BPS 338 - Antiplatelets Lecture Notes Sp2024 PDF

BPS 338 – Module 1 Hemostasis (blood clotting) Inhibitors of blood clot formation: - Antiplatelets (prevent platelet plug) - Antithrombotics (prevent fibrin clot) Enhancers of blood clot degradation: - Thrombolytics - These will be covered...

BPS 338 – Module 1 Hemostasis (blood clotting) Inhibitors of blood clot formation: - Antiplatelets (prevent platelet plug) - Antithrombotics (prevent fibrin clot) Enhancers of blood clot degradation: - Thrombolytics - These will be covered in P3 (acute, hospital use, intravenous) Dr. King 1  Hemostasis - Definition Hemostasis is the process responsible for maintaining circulatory system integrity following blood vessel damage--delicate balance between clotting and bleeding Hemostatic clots remain localized to the vessel wall and do not greatly impair blood flow. – These are “good” clots, and they prevent unwanted bleeding Pathologic clots result in blood flow impairment and often cause complete vessel occlusion. – Also called thrombosis – These are “bad” (pathologic) clots 2  Types of Pathologic clots Two main types (and a few subtypes) of pathologic clots Venous thrombosis is a clot blocking a vein – Remember, veins carry un-oxygenated blood back to the heart, flow is rather slow (venous return) – Typically started by endothelial damage and contain mostly fibrin – Deep Vein Thrombosis, DVT, occurs when a blood clot forms in a deep vein, usually in the lower leg, thigh, pelvis, or arms with i.v. lines – Pulmonary embolism, PE, occurs when a DVT clot breaks loose and travels through the bloodstream to an artery in the lungs. – DVT and PE together are called Venous Thromboembolism (VTE) Arterial thrombosis is a blockage of an artery. – Arteries carry oxygen-rich blood away from the heart, flow is rather fast. – Typically started by atherosclerosis and contain platelets and fibrin – Coronary thrombosis is a blockage of an artery in the heart, may lead to a heart attack (MI, myocardial infarction, angina). – Stroke results when arterial thrombosis occurs in an artery in the brain. 3  Watch these videos AFTER the lecture What is the difference between arterial thrombosis and venous thrombosis? Arterial Thrombosis Explained (1:39): https://www.youtube.com/watch?v=FM0vJTkT60Y Venous thrombosis Explained (1:53): https://www.youtube.com/watch?v=QTuN60hgL5Y These come from a group sponsored by Bayer Pharmaceutical company called: ThrombosisAdvisor.com 4  Watch these videos AFTER the lecture 1st : Platelet Activation and Factors for Clot Formation: https://www.youtube.com/watch?v=R8JMfbYW2p4, 2:08 min 2nd : Coagulation Cascade Animation - Physiology of Hemostasis: https://www.youtube.com/watch?v=cy3a__OOa2M , 2:29 min, 3rd : Coagulation Cascade Explained: https://www.youtube.com/watch?v=DKFSH5MMPLM , 2:44 min 4th : PAR-1 Inhibitor, Vorapaxar: https://www.youtube.com/watch?v=uYpv8T7Qoh0, 1:57 min, These come from a group sponsored by Bayer Pharmaceutical company called: ThrombosisAdvisor.com 5 Hemostasis is a delicate balance: Prevents or terminates blood loss from a disrupted intravascular space, but allows blood to flow unrestricted under normal circumstances 4 physiologic processes, occur in this general order 1. Vascular constriction (slow blood flow to damaged site), 2. Platelet plug formation (clot formation), 3. Fibrin formation (clot formation), 4. Fibrinolysis (clot degradation, after damaged site is healed) Endogenous Clots once formed, need to be degraded But the components of each of these four processes are highly connected, and the processes are happening at the same time with multiple reinforcements 6  First, how do clots normally form? Formation of blood clots… Occurs by two fundamental processes: 1. Platelet activation/aggregation, called the Platelet Plug 2. 2. Fibrin formation 1. Platelet plug plus fibrin Is called Thrombus Arteriosclerosis, Thrombosis, and Vascular Biology. 2019;39:7–12 7  Preview Drugs altering blood clot formation and degradation Inhibitors of clot formation (this slidepack) I. Antiplatelets (inhibit platelet activation and/or aggregation) (inhibit clot formation) A. COX inhibitor, irreversible, decreases Thromboxane A2 synthesis: Aspirin B. Glycoprotein IIb/IIIa antagonists: abciximab, tirofiban, eptifibatide C. Purinergic receptor antagonists (ADP-receptor, P2Y12): clopidogrel, prasugrel, ticagrelor, cangrelor D. Phosphodiesterase 3 inhibitor: cilostazol E. Adenosine Reuptake inhibitor and PDE inhibitor: dipyridamole F. Inhibition of protease-activated receptor-1: vorapaxar II. Anticoagulants (inhibit fibrin formation) (inhibit clot formation) (next slidepack) A. Direct Factor Xa inhibiors apixaban, rivaroxaban, edoxaban B. Direct Thrombin inhibitors: dabigatran C. Antithrombin enhancers: Unfractionated Heparin, Low MW heparin, fondaparinux (Arixtra) D. γ-Glutamate carboxylation inhibitors: Warfarin 8 Platelet Activation & Aggregation mechanisms and Drugs causing antiplatelet effect So many mechanisms… 9  Platelet Activation & Aggregation So many mechanisms Molecular targets of FDA-approved Gq antiplatelet agents are shown in blue & green. Gi Cytosolic Ca2+ Investigational Signal transduction strategies are marked in red. Vorapaxar Integrin GPIIb-GPIIIa Copyright © 2022 American Society of Binding to another Hematology TP receptor Advances in Antiplatelet Therapy, platelet is “aggregation” (TXA2 receptor) Hematology Am Soc Hematol Educ Program, 2011, Figure 1. 10  Start with Aspirin, MOA: irreversible inhibitor of cyclooxygenase, results in inhibition of synthesis of TXA2 in platelet II. Antiplatelets (inhibit platelet aggregation) (inhibit clot formation) A. Thromboxane A2 synthesis inhibitor: Aspirin B. Glycoprotein IIb/IIIa inhibitors: abciximab, tirofiban C. Purinergic receptor inhibitors (ADP-receptor, P2Y12) D. Phosphodiesterase 3 inhibitor E. Inhibition of platelet uptake of adenosine F. Inhibition of protease-activated receptor-1 11  Aspirin You may already know that aspirin inhibits cyclooxygenase (COX-1 and COX-2). That is how it has its anti-inflammatory & analgesic actions. But, How does aspirin inhibit thromboxane A2 biosynthesis, and how does this affect platelet aggregation? Why is aspirin the only NSAID that effectively inhibits platelet aggregation at relatively low once daily dose? This can be understood by thinking about the arachidonic (aa) acid cascade and understanding the specific role of aa in the platelet in thromboxane A2 synthesis. 12  Do not learn the structures of these, just that they are Arachadonic acid cascade- variations of each other and that they are a pathway. in all tissues Inhibited by aspirin, Irreversible, COX-1 Different PGE2 tissues have proinflammatory different combinations PGI2 (prostacyclin) of the Proinflammatory At GPCR Gi enzymes. Results in different products being TXA synthase, formed in Only in platelets!!!! different Inhibited by Ridogrel (not FDA) tissues TXA (TXA2) is synthesized ONLY in the platelet. It is then released to activate more platelets. TXA2 is a strong platelet activator! 13  Cytosolic Ca2+ Phospholipase A2 In platelets only: Thromboxane A2 (TXA2) Arachadonic acid Thromboxane A2 is an COX-1 inducer of platelet cyclooxygenase aspirin aggregation PGH2 It is synthesized in the platelet after activation of Thromboxane synthase Ridogrel phospholipase A2 (not FDA-approved) Cyclooxygenase is TXA2 present in nearly all tissues, but Inside platelet TXA2 binds to thromboxane synthase is other platelets to present only in platelets TXA2 activate them Thus, TXA2 only outside platelet synthesized in O COOH platelets! CH3 O OH Thromboxane A2 Figure from: Am J Cardiovasc Drugs, 2002, 2(4): 227-235 14 How does aspirin inhibit thromboxane A2 biosynthesis? Aspirin irreversibly inhibits cyclooxygenase – Aspirin permanently (irreversibly) inactivates COX-1 and COX-2 through covalent acylation of the catalytic serine residue in the active site of the enzyme. Prevents formation of TXA2. COOH COX protein backbone near active site-serine O CH3 O O CH3 O OH Acetylated COX (inactive) Irreversible inhibition Why is irreversible inhibition important? Next slide 15  Why is aspirin the only NSAID that effectively inhibits platelet aggregation? – Since platelets have no nucleus, they cannot synthesize new cyclooxygenase. New platelets have to be synthesized instead (takes several days to week). – In practical terms, this means that a daily, relatively low dose, of aspirin will completely inhibit TXA2-dependent platelet aggregation. – Other NSAIDs (example ibuprofen) also inhibit platelet cyclooxygenase (and inhibit TXA2 production), but these are reversible COX inhibitors and inhibition of aggregation is lost with clearance of the drug (few hours). 16  Done with Aspirin, Next group… II. Antiplatelets (inhibit platelet aggregation) (inhibit clot formation) A. Thromboxane A2 synthesis inhibitor: Aspirin B. Glycoprotein IIb/IIIa inhibitors: abciximab, tirofiban, eptifibatide C. Purinergic receptor inhibitors (ADP-receptor, P2Y12) D. PDE inhibitors and other 17  Cytosolic Ca2+ From slide 10 Signal transduction From slide 20: detail GPIIb-GPIIIa Integrin αIIbβ3 Zoom in to show Resting active Platelet detail (inactive) membrane Inside platelet PDB.org, https://pdb101.rcsb.org/motm/134 Feb 2011 II. Antiplatelets (inhibit platelet aggregation) (inhibit clot formation) A. Thromboxane A2 synthesis inhibitor: Aspirin B. Glycoprotein IIb/IIIa inhibitors: abciximab, tirofiban, eptifibatide C. Purinergic receptor inhibitors (ADP-receptor, P2Y12) D. PDE inhibitors and other fiba = Fibrinogen antagonist” AS of 2019, Abciximab is not manufactured 18  Glycoprotein IIb/IIIa inhibitors: abciximab, tirofiban, eptifibatide Glycoprotein IIb/IIIa receptor – modern name is Integrin αIIbβ3. (alpha two bee, beta three) On platelets, – αIIbβ3 is the major integral plasma membrane protein. – 3% of the total protein mass of the platelet. – 17% of the platelet membrane protein mass. – Altogether, there are 80,000–100,000 copies of αIIbβ3 per platelet! (on the surface and more in storage). Important function is in adhesion and aggregation of one platelet to another Alan D. Michelson, Platelets, Fourth Edition, 2019, Chapter 12, Integrin αIIbβ3, K.Blezdka 19 Major shape (conformation) change upon activation of Integrin αIIbβ3 (GPIIb/IIIa) receptor Resting (inactive) state is folded (bent) – see the image below Active state is unfolded (open) Important Active Resting binding site is (inactive) here Platelet membrane Inside platelet PDB.org, https://pdb101.rcsb.org/motm/134 Feb 2011 20 Major shape (conformation) change upon activation of Integrin αIIbβ3 (GPIIb/IIIa) receptor There are animations on this slide Fibrinogen normally binds to the active (open) receptor Active Resting (inactive) And binds to another platelet at the other end Platelet membrane Inside platelet This results in platelet aggregation PDB.org, https://pdb101.rcsb.org/motm/134 Feb 2011 21 Major shape (conformation) change upon activation of Integrin αIIbβ3 (GPIIb/IIIa) receptor Abciximab is a monoclonal antibody that blocks fibrinogen binding to open Integrin αIIbβ3 Abciximab Active Resting (inactive) Blocks fibrinogen binding and blocks aggregation Abciximab structure from: 23 Jan 2020https://doi.org/10.1161/ATVBAHA.119.313671 There are animations Arteriosclerosis, Thrombosis, and Vascular Biology. 2020;40:624–637 on this slide 22 Major shape (conformation) change upon activation of Integrin αIIbβ3 (GPIIb/IIIa) receptor Eptifibatide is a small molecule, 6 amino acid cyclic peptide Eptifibatide Resting (inactive) Blocks fibrinogen binding and blocks aggregation There are animations Eptifibatide bound to Integrin αIIbβ3 structure from: on this slide 2VDN, rcsb.org. PDBID: 2VDN, figure made by Dr. King with UCSF Chimera 1.15 23 Major shape (conformation) change upon activation of Integrin αIIbβ3 (GPIIb/IIIa) receptor Tirofiban is a small molecule, non-peptide, synthetic tirofiban Resting (inactive) Blocks fibrinogen binding and blocks aggregation Tirofiban bound to Integrin αIIbβ3 structure from: There are animations 2VDN, rcsb.org. PDBID: 2VDN, figure made by Dr. King with UCSF Chimera 1.15 on this slide 24 glycoprotein IIb/IIIa antagonists, fibrinogen antagonists, Integrin αIIbβ3 inhibitors Abciximab (ReoPro): Monoclonal antibody – Antibody is large protein, must be administered intravenous. – Active until about 48 hr after infusion – FDA approved in 1994 – Since 2019, Not manufactured, not available! Eptifibatide (Integrilin) – 6-amino acid peptide linked into a circle – Short-acting, reversible, only effective until cleared from plasma – FDA-approved 1998, administered i.v. bolus or infusion Tirofiban (Aggrastat) is a small molecule, non-peptide, synthetic – Administered i.v. even though small molecule could survive GI tract acidity. – FDA-approved 1998 25 II. Antiplatelets (inhibit platelet aggregation) (inhibit clot formation) A. Thromboxane A2 synthesis inhibitor: Aspirin B. Glycoprotein IIb/IIIa inhibitors: abciximab, tirofiban C. Purinergic receptor inhibitors (ADP-receptor, P2Y12) P2Y12 antagonists D. PDE inhibitors and other Platelet activation 26  Preview: prasugrel, clopidogrel, ticagrelor, cangrelor Journal of Thrombosis and Haemostasis, 2013, 11 (Suppl. 1): 316–329 2011 2015 2009 1997 (iv) Esterase, Intestinal CES1 Esterase, CES2 CYP2C19 They are all antagonists at the Platelet P2Y12 receptor membrane 27 27  P2Y12 is a GPCR (Gαi) P2Y12 antagonists ADP Agonist, Gi mechanism prevent platelet activation and – Alpha subunit leads to adenylyl aggregation cyclase (AC) inhibition – and to less cAMP Less phosphorylation of VASP – Activation of GPIIb/IIIa – More platelet aggregation Tissue-specific βγ response – Through PI3K (phosphatidyl-inositol-3- kinase) and PKB (protein-kinase B) – Activation of GPIIb/IIIa – More platelet aggregation Antagonists block the above and inhibit platelet aggregation 28 Irreversible ADP/Purinergic receptor antagonist (P2Y12) Clopidogrel (Plavix, 1997), ticlopidine (Ticlid, 1991; discontinued 2015), prasugrel (Effient, 2009): These drugs are also known by their chemical class, thienopyridines. O OCH3 discontinued 2015 N N S Cl S Cl Clopidogrel Ticlopidine The Thienopyridine is important!! Prasugrel Which part of structure is thienopyridine? 29 Irreversible: Clopidogrel, prasugrel, (ticlopidine) Biotransformation is necessary (and thus prodrug) to produce inhibition of platelet aggregation, via a reactive metabolite. O OCH3 H COOCH3 H Z S HOOC N N CYP CYP ? S Cl 2C19 HS Cl Clopidogrel Active drug, reactive DMD, 2002, Pereillo metabolite, binds irreversibly to P2Y12 Act by irreversibly modifying the platelet ADP receptor. Consequently, platelets exposed are affected for the remainder of the platelet lifespan (~10 days). 30  Prasugrel Prasugrel activated by ester hydrolysis (CES2) then by CYPs to make the active drug Active drug, reactive metabolite, binds irreversibly to P2Y12 Esterase, Multiple CYPs CES2 31  CES1 leads to LESS clopidogrel active drug; CES2 leads to MORE prasrugrel active drug clopidogrel CES1, activatedaby carboxyesterase CYP2C19 + several CYPs Prasugrel activated by ester hydrolysis (CES2) CES1 leads to inactive then by several CYPs; CES1 CYP2C19 INactive CES2 CES1 --OH INactive Multiple Multiple CYPs CYPs CES1 Active INactive Active 32  more Clopidogrel oxidized mainly by CYP2C19: black box warning for 2-14% of US population with low CYP2C19 activity – Also CYP2C19 inhibitors: Avoid concomitant use of omeprazole or esomeprazole. (5.1) Prasugrel (after ester hydrolysis) converted to the active metabolite, primarily by CYP3A4 and CYP2B6 and to a lesser extent by CYP2C9 and CYP2C19. CYP inhibitors & CYP genetic polymorphism not a problem with prasugrel (because multiple CYPs capable). Under research: affect of CES1 and CES2 (carboxyesterase) variation in activity on both prasugrel and clopidogrel 33  Reversible: Ticagrelor ADP/Purinergic receptor antagonist (P2Y12) Ticagrelor is a nucleoside (adenosine) analogue: adenosine Does not need hepatic activation, so CYP2C19 variation causes no concern for ticagrelor, not a prodrug Reversible, so no concern about extra long duration of action FDA approved 2011 Orally active 34 Cangrelor, another reversible P2Y12 antagonist Differences – Not a prodrug, reversible – IV injection only – Very short acting (during infusion only) designed to be this way – Approved 2015 – Structural mimic of ADP (competitive antagonist) 35  Review: prasugrel, clopidogrel, ticagrelor, cangrelor Journal of Thrombosis and Haemostasis, 2013, 11 (Suppl. 1): 316–329 2011 2015 2009 1997 (iv) Esterase, Intestinal CES1 Esterase, CES2 CYP2C19 They are all antagonists at the Platelet P2Y12 receptor membrane 36 36 I. Antiplatelets (inhibit platelet aggregation) (inhibit clot formation) A. COX inhibitor, irreversible, decreases Thromboxane A2: Aspirin B. Glycoprotein IIb/IIIa antagonists: abciximab, tirofiban, eptifibatide C. Purinergic receptor antagonists (ADP-receptor, P2Y12): clopidogrel, prasugrel, ticagrelor, cangrelor D. Phosphodiesterase 3 inhibitor: cilostazol E. Adenosine Reuptake inhibitor and PDE inhibitor: dipyridamole F. Inhibition of protease-activated receptor-1: vorapaxar 37  Platelet Activation Focus on So many mechanisms Cilostazol, Dipyridamole Vorapaxar Gq Gi Cytosolic Ca2+ Signal transduction Vorapaxar GPIIb-GPIIIa Copyright © 2022 American Society of Hematology Advances in Antiplatelet Therapy, Hematology Am Soc Hematol Educ Program, 2011, Figure 1. 38  Cilostazol (Pletal) Decrease platelet aggregation – Phosphodiesterase 3 (PDE3) inhibitor – PDE3 inhibition Increases cyclic guanosine monophosphate (cGMP) which inhibits platelet activation Also a Vasodilator – Especially, Dilation of arteries supplying blood to the legs ORAL administration 39  Dipyridamole Dipyridamole inhibits the phosphodiesterase enzymes that normally break down cAMP (increasing cellular cAMP levels causing greater inhibition of platelet activation. It also inhibits the cellular reuptake of adenosine into platelets, red blood cells, and endothelial cells leading to increased extracellular concentrations of adenosine. – inhibits blood clot formation when given chronically – causes blood vessel dilation when given at high doses over a short time Dipyridamole also potentiates the antiaggregating action of prostacyclin (PGI2). ORAL administration 40  Vorapaxar: PAR-1 inhibitor (antagonist): Watch the movie: https://www.youtube.com/watch?v=uYpv8T7Qoh0 FDA-Approved in 2014, first-in-class Zontivity (vorapaxar) is a protease- activated receptor-1 (PAR-1) antagonist reversible, but long half-life extends its action ORAL administration PAR-1 is the main Thrombin-receptor on human platelets (Gαq to↑Ca2+) – antagonist causes less platelet activation Much more about Thrombin in the next slide-pack 41  Vorapaxar (Zontivity) Great movie showing mechanism of action: https://www.youtube.com/watch?v=uYpv8T7Qoh0 Also shows nice, simplified view of thrombus formation 42  Go back to slide #10 (copied next slide) and see a few additional mechanisms – PGI2, PGE2, from arachidonic acid cascade – NO, – VWF, collagen, – 5HT2A, – P2Y1 (a Gq GPCR) 43  Platelet Activation & Aggregation So many mechanisms Molecular targets of FDA-approved Gq antiplatelet agents are shown in blue & green. Gi Cytosolic Ca2+ Investigational Signal transduction strategies are marked in red. Vorapaxar GPIIb-GPIIIa Copyright © 2022 American Society of Binding to another Hematology TP receptor Advances in Antiplatelet Therapy, platelet is “aggregation” (TXA2 receptor) Hematology Am Soc Hematol Educ Program, 2011, Figure 1. 44  Study Questions-antiplatelets Why does the action of aspirin in platelets cause inhibition of aggregation? Why does the inhibition occur after clearance of aspirin? Why is aspirin the only NSAID that inhibits platelet aggregation? How does aspirin inhibit thromboxane A2 biosynthesis, and how does this affect platelet aggregation? What is the therapeutic effect of glycoprotein IIb/IIIa antagonists? How do clopidogrel and ticlopidine bind the the P2Y12 receptor? How does this explain their continued effect after stopping treatment? How is P2Y12 receptor inactivation related to decreased platelet aggregation? Structures important for Clopidogrel, Prasugrel, and Aspirin NOT for the other medications. 45 Which part of prasugrel is the important thienopyridine? A. D. C. B. 46