How do tissue plasminogen activator tPA treat stroke?

Mechanism of Action of Tissue Plasminogen Activator

t-PA, a serine protease, contains five discrete domains: a fibronectin-like finger domain, an epidermal growth factor domain, two kringle domains, and a protease domain. Synthesized as a single-chain polypeptide, plasmin converts single-chain t-PA into a two-chain form. Both forms of t-PA convert plasminogen to plasmin. Native Glu-plasminogen is a single-chain polypeptide with a Glu residue at its amino-terminal. Plasmin cleavage near the amino-terminal generates Lys-plasminogen, a truncated form with a Lys residue at its new amino-terminus. t-PA cleaves a single peptide bond to convert single-chain Glu- or Lys-plasminogen into two-chain plasmin, which is composed of a heavy chain containing five kringle domains and a light chain containing the catalytic domain. Because its open conformation exposes the t-PA cleavage site, Lys-plasminogen is a better substrate for t-PA than Glu-plasminogen is, which assumes a circular conformation that renders this bond less accessible.

t-PA has little enzymatic activity in the absence of fibrin, but its activity increases by at least three orders of magnitude when fibrin is present.10 This increase in activity reflects the capacity of fibrin to serve as a template that binds t-PA and plasminogen and promotes their interaction. t-PA binds fibrin via its finger and second kringle domains, whereas plasminogen binds fibrin via its kringle domains. Kringle domains are loop-like structures that bind Lys residues on fibrin. Degradation of fibrin exposes more Lys residues, which provides additional binding sites for t-PA and plasminogen. Consequently, degrading fibrin stimulates activation of plasminogen by t-PA more than intact fibrin does.

Alpha2-antiplasmin rapidly inhibits circulating plasmin by docking to its first kringle domain and then inhibiting the active site.10 Because plasmin binds to fibrin via its kringle domains, plasmin generated on the fibrin surface resists inhibition by alpha2-antiplasmin. This phenomenon endows fibrin-bound plasmin with the capacity to degrade fibrin. Factor XIIIa cross-links small amounts of alpha2-antiplasmin onto fibrin, which prevents premature fibrinolysis.

Like fibrin, annexin II on endothelial cells binds t-PA and plasminogen and promotes the interaction of these proteins. Cell surface gangliosides and alpha-enolase may also bind plasminogen and promote its activation by altering its conformation into the more readily activated open form. Plasminogen binds to endothelial cells via its kringle domains. Lipoprotein(a), which also possesses kringle domains, impairs cell-based fibrinolysis by competing with plasminogen for cell surface binding (see alsoChapter 48). This phenomenon may explain the association between elevated levels of lipoprotein(a) and atherosclerosis (see alsoChapters 45and48).27

Tissue Plasminogen Activator

t-PA exists in two forms in plasma: (1) free t-PA capable of binding to and activating plasminogen, and (2) t-PA/PAI-1 complex an inactive form of inhibited t-PA. t-PA may be measured by either functional or antigenic assays. t-PA activity measures only the free, functional form of t-PA. t-PA in complex with PAI-1 and other inhibitors is not measured. Free t-PA in blood must be stabilized by drawing the sample into an acidified citrate solution to prevent inactivation of free t-PA by active PAI-1 in blood. t-PA activity is measured by capturing t-PA on a microtiter plate using monoclonal antibodies, washing off PAI-1 and antiplasmin inhibitors, and then measuring activity by adding plasminogen, a fibrinlike activator that enhances plasminogen activation by t-PA and a plasmin sensitive chromogenic substrate. t-PA converts plasminogen to plasmin, and the plasmin cleaves the substrate. Total t-PA antigen representing both active and inactive fractions can be measured using an enzyme immunoassay.

Tissue Plasminogen Activator in Vitreoretinal Surgery

AMD is one of the leading causes of blindness in the Western world. New pharmacologic treatment options have revolutionized the treatment armamentarium and provided true functional improvement in neovascular forms of the disease. However, complications of neovascular AMD such as major subretinal hemorrhages are still associated with a limited visual prognosis that could require vitreoretinal intervention.

This problem may be related to toxic effects of iron released from subretinal hemoglobin as well as an increased physical barrier for retinal diffusion and fibrotic changes.46–48 Up to now, there is no consensus regarding how to treat subretinal hemorrhages associated with neovascular AMD, and most surgical treatment strategies seem not efficient in restoring or improving vision.49

A displacement of the hemorrhage may be achieved by intravitreally applied expansile gas combined with intravitreal injection of recombinant plasminogen activator (rTPA). However, the subretinal lytic effect of intravitreally applied rTPA was challenged by some authors due to its molecular size and the reduced retinal diffusion.50,51 Therefore, a combination of intravitreal VEGF inhibitors to treat the underlying neovascular process combined with an injection of expansible gas is favored by some authors.52

There is clinical evidence that intravitreal rTPA in combination with expansile gas was in the long term more effective if combined with subsequent anti-VEGF treatment than intravitreal bevacizumab in combination with expansile gas alone.53 Other groups have suggested triple therapies using using rtPA, bevacizumab or ranibizumab, and have shown a successful management of the disease with this approach.54 If rTPA and pneumatic displacement combination is contraindicated, an anti-VEGF monotherapy may be performed to prevent further visual loss.55 However, after intravitreal injection of any agent combined with expansible gas a vitreous hemorrhage may occur, very often due to a displacement of the subretinal blood into the vitreous cavity. In these cases vitrectomy is indicated and may lead to good functional results.

An additional strategy is to perform a vitrectomy and apply rtPA subretinally followed by fluid gas exchange56 to displace the hemorrhage. This approach led to visual improvement in some cases. The authors also reported that vitrectomy with subretinal injection of rtPA and intravitreal gas tamponade was more effective than vitrectomy with intravitreal injection of rtPA and gas in terms of complete displacement of the submacular blood.57 Although functional improvement in the majority of patients suggests the absence of direct retinal toxicity of subretinally applied rtPA, vitrectomy and subretinal injection of rtPA carry a greater risk for postoperative complications.57

Tissue plasminogen activator assay

The two physiologic human plasminogen activators are TPA and urokinase.124,125 TPA is synthesized in vascular endothelial cells and released into the circulation, where its half-life is approximately 3 minutes and its plasma concentration averages 5 ng/mL. Urokinase is produced in the kidney and vascular endothelial cells and has a half-life of approximately 7 minutes and a concentration of 2 to 4 ng/mL. Both activators are serine proteases that form ternary complexes with bound plasminogen at the surface of fibrin, activating plasminogen to form plasmin and initiating thrombus degradation. The endothelial-secreted PAI-1 covalently inactivates both.

Tissue Plasminogen Activator

Thrombolytic agents have been proposed as a treatment of a suspected thrombus in the central retinal vein. If a thrombus is indeed etiologic, lysis is recommended within 21 days of its formation. Recombinant tissue plasminogen activator (r-tPA) is a synthetic fibrinolytic agent that converts plasminogen to plasmin and destabilizes intravascular thrombi. Reduction in clot size may facilitate dislodging of the entire thrombus or recanalization of the occluded retinal vein. Recombinant tissue plasminogen activator has been administered by several routes: systemic, intravitreal, and by endovascular cannulation of retinal vessels.

Systemic administration of low-dose (50 µg) front-loaded r-tPA has been attempted in two pilot studies with visual acuity improvement in 30–73% of patients.113,114 In a prospective, multicenter randomized trial of 41 patients with CRVO, Hattenbach and colleagues demonstrated significant 1-year improvement of 3 lines in 45% of patients undergoing low dose r-tPA compared to 21% of patients undergoing hemodilution.115 Another study examining high-dose (<100 µg) systemic tPA for the treatment of 96 patients reported development of intraocular hemorrhage in three patients and a fatal stroke in one patient.116 While Hattenbach did not observe any serious adverse events in his trial of low-dose r-tPA, these complications highlight the importance of approaching systemic administration of r-tPA with caution.

Intravitreal delivery of r-tPA has potential advantages, including decreased risk of systemic complications, directed delivery to the vitreous cavity, and subsequent access to the retinal vessels with low risk of ocular morbidity from the procedure. Of 47 persons in three noncontrolled studies of intravitreal r-tPA for both ischemic and nonischemic CRVO of less than 21 days' duration, 28–44% had 3 lines of visual acuity improvement with 6 months follow-up.117–119 Administration of r-tPA did not significantly alter final perfusion status, especially in pretreatment ischemic eyes.120 Although there were no significant treatment-related complications, differences in inclusion criteria and dosage of r-tPA used (between 66 and 100 µg) limits generalizations from these studies.

Endovascular delivery of r-tPA involves cannulation of retinal vessels, either through a neuroradiologic or a vitreoretinal approach, with delivery of minute quantities of r-tPA directly into the occluded vein to dissolve the suspected thrombus.121,122 Weiss and Bynoe reported their technique of vitrectomy followed by cannulation of a branch vein and infusion of r-tPA towards the optic nerve head.123 In their uncontrolled study, 50% of 28 eyes with CRVO of greater than 1-month duration and worse than 20/400 preoperative acuity recovered more than 3 lines by a mean follow-up of 12 months. Complications included vitreous hemorrhage in seven eyes and treated retinal detachment in one eye. In another prospective study of 13 patients undergoing endovascular r-tPA delivery, visual recovery did not correspond with successful thrombolysis, and complications including retinal detachment, phthisis, neovascular glaucoma, and cataract were considered unacceptably high.124

B. Tissue Plasminogen Activator

Tissue plasminogen activator is a thrombolytic protease that converts inactive plasminogen into active plasmin, which then degrades fibrin complexes, a major component of a thrombus. This enzyme can be overexpressed by ECs and SMCs following intracellular transfer of the tPA gene (11, 12). Overexpression of tPA has been shown to further decrease retention of seeded ECs on prosthetic grafts. The higher tPA production induces nonspecific proteolysis of the supporting extracellular matrix (ECM), thus reducing the adhesion of ECs to the graft surface (13, 14). A zymogen tPA variant (15) can be used to overcome the adverse effects of tPA on cell adhesion. Wild-type tPA is secreted from cells as an active, single-chained enzyme with a catalytic efficiency only slightly less than that of the proteolytically cleaved form. The zymogen mutant tPA (R275E, A292S, F305H) has been reported to have reduced catalytic efficiency by a factor of 200 in a single-chain form, while retaining full activity in its cleaved form, which is accomplished by binding fibrin. The zymogen tPA mutant can be used to advantage: its low protease activity, once secreted from the cell, will limit digestion of the ECM; therefore, the increased tPA expression will have limited adverse effects on cellular adhesion. After the secreted zymogen tPA is bound to fibrin within the thrombus, the mutant tPA will be cleaved into its active form, which will activate plasminogen as efficiently as wild-type tPA.

Interpretation

tPA is secreted into plasma in its active form but rapidly complexes with its principal inhibitor, PAI-1. The amount of active tPA in the plasma is the result of this equilibrium and represents only a small fraction of the total (antigenic) tPA. This process continues after blood sampling unless blood is taken into an appropriate acidic anticoagulant (see above). tPA levels are elevated by exercise and endothelial activation as well as in cirrhosis. The presence of heparin will augment tPA activity and lead to a misleadingly high estimate of activity.58 Deficiency of tPA in mice produces a number of effects on repair, angiogenesis, cell migration and tissue organisation, but a deficiency state in humans has not been described.59–61

tPA can also be measured by ELISA using monoclonal antibodies on microtitre plates, although this closely parallels the PAI-1 concentration and says little about the proportion of free, active tPA.

Identification of Protein Receptors

Tissue plasminogen activator (tPA) is associated with pancreatic tumor growth and invasion, and its interaction with cell membrane receptors has been related to increased proteolytic activity and transduction of tPA signaling in pancreatic tumor [76]. A proteomics approach utilizing antibody affinity capturing was applied to characterize the tPA receptors in pancreatic cancer cell lines [77]. Using nonpancreatic cancer endothelial cells as a comparison, 31 proteins were identified in the pull-down of tPA; and annexin A2 and galectin 1 were verified to be the functional receptors of tPA in pancreatic cancer [30,76,77].

1.1.1 Tissue Plasminogen Activator

tPA catalyzes conversion of plasminogen to plasmin and is mainly involved in thrombolysis. However, recent evidence indicates that tPA has additional diverse physiological and pathological roles in the brain. tPA is synthesized and released by neurons, glial cells, and endothelial cells, and it is constitutively expressed in various brain regions. In neurons, tPA mRNA expression is rapidly induced. In addition to regulation via the control of release and expression, tPA activity can be regulated by the interaction with endogenous serine protease inhibitors (serpins) such as neuroserpin, protease nexin I, and plasminogen activator inhibitor-1 (for a review, see Melchor and Strickland, 2005).

TISSUE PLASMINOGEN ACTIVATOR (tPA)

tPA converts plasminogen to plasmin and destabilizes intravascular thrombi. tPA reduces clot size in occlusive vessels, resulting in dislodgment of the entire thrombus or recanalization of the occluded retinal vein. When used to treat CRVO, tPA has been administered by several routes: systemic, intravitreal, and by endovascular cannulation of retinal vessels. Systemic administration of low-dose (50 mg) tPA resulted in a VA increase of one line or more in 10 (7 CRVO, 3 BRVO) of 14 patients (8 CRVO, 6 BRVO) with RVO.24 The VA improved 2 lines or more in 10 of 23 patients with CRVO.25 However, one patient died of an intracranial hemorrhage following tPA administration.26 Intravitreal administration of tPA is associated with less risk of systemic complications. tPA injected into the vitreous cavity subsequently reaches the retinal vessels. tPA (65–110 µg) was injected intravitreally in 23 patients with CRVO with recent onset of visual symptoms and the VA improved or stabilized in 16 eyes (70%).27 Another study reported that 4 of 9 patients with CRVO treated with 100 µg of tPA intravitreally had 3 lines or more of VA improvement.28 However, tPA is associated with retinal toxicity.29 Endovascular delivery of tPA involved cannulation of retinal vessels and tPA was injected directly into the occluded retinal vein, resulting in release of the suspected thrombus. Pars plana vitrectomy followed by cannulation of a branch vein and, with the help of a stabilization arm, injection of a bolus of 200 µg/ml of tPA toward the optic nerve head improved the VA more than 3 lines in 50% of 28 eyes after a mean follow-up of 12 months. Complications included vitreous hemorrhage in 7 eyes and retinal detachment in 1 eye.30