Collaterals improve recanalization in acute ischemic stroke patients treated with intravenous thrombolysis, but the mechanisms are poorly understood. To investigate it, an in vitro flow model of the middle cerebral artery was developed with or without collaterals. An occlusion was achieved using human blood clots. Recanalization time, thrombolysis (clot length decrease and red blood cell (RBC) release), pressure gradient across the clot and clot compaction were measured. Results showed that with or without collateral alteplase-treated RBC dominant clots showed recanalization time 98±23 min vs 130±35 min (difference 32 min, 95% CI -6-58 min), relative clot reduction 31.8±14.9% vs 30.3±13.2% (difference 1.5%, 95% CI 10.4-13.4%) and RBC release 0.30±0.07 vs 0.27±0.09 (difference 0.03, 95% CI 0.04-0.10). Similar results were observed with fibrin-dominant clots. In RBC dominant clots, the presence vs absence of collateral caused different pressure gradients across the clot 0.41±0.09 vs 0.70±0.09 mmHg (difference 0.29 mmHg, 95% CI -0.17-0.41 mmHg), and caused the reduction of initial clot compaction by 5%. These findings align with observations in patients, where collaterals shortened recanalization time. However, collaterals did not increase thrombolysis. Instead, they decreased the pressure gradient across the clot, resulting in less clot compaction and easier distal displacement of the clot.

• MeSH
• Middle Cerebral Artery drug effects   physiopathology   diagnostic imaging   MeSH
• Erythrocytes drug effects   MeSH
• Fibrinolytic Agents therapeutic use   pharmacology   MeSH
• Ischemic Stroke * drug therapy   physiopathology   MeSH
• Collateral Circulation * drug effects   MeSH
• Humans MeSH
• Tissue Plasminogen Activator therapeutic use   pharmacology   MeSH
• Thrombolytic Therapy methods   MeSH
• Thrombosis drug therapy   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fibrinolytic Agents MeSH
• Tissue Plasminogen Activator MeSH

Intravenous thrombolysis with a recombinant tissue plasminogen activator (rt-PA) is the first-line treatment of acute ischemic stroke. However, successful recanalization is relatively low and the underlying processes are not completely understood. The goal was to provide insights into clinically important factors potentially limiting rt-PA efficacy such as clot size, rt-PA concentration, clot age and also rt-PA in combination with heparin anticoagulant. We established a static in vitro thrombolytic model based on red blood cell (RBC) dominant clots prepared using spontaneous clotting from the blood of healthy donors. Thrombolysis was determined by clot mass loss and by RBC release. The rt-PA became increasingly less efficient for clots larger than 50 μl at a clinically relevant concentration of 1.3 mg/l. A tenfold decrease or increase in concentration induced only a 2-fold decrease or increase in clot degradation. Clot age did not affect rt-PA-induced thrombolysis but 2-hours-old clots were degraded more readily due to higher activity of spontaneous thrombolysis, as compared to 5-hours-old clots. Finally, heparin (50 and 100 IU/ml) did not influence the rt-PA-induced thrombolysis. Our study provided in vitro evidence for a clot size threshold: clots larger than 50 μl are hard to degrade by rt-PA. Increasing rt-PA concentration provided limited thrombolytic efficacy improvement, whereas heparin addition had no effect. However, the higher susceptibility of younger clots to thrombolysis may prompt a shortened time from the onset of stroke to rt-PA treatment.

• MeSH
• Stroke drug therapy   MeSH
• Erythrocytes drug effects   metabolism   MeSH
• Fibrinolytic Agents therapeutic use   MeSH
• Blood Coagulation drug effects   MeSH
• Heparin * therapeutic use   MeSH
• Ischemic Stroke * drug therapy   MeSH
• Humans MeSH
• Recombinant Proteins * therapeutic use   MeSH
• Tissue Plasminogen Activator * therapeutic use   MeSH
• Thrombolytic Therapy * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fibrinolytic Agents MeSH
• Heparin * MeSH
• Recombinant Proteins * MeSH
• Tissue Plasminogen Activator * MeSH

Cardio- and cerebrovascular diseases are leading causes of death and disability, resulting in one of the highest socio-economic burdens of any disease type. The discovery of bacterial and human plasminogen activators and their use as thrombolytic drugs have revolutionized treatment of these pathologies. Fibrin-specific agents have an advantage over non-specific factors because of lower rates of deleterious side effects. Specifically, staphylokinase (SAK) is a pharmacologically attractive indirect plasminogen activator protein of bacterial origin that forms stoichiometric noncovalent complexes with plasmin, promoting the conversion of plasminogen into plasmin. Here we report a computer-assisted re-design of the molecular surface of SAK to increase its affinity for plasmin. A set of computationally designed SAK mutants was produced recombinantly and biochemically characterized. Screening revealed a pharmacologically interesting SAK mutant with ∼7-fold enhanced affinity toward plasmin, ∼10-fold improved plasmin selectivity and moderately higher plasmin-generating efficiency in vitro. Collectively, the results obtained provide a framework for SAK engineering using computational affinity-design that could pave the way to next-generation of effective, highly selective, and less toxic thrombolytics.

• Keywords
• Acute myocardial infarction, AffiLib, Affinity engineering, Enzyme kinetics, Plasminogen activators, Rational design, SAK, Staphylokinase, Staphylokinase, Stroke treatments, Thrombolytics,
• Publication type
• Journal Article MeSH