Platelet-derived lysophosphatidic acid mediated LPAR1 activation as a therapeutic target for osteosarcoma metastasis

Osteosarcoma is the most common primary malignant bone cancer, with high rates of pulmonary metastasis. Osteosarcoma patients with pulmonary metastasis have worse prognosis than those with localized disease, leading to dramatically reduced survival rates. Therefore, understanding the biological characteristics of metastatic osteosarcoma and the molecular mechanisms of invasion and metastasis of osteosarcoma cells will lead to the development of innovative therapeutic intervention for advanced osteosarcoma. Here, we identified that osteosarcoma cells commonly exhibit high platelet activation-inducing characteristics, and molecules released from activated platelets promote the invasiveness of osteosarcoma cells. Given that heat-denatured platelet releasate maintained the ability to promote osteosarcoma invasion, we focused on heat-tolerant molecules, such as lipid mediators in the platelet releasate. Osteosarcoma-induced platelet activation leads to abundant lysophosphatidic acid (LPA) release. Exposure to LPA or platelet releasate induced morphological changes and increased invasiveness of osteosarcoma cells. By analyzing publicly available transcriptome datasets and our in-house osteosarcoma patient-derived xenograft tumors, we found that LPA receptor 1 (LPAR1) is notably upregulated in osteosarcoma. LPAR1 gene KO in osteosarcoma cells abolished the platelet-mediated osteosarcoma invasion in vitro and the formation of early pulmonary metastatic foci in experimental pulmonary metastasis models. Of note, the pharmacological inhibition of LPAR1 by the orally available LPAR1 antagonist, ONO-7300243, prevented pulmonary metastasis of osteosarcoma in the mouse models. These results indicate that the LPA–LPAR1 axis is essential for the osteosarcoma invasion and metastasis, and targeting LPAR1 would be a promising therapeutic intervention for advanced osteosarcoma.

Allogeneic Platelet-Rich Plasma: Is It Safe and Effective for Wound Repair?

Background: Platelet-rich plasma (PRP) and its derivatives are an emerging biotechnology whereby concentrated platelets provide damaged tissue with growth factors, cytokines, and other mediators to improve healing outcomes. Although there is strong evidence in the benefits of autologous PRP for both acute and chronic wounds, allogeneic PRP has been studied far less in comparison. Summary: In this mini-review, we discuss critical steps of allogenic PRP (and its derivatives) preparation. We performed a non-systematic review of the literature to identify animal and human subject studies testing allogenic PRP for wound treatment. We searched OVID Medline and PubMed for articles using the keywords “wound, ulcer, lesion, skin, and cutaneous” and “PRP, or platelet-rich plasma, or platelet-rich fibrin, or PRF, or platelet releasate” and “homologous, allogeneic or allogenic,” which were limited to non-review articles and English language. Two studies in animal models and 8 studies in patients were reviewed. There were inconsistencies in preparation methods, treatment regimens, and some lacked a control group in their studies. Despite these variations, none of the studies identified any major side effects or adverse events. The treatment resulted in a reduced time to heal and/or reduced wound size in most cases. Key Messages: In situations where autologous PRP is not available or suitable, allogeneic PRP appears to provide a safe alternative. Its efficacy, however, requires larger-scale studies with appropriate controls. Standardization in PRP preparation and treatment regime are also needed to be able to interpret allogenic PRP efficacy.

Platelet Transforming Growth Factor-β1 Induces Liver Sinusoidal Endothelial Cells to Secrete Interleukin-6

The roles and interactions of platelets and liver sinusoidal endothelial cells in liver regeneration are unclear, and the trigger that initiates hepatocyte proliferation is unknown. We aimed to identify the key factors released by activated platelets that induce liver sinusoidal endothelial cells to produce interleukin-6 (IL-6), a cytokine implicated in the early phase of liver regeneration. We characterized the releasate of activated platelets inducing the in vitro production of IL-6 by mouse liver sinusoidal endothelial cells and observed that the stimulating factor was a thermolabile protein. Following gel filtration, a single fraction of activated platelet releasate induced a maximal IL-6 secretion by liver sinusoidal endothelial cells (90.2 ± 13.9 versus control with buffer, 9.0 ± 0.8 pg/mL, p < 0.05). Mass spectroscopy analysis of this fraction, followed by in silico processing, resulted in a reduced list of 18 candidates. Several proteins from the list were tested, and only recombinant transforming growth factor β1 (TGF-β1) resulted in an increased IL-6 production up to 242.7 ± 30.5 pg/mL, which was comparable to non-fractionated platelet releasate effect. Using neutralizing anti-TGF-β1 antibody or a TGF-β1 receptor inhibitor, IL-6 production by liver sinusoidal endothelial cells was dramatically reduced. These results support a role of platelet TGF-β1 β1 in the priming phase of liver regeneration.

Thrombin contributes to cancer immune evasion via proteolysis of platelet-bound GARP to activate LTGF-β

Cancer-associated thrombocytosis and high concentrations of circulating transforming growth factor–β1 (TGF-β1) are frequently observed in patients with progressive cancers. Using genetic and pharmacological approaches, we show a direct link between thrombin catalytic activity and release of mature TGF-β1 from platelets. We found that thrombin cleaves glycoprotein A repetitions predominant (GARP), a cell surface docking receptor for latent TGF-β1 (LTGF-β1) on platelets, resulting in liberation of active TGF-β1 from the GARP–LTGF-β1 complex. Furthermore, systemic inhibition of thrombin obliterates TGF-β1 maturation in platelet releasate and rewires the tumor microenvironment toward favorable antitumor immunity, which translates into efficient cancer control either alone or in combination with programmed cell death 1–based immune checkpoint blockade therapy. Last, we demonstrate that soluble GARP and GARP–LTGF-β1 complex are present in the circulation of patients with cancer. Together, our data reveal a mechanism of cancer immune evasion that involves thrombin-mediated GARP cleavage and the subsequent TGF-β1 release from platelets. We propose that blockade of GARP cleavage is a valuable therapeutic strategy to overcome cancer’s resistance to immunotherapy.

P2589Platelets serve as carriers of cardioprotective factors after remote ischemic conditioning in humans

Background/Introduction Brief episodes of ischemia/reperfusion (I/R) in a tissue or organ remote from the heart reduce myocardial infarct size after sustained severe myocardial I/R in all species tested so far, including humans. Remote ischemic conditioning can be induced before (pre-), during (per-) or following (post-) myocardial ischemia. Signal transfer from the remote tissue/organ to the heart is both, neuronal and humoral. Humoral signal transfer has been evidenced by the transfer of cardioprotection via plasma or plasma derivatives from one individual to another individual's heart, even across species. Circulating blood cells have been considered as targets for cardioprotection, but so far not as carriers of cardioprotective signals. Purpose To investigate the role of platelets as potential carriers of cardioprotection by remote ischemic preconditioning (RIPC). Methods Peripheral venous blood samples were collected from healthy volunteers (5 male/5 female, mean age 26±5 years) before and 60 min after RIPC (3×5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/5 min deflation) or placebo (PLA) protocol (blood pressure cuff uninflated). RIPC and PLA protocols, respectively, were performed in randomized sequence at an interval of one week. Blood (80 mL) was drawn into tubes containing sodium citrate, apyrase and prostaglandin E1. Blood cells were counted using a hematology analyzer. Blood was centrifuged (100 g, 15 min, at room temperature) to obtain platelet-rich plasma (PRP). PRP was washed twice with buffer (pH 6.5), and the pellet was re-suspended in suspension buffer (pH 7.35); the platelet amount was adjusted to 2.5x103 platelets/μL. The platelet suspension was supplemented with 1 mol/L CaCl2 and centrifuged (14,000 g) at 4°C for degranulation. The supernatant, i.e. the platelet releasate, was retrieved. Male Lewis rats were sacrificed, their hearts isolated and perfused at constant pressure. Diluted platelet releasate (1:10) was infused into the isolated perfused rat hearts for 8 min followed by 2 min washout before 30/120 min global I/R. Infarct size (percentage of ventricular mass) was demarcated with staining by triphenyltetrazolium chloride. Data are presented as mean±SD. Results The platelet count was increased after RIPC, but unchanged after PLA (RIPC: from 204±19x103 platelets/μL to 247±16x103 platelets/μL; PLA: from 230±16x103 cells/μL to 222±18x103 platelets/μL; PLA vs. RIPC p<0.01, RIPC before vs. after p<0.01, two-way ANOVA for repeated measures with Fisher's least significant differences post-hoc tests), whereas all other blood cell counts remained unchanged. Infarct size was less with infusion of platelet releasate after RIPC in comparison to platelet releasate before RIPC and to platelet releasate before and after PLA, respectively (see Figure). Conclusion In response to RIPC in healthy volunteers, platelets carry soluble cardioprotective factor(s). Their precise nature must still be identified.

Current Insights into the Potential Misuse of Platelet-based Applications for Doping in Sports

Platelet-based applications are currently used for the delivery of growth factors and other biomolecules as autologous biomaterials in regenerative medicine and cosmetic therapies. Many studies have revealed that platelet-based applications such as platelet-rich plasma and platelet releasate exhibit beneficial biological effects after a sports injury or trauma when administered locally by intramuscular injections. At present, treatment of the public, patients and athletes with platelet-based applications is permitted and regulated by the Food and Drug Administration and the World Anti-Doping Agency. Since 2011 the use of autologous platelet-rich plasma is permitted in competitive sports by the World Anti-Doping Agency, due to the lack of evidence in performance enhancement and anabolic effects. However, accumulating research has recently shed light on the role of platelet-derived growth factors in wound healing, skeletal myogenesis, muscle stem cell function and tissue regeneration. Although any ergogenic potential of platelet-rich plasma and platelet releasate on intact skeletal muscle and human sports performance remain to be established, novel evidence suggests that platelet-derived growth factors can modulate muscle, tendon, ligament, protein synthesis/degradation, vascularization, energy utilization and regenerative capacity in various experimental settings. Since platelet-based applications are currently not prohibited, they constitute a tool for potential abuse and doping in sports. The aim of this review is to critically discuss and assimilate current insights and biological evidence that set the ground for exploitation and misuse in competitive sports, and develop strategies to combat these activities.