Platelet-targeted dual pathway antithrombotic inhibits thrombosis with preserved hemostasis
Donny Hanjaya-Putra, … , Karlheinz Peter, Elliot L. Chaikof
Donny Hanjaya-Putra, … , Karlheinz Peter, Elliot L. Chaikof
Published August 9, 2018
Citation Information: JCI Insight. 2018;3(15):e99329. https://doi.org/10.1172/jci.insight.99329.
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Research Article Cardiology Hematology

Platelet-targeted dual pathway antithrombotic inhibits thrombosis with preserved hemostasis

Abstract

Despite advances in antithrombotic therapy, the risk of recurrent coronary/cerebrovascular ischemia or venous thromboembolism remains high. Dual pathway antithrombotic blockade, using both antiplatelet and anticoagulant therapy, offers the promise of improved thrombotic protection; however, widespread adoption remains tempered by substantial risk of major bleeding. Here, we report a dual pathway therapeutic capable of site-specific targeting to activated platelets and therapeutic enrichment at the site of thrombus growth to allow reduced dosing without compromised antithrombotic efficacy. We engineered a recombinant fusion protein, SCE5-TAP, which consists of a single-chain antibody (SCE5) that targets and blocks the activated GPIIb/IIIa complex, and tick anticoagulant peptide (TAP), a potent direct inhibitor of activated factor X (FXa). SCE5-TAP demonstrated selective platelet targeting and inhibition of thrombosis in murine models of both carotid artery and inferior vena cava thrombosis, without a significant impact on hemostasis. Selective targeting to activated platelets provides an attractive strategy to achieve high antithrombotic efficacy with reduced risk of bleeding complications.

Authors

Donny Hanjaya-Putra, Carolyn Haller, Xiaowei Wang, Erbin Dai, Bock Lim, Liying Liu, Patrick Jaminet, Joy Yao, Amy Searle, Thomas Bonnard, Christoph E. Hagemeyer, Karlheinz Peter, Elliot L. Chaikof

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Figure 1

Characterization of SCE5-TAP antiplatelet and anticoagulant activity.

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Characterization of SCE5-TAP antiplatelet and anticoagulant activity. Se...
Selective targeting to ADP-activated human (A–C) or mouse (D–F) platelets was assessed by flow cytometry. Construct binding to activated platelets (white histogram) or nonactivated platelets (gray histogram) was detected by AF488 anti-His antibody. SCE5 and SCE5-TAP target human (A and B) or mouse (D and E) activated platelets, while MUT-TAP displays no binding (C and F). Representative histograms are shown from 4 experiments. (G) GPIIb/IIIa blocking activity was examined through flow cytometry analysis of fibrinogen binding to human and mouse ADP-activated platelets. Fibrinogen binding was quantified as mean fluorescent intensity, and % inhibition was calculated relative to vehicle control; n = 4. (H and I) Anti-FXa activity was monitored using chromogenic FXa substrate in solution (H) and after targeting constructs to fibrinogen-adherent activated platelets (I). Percent inhibition of FXa was calculated relative to vehicle control with measurements performed in triplicate; n = 4 experiments. (J and K) Flow chamber adhesion assay was performed with perfusion (500 s–1) of whole blood over collagen-coated glass capillaries (J). Phase contrast images of microthrombi formed in presence of SCE5 (5 and 15 μg/ml), SCE5-TAP (5 and 15 μg/ml), MUT-TAP (15 μg/ml), or saline vehicle. Scale bars: 20 μm. (K) Microthrombi were captured at 20× and area quantified with ImageJ; n = 4 per group. Data represent mean ± SD, ***P ≤ 0.001 (ANOVA and Bonferroni’s multiple comparison test).