Lessons to learn from tumor-educated platelets.

Platelets have long been known to play important roles beyond hemostasis and thrombosis. Now recognized as a bona fide mediator of malignant disease, platelets influence various aspects of cancer progression, most notably tumor cell metastasis. Interestingly, platelets isolated from cancer patients often display distinct RNA and protein profiles, with no clear alterations in hemostatic activity. This phenotypically distinct population, termed tumor-educated platelets, now receive significant attention for their potential use as a readily available liquid biopsy for early cancer detection. Although the mechanisms underpinning platelet education are still being defined, direct uptake and storage of tumor-derived factors, signal-dependent changes in platelet RNA processing, and differential platelet production by tumor-educated megakaryocytes are the most prominent scenarios. This article aims to cover the various modalities of platelet education by tumors, in addition to assessing their diagnostic potential.

Cardiac megakaryocytes in SARS-CoV-2-positive autopsies.

Thromboembolic phenomena are an important complication of infection by severe acute respiratory coronavirus 2 (SARS-CoV-2). Increasing focus on the management of the thrombotic complications of Coronavirus Disease 2019 (COVID-19) has led to further investigation into the role of platelets, and their precursor cell, the megakaryocyte, during the disease course. Previously published postmortem evaluations of patients who succumbed to COVID-19 have reported the presence of megakaryocytes in the cardiac microvasculature. Our series evaluated a cohort of autopsies performed on SARS-CoV-2-positive patients in 2020 (n = 36) and prepandemic autopsies performed in early 2020 (n = 12) and selected to represent comorbidities common in cases of severe COVID-19, in addition to infectious and noninfectious pulmonary disease and thromboembolic phenomena. Cases were assessed for the presence of cardiac megakaryocytes and correlated with the presence of pulmonary emboli and laboratory platelet parameters and inflammatory markers. Cardiac megakaryocytes were detected in 64% (23/36) of COVID-19 autopsies, and 40% (5/12) prepandemic autopsies, with averages of 1.77 and 0.84 megakaryocytes per cm2 , respectively. Within the COVID-19 cohort, autopsies with detected megakaryocytes had significantly higher platelet counts compared with cases throughout; other platelet parameters were not statistically significant between groups. Although studies have supported a role of platelets and megakaryocytes in the response to viral infections, including SARS-CoV-2, our findings suggest cardiac megakaryocytes may be representative of a nonspecific inflammatory response and are frequent in, but not exclusive to, COVID-19 autopsies.

NEDD9 Is a Novel and Modifiable Mediator of Platelet-Endothelial Adhesion in the Pulmonary Circulation.

Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications. Objectives: To identify a hypoxia-sensitive, modifiable mediator of platelet-pulmonary artery endothelial cell adhesion and thrombotic remodeling. Methods: Network medicine was used to profile protein-protein interactions in hypoxia-treated human pulmonary artery endothelial cells. Data from liquid chromatography-mass spectrometry and microscale thermophoresis informed the development of a novel antibody (Ab) to inhibit platelet-endothelial adhesion, which was tested in cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH) and three animal models in vivo. Measurements and Main Results: The protein NEDD9 was identified in the hypoxia thrombosome network in silico. Compared with normoxia, hypoxia (0.2% O2) for 24 hours increased HIF-1α (hypoxia-inducible factor-1α)-dependent NEDD9 upregulation in vitro. Increased NEDD9 was localized to the plasma-membrane surface of cells from control donors and patients with CTEPH. In endarterectomy specimens, NEDD9 colocalized with the platelet surface adhesion molecule P-selectin. Our custom-made anti-NEDD9 Ab targeted the NEDD9-P-selectin interaction and inhibited the adhesion of activated platelets to pulmonary artery endothelial cells from control donors in vitro and from patients with CTEPH ex vivo. Compared with control mice, platelet-pulmonary endothelial aggregates and pulmonary hypertension induced by ADP were decreased in NEDD9-/- mice or wild-type mice treated with the anti-NEDD9 Ab, which also decreased chronic pulmonary thromboembolic remodeling in vivo. Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.

Approach to the Patient with COVID-19-Associated Thrombosis: A Case-Based Review.

Coronavirus disease 2019 (COVID-19) is a current global pandemic caused by the novel coronavirus SARS-CoV-2. Alongside its potential to cause severe respiratory illness, studies have reported a distinct COVID-19-associated coagulopathy that is characterized by elevated D-dimer levels, hyperfibrinogenemia, mild thrombocytopenia, and slight prolongation of the prothrombin time. Studies have also reported increased rates of thromboembolism in patients with COVID-19, but variations in study methodologies, patient populations, and anticoagulation strategies make it challenging to distill implications for clinical practice. Here, we present a practical review of current literature and uses a case-based format to discuss the diagnostic approach and management of COVID-19-associated coagulopathy. IMPLICATIONS FOR PRACTICE: Coronavirus disease 2019 (COVID-19)-associated coagulopathy is characterized by elevated D-dimer levels, hyperfibrinogenemia, and increased rates of thromboembolism. Current management guidelines are based on limited evidence from retrospective studies that should be interpreted carefully. At this time, all hospitalized patients with suspected or confirmed COVID-19 should receive coagulation test surveillance and standard doses of prophylactic anticoagulation until prospective randomized controlled trials yield definitive information in support of higher prophylactic doses.

CCL5 derived from platelets increases megakaryocyte proplatelet formation.

In times of physiological stress, platelet count can transiently rise. What initiates this reactive thrombocytosis is poorly understood. Intriguingly, we found that treating megakaryocytes (MKs) with the releasate from activated platelets increased proplatelet production by 47%. Platelets store inflammatory cytokines, including the chemokine ligand 5 (CCL5, RANTES); after TRAP activation, platelets release over 25 ng/mL CCL5. We hypothesized that CCL5 could regulate platelet production by binding to its receptor, CCR5, on MKs. Maraviroc (CCR5 antagonist) or CCL5 immunodepletion diminished 95% and 70% of the effect of platelet releasate, respectively, suggesting CCL5 derived from platelets is sufficient to drive increased platelet production through MK CCR5. MKs cultured with recombinant CCL5 increased proplatelet production by 50% and had significantly higher ploidy. Pretreating the MK cultures with maraviroc prior to exposure to CCL5 reversed the augmented proplatelet formation and ploidy, suggesting that CCL5 increases MK ploidy and proplatelet formation in a CCR5-dependent manner. Interrogation of the Akt signaling pathway suggested that CCL5/CCR5 may influence proplatelet production by suppressing apoptosis. In an in vivo murine acute colitis model, platelet count significantly correlated with inflammation whereas maraviroc treatment abolished this correlation. We propose that CCL5 signaling through CCR5 may increase platelet counts during physiological stress.

Tamoxifen Directly Inhibits Platelet Angiogenic Potential and Platelet-Mediated Metastasis.

OBJECTIVE: Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in metastasis. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment of breast cancer. Tamoxifen and its metabolites have been shown to directly impact platelet function, suggesting that this drug has additional mechanisms of action. The purpose of this study was to determine whether tamoxifen exerts antitumor effects through direct platelet inhibition. APPROACH AND RESULTS: This study found that pretreatment with tamoxifen leads to a significant inhibition of platelet activation. Platelets exposed to tamoxifen released significantly lower amounts of proangiogenic regulator vascular endothelial growth factor. In vitro angiogenesis assays confirmed that tamoxifen pretreatment led to diminished capillary tube formation and decreased endothelial migration. Tamoxifen and its metabolite, 4-hydroxytamoxifen, also significantly inhibited the ability of platelets to promote metastasis in vitro. Using a membrane-based array, we identified several proteins associated with angiogenesis metastasis that were lower in activated releasate from tamoxifen-treated platelets, including angiogenin, chemokine (C-X-C motif) ligand 1, chemokine (C-C motif) ligand 5, epidermal growth factor, chemokine (C-X-C motif) ligand 5, platelet-derived growth factor dimeric isoform BB, whereas antiangiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished vascular endothelial growth factor release, and lower angiogenic and metastatic potential. CONCLUSIONS: We demonstrate that tamoxifen and its metabolite 4-hydroxytamoxifen directly alter platelet function leading to decreased angiogenic and metastatic potential. Furthermore, this study supports the idea of utilizing targeted platelet therapies to inhibit the platelet's role in angiogenesis and malignancy.

Lupus anticoagulant testing using two parallel methods detects additional cases and predicts persistent positivity.

BACKGROUND: Antiphospholipid antibody syndrome (APS) is characterized by laboratory evidence of antiphospholipid antibodies (aPL) [e.g. lupus anticoagulant (LA), anticardiolipin (ACL), and/or antiß2-glycoprotein I (aB2GPI)] in a clinical setting of thrombosis or pregnancy morbidity. The International Society on Thrombosis and Haemostasis recommends two different testing modalities to detect LA. To evaluate these recommendations in a clinical setting, our hospital, a tertiary care center with a specialized coagulation laboratory, added the dilute Russell's viper venom time to be performed in parallel with the PTT-lupus anticoagulant to detect LA. METHODS: Results of aPL testing were collected on all patients who had LA testing for one year. Chart review was performed to correlate LA results with ACL, aB2GPI, and clinical history. RESULTS: Patients who were initially LA positive by both PTT-lupus anticoagulant and dilute Russell's viper venom time were more likely to be persistently positive. Patients who were positive for ACL and aB2GPI were likely to be positive by both LA methodologies. No single method was absolutely sensitive, as cases of APS were detected by PTTLA only, DRVVT only, and both methods. CONCLUSIONS: The addition of a second testing method for LA provides additional diagnostic information and may be helpful in stratifying risk of thrombosis.

Anticoagulation inhibits tumor cell-mediated release of platelet angiogenic proteins and diminishes platelet angiogenic response.

Platelets are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. Because of the propensity for clotting, patients with malignancy are often anticoagulated with heparin products, which paradoxically offer a survival benefit by an unknown mechanism. We hypothesized that antithrombotic agents alter the release of angiogenesis regulatory proteins from platelets. Our data revealed that platelets exposed to heparins released significantly decreased vascular endothelial growth factor (VEGF) in response to adenosine 5'-diphosphate or tumor cells (MCF-7 cells) and exhibited a decreased angiogenic potential. The releasate from these platelets contained decreased proangiogenic proteins. The novel anticoagulant fondaparinux (Xa inhibitor) demonstrated a similar impact on the platelet angiogenic potential. Because these anticoagulants decrease thrombin generation, we hypothesized that they disrupt signaling through the platelet protease-activated receptor 1 (PAR1) receptor. Addition of PAR1 antagonists to platelets decreased VEGF release and angiogenic potential. Exposure to a PAR1 agonist in the presence of anticoagulants rescued the angiogenic potential. In vivo studies demonstrated that platelets from anticoagulated patients had decreased VEGF release and angiogenic potential. Our data suggest that the mechanism by which antithrombotic agents increase survival and decrease metastasis in cancer patients is through attenuation of platelet angiogenic potential.

Release of angiogenesis regulatory proteins from platelet alpha granules: modulation of physiologic and pathologic angiogenesis.

An association between platelets, angiogenesis, and cancer has long been recognized, but the mechanisms linking them remains unclear. Platelets regulate new blood vessel growth through numerous stimulators and inhibitors of angiogenesis by several pathways, including differential exocytosis of angiogenesis regulators. Herein, we investigated the differential release of angiogenesis stimulators and inhibitors from platelets. Activation of human platelets with adenosine diphosphate (ADP) stimulated the release of VEGF, but not endostatin whereas, thromboxane A(2) (TXA(2)) released endostatin but not VEGF. Platelet releasates generated by activation with ADP promoted migration and formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in in vitro angiogenesis models. Conversely, TXA(2)-stimulated platelet releasate inhibited migration and formation of capillary structures. Because tumor growth beyond 1-2 mm(3) is angiogenesis-dependent, we hypothesized that cancer cells preferentially stimulate platelets to secrete their pro-angiogenic payload. In support of this, the breast cancer cell line MCF-7 stimulated secretion of VEGF and a pro-angiogenic releasate from platelets. Furthermore, the antiplatelet agent aspirin inhibited platelet-mediated angiogenesis after exposure to ADP or MCF-7 cells providing a potential mechanism for how aspirin may impact malignancy. Manipulation of differentially mediated release of angiogenic factors from platelets may provide a new modality for cancer treatment.

Platelets and (Lymph)angiogenesis.

The formation of new blood and lymphatic vessels is essential for both the development of multicellular organisms and (patho)physiological processes like wound repair and tumor growth. In the 1990s, circulating blood platelets were first postulated to regulate tumor angiogenesis by interacting with the endothelium and releasing angiogenic regulators from specialized α granules. Since then, many studies have validated the contributions of platelets to tumor angiogenesis, while uncovering novel roles for platelets in other angiogenic processes like wound resolution and retinal vascular disease. Although the majority of (lymph)angiogenesis occurs during development, platelets appear necessary for lymphatic but not vascular growth, implying their particular importance in pathological cases of adult angiogenesis. Future work is required to determine whether drugs targeting platelet production or function offer a clinically relevant tool to limit detrimental angiogenesis.

Rapid Onset Severe Immune Thrombocytopenia following mRNA COVID-19 Vaccine in a Young Patient.

The coronavirus disease 2019 (COVID-19) pandemic has affected millions of people around the world. Vaccination against COVID-19 has been approved for the following three vaccines in the United States: Pfizer-BioNTech, Moderna, and Janssen. Hematological complications of vaccination have been reported in the literature but remain as a rare phenomenon. We present the case of a patient who developed severe thrombocytopenia within twenty-four hours following the Pfizer-BioNTech vaccination. Commonly encountered differentials including heparin-induced thrombocytopenia and common viral etiologies were ruled out, and other causes such as drug reactions deemed unlikely as the etiology of this presentation after a broad workup. Nucleocapsid antibodies against COVID-19 were found to be positive which indicated that vaccination was at least the second encounter with this virus for our patient, which has been reported previously as the cause of immune thrombocytopenia (ITP), and this might be the culprit for sudden onset. He responded to the first-line ITP treatment with corticosteroids and intravenous immunoglobulin (IVIG) as evidenced by the fast recovery of platelet count and lack of recurrence of thrombocytopenia.

One-year retrospective analysis of anti-FXa apixaban and rivaroxaban levels demonstrates utility for management decisions in various urgent and nonurgent clinical situations.

OBJECTIVES: Quantification of direct oral anticoagulant (DOAC) plasma levels can guide clinical management, but insight into clinical scenarios surrounding DOAC-calibrated anti-FXa assays is limited. METHODS: Apixaban- and rivaroxaban-calibrated chromogenic anti-Xa assays performed over a 1-year period were retrospectively analyzed. Patient demographics, DOAC history, concomitant medications, and renal/liver comorbidities were obtained. Indications for testing and associated clinical actions were reviewed. Machine learning (ML) models predicting clinical actions were evaluated. RESULTS: In total, 371 anti-FXa apixaban and 89 anti-FXa rivaroxaban tests were performed for 259 and 67 patients in recurring urgent (acute bleeding, unplanned procedures) and nonurgent situations, including several scenarios not captured by existing testing recommendations (eg, drug monitoring, recurrent thromboembolic events, bleeding tendency). In urgent settings, andexanet reversal was guided by radiologic and clinical findings over DOAC levels in 14 of 32 instances, while 51% of apixaban patients qualified for nonreversal strategies through the availability of levels. Levels also informed procedure/intervention timing and supported management decisions when DOAC clearance or DOAC target levels were in question. The importance of clinical context was emphasized by exploratory ML models predicting particular clinical actions. CONCLUSIONS: Although clinical situations are complex, DOAC testing facilitates clinical decision-making, including reversal, justifying more widespread implementation of these assays.

Platelets upregulate tumor cell programmed death ligand 1 in an epidermal growth factor receptor-dependent manner in vitro.

Programmed death ligand 1 (PD-L1) is an immune checkpoint protein that suppresses cytotoxic T lymphocytes and is often overexpressed in cancers. Due to favorable clinical trial results, immune checkpoint inhibition (ICI) is part of Food and Drug Administration approved immuno-oncology therapies; however, not all patients benefit from ICI therapy. High blood platelet-to-lymphocyte ratio has been associated with failure of ICI treatment, but whether platelets have a role in hindering ICI response is unclear. Here, we report that coculturing platelets with cancer cell lines increased protein and gene expression of tumor cell PD-L1, which was reduced by antiplatelet agents, such as aspirin and ticagrelor. Platelet cytokine arrays revealed that the well-established cytokines, including interferon-γ, were not the main regulators of platelet-mediated PD-L1 upregulation. Instead, the high molecular weight epidermal growth factor (EGF) is abundant in platelets, which caused an upregulation of tumor cell PD-L1. Both an EGF-neutralizing antibody and cetuximab (EGF receptor [EGFR] monoclonal antibody) inhibited platelet-induced increases in tumor cell PD-L1, suggesting that platelets induce tumor cell PD-L1 in an EGFR-dependent manner. Our data reveal a novel mechanism for platelets in tumor immune escape and warrant further investigation to determine if targeting platelets improves ICI therapeutic responses.

Pro-inflammatory megakaryocyte gene expression in murine models of breast cancer.

Despite abundant research demonstrating that platelets can promote tumor cell metastasis, whether primary tumors affect platelet-producing megakaryocytes remains understudied. In this study, we used a spontaneous murine model of breast cancer to show that tumor burden reduced megakaryocyte number and size and disrupted polyploidization. Single-cell RNA sequencing demonstrated that megakaryocytes from tumor-bearing mice exhibit a pro-inflammatory phenotype, epitomized by increased Ctsg, Lcn2, S100a8, and S100a9 transcripts. Protein S100A8/A9 and lipocalin-2 levels were also increased in platelets, suggesting that tumor-induced alterations to megakaryocytes are passed on to their platelet progeny, which promoted in vitro tumor cell invasion and tumor cell lung colonization to a greater extent than platelets from wild-type animals. Our study is the first to demonstrate breast cancer-induced alterations in megakaryocytes, leading to qualitative changes in platelet content that may feedback to promote tumor metastasis.