The Hemostatic Pathway & Novel Anticoagulants

1. The Hemostatic Pathway & Novel Anticoagulants David Bolos, PGY5 Fellow Talk 9/6/2016

2. Overview • Phases of the Hemostatic Process • Control Mechanisms of Clotting • Novel Anticoagulants/Oral Anticoagulants • Transitioning between Anticoagulants • Management of Bleeding • Anticoagulants in Development

3. Phases of the Hemostatic Process • Endothelial Injury and Formation of the Platelet Plug • Propagation of the Clotting Process by the Coagulation Cascade • Termination of Clotting by Anti-thrombotic Control Mechanisms • Removal of the Clot by Fibrinolysis

4. Formation of the Platelet Plug • Platelets are activated at the site of vascular injury to form a “plug” • Injury to endothelium  exposure of blood to subendothelial elements  endothelial cell activation promotes further recruitment • Functional response of activated platelets (FOUR) • Adhesion: deposition to subendothelial matrix • Aggregation: platelet-platelet cohesion • Secretion: release of platelet granule proteins • Procoagulant activity: enhancement of thrombin generation

5. Platelet Activation • There are a number of physiologic platelet stimuli • Adenosine Diphosphate (ADP) • ADP binds to two G-protein coupled purinergic receptors, P2Y1 & P2Y12. • P2Y1  Calcium mobilization, platelet shape change, reversible aggregation • P2Y12  platelet secretion and more stable aggregation (Clopidogrel blocks activation) • Epinephrine • Thrombin (potent) • Thrombin activation is mediated by a family of G-protein coupled protease-activated receptors (PARs). Platelets have a dual receptor system for thrombin, with 2 distinct receptors PAR1 & PAR4. • Collagen (potent) • Integrin glycoproteins GPIa/IIa and GPVI are the two most important platelet collagen receptors (bleeding diathesis with deficiencies)

6. Platelet Adhesion • Following activation  platelets undergo shape changes (elongated pseudopods) making them “sticky” • Adhesion Primarily mediated by binding of platelet surface receptor GP Ib/IX/V complex to VWF in subendothelial matrix. • Deficiency of any component of GP Ib/IX/V complex or VWF leads to congenital bleeding disorders • Bernard-Soulier Disease • Von Willebrand Disease • Other adhesive interactions contribute  GP Ia/IIa to collagen fibrils in the matrix.

7. Platelet Aggregation • Results in both exposure and conformational changes in the GP IIb/IIIa receptor on platelet surface  binding of immobilized VWF and Fibrinogen • GP IIb/IIIa (integrin) undergoes a conformation change following platelet stimulation  higher affinity fibrinogen receptor • When GP IIb/IIIa binds to immobilized VWF, the cytosolic portion of the activated complex binds to cytoskeleton  mediating platelet spreading & clot retraction • Glanzmann Thrombasthenia  mutations in the gene for either the alpha IIb or beta-3 subunit • GP IIb/IIIa antagonists in treatment of CAD (Abciximab, Eptifibatide)

8. Platelet Secretion • Two types of Granules: alpha & dense • Alpha Granules: many proteins including: • Fibrinogen (source at sites of injury in addition to source in plasma) • VWF: Von Willebrand Factor • Thrombospondin & Fibronectin (adhesive proteins, stabilize aggregates) • PDGF (mediates tissue repair), • Platelet Factor 4 (chemokine moderating effects of heparin-like molecules) • P-selectin (cell adhesion molecule) • Dense Granules: • ADP, ATP, iCal, Histamine, Serotonin

9. Procoagulant Activity • Involves both exposure of procoagulant phospholipids (primarily phosphatidylserine), and • the subsequent assembly of the enzyme complexes in the clotting cascade on the platelet surface.

10. Formation of Platelet Plug Overview • https://www.youtube.com/watch?v=0pnpoEy0eYE

11. Clotting Cascade & Propagation of the Clot • The central feature is the sequential activation of a series of proenzymes (zymogens) to active enzymes • Consists of Intrinsic & Extrinsic pathways (see handout 1) • The function of the active enzymes is markedly facilitated by the formation of multiple component macromolecular complexes (The X-ases, Prothrombinase) • All of the procoagulants are synthesized in Liver, • Except VWF (megakaryocytes & endothelial cells) and • Factor VIII (produced by endothelial cells) • Post-translational modification Occurs • Vitamin K dependent Procoagulants (II, VII, IX, X) and anticoagulants (Proteins C & Protein S) • Vitamin K dependent carboxylated glutamic acid residues function as Ca-binding sites that are important in the assembly of the membrane bound macromolecular procoagulant complexes

12. Coagulation Cascade • https://www.youtube.com/watch?v=s4FoSf6Yi_s • Handout 1

13. Clotting Cascade & Propagation of the Clot • Intrinsic Pathway: initiated by exposure of blood to a negatively charged surface (such as celite, kaolin, or silica in the in vitro activated partial thromboplastin clotting time, aPTT) • Extrinsic Pathway: activated by tissue factor exposed at the site of injury or tissue factor-like material (thromboplastin, in the in vitro prothrombin clotting time, PT) • Both pathways converge on the activation of Factor X • X, as a component of Prothrombinase  thrombin • Thrombin  converts fibrinogen from a soluble plasma protein into an insoluble fibrin clot (testing in the in vitro thrombin time, TT)

14. Thrombin Generation • Primary physiologic event in initiating clotting is exposure of TF at wound site and interaction with Factor VIIa. • Small initial amount of Thrombin generated then activates Factor XI (in a feedback manner)  amplification of thrombin generation • This then further leads to downstream amplification of factors V, VIII, IX, platelets, etc.  explosive thrombin generation (see Handout 2)

15. Multicomponent Complexes • Extrinsic X-ase: • activated Factor VIIa (protease), TF (cofactor), Factor X (substrate)  activates Factor X and Factor IX • Intrinsic X-ase: • Factor IXa (protease), activated factor VIIIa (cofactor), Factor X (substrate)  activates Factor X • Prothrombinase: • Factor Xa (protease), Factor Va (cofactor), Prothrombin/Factor II (substrate)  Thrombin (IIa) • Protein C Anticoagulant Complex: • Thrombin (enzyme), Thrombomodulin (cofactor), Protein C (substrate)  anticoagulation control mechanisms

17. Critical Role of Polyphosphate (PolyP) • Inorganic polyphosphate (PolyP) is a linear highly anionic polymer of orthophosphates linked by high-energy phosphoanhydride bonds. • In microorganisms, PolyP is synthesized from ATP and may serve as an energy store allowing bacteria to resynthesize ATP in times of stress. • PolyP is also stored in human platelet dense granules and is efficiently released upon platelet activation

18. Critical Role of Polyphosphate (PolyP) • Multiple anion charges, PolyP is a potent procoagulant  physiological negatively charged surface that triggers blood coagulation via intrinsic pathway. • Source in this setting may be derived from injured tissue • PolyP’s other effects on clotting: • accelerates Factor V activation, • Dampens the inhibitory effects of Tissue Factor Pathway Inhibitor (TFPI), • enhances fibrin polymerization, • accelerates Factor XI activation by Thrombin

19. Deficiencies of Initial Intrinsic Pathway Proteins • Physiological relevance of the initial complex of intrinsic pathway is not established • Deficiencies in these proteins (Prekallikrein, HMWK, and Factor XII) are not associated with bleeding tendencies • Mutations in Factor XII have been linked in a subset of patients with Hereditary Angioedema and normal C1 inhibitor levels • However, injury-related bleeding is seen with deficiency of Factor XI, • suggesting it plays important hemostatic role, • independent of contact activation and Factor XII (thrombin feedback activates Factor XI, with PolyP as cofactor).

20. Continuation of the Cascade • Factor Va binds Factor Xa prothrombinase complex  Thrombin (IIa) • Thrombin converts Fibrinogen  Fibrin  polymerization • Activated Factor XIIIa stabilizes and crosslinks overlapping Fibrin strands • Factor XIIIa with fibrinogen controls volume of RBC’s trapped within a thrombus  Clot Size

21. Control Mechanisms & Termination of Clotting • Hemostatic response is rapid and if left unchecked would lead to thrombosis, vascular inflammation, and tissue damage • Modulated by number of mechanisms • Dilution of procoagulants in flowing blood • Removal of activated factors through the reticuloendothelial system (esp in Liver), • Control by natural antithrombotic pathways anchored on the vascular endothelial cells (maintaining fluidity of blood)

22. Control Mechanisms & Termination of Clotting • Termination Phase of Coagulation Process • Involves two circulating enzyme inhibitors Antithrombin and Tissue Factor Pathway Inhibitor (TFPI) • & clotting-initiated inhibitory process, The Protein C pathway • Prostacyclin, Thromboxane, and Nitric Oxide modulate vascular and platelet reactivity • Termination phase is critical  problems lead to thrombotic disorders such as Antithrombin, Protein C, and Protein S deficiency

23. Antithrombin and Heparin • Antithrombinis a circulating plasma protease inhibitor, neutralizing many enzymes including thrombin, factors Xa & IXa, XIIa & XIa. • Two active functional Sites: the reactive center (Arg393-Ser394), and the Heparin Binding Site located at the amino-terminus • The binding of endogenous/exogenous heparins to the Heparin Binding Site on AT produces a conformational change in AT which accelerates the inactivating process 1000x to 4000x. • See Handout 4

24. Activated Protein C & Protein S • APC inactivates Factors Va & VIIIa, thereby inactivating the Prothrombinase and the intrinsic X-ase, respectively • Factor Va is firstcleaved at Arg506 and then at Arg306 & Arg679 by APC • Peptide bone cleavage at Arg506 is essential for the exposure of cleavage sites at Arg306 & Arg 679

25. Activated Protein C & Protein S • Factor V Leiden, in which Arginine at position 506 is replaced by Glutamine, is not susceptible to cleavage at position 506 by APC and is therefore inactivated more slowly hypercoagulable state • Protein S circulates in two forms, free (active) and bound to C4b binding protein of complement system • C4b is an acute phase reactant, inflammatory states increase its concentration, decrease free Protein S  enhancing likelihood of thrombosis in these states

26. Tissue Factor Pathway Inhibitor • Synthesized by the microvascular endothelium • TFPI circulates in the plasma, low concentrations compared to AT • Inhibits Factor X activation in two ways: • Direct inhibition of Factor Xa • Complexes with Factor Xa and the complex inhibits TF/FVIIa  impairing the triggering mechanism of the Extrinsic pathway

27. Clot Elimination & Fibrinolysis • To restore vessel patency following hemostasis, the clot must be organized and removed by the proteolytic enzyme Plasmin in conjunction with wound healing and tissue remodeling • Plasminogen binds fibrin and tissue plasminogen activator (tPA). • This ternary complex leads to conversion of plasminogen  Plasmin (active) • Plasmin has broad substrate specificity  cleaves fibrin, fibrinogen, plasma proteins, clotting factors

28. Clot Elimination & Fibrinolysis • Plasmin cleaves fibrin strands  Fibrin degradation products  one major product is D-dimer • (consists of two D-domains from fibrin adjacent monomers that have been crosslinked by Factor XIIIa) • Plasmin also cleaves Factor XIIIa  reduces fibrin crosslinking • Plasmin-plasminogen activator system is complex, and is regulated with inhibitors as well (plasminogen activator inhibitors, PAI-1/PAI-2)

30. https://www.youtube.com/watch?v=YnG3UTpWwW4 • https://www.youtube.com/watch?v=uE3AfRT4Vsw

31. Novel Anticoagulants/Oral Anticoagulants • Options for anticoagulation have been expanding • In addition to heparins and vitamin K antagonists, anticoagulants that directly target the enzymatic activity of thrombin and Factor Xa are available • Appropriate use requires knowledge of their individual characteristics, risks, benefits • Here are practical aspects of Novel Anticoagulants • Common uses include VTE treatment/ppx, Afib, Unstable Angina, MI, CVA, PCI, and HIT

32. Direct Thrombin Inhibitors (DTIs) • Bind to the active site of the thrombin enzyme or to two sites: the active site and “exosite I”, a positively charged region of the thrombin molecule that is physically separated from the active site. See Handout 3 • “Exosite I” is also the site of interaction of many physiologic thrombin substrates (fibrinogen, Factor V, Protein C, Thrombomodulin, and thrombin receptors (PAR1/PAR4) on platelets) • Thrombin is activated in both circulating and clot-bound forms. DTIs are able to block BOTH forms • (Heparins are only able to inactivate Thrombin in the fluid phase, via antithrombin)

33. Direct Thrombin Inhibitors (DTIs) • Parenteral DTIs include Bivalirudin, Argatroban, and Desirudin • The ONLY oral DTI available for clinical use is Dabigatran (Pradaxa)

34. Direct Factor Xa Inhibitors • Acts immediately upstream to of thrombin, at convergence point of intrinsic/extrinsic pathways • Prevents amplified thrombin generation • (one molecule of Xa can cleave 1000 molecules of prothrombin) • Xa inhibitors bind to the active site of Xa and inhibit factor Xa activity without requirement of cofactors • Similar to thrombin, Xa is active in circulating and clot forms, and Xa inhibitors block BOTH forms

35. Diret Factor Xa Inhibitors • No parenteral Factor Xa inhibitors in clinical use • Severeal oral direct Factor Xa inhibitors available: • Rivaroxaban (Xarelto) • Apixaban (Eloquis) • Edoxaban (Lixiana) • Nomenclature: -xaban (Xa-Ban)

36. Comparison with Heparin/Warfarin • Differ in efficacy depending on clinical setting • Dosing • Dietary restrictions • Ability to tolerate oral intake • Monitoring Therapy • Drug adherence • Drug interactions • Time in therapeutic range • Cost $$$ • Risks & Reversal Agents • Monitoring reversal (INR/PTT/PT/TT/Xa) • Advantages/disadvantages must be individualized to the patient and clinical setting

37. Settings in which Heparin/Warfarin Preferred • Prosthetic heart valves • Pregnancy • Renal Impairment • Antiphospholipid Syndrome • (randomized study comparing Rivaroxaban to Warfarin in progress) • Compliance • (lack of monitoring, short half lives) • Gastrointestinal Disease • Dosing Convenience • (dabigatran & Apixaban require BID dosing) • Cost $$$

38. Novel Oral Anticoagulants See Handout 5

39. Novel Oral Anticoagulants

40. Novel Oral Anticoagulants

41. Bivalirudin • Synthetic 20 amino acid peptide that binds to the thrombin catalytic site and exosite I, reversibly inhibiting • Peptide sequence is analog of hirudin, • protein extracted from salivary gland of medicinal leech • Indications are PCI and HIT • Administered at dose of 0.75mg/kg IV bolus, followed by 1.75mg/kg/hr during procedure (CrCl<30, 1 mg/kg/hr rate) • Metabolized in Kidney/Liver • Can be hemodialyzed

42. Argatroban • Synthetic peptide-based direct thrombin inhibitor that interacts with active site of thrombin • Short in vivo plasma half life (40-50 min) • Hepatically metabolized • (dosing adjustments for hepatic impairment, not for renal) • HIT: 2mcg/kg/min CIVI, monitoring q2hrs with aPTT • Dose adjusted to achieve target 1.5 to 3x the initial baseline aPTT, • not to exceed 100 seconds • Also affects PT, so when transitioning to warfarin must use adjusted INR target • Also used for PCI in patients with HIT, bolus of 350 mcg/kg over 3-5 minuts, followed by 25 mcg/kg/min

43. Dabigatran (Pradaxa) • Orally administered prodrug converted in liver to dabigatran, an active direct thrombin inhibitor • (inhibts clot and circulating thrombin) • Half life ~ 12 to 17 hours (normal renal function) • Capsules should only be dispensed and stored in original bottle (with desiccant) or blister package • (product breakdown from moisture) • Not to be crushed or have capsule opened (increases)

44. Dabigatran (Dosing) • Fixed dose without monitoring • Maximum effects achieved within 2-3 hours of ingestion • Dosing differs based on indication and renal function • VTE ppx in surgical patients • 110mg 1-4 hours after surgery, followed by 220mg daily for 28-35 days (hip) or 10 days (knee replacement) • Treatment VTE • 150mg BID (CrCl>30) • Stroke Prevention in Afib • 110mg BID or 150mg BID (CrCl>30)

45. Dabigatran (Dose Modifications) • Renal Insufficiency • Dose reduction for CrCl 15–30, generally by 50% • CrCl<15, avoid use • P-glycoprotein Inhibitors/Inducers (See Handout 5 & 6) • Dabigatran is a Substrate for P-glycoprotein, • inducers reduce anticoagulation effect, • inhibitors increase anticoagulation effect • NOTE: not metabolized by Cytochrome P450 system • Obesity • Avoid if BMI > 40 kg/m2 or Weight > 120 kg (same for all DOAC’s) • European labeling: Age>75, dose reduce

46. Dabigatran (Risks) • As with all anticoagulants, bleeding risk increased • Antidote is available  PraxBind (Idarucizumab) • Overall bleeding rates similar to that of warfarin • Dabigatran may be associated with slightly lower rate of intracranial hemorrhage and death, and • Slightly higher risk of GI bleed at 150mg BID dose, but not the 100mg BID dosage • Non-bleeding GI events (dyspepsia, dysmotility, GERD) were twice as common compared to Warfarin (RE-LY trial) • Black Box Warning regarding the risk of thrombotic events following premature discontinuation

47. Rivaroxaban (Xarelto) • Orally available direct factor Xa inhibitor • Half life ~7-17 hours • Given at a fixed dose without monitoring • 15mg and 20mg tablets are to be taken with food • VTE ppx in surgical patients • 10mg daily for 35 days (hip), 12 days (knee) • Treatment VTE • 15mg BID x21 days, then 20mg daily w/food • Stroke prevention in Afib • 20mg daily with evening meal (CrCl>50), or 15mg daily with evening meal (CrCl 30-49)

48. Rivaroxaban (Dose Modifications) • Not recommend for CrCl<30 • Not to be used with CrCl<15 • Avoid use in patients with significant hepatic impairment, • Child-Pugh Class B and C with coagulopathy • Interacts with drugs that are potent dual inhibitors of CYP-3A4 and P-glycoprotein (see handouts 5 & 6) • (ketoconazole, itraconazole, voriconazole, posaconazole, clarithromycin, protease inhibitors (ritonavir)) • Potent inducers of CYP-3A4  reduce effects • No specific targeted antidote • Black Box warnings similar to Dabigatran

49. Apixaban (Eliquis) • Orally available direct factor Xa inhibitor • Half life ~5-9 hours • Less renal excretion than others, better for CKD pts • Given at a fixed dose without monitoring • VTE ppx in surgical patients • 2.5 mg BID for 35 days (hip), 12 days (knee) • Treatment VTE • 10mg BID x7 days, then 5mg BID • Stroke prevention in Afib • 5 mg BID(CrCl>50), or 2.5 mg BID if Age>80, Weight <60kg, and/or Cr > 1.5 • Dose reduce for CYP 3A4 inhibitors and P-glycoprotein inhibitors • Similar black box warnings as stated previously • No direct antidote

50. Edoxaban (Lixiana) • Orally available direct factor Xa inhibitor • Half life ~6-11 hours • Given at a fixed dose without monitoring, absorption unaffected by food • VTE ppx in surgical patients, VTE treatment, Afib stroke prevention • 30 to 60 mg daily (depending on risk factors) • Dose reduce for P-glycoprotein inhibitors, caution with CYP 3A4 inhibitors • Similar black box warnings as stated previously • No direct antidote