Background: Patients with thrombotic thrombocytopenic purpura (TTP) experience attacks of microvascular thrombosis, when platelets complex to von Willebrand Factor (VWF). This is caused by ADAMTS13 deficiency, which normally regulates VWF thrombogenicity. Unfortunately, ADAMTS13 re-supplemental therapy can be hampered by persistent autoantibodies. We previously reported that systemic plasminogen activation (with streptokinase) was therapeutic in a TTP mouse model, suggesting that plasmin can act as an alternative to ADAMTS13. For optimal therapeutic efficacy and safety, it is wishful to localize plasminogen activation to sites of microvascular occlusion. While plasmin(ogen) can directly bind to VWF in microthrombi, its natural activators (tissue- and urokinase type plasminogen activator; tPA, uPA) cannot.

Aims: Development of fusion proteins (microlyse; fig.1) for targeted delivery of plasminogen activators to platelet-VWF complexes.

Methods: We fused the catalytic domain of uPA to nanobodies against VWF or a non-targeting control nanobody.

Results: Microlyse variants are well expressed in HEK293 cells, show no spontaneous activity, but develop enzymatic activity upon incubation with plasmin(ogen). In the presence of open VWF, plasminogen activation is accelerated 6-fold for the anti-VWF microlyse variants. In VWF-dependent platelet agglutination experiments, both anti-VWF and -GP1b microlyse variants accelerate destruction of microthrombi, as compared its non-targeting control. At equimolar concentrations, targeting microlyse variants degrade microthrombi 7-fold more rapidly than the bivalent humanized nanobody Cablivi. In a flow model for TTP, targeting fusion proteins accelerate the removal of VWF-platelet strings from the endothelial surface in ADAMTS13 deficient plasma. Finally, in a ADAMTS13(-/-) mice in a model of thrombotic thrombocytopenic purpura, anti-VWF but not the non-targeted microlyse variant, attenuate thrombocytopenia within 24 hours with an efficacy that is comparable to that of cablivi. However, where we find evidence for circulating microthrombi after cablivi treatment, these are not present after microlyse treatment.

Conclusions: Targeted delivery of plasminogen activators to microthrombi is a promising approach for the treatment of TTP.

[Figure1. Microlyse: a nanobody-UPA fusion protein]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/microlyse-a-vwf-targeting-thrombolytic-agent-for-thrombotic-thrombocytopenic-purpura/