Kerns Anticoagulant Antiplatelet Agents PDF

Fibrinolytic, Coagulants, and Antiplatelet Agents Robert J. Kerns, PhD Professor Department of Pharmaceutical Sciences & Experimental Therapeutics August 22, 2023 Readings: Chapter 26 Foye’s Chapter 36 Goodman and Gillman’s (14e edition) 1 I. Types of antithrombotic drugs in clinical use: Anti...

Fibrinolytic, Coagulants, and Antiplatelet Agents Robert J. Kerns, PhD Professor Department of Pharmaceutical Sciences & Experimental Therapeutics August 22, 2023 Readings: Chapter 26 Foye’s Chapter 36 Goodman and Gillman’s (14e edition) 1 I. Types of antithrombotic drugs in clinical use: Anticoagulants attenuate fibrin clot formation by blocking fibrin crosslinking Fibrinolytic agents breakdown fibrin clots Antiplatelet agents inhibit activation of platelets inhibit aggregation of platelets 2 Learning Objectives and Study Guide: -Describe the fibrinolytic process/mechanism, understand the role of tPA and plasminogen/plasmin in breakdown of fibrin, and compare the differences in mechanism and action for the different fibrinolytic agents. -Understand the mechanism by which coagulant agents act to promote coagulation -Understand fundamental processes of platelet activation and platelet aggregation as well as the underlying biochemical and physiological processes with an emphasis on those where pharmaceutical agents can act to alter/inhibit platelet activation and/or platelet aggregation. -Understand the mechanism of action for antiplatelet agents discussed and how the structures, differences in structures, and properties of the agents (when discussed) impact their use or activity. -Describe the mechanism of action for agents that are irreversible inhibitors of platelet activation/aggregation and differences in effects between reversible and irreversible inhibitors. -While not discussed for every agent, when discussed in class understand structure-function relationships (activity, PK/PD, metabolism, etc.). 3 II. FIBRINOLYSIS *As wound healing progresses, the clot needs to be removed (broken down). Tissue Plasminogen Activator (t-PA) is released from endothelial cells. t-PA acts to activate plasminogen to plasmin, and plasmin is active protease that cleaves cross-linked fibrin to break down clot (also restores some soluble fibrin monomer back to circulation). Effect of t-PA is regulated to be at site of clot… *Circulating t-PA is rapidly cleared from blood and inhibited by PAI-1 and PAI-2. Any circulating plasmin formed is rapidly inhibited by α2-antiplasmin. Therefore, t-PA exerts little effect on circulating plasminogen. *The catalytic efficiency of t-PA activation of plasminogen to active plasmin increases more than 300-fold in the presence of (when attached to) fibrin, which promotes plasmin generation on its surface. Fibrinolysis. Endothelial cells secrete t-PA at sites of injury. t-PA binds to fibrin and converts plasminogen to plasmin, which digests fibrin. PAI-1 and PAI-2 inactivate t-PA; α2-AP inactivates plasmin. Plasminogen Activator Inhibitor = PAI 4 III. THROMBOLYTIC DRUGS (Promote Fibrinolysis) Thrombolytic Agents: (Given IV to rapidly dissolve bloods clots by proteolytic cleavage.) Each acts in some way to facilitate the conversion of plasminogen to plasmin. Urokinase (Abbokinase) – protease that directly activates plasminogen to plasmin to degrade fibrin…. (NO t-PA NEEDED) -Half-life of 15 min. -Human protein that does not show antigenicity Streptokinase (Streptase) –thrombolytic enzyme (protease) purified from streptococci; a net result of its action is proteolytic dissolution of fibrin. -not active on its own. Streptokinase forms a complex with plasminogen (1:1). The streptokinase-plasminogen complex (t 1/2 = 23 minutes) converts uncomplexed plasminogen to plasmin. (Fibrin non-specific action because the complex also catalyzed the breakdown of fibrinogen and other coagulation factors). -Bacterial protein and thus hypersensitivity (allergic) reaction to the drug is a significant problem. No Tissue Plasminogen Activator (t-PA) needed for these two fibrinolytic agents. 5 THROMBOLYTIC DRUGS (Promote Fibrinolysis) Thrombolytic Agents: (Given IV to rapidly dissolve bloods clots by proteolytic cleavage.) Each acts in some way to facilitate the conversion of plasminogen to plasmin. Streptokinase (Streptase) vs. Eminase (Anistreplase) Anistreplase is an anisoylated plasmin streptokinase activator complex. Chemical methods used to prepare, in vitro, a plasmin-streptokinase complex where the active site of the complex, has a panisoylated lysine residue. This p-anisoyl group in the catalytic site inactivates the complex. In solution or in the body, the anisoyl group is cleaved, deacylated, by non-enzymatic first order kinetics to give back the active form of plasmin in the streptokinase-plasmin complex. The t 1/2 for this complex is ~2 hours. Thus, Anistreplase has a longer active lifetime in the body than streptase. In addition, this gives the complex time to bind to the fibrin clot before it is activated thus reducing nonspecific proteolysis elsewhere in the vasculature. H20 6 Tissue Plasminogen Activator (tPA) for Fibrinolysis *tPA is a serine protease that converts plasminogen to plasmin Alteplase (Activase) – unmodified recombinant tPA -Protease that is selective for converting “fibrin-bound” plasminogen to plasmin -IV dose, 5 minute half-life (given by bolus followed by infusion) Tenectaplase (TNKase) - a recombinant tPA that contains 3 modifications (point mutations) from native tPA that give TNKase greater resistance to inactivation. P is the plasmin domain -Protease that is selective for converting “fibrin-bound” plasminogen to plasmin -IV bolus dose, 17 minute half-life Reteplase (Retavase) - a recombinant form of tPA having the first 172 amino acids removed, which does reduce fibrin binding selectivity -longer half-life than alteplase due to reduced hepatic elimination (IV bolus dose, 14-18 minute half-life) (Coagulants that block lysine binding site of plasmin can reverse activity of tPA) 7 IV. Coagulants (Revisited) Antifibrinolytic Agents: Mimics of the lysine found on Fibrin *Plasmin binds to fibrin through lysine binding site in fibrinolysis (degradation of fibrin). -Plasmin/plasminogen has 5 of these lysine binding sites. - Structurally resemble lysine, bind plasmin/plasminogen and occupy lysine-binding sites, thereby blocking plasmin-mediated fibrinolysis by blocking binding and degradation of fibrin ……………………………………............................ Prevent Bleeding: Trauma Post Partum Bleeding Tooth/Dental Work (Tranexamic acid in a mouth wash form) Others…. 8 V. Replacement of coagulation factors etc. to treat diseases of coagulation… Emicizumab (Hemlibra) for hemophilia, a bispecific factor IXa- and factor X-directed antibody made to activate the natural coagulation cascade and restore the blood clotting process for those with hemophilia A (a hereditary bleeding disorder caused by a lack of blood clotting factor VIII). (QUITE A NUMBER OF ADDITIONAL COAGULATION FACTORS ARE GIVEN IV TO PROMOTE COAGULATION IN CERTAIN DISEASES; PARTICULARLY DISEASES WHERE DIRECT REPLACEMENT OF A COAGULATION FACTOR IS NEEDED) -Factor IX , Factor VIIa -Antihemophilic Factor and Complex with Von Willebrand factor -ETC… 9 VIa. Role of Platelets in Hemostasis: Platelets first adhere to macromolecules in the subendothelial regions of injured blood vessel and become activated, release substances that activate nearby platelets to recruit them to the injury site. The activated platelets aggregate to form the primary hemostatic plug. Vessel wall injury exposes tissue factor (TF), which initiates coagulation (extrinsic pathway leading to fibrin cross-linking). Activated platelets stimulate coagulation by providing a surface for clotting factors to assemble onto and by releasing stored clotting factors, in particular a burst of thrombin, which converts soluble fibrinogen to fibrin, activates additional platelets, and feeds back to promote additional thrombin generation. The fibrin strands tie the platelet aggregates together to form a stable clot. 10 Platelets and Platelet Aggregation Thrombin: central to fibrin crosslinking and platelet aggregation: Thrombin cleaves PAR receptors causing mobile, non-adhesive platelets to change shape, to aggregate (stick together), and to secrete the contents of their storage granules. Platelet adhesion and aggregation: GPVI and GPIb are platelet receptors that bind to collagen and vWF, causing platelets to adhere to the subendothelium of a damaged blood vessel. PAR-1 and PAR-4 are PARs that respond to thrombin (IIa); P2Y1 and P2Y12 are receptors for ADP; when stimulated by agonists, these receptors activate the fibrinogen-binding protein GPIIb/IIIa and COX-1 to promote platelet aggregation and secretion. TxA2 is the major product of COX-1 involved in platelet activation. Prostacyclin (PGI2), synthesized by endothelial cells, inhibits platelet activation. PAR (Protease Activated Receptor) P2Y (Purine Receptors, ADP is a Purine) 11 VIb: Platelets, Platelet Activation and Agents that Block Platelet Function *Targets for Blocking Platelet Activation and aggregation PAR (Protease Activated Receptor) P2Y (Purine Receptors, ADP is a Purine) 12 VII. ANTIPLATELET DRUGS VIIa. Aspirin and COX inhibitors Thromboxane A2 (TXA2) is an eicosanoid that induces platelet aggregation and is a potent vasoconstrictor. In platelets, an enzyme called cyclooxygenase produces as its major product, TXA2. Therefore, inhibition of TXA2 production has an antiplatelet effect. The chemistry of how Aspirin inhibits production of TXA2 is shown below. The action of aspirin on cyclooxygenase1 (COX-1) is irreversible. This irreversible inhibition lasts until new platelets are produced because platelets don’t synthesize new protein. It takes 710 days for turnover of all platelets in blood stream (to make new platelets). 13 VIIa. Aspirin and COX inhibitors: Additional, COX inhibitors that have a number of clinical uses. As compared to Aspirin: Comparison of COX-1 inhibitor structures: Triflusal (irreversible COX-1 inhibition) has additional actions that appear to be important for its activity profile for blocking platelet aggregation. In particular, Triflusal inhibits phosphodiesterase (PDE) that leads to increases in cAMP which inhibits platelet aggregation (see later slide). Triflusal also increases nitric oxide synthesis in neutrophils, resulting in increased vasodilation. Indobufen (reversible COX-1 inhibition) is much more potent COX-1 inhibitor than the many other NSAIDs that are reversible (competitive binding) COX-1 inhibitors. It is therefore unique among NSAIDs in that it does impart significant inhibition of platelet aggregation and is in clinical use as a reversible antiplatelet agent. 14 VIIb. Dipyridamole (Persantine) and Cilostazol (Pletal) An increase in cAMP in platelets increases the amount of active form of protein kinase A (PKA) present which correlates with inhibition in platelet aggregation. PKA also prevents the activation of myosin light-chain kinase, which plays a role in the contraction of smooth muscle cells, and therefore an increase in cAMP also produces a vasodilatory effect. -Block adenosine uptake in platelets, epithelial cells and eryrthrocytes (red blood cells), which increases adenosine in plasma that can then bind Adenosine A2 receptors on platelets resulting in increased cAMP production. -Inhibit Phosphodiesterases that break down cAMP in platelets and blood. Therefore, Dipyridamole and Cilostazol increase levels of cAMP by blocking PDE-mediated degradation of cAMP. Dipyridamole Cilostazol is selective for phosphodiesterase-3 (PDE3) Increased cAMP levels inhibits platelet aggregation and promotes vasodilation. Cilostazol Aspirin with Dipyridamole = (Aggrenox). Combined, additive, antiplatelet15effects. VIIb. Dipyridamole (Persantine) and Cilostazol (Pletal) IIIbi.Cilostazol and Dipyridamole Mechanism Pathway Note: Triflusal, previously discussed as a COX-1 inhibitor, is also believed to inhibit PDE3, which would further contribute to the anti-platelet effect of triflusal. 16 VIIc. Covalent Inhibitors of P2Y12 receptors (the thienopyridines) Ticlopidine (Ticlid) and Clopidogrel (Plavix) and Prasugrel (Effient): Irreversible inhibitors of ADP-mediated activation of the GPIIb/IIIa complex in platelets. They are given ‘Orally’. ADP is one of a number of chemical or physical stress mediators that activates platelets. ADP activates platelets by activating G-protein coupled receptors (P2Y purinergic receptors). This activation results in changes in platelet shape and initiates platelet aggregation events, including GPIIb/IIIa complex activation. Clopidogrel, Ticolpidine and Prasugrel irreversibly modify the platelet ADP receptor, the P2Y12 receptor, on the platelet surface, thus inhibiting ADPreceptor binding and blocking ADP-mediated activation of the GPIIb/IIIa complex, and blocking activation of the GPIIb/IIIa complex blocks ADPinduced platelet-fibrinogen binding and therefore net effect is inhibition of platelet aggregation. Each of these agents requires metabolic conversion to an active metabolite: (prodrugs or bioprecursor drugs) that require P450 metabolic activation). 17 VIIc. Covalent Inhibitors of P2Y12 Mechanism of Action H20 HS-P2Y12 (Thiol of cysteine in P2Y12 receptor is the –SH for disulfide formation) P2Y12 H20 Active Inactive Inactive 18 VIIc. Covalent Inhibitors of P2Y12 Mechanism of Action Prasugrel does not require P450 activation and therefore less interpatient variability Esterase H20 Esterase Inactive 19 VIIci. Reversible Inhibitors of P2Y12 receptors -Reversible binding nucleotide analogs (ATP analogs) -Do not require metabolic activation like the original thienepyridine covalent Inhibitors of P2Y12, but are metabolized and therefore have drug-drug interactions too. -Ticagrelor binds at site different from ADP, therefore it is an allosteric antagonist. Brilinta (oral) Kengreal (IV) -More rapid onset and offset of action as well as greater and more predictable inhibition of ADPinduced platelet aggregation than clopidogrel. 20 VIId. Direct GPIIb/IIIa Antagonists IIIdi) Abciximab (Reopro) This agent is the Fab fragment of a monoclonal antibody that binds to the GPIIb/IIIa complex on the platelet surface. This binding may sterically inhibit the GPIIb/IIIa complex from interacting with fibrinogen or cause conformation changes in the GPIIb/IIIa complex thus inhibits the GPIIb/IIIa complex from interacting with fibrinogen. The net result of inhibiting the GPIIb/IIIa - fibrinogen interaction is blockage of platelet aggregation. This antibody also binds to other cellular adhesive molecules and von Willebrand factor. As one would expect with an antibody, administration is via IV drip. 21 VIIdii. Eptifibatatide (Integrelin) Eptifibatatide is a cyclic heptapeptide that contains the RGD tripeptide sequence in a structurally more rigid cyclic structure. By using an appropriately designed cyclic system, the conformation of the RGD sequence is more rigid and more closely locked into the conformation that is required for selective binding to the RGD binding pocket in the GPIIb/IIIa complex. This binding blocks fibrinogen - GPIIb/IIIa interactions resulting in the inhibition of platelet aggregation. Cyclic peptide/peptide mimic: more stable to proteases and peptidases than liner peptides. 22 VIIdiii. Tirofiban (Aggrastat); fiban class of agents The interaction between fibrinogen and the GPIIb/IIIa complex is mediated by the binding of the GPIIb/IIIa complex to an RGD tripeptide sequence found on fibrinogen. Tirofiban is a structural mimic (non-peptide mimic) of the RGD tripeptide sequence (shown to the right). Tirofiban competitively binds to the RGD binding sites in the GPIIb/IIIa complex and therefore inhibits fibrinogen-GPIIb/IIIa complex interactions and blocks platelet aggregation. (Peptidomimetic of the RGD tripeptide) Future?: Tirofiban is administered parenterally. A number of “orally active” fibans were under development but after a failed clinical trial with increased mortality rates it is yet unclear if an orally available GPIIb/IIIa antagonist will make it to23market. VIIe. Vorapaxar (Zontivity) (History) Analog of an alkaloid natural product (Himbacine) isolated from bark of Australian magnolias. Work to find analogs of Himbacine as potential treatments of Alzheimer’s disease failed, but led to discovery of such analogs as inhibitors of platelet aggregation. Blocks platelet aggregation by inhibiting thrombinmediated platelet aggregation; it blocks (is an antagonist of) protease-activated receptor-1, PAR-1. This is a newer agent with mechanism that is different from the other anti-platelet agents. Metabolized by CYP3A enzymes so drug-drug interactions need to be monitored. 24 Summary: Review Sites of Action 25