The History of Armand Trousseau and Cancer-Associated Thrombosis.

"Je suis perdu; une phlegmatia qui vient de se déclarer cette nuit, ne me laisse aucun doute sur nature de mon mal [...].

Cancer-Associated Thrombosis: An Overview of Mechanisms, Risk Factors, and Treatment.

Cancer-associated thrombosis is a major cause of mortality in cancer patients, the most common type being venous thromboembolism (VTE). Several risk factors for developing VTE also coexist with cancer patients, such as chemotherapy and immobilisation, contributing to the increased risk cancer patients have of developing VTE compared with non-cancer patients. Cancer cells are capable of activating the coagulation cascade and other prothrombotic properties of host cells, and many anticancer treatments themselves are being described as additional mechanisms for promoting VTE. This review will give an overview of the main thrombotic complications in cancer patients and outline the risk factors for cancer patients developing cancer-associated thrombosis, focusing on VTE as it is the most common complication observed in cancer patients. The multiple mechanisms involved in cancer-associated thrombosis, including the role of anticancer drugs, and a brief outline of the current treatment for cancer-associated thrombosis will also be discussed.

The Role of Platelet-Derived ADP and ATP in Promoting Pancreatic Cancer Cell Survival and Gemcitabine Resistance.

Platelets have been demonstrated to be vital in cancer epithelial-mesenchymal transition (EMT), an important step in metastasis. Markers of EMT are associated with chemotherapy resistance. However, the association between the development of chemoresistance, EMT, and the contribution of platelets to the process, is still unclear. Here we report that platelets regulate the expression of (1) human equilibrative nucleoside transporter 1 (hENT1) and (2) cytidine deaminase (CDD), markers of gemcitabine resistance in pancreatic cancer. Human ENT1 (hENT1) is known to enable cellular uptake of gemcitabine while CDD deactivates gemcitabine. Knockdown experiments demonstrate that Slug, a mesenchymal transcriptional factor known to be upregulated during EMT, regulates the expression of hENT1 and CDD. Furthermore, we demonstrate that platelet-derived ADP and ATP regulate Slug and CDD expression in pancreatic cancer cells. Finally, we demonstrate that pancreatic cancer cells express the purinergic receptor P2Y12, an ADP receptor found mainly on platelets. Thus ticagrelor, a P2Y12 inhibitor, was used to examine the potential therapeutic effect of an ADP receptor antagonist on cancer cells. Our data indicate that ticagrelor negated the survival signals initiated in cancer cells by platelet-derived ADP and ATP. In conclusion, our results demonstrate a novel role of platelets in modulating chemoresistance in pancreatic cancer. Moreover, we propose ADP/ATP receptors as additional potential drug targets for treatment of pancreatic cancer.

Targeting Platelets for the Treatment of Cancer.

The majority of cancer-associated mortality results from the ability of tumour cells to metastasise leading to multifunctional organ failure and death. Disseminated tumour cells in the blood circulation are faced with major challenges such as rheological shear stresses and cell-mediated cytotoxicity mediated by natural killer cells. Nevertheless, circulating tumour cells with metastatic ability appear equipped to exploit host cells to aid their survival. Despite the long interest in targeting tumour-associated host cells such as platelets for cancer treatment, the clinical benefit of this strategy is still under question. In this review, we provide a summary of the latest mechanistic and clinical evidence to evaluate the validity of targeting platelets in cancer.

Current state and novel approaches of antiplatelet therapy.

An unresolved problem with clinical use of antiplatelet therapy is that a significant number of individuals either still get thrombosis or run the risk of life-threatening bleeding. Antiplatelet drugs are widely used clinically, either chronically for people at risk of athero/thrombotic disease or to prevent thrombus formation during surgery. However, a subpopulation may be resistant to standard doses, while the platelet targets of these drugs are also critical for the normal hemostatic function of platelets. In this review, we will briefly examine current antiplatelet therapy and existing targets while focusing on new potential approaches for antiplatelet therapy and improved monitoring of effects on platelet reactivity in individuals, ultimately to improve antithrombosis with minimal bleeding. Primary platelet adhesion-signaling receptors, glycoprotein (GP)Ib-IX-V and GPVI, that bind von Willebrand factor/collagen and other prothrombotic factors are not targeted by drugs in clinical use, but they are of particular interest because of their key role in thrombus formation at pathological shear.

The functional role of platelets in the regulation of angiogenesis.

Functionally, platelets are primarily recognized as key regulators of thrombosis and hemostasis. Upon vessel injury, the typically quiescent platelet interacts with subendothelial matrix to regulate platelet adhesion, activation and aggregation, with subsequent induction of the coagulation cascade forming a thrombus. Recently, however, newly described roles for platelets in the regulation of angiogenesis have emerged. Platelets possess an armory of pro- and anti-angiogenic proteins, which are actively sequestered and highly organized in α-granule populations. Platelet activation facilitates their release, eliciting potent angiogenic responses through mechanisms that appear to be tightly regulated. In conjunction, the release of platelet-derived phospholipids and microparticles has also earned merit as synergistic regulators of angiogenesis. Consequently, platelets have been functionally implicated in a range of angiogenesis-dependent processes, including physiological roles in wound healing, vascular development and blood/lymphatic vessel separation, whilst facilitating aberrant angiogenesis in a range of diseases including cancer, atherosclerosis and diabetic retinopathy. Whilst the underlying mechanisms are only starting to be elucidated, significant insights have been established, suggesting that platelets represent a promising therapeutic strategy in diseases requiring angiogenic modulation. Moreover, anti-platelet therapies targeting thrombotic complications also exert protective effects in disorders characterized by persistent angiogenesis.

Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

BACKGROUND: We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI. AIMS: To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway. METHODS AND RESULTS: Human and mouse washed platelets (from WT or Pyk2 KO mice) were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively) and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P) surface expression) and integrin αIIbß3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk), PI3-K and Bruton's tyrosine kinase (Btk) and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbß3 activation. CONCLUSION: Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

The role of Rac1 in glycoprotein Ib-IX-mediated signal transduction and integrin activation.

OBJECTIVE: The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, mediates platelet adhesion at sites of vascular injury and transmits signals leading to platelet activation. von Willebrand factor/GPIb-IX interaction sequentially activates the Src family kinase Lyn (SFK), phosphoinositide 3-kinase (PI3K), and Akt, leading to activation of integrin α(IIb)ß(3) and integrin-dependent stable platelet adhesion and aggregation. It remains unclear how Lyn activates the PI3K/Akt pathway after ligand binding to GPIb-IX. METHODS AND RESULTS: Using platelet-specific Rac1(-/-) mice and the Rac1 inhibitor NSC23766, we examined the role of Rac1 in GPIb-IX-dependent platelet activation. Rac1(-/-) mouse platelets and NSC23766-treated human platelets were defective in GPIb-dependent stable adhesion to von Willebrand factor under shear stress, integrin activation, thromboxane A(2) synthesis, and platelet aggregation. Interestingly, GPIb-induced activation of Rac1 and the guanine nucleotide exchange factor for Rac1, Vav, was abolished in both Lyn(-/-) and SFK inhibitor-treated platelets but was unaffected by the PI3K inhibitor LY294002, indicating that Lyn mediates activation of Vav and Rac1 independently of PI3K. Furthermore, GPIb-induced activation of Akt was abolished in Rac1-deficient platelets, suggesting that Rac1 is upstream of the PI3K/Akt pathway. CONCLUSIONS: A Lyn-Vav-Rac1-PI3K-Akt pathway mediates von Willebrand factor-induced activation of integrin α(IIb)ß(3) to promote GPIb-IX-dependent platelet activation.

An acquired defect associated with abnormal signaling of the platelet collagen receptor glycoprotein VI.

INTRODUCTION: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. AIM: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. METHODS: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. RESULTS: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ∼40-kDa Syk fragment. CONCLUSIONS: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.

Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood.

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.

Thrombotic thrombocytopenic purpura: reducing the risk?

von Willebrand factor (vWF) has a key role in initiating platelet aggregation, and thereby thrombus formation, that is dependent on its ability to form appropriately sized multimers. Ultralarge multimers promote the formation of the microvascular thrombi that are hallmarks of the life-threatening condition thrombotic thrombocytopenic purpura (TTP). In this issue of the JCI, Chen et al. show that the drug N-acetylcysteine (NAC) can decrease the size of vWF multimers in vitro and in vivo, resolving thrombi in mice. These data suggest that NAC could potentially be used to treat thrombotic conditions such as TTP.

Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha.

Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIbα, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIbα sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIbα, GPIbß, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIbß is critical for ADAM17-dependent shedding of GPIbα induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIbα and GPIbß subunits further lessened the restraint of GPIbß on GPIbα shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIbß cytoplasmic domain disrupted calmodulin binding to GPIbß but had little effect on GPIbα shedding. Analysis of induction of GPIbα shedding by membrane-permeable GPIbß-derived peptides implicated the association of GPIbß with an unidentified intracellular protein in mediating regulation of GPIbα shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.

GPIbalpha-selective activation of platelets induces platelet signaling events comparable to GPVI activation events.

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbalpha (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbalpha-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbalpha- and alpha(IIb)beta(3)-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbalpha and anti-alpha(IIb)beta(3) antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbalpha-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbalpha engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbalpha can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.

Nerve growth factor inhibits metalloproteinase-disintegrins and blocks ectodomain shedding of platelet glycoprotein VI.

Nerve growth factor (NGF) plays an important role in regulating mammalian neuronal/embryonic development, angiogenesis, and other physiological processes and has recently been investigated as a potential treatment for the neurodegenerative disorder, Alzheimer disease. In this study, we provide evidence that human NGF may also function as a metalloproteinase inhibitor, based on studies of NGF from snake venom. Originally, our aim was to isolate snake venom metalloproteinases targeting platelet receptors and/or ligands relevant to hemostasis and thrombosis, using Ni(2+)-agarose as a purification step based on the conserved metal ion-coordination motif in venom metalloproteinases. However, subsequent analysis of cobra (Naja kaouthia) venom led to the unexpected discovery that cobra venom NGF bound to Ni(2+)-agarose, eluting at approximately 15 mm imidazole, enabling a one-step purification. The identity of the purified protein was confirmed by mass spectrometry and N-terminal sequence analysis. Partial co-purification of NGF within metalloproteinase-enriched venom fractions led us to test whether NGF affected metalloproteinase activity. Venom NGF potently inhibited metalloproteinases isolated from the same or different venom and specifically bound to purified Nk metalloproteinase immobilized on agarose beads. Human NGF also interacted with human metalloproteinases because it blocked metalloproteinase-mediated shedding of the platelet collagen receptor, glycoprotein (GP)VI, and associated with recombinant ADAM10 by surface plasmon resonance. Together, these results suggest that NGF can function as a metalloproteinase inhibitor.

Measuring soluble platelet glycoprotein VI in human plasma by ELISA.

Recent experimental evidence demonstrates that the platelet-specific collagen receptor, glycoprotein (GP)VI is essentially all uncleaved on normal circulating platelets, but is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligands (including collagen), anti-GPVI antibodies or activation at the platelet Fc receptor, FcgammaRIIa. This raises the question of whether shed ectodomain fragment in plasma could be a useful biomarker of thrombotic risk and/or autoimmune thrombocytopenia. In this study, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring soluble GPVI in human plasma, using rabbit anti-GPVI polyclonal antibody in the solid-phase, murine anti-GPVI monoclonal antibody (1A12) in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimized for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of approximately 1 ng/mL. Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. In ten healthy donors, soluble plasma GPVI levels were 18.9 +/- 4.1 ng/mL. Treating normal platelet-rich plasma with a GPVI ligand (collagen-related peptide, CRP), calmodulin inhibitor W7 (that induces GPVI shedding without platelet activation) or N-ethylmaleimide (that directly activates platelet sheddases), under conditions previously shown to induce GPVI shedding, also increased plasma GPVI levels by up to approximately 7-fold, compared to previously reported autoimmune (anti-GPVI) patient plasma where soluble GPVI was approximately 10-fold higher than normal. Characterization of this sensitive ELISA should facilitate analysis of functional/diagnostic role(s) for soluble GPVI in human plasma associated with thrombotic/immune dysfunction.

Platelet receptor redox regulation.

Several recent findings point to an important role for redox regulation of platelet responses to collagen involving the receptor, glycoprotein (GP)VI. First, the antioxidant dietary compound, quercetin, was shown to inhibit GPVI-dependent platelet activation and signaling responses to collagen. Second, collagen increased platelet production of the oxygen radical, superoxide anion (O2-), mediated by the multi-subunit enzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase. In that case, O2- was implicated in regulating not initial aggregation, but collagen-induced thrombus stabilization involving release of ADP. Third, our laboratory showed that an unpaired thiol in the GPVI cytoplasmic tail undergoes rapid oxidation to form GPVI homodimers following ligand binding, preceding GPVI signaling and ectodomain metalloproteolysis, and indicating formation of an oxidative submembranous environment in activated platelets. This review examines receptor/redox regulation in other cells, and relevance to the pathophysiological function of GPVI and other platelet receptors initiating thrombus formation in haemostasis or thrombotic diseases such as heart attack and stroke.

The 14-3-3zeta-GPIb-IX-V complex as an antiplatelet target.

The functional nexus involving the platelet adhesion receptor, the glycoprotein (GP)Ib-IX-V complex, and the signaling protein, 14-3-3zeta, is emerging as a new drug target for control of GPIb-IX-V-dependent thrombotic diseases such as heart attack and stroke. Together, advances in understanding mechanisms of regulation and functional consequences of the GPIb-IX-V/14-3-3zeta interaction, and the growing potential of 14-3-3-targeting therapeutic approaches used for 14-3-3-dependent processes in other cells--principally involving cell cycling and cancer--point to 14-3-3zeta as a promising antithrombotic target. The unique recognition sequences and arrangement of functional 14-3-3zeta-binding sites found within the cytoplasmic domain of GPIb-IX-V increase the likelihood of selective targeting of this interaction.