ALBERT

Description: Key Points Mice constitutively developing a JAK2V617F-induced PV exhibit a bleeding tendency combined with the accelerated formation of unstable clots. Hemostatic defects are not concomitant with JAK2V617F expression, suggesting they are not directly caused by the mutation.

Description: Damage of blood vessels exposes the subendothelial matrix and results in the adhesion of platelets and monocytes at the site of injury. On eroded atherosclerotic plaques, platelets avidly bind to abundant type I collagen and monocytes are recruited by activated platelets. The purpose of the current investigation was to examine reciprocal interaction between platelets and monocytes upon exposure to a collagen surface. CD14 isolated monocytes and washed platelets were incubated separately or together in a 1/100 ratio in plates coated with type I fibrillar collagen. Platelet activation was assessed by measuring P-selectin expression by flow cytometry and RANTES secretion by ELISA. Platelet adherence and activation on immobilized collagen was analysed by confocal microscopy using FITC-phalloidin. Alternatively, cell-cell contacts were prevented by incubating platelets and monocytes in transwell coculture dishes, both parts of which were coated with collagen. In selected experiments cells were pretreated with the anti-PECAM 1.3 monoclonal antibody or with L-NMMA (NG-methyl-L-arginine, inhibitor of NO synthesis). IL8 was measured as an activation marker of monocytes. In co-incubation studies, collagen-activated platelets triggered IL8 secretion (6-fold increased) by monocytes, in agreement with previous observations indicating that platelets adherent to collagen delivers activating signals to monocytes. We have then focused our attention on the effect of monocytes on platelet activation. Unexpectedly, when monocytes were added five minutes after platelets to the collagen-coated plates, we obtained a decreased platelet expression of P-selectin by 42% (15.2 ± 9.1% positive platelets versus 26.3 ± 11.7% in the absence of monocytes, p = 0.0053, n = 16) and RANTES secretion by 39% (p 〈 0.0001, n = 6). The inhibitory effect of monocytes on platelet secretion decreased when the time to which they were added to platelets and collagen increased. Platelets incubated with immobilized collagen adhered and formed large aggregates consistent with a strong activation state. When monocytes were added, they established contacts with platelets while the number and size of the aggregates were dramatically decreased and isolated platelets were observed. In experiments performed in transwell coculture dishes, platelet P-selectin expression and RANTES secretion returned to the levels obtained in the absence of monocytes indicating that cell-cell contacts were required to inhibit platelet secretion induced by collagen. Preincubation of monocytes with anti-PECAM 1.3 reduced the inhibition of collagen-induced P-selectin expression and of RANTES secretion by ~ 40 %. Moreover, the inhibitory effect of monocytes on platelet aggregation appeared to be reversed by the anti-PECAM 1.3 antibody with a loss of individual platelets and the presence or large aggregates. In the presence of L-NMMA pre-treated monocytes, RANTES secretion was similar to the value obtained in the absence of monocytes. Together, our data provide evidence that, monocytes limit the initial phase of platelet activation by a collagen surface. The mechanism of this effect is dependent on cell-cell contacts. It is, at least in part, mediated by PECAM-1 with a contribution of NO. The interaction of platelets with monocytes at the surface of a damaged vessel would thus have two different effects:it would limit platelet activation and recruitment andit would increase the contribution of monocytes in inflammatory and procoagulant responses.

Description: 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.

Description: The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

Description: Acquired hemophilia is a rare bleeding disorder characterized by the spontaneous occurrence of inhibitory antibodies against endogenous factor VIII (FVIII). IgG from some patients with acquired hemophilia hydrolyze FVIII. Because of the complex etiology of the disease, no clinical parameter, including the presence of FVIII-hydrolyzing IgG, has been associated with patient's survival or death. Here, we demonstrate the presence of anti-FIX antibodies in acquired hemophilia patients. IgG from some patients were found to hydrolyze FIX. In most cases, IgG-mediated FIX-hydrolysis resulted in FIX activation. IgG-mediated hydrolysis of FIX thus led to the significant generation of activated FIX in 25 of 65 patients. Based on the estimated kinetic parameters, patients' IgG activated up to 0.3nM FIX in 24 hours, an amount that restored thrombin generation in vitro provided the presence of more than or equal to 3% residual FVIII activity in plasma. This work identifies proteolytic IgG as novel molecules able to activate FIX under pathologic conditions. IgG-mediated FIX activation is a prevalent phenomenon among acquired hemophilia patients. The presence of FIX-activating IgG may partly compensate for the antibody-mediated inhibition of endogenous FVIII in restoring thrombin generation. This clinical trial was registered at www.clinicaltrials.gov as #NCT00213473.

Description: The glycoprotein VI (GPVI)/FcRγ complex is a key receptor for platelet activation by collagen. We describe, for the first time, 2 genetic abnormalities in one patient. This 10-year-old girl presented ecchymoses since infancy, a prolonged bleeding time despite a normal platelet count and no antiplatelet antibodies. Collagen-induced platelet activation was null, whereas GPVI quantification by flow cytometry evidenced an incomplete deficiency. Immunoblotting showed an abnormal migration of residual GPVI, and no FcRγ defect. GPVI DNA sequencing revealed (1) an R38C mutation in exon 3 of one allele and (2) an insertion of 5 nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Introduction of the R38C mutation into recombinant GPVI-Fc resulted in abnormal protein migration and a loss of collagen binding. Thus, this composite genetic GPVI deficiency and dysfunction cause absence of platelet responses to collagen and a mild bleeding phenotype.

Description: Protease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

Description: Abstract 618 Disorders of hemostasis are frequent in patients suffering from classic myeloproliferative neoplasm (MPN) i.e. essential thrombocytemia (ET) or polycythemia vera (PV). They include high incidence of thrombosis, which represents a leading cause of morbidity and mortality, and a lower incidence of hemorrhagic events. The pathogenesis of hemostatic disorders is complex. The JAK2V617F mutation is found in 95% of PV and in 50% of ET. Since receptors coupled to JAK2V617F are hyper-responsive to growth factors, it is proposed that JAK2V617F primes platelets and, as a consequence, directly contributes to the thrombotic risk. However, the relation between the mutation and thrombosis has yet not clearly been established due to variable JAK2V617F allele burden and antithrombotic prophylaxis. Novel MPN animal models expressing the mutation have been developed with reported hemostasis disorders from unknown origin. Therefore, we took the opportunity to use these JAK2V617F knock-in (KI) mice to investigate the effects of JAK2V617F on platelet functions. We used irradiated WT recipient mice grafted with bone marrow (BM) cells from JAK2+/V617F KI donor mice. JAK2+/V617F KI mice were conditional floxed JAK2+/fl mice crossed with transgenic mice expressing Cre under the control of the vav promoter (vavCre/JAK2+/V617F) or Cre-tamoxifen-inducible transgenic mice expressing CreERt under the HSC-SCL promoter (sclCreERt/JAK2+/V617F). All recipient mice developed 4 weeks after transplantation (vavCre/JAK2+/V617F) and tamoxifen-induction (sclCreERt/JAK2+/V617F) a MPN mimicking PV and characterized by elevated hematocrit, platelet and leucocyte levels. Mice were studied between 4 and 9 weeks after the graft or 7, 14, 32 and 60 days after tamoxifen-induction (sclCreERt/JAK2+/V617F). Platelet functions were analyzed in vitro by flow cytometry and light transmission aggregometry, ex vivo by measuring thrombus formation in whole blood and flow conditions on collagen and vWF, and in vivo in a FeCl3-induced model of thrombosis. The analysis of platelet glycoprotein expression was focused on two ITAM receptors, GPVI and CLEC2. The principal observation is a deficiency in GPVI up to 50% in vavCre/JAK2+/V617F mice (p

Description: Key Points GPVI-dependent platelet binding and activation contribute to seal neutrophil-induced vascular damage in IC-mediated inflammation. Inflammation represents an uncommon hemostatic situation in which adhesion and activation of single platelets prevent bleeding.

Description: Key PointsGPVI interaction with polymerized fibrin triggers a new loop amplifying thrombin generation and platelet recruitment at the clot surface.