1. Thrombolytic Agents Benedict R. Lucchesi, M.D., Ph.D. Department of Pharmacology University of Michigan Medical School

2. Thrombolytic Therapy Streptokinase Tissue Plasminogen Activator (rt-PA) Urokinase Retavase Tenecteplase, TNK-tPA ( TNKase™)

3. Anticoagulant Therapy Thrombolysis

4. The primary action of all thrombolytic agents is to convert plasminogen to plasmin. Plasmin possess enzymatic activity that brings about the degradation of fibrin (fibrinolysis) that results in clot lysis.

5. Thrombolytic Therapy The plasminogen molecule contains structures called LYSINE- BINDING SITES (kringles). The lysine-binding sites bind plasminogen to fibrin. The inactive pro-enzyme, plasminogen, is associated with the fibrin in the thrombus or blood clot. The lysine-binding sites play an important role in regulating FIBRINOLYSIS.

6. Thrombolytic Therapy PLASMIN has the ability to “digest” FIBRIN to soluble degradation products. The blood contains two physiologically active plasmin- ogen activators: 1.Tissue type plasminogen activator 2.Single-chain urokinase-type plasminogen activator In addition there are two inhibitors of the fibrinolytic system present in blood: 1.Plasminogen activator inhibitor - 1 2.alpha-2 - antiplasmin

7. The principal functional domains of plasminogen include: 1. Fibrin binding sites or "kringles" on the heavy chain 2. Serine-histidine-arginine catalytic site on the light chain. The activation of plasminogen to plasmin is accomplished by : cleavage of the arginine-560 - valine 561 bond. The reaction splits the molecule into disulfide-linked heavy and light chains. The light chain assumes the conformation capable of proteolytic activity. The various plasminogen activators have unique properties, but all lead to this peptide cleavage of the plasminogen molecule. In addition, the plasmin that is formed may cleave the amino N- terminal 76 residue activation peptide from plasminogen, converting the latter from the Glutamic acid-1 to the Lysine-77 form. The latter is a better substrate for plasminogen activators and for subsequent conversion to plasmin. Plasminogen

9. Streptokinase Streptokinase Nonenzyme protein produced by several strains of hemolytic streptococci.

10. Streptokinase is a nonenzyme protein produced by several strains of hemolytic streptococci. It consists of a single polypeptide chain with a Mr of 47,000 to 50,000. Streptokinase cannot directly cleave peptide bonds. It activates plasminogen to plasmin indirectly following a three step process. The first step involves the formation of an equimolar complex with plasminogen. The complex undergoes a conformational change resulting in the exposure of an active site in the plasminogen moiety. In the second step, this active site catalyzes the activation of plasminogen to plasmin. In the third step, plasminogen-streptokinase molecules are converted to plasmin-streptokinase complex. Streptokinase

11. INDIRECTLY activates plasminogen (Plg) to plasmin Pl) in 3 steps. – Step 1 Streptokinase forms an equimolar complex with plasminogen (Plg-SK complex). The conformational change takes place in plasminogen and exposes an “active site” – Step 2 Active site catalyzes the activation of plasminogen (Plg) to plasmin (Pl) – Step 3 Plg-SK is converted to Pl-SK

12. Streptokinase Plasma half-life of 20 min Neutralized by anti-streptokinase antibodies due to previous infections with ß-hemolytic streptococci Anti-streptokinase titer increases 50 - 100 times within a few days after administration, remains high for 4 - 6 months Repeated therapy within this time is impractical

13. Tissue Plasminogen Activator Native tissue-type plasminogen activator (t-PA) is a serine proteinase with a molecular weight of 70,000. t-PA is composed of a single polypeptide chain that is converted by plasmin to a two chain form by hydrolysis of the Agr275-Ile276 peptide bond. The form of the compound used clinically is made by recombinant DNA technology. It consists mainly of the single chain form. The amino terminal portion of t-PA is composed of several domains with homologies to other proteins such as fibronectin and epidermal growth factor. The region comprising residues 276 to 527 is homologous with that of other serine proteinases and contains the catalytic site containing His322-Asp 371-and serine 478.

14. Tissue Plasminogen Activator t-PA has affinity for fibrin and is mediated the finger domain via the second kringle region. Structures involved in fibrin-binding are contained within the heavy A chain. A lysine binding site is involved in the interaction of the kringle 2 domain with fibrin. The structures involved in the enzymatic domain are in the B chain. The presence of fibrin enhances, by 2-3 orders of magnitude, the efficacy with which t-PA activates plasminogen. Therefore, plasminogen bound to fibrin in the clot will be preferentially activated over plasminogen that is free in plasma. Fibrin essentially enhances the local concentration of t-PA by creating an additional interaction between t-PA and its substrate, plasminogen. The plasmin that is formed on the surface of fibrin within the clot has both of its lysine binding sites occupied and is protected therefore from rapid inactivation by alpha-2 antiplasmin (t 1/2 is about 10 to 100 seconds).

15. Tissue Plasminogen Activator it shows a specificity for fibrin activation of plasminogen is 2 to 3 orders of magnitude greater when plasminogen is bound to fibrin. plasmin is formed on the fibrin surface within the clot. plasmin bound to fibrin is protected from the rapid inactivation of its enzymatic activity. free plasmin in the plasma has a t 1/2 of 0.1 seconds as compared to plasmin that is bound to fibrin (10 to 100 secs). rt-PA (Alteplase; Activase™) approved for use in: myocardial infarction and stroke. Facts about t-PA

16. Guidelines for use of rt-PA in stroke - 1 Intravenous rt-PA should be given within 3 hours of onset of ischemic stroke in a dose of 0.9 mg/kg (max 90 mg), with 10% of the dose given as a bolus followed by a 60 min infusion. rt-PA can not be recommended for use beyond 3 hours after stroke onset. Data are inadequate to permit recommendation of i.v. streptokinase for ischemic stroke.

17. Guidelines for use of rt-PA in stroke - 2 Thrombolysis is not recommended unless the proper diagnosis (with CT of the brain) made by physicians expert in diagnosing stroke and reading CT. Avoid thrombolysis where there is evidence of recent major infarction, mass effect, edema or possible hemorrhage, on heparin in the last 48 hrs or warfarin or with a platelet count <100,000. Patients treated with rt-PA for stroke, should not be given aspirin, ticlopidine, clopidogrel, heparin, or warfarin.

18. Anisoylated Plasminogen Streptokinase Activator Complex (APSAC) APSAC is an equimolar noncovalent complex between human plasminogen and streptokinase. It has a catalytic center located in the COOH-terminal region of the molecule, whereas the lysine-binding sites are contained within the amino terminal region of plasminogen. Reversible acylation of the catalytic center would thus not affect the fibrin binding capacity of the complex. The plasmin(ogen)-streptokinase complex is an efficient activator of plasminogen. APSAC binds to fibrin via the lysine-binding sites of plasminogen, however the affinity of plasminogen for fibrin is weak. Deacylation of APSAC uncovers the catalytic center, which converts plasminogen to plasmin. Deacylation of APSAC occurs in the circulation as well as at the surface of the clot. Fibrin specificity, therefore is marginal.

21. Comparative Pharmacologic Features Feature SK APSAC UK SCUPA rtPA Half-Life (min) 23 90 16 7 5 Fibrin-Selective + + ++ ++++ +++ Duration of Infusion 60 min 2-5 m 5-15 m Hours Hours Antigenicity Yes Yes No No? No? Incidence of Reperfusion (%) 60-70 60-70 60-70 60-70 60-70 Frequency of Reocclusion (%) 15 10 10 NA 20 Fibrinogenolysis ++++ ++++ +++ ++ ++ Platelet Activation +++ +++ 0 ? ++++

22. Reteplase (Retavase™) Non-glycosylated deletion mutant of t-PA containing 355 of the 527 amino acids of native t-PA. Does not bind fibrin as tightly, thus allowing the drug to diffuse more freely through the clot rather than binding to the surface as does t-PA. Does not compete with plasminogen for fibrin-binding sites, thus allowing plasminogen at the site of the clot to be transformed into clot-dissolving plasmin. Acts more rapidly than t-PA, has more rapid plasma clearance, shorter half-live (11-19 min). More convenient administration - 10 U over <2 min followed in 30 min by a second iv bolus.

23. Tenecteplase TNK-tPA (TNKase™) Acts more rapidly than t-PA, has more rapid plasma clearance, shorter half-live (11-19 min). More convenient administration - 10 U over <2 min followed in 30 min by a second iv bolus.

24. Tenecteplase TNK-tPA (TNKase™) Description: – tenecteplase is a bio-engineered plasminogen activator, – tenecteplase is also known as TNK-tPA. "TNK" refers to the sites of the tPA molecule that have been modified (T103N, N117Q, KHRR 296—299 AAAA). Actions: – tenecteplase is a variant of tPA. – tenecteplase reduces fibrinogen by 5—10% and plasminogen by 10—15%, compared to decreases of 40% and 50%, respectively, for alteplase; because of this, TNK-tPA may be more fibrin specific than alteplase.

25. Tenecteplase TNK-tPA (TNKase™) Uses: – for the treatment of acute MI. Distinguishing Features: – compared to alteplase, tenecteplase has a prolonged half- life (alpha half-life 11—20 min; beta half-life 41—138 min), increased specificity for fibrin, and increased resistance to plasminogen activator inhibitor-1 (PAI-1) – its main advantage might be that it can be administered as a single IV bolus injection.

26. Tenecteplase TNK-tPA (TNKase™) Major Adverse Reactions: similar to other thrombolytic agents: bleeding is the most serious ADR, TNK-tPA does not affect coagulation parameters to the same degree as alteplase; serious bleeding was not different. Usual Adult Dosage: weight-adjusted 30—50 mg IV bolus injection.

27. rNPA : a New Thrombolytic Agents Under Study rNPA is a modified form of tissue plasminogen activator (tPA), a naturally occurring clot-dissolving protein found normally in only small quantities in the body. A first generation tPA product is now available to treat heart attack patients. However, this product must be administered by a three-step procedure which takes at least 90 minutes to deliver a full therapeutic dose by intravenous infusion. Genetics Institute scientists engineered rNPA to significantly extend the period of activity (half-life) in circulation, potentially resulting in substantial increases in "clot-busting" activity compared to tPA. Thus, rNPA may be administered in a single bolus injection which eliminates the need for the slower IV treatment and may result in a more rapid opening of clogged blood vessels.