The Physiology and Pathology of Coagulation Factor XI Dr. David Gailani

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1 The Physiology and Pathology David Gailani, MD Departments of Pathology and Medicine Vanderbilt University, Nashville, TN 1 Discovery of Factor XI Robert Rosenth al and cow orkers charact eriz ed a b leed ing disord er in tw o sist ers and their uncle As in hemophilia, the plasma clotting time (similar to the partial thromboplastin time [PTT]) was prolonged, while th e prothrombin time and b leeding t ime were normal; Th eir was no evid ence of an inhibitory anticoagulant Unlike hemophilia, bleeding was relatively mild, (primarily occurring with tooth extraction), and males and females w ere affected Mixing plasma from these patients with plasma from patients with true hemoph ilia (factor VIII d eficiency), or th e clinically similar disorder Ch ristmas disease (factor IX deficiency), corrected the prolonged clotting time Based on th ese observations, a th ird plasma factor, tentat ively named plasma thromboplastin ant eced ent (PTA), was proposed th at, when missing, could confer a hemoph ilia-lik e disorder Congenital deficiency of PTA - Rosenth al syndrome or hemoph ilia C Th e plasma factor missing in congenital PTA deficiency was designated factor XI 2 XII Prekallikrein HMWK XIIa Coagulation Cascade XI XIa X Extrinsic Intrinsic IX IXa PL VIIa/ TF (III) VIIIa Common Xa PL Phospholipid Calcium ions II PL Va Thrombin (IIa) Proteases cofactors Fibrinogen (I) Fibrin (Ia) 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Apple 1 Apple 2 Apple 3 After McMullen et al., Biochemistry 3:256-26; 1991 Apple 4 4 Factor XI - Gel Filtration Plasma fxi Recomb. fxi fxi-ala 321 Rabbit fxi Prekallikrein fxi - reduced kda Gailani et al., Blood 97: ; 21 Sinha et al., Biochem. J. 367:49-56; 22 OD 28 nm Retention time (min) 5 XII Prekallikrein HMWK XIIa Intrinsic Pathway Protease Deficiencies XI XIa X IX IXa VIIIa PL Xa VIIa/ TF (III) PL Phospholipid Calcium ions Prot eases cofact ors II PL Va Thrombin (IIa) Fibrinogen (I) Fibrin (Ia) 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Tissue Factor 7 Phenotypes of Plasma Coagulation Coagulation Factor Null Mice Vessel Injury IX IX VIIa TF X TFPI IXa VIIIa X XIa? Xa Va Prothrombin Thromb in Fibrinogen Fibrin XI Activation Inhibition Intrauterine Cofactor lethal Viable -Protease bleeding Viable - little bleeding 8 Factor XI Activation by Thrombin on Platelets 6 5 Factor XIa (nm) FXI + IIa FXI + IIa + APt FXI + IIa + APt + HK/ Zn FXI + IIa + APt + II/Ca FXI + UPt + II/Ca Baglia FA and Walsh PE, Biochemistry : The screen versions of these slides have full details of copyright and acknowledgements 3

4 A2 A3 A2 A3 FXI FXI HK FXI A1 A3 Zn 2+ A2 A3 FXI FXI A3 FXI PT A1 A3 IIa, XIIa, XIa FIX FIX A2 A3 FXIa FXIa FIXa A3 After Gailani et al., Blood 97: ; 21 1 Thrombin Generation in Platelet Rich Plasma 2 Thrombin (nm) Normal plasma XII dp XI dp XI dp + 1% fxi XI dp + 1% fxi Fibrin Clot Resistance to Fibrinolysis Microtiter Plate Assay Plasma Tissue factor Urokinase Clot formation and integrity are d etermined by ch anges in OD 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Fibrin Clot Resistance to Fibrinolysis.5.4 A 45 nm Normal plasma XII dp XI dp XI dp + XI XI dp - urokinase Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Fibrinogen Plasminogen Thrombin TAFI ( ) tpa Plasmin TAFIa ( ) fibrinolysis D E D Factor XIIIa Fibrin 14 Physiology of Factor XI Summary Classical intrinsic pathw ay (fact or IX activation by fact or X Ia) is prob ably not a mechanism for initiat ion of fibrin formation in vivo Factor XI is now thought to function in sustaining factor IX activation, and ultimately thrombin generation after initiation of fibrin clot formation Th e physiologic mechanism for factor XI activation is uncertain, but thrombin mediated activation on the platelet surface is a possibility Sustained th rombin production through fact or XIa and factor IX may b e required in areas where tissue factor is limiting, or for maintaining fibrin integrity in tissues with high fibrinolytic activity 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Factor XIc (U/dl) Bleeding in Bleeders Bolton-Maggs Haemophilia 2, sup 1:1-19 Non-bleeders Normal Mild Severe 54 families 249 individuals 128 partial deficiency 45/128 (35%) bleed 16 Apple 1 Type II E117X X Apple 2 After McMullen et al., Biochemistry 3:256-26; 1991 Asaki et al., Proc. Natl. Acad. Sci. (USA) 86: ; 1989 L Apple 4 Apple 3 Type III F283L 17 Bleeding in Type II a nd III Mutations Patient Gen otype # of Subjects Factor XIc aptt (% normal) (seconds) II / II 16 ±.5 18 ± 18 II / III ± ± 22 III / III ± ± 18 II / WT ± ± 7.4 III / WT ± ± 7.4 Co ntrols ± 3 ND Genotype Tonsillectomy, Nasopharynx, or Urinary tract Asakai et al., N. Engl. J. Med. 1991; 325:153-8 Other sur gical procedures Tooth Extraction Circumcision II / II 5/6 (5)* 4/8 (6) 7/11/ (11) /5 II / III 5/7 (7) 3/2 (8) 11/19 (19) 1/13 III / III 2/3 (3) 1/1 (6) 8/1 (1) /4 Total 12/16 (15) 8/38 (2) 26/4 (4) 1/22 * procedures with bleeding/total procedures (no. of patients) 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Type II Wild type Normal Heterozygote Homozygote Activity 1% Antigen 1% 5% 5% 19 Factor XI Domain Mutations Control Wild type Phe 283 L eu Gly 35 Glu F283LType III L S-Cysteine E G35E Nagoya II After Meijers et al., Blood 79: ; Types II a nd III Mutations Type II mutations Type III mutations Activity 1% Antigen 1% 5% 5% 6% 6% 1% 1% Wild type Type III mutant 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 G155E P52L V2A Q226 R S248N C321 F G555E T57 5M P52L patients from 8 kindreds 96 Jewish patients (7 k indreds), 29 non-jewish patients (1 k indreds) 78 h omozygotes, 47 h eterozygotes No CRM+ patients Homozygotes - Jewish Homozygotes - non-jewish Heterozygotes - Jewish Heterozygotes - non-jewish No. of patients Factor XI:C % 3.5 ± ± ± ± 1.5 Saito et al., J. Lab. Clin. Med. 16: , Autosomal Dominant? S-S 25% Wild type allele Mutant allele Wild type peptide Mutant peptide S-S 5% S-S 25% 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 Dominant Mutations CRM + mutation CRM - mutation Activity 1 % 25+ % <1+ % 25% Antigen 1 % 1 % 1 % 25% Wild type Mutant25 Factor XI Deficient Kindred 12? Male Female After Litz et al., Am. J. Hematol. 28:8-12; 1988 Bleeding Control Wild type - CM Wild type - Lysate Tyr 398 Cys Trp 569 Ser Control Wild Type Phe 283 Leu Gly 35Glu Factor XI Mutations Gly 4Val Catalytic Domain Nagoya III G 4 V V S W569S 35 S-Cysteine Kravtsov et al., Blood 14: ; 15: ; The screen versions of these slides have full details of copyright and acknowledgements 9

10 pjvcmv + FXI cdna Transfection 293 fibroblasts Renilla Luciferase Media Lysate Factor XI ELISA 28 Relative factor XI concentration (%) Relative factor XI concentration (%) WT Type II E117X Kravtsov et al., Blood 14: ; 24 fxi in media fxi in lysates Type Nagoya Nagoya W569 S C III II III P283L G35E G4V fibroblasts pjvcmv + FXI cdna pjvcmv + FXI mutant cdna Transfection Renilla Luciferase Med ia Lysat e Factor XI ELISA 3 The screen versions of these slides have full details of copyright and acknowledgements 1

11 14 Relative factor XI concentration (%) fxi in media Relative factor XI concentration (%) Kravtsov et al., Blood 14: ; 24 C WT type II type III Nagoya Nagoya W569 II III S - C fxi in lysates 31 Dominant Mutations Normal Gly 4 Val and Trp 569 Ser Heterozygote Homozygote Activity 1% 25% Antigen 1% 25% Wild type Mutant 32 Classification System for CRM- Category 1: mutat ions that prevent synth esis of th e fx I polypeptide (deletions, premature stop codons, splice variants promoter d efect s) Category 2: mutations that result in a defect in protein dimer formation (type III, Nagoya II) Category 3: mutations that result in peptides that can not be secret ed, b ut th at d o form dimers Kravtsov et al., Blood 15: ; The screen versions of these slides have full details of copyright and acknowledgements 11

12 Summary Excessive bleeding in factor XI deficient patients may be influenced by: factor level site and type of injury other coagulation defects or procoagulant factors Th e t ype II and t ype III mut ations prob ably follow an aut osomal recessive inh eritance patt ern, with symptoms primarily in those with severe deficiency (h omozygotes) Th e d imeric structure of factor X I predicts that d ominant negative forms of fact or XI d eficiency should occur through h eterodimer formation Dominant negat ive mut ations are likely t o be responsib le for cases of families with apparent autosomal dominant factor X I deficiency 34 Treatment Bleeding in factor XI deficiency is usually trauma or surgery induced and may occur immediately after injury or after several hours delay Bleeding, once it occurs, tends to persist in the absence of treatment It is recommended that all factor XI deficient patients undergoing surgery receive replacement therapy, regardless of past bleeding history The target level for replacement is 3-4% of the normal level prior to surgery, with the goal of maintaining this level for about one week fresh frozen plasma factor XI concentrates (thrombosis in 1%) Anti-fibrinolytic agents such as tranexamic acid may be used for tooth extraction without plasma replacement Replacement therapy is not routinely recommended prior to vaginal delivery or caesarean section (reserve for post-partum hemorrhage) After Salomon and Seligsohn, Haemophilia 1: ; Inhibitors Alloantibody factor XI inhibitors may complicate factor replacement in factor XI deficient patients A study by Salomon and coworkers in Israel demonstrated that 33% of homozygotes for the type II (Glu117Stop) mutation develop these inhibitors Inhibitors were associated with bleeding during surgery It may be prudent to identify these patients by DNA analysis, and avoid plasma replacement therapy where possible (anti-fibrinolytic therapy for tooth extraction, for example) In bleeding factor XI deficient patients who do not respond to replacement therapy because of inhibitors, recombinant factor VIIa has been used in several cases to stop bleeding Salomon et al., Blood 11; 88: ; 23 After Salomon and Seligsohn, Haemophilia 1: ; The screen versions of these slides have full details of copyright and acknowledgements 12

13 Factor XI and Thrombotic Disease Leiden thromboph ilia study (Meijers et al., NEJM 2; 342:696-71) - individ uals in the upper 1% of th e normal population for factor IX or fact or X I levels h ave a ~ 2-fold increased risk of venous thromboembolic disease Elevat ed factor X I levels may increase risk of art erial cerebrovascular thrombotic events (Yang et al., Blood 25; 16:737a) Severe fact or X I d eficiency d oes not provid e protection from myocardial infarction (Salomon et al., J. Thromb. Haemost. 23; 1:658-61) Thrombus formation in tissue factor coated sh unts in primates is inh ibited by blocking factor XI (Gruber and Hansen, Blood 23; 12:953-5) Fact or XI d eficiency allows mice with t otal d eficiency of prot ein C to survive into adulthood (Chan et al., Am J. Pathol. 21; 158:469-79) 37 Factor IX and Factor XI Deficient Mice Factor IX null mice - Lin et al., Blood 9; ; 1997 Factor XI null mice - Gailani et al., Blood Coag. Fibrinol. 8; ; 1997 Assay Wild Type Factor IX -/- Factor XI -/- Partial Thromboplastin Time (sec) Prothrombin time (sec) Tail Bleeding Time (sec) 265 ± ± ± Carotid Artery Occlusion Ferric Chloride Model Doppler flow probe Ferric chloride Blood flow 39 The screen versions of these slides have full details of copyright and acknowledgements 13

14 Carotid Artery Injury Model Ferric Chloride Model Relative Blood Flow [FeCl 3 ] 2.5% 3.% 3.5% 5% 7.5% 1% Wang et al., J. Thromb. Haemost. 3:695-72; 25 4 Relative Blood Flow Factor XI Deficient Mice Ferric Chloride Model % 2 * * % WT FXI -/- FIX -/ % * * % WT FXI -/- FIX -/- 6 1% Area under the curve Wild type Factor XI -/- Factor IX -/- * Statistical significant difference from wild type % Wang et al., J. Thromb. Haemost. 3:695-72; 25 WT FXI -/- FIX -/- 41 Relative Blood Flow Relative Blood Flow Factor XI Deficient Mice Ferric Chloride Model Aspirin Contro l Contro l 1 mg /kg 1. 1 mg /kg 3 mg/ kg.8 3 mg/ kg 1 mg/ kg 1 mg/ kg %.2 5% Heparin 2 U/kg U/kg U/kg.4 2 U/kg.2 3.5%.2 Control 5% Control Wang et al., J. Thromb. Haemost. 3:695-72; The screen versions of these slides have full details of copyright and acknowledgements 14

15 Factor XI and Thrombotic Disease Summary Epidemiologic studies in humans indicates that factor XI may contribute to venous and arterial thrombotic disease Th e procoagulant and anti-fibrinolytic activities of factor XI could contribute to thrombus formation Factor IX deficient mice have a significant bleeding disorder while factor XI deficient mice have little excessive bleeding In a carot id art ery thromb osis model fact or X I and fact or IX deficiency appear to have comparable protective effects This suggests that the role of factor X I in path ologic thromb us formation differs substantially from its role in normal h emostasis As fact or XI d eficient pat ients d o not suffer from spontaneous bleeding, therapeutic inhib ition of factor X I may be a safe and effect ive method for t reating or preventing thromboembolic disease The screen versions of these slides have full details of copyright and acknowledgements 15