Integrins

Integrin - structure , superfamily members , diversity of function and mutation causing disorders with possible therapies

1. INTEGRINS : STRUCTURE SUPERFAMILY MEMBERS DIVERSITY OF FUNCTION EXAMPLES OF MUTATED INTEGRIN GENES CAUSING DISORDERS AND THEIR POSSIBLE THERAPY MADE BY : DIVYA SONI ( M.Sc )

2. INDEX : TOPIC NAME PAGE NO INTRODUCTION 3 CLASSIFICATION OF CELL ADHESION MOLECULE 4 STRUCTURE 5-9 SUPERFAMILY/TYPES OF INTEGRIN 10-16 INTEGRIN DEFECTS RESPONSIBLE FOR VARIOUS GENETIC DISEASES 17-19 INTEGRIN ACTIVATION 20-25 FUNCTIONS 26-34 DISORDERS 34-57 GLANZMANN THROMBASTHENIA (GT) 34-46 CAUSE 35 DETECTION 35 DIAGNOSIS 36-40 TREATMENT 40-46 EPIDERMOLYSIS BULLOSA (EB) 47-57 CAUSE 47 DETECTION 47-48 DIAGNOSIS 48-50 TREATMENT 51-57 2

3. INTRODUCTION : Integrins are a large family of type 1 transmembrane heterodimeric glycoprotein receptors that function as the major metazoan receptors for cell adhesion and connect the intracellular and extracellular environments i.e, functions as a transmembrane linkers between the extracellular matrix and the cytoskeleton . Integrins can bind to extracellular matrix ( ECM ) glycoproteins including collagens , fibronectins , laminins and cellular receptors such as V ascular C ell A dhesion M olecule 1 ( VCAM -1 ) and the I ntercellular C ell A dhesion M olecule ( ICAM ) family showing heterophilic binding . It is the principal receptor used by animal cell to bind to extracellular matrix . Thus integrin functions as a cell adhesion molecule along with cadherins , selectins and I mmuno g lobulin s uper f amily ( IgSF ) . 3

4. CLASSIFICATION OF CELL ADHESION MOLECULES : 4 CELL ADHESION MOLECULES ( proteins located on cell surface involved in binding with other cells or ECM by cell adhesion process ) INTEGRINS CADHERINS SELECTINS IMMUNOGLOBULIN SUPERFAMILY ( IgSF )

5. STRUCTURE : Heterodimer composed of two non-covalently associated glycoprotein subunits called alpha and beta that spans cell membrane . Molecular mass of the subunits vary from 90-100 kDa . A lpha and beta chains lie close together outside cell membrane along length of about 23 nm . DOMAINS : EXTRACELLULAR DOMAIN : interacts with other cell adhesion molecules or extracellular matrix ( ECM ) and binds to specific amino acid sequence motifs in extracellular matrix proteins or , in some cases , in proteins on the surfaces of other cells . Transmembrane domain INTRACELLULAR DOMAIN : Interacts with cytoskeleton and intracellular signaling molecules . Both subunits have short intracellular C terminal tails . Intracellular portion of integrin dimer binds to a complex of several different proteins which together form linkages to cytoskeleton . 5

6. For all but one of the 24 varieties of human integrins , this intracellular linkage is to actin filament . These linkages depend on proteins that assemble at the short cytoplasmic tails of the integrin subunits . A large adaptor protein talin is a component of the linkage in many cases but numerous additional proteins are also involved . Like the actin-linked cell-cell junctions formed by cadherins , actin linked cell matrix junctions formed by integrins may be small , inconspicuous and transient or large , prominent and durable . Examples of latter include focal adhesions that form when fibroblasts have sufficient time to establish strong attachments to the rigid surface of a culture dish and the myotendinous junctions that attach muscle cells . 5) Alpha and beta subunits are type 1 trasmembrane proteins with large N- terminal extracellular domains (700 or more amino acids) and relatively small cytoplasmic domains (30-50 amino acids) Beta subunit is provided with four cysteine rich repeated sequences . Cytoplasmic tail is connected to linker protein that connects to the cytoskeleton . 6

7. BINDING SITE : The best binding site for integrins is RGD sequence which is found in fibronectin and other extracellular matrix proteins . Some integrins bind Leu-Asp-Val ( LDV ) sequence in fibronectin and other proteins . Since same integrin molecule in different cell types can have different ligand binding specificities , it seems that additional cell type specific factors can interact with integrins to modulate their binding activity . The specificity of integrin binding to ECM components including laminins , collagens and fibronectin depends on the extracellular domains of alpha and beta subunits . The binding of integrins to their matrix ligands is also affected by concentration of calcium (Ca+2) and magnesium (Mg+2) in the extracellular medium reflecting the presence of divalent cation binding domains in alpha and beta subunits . The divalent cations can influence both the affinity and the specificity of binding of integrins to its extracellular ligands . The final 5nm N-termini of each chains form ligand binding region of extracellular matrix (ECM) . 7

8. h) Both alpha and beta subunits bind several divalent cations whose role in the subunit is unknown but involved in stabilization of the protein folds . The cations in the beta subunit is directly involved in the coordinating atleast some of the ligands that integrins bind to . Additional integrin binding sequences as yet poorly defined exist in laminins and collagens . 8

9. REFERENCE : Molecular biology of the cell by Bruce Alberts , Alexander Johnson , Julian Lewis , David Morgan , Martin Raff , Keith Roberts , peter Walter ; 6 th edition (page no – 1075) 9

10. SUPER FAMILY OR TYPES OF INTEGRINS : Human contain 24 types of integrins formed from the products of 8 different beta chain genes and 18 different alpha chain genes dimerized in different combinations . Drosophila sp. consists of 5 alpha and 2 beta subunits . Caenorhabditis sp. ( nematode ) consists of 2 alpha and 1 beta subunits . The 8 different beta chain genes and 18 different alpha chains genes seen in mammals are listed below : 10 GENE PROTEIN SYNONYM GENE PROTEIN SYNONYM ITGA1 CD49a VLA1 ITGA8 ITGA8 - ITGA2 CD49b VLA2 ITGA9 ITGA9 RLC ITGA3 CD49c VLA3 ITGA10 ITGA10 - ITGA4 CD49d VLA4 ITGA11 ITGA11 HsT18964 ITGA5 CD49e VLA5 ITGAD’ CD11D FL J39841 ITGA6 CD49f VLA6 ITGAE CD103 HUMINAE ITGA7 ITGA7 FL J25220 ITGAL CD11a LFA1A

11. Integrins can be categorized in multiple ways : ALPHA CHAIN : Some alpha chains have additional structural element or domain inserted towards the N-terminal called alpha-A domain ( called because it has similar structure to the A domain found in the protein Von Willebrand factor ) also termed as alpha-1 domain . Integrins carrying this domain either bind to collagens ( e.g. integrins alpha-1-beta-1 and alpha-2-beta-1 ) or act as cell-adhesion molecules ( integrins of beta-3 family ) . 11 GENE PROTEIN SYNONYM GENE PROTEIN SYNONYM ITGAM CD11b MAC-1 ITGB3 CD61 GP3A ITGAV CD51 VNRA , MSK8 ITGB4 CD104 - ITGA2B CD41 GP11b ITGB5 ITGB5 FL J26658 ITGAX CD11c - ITGB6 ITGB6 - ITBG1 CD29 FNRB , MDF2 ITGB7 ITGB7 - ITGB2 CD18 MACC-1 ITGB8 ITGB8 -

12. A domain carry 3 divalent cation binding sites . One is permanently occupied in physiological concentration of divalent cations and carries either calcium or magnesium ion , the principle divalent cations in blood at median concentrations of 1.4 mM (calcium) and 0.8 mM (magnesium) . Other sites become occupied by cations when ligands bind atleast for those ligands involving an acidic amino acid in their interaction sites . BETA CHAIN : Beta 1 subunit : form dimers with atleast 12 distinct alpha subunits found in almost all vertebrate cells . Beta 2 subunit : form dimers with atleast 4 types of alpha subunit and is expressed exclusively on the surface of white blood cells where it has an essential role in enabling these cells to fight infection . Beta 3 subunit : bind several matrix proteins including blood clotting factor fibrinogen and is found on blood platelets as well as various other cell 12

13. INTEGRIN FAMILY - various associations that occur between 18 types of integrin alpha chain and 8 types of beta chain allow formation of atleast 24 integrin types . Alpha 1 beta 1 , alpha 2 beta 1 , alpha 10 beta 1 , alpha 11 beta 1 represent collagen receptors . Alpha 3 beta 1 , alpha 6 beta 1 , alpha 6 beta 4 , alpha 7 beta 1 represent laminin receptors . Alpha 5 beta 1 , alpha 8 beta 1 , alpha 11b beta 3 , alpha v beta 1 represent fibronectin receptor that binds in RGD dependent manner. 13

14. TYPES OF INTEGRINS ALONG WITH THEIR LIGANDS OR COUNTER – RECEPTORS AND FUNCTIONS : 14 SUBUNITS NAME LIGANDS / COUNTER – RECEPTORS FUNCTIONS Beta 1 Alpha 1 VLA -1 ( CD 49a / 29 ) Collagen , laminin Cell matrix adhesion Alpha 2 VLA -2 ( CD 49b / 29 ) Collage n , laminin Alpha 3 VLA-3 ( CD 49c / 29 ) Fibronectin , collage n , laminin Alpha 4 VLA-4 ( CD 49d / 29 ) Fibronectin , VCAM-1 , MadCAM-1 Alpha 5 VLA-5 ( CD 49e / 29 ) Fibronectin Alpha 6 VLA-6 ( CD 49f / 29 ) Laminin Alpha 7 CD 49g / 29 Laminin Alpha 8 CD 49h / 29 - - Alpha v - Fibronectin , vitronectin Cell matrix adhesion

15. SUBUNITS NAME LIGANDS / COUNTER – RECEPTORS FUNCTIONS Beta 2 Alpha L CD 11a /18 ( LFA-1 ) ICAM - 1, 2,3 Leukocyte adhesion to endothelium Alpha M CD 11b/18 ( MAC-1 , CR3 ) iC3b , fibrinogen factor V , ICAM-1 Leukocyte adhesion , phagocytosis , cell matrix adhesion Alpha X CD 11c/18 ( p150 , 95 ; CR4 ) iC3b , fibrinogen Beta 3 Alpha 11b - Fibrinogen , fibronectin , Willebrand factor , vitronectin , thrombospondin Platelet adhesion and aggregation Alpha v Vitronectin receptor ( CD51/61 ) Vitronectin , fibrinogen , Willebrand factor , thrombospondin , fibronectin , osteoportin , collagen Cell matrix adhesison Beta 4 Alpha 6 - Laminin Beta 5 Alpha v - Vitronectin 15

16. ABBREVIATIONS : ICAM : I ntercellular A dhesion M olecule LFA : L eukocyte F unction A ssociated A ntigen MadCAM – 1 : M ucosal A ddressin C ell A dhesion M olecule – 1 VCAM-1 : V ascular C ell A dhesion M olecule – 1 REFERENCE : Hynyes RO . Integrins – versatility , modulation and signaling in cell adhesion . Cell 69 : 11-25 , 1992 16 SUBUNITS NAME LIGANDS / COUNTER – RECEPTORS FUNCTIONS Beta 6 Alpha v - Fibronectin Cell matrix adhesison Beta 7 Alpha 4 LPAM-1 Fibronectin , VCAM-1 , MadCAM-1 Lymphocyte homing to mucosal lymphoid tissues Alpha E HML-1 E- cadherin Retention to intraepithelial T cells

17. INTEGRIN DEFECTS ARE RESPONSIBLE FOR VARIOUS GENETIC DISEASES : Some varieties of integrins and the problems that result when individual integrin alpha and beta chains are defective . [ REFERENCE : Molecular biology of the cell by Bruce Alberts , Alexander Johnson , Julian Lewis , David Morgan , Martin Raff , Keith Roberts , peter Walter ; 6 th edition (page no – 1076) ] 17 INTEGRIN LIGAND DISTRIBUTION PHENOTYPE WHEN SUBUNIT MUTATED ALPHA BETA Alpha 5 Beta 1 Fibronectin Ubiquitous Death of embryo , defects in blood vessels , somites , neural crest Early death of embryo at implantation Alpha 6 Beta 1 Laminin Ubiquitous Severe skin blistering , defects in other epithelia Alpha 7 Beta 1 Laminin Muscle Muscular dystrophy , defective myotendinous junctions

18. 18 INTEGRIN LIGAND DISTRIBUTION PHENOTYPE WHEN SUBUNIT MUTATED ALPHA BETA Alpha L Beta 2 (LFA -1) Ig superfamily counter receptors (ICAM -1) White Blood Cells Impaired recruitment of leucocytes Leukocyte adhesion deficiency (LAD) , impaired inflammatory response , recurrent life threatening infections Alpha 11b Beta 3 Fibrinogen Platelets Bleeding , no platelet aggregation (Glanzmann’s Disease) Bleeding , no platelet aggregation (Glanzmann’s Disease) , mild osteoporosis Alpha 6 Beta 4 Laminin Hemidesmosomes in epithelia Severe skin blistering , defects in other epithelia

19. REFERENCE : Molecular biology of the cell by Bruce Alberts , Alexander Johnson , Julian Lewis , David Morgan , Martin Raff , Keith Roberts , peter Walter ; 6 th edition (page no – 1076) 19

20. INTEGRIN ACTIVATION : Integrin molecules that span the membrane and mediate the attachments cannot simply be passive , rigid objects with sticky patches at their two ends . They must be able to switch between an active state where they readily form attachments and an inactive state where they do not . Structural studies using combination of electron microscopy and X-ray crystallography suggest that integrins exist in multiple structural conformations that reflect different states of activity . INACTIVE STATE : External segments of integrin dimer are folded together into a compact structure that cannot bind matrix proteins . Cytoplasmic tails of dimer are hooked together preventing their interaction with cytoskeletal linker proteins . 20

21. ACTIVE STATE : Two integrin subunits are unhooked at the membrane to expose the intracellular binding sites of cytoplasmic adaptor proteins and the external domains unfold and extend like a pair of legs to expose high affinity matrix binding sites at the tips of the subunits . Thus , the switch from inactive to active state depends on major conformational change that simultaneously exposes the external and internal ligand binding sites at the end of integrin molecule . 21

22. SWITCHING BETWEEN INACTIVE AND ACTIVE STATES IS REGULATED BY A VARIETY OF MECHANISMS THAT VARY DEPENDING ON THE NEEDS OF THE CELL : OUTSIDE IN MECHANISM : Binding of external matrix protein such as RGD sequence of fibronectin Integrins switch from low affinity inactive state to high affinity active state Binding sites for talin and other cytoplasmic adaptor proteins are exposed on the tails of beta chain Binding of these adaptor proteins Attachment of actin filaments to the intracellular end of the integrin molecule 22

23. In this way , when integrin catches hold of its ligand outside the cell , the cell reacts by tying integrin molecule to the cytoskeleton so that force can be applied at the point of cell attachment . INSIDE OUT MECHANISM : Intracellular regulatory signal Stimulates ability of talin and other proteins to interact with beta chain of integrin Talin competes with integrin’s alpha chain for its binding site on beta chain tail Talin binds to beta chain Blocks intracellular alpha beta linkage 2 legs of integrin molecule springs apart 23

24. The regulation of inside out integrin activation is particularly well understood in platelets where an extracellular signal protein thrombin binds to specific G p rotein c oupled r eceptors ( GPCR ) on the cell surface and thereby activates an intracellular signaling pathway that leads to integrin activation . 24

25. REFERENCE : Molecular biology of the Cell by Bruce Alberts , Alexander Johnson , Julian Lewis , David Morgan , Martin Raff , Keith Roberts , peter Walter ; 6 th edition (page no – 1078) 25

26. FUNCTIONS : Attachment of the cell to ECM : Integrins couple ECM outside cell to cytoskeleton (in particular microfilaments) inside the cell . Integrin-ligand binding is defined by alpha and beta subunits of integrin . Among ligands are fibronectin , vitronectin , collagen and laminin . Cell attachment takes place through formation of cell-adhesion complexes which consist of integrins and many cytoplasmic proteins such as talin , paxillin and alpha- actinin which act by regulating kinases such as FAK ( F ocal A dhesion K inase) and Src kinase family . These adhesion molecules attach to actin cytoskeleton . Thus , integrin links two networks across plasma membrane – extracellular ECM and intracellular actin filaments . 26

27. FIGURE : Model for how integrins in the plasma membrane connect intracellular actin filaments to extracellular matrix at a focal contact formed when binding of matrix glycoproteins such as fibronectin on outside of cell , clusters integrin molecules at the contact site . 27

28. Integrins do more than just create attachments . They also activate intracellular signaling pathways controlling almost any aspect of cell’s behaviour according to nature of surrounding , matrix and state of cell’s attachments to it . Many cells will not grow or proliferate in culture unless they are attached to extracellular matrix as nutrients and soluble growth factors in the culture medium are not enough . For some cell types including epithelial , endothelial and muscle cells , cell survival depends on such attachments as they undergo apoptosis when they loose contact with extracellular matrix . This dependence of cell growth , proliferation and survival on attachment to a substratum is called anchorage dependence mediated by integrins and intracellular signals they generate . 28

29. 2) SIGNAL TRANSDUCTION : Integrins play important role in cell signaling by modulating cell signaling pathways of transmembrane protein kinases such as receptor tyrosine kinase (RTK) . Integrins regulate receptor tyrosine kinase signaling by recruiting specific adaptors to the plasma membrane . e.g . Beta 1 integrin recruits Gab1/shp2 and presents shp2 to IGF 1R results in dephosphorylation of receptor . Depending on integrin’s regulatory impact on specific receptor tyrosine kinase , cell can experience cell growth , cell division , cell survival , cellular differentiation , apoptosis (programmed cell death) . Knowledge of the relationship between integrins and receptor tyrosine kinase has laid a foundation for new approaches to cancer therapy . Specifically , targeting integrin’s associated with RTK’s is an emerging approach for inhibiting angiogenesis . 29

30. SOME OF THE RECEPTOR TYROSINE KINASE : FOCAL ADHESION KINASE (FAK) : One of the best studied and first integrin signaling molecules to be identified which acts as phosphorylation regulated signaling scaffold important for adhesion turn-over , rho-family GTPase activation , cell migration and cross-talk between growth-factor signaling and integrins . [ Mitra et al .,2005 ] DOMAINS : FERM domain , Central kinase domain , Proline-rich regions and C-terminal focal adhesion targeting (FAT) domain that interacts with paxillin and talin . Integrins cluster at cell matrix contacts FAK recruit to integrin beta subunit by talin/paxillin Clustered FAK molecule autophosphorylate on specific tyrosine 30

31. Create phosphotyrosine docking sites for SH2 domain containing proteins which include Src kinase Src kinase activated Phosphorylates FAK Promote kinase activity and interaction with other proteins Src family protein tyrosine kinase (SFK’s) : Rapidly activated following integrin-ligand interactions and SFK activity , contributes to reinforcement of initial integrin mediated adhesions by activating downstream kinases and adaptors ( Giannone and Sheetz , 2006 ; Ginsberg et al .,2005 ) . SFK’s bind directly to beta integrin tails and this interaction contributes to activation of kinase activity and controls cell spreading . ( Arias-Salgado et al ., 2003 ; Reddy et al ., 2008 ) 31

32. 3) INTEGRIN-LINKED KINASE (ILK) : Essential protein that has a major role as signaling scaffold at integrin adhesions . Forms heterotrimeric complex with LIM domain protein PINCH , actin and paxillin binding protein parvin . This complex serves as hub in integrin signaling networks and is required for correct targeting of its components to integrin mediated adhesions . This complex also protects from proteosomal degradation ( Legate et al .,2006 ) DIAGRAM FOR INTEGRIN SIGNALLING : Activation of integrins Engagement of integrins Clustering of integrins Disengagement of integrins Refer Journal of Cell Science by David S.Harburger and David A.Calderwood ,2009 ( 122, pp.159-163) https://jcs.biologists.org/content/122/2/159 32

33. 3) NEUROREGULATION : Integrins have important function in neuroregulation after peripheral nervous system injury . Integrins are present at the growth cone of damaged PNS neurons and attach to ligands in ECM to promote axon regeneration . It is unclear whether integrins promote axon regeneration in adult central nervous system (CNS) because of two obstacles that prevent integrin mediated regeneration in the CNS – Integrins are not localized in axon of most adult CNS neurons . Integrins become inactivated by molecules in scar tissue after injury . Cancer metastasis Embryogenesis Immunity ( migration of immune cells to inflammation centre ) 33

34. g) Cell tissue organ development ROLE OF FEW IMPORTANT INTEGRINS : DISORDERS CAUSED DUE TO MUTATED INTEGRIN GENES : GLANZMANN THROMBASTHENIA (GT) : This was first described by Edward Glanzmann in 1918 as “ hereditary hemorrhagic thrombasthenia ” . 34 INTEGRINS FUNCTIONS Beta 1 Development Alpha V Vasculogenesis ( blood vessel formation in embryo ) Alpha 9 Beta 1 Lymphangiogenesis (lymphatic vessel formation from pre-existing lymphatic vessel) Alpha 11b Beta 3 Thrombus formation Alpha 6 Beta 4 Integrity of skin

35. Glanzmann thrombasthenia is a rare autosomal recessive bleeding syndrome affecting the megakaryocyte lineage characterized by lack of platelet aggregation causing life long bleeding . CAUSE : Mutations in alpha 11b beta 3 integrin that is the qualitative and quantitative deficiencies or abnormalities in alpha 11b beta 3 formation when bleeding occurs . DETECTION : Mucosal bleeding Gingival bleeding ( bleeding gums ) Petechiae (tiny , round , brown-purple spots due to bleeding under skin) Ecchymoses (skin discoloration due to bleeding underneath) Mennorhagia (heavy or prolonged vaginal bleeding with menstrual cycles) Gastrointestinal bleeding Hematuria (internal bleeding and blood in urine) Brain hemorrhages Epistaxis (nose bleeds) 35

36. Postpartum bleeding (excessive bleeding after childbirth) Increased bleeding post-operatively The symptoms usually begin at birth or shortly after with some affected individuals having mild bruising while others having severe hemorrhages that can be life threatening . DIAGNOSIS : CLINICAL EVALUATION : History is a cornerstone in the diagnosis of patients with hereditary platelet disorders . Bleeding history : Manifestations of a typical platelet function include unexplained extensive bruising , epistaxis causing anemia or requiring hospital admission , menorrhagia , oral cavity bleeding , bleeding during childbirth . Site , severity , frequency of bleeding episodes and response to therapies . Requirement for blood transfusion . Family history of bleeding disorder and consanguinity . 36

37. Evaluation of drugs and foods possibly interfering with platelet function . Bleeding from hereditary platelet disorder occurring at times of trauma rather than delayed onset . Severe diseases likely to present at early childhood including the neonatal period . Presentation may include intracranial hemorrhage (ICH) following delivery . Excessive bleeding from umbilical stump or postcircumcision , easy or extensive bruising . Later in life prolonged epistaxis and menorrhagia may encountered . SCREENING TESTS : Complete blood count (CBC) and peripheral blood morphology : examines platelet count , size , morphology . Routine coagulation test : such as prothrombin time , activated partial thromboplastin time and thrombin time are usually normal . Screening of platelet function : best done by in-vitro techniques such as PFA-100 measuring closure time using cartridges first coated with collagen and adenosine diphosphate (ADP) and then collagen and epinephrine . 37

38. Once the initial screening test suggest inherited platelet disorder , more specific tests are needed to confirm the diagnosis and classify the disease . SPECIFIC TESTS FOR PLATELETS DYSFUNCTION INCLUDE : Light transmission aggregometry (LTA ) : considered to be gold standard method that uses p latelet r ich p lasma ( PRP ) with agonists such as ADP , collagen , arachidonic acid , ristocetin added to PRP or washed platelets . Limitation: It is technically demanding and requires a significant amount of blood which can be difficult for young children . Multiple electrode aggregometry (MEA ) : recently developed for rapid assessment of platelet function using pairs of electrode to measure electrical resistance . Advantage : requires small blood volumes with standard reagents . Recently published research has validated using MEA to diagnose GT . 38

39. Platelet flow cytometry : evaluates glycoproteins GP1a/11a (CD31 , CD49b) , GPIV (CD36) and GPVI to identify deficiency and identify GT . Molecular analysis of ITGA2B and ITGB3 genes : used for confirmation of difficult cases particularly GT variants , prenatal screening , prenatal genetic diagnosis and prevention of GT . CLASSIFICATION OF GT BASED ON DIAGNOSTIC RESULTS : 39 Type Proportion of patients Alpha 11b Beta 3 expression (platelet membrane) Alpha 11b Beta 3 % (flow cytometry CD41 – Alpha 11b , CD61 – Beta 3) Platelet aggregation (ADP , EP , collagen , thrombin) I 75 % Absent or trace expression 0-5 Nil II 15 % Substantially reduced 5-20 Nil Variant 10 % Abnormal alpha 11b beta 3 cannot bind fibrinogen >20 Nil/ normal

40. TREATMENT : PHARMACOLOGICAL METHODS : Antifibrinolytic agents : acts through inhibition of plasminogen conversion to plasmin and subsequent fibrinolysis inhibition . They have shown effectiveness in controlling surgical and non-surgical bleeds particularly mouth bleeds and menorrhagia . It is not recommended for treatment of renal bleeding or in case of disseminated intravascular coagulation . 40 Type Platelet agglutination (ristocetin) Bleeding time/closure time (PFA-100) Clot retraction Alpha granule pool fibrinogen I Normal Prolonged Nil Nil II Normal Prolonged Residual Sub-normal Variant Normal Prolonged Variable Variable

41. ANTIFIBRINOLYTIC AGENTS INCLUDE : Epsilon aminocaproic acid : Dosage - 50-100 mg/kg (maximum 6g) every 4-6 hours (maximum 24 g per 24 hours) Tranexamic acid : Oral - 25 mg/kg every 8 hours Intravenous - 10 mg/kg every 8 hours Mouthwashes – prepared by diluting 5 ml of tranexamic acid with 5 ml of sterile water or 500 mg tablets placed into 10-20 ml of water used for every 6-8 hours with 10 ml quantity . 2) Desmopresin : effective in many bleeding disorders including inherited platelet function disorder . Dosage - Intravenous infusion : 0.3 ug/kg diluted to 30-50 ml in saline over 30 minutes Subcutaneous injection : 0.3 ug/kg 41

42. Intranasal spray : Adults (300 ug) and children (150 ug) Fibrin sealants : treat local bleeding . Epistaxis and gingival bleeding are treated by nasal packing or application of gel foam soaked in tropical thrombin . NON-PHARMACOLOGICAL METHODS : Platelet transfusion : It is the gold standard treatment for uncontrolled bleeding in GT preferably h uman l eukocyte a ntigen ( HLA ) matched single-donor and leukocyte-reduced aphaeresis platelets . Between 25 % and 80 % of GT patients , develop antiparallel antibodies particularly against alpha 11b beta 3 developing more frequently in homozygous patients . This development of platelet specific antibodies should be minimized by avoidance of platelet transfusions particularly for minor bleeds . In GT patients , consumption of platelets due to surgery or bleeding is increased and platelet transfusions need to be given more frequently . 42

43. Dosage : Adults : S ingle donor aphaeresis – one unit Random donor – 4-6 units Children : 10-15 ml/kg Recombinant activated factor VII (rFVIIa ) : Due to multiple issues related to platelet transfusion and the desire of patients to conceive , rFVIIa was elected as the first line of therapy to save platelets for ,ore severe or non-responsive bleeds . Dosage : Conventional dose : >=80 ug/kg repeated at interval of 2.5 hours . High dose : 270 ug/kg Hematopoietic stem cell transplant (HSCT ) : successfully performed in case of severe current hemorrhages and found to improve quality of life . This is recommended for GT patients having refractoriness to platelet transfusions . Hormonal therapy is used to suppress menstrual periods . 43

44. TREATMENT OF MINOR BLEEDING EPISODES : Local measures and antifibrinolytic drugs . If antifibrinolytic drug fails to function then , rFVIIa >= 80 ug/kg single dose and repeated as necessary . Platelet transfusion usually unnecessary . TREATMENT OF SEVERE BLEEDING EPISODES : Local measures , antifibrinolytic drug started at home and continued in hospital . Early start of rFVIIa either single dose (high) or conventional dose every 2.5 hours for 3 doses . If above fails then , rFVIIa >= 80 ug/kg every 2-4 hours to control bleeding (dose spacing made once bleeding is controlled) . Platelet transfusion can be done as a single dose and repeated as necessary with continuation of rFVIIa if rFVIIa cannot stop bleeding . 44

45. TREATMENT OF MINOR SURGERY : minor surgery means surgery that only manipulates skin , mucous membrane or superficial connective tissue . Preoperative : Antifibrinolytic should be started 2 hours preoperative . Single dose rFVIIa>= 80 ug/kg 10 minutes preoperative . Intraoperative : rFVIIa >= 80 ug/kg repeated as necessary at intervals of 2.5 hours . Postoperative : Antifibrinolytic should be continued until hemostasis is maintained . rFVIIa >= 80 ug/kg every 2-4 hours if needed . Platelet transfusions as single dose with rFVIIa if rFVIIa cannot stop bleeding . 45

46. TREATMENT OF MAJOR SURGERY : surgery that cross a mesenchymal barrier , remove an organ , alter anatomy , open facial plane or enter body cavity . Preoperative : Antifibrinolytic started 2 hours preoperative . rFVIIa >= 80 ug/kg 10 minutes preoperative . Platelet transfusion 1-2 hours presurgery . 3 doses of platelet units ready . Intraoperative : rFVIIa >= 80/kg every 2.5 hours . Platelet transfusion if necessary . Postoperative : Antifibrinolytic should be continued until hemostasis is maintained . rFVIIa >= 80 ug/kg every 2-4 hours . Continuation of rFVIIa >= 80 ug/kg with decreasing frequency until proper hemostasis achieved . Platelet transfusion daily or when necessary for first 3 days . 46

47. EPIDERMOLYSIS BULLOSA (EB) : It is a rare genetic disorder characterized by skin fragility with blister formation occurring spontaneously or following minor trauma such as gentle pressure or friction . It is referred to as the “ worst disease one has ever heard of “ . CAUSE : Mutations in alpha 6 beta 4 integrin causes EB which can be inherited by the offsprings in either of the following ways – Passed on from one parent who has autosomal dominant inheritance disease Passed on from both parents showing autosomal recessive inheritance . Arise in the affected person as a new mutation that can be passed on . DETECTION : Usually signs of EB first appear in babies or toddlers . They include – Fragile skin that blisters easily , especially on hands and feet . Nails that are thick or don’t form . Blisters inside the mouth and throat . 47

48. d) Thickened skin on the palms and soles of the feet . Scalp blistering , scarring and hair loss ( scarring alopecia ) . Thin appearing skin ( atrophic scarring ) Tiny white skin bumps or pimples ( milia ) Dental problems such as tooth decay from poorly formed enamel Difficulty in swallowing ( dysphagia ) Itchy painful skin Pigmented (coloured) areas of skin This blisters may appear in response to minor injury , even from heat , rubbing , scratching or adhesive tape . In severe cases , blisters occur inside body such as lining of mouth or stomach . Adults with EB have increased risk of developing skin cancer while some patients develop problem with urinary systems such as blistering or scarring of tube that carries urine out of the bladder (urethra) . DIAGNOSIS : The doctor may suspect of the EB disease by its characteristic degree of affected skin. The laboratory tests undergone to confirm disease includes - 48

49. SKIN BIOPSY FOR IMMUNOFLUORESCENT MAPPING : Small sample of affected skin Removed Examined with microscope Identify layers of skin involved Reflected light This test also identifies whether the proteins needed for skin growth are functioning or not . GENETIC TESTING : Sometimes used to confirm diagnosis as most forms of Epidermolysis Bullosa are inherited . In this , small blood sample is taken and sent to lab for analysis . PRENATAL TESTING : Families with history of EB may want to consider prenatal testing and genetic counseling . This is done usually about 11 weeks of pregnancy . 49

50. FIGURE : Laboratory diagnosis of EB ABBREVIATIONS : IFM – Immunofluorescence mapping MLPA : Multiple ligation dependent probe amplification NGS : Next generation sequencing qPCR : Quantitative polymerase chain reaction TEM : Transmission electron microscopy WES : Whole genome sequencing REFERENCE : Clinical practise guidelines for laboratory diagnosis of epidermolysis bullosa by C.Has et al., 2019 https://pubmed.ncbi.nlm.nih.gov/31090061/ 50

51. TREATMENT : PHARMACOLOGICAL METHODS : Medications can help control pain and itching and treat complications such as infection in bloodstream ( sepsis ) . Doctor may prescribe oral antibiotics if wounds show signs of widespread infection (fever , weakness , swollen lymph glands) . NON- PHARMACOLOGICAL METHODS : SURGERY : Widening esophagus : Surgical dilation of esophagus relieves narrowing caused due to blistering and scarring making difficult to eat . This makes easier to travel food from mouth to stomach . Placing a feeding tube : Gastronomy or feeding tube may be implanted to deliver food directly to stomach . Grafting skin : Suggested when scarring affects function of hand . Restoring mobility : Repeated blistering and scarring can cause fusing of fingers or toes or abnormal bends in the joints (contractures) so the doctor 51

52. may recommend surgery to correct these conditions if they interfere with normal function . GENE THERAPY : At the beginning of the therapeutic era for more than 10 years ago , most obvious consideration was to replace a defective gene with gene therapy using retrovirus mediated gene correction in keratinocytes and grafting which was indeed successful . However , it turns out that this form of gene therapy brings with it complex issues and questions relating to technological development of gene vectors , oncogenic potential and future risk of malignancy , duration of therapeutic effects etc . Current research on EB focused on different approaches including use of gene- corrected patient’s own iPS cells , gene editing technologies and polymer mediated DNA delivery systems . It is important to note that no gene therapy approaches have been approved for EB as all these therapy developments are currently experimental and in in-vitro or preclinical state , with a few exceptions that are tested in early clinical trials on individuals with EB . An interesting option is so called “natural gene therapy” based on use of cells or tissue derived from revertant mosaic patches in patient’s skin . Revertant mosaicsm is a relatively common phenomenon in human genetic disorders and is based on the fact that in individual cells , the inherited mutation is compensated by a second somatic mutation that restores the expression of lacking protein and reverts the disease phenotype in the cell . 52

53. Revertant cells proliferate clonally and mosaic skin patches can be found in all EB types . 3) CELL THERAPIES : Intradermal injections of fibroblast improved dermal-epidermal adhesion in EB mouse model for several months but treatments for patients revealed this therapeutic modality to be very painful , not well tolerated and not consistently efficacious . Indeed , one study found similar effects on wound healing with both fibroblast injections and vehicle injections . Systemic therapy for severe EB by bone marrow transplantation improved symptoms in some patients but did not cure EB . The rate of adverse effects was high and some patients died from complications of bone marrow transplantation . Intravenous infusions with mesenchymal stromal stem cells (MSC) from bone marrow relieved symptoms such as itch and improved the general well-being of children but did not increase collagen V in skin . Current clinical trials are studying the effects of different kinds of MSC in adults with EB . 53

54. PROTEIN THERAPY : Protein therapy approach using intravenous or intradermal recombinant collagen VII injections for treatment of EB seemed promising at preclinical level but further development and clinical assessment are still required before its suitability for treatment of patients can be determined . ANTISENSE OLIGONUCLEOTIDES : Antisense oligonucleotides treatment of cells lead to skipping of mutated exons at RNA level thus restoring synthesis of nearly normal protein that lacks a small fragment encoded by deleted exon. Preclinical testing in mice suggested that antisense oligonucleotides based exon skipping improve skin stability by partially functional collagen VII . PTC READ THROUGH : This is based on the fact that approximately 10% of genetic diseases are caused by mutations that generate premature termination codons (PTC) and lead to nonsense mediated mRNA decay . Efficiency of read through depends on local nucleotide micro environment of mutations and careful selection of suitable mutations as an essential prerequisite for successful treatment . 54

55. Gentamicins have demonstrated some efficacy for COL7A1 and LAMB3 mutations in vitro and clinical study on EB patients treated with topical gentamicin showed increased collagen VII in skin . Amlexanox , anti-inflammatory drug approved in some countries can induce PTC read through and enhances collagen VII protein synthesis in vitro in EB cells carrying nonsense mutations . CURRENTLY RECRUITING CLINICAL THERAPY TRIALS FOR EB : 55 THERAPY INVESTIGATIONAL DRUG EB TYPE Gene therapy Transplantation surgery of genetically corrected cultured epidermal autograft (ATMP) Genetically corrected cultured epidermal autograft (ATMP) KB103 , non-integrating replication - incompetent herpes simplex virus vector expressing human collagen VII protein FCX-007 , genetically modified autologous human dermal fibroblasts Junctional EB (JEB) with COL17A1 mutations Recessive dystrophic EB (RDEB) DEB RDEB

56. THERAPY INVESTIGATIONAL DRUG EB TYPE Antisense oligonucleotides QR-313 , an antisense oligonucleotide (AON) DEB with mutations in exon 73 of COL7A1 PTC read through Gentamicin (intravenous) RDEB Cell therapy Serial mesenchymal stem cell (MSC) infusions from a related donor . Allogenic stem cell transplantation and off the shelf mesenchymal stem cells All EB types All EB types Symptom relief therapies Antifibrotic Anti -inflammatory Losartan , systemic Diacerein , topical Pharmacokinetics , safety of diacerein after maximum use Oleogel , topical RDEB EB simplex (ESB) EBS All EB types 56

57. REFERENCE : Indian Dermatology Online Journal THANK YOU MADE BY : DIVYA SONI (M.Sc) 57 THERAPY INVESTIGATIONAL DRUG EB TYPE d) BPM31510 , 3% cream , topical e) Sirolimus , topical All EB types EBS