Platelets are anuclear cells released from megakaryocytes, the rare myeloid cells that undergo multiple rounds of endomitosis before releasing platelets. Platelets, known as the mediator of thrombosis, are emerging as cellular mediators of type Ⅱ diabetes, atherosclerosis, cancer cell metastasis, and immune responses [1,2]. Despite the lack of a nucleus, they express a large number of transcription factors including peroxisome proliferator-activated receptor (PPAR). PPAR needs ligand-binding to be activated [3].

PPAR, a ligand-activated transcription factor, consists of 3 isoforms: α, β/δ, γ, and requires heterodimerization with retinoid X receptor to modulate transcription of target genes [4]. PPARγ is involved in glucose metabolism, inflammation, and differentiation and functions of adipose tissue [5-7]. Recent studies have reported that the PPARγ inhibits platelet activation and aggregation [8-11]. Also, studies have reported the PPARγ and ligand of PPARγ as the factor influences the hematopoietic system. They could affect the roles of erythroid, myeloid, monocytic, and lymphocytic cell function during an immune response, and modulate proliferation and maturation of erythroid progenitor cells [12-14]. The endogenous 15-deoxy-Δ [12,14] prostaglandin J₂ (15d-PGJ₂) is a ligand of PPARγ. Biologically, 15d-PGJ₂ is the most active metabolite of prostaglandin D2 [15].

Fucoidans, fucose-containing sulfated polysaccharides, are constituents of brown seaweed and some marine invertebrates. Extensive studies have reported their various biological effects including immunostimulation, anti-tumor, antiviral, antithrombotic and anticoagulant activities. By far the anticoagulant and antithrombotic actions of fucoidans are the most widely recognized bioactivities, but the basis for these activities is not completely understood [16-19].

We are interested in the influences of 15d-PGJ₂ and fucoidans to the megakaryocytic differentiation and the platelet production, as well as the influences of 15d-PGJ₂ and fucoidans to the thrombosis and coagulation system. Due to their antithrombotic and anticoagulant activities, they have the probability of the application as the novel therapeutic agents to cardiovascular disease. Also, if they have the abilities inducing the production of platelet, they could be the stable solution of platelet supply to the thrombocytopenic patients.

In this study, we evaluated the effects of 15d-PGJ₂ and fucoidans on megakaryocytes and platelets with diverse methods from protein to the molecular level. This study aims to determine 1) whether the 15d-PGJ₂ and fucoidans encourage the megakaryocytes to produce platelets actively; 2) whether the 15d-PGJ₂ and fucoidans possess the anticoagulant and antithrombotic actions, and 3) whether specific factors, such as prostaglandins or genes, explain the mechanism of how the 15d-PGJ₂ and fucoidans influence differentiation and function of the platelets. To search these factors, we also included PPARα, which activates fatty acid catabolism and stimulates gluconeogenesis, attenuates inflammatory responses, and participates in the amino acid metabolism, and COX-2, which is responsible for the formation of prostanoids, including thromboxane and prostaglandins such as prostacyclin, from arachidonic acid.

Megakaryocytes are the biggest cell of the bone marrow, and platelets are derived from the cytoplasm of megakaryocytes. Platelets are metabolically active cellular mediators of various vascular biology containing copious amounts of bioactive materials including thromboxanes, prostaglandins, chemokines, and cytokines. Besides their crucial function of initiating thrombosis and maintaining hemostasis, platelets are also revealed as key mediators of inflammation and immune cell activation [1,2].

While the clear importance of the platelets in various conditions and diseases has been emerging, diverse proteins to be expressed or released from platelets have been identified through proteomics. Although mature platelets are anucleated, they were discovered to express a large number of transcription factors including PPAR which is involved in the regulation of metabolism and inflammation and regulated by activation or treatment with PPAR ligands [3]. PPARγ ligands have been suggested to influence platelet production and function.

There are researches aimed to reveal the functions of the PPAR and ligand of PPAR influencing the hematopoietic system. The interesting thing is that the reports mentioned it is unclear if the PPARγ itself modulates the differentiation of megakaryocytes, whereas the PPARγ ligand enhances platelet production from megakaryocytes [12,14]. In our study, 15d-PGJ₂ induced morphological differentiation in DAMI and Meg-01 cells, reflective of megakaryocyte maturation. Increased levels of CD41, CD42b, CD62p, and PAC-1 by 15d-PGJ₂ in DAMI and Meg-01 cells also indicate differentiation of megakaryocytes into mature cells and platelet release. These results suggest 15d-PGJ₂ could lead to enhanced platelet production. However, we couldn’t conclude it is dependent on the PPARγ because of the effects of GW9662, the PPARγ antagonist, on megakaryocytes. Although it induced a few changes on the morphology and flow cytometry analysis, the lower N (nucleus)/C (cytoplasm) ratio of megakaryocytes and significantly increased the level of CD41 indicate GW9662 could influence the differentiation of megakaryocytes slightly. So, the effects of 15d-PGJ₂ on megakaryocytes are clear, but the probability of intermediation of PPARγ is somewhat lower.

15d-PGJ₂ significantly decreased COX-2 expression of megakaryocytes. A study suggested 15d-PGJ₂ use both receptor-dependent and -independent mechanisms to influence COX2 gene expression [20]. In the study, it was observed that COX2 expression was significantly abrogated by 15d-PGJ₂. But they made an experiment on the influence of 15d-PGJ₂ in Human WISH, the cells originated from amniotic sac, and amnion cells, further studies using megakaryocytes would be helpful to understand the precise mechanisms.

In our study, fucoidan had no significant effect on megakaryocytes in morphology, flow cytometry analysis, and Western blotting. But in the microarray analysis, the five genes were significantly upregulated in fucoidan-treated DAMI cells. The protein encoded by EGR1 is a nuclear protein and functions as a transcriptional regulator. FCGR, the receptors for the Fc region of IgG, is expressed by leukocytes and platelets. YME1L1 encodes the human ortholog of yeast mitochondrial AAA metalloprotease Yme1p, which has been proposed that the gene plays a role in mitochondrial protein metabolism. STC1 is a homodimeric glycoprotein that may play a role in the regulation of calcium and phosphate transport and cell metabolism. STC1 gene expression has altered in tumors. CYP11B2 encodes a member of the cytochrome P450 superfamily of enzymes, which synthesize aldosterone.

EGR1 has been reported to contribute to suppression of PPARγ expression [21]. To know the more accurate mechanism related to the PPARγ, the microarray analysis using ago- and antagonists of PPARγ along with fucoidan could be helpful. EGR1 has also been reported to regulate the other gene in megakaryocytes [22]. So, EGR1 could influence megakaryocytes with other substances as well as PPARγ.

It has been reported that the expression of STC1 was strongly upregulated during megakaryocytic differentiation and accumulation of STC1 protein in normal megakaryocytes and platelet was observed [23].

CYP11B2 has been reported to be suppressed by PPARγ [24]. We suggest there is a negative correlation between fucoidan and PPARγ, but as we said earlier, the effect of PPARγ on megakaryocytes is not explained obviously, this correlation should be further studied. Functions of FCGR1 and YME1L1 in platelets are studied well, but their roles in megakaryocytes are not fully understood.

We couldn’t have meaningful results with platelets. There are several studies reporting PPARγ ligand to have an inhibitory effect on platelet activation. And fucoidan has been reported possessing anticoagulant and antithrombotic actions. In our study, the ligand and antagonist of PPARγ showed no significant effects in mature platelets. Fucoidan significantly elevated the level of CD62p and CD63 and decreased PPARα expression. With or without GW6471 or celecoxib, fucoidan increased COX-2 expression than control. In PTF-assay with T-TAS, GW6471 appeared to enhance thrombogenicity, whereas fucoidan appeared to suppress thrombogenicity. We guess the platelets isolated from discarded plateletpheresis products could have some limitations such as partial activation of platelets influencing the results.

Thrombocytopenia is a critical problem causing significant morbidity and mortality, and platelet transfusion is the most commonly used therapy but has limitations of alloimmunization, availability, and expense. In this study, we found that 15d-PGJ₂ and fucoidan influence the development of megakaryocytes through regulation of PPARγ and genes including EGR1 and STC1. This finding suggests the possibility of development of more safe and convenient method using the small molecules to treat thrombocytopenic patients.

The PPARγ ligand could enhance megakaryocytic maturation independent on PPARγ, so studies for another mechanism of PPARγ ligand influencing megakaryocytes are needed. And the relationship between megakaryocytic differentiation and decrease of COX-2 expression by PPARγ ligand should be investigated further. Also, the hidden abilities of genes upregulated by fucoidan should be studied as well. In summary, these findings support PPAR ligand, and brown seaweed based compound plays a role in megakaryocytic differentiation even in genomic level.