SCD Coagulation 2024 PDF

Crosstalk between coagulation and inflammation in sickle cell disease Claire Auditeau [email protected] Master 2 D2HP - 19/11/2024...

Crosstalk between coagulation and inflammation in sickle cell disease Claire Auditeau [email protected] Master 2 D2HP - 19/11/2024 1  Contents I. Sickle cell disease II. Coagulation in sickle cell disease 1. Hypercoagulable state 2. Role of coagulation activation in SCD pathophysiology III. Targeting protein S in sickle cell disease 1. Rationale 2. PROSICK study 2  Sickle cell disease : epidemiology One of the most common genetic diseases : global health problem and also if they are infected with malaria ~ 3 million people affected worldwide 3 Piel, NEJM 2017  Sickle cell disease : definition Single base change GAG GTG Beta chain of human Autosomal recessive haemoglobin gene transmission Homozygosity AS AS AA AS AS SS Glutamic acid  Valine Hemoglobin A  Hemoglobin S Chromosome 11 hem trasports the O2 Conran & De Paula. Haematologica 2020 4  Sickle cell disease : definition Hemoglobin S (HbS)  Unique property of polymerising on delivering up the oxygen it carries provoked by polymers that are in the Hem s  Forming long rigid fibers which distort the red cells 5 Conran & De Paula. Haematologica 2020  Sickle cell disease : pathophysiology issues that are outside cell  A self perpetuating vicious cycle  Leading to acute complications ROS formation  Vaso-occlusive painful crises  Acute chest syndrome  Stroke  Thrombo-embolic events  Infections  Leading to chronic complications  Chronic anemia  Spleen infarction  Organ damages multiple Sundd et al, Annu Rev6Pathol 2019  Major acute symptoms Vaso-occlusive Acute chest syndrome Stroke Painful crises Sudden, throbbing, and sharp pain Presence of a new pulmonary Blood vessel that carries oxygen following vaso-occlusion infiltrate and nutrients to the brain is either blocked by a clot or bursts Common sites : Respiratory signs including - Joints - chest pain Both ischemic and hemorrhagic Definition - Extremities - fever strokes - Lower back - dyspnea - cough Leading cause of hospitalization Leading cause of death. Major cause of morbidity and Consequence mortality Hypoxia, infection, fever, acidosis, Infection, fat embolism and Transient ischemic attack, low dehydration, pregnancy, menstruation, possibly pulmonary infarction steady-state hemoglobin level, Triggers and obstructive sleep apnea rate per year of acute chest syndrome 7 Rees et al. Lancet 2010  Major acute symptoms Vaso-occlusive Acute chest syndrome Stroke Painful crises Sudden, throbbing, and sharp pain Presence of a new pulmonary Blood vessel that carries oxygen following vaso-occlusion infiltrate and nutrients to the brain is either blocked by a clot or bursts Common sites : Respiratory signs including - Joints - chest pain Both ischemic and hemorrhagic Definition - Extremities - fever strokes - Lower back - dyspnea - cough Leading cause of hospitalization Leading cause of death Major cause of morbidity and Consequence mortality Hypoxia, infection, fever, acidosis, Infection, fat embolism and Transient ischemic attack, low dehydration, pregnancy, menstruation, possibly pulmonary infarction steady-state hemoglobin level, Triggers and obstructive sleep apnea rate per year of acute chest syndrome 8 Rees et al. Lancet 2010  Major acute symptoms Vaso-occlusive Acute chest syndrome Stroke Painful crises Sudden, throbbing, and sharp pain Presence of a new pulmonary Blood vessel that carries oxygen following vaso-occlusion infiltrate and nutrients to the brain is either blocked by a clot or bursts Common sites : Respiratory signs including - Joints - chest pain Both ischemic and hemorrhagic Definition - Extremities - fever strokes - Lower back - dyspnea - cough Leading cause of hospitalization Leading cause of death Major cause of morbidity and Consequence mortality Hypoxia, infection, fever, acidosis, Infection, fat embolism and Transient ischemic attack, low dehydration, pregnancy, menstruation, possibly pulmonary infarction steady-state hemoglobin level, Triggers and obstructive sleep apnea rate per year of acute chest syndrome 9 Rees et al. Lancet 2010  Sickle cell disease : symptoms Almost every organ in the body can be affected bcs it linked to the microvessels  life expectancy : reduced by about 30 years Poor quality of life 10 Piel, NEJM 2017  Treatments Treatment Type Mechanism of action Clinical outcome Hematopoietic Replacement of bone marrow with stem cells frome healthy stem cell Curative Cure donor transplantation  VOC Hydroxyurea Supportive  Hemoglobin F  Need for transfusion Prevents oxidative damage to RBCs  VOC L-glutamine Supportive Decreases endothelial cell adhesion Modulator of hemoglobin-oxygen affinity Voxelotor Supportive  Anemia Inhibiting HbS polymerization Antibody anti P-selectin  VOC Crizanlizumab Supportive Blocks interactions between endothelial cells, platelets, RBCs, and leukocytes Blood  Hemoglobin A Anemia Supportive transfusion  Hemoglobin S Stroke prevention 11  Treatments Hydroxyurea Voxelotor Transfusion Crizanlizumab L-glutamine 12  Contents I. Sickle cell disease II. Coagulation in sickle cell disease 1. Hypercoagulable state 2. Role of coagulation activation in SCD pathophysiology III. Targeting protein S in sickle cell disease 1. Rationale 2. PROSICK study 13  Hypercoagulable state FVIIa FIXa FXIa TF FVIIIa PCa+ PS FX FXa TFPI FVa Antithrombine FII FIIa Plaquettes PCa : protéine C activée PS : protéine S Clot 14  Hypercoagulable state : biomarkers   biomarkers of coagulation - Thrombin-Antithrombin complexes - Prothrombin fragment 1+2 - D-dimer  Exposition of phophatidylserine on RBC surface and microparticules  docking site for enzymatic complexes involved in coagulation Contents  Abnormal expression of tissue factor (TF) on circulating endothelial cells  expression is increased further during pain episodes. 15 Tomer et al, J Lab Clin Med 2001  Hypercoagulable state : biomarkers  Decreased levels of natural anticoagulant proteins  protein C : activity and antigen  protein S : activity and antigen  Maybe even more during VOC crisis protein C is activated by the tissue factor in the plasma add endothelial cells the florescnse -to foloow the  Resistance to activated protein C thombin formation  Thrombin generation assay without activated protein C  Similar thrombin generation between SCD and control patients  TGA with aPC  Control : 80% reduction of thrombin generation  SCD patients : only 60% reduction of thrombin generation resistance in the coagulation Whelihan et al. JTH 2016 Westerman, et al J Lab Clin Med 1999 16  Hypercoagulable state : biomarkers global throbin generation Similar results in pediatric patients  Protein C  Protein S  Thrombin generation parameters in presence of thrombomodulin  Resistance to activated protein C Noubouossie et al, AJH 2011 17  Hypercoagulable state : biomarkers  Platelets  Chronic activation of circulating platelets  Acute pain episodes : increased platelet activation  TF-positive microparticules  Small membrane–derived vesicles released by cells following activation or apoptosis  May be derived from RBC, platelets, endothelial cells and monocytes  TF-positive MP are increased in patients with SCD, with further increases during acute pain episodes.  MP are derived from endothelial cells and monocytes  Correlation with coagulation activation biomarkers Lee et al, Arterioscler Thromb Vasc Biol 2006 18 Shet et al, Blood 2003  Hypercoagulable state : clinical data  thromboembolic events in patients with SCD  mortality  Incidence of venous thromboembolism (pulmonary embolism and deep vein thrombosis) : up to 25%  High recurrence risk but also high bleeding risk Large retrospective cohort study : 6423 patients 877 (13,7%) developped VTE 1 year follow up 1 year follow up 13,2% reccurent VTE 7,9% bleeding 5 year follow up 24,1% reccurence Naik et al, JTH 2014 Fatal VTE : 3,1% Fatal bleeding : 7,4% 19 Brunson et al, AJH 2019 Coagulation in SCD pathophysiology FVIIa FIXa FXIa TF FVIIIa FX FXa FVa Platelets FII FIIa Clot 20  Coagulation in SCD pathophysiology control IgG- 1H1 : anti-TF antibody 1H1 : anti-TF antibody accumulation of IgG : control IgG IgG : control IgG the coagulation 1. inhibit the TF Hb- leads to Townes 2. Measure thrombin and antithromin Townes sickle mice control mice TWSS TWAA  Activation of coagulation in sickle cell mice is TF dependent  Inhibition of TF has no effect on hemolysis or red blood cell maturation and turnover in sickle cell mice 21 Chantrathammachart, Blood 2012  Coagulation in SCD pathophysiology sVCAM-1 (ng/mL) 1H1 : anti-TF antibody 1H1 : anti-TF antibody IgG : control IgG IgG : control IgG Townes Townes TWAA TWSS sickle mice inhibiting the TF- control mice decreaseth the TWSS TWAA endothelial cell activation IL-6 (pg/mL) VCAM In mouse models of SCD, inhibition of TF reduces - inflammation - endothelial cell activation TWAA TWSS 22 Chantrathammachart, Blood 2012  Coagulation in SCD pathophysiology inflammation in the tussues WT mouse OR Berkeley sickle mouse  Infiltration of neutrophils in the lungs and livers BERK mice compared with WT controls  Levels of MPO were increased in the lungs and livers of BERK mice brown- infiltration 23 Chantrathammachart, Blood 2012  Coagulation in SCD pathophysiology WT mouse or Berkeley 1H1 : anti-TF antibody sickle mouse IgG : control IgG WTBM BERKBM WTBM BERKBM WT mouse WTBM or BERKBM transformed with the sickle dieasese to make two groups comparable Blocking TF attenuates neutrophil infiltration/activation into the lungs of BERKBM mice In sickle cell mice TF plays an important role in : - the activation of coagulation - inflammation Neutrophils are important in the nutrophil infiltration? - vascular injury 24 Chantrathammachart, Blood 2012 Coagulation in SCD pathophysiology FVIIa FIXa FXIa TF FVIIIa FX FXa FVa Platelets FII FIIa Clot 25  Coagulation in SCD pathophysiology anti-thrombin Mice were fed with chow containing placebo or rivaroxaban for 10 days. anti-coagulant anti 10a no difference Inhibition of factor Xa decreased: - Plasma TAT levels - Plasma IL-6 neutrophils biomarkers - MPO levels in the lung - Numbers of neutrophils in the lungs in BERKSS mice 26 Sparkenbaugh, Blood 2014 Coagulation in SCD pathophysiology FVIIa FIXa FXIa TF FVIIIa FX FXa FVa Platelets FII FIIa Clot 27  Coagulation in SCD pathophysiology FII ASO Control ASO = anti-sense oligonucleotide = ∼10% of normal FII levels decrease the synthesis of the pro-thrombin or Berkeley Berkeley sickle mouse sickle mouse increasing the survival rate during the decreasing of the pro- thrombin Sickle mice treated with FII ASO showed significantly improved survival compared with sickle mice administered control ASO Thrombin is both - a central protease in hemostasis - a key modifier of inflammatory processes 28 Arumugam et al Blood 2015  Coagulation in SCD pathophysiology sickle cells- increased the WBCs with no factor 2 expression In FIIlox/- sickle mice : -  leucocytosis (neutrophils + monocytes) -  Thrombocytosis - Inflammatory syndome :  IL-6 - Endothelial cells activation :  sVCAM-1 29 Arumugam et al Blood 2015  Coagulation in SCD pathophysiology Lung histology HbS/FIIWT : - Foamy alveolar macrophages - Thickened arterial/arteriolar tunica media - Increased lymphocyte and macrophage infiltration - Edema around the blood vessels - Vascular congestion was also pronounced HbS/FIIlox/−mice: all of these pathologies were remarkably reduced Significant improvement in SCD-associated end-organ damage (nephropathy, pulmonary hypertension, pulmonary inflammation, liver function, inflammatory infiltration, and microinfarctions) 30 Arumugam et al Blood 2015  Thrombo-inflammation in SCD 31 Conran et al, Haematologica 2020  Treatments Hydroxyurea Voxelotor Transfusion Crizanlizumab L-glutamine 32 Anticoagulants?  Anticoagulation Ataga et al (2021 HbSS 14 Rivaroxaban vs Yes 4 weeks Safe placebo Did not significantly  - coagulation activation, - endothelial activation, - inflammation Did not improve microvascular blood flow. Conflincting results on anticoagulation efficiency in SCD patients Noubouossie et al. Blood Rev 2017 33 Ataga et al, Transfusion 2021  Contents I. Sickle cell disease II. Coagulation in sickle cell disease 1. Hypercoagulable state 2. Role of coagulation activation in SCD pathophysiology III. Targeting protein S in sickle cell disease 1. Rationale 2. PROSICK study 34  Targeting protein S : rationale FVIIa FIXa FXIa  72 kDa glycoprotein TF  Synthesis : liver, endothelial cells, megacaryocytes… FVIIIa aPC+ ProS  Plasma concentration : 25 µg/mL = 350 nM FX FXa FVa  Free active form : 120 nM  Cofactor of activated protein C Major anticogulant pathway has the cellular properties FII FIIa Platelets  Binds to phosphatidylserine in presence of calcium NH2- -COOH Clot EGFs SHBG Gla TSR 1 2 3 4 LG1 LG2 aPC : activated protein C 35 Phosphatidylsérine (PdSer) ProS : protein S  Targeting protein S : rationale  Protein S deficiency in SCD patients is frequent  Associated with resistance to activated protein C Whelihan, JTH 2016 36  Targeting protein S : rationale  Protein S deficiency in SCD patients is frequent  Associated with resistance to activated protein C  Why?  Chronic consumption because of ongoing activation of coagulation?  Accelerated clearance because of binding to RBC exposing phosphatidylserine?  Protein S binds to 6% of RBC in SCD patients  Binding removed by addition of calcium Whelihan, JTH 2016 37 Targeting protein S : rationale no loctaderine unlabelled octodrine was in competition with the protein S Protein S binds to phosphatdylserine exposing RBC Whelihan, JTH 2016 38  Targeting protein S : rationale enhances the phosphatidylserine Protein S is also involved in phagocytosis phosphatidylserine exposing cells  Binding of SHBG domain of protein S to TAM receptor on macrophages Phagocytosis Macrophage PtdSer MerTK PS Is protein S involved in the clearance of phosphatidylserine procoagulant exposing RBC or microparticules ? Lu et al. Nature 1999 McColl et al. J Immunol 2009 Toda et al. Blood 2014  Targeting protein S : rationale Ab taken from lama has one part of heavy chain PS003biv : a single domain antibodytargting protein S Thrombin generation assay in Size (~ 15 kDa) presence of aPC and PS Production Stability Solubility Cryptic epitopes Anti-coagulant Ab PS003biv enhances protein S cofactor activity of aPC Peyron et al. 2020 Sedzro et al, 2022  Targeting protein S : rationale FeCl3-induced murine thrombosis model Bleeding model PS003biv (IV 10 mg/kg) or LMWH (SC 200 UI/kg) PS003biv recognize murine PS Venule PS003biv Ctl sdAb Sedzro J et al, JTH 2022  PROSICK : role of protein S in sickle cell disease I. Role of PS deficiency in a mouse model of SCD (HbSS mice) II. Protein S in the pathogenesis of SCD in humans: cellular and ex-vivo approaches III. Pharmacological approaches targeting PS  PROSICK : role of protein S in sickle cell disease I. Role of PS deficiency in a mouse model of SCD (HbSS mice) Expected results : Protein S levels decrease may induce:  Regulation of thrombin generation Townes  Clearance of phopshatidylserine exposing RBC Sickle mice and microparticules = Exacerbation of ongoing coagulation activation and thromboinflammation Tests to be performed : - Coagulation activation : TAT complexes PROS1+/- mice - Inflammation : IL-6 PROS1+/- - Endothelial cell activation : sVCAM-1 Sickle mice further decareased the protein S expression - Organ damage : neutrophils infiltration, MPO - Measurement of survival  PROSICK : role of protein S in sickle cell disease II. Protein S in the pathogenesis of SCD in humans: cellular and ex-vivo approaches RBC + Ionomycin protein S important for the phagocytosis CFSE labelling Wash steps (X5) Tryspin Centrifugation if phagocytosis- increased fluorescence  PROSICK : role of protein S in sickle cell disease II. Protein S in the pathogenesis of SCD in humans: cellular and ex-vivo approaches Phagocytosis of PdSer Phagocytosis is dose-dependently enhanced by expressing RBC by THP1 protein S macrophages  PROSICK : role of protein S in sickle cell disease II. Protein S in the pathogenesis of SCD in humans: cellular and ex-vivo approaches BF APC+ Merge BF APC+ Merge BF APC+ BF APC+ BF APC+ Merge gAPC  PROSICK : role of protein S in sickle cell disease II. Protein S in the pathogenesis of SCD in humans: cellular and ex-vivo approaches Phagocytosis Macrophage PtdSer MerTK PS MER expressed in activation on phagocytosis Inhibition of MERTK receptors blocks protein S effect on phagocytosis  Targeting protein S : rationale Phagocytosis Phagocytosis Macrophage PtdSer ? Macrophage PtdSer MerTK PS MerTK PS Is protein S involved in the clearance of phosphatidylserine exposing sickle RBC? Lu et al. Nature 1999 McColl et al. J Immunol 2009 Toda et al. Blood 2014  PROSICK : role of protein S in sickle cell disease III. Pharmacological approaches targeting PS Townes sickle mice (16 semaines) knock-in: human -globine and -globine S Heme injection vaso-occlusive crisis model Two groups : SCD control (n=4) and SCD PS003biv (n=4) Lungs 1h 3h Sacrifice Liver Intravenous Intravenous Blood PBS injection PS003biv 10mg/kg Heme collection Left heart or vehicle (50µmol/kg) ventricule  PS003biv decreased vaso-occlusion in the liver Modèle hème : résultats and the lungs stained RBCs SCD – steady state Liver Lungs Light sheet microscopy : marquage TER 119 SCD steady state 50  PS003biv decreased vaso-occlusion in the liver Modèle hème : résultats and the lungs Provoke the vasooclusion SCD – steady state SCD Heme - Control Liver Lungs Light sheet microscopy : marquage TER 119 51 SCD steady state  PS003biv decreased vaso-occlusion in the liver Modèle hème : résultats and the lungs SCD – steady state SCD Heme - Control SCD Heme – PS003biv Liver Lungs Light sheet microscopy : marquage TER 119 52 SCD steady state  PS003biv decreased hemolysis and coagulation activation III. Pharmacological approaches targeting PS Hemolysis biomarkers Coagulation activation Free-hemoglobin: spectrophotometry, Fairbanks et al., Clin Chem 1992 Bilirubin: spectrophotometry, Fairbanks et al., Clin Chem 1992 SCD steady state 53 Thrombin-antithrombin TAT: ELISA (Abcam)  Conclusion  Ongoing activation of coagulation is a hallmark of SCD  Coagulation is triggered by TF-exposing cells and phosphatidylserine exposure in microparticles and red blood cells  Activation of coagulation increases inflammation and endothelial cell activation, leading to a vicious self perpetuating cycle : thromboinflammation  The inhibition of coagulation showed conflicting first results that have to be confirmed in large prospective studies  The enhancement of protein S is an innovative mechanism of action and showed promising first results in a murine model of sickle cell disease