Development and Testing of Thrombolytics in Stroke

Abstract

Despite recent advances in recanalization therapy, mechanical thrombectomy will never be a treatment for every ischemic stroke because access to mechanical thrombectomy is still limited in many countries. Moreover, many ischemic strokes are caused by occlusion of cerebral arteries that cannot be reached by intra-arterial catheters. Reperfusion using thrombolytic agents will therefore remain an important therapy for hyperacute ischemic stroke. However, thrombolytic drugs have shown limited efficacy and notable hemorrhagic complication rates, leaving room for improvement. A comprehensive understanding of basic and clinical research pipelines as well as the current status of thrombolytic therapy will help facilitate the development of new thrombolytics. Compared with alteplase, an ideal thrombolytic agent is expected to provide faster reperfusion in more patients; prevent re-occlusions; have higher fibrin specificity for selective activation of clot-bound plasminogen to decrease bleeding complications; be retained in the blood for a longer time to minimize dosage and allow administration as a single bolus; be more resistant to inhibitors; and be less antigenic for repetitive usage. Here, we review the currently available thrombolytics, strategies for the development of new clot-dissolving substances, and the assessment of thrombolytic efficacies in vitro and in vivo.

Keywords: Protein engineering; Stroke; Thrombolytic therapy; Tissue plasminogen activator.

Figure 1.

Molecular structure and fibrinolytic function of tissue plasminogen activator (tPA). (A) The F domain (green) is involved in binding to fibrin, which stimulates the activity of tPA. Binding of F and epidermal growth factor (EGF)-domain (violet) to the low-density lipoprotein receptor-related protein 1 (LRP1) receptor on the surface of a hepatocyte is involved in the clearance of tPA. The K1 domain (orange) is homologous to the K domains of urokinase and desmoteplase, and glycosylation at Asn117 on the K1 domain influences tPA uptake in the liver [34]. The K2 domain (red) has a lysine-binding site, which binds to partially degraded fibrin and other proteins containing C-terminal lysines. This could be the basis of enhancement of plasminogen activating activity of tPA by interacting with cofactors other than fibrin. The trypsin-like serine protease P domain (steelblue) is responsible for the catalytic activity of tPA. Upon tPA binding to fibrin, the catalytic activity is increased by both colocalization of plasminogen and conformational change of tPA into a more active state. (B) Carbohydrate chains (white+red) affect the half-life of tPA by interacting with the mannose receptor and asialoglycoprotein receptor on the endothelial cells in the liver [33,35]. In addition, carbohydrate chains influence the catalytic activity by preventing interactions between the domains of tPA [35]. (C) The cartoon in the lower right panel illustrates the mechanism of tPA-mediated fibrinolysis in thrombi and circulating blood. Fibrinolysis (1º) occurs after tPA binds to fibrin-associated plasminogen (PLG) on the surface of the clot by generating plasmin (PLA), and thereby breaking the cross-links between fibrin molecules. PLA could also break down circulating fibrinogen (2º), and thus potentially causing bleeding complications. Plasmin activity is inhibited by α₂-antiplasmin. Images of molecular structures of tPA that were adapted from Mican et al. [36], with permission from the Elsevier.

Figure 2.

Scheme depicting the development of novel thrombolytics. The overall workflow is separated into four main steps, which are thoroughly discussed in the main text. (A) Development of novel molecules using protein engineering. (B) In vitro testing by biochemical assays. (C) In vivo testing in animal models. (D) Several phases of clinical testing. F, finger domain; EGF, epidermal growth factor-like domain; K1, kringle 1 domain; K2, kringle 2 domain; P, protease domain; RGD, arginine (R), glycine (G), aspartic acid (D); TTI, tsetse thrombin inhibitor; SAK, staphylokinase; SPR, surface plasmon resonance; mCT, micro computed tomography; MRI, magnetic resonance imaging; ICH, intracerebral hemorrhage; mRS, modified Rankin Scale; NIHSS, National Institutes of Health Stroke Scale.

Figure 3.

Overview of thrombolytic enzymatic activity assays. (A) The physiological thrombolytic pathway without any modification can be monitored by fibrin plates, providing clear zones through the dissolution of fibrin. (B) Direct chromogenic/fluorogenic assay uses a synthetic substrate instead of plasminogen. The assay allows easy and straightforward analysis of thrombolytic enzyme kinetics by a spectrophotometer. The direct cleavage by plasminogen activators results in a linear increase of the signal over time. (C) Coupled chromogenic/fluorogenic assay uses a synthetic substrate instead of fibrin, while the analyzed step of activation involves the physiological plasminogen substrate. The measurement is thus more robust, reliable, and closer to real thrombolytic events than the direct assay. The coupled nature of the assay results in a quadratic increase of the signal over time.