Search

Background Cholesterol crystal (CC) embolism causes acute kidney injury (AKI) and ischaemic cortical necrosis associated with high mortality. We speculated that sustaining the fibrinolytic system with Glu-plasminogen (Glu-Plg) could be a safe way to attenuate AKI and prevent ischaemic infarction upon CC embolism. Methods We induced CC embolism by injecting CC into the left kidney artery of C57BL/6J mice. The primary endpoint was glomerular filtration rate (GFR). Results Starting as early as 2 h after CC embolism, thrombotic angiopathy progressed gradually in the interlobular, arcuate and interlobar arteries. This was associated with a decrease of GFR reaching a peak at 18 h, i.e. AKI, and progressive ischaemic kidney necrosis developing between 12–48 h after CC injection. Human plasma Glu-Plg extracts injected intravenously 4 h after CC embolism attenuated thrombotic angiopathy, GFR loss as well as ischaemic necrosis in a dose-dependent manner. No bleeding complications occurred after Glu-Plg injection. Injection of an intermediate dose (0.6 mg/kg) had only a transient protective effect on microvascular occlusions lasting for a few hours without a sustained protective effect on AKI at 18–48 h or cortical necrosis, while 1.5 mg/kg were fully protective. Importantly, no bleeding complications occurred. Conclusions These results provide the first experimental evidence that Glu-Plg could be an innovative therapeutic strategy to attenuate thrombotic angiopathy, AKI, kidney necrosis and potentially other clinical manifestations of CC embolism syndrome.

Cancer-associated inflammation has been established as a hallmark feature of almost all solid cancers. Tumor-extrinsic and intrinsic signaling pathways regulate the process of cancer-associated inflammation. Tumor-extrinsic inflammation is triggered by many factors, including infection, obesity, autoimmune disorders, and exposure to toxic and radioactive substances. Intrinsic inflammation can be induced by genomic mutation, genome instability and epigenetic remodeling in cancer cells that promote immunosuppressive traits, inducing the recruitment and activation of inflammatory immune cells. In RCC, many cancer cell-intrinsic alterations are assembled, upregulating inflammatory pathways, which enhance chemokine release and neoantigen expression. Furthermore, immune cells activate the endothelium and induce metabolic shifts, thereby amplifying both the paracrine and autocrine inflammatory loops to promote RCC tumor growth and progression. Together with tumor-extrinsic inflammatory factors, tumor-intrinsic signaling pathways trigger a Janus-faced tumor microenvironment, thereby simultaneously promoting or inhibiting tumor growth. For therapeutic success, it is important to understand the pathomechanisms of cancer-associated inflammation, which promote cancer progression. In this review, we describe the molecular mechanisms of cancer-associated inflammation that influence cancer and immune cell functions, thereby increasing tumor malignancy and anti-cancer resistance. We also discuss the potential of anti-inflammatory treatments, which may provide clinical benefits in RCCs and possible avenues for therapy and future research.

Transient receptor potential (TRP) proteins form a superfamily of cation channels that are expressed in a wide range of tissues and cell types. During the last years, great progress has been made in understanding the molecular complexity and the functions of TRP channels in diverse cellular processes, including cell proliferation, migration, adhesion and activation. The diversity of functions depends on multiple regulatory mechanisms by which TRP channels regulate Ca

Cytosolic free calcium (Ca2+) is a second messenger regulating a wide variety of functions in blood cells, including adhesion, activation, proliferation and migration. Store-operated Ca2+ entry (SOCE), triggered by depletion of Ca2+ from the endoplasmic reticulum, provides a main mechanism of regulated Ca2+ influx in blood cells. SOCE is mediated and regulated by isoforms of the ion channel proteins ORAI and TRP, and the transmembrane Ca2+ sensors stromal interaction molecules (STIMs), respectively. This report provides an overview of the (patho) physiological importance of SOCE in blood cells implicated in thrombosis and thrombo-inflammation, i.e. platelets and immune cells. We also discuss the physiological consequences of dysregulated SOCE in platelets and immune cells and the potential of SOCE inhibition as a therapeutic option to prevent or treat arterial thrombosis as well as thrombo-inflammatory disease states such as ischemic stroke.

Background and Aims Numerous observational studies have reported an association between hyperuricemia (HU) and cardiovascular disease where atherosclerosis is a leading cause. In advanced atherosclerosis, cholesterol crystal (CC) embolism is a life-threatening complication with an average mortality of 62.8%. Clinical manifestations include skin necrosis, intestinal injury and acute kidney injury (AKI). Autopsies and tissue biopsies reveal CC inside the arterial lumen. In a new model of CC-induced AKI, we recently found that fibrin clots formed around CC obstruct peripheral arteries and cause tissue infarction and organ failure. However, the role of asymptomatic HU in CC-induced AKI is currently unknown. Thus, we hypothesized that asymptomatic HU improves the outcomes after CC-induced AKI. Method In vivo, 6 weeks old Alb-creERT2;Glut9lox/lox and Glut9lox/lox control mice were intraperitoneally injected with tamoxifen to deplete hepatic Glut9 expression. Both groups of mice were placed on a standard chow diet enriched with inosine for two weeks. Afterward, a solution of CCs was injected into the left kidney arteries of mice to induce AKI. Serum and kidneys were collected 24 hours later, and kidney function, infarct size and kidney injury evaluated using GFR measurement, colorimetric assays, ELISA and immunohistochemistry of kidney sections. For in vitro studies, we isolated platelets from healthy mice and cultured them in the presence or absence of soluble uric acid (sUA) prior to CC activation. After stimulation, platelet aggregation assays and flow cytometry were performed. Results Our in vivo data showed that Alb-creERT2;Glut9lox/lox mice developed asymptomatic HU without kidney impairment (serum UA levels 9-15 mg/dL), while Glut9lox/lox control mice remained healthy. HU mice displayed a rapid decline in GFR of approx. 80% as compared to only 30% in healthy mice after CC-induced AKI, which was in line with increased serum BUN and IL-6 levels. The rapid GFR decline was due to vasoconstriction in arteries of the contralateral kidney as determined by αSMA/fibrin staining and a reduced ratio of lumen versus artery area in mice with HU after AKI. HU mice also had significantly less kidney infarct size as well as reduced kidney injury (necrosis and edema) and arterial occlusion. The in vitro data showed that sUA had no impact on platelet aggregation, activation and degranulation as compared to medium control in response to CCs. Conclusion We now show that HU has vasoconstrictive effects in the contralateral kidney by reducing GFR, while at the same time protects mice from CC-induced AKI suggesting a compensatory physiological mechanism of autoregulation to protect nephrons. On the other hand, in patients with chronic kidney disease, HU-related vasoconstriction might lead to increased arterial blood pressure; thus, increasing the risk for cardiovascular complications due to platelet activation and aggregation.

Background: InformationTumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma. Results: By employing mouse models expressing high and low levels of the laminin 1 chain (LM1), we highlighted its implication in a tumor-stroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LM1, VEGFA expression triggered by the complex integrin 21-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer. Conclusions: Together, we have identified VEGFA, CXCR4 and 21 integrin downstream of LM1 in colon cancer as of bad prognostic value for patient survival. Significance: This information opens novel opportunities for diagnosis and treatment of colon cancer.

Lysosome-related organelles (LROs) are a category of secretory organelles enriched with ions such as calcium, which are maintained by ion transporters or channels. Homeostasis of these ions is important for LRO biogenesis and secretion. Hermansky-Pudlak syndrome (HPS) is a recessive disorder with defects in multiple LROs, typically platelet dense granules (DGs) and melanosomes. However, the underlying mechanism of DG deficiency is largely unknown. Using quantitative proteomics, we identified a previously unreported platelet zinc transporter, transmembrane protein 163 (TMEM163), which was significantly reduced in BLOC-1 (Dtnbp1(sdy) and Pldn(pa))–, BLOC-2 (Hps6(ru))–, or AP-3 (Ap3b1(pe))–deficient mice and HPS patients (HPS2, HPS3, HPS5, HPS6, or HPS9). We observed similar platelet DG defects and higher intracellular zinc accumulation in platelets of mice deficient in either TMEM163 or dysbindin (a BLOC-1 subunit). In addition, we discovered that BLOC-1 was required for the trafficking of TMEM163 to perinuclear DG and late endosome marker–positive compartments (likely DG precursors) in MEG-01 cells. Our results suggest that TMEM163 is critical for DG biogenesis and that BLOC-1 is required for the trafficking of TMEM163 to putative DG precursors. These new findings suggest that loss of TMEM163 function results in disruption of intracellular zinc homeostasis and provide insights into the pathogenesis of HPS or platelet storage pool deficiency.

Zn2+ deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn2+-deficient diets, accounting for 1−4% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn2+ deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn2+ status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn2+ uptake in the gut using different nutritional supplementation of Zn2+ could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn2+ diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn2+ in hemostasis. Storage protein metallothionein maintains or releases Zn2+ in the cytoplasm, and the dynamic change of this cytoplasmic Zn2+ pool is regulated by the redox status of the cell. An increase of labile Zn2+ pool can be toxic for the cells, and therefore cytoplasmic Zn2+ levels are tightly regulated by several Zn2+ transporters located on the cell surface and also on the intracellular membrane of Zn2+ storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn2+ is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn2+ transport and the physiological role of Zn2+ store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn2+ to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn2+ homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.

Increasing evidence suggests that platelets play a predominant role in colon and breast cancer metastasis but the underlying molecular mechanisms remain elusive. Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin that triggers platelet activation through immunoreceptor tyrosine-based activation motif (ITAM)-signaling and thereby regulates diverse functions including platelet adhesion, aggregation and procoagulant activity. GPVI has been proposed as a safe antithrombotic target as its inhibition is protective in models of arterial thrombosis with only minor effects on hemostasis. Here, we demonstrate that genetic deficiency of platelet GPVI in mice decreases experimental and spontaneous metastasis of colon and breast cancer cells. Similar results were obtained with mice lacking the spleen-tyrosine kinase Syk in platelets, an essential component of the ITAM-signaling cascade. In vitro and in vivo analyses show that mouse, as well as human GPVI, supports platelet adhesion to colon and breast cancer cells. Using a CRISPR/Cas9-based gene knock-out approach, we identified Galectin-3 as the major counter-receptor of GPVI on tumor cells. In vivo studies demonstrated that the interplay between platelet GPVI and tumor cell-expressed Galectin-3 utilizes ITAM-signaling components in platelets and favors the extravasation of tumor cells. Finally, we showed that JAQ1 F(ab)2-mediated inhibition of GPVI efficiently impairs platelet-tumor cell interaction and tumor metastasis. Our study reveals a new mechanism by which platelets promote the metastasis of colon and breast cancer cells and suggests that GPVI represents a promising target for antimetastatic therapies.

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.

Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively rendered tumor vessels highly permeable and caused massive intratumoral hemorrhage. While these results established platelets as potential targets for antitumor therapy, their depletion is not a treatment option due to their essential role in hemostasis. Thus, a detailed understanding of how platelets safeguard vascular integrity in tumors is urgently demanded. Here, we show for the first time that functional inhibition of glycoprotein VI (GPVI) on the platelet surface with an antibody (JAQ1) F(ab)2 fragment rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion while not inducing systemic bleeding complications. The intratumor bleeding and tumor growth arrest could be reverted by depletion of Ly6G+ cells, confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, JAQ1 F(ab)2–mediated GPVI inhibition increased intratumoral accumulation of coadministered chemotherapeutic agents, such as Doxil and paclitaxel, thereby resulting in a profound antitumor effect. In summary, our findings identify platelet GPVI as a key regulator of vascular integrity specifically in growing tumors and could serve as a basis for the development of antitumor strategies based on the interference with platelet function.

Tumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma.By employing mouse models expressing high and low levels of the laminin α1 chain (LMα1), we highlighted its implication in a tumor-stroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LMα1, VEGFA expression triggered by the complex integrin α2β1-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer.Together, we have identified VEGFA, CXCR4 and α2β1 integrin downstream of LMα1 in colon cancer as of bad prognostic value for patient survival.This information opens novel opportunities for diagnosis and treatment of colon cancer.

Metastatic dissemination of cancer cells, which accounts for 90% of cancer mortality, is the ultimate hallmark of malignancy. Growing evidence suggests that blood platelets have a predominant role in tumor metastasis; however, the molecular mechanisms involved remain elusive. Here, we demonstrate that genetic deficiency of integrin α6β1 on platelets markedly decreases experimental and spontaneous lung metastasis. In vitro and in vivo assays reveal that human and mouse platelet α6β1 supports platelet adhesion to various types of cancer cells. Using a knockdown approach, we identified ADAM9 as the major counter receptor of α6β1 on both human and mouse tumor cells. Static and flow-based adhesion assays of platelets binding to DC-9, a recombinant protein covering the disintegrin-cysteine domain of ADAM9, demonstrated that this receptor directly binds to platelet α6β1. In vivo studies showed that the interplay between platelet α6β1 and tumor cell–expressed ADAM9 promotes efficient lung metastasis. The integrin α6β1–dependent platelet-tumor cell interaction induces platelet activation and favors the extravasation process of tumor cells. Finally, we demonstrate that a pharmacological approach targeting α6β1 efficiently impairs tumor metastasis through a platelet-dependent mechanism. Our study reveals a mechanism by which platelets promote tumor metastasis and suggests that integrin α6β1 represents a promising target for antimetastatic therapies.

Metastatic dissemination of cancer cells, which accounts for 90% of cancer mortality, is the ultimate hallmark of malignancy. Growing evidence suggests that blood platelets have a predominant role in tumor metastasis; however, the molecular mechanisms involved remain elusive. Here, we demonstrate that genetic deficiency of integrin α6β1 on platelets markedly decreases experimental and spontaneous lung metastasis. In vitro and in vivo assays reveal that human and mouse platelet α6β1 supports platelet adhesion to various types of cancer cells. Using a knockdown approach, we identified ADAM9 as the major counter receptor of α6β1 on both human and mouse tumor cells. Static and flow-based adhesion assays of platelets binding to DC-9, a recombinant protein covering the disintegrin-cysteine domain of ADAM9, demonstrated that this receptor directly binds to platelet α6β1. In vivo studies showed that the interplay between platelet α6β1 and tumor cell-expressed ADAM9 promotes efficient lung metastasis. The integrin α6β1-dependent platelet-tumor cell interaction induces platelet activation and favors the extravasation process of tumor cells. Finally, we demonstrate that a pharmacological approach targeting α6β1 efficiently impairs tumor metastasis through a platelet-dependent mechanism. Our study reveals a mechanism by which platelets promote tumor metastasis and suggests that integrin α6β1 represents a promising target for antimetastatic therapies.

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbβ3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization.

SummaryPlatelets are well-known for their major role in primary hemostasis and thrombosis. Cancer patients frequently manifest thrombotic events and present abnormalities in blood coagulation which appear to be linked to altered platelet function and turnover. Moreover, numerous studies indicate an intimate cross-talk between platelets and tumor growth, angiogenesis and metastatic dissemination. Finally, several experimental data and clinical trials suggest possible benefits of anti-platelet drugs on some cancers. Here, we will review the current state of basic biological research regarding the role of platelets in cancer progression. We also critically review the possible clinical applicability of some anti-platelet therapies to limit tumor growth and prevent metastatic dissemination.

The interaction of endothelial cells and pericytes with their microenvironment, in particular with the basement membrane, plays a crucial role during vasculogenesis and angiogenesis. In this review, we focus on laminins, a major family of extracellular matrix molecules present in basement membranes. Laminins interact with cell surface receptors to trigger intracellular signalling that shapes cell behaviour. Each laminin exerts a distinct effect on endothelial cells and pericytes which largely depends on the adhesion receptor profile expressed on the cell surface. Moreover, proteolytic cleavage of laminins may affect their role in angiogenesis. We report in vitro and in vivo data on laminin-111, -411, -511 and -332 and their associated signalling that regulates cell behaviour and angiogenesis under normal and pathological conditions. We also discuss how tissue-specific deletion of laminin genes affects the behaviour of endothelial cells and pericytes and thus angiogenesis. Finally, we examine how coculture systems with defined laminin expression contribute to our understanding of the roles of laminins in normal and pathological vasculogenesis and angiogenesis.

Background: Fibrin, the end product of the coagulation cascade, consolidates the platelet plug at site of thrombosis: polymerized fibrin supports platelet adhesion under low and high shear rate conditions (Hantgan RR et al., Thromb Haemost 1992) and triggers platelet procoagulant activity (Beguin S et al., Blood 1999). These responses are largely independent of the integrin αIIβ3 and are carried by a yet ill-defined receptor. Platelet glycoprotein VI (GPVI) has a well-established key role in the initiation of thrombosis since it supports collagen-mediated platelet activation but it has recently been recognized to interact with other macromolecules such as fibronectin, vitronectin and laminins. We hypothesized GPVI could be the “missing” platelet receptor of fibrin. Aim of the study: to challenge the hypothesis that glycoprotein VI (GPVI) could be a functional fibrin receptor Methods: Thrombin generation was measured using calibrated automated thrombogram (CAT) in PRP from healthy volunteers, four GPVI-deficient patients and one patient with a fibrinogen deficiency. CAT was also performed on washed platelets mixed with prothrombin complex (FII, FVII, FIX, FX), antithrombin and fibrinogen. GPVI was blocked using the Fab of the monoclonal antibody 9O12. Fibrin polymerization was blocked using the GPRP peptide. GPVI binding to fibrin was measured in vitro using recombinant soluble GPVI (GPVI-Fc). Flow based adhesion assays were performed in capillary chambers coated with polymerized fibrin at variable shear rates and platelet morphological changes analyzed by scanning electron microscopy. The formation of fibrin-platelet thrombi was visualized by perfusing recalcified blood containing A647 fibrinogen in flow chambers (Vena8 Fluoro+ Cellix) coated with collagen and tissue factor. In a second step, the perfusion of hirudinated blood in which platelets were stained by A488-RAM1 allowed to visualize platelet recruitment by fibrin rich clots. Results: Thrombin generation triggered by tissue factor was impaired in the PRP of patients with a GPVI deficiency or in the presence of the Fab 9O12 as indicated by a respective decrease in the peak height of 45 and 25% as compared to controls. This effect was observed regardless the trigger of thrombin generation and required platelet activation. Measuring thrombin generation in a purified system showed that fibrinogen dose-dependently increased the thrombin peak by up to 150% at 3 mg/mL but the Fab 9O12 blunted this effect. Moreover, the Fab 9O12 had no effect on thrombin generation in the PRP of a fibrinogen-deficient patient confirming a GPVI/fibrin(ogen)interplay. Blocking fibrin polymerization by GPRP reduced the thrombin peak in normal PRP, in fibrinogen-supplemented PRP of the fibrinogen-deficient patient and in purified conditions. In contrast GPRP had no effect on the thrombin peak in normal PRP containing the Fab 9O12 and in the PRP of GPVI-deficient patients. The proof that GPVI specifically interacts with fibrin was obtained in a binding assay showing a dose-dependent binding of GPVI-Fc to fibrin polymers that was reversed by the Fab 9O12. Platelets adhered to polymerized fibrin resulting in platelet shape change and exposure of phosphatidylserine. Platelet adhesion on a fibrin network was observed at low (300 s-1) and high (1500 s-1) shear rates with the formation of small contractile thrombi. Adhesion was decreased by 62% for 9O12-treated platelets and by 43% with the blood of GPVI-deficient mice as compared to controls. Importantly, lack of GPVI or its blockade decreased stationary adhesion indicating that GPVI is required to stabilize the interactions between platelets and fibrin. Finally when hirudinated blood was perfused at a shear rate of 1500 s-1 onto preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 93%. Conclusions: Here we show for the first time that GPVI acts as a receptor for polymerized fibrin with two major functions: GPVI interaction with polymerized fibrin triggers (i) a new loop amplifying thrombin generation and (ii) platelet recruitment at the clot surface. These, so far, unrecognized properties of GPVI confer it a key role in the maturation of the thrombus by facilitating its growth and stabilization in addition to its well-known effect in the initiation of thrombus formation. Disclosures Jandrot-Perrus: Acticor Biotech: Other. Gachet:Acticor Biotech: Other.