Role of Nitric Oxide in Megakaryocyte Function.

Megakaryocytes are the main members of the hematopoietic system responsible for regulating vascular homeostasis through their progeny platelets, which are generally known for maintaining hemostasis. Megakaryocytes are characterized as large polyploid cells that reside in the bone marrow but may also circulate in the vasculature. They are generated directly or through a multi-lineage commitment step from the most primitive progenitor or Hematopoietic Stem Cells (HSCs) in a process called "megakaryopoiesis". Immature megakaryocytes enter a complicated development process defined as "thrombopoiesis" that ultimately results in the release of extended protrusions called proplatelets into bone marrow sinusoidal or lung microvessels. One of the main mediators that play an important modulatory role in hematopoiesis and hemostasis is nitric oxide (NO), a free radical gas produced by three isoforms of nitric oxide synthase within the mammalian cells. In this review, we summarize the effect of NO and its signaling on megakaryopoiesis and thrombopoiesis under both physiological and pathophysiological conditions.

The role of platelets in the tumor microenvironment: From solid tumors to leukemia.

Platelets are increasingly being recognized for promoting tumor growth and metastasis. Many cells derived from solid tumors have the ability to aggregate platelets, and this ability correlates with their metastatic potential. Over the past half century, our understanding of tumor cell-induced platelet aggregation (TCIPA) has grown beyond the simple concept that tumor cell-containing microthrombi mechanically embolize the microvasculature. Tumor cell-activated platelets secrete a multitude of factors that reciprocally act on tumor cells, as well as other cells within the tumor microenvironment; thus, affecting both parenychma and tumor-associated stroma. In this review, we summarize the current knowledge of tumor cell-platelet interactions and their influence on the tumor microenvironment, including how these interactions impact neoplastic epithelial cells, endothelial cells, pericytes, fibroblasts, immune cells, and early metastatic niches. In addition, we review the current knowledge of platelet-cancer cell interactions within hematological malignancies and speculate on how platelets may influence the leukemic microenvironment. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.

CYP1B1 inhibition attenuates doxorubicin-induced cardiotoxicity through a mid-chain HETEs-dependent mechanism.

Doxorubicin (DOX) has been reported to be a very potent and effective anticancer agent. However, clinical treatment with DOX has been greatly limited due to its cardiotoxicity. Furthermore, several studies have suggested a role for cytochrome P450 1B1 (CYP1B1) and mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) in DOX-induced cardiac toxicity. Therefore, we hypothesized that DOX induced cardiotoxicity is mediated through the induction of CYP1B1 and its associated mid-chain HETEs metabolite. To test our hypothesis, Sprague-Dawley rats and RL-14 cells were treated with DOX in the presence and absence of 2,3',4,5'-tetramethoxystilbene (TMS), a selective CYP1B1 inhibitor. Thereafter, cardiotoxicity parameters were determined using echocardiography, histopathology, and gene expression. Further, the level of mid-chain HETEs was quantified using liquid chromatography-electron spray ionization-mass spectrometry. Our results showed that DOX induced cardiotoxicity in vivo and in vitro as evidenced by deleterious changes in echocardiography, histopathology, and hypertrophic markers. Importantly, the TMS significantly reversed these changes. Moreover, the DOX-induced cardiotoxicity was associated with a proportional increase in the formation of cardiac mid-chain HETEs both in vivo and in our cell culture model. Interestingly, the inhibition of cardiotoxicity by TMS was associated with a dramatic decrease in the formation of cardiac mid-chain HETEs suggesting a mid-chain HETEs-dependent mechanism. Mechanistically, the protective effect of TMS against DOX-induced cardiotoxicity was mediated through the inhibition of mitogen activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that the inhibition of CYP1B1 and mid-chain HETE formation attenuate DOX-induced cardiotoxicity.

Occlusive lung arterial lesions in endothelial-targeted, fas-induced apoptosis transgenic mice.

Pulmonary arterial hypertension (PAH) is a lethal disease that is characterized by functional and structural abnormalities involving distal pulmonary arterioles that result in increased pulmonary vascular resistance and ultimately right heart failure. In experimental models of pulmonary hypertension, endothelial cell (EC) apoptosis is a necessary trigger for the development of obliterative lung arteriopathy, inducing the emergence of hyperproliferative and apoptosis-resistant vascular cells. However, it has not been established whether EC apoptosis is sufficient for the induction of complex lung arteriolar lesions. We generated a conditional transgenic system in mice to test the hypothesis that lung endothelial cell apoptosis is sufficient to induce a PAH phenotype. The Fas-induced apoptosis (FIA) construct was expressed under the control of endothelial-specific Tie2 promoter (i.e., EFIA mice), and administration of a small molecule dimerizing agent, AP20187, resulted in modest pulmonary hypertension, which was associated with obliterative vascular lesions localized to distal lung arterioles in a proportion of transgenic mice. These lesions were characterized by proliferating cells, predominantly CD68 macrophages. Although endothelial cell apoptosis was also seen in the kidney, evidence of subsequent arteriopathy was seen only in the lung. This model provides direct evidence that lung endothelial cell apoptosis acts as a trigger to initiate a PAH phenotype and provides initial insight into the potential mechanisms that underlie a lung-specific arterial response to endothelial injury.

Hypoxia results in upregulation and de novo activation of von Willebrand factor expression in lung endothelial cells.

OBJECTIVE: Increased von Willebrand factor (VWF) levels in lungs are associated with diseases such as pulmonary hypertension. The objective of our study was to determine the mechanism of increased VWF levels in conditions, such as hypoxia, which contribute to pulmonary hypertension. APPROACH AND RESULTS: We have previously reported generation of transgenic mice that express LacZ transgene under the regulation of lung- and brain-specific transcriptional regulatory elements of the VWF gene. Hypoxia exposure of these transgenic mice resulted in increased VWF and LacZ mRNA levels as well as redistribution of their expression from primarily larger vessels in the lungs to microvessels. Exposure of cultured lung microvascular endothelial cells to hypoxia demonstrated that VWF upregulation was accompanied by increased platelet binding. Transcription upregulation was mediated through inhibition of the repressor nuclear factor-IB association with the VWF promoter, and increased nuclear translocation of the transcription factor YY1 and association with its cognate binding site on the VWF gene. Knockdown of YY1 expression abolished the hypoxia-induced upregulation and reduced basal level of VWF. CONCLUSIONS: These analyses demonstrate that hypoxia induces a phenotypic shift, accompanied by modulation of nuclear factor-IB and YY1 activities, in microvascular endothelial cells of the lungs to support VWF promoter activation.

Elevated levels of pro-thrombotic eNOS-negative platelets in COVID-19 patients.

BACKGROUND: Platelet-rich microvascular thrombi are common in severe COVID-19. Endogenous nitric oxide (NO)-signaling limits thrombus formation and previously we identified platelet subpopulations with a differential ability to produce NO based on the presence or absence of endothelial nitric oxide synthase (eNOS). eNOS expression is counter-regulated by cytokines, and COVID-19-associated immune/inflammatory responses may affect the transcriptome profile of megakaryocytes and their platelet progeny. OBJECTIVES: We investigated whether the percentage of eNOS-negative to eNOS-positive platelets increases in COVID-19 patients and whether this change may be due to the actions of pro-inflammatory cytokines on megakaryocytes. METHODS: Platelets were isolated from hospitalized COVID-19 patients and COVID-19-negative controls. Platelet eNOS was measured by flow cytometry and plasma inflammatory cytokines by ELISA. Megakaryocytes from eNOS-GFP transgenic mice and the Meg-01 cell line were characterized to identify an appropriate model to study eNOS-based platelet subpopulation formation in response to inflammatory cytokines. RESULTS: COVID-19 patients demonstrated a significant increase in eNOS-negative and a concomitant decrease in eNOS-positive platelets compared to controls, and this change was associated with disease severity as assessed by ICU admission. A higher eNOS-negative to -positive platelet percentage was associated with enhanced platelet activation as measured by surface CD62P. Accordingly, COVID-19 patients demonstrated higher TNF-α, IL-6, and IL-1ß plasma concentrations than controls. Inflammatory cytokines associated with COVID-19 promoted eNOS-negative Meg-01 formation and enhanced subsequent eNOS-negative platelet-like particle formation. CONCLUSIONS: COVID-19 patients have a higher percentage of eNOS-negative to -positive platelets, likely as a result of inflammatory response reducing megakaryocyte eNOS expression, which predisposes to thrombosis.

Pharmacologic protein kinase Cα inhibition uncouples human platelet-stimulated angiogenesis from collagen-induced aggregation.

Platelets promote angiogenesis by releasing angiogenesis-regulating factors from their α-granules upon aggregation. This effect has both physiologic and pathologic significance as it may contribute to carcinogenesis. Platelet α-granule release and aggregation are regulated, in part, via protein kinase C (PKC) α and ß signaling. Our study investigated the effects of PKC inhibition on aggregation, angiogenesis-regulator secretion from α-granules, and platelet-stimulated angiogenesis. We hypothesized that selective PKCα inhibition may preferentially suppress angiogenesis-regulator secretion from α-granules but not aggregation, limiting platelet-stimulated angiogenesis. Human platelets were aggregated in the presence of conventional PKC inhibitors myr-FARKGALRQ and Ro 32-0432 (2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-α]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide). Immunofluorescence microscopy of PKC translocation was used to determine the specificity of PKC-inhibitor targeting. Enzyme-linked immunosorbent assay was used to measure vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) release from platelets. Platelet effects on angiogenesis were tested using a capillary-formation assay. Ro 32-0432, but not the peptide inhibitor myr-FARKGALRQ (myristoylated-pseudosubstrate peptide inhibitor), inhibited aggregation in a concentration-dependent manner, while both Ro 32-0432 and myr-FARKGALRQ preferentially suppressed VEGF over TSP-1 secretion. Suppression of angiogenesis-regulator release occurred at inhibitor concentrations that did not significantly affect aggregation. Immunofluorescence microscopy revealed that PKCα targeting to α-granules is inhibited when angiogenesis-regulator secretion is uncoupled from aggregation. At concentrations that uncoupled α-granule release from aggregation, Ro 32-0432 and myr-FARKGALRQ inhibited platelet-stimulated angiogenesis. Hence, selective PKCα inhibition suppresses angiogenesis-regulator release from platelet α-granules with minimal effects on aggregation. Thus, selective PKCα inhibitors may have pharmacologic significance to regulate platelet-promoted angiogenesis.

Platelet-associated angiogenesis regulating factors: a pharmacological perspective.

Platelets, in addition to maintaining hemostasis, also stimulate angiogenesis by generating and releasing, upon activation, factors that promote the growth of new blood vessels. To date, at least 20 angiogenesis-regulating factors have been identified in platelets, including both promoters and inhibitors. Platelet-derived angiogenesis regulators promote angiogenesis during wound healing, tumor growth, and in response to ischemia. Within platelets, angiogenesis regulators are primarily stored in α-granules, but are also found in the cytosol or derived from membrane lipids. Their release can be inhibited pharmacologically by anti-platelet agents, which consequently suppress platelet-stimulated angiogenesis. Several years ago, our research group discovered that platelets generate the angiogenesis inhibitor angiostatin independent of the activation state of platelets, and that platelet-derived angiostatin serves to limit the angiogenesis-stimulating effects of platelets. In this review, we summarize the current knowledge of platelet-associated angiogenesis regulators, how they impact angiogenesis, and how they are controlled pharmacologically.

Inhibition of platelet aggregation by activation of platelet intermediate conductance Ca2+ -activated potassium channels.

BACKGROUND: Within the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca2+ -activated K+ (IKCa ) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IKCa channels in platelet function and NO generation has not yet been explored. OBJECTIVES: We investigated whether IKCa channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production. METHODS: Platelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin αIIb /ß3 activation, granule release, and procoagulant platelet formation. RESULTS: Platelet IKCa channel activation with SKA-31 inhibited aggregation in a concentration-dependent manner, an effect reversed by the selective IKCa channel blocker TRAM-34. The QCM model along with confocal microscopy demonstrated that SKA-31 inhibited platelet aggregation under flow conditions. Surprisingly, IKCa activation by SKA-31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IKCa activation by SKA-31 also inhibited dense and alpha-granule secretion and integrin αIIb /ß3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response. CONCLUSIONS: Platelet IKCa channel activation inhibits aggregation by reducing calcium-signaling and granule secretion, but not by enhancing platelet NO generation. IKCa channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation.

VEGF masks BNIP3-mediated apoptosis of hypoxic endothelial cells.

Hypoxia results in the apoptotic death of myocytes, neurons, and epithelial cells, through the actions of Bcl-2 and Nineteen kilodalton Interacting Protein-3 (BNIP3). On the contrary, endothelial cells are especially adept at surviving conditions of oxygen deprivation via up-regulation of vascular endothelial growth factor (VEGF) the most potent endothelial survival factor. Both VEGF and BNIP3 expression are transcriptionally regulated by hypoxia inducible factor and may antagonize each other's affects in endothelial cells (ECs). Since factors that promote and inhibit apoptosis may be expressed at the same time in endothelial cells under hypoxic conditions, we decided to investigate whether VEGF and BNIP3 have opposing actions in endothelial cells. Human microvascular endothelial cells were exposed to hypoxic conditions in a Billups-Rothenburg chamber. Under hypoxic conditions BNIP3 expression by endothelial cells increased as measured by real-time PCR and immunoblot. After 48 h of hypoxia, EC apoptosis was assessed by flow cytometry and was lower than in corresponding normoxia serum starved controls. The increase in EC survival under hypoxic conditions corresponded with an increase in the expression of VEGF. Under normoxic conditions adenoviral BNIP3 over-expression promoted apoptosis of ECs; however, recombinant VEGF (100 pg/ml) antagonized the BNIP3 apoptosis promoting affects. SiRNA knockdown of VEGF expression by hypoxic ECs resulted in increased apoptosis with a concomitant increase in BNIP3 expression. SiRNA knockdown of BNIP3 expression by hypoxic ECs reduced the increase in EC apoptosis as a result of VEGF knockdown. We conclude that under hypoxic conditions VEGF counteracts and masks the apoptosis promoting affects of BNIP3.

Platelets stimulate programmed death-ligand 1 expression by cancer cells: Inhibition by anti-platelet drugs.

BACKGROUND: Platelets facilitate hematogenous metastasis in part by promoting cancer cell immunoevasion, although our understanding of platelet function in modulating the adaptive immune system in cancer is limited. A major negative regulator of the adaptive response is the immune checkpoint protein Programmed Death Ligand 1 (PD-L1). OBJECTIVES: As platelets secrete factors that may increase PD-L1 expression, we investigated whether they up-regulate cancer cell PD-L1, thus promoting immunoevasion, and whether common anti-platelet drugs inhibit this process. METHODS: Platelets were isolated from human volunteers. A549 lung, PD-L1 null A549, and 786-O renal cancer cells were incubated with and without platelets, and cancer cell PD-L1 expression was measured by qPCR and flow cytometry. Additionally, platelet-cancer cell incubations were performed in the presence of common anti-platelet drugs, and with growth factor neutralizing antibodies. Following incubation with platelets, A549 were co-cultured with T-cells and interleukin-2 (IL-2) levels were measured by flow cytometry as a marker of T-cell activation. RESULTS: Platelets increased PD-L1 mRNA and surface protein expression by A549 and 786-0 cells. Combined neutralization of VEGF and PDGF prevented the platelet-induced up-regulation of PD-L1 by A549, as did the anti-platelet drug eptifibatide. A549 incubated with platelets demonstrated a reduced ability to activate human T-cells, an effect reversed by eptifibatide. CONCLUSIONS: As platelets promote immunoevasion of the adaptive immune response by increasing cancer cell PD-L1 expression and as anti-platelet drugs prevent this immunoevasive response, the investigation of anti-platelet drugs as adjuvant therapy to immune checkpoint inhibitors may be warranted in the treatment of cancer.

Preferential interaction of platelets with prostate cancer cells with stem cell markers.

BACKGROUND: Prostate cancer (PCa) may be initiated by CD133+/CD44+ expressing stem cell-like cells (PCSC), which are also thought to drive metastasis. Platelets also contribute to metastasis via tumor cell-induced platelet aggregation (TCIPA), which in part enhances cancer cell invasion. Moreover, activated platelets secrete stromal derived growth factor-1α (SDF-1α) that can mobilize CSCs via the CXCR4 receptor. However, the potential reciprocal interactions between CSCs and platelets have not been investigated. OBJECTIVE: To characterize the mechanisms behind PCSC-platelet interaction. METHODS: Fluorescence Activated Cell Sorting was utilized to separate DU145 and PC3 PCa cells into CD133+/CD44+, CD133+/CD44-, CD44+/CD133-, and CD133-/CD44- subpopulations and to measure their CXCR4 surface expression. PCa subpopulation TCIPA experiments were performed using aggregometry and immunoblot was used to measure prothrombin. Platelet SDF-1α secretion was measured by ELISA. Modified-Boyden chamber assays were used to assess the role of SDF-1α:CXCR4 pathway in platelet-PCSC interactions. RESULTS: DU145 and PC3 expressing both CD133 and CD44 stem cell markers accounted for only small fractions of total cells (DU145: CD133+/CD44+ 3.44 ± 1.45% vs. CD133+/CD44- 1.56 ± 0.45% vs. CD44+/CD133- 68.19 ± 6.25% vs. CD133-/CD44- 20.36 ± 4.51%). However, CD133+ subpopulations induced the greatest amount of aggregation compared to CD44+/CD133- and double-negative DU145, and this aggregation potency of CD133+ PCa cells corresponded with high levels of prothrombin expression. Additionally, CD133+ subpopulations expressed significantly higher level of CXCR4 compared to CD133-/CD44- and CD44+/CD133-. Disruption of SDF-1α:CXCR4 pathway reduced platelet-induced PCSC invasion. CONCLUSIONS: CD133+/CD44+ and CD133+/CD44- PCSCs have highest platelet aggregation potency, which could be attributed to their increased prothrombin expression. Reciprocally, platelet-derived SDF-1α stimulates PCSC invasion.

The role of the androgen receptor in prostate cancer-induced platelet aggregation and platelet-induced invasion.

BACKGROUND: Metastatic prostate cancer progresses from a hormone sensitive androgen receptor expressing phenotype to a hormone insensitive androgen receptor-independent subtype with low overall survival. Human platelets contribute to metastasis via tumor cell-induced platelet aggregation, which in part enhances cancer cell invasion. Given the more aggressive nature of hormone insensitive prostate cancer, we hypothesized that androgen receptor-negative prostate cancer cells exhibit higher platelet aggregation potency and invasive response compared to cells with androgen receptor. OBJECTIVE: To characterize the role of androgen receptors in prostate cancer-induced platelet aggregation and platelet-induced invasion. METHODS: Tumor cell-induced platelet aggregation experiments were performed with platelets from healthy human donors and benign prostate (RWPE-1) and prostate cancer cell lines positive (LNCaP) and negative for androgen receptor (DU145 and PC3). Immunoblot measured prostate cancer prothrombin. Modified Boyden chamber invasion assays and zymography were performed to assess the effects of platelets on prostate cancer cell invasion and matrix metalloproteinase (MMP) expression, respectively. RESULTS: Androgen receptor-positive prostate cancer cell lines failed to induce platelet aggregation. However, androgen receptor-inhibited and -negative cell lines all induced platelet aggregation, which was abolished by dabigatran. Androgen receptor-inhibited and -negative cell lines demonstrated greater expression of prothrombin than androgen receptor-positive cells. Platelets enhanced invasion and MMP-2 and -9 expression by androgen receptor-inhibited and negative prostate cancer cells, but not that of the androgen receptor-positive cells. CONCLUSIONS: Androgen receptor loss within prostate cancer results in increased thrombogenicity due to upregulation of prothrombin expression. Reciprocally, platelets enhance invasion of androgen receptor-negative prostate cancer cells via increased MMP expression.

Tyrosine phosphatase beta regulates angiopoietin-Tie2 signaling in human endothelial cells.

OBJECTIVES: The endothelial cell (EC)-selective receptor tyrosine kinase, Tie2, and its ligands angiopoietin Ang-1 and Ang-2, are essential for blood vessel maintenance and repair. Ang-1 is an agonist of Tie2 receptor activation, whereas Ang-2 is a context-dependent antagonist/agonist. Therefore, we investigated the role of the EC-selective phosphatase, human protein tyrosine phosphatase beta (HPTPbeta), in regulating Tie2 activity. METHODS AND RESULTS: siRNA silencing of HPTPbeta enhanced Ang-1 and Ang-2-induced Tie2 phosphorylation at 10 min (2.5-fold, P < 0.001; and 1.8-fold, P < 0.05, respectively). The cell survival response to Ang-1, but not Ang-2, was enhanced by HPTPbeta silencing as measured by flow cytometry (0.85-fold to 0.66-fold, P < 0.05) and ELISA (0.88-fold to 0.53-fold, P < 0.01). Hypoxia, which upregulated HPTPbeta expression in endothelial cells, impaired Ang-1-induced Tie2 phosphorylation. CONCLUSIONS: These results reveal a novel role for HPTPbeta in modulating Ang-1-Tie2 signaling and endothelial cell survival.

Advances in Platelet Subpopulation Research.

Although lacking a nucleus, platelets are increasingly recognized not only for their complexity, but also for their diversity. Some 50 years ago platelet subpopulations were characterized by size and density, and these characteristics were thought to reflect platelet aging. Since, our knowledge of platelet heterogeneity has grown to recognize that differences in platelet biochemistry and function exist. This includes the identification of vanguard and follower platelets, platelets with differing procoagulant ability including "COAT-platelets" which enhance procoagulant protein retention on their surface, and most recently, the identification of platelet subpopulations with a differential ability to generate and respond to nitric oxide. Hence, in this mini-review, we summarize the current knowledge of platelet subpopulation diversity focusing on their physical, biochemical, and functional heterogeneity. In addition, we review how platelet subpopulations may change between health and disease and how differences among platelets may influence response to anti-platelet therapy. Finally, we look forward and discuss some of the future directions and challenges for this growing field of platelet research.

Potential Antimigraine Effects of Warfarin: An Exploration of Biological Mechanism with Survey of Patients.

Case reports suggest a link between anticoagulant use and improved migraine symptoms, and a role for platelet-induced cerebral vasoconstriction in migraine pathobiology. Hence, we investigated the mechanism by which warfarin may affect migraine symptoms and whether there is a change in migraine symptomology in patients initiating oral anticoagulants, most commonly warfarin. The effects of warfarin on human platelet aggregation and secretion as well as platelet-induced rat cerebral artery vasoconstriction were studied. A survey of migraine and symptom change after starting or stopping oral anticoagulants was also conducted. Warfarin inhibited platelet aggregation and 5-hydroxytryptamine (5-HT) secretion in a concentration-dependent manner. Warfarin-inhibited platelet secretion products constricted middle cerebral arteries from male but not from female rats. For the survey, patient demographic information, migraine and medical history, and Migraine Disability Assessment Score (MIDAS) changes were collected. Out of 175 consenting, 40 respondents met the criteria for migraine and completed the survey. A total of 11 patients reported migraine symptom change, all coinciding with starting warfarin. Of those having symptom and MIDAS improvement, most were female with migraines with aura, whereas those worsening were male with fewer having migraine with aura. Of those reporting migraine symptom change with warfarin, female sex may be associated with improved MIDAS, and those experiencing an aura component are more likely to report a symptom change. Warfarin-mediated symptom improvement in females may occur due to inhibition of platelet 5-HT secretion and a lower sensitivity of female cerebral blood vessels to platelet-derived 5-HT-induced vasoconstriction.

Role of metalloproteinases in platelet function.

Platelets contain and release matrix metalloproteinases (MMPs), their inhibitors (TIMPs) and disintegrin metalloproteinases (ADAMs) including MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP (MMP-14), ADAM-10, ADAM-17, ADAMTS-13, TIMP-1, TIMP-2 and TIMP-4. These proteins exert several effects regulating platelet functions such as agonist-stimulated platelet adhesion and aggregation, tumour cell-induced platelet aggregation and platelet-leukocyte aggregation. In this review, mechanisms of MMPs, TIMPs and ADAMs on platelets are discussed.

STAT3 but Not HIF-1α Is Important in Mediating Hypoxia-Induced Chemoresistance in MDA-MB-231, a Triple Negative Breast Cancer Cell Line.

Hypoxia-induced chemoresistance (HICR) is a well-recognized phenomenon, and in many experimental models, hypoxia inducible factor-1α (HIF-1α) is believed to be a key player. We aimed to better understand the mechanism underlying HICR in a triple negative breast cancer cell line, MDA-MB-231, with a focus on the role of HIF-1α. In this context, the effect of hypoxia on the sensitivity of MDA-MB-231 cells to cisplatin and their stem-like features was evaluated and the role of HIF-1α in both phenomena was assessed. Our results showed that hypoxia significantly increased MDA-MB-231 resistance to cisplatin. Correlating with this, intracellular uptake of cisplatin was significantly reduced under hypoxia. Furthermore, the stem-like features of MDA-MB-231 cells increased as evidenced by the significant increases in the expression of ATP-binding cassette (ABC) drug transporters, the proportion of CD44⁺/CD24- cells, clonogenic survival and cisplatin chemoresistance. Under hypoxia, both the protein level and DNA binding of HIF-1α was dramatically increased. Surprisingly, siRNA knockdown of HIF-1α did not result in an appreciable change to HICR. Instead, signal transducer and activator of transcription 3 (STAT3) activation was found to be important. STAT3 activation may confer HICR by upregulating ABC transporters, particularly ABCC2 and ABCC6. This study has demonstrated that, in MDA-MB-231 cells, STAT3 rather than HIF-1α is important in mediating HICR to cisplatin.

Functional assessment of von Willebrand factor expression by cancer cells of non-endothelial origin.

Von Willebrand factor (VWF) is a highly adhesive procoagulant molecule that mediates platelet adhesion to endothelial and subendothelial surfaces. Normally it is expressed exclusively in endothelial cells (ECs) and megakaryocytes. However, a few studies have reported VWF detection in cancer cells of non-endothelial origin, including osteosarcoma. A role for VWF in cancer metastasis has long been postulated but evidence supporting both pro- and anti-metastatic roles for VWF has been presented. We hypothesized that the role of VWF in cancer metastasis is influenced by its cellular origin and that cancer cell acquisition of VWF expression may contribute to enhanced metastatic potential. We demonstrated de novo expression of VWF in glioma as well as osteosarcoma cells. Endothelial monolayer adhesion, transmigration and extravasation capacities of VWF expressing cancer cells were shown to be enhanced compared to non-VWF expressing cells, and were significantly reduced as a result of VWF knock down. VWF expressing cancer cells were also detected in patient tumor samples of varying histologies. Analyses of the mechanism of transcriptional activation of the VWF in cancer cells demonstrated a pattern of trans-activating factor binding and epigenetic modifications consistent overall with that observed in ECs. These results demonstrate that cancer cells of non-endothelial origin can acquire de novo expression of VWF, which can enhance processes, including endothelial and platelet adhesion and extravasation, that contribute to cancer metastasis.