction of target genes (filamin A and Hic-5) by the inhibitors, and 3) loss of response to inhibitors in Mkl1-ko murine progenitors. Supporting Dyrk1a as a relevant target, mice with Mk-specific loss of one Dyrk1a allele (Dyrk1aflox/wt;Pf4-Cre) displayed increases in platelet counts (p = 0.037) and marrow Mk polyploidization (p = 0.02). In addition, retroviral expression in human progenitors of a dominant negative Dyrk1a mutant K188R promoted Mk enlargement (p = 0.014). shRNA knockdowns could not be obtained due to toxicity of > ~60% loss of Dyrk1a. To determine mechanisms for Dyrk1a control of morphogenesis, we analyzed the actin cytoskeleton, a key regulator of MKL1. Dyrk1 inhibition in all types of Mk progenitors (adult, infantile, and iPSC) induced assembly of cortical filamentous actin (F-actin), as detected by Alexa594-phalloidin staining. Prior studies showed cytoskeletal binding by Dyrk1a and direct phosphorylation of F-actin regulators N-WASP and Ablim1. A survey of human marrow expression patterns for candidate Dyrk1a substrates (The Human Protein Atlas) identified Ablim2, as showing a Mk-specific, cortical staining pattern. Dyrk1 inhibition increased Ablim2 levels ~5-fold in CB Mk (p < 0.005), and immunofluorescence displayed a cortical distribution similar to F-actin. Lentiviral shRNA knockdown of Ablim2 abrogated all effects of Dyrk1 inhibition, blocking: F-actin formation, MKL1 nuclear translocation, activation of the MKL1 targets, and Mk morphogenesis. These findings thus delineate a novel Dyrk1a-Ablim2-MKL1 regulatory module in Mk morphogenesis that can be manipulated to address the problem of scaling ex vivo production and might also serve as a future in vivo therapeutic target for thrombocytopenia. Disclosures Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Platelet Biogenesis in the Lung Circulation

Megakaryocytes are normal cellular components of the blood returning to the heart and entering the lungs, and historical data has pointed to a role of the lungs in platelet production. Recent studies using intravital microscopy have demonstrated that platelet release occurs in the lung from bone marrow megakaryocytes that embolize into the lung circulation.

Endothelial O-Glycoproteins Cooperate with Platelet CLEC-2 Signaling to Maintain Cerebrovascular Integrity during Mouse Development

Protein glycosylation plays vital roles in many biological processes. We previously discovered that embryos with global deficiencies of mucin-type O-glycans (i.e. C1galt1-/- mice) develop fatal brain hemorrhage during embryogenesis. However, mice lacking endothelial cell O-glycans exhibit blood-lymphatic mixing with no brain bleeding phenotype, whereas mice lacking either O-glycoprotein podoplanin or its receptor CLEC-2 develop similar spontaneous bleeding in developing brain. To further investigate whether endothelial O-glycans contribute to cerebrovascular barrier development and/or maintenance, we generated two independent transgenic mouse lines, in which the expression of C1galt1 is under the control of endothelial-specific Tie2 promoter/enhancer (EC-C1galt1 Tg mice). When breeding the EC-C1galt1 Tg mice into C1galt1-/- background, we found that the embryonic lethality of C1galt1-/- mice was completely rescued, supporting a critical role of endothelial O-glycans in governing the cerebral vascular integrity during embryogenesis. Platelets have been identified as one of the key players for vascular integrity under physiological and pathological conditions although the underlying mechanisms remain elusive. Based on the existing knowledge, we hypothesize that endothelial O-glycoproteins cooperate with neural podoplanin, an O-glycoprotein highly expressed on neural progenitor cells in early developing brain, through activating its platelet receptor CLEC-2, to maintain cerebral vascular integrity during active sprouting angiogenesis. To test the hypothesis, we generated mice with compound deficiencies of endothelial O-glycans and podoplanin. Interestingly, double knockout mice faithfully phenocopied the global C1galt1-/- mice, including a full penetration of fatal hemorrhage. These results strongly support a cooperative role between endothelial O-glycans and neural podoplanin. Mechanistically, we demonstrated that loss of podoplanin or CLEC-2 in vivo leads to an impaired expression of VE-cadherin, a major endothelial intercellular junctional protein. Moreover, in vitro cell-based analysis showed that podoplanin-CLEC-2 interaction and subsequent platelet release counteracts the effects of VEGF on VE-cadherin expression and endothelial permeability, indicating that podoplanin-dependent activation of platelet CLEC-2 signaling is critical for the stability of newly formed fragile vessels in the early developing brain by promoting endothelial junctions via platelet release. Taken together, our results provide a new model for the maintenance of cerebrovascular integrity during early brain development, in which the interactions between endothelial cells, neural cells and platelets are essential for the brain vascular homeostasis. These findings not only provide new insights into the regulation of cerebrovascular development, but could also allow us to test new therapies for hemorrhagic disorders with pathway-targeted drugs. Disclosures No relevant conflicts of interest to declare.

ADAP deficiency impairs megakaryocyte polarization with ectopic proplatelet release and causes microthrombocytopenia

Key Points ADAP deficiency in mice leads to microthrombocytopenia caused by a reduced platelet life span and ectopic (pro)platelet release. Lack of ADAP in MKs impairs demarcation membrane system polarization and podosome formation.