Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

BACKGROUND: Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. METHODS: Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. RESULTS: Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. CONCLUSIONS: Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Fitusiran reduces bleeding in factor X-deficient mice.

ABSTRACT: Factor X (FX) deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aim to explore the use of fitusiran (an investigational small interfering RNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX deficiency. We therefore developed a novel model of inducible FX deficiency, generating mice expressing <1% FX activity and antigen (f10low mice). Compared with control f10WT mice, f10low mice had sixfold and fourfold prolonged clotting times in prothrombin time and activated partial prothrombin time assays, respectively (P < .001). Thrombin generation was severely reduced, irrespective of whether tissue factor or factor XIa was used as an initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury model. Furthermore, in 2 distinct bleeding models, f10low mice displayed an increased bleeding tendency compared with f10WT mice. In the tail-clip assay, blood loss was increased from 12 ± 16 µL to 590 ± 335 µL (P < .0001). In the saphenous vein puncture (SVP) model, the number of clots generated was reduced from 19 ± 5 clots every 30 minutes for f10WT mice to 2 ± 2 clots every 30 minutes (P < .0001) for f10low mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low mice with fitusiran (2 × 10 mg/kg at 1 week interval) resulted in 17 ± 6% residual antithrombin activity and increased thrombin generation (fourfold and twofold to threefold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP model, the number of clots was increased to 8 ± 6 clots every 30 minutes (P = .0029). Altogether, we demonstrate that reduction in antithrombin levels is associated with improved hemostatic activity under conditions of FX deficiency.

A thrombus is formed by a gradient of platelet activation and procoagulant endothelium.

Background: The contribution of platelets in thrombosis within microcirculation has been extensively documented in the literature. We previously showed, in vivo, that platelet activation revealed by intracellular calcium mobilization was a crucial step in the growth of thrombi following laser-induced injury, a model of thromboinflammation. Objective: Our goal was to investigate the extent of platelet activation and the spatial distribution of platelets throughout a growing thrombus. Methods: We employed a multimodal, correlative microscopy approach and computational biology to study the state of platelets on a growing thrombus obtained after a laser injury. Results: We observed a reversible intracellular platelet calcium mobilization that correlates with the time a platelet resides during thrombus growth. Our bioinformatics analysis displayed the following 3 distinct platelet subpopulations resident within a thrombus: (1) resting, (2) partially activated, and (3) "fully" activated platelets. The spatial distribution of the platelet subpopulations in the thrombus creates a double gradient in both the transversal and longitudinal axis, with the maximal percentage of fully activated platelets close to the site of injury. However, these activated platelets did not express negative phospholipids. The injured endothelium was identified to play a vital role in activating the blood coagulation cascade in this model of thrombosis. Conclusion: Following a laser-induced injury, thrombi are formed by a gradient of activated platelets from the injury site to the periphery of the thrombus. These different activation states of platelets throughout the thrombi regulate the biomechanics of the thrombus. The injured endothelium, rather than platelets, was identified to play a key role in the activation of the blood coagulation cascade in this model of thromboinflammation.

An OMA1 redox site controls mitochondrial homeostasis, sarcoma growth, and immunogenicity.

Aggressive tumors often display mitochondrial dysfunction. Upon oxidative stress, mitochondria undergo fission through OMA1-mediated cleavage of the fusion effector OPA1. In yeast, a redox-sensing switch participates in OMA1 activation. 3D modeling of OMA1 comforted the notion that cysteine 403 might participate in a similar sensor in mammalian cells. Using prime editing, we developed a mouse sarcoma cell line in which OMA1 cysteine 403 was mutated in alanine. Mutant cells showed impaired mitochondrial responses to stress including ATP production, reduced fission, resistance to apoptosis, and enhanced mitochondrial DNA release. This mutation prevented tumor development in immunocompetent, but not nude or cDC1 dendritic cell-deficient, mice. These cells prime CD8+ lymphocytes that accumulate in mutant tumors, whereas their depletion delays tumor control. Thus, OMA1 inactivation increased the development of anti-tumor immunity. Patients with complex genomic soft tissue sarcoma showed variations in the level of OMA1 and OPA1 transcripts. High expression of OPA1 in primary tumors was associated with shorter metastasis-free survival after surgery, and low expression of OPA1, with anti-tumor immune signatures. Targeting OMA1 activity may enhance sarcoma immunogenicity.

Neutrophils, Cancer and Thrombosis: The New Bermuda Triangle in Cancer Research.

Spontaneous venous thrombosis is often the first clinical sign of cancer, and it is linked to a worsened survival rate. Traditionally, tumor-cell induced platelet activation has been the main actor studied in cancer-associated-thrombosis. However, platelet involvement alone does not seem to be sufficient to explain this heightened pro-thrombotic state. Neutrophils are emerging as key players in both thrombus generation and cancer progression. Neutrophils can impact thrombosis through the release of pro-inflammatory cytokines and expression of molecules like P-selectin and Tissue Factor (TF) on their membrane and on neutrophil-derived microvesicles. Their role in cancer progression is evidenced by the fact that patients with high blood-neutrophil counts have a worsened prognosis. Tumors can attract neutrophils to the cancer site via pro-inflammatory cytokine secretions and induce a switch to pro-tumoral (or N2) neutrophils, which support metastatic spread and have an immunosuppressive role. They can also expel their nuclear contents to entrap pathogens forming Neutrophil Extracellular Traps (NETs) and can also capture coagulation factors, enhancing the thrombus formation. These NETs are also known to have pro-tumoral effects by supporting the metastatic process. Here, we strived to do a comprehensive literature review of the role of neutrophils as drivers of both cancer-associated thrombosis (CAT) and cancer progression.

Role of Neutrophils and NETs in Animal Models of Thrombosis.

Thrombosis is one of the major causes of mortality worldwide. Notably, it is not only implicated in cardiovascular diseases, such as myocardial infarction (MI), stroke, and pulmonary embolism (PE), but also in cancers. Understanding the cellular and molecular mechanisms involved in platelet thrombus formation is a major challenge for scientists today. For this purpose, new imaging technologies (such as confocal intravital microscopy, electron microscopy, holotomography, etc.) coupled with animal models of thrombosis (mouse, rat, rabbit, etc.) allow a better overview of this complex physiopathological process. Each of the cellular components is known to participate, including the subendothelial matrix, the endothelium, platelets, circulating cells, and, notably, neutrophils. Initially known as immune cells, neutrophils have been considered to be part of the landscape of thrombosis for more than a decade. They participate in this biological process through their expression of tissue factor (TF) and protein disulfide isomerase (PDI). Moreover, highly activated neutrophils are described as being able to release their DNA and thus form chromatin networks known as "neutrophil extracellular traps" (NETs). Initially, described as "dead sacrifices for a good cause" that prevent the dissemination of bacteria in the body, NETs have also been studied in several human pathologies, such as cardiovascular and respiratory diseases. Many articles suggest that they are involved in platelet thrombus formation and the activation of the coagulation cascade. This review presents the models of thrombosis in which neutrophils and NETs are involved and describes their mechanisms of action. We have even highlighted the medical diagnostic advances related to this research.

Platelet and Cancer-Cell Interactions Modulate Cancer-Associated Thrombosis Risk in Different Cancer Types.

The first cause of death in cancer patients, after tumoral progression itself, is thrombo-embolic disease. This cancer-associated hypercoagulability state is known as Trousseau's syndrome, and the risk for developing thrombotic events differs according to cancer type and stage, as well as within patients. Massive platelet activation by tumor cells is the key mediator of thrombus formation in Trousseau's syndrome. In this literature review, we aimed to compare the interactions between cancer cells and platelets in three different cancer types, with low, medium and high thrombotic risk. We chose oral squamous cell carcinoma for the low-thrombotic-risk, colorectal adenocarcinoma for the medium-thrombotic-risk, and pancreatic carcinoma for the high-thrombotic-risk cancer type. We showcase that understanding these interactions is of the highest importance to find new biomarkers and therapeutic targets for cancer-associated thrombosis.

The P2Y12 Receptor Antagonist Selatogrel Dissolves Preformed Platelet Thrombi In Vivo.

Selatogrel, a potent and reversible antagonist of the P2Y12 receptor, inhibited FeCl3-induced thrombosis in rats. Here, we report the anti-thrombotic effect of selatogrel after subcutaneous applications in guinea pigs and mice. Selatogrel inhibited platelet function only 10 min after subcutaneous application in mice. In addition, in a modified Folts thrombosis model in guinea pigs, selatogrel prevented a decrease in blood-flow, indicative of the inhibition of ongoing thrombosis, approximately 10 min after subcutaneous injection. Selatogrel fully normalised blood flow; therefore, we speculate that it may not only prevent, but also dissolve, platelet thrombi. Thrombus dissolution was investigated using real-time intravital microscopy in mice. The infusion of selatogrel during ongoing platelet thrombus formation stopped growth and induced the dissolution of the preformed platelet thrombus. In addition, platelet-rich thrombi were given 30 min to consolidate in vivo. The infusion of selatogrel dissolved the preformed and consolidated platelet thrombi. Dissolution was limited to the disintegration of the occluding part of the platelet thrombi, leaving small mural platelet aggregates to seal the blood vessel. Therefore, our experiments uncovered a novel advantage of selatogrel: the dissolution of pre-formed thrombi without the disintegration of haemostatic seals, suggesting a bipartite benefit of the early application of selatogrel in patients with acute thrombosis.

P2RY12-Inhibitors Reduce Cancer-Associated Thrombosis and Tumor Growth in Pancreatic Cancers.

Platelet function can be modified by cancer cells to support tumor growth, causing alterations in the delicate hemostatic equilibrium. Cancer-cell and platelet interactions are one of the main pillars of Trousseau's syndrome: a paraneoplastic syndrome with recurring and migrating episodes of thrombophlebitis. Altogether, this leads to a four-fold risk of thrombotic events in cancer patients, which in turn, portend a poor prognosis. We previously demonstrated that anti-P2RY12 drugs inhibit cancer-associated-thrombosis and formation of tumor metastasis in pancreatic cancer models. Here, we aimed to (1) compare the effects of aspirin and clopidogrel on pancreatic cancer prevention, (2) characterize the effects of clopidogrel (platelet P2RY12 inhibitor) on cancer-associated thrombosis and cancer growth in vivo, (3) determine the effect of P2RY12 across different digestive-tract cancers in vitro, and (4) analyze the expression pattern of P2RY12 in two different cancer types affecting the digestive system. Clopidogrel treatment resulted in better survival rates with smaller primary tumors and less metastasis than aspirin treatment. Clopidogrel was also more effective than aspirin at dissolving spontaneous endogenous thrombi in our orthotopic advanced cancer mouse model. P2RY12 expression gives pancreatic adenocarcinomas proliferative advantages. In conclusion, we propose the hypothesis that clopidogrel should be further studied to target and prevent Trousseau's syndrome; as well as diminish cancer growth and spread. However, more studies are required to determine the implicated pathways and effects of these drugs on cancer development.

DNAse-dependent, NET-independent pathway of thrombus formation in vivo.

The contribution of NETs (neutrophil extracellular traps) to thrombus formation has been intensively documented in both arterial and venous thrombosis in mice. We previously demonstrated that adenosine triphosphate (ATP)-activated neutrophils play a key role in initiating the tissue factor-dependent activation of the coagulation cascade, leading to thrombus formation following laser-induced injury. Here, we investigated the contribution of NETs to thrombus formation in a laser-induced injury model. In vivo, treatment of mice with DNase-I significantly inhibited the accumulation of polymorphonuclear neutrophils at the site of injury, neutrophil elastase secretion, and platelet thrombus formation within seconds following injury. Surprisingly, electron microscopy of the thrombus revealed that neutrophils present at the site of laser-induced injury did not form NETs. In vitro, ATP, the main neutrophil agonist present at the site of laser-induced injury, induced the overexpression of PAD4 and CitH3 but not NETosis. However, compared to no treatment, the addition of DNase-I was sufficient to cleave ATP and adenosine diphosphate (ADP) in adenosine. Human and mouse platelet aggregation by ADP and neutrophil activation by ATP were also significantly reduced in the presence of DNase-I. We conclude that following laser-induced injury, neutrophils but not NETs are involved in thrombus formation. Treatment with DNase-I induces the hydrolysis of ATP and ADP, leading to the generation of adenosine and the inhibition of thrombus formation in vivo.

Illustrated State-of-the-Art Capsules of the ISTH 2020 Congress.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person. An added feature of the program is that talks streamed at the designated times will then be available on-line for asynchronous viewing. The program included 77 State of the Art (SOA) talks, thematically grouped in 28 sessions, given by internationally recognized leaders in the field. The SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. The topics, across the main scientific themes of the congress, include Arterial Thromboembolism, Coagulation and Natural Anticoagulants, COVID-19 and Coagulation, Diagnostics and Omics, Fibrinogen, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostasis in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Angiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the congress.

Oral Squamous Cell Carcinoma Is Associated with a Low Thrombosis Risk Due to Storage Pool Deficiency in Platelets.

Venous thrombo-embolism (VTE) disease is the second most common cause of mortality in cancer patients, and evaluation and prevention of thrombosis risk is essential. VTE-associated risk varies according to the type of tumor disease. Oral cancer is the most frequent type of head and neck cancer, and it represents approximately 2.1% of all cancers worldwide. Most tumors are squamous cell carcinomas and are mainly due to tobacco and alcohol abuse. VTE risk associated with oral squamous cell carcinoma (OSCC) is low. However, many studies have shown that OSCC has the following biological features of cancers associated with a high thrombosis risk: modified thrombosis and fibrinolysis mechanisms; strong expression of procoagulant proteins; secretion of procoagulant microparticles; and production of procoagulant cytokines. Using an original mouse model of tongue squamous cell carcinoma, our study aimed to clarify this paradoxical situation. First, we showed that OSCC tumors have a pro-aggregatory phenotype and a high local thrombosis risk. Second, we found that tongue tumor mice do not have an elevated systemic thrombosis risk (the risk of an "at distance" thrombosis event such as lower extremity deep venous thrombosis or pulmonary embolism) and even show a reduction in risk. Third, we demonstrated that tongue tumor mice show a reduction in platelet reactivity, which explains the low systemic thrombosis risk. Finally, we found that tongue tumor mice present granule pool deficiency, thereby explaining the reduction in platelet reactivity and systemic thrombosis risk.

Selatogrel, a reversible P2Y12 receptor antagonist, has reduced off-target interference with haemostatic factors in a mouse thrombosis model.

INTRODUCTION: Selatogrel is a reversible antagonist of the P2Y12 receptor. In rat thrombosis/haemostasis models, selatogrel was associated with lower blood loss than clopidogrel or ticagrelor at equivalent anti-thrombotic effect. MATERIAL AND METHODS: We sought to elucidate the mechanism underlying the observed differences in blood loss, using real-time intravital microscopy in mouse. RESULTS: Selatogrel, ticagrelor and clopidogrel dose-dependently inhibited laser-induced platelet thrombus formation. At maximal antithrombotic effect, only small mural platelets aggregates, corresponding to hemostatic seals, were present. The phenotype of these hemostatic seals was dependent on the type of P2Y12 receptor antagonist. In the presence of clopidogrel and ticagrelor, detachment of platelets from the hemostatic seals was increased, indicative of reduced stability. In contrast, in the presence of selatogrel, platelet detachment was not increased. Moreover, equivalent antithrombotic dosing regimens of ticagrelor and clopidogrel reduced laser-induced calcium mobilization in the endothelium, restricted neutrophil adhesion and subsequent fibrin formation and thus reduced fibrin-mediated stabilization of the hemostatic seals. The effects of ticagrelor were also observed in P2Y12 receptor deficient mice, indicating that the effects are off-target and independent of the P2Y12 receptor. In contrast, selatogrel did not interfere with these elements of haemostasis in wild-type or in P2Y12 receptor deficient mice. CONCLUSION: In the presence of selatogrel the stability of hemostatic seals was unperturbed, translating to an improved blood loss profile. Our data suggest that the mechanism underlying the differences in blood loss profiles of P2Y12 receptor antagonists is by off-target interference with endothelial activation, neutrophil function and thus, fibrin-mediated stabilization of haemostatic seals.

Cancer animal models in thrombosis research.

The cancer-thrombosis relationship has been established for decades, in both cancer biology and in the clinical signs and symptoms seen in cancer patients (thrombosis in cancer patients has been associated with a worse prognosis and survival). As the link between the pathologies becomes clearer, so does the need to develop models that enable researchers to study them simultaneously in vivo. Mouse models have often been used, and they have helped determine molecular pathways between cancer spread and thrombosis in humans. This review is a summary of the current literature that describes the use of cancer mouse models in thrombosis research. We included cancer models that are not yet used in thrombosis research, but that can positively impact this area of research in the near future. We describe the most commonly used techniques to generate thrombosis as well as the mouse strains and cancer cell types that are commonly used along with inoculation techniques. We endeavoured to create a compendium of the different mouse models that are beneficial for cancer-thrombosis research, as understanding these mechanisms is crucial for creating better and more effective treatments for thrombosis in cancer patients.

Intrinsic control of muscle attachment sites matching.

Myogenesis is an evolutionarily conserved process. Little known, however, is how the morphology of each muscle is determined, such that movements relying upon contraction of many muscles are both precise and coordinated. Each Drosophila larval muscle is a single multinucleated fibre whose morphology reflects expression of distinctive identity Transcription Factors (iTFs). By deleting transcription cis-regulatory modules of one iTF, Collier, we generated viable muscle identity mutants, allowing live imaging and locomotion assays. We show that both selection of muscle attachment sites and muscle/muscle matching is intrinsic to muscle identity and requires transcriptional reprogramming of syncytial nuclei. Live-imaging shows that the staggered muscle pattern involves attraction to tendon cells and heterotypic muscle-muscle adhesion. Unbalance leads to formation of branched muscles, and this correlates with locomotor behavior deficit. Thus, engineering Drosophila muscle identity mutants allows to investigate, in vivo, physiological and mechanical properties of abnormal muscles.

Each muscle in the body has a unique size, shape and set of attachment points. Animals need all of their muscles to have the correct identity to help maintain posture and control movement. A specific set of proteins, called transcription factors, co-ordinate and regulate gene activity in cells so that each muscle develops in the right way. To create a muscle, multiple precursor cells fuse together to form a muscle fibre, which then elongates and attaches to specific sites. Correct attachment is critical so that the fibre is properly oriented. When this process goes wrong, for example in disease, muscle fibres sometimes attach to the wrong site; they become branched and cannot work properly. Collier is a transcription factor protein that controls muscle identity in the fruit fly Drosophila melanogaster. However, like many transcription factors, Collier also has several other roles throughout the body. This made it difficult to evaluate the effect of the protein on the formation of specific muscles. Here, Carayon et al. managed to selectively deactivate Collier in just one muscle per body section in the larvae of fruit flies. This showed that the transcription factor is needed throughout muscle development; in particular, it is required for muscle fibres to select the correct attachment sites, and to be properly oriented. Affected muscles showed an altered orientation, with branched fibres attaching to the wrong site. Even minor changes, which only affect a single muscle from each body segment, greatly impaired the movement of the larvae. The work by Carayon et al. offers a new approach to the study of muscular conditions. Branched muscles are seen in severe human illnesses such as Duchenne muscular dystrophy. Studying the impact of these changes in a living animal could help to understand how this disease progress, and how it can be prevented.

Assessment of Thrombotic and Bleeding Tendency in Two Mouse Models of Chronic Kidney Disease: Adenine-Diet and 5/6th Nephrectomy.

The coexistence of bleeding and thrombosis in patients with chronic kidney disease (CKD) is frequent and poorly understood. Mouse models are essential to understand complications of CKD and to develop new therapeutic approaches improving the health of patients. We evaluated the hemostasis in two models of renal insufficiency: adenine-diet and 5/6th nephrectomy (5/6Nx). Compared with 5/6Nx mice, mice fed with 0.25% adenine had more severe renal insufficiency and so higher levels of prothrombotic uremic toxins like indoxyl sulfate. More severe renal inflammation and fibrosis were observed in the adenine group, as demonstrated by histological and reverse transcription quantitative polymerase chain reaction experiments. Liver fibrinogen γ chain expression and level of plasma fibrinogen were increased only in adenine mice. In both CKD mouse models, tissue factor (TF) expression was increased in kidney and aorta extracts. Immunochemistry analysis of kidney sections showed that TF is localized in the vascular walls. Thrombin-antithrombin complexes were significantly increased in plasma from both adenine and 5/6Nx mice. Tail bleeding time increased significantly only in adenine mice, whereas platelet count was not significant altered. Finally, results obtained by intravital microscopy after laser-induced endothelial injury showed impaired platelet function in adenine mice and an increase in fibrin generation in 5/6Nx mice. To summarize, adenine diet causes a more severe renal insufficiency compared with 5/6Nx. The TF upregulation and the hypercoagulable state were observed in both CKD models. Bleeding tendency was observed only in the adenine model of CKD that recapitulates the whole spectrum of hemostasis abnormalities observed in advanced human CKD.

The Interaction of Platelets with Colorectal Cancer Cells Inhibits Tumor Growth but Promotes Metastasis.

Platelets promote metastasis, however, their role in tumor growth remains controversial. Here, we investigated the effect of platelet interactions with colorectal tumor cells. Platelets extravasated into the tumor microenvironment and interacted with tumor cells in a cadherin-6-dependent manner. The interaction induced platelet spreading, release of their granule content, and the generation of three types of microparticles (iMP) that expressed platelet markers, tumor markers, or both. The presence of iMPs was confirmed in colorectal cancer tissue specimens. Platelets significantly reduced tumor growth and increased intratumoral macrophages. This was mediated by iMP recruitment of macrophages via the chemoattractants RANTES, MIF, CCL2, and CXCL12 and activation of their tumor cell killing capacity through IFNγ and IL4, which led to cell-cycle arrest of tumor cells in a p21-dependent manner. In contrast, in the bloodstream, iMPs activated endothelial cells and platelets and induced epithelial-to-mesenchymal transition of tumor cells, promoting metastasis. Altogether, these results indicate that depending on the environment, local or bloodstream, the consequences of the interactions between platelets and a tumor may promote or prevent cancer progression. SIGNIFICANCE: Tumor cell interaction with platelets produces chimeric extracellular vesicles that suppress primary tumor growth by activating tumor-eliminating macrophages, while promoting metastasis through EMT and endothelial activation.

A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A.

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.

Microvesicles and Cancer Associated Thrombosis.

Microvesicles (MVs) are small membrane enclosed structures released into the extracellular space by virtually all cell types. Their composition varies according to the cell origin and the stimulus which caused their formation. They harbor functional molecules and participate in intercellular communication. Endothelium, inflammatory cells, and cancer cells produce procoagulant MVs which contribute to cancer-associated thrombosis (CAT) in animal models. The tissue factor (TF) conveyed by these MVs was shown to play a key role in different animal models of experimental CAT. Alternatively, other molecular mechanisms involving polyphosphates or phosphatidylethanolamine could also be involved. In clinical practice, an association between an increase in the number of TF-positive or the procoagulant activity of these MVs and the occurrence of CAT has indeed been demonstrated in pancreatic-biliary cancers, suggesting that they could behave as a biomarker predictive for CAT. However, to date, this association was not confirmed in other types of cancer. Potential causes explaining this limited associated between MVs and CAT are (1) the diversity of mechanisms associating MVs and different types of cancer; (2) a more complex role of MVs in hemostasis integrating their anticoagulant and fibrinolytic activity; and (3) the lack of sensitivity, reproducibility, and standardization of current methodologies permitting measurement of MVs. Each of these hypotheses constitutes an interesting exploration path for a future reassessment of the clinical interest of the MVs in CAT.

Platelets, Thrombo-Inflammation, and Cancer: Collaborating With the Enemy.

Platelets are small anucleate cells present in the blood stream, their typical role in primary hemostasis has been well-described. However, new evidence suggests that they have critically important roles in cancer progression and inflammation. Cancer cells can activate platelets, thus using them as physical shields from blood shear forces and natural killer (NK) cells. The activated platelets may also regulate hematopoietic and immune cell migration toward the tumor site; therefore, contributing to the cancer-associated inflammation. The activation of platelets by cancer cells may also contribute to metastasis and cancer progression by stimulating deep venous thrombosis and neutrophil extracellular trap formations (NETs) that "hide" cancer cells. We strived to review the current literature to dissect the role of platelets in cancer-associated thrombosis and tumor microenvironment inflammation.