Key Points• GPVI-dependent platelet binding and activation contribute to seal neutrophilinduced vascular damage in IC-mediated inflammation.• Inflammation represents an uncommon hemostatic situation in which adhesion and activation of single platelets prevent bleeding.Platelets protect vascular integrity during inflammation. Recent evidence suggests that this action is independent of thrombus formation and requires the engagement of glycoprotein VI (GPVI), but it remains unclear how platelets prevent inflammatory bleeding. We investigated whether platelets and GPVI act primarily by preventing detrimental effects of neutrophils using models of immune complex (IC)-mediated inflammation in mice immunodepleted in platelets and/or neutrophils or deficient in GPVI. Depletion of neutrophils prevented bleeding in thrombocytopenic and GPVI 2/2 mice during IC-mediated dermatitis. GPVI deficiency did not modify neutrophil recruitment, which was reduced by thrombocytopenia. Neutrophil cytotoxic activities were reduced in thrombocytopenic and GPVI 2/2 mice during IC-mediated inflammation. Intravital microscopy revealed that in this setting, intravascular binding sites for platelets were exposed by neutrophils, and GPVI supported the recruitment of individual platelets to these spots. Furthermore, the platelet secretory response accompanying IC-mediated inflammation was partly mediated by GPVI, and blocking of GPVI signaling impaired the vasculoprotective action of platelets. Together, our results show that GPVI plays a dual role in inflammation by enhancing neutrophil-damaging activities while supporting the activation and hemostatic adhesion of single platelets to neutrophil-induced vascular breaches. (Blood. 2015;126(8):1017-1026
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.
Myeloproliferative neoplasms (MPN) are associated with an increased risk of arterial and venous thrombosis. Pegylated-interferon alpha (IFN) and hydroxyurea (HU) are commonly used to treat MPN, but their effect on hemostasis has not yet been studied. The aim of our study was to determine whether IFN and HU impact the biological hemostatic profile of MPN patients by studying markers of endothelial, platelet, and coagulation activation. A total of 85 patients (50 polycythemia vera and 35 essential thrombocythemia) were included: 28 treated with IFN, 35 with HU, and 22 with no cytoreductive drug (non-treated, NT). Von Willebrand factor, shear-induced platelet aggregation, factor VIII coagulant activity (FVIII:C), fibrinogen, and thrombin generation with and without exogenous thrombomodulin were significantly higher in IFN-treated patients compared to NT patients, while protein S anticoagulant activity was lower. In 10 patients in whom IFN therapy was discontinued, these hemostatic biomarkers returned to the values observed in NT patients, strongly suggesting an impact of IFN therapy on endothelial and coagulation activation. Overall, our study shows that treatment with IFN is associated with significant and reversible effects on the biological hemostatic profile of MPN patients. Whether they could be associated with an increased thrombotic risk remains to be determined in further randomized clinical studies.
Introduction: Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. They are due to three main recurrent mutations affecting the JAK/STAT signaling pathway: JAK2V617F and mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only drug that not only induces a hematological response in ET, PV and early MF, but also a significant molecular response on both JAK2V617F or CALR-mutated cells. Our broad aim was to understand the mechanism of action of IFNα. Previously, our group and others have shown that IFNα specifically targets JAK2V617F HSC in a chimeric JAK2V617F knock-in mouse model. In this study, we wanted to know how and how fast IFNα impacts the different mutated human hematopoietic compartments. Methods: A prospective study was performed with a cohort of 47 patients treated by IFNα for 3-5 years. The MPN disease distribution was 40% ET, 49% PV and 11% MF. This cohort included 33 JAK2V617F-mutated patients, 11 CALR-mutated patients (7 type 1/type 1-like and 4 type 2/type 2-like), 2 both JAK2V617F- and CALR-mutated patients and 1 MPLW515K-mutated patient. At 4-month intervals, the JAK2V617F or/and CALR mutation allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was also determined by studying the presence of the JAK2V617F or CALR mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38- HSC-enriched progenitors, CD90-CD34+CD38- immature progenitors and CD90- CD34+CD38+ committed progenitors). Results: After a median follow-up of 33 months, IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p<.05). Moreover, homozygous JAK2V617F clones responded more rapidly than heterozygous clones in all hematopoietic cell compartments showing that the intensity of JAK2V617F signaling is correlated with the efficacy of IFNα. This efficacy was slightly increased after a median follow-up of 51 months. In contrast, during a median follow-up of 33 months for CALR-mutated patients, IFNα targeted similarly the HSPC and the mature cells. Moreover, IFNα induced a slower response in targeting CALR-mutated HSPC than the JAK2V617F HSPC (p<.05) (see Figure). The role of associated mutations at diagnosis was also investigated in the IFNα-mediated HSPC molecular responses using a NGS targeted myeloid panel. In JAK2V617F-mutated patients, the number of associated mutations did not impact the HSPC molecular response. In contrast, in CALR-mutated patients, the only molecular responders were not associated with other mutations, although the lower number of cases should be expanded. Using Ba/F3-MPL cellular models and primary cells, we observed that JAK2V617F was more prone to sensitize to IFNα signaling (increased Phospho-STAT1 and IFN-stimulating genes (ISGs)) compared to controls or CALRdel52 mutated cells. Conclusion: Altogether, our results show that IFNα targets more efficiently the human JAK2V617F-HSPCthan the mature cells. Moreover, IFNα has a greater efficacy on JAK2V617F HSPC thanCALR-mutated HSPC. This former result was associated with a greater priming of the IFNα signaling by JAK2V617F than by CALRdel52. The molecular response was dependent not only on mutational status, but also on the presence of other associated mutations for the CALR-mutated HSPC. Patient data are currently incorporated into a mathematical model taking into account clonal architecture and associated mutations to develop an algorythm able to predict patient response. Figure. Figure. Disclosures Kiladjian: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Background Filamin (FLN) regulates many cell functions through its scaffolding activity cross‐linking cytoskeleton and integrins. FLN was shown to inhibit integrin activity, but the exact mechanism remains unclear. Objectives The aim of this study was to evaluate the role of filamin A (FLNa) subdomains on the regulation of integrin αIIbβ3 signaling. Methods Three FLNa deletion mutants were overexpressed in the erythro‐megakaryocytic leukemic cell line HEL: Del1, which lacks the N‐terminal CH1‐CH2 domains mediating the FLNa‐actin interaction; Del2, lacking the Ig‐like repeat 21, which mediates the FLNa‐β3 interaction; and Del3, lacking the C‐terminal Ig repeat 24, responsible for FLNa dimerization and interaction with the small Rho guanosine triphosphatase involved in actin cytoskeleton reorganisation. Fibrinogen binding to HEL cells in suspension and talin‐β3 proximity in cells adherent to immobilized fibrinogen were assessed before and after αIIbβ3 activation by the protein kinase C agonist phorbol 12‐myristate 13‐acetate. Results Our results show that FLNa‐actin and FLNa‐β3 interactions negatively regulate αIIbβ3 activation. Moreover, FLNa‐actin interaction represses Rac activation, contributing to the negative regulation of αIIbβ3 activation. In contrast, the FLNa dimerization domain, which maintains Rho inactive, was found to negatively regulate αIIbβ3 outside‐in signaling. Conclusion We conclude that FLNa negatively controls αIIbβ3 activation by regulating actin polymerization and restraining activation of Rac, as well as outside‐in signaling by repressing Rho.
Background: Filaminopathies A are rare disorders affecting the brain, intestine, or skeleton, characterized by dominant X-linked filamin A (FLNA) gene mutations.Macrothrombocytopenia with functionally defective platelets is frequent. We have described a filaminopathy A male patient, exhibiting a C-terminal frame-shift FLNa mutation (Berrou et al., Arterioscler Thromb Vasc Biol. 2017;37:1087-1097. Contrasting with female patients, this male patient exhibited gain of platelet functions, including increased platelet aggregation, integrin αIIbβ3 activation, and secretion at low agonist concentration, raising the issue of thrombosis risk.Objectives: Our goal is to assess the thrombotic potential of the patient FLNa mutation in an in vivo model. Methods:We have established a mutant FlnA knock-in mouse model. Results:The mutant FlnA mouse platelets phenocopied patient platelets, showing normal platelet count, lower expression level of mutant FlnA, and gain of platelet functions: increased platelet aggregation, secretion, and αIIbβ3 activation, as well as increased spreading and clot retraction. Surprisingly, mutant FlnA mice exhibited a normal bleeding time, but with increased re-bleeding (77%) compared to wild type (WT) FlnA mice (27%), reflecting hemostatic plug instability. Again, in an in vivo thrombosis model, the occlusion time was not altered by the FlnA mutation, but arteriolar embolies were increased (7-fold more frequent in mutant FlnA mice versus WT mice), confirming thrombus instability. Conclusions:This study shows that the FlnA mutation found in the male patient induced gain of platelet functions in vitro, but thrombus instability in vivo. Implications for the role of FLNa in physiology of thrombus formation are discussed.
Background: Myeloproliferative neoplasms (MPN) are associated with an increased risk of arterial and venous thrombosis with an annual incidence varying from 1.1% to 6.6% (Patrono, Blood 2013). Comparable to that recommended in non-MPN high-risk patients, low-dose aspirin (81-100 mg/day) is recommended in primary and secondary prophylaxis of thrombosis in polycythemia vera and essential thrombocytemia (Tefferi, Am J Hematol 2012). However, the daily dose of aspirin to optimize efficacy and safety for MPN patients has never been challenged. Aim of the study: Determine the optimal dose of aspirin to achieve biological efficacy in Philadelphia-negative MPN patients. Methods: Patients with Philadelphia-negative MPN who were treated with low-dose aspirin 75 or 100 mg/day were enrolled in this observational study. Patients without any cytostatic drug and patients treated with either pegylated interferon alpha or hydroxyurea were eligible. Major exclusion criteria included inability to adhere to aspirin therapy and chronic oral anticoagulation. Biological efficacy to aspirin was evaluated by platelet aggregation induced by arachidonic acid 1.33 mM on platelet rich plasma and tested at trough level and 24 hours after last aspirin intake. Resistance to aspirin was defined as a maximal platelet aggregation over 20%. According to the results of platelet aggregation, aspirin dose and dosing regimen were modified as follows: from 100 mg/day to 160 mg/day or 75 mg x2/day. Patients enrolled in this have a median follow up of at least 6 months after the analysis. Results: Between January 2012 and February 2014, 77 patients with Philadelphia-negative MPN were included. 53 were treated with aspirin 75 mg/day (69%) and 24 with aspirin 100 mg/day (31%). Out of the 53 patients treated with aspirin 75 mg/day, 12 patients (23%) were resistant to aspirin. Resistance to aspirin was not correlated to sex, age, presentation, JAK2 status, treatment, history of thrombotic or bleeding and hematologic values (see following table). Table 1:The MPN Grade 1 Fibrosis PhenotypePMF (N:33)PV/ET (N:58)Total (N:91)Median age63 yrs55 yrs58 yrsSex1:31:11:2JAK V617F +16/33: (49%)40/58: (69%)56/91: (62%)Median Hgb(g/dL)11.9 (range 7.9-16.4)12.5 (range 8.0-19.8)12.2 (range 7.9-19.8)Median WBC(X10 (9))26.9 (range 1.3-188)8.9 (range 3.5-51.3)10.8 (range 1.3-188 )Median Platelet(X10 (9))179 (range 18.0-1194)505 (range 67-2286)370 (range 18-2286)Leukoerythroblastic Blood Smear15/33 (45%)PV: 17 ET: 5 Total: 22/58 (38%)37/91 (41%)Splenomegaly(cm below costal margin)18/33: (55%) Median: 1025/58: (43%) Median: 643/91(48%) Median: 4Transfusion dependence6/33: (18%)2/58: (4%)8/91: (9%)Presence Of ³ 1 symptom 17/33: (52%)33/58: (57%)50/91: (55%)DIPSS risk intermediate 2 or higher13/33: (39%)17/58: (29%)30/91: (33%)2 or more prior therapies9/33: (27%)36/58: (62%)45/91: (49%)Vital Status (Alive)25/33: (76%)53/58: (91%)78/90: (86%)Median follow up time (yrs)1.9 (range 0.1-9.8)5.7 (range 0-34.5)3.1 (range 0-34.5) An increased dose of 100 mg/day for at least 7 days overcame this biological resistance in 8 out of 8 re-tested patients. The 2 remaining 75mg resistant patients received an increased dose of aspirin but were not retested. Out of the 24 patients under 100 mg/day, only two (8%) were resistant to aspirin. In these patients, increasing the dose to 160 mg/day did not modify their biological response. However a 75 mgx2 /day was effective to overcome biological resistance. No thrombotic or bleeding event was observed during the 6-month follow-up regardless of the aspirin dose. Conclusions: This is the first study to measure in standardized conditions the biological resistance of aspirin in 77 well-characterized Philadelphia-negative MPN patients. Although this does not reach statistical significance a higher proportion of patients treated with aspirin 75mg/day was resistant to anti-platelet therapy compared to patients treated with aspirin 100 mg/day. Increasing the dose of aspirin from 75 to 100 mg once daily overcomes this biological resistance without increasing bleeding side effects and seems the best compromise. Interestingly in rare cases of extreme resistance to 100mg doubling the dose twice daily (75mgX2) was better than an increase of 160mg in one take. This pilot study on a small number of patients with a limited 6-month follow up compared to the low annual incidence of thromboses in these MPNs will be followed by a prospective study on a larger number of patients with an extended follow-up period to determine if biological resistance to aspirin is correlated to the occurrence of thrombotic events. Disclosures No relevant conflicts of interest to declare.
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