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Combined Antiplatelet/Anticoagulant Drug for Cardiac Ischemia/Reperfusion Injury

Originally publishedhttps://doi.org/10.1161/CIRCRESAHA.120.317450Circulation Research. 2020;127:1211–1213

Meet the First Author, see p 1121

Myocardial infarction is typically caused by rupture of atherosclerotic plaques leading to thrombotic occlusion of coronary arteries. Major progress has been achieved by reducing the time needed and increasing the success rate of reperfusion of blocked coronary arteries. Paradoxically, reperfusion itself results in further tissue damage, known as ischemia/reperfusion (I/R) injury, by provoking major inflammatory and microthrombotic responses, leading to loss of cardiac function.

Platelets accumulate very early in ischemic/reperfused myocardium mediating I/R injury.1 Current antiplatelet/anticoagulant drugs, particularly in combination, are associated with bleeding complications representing a major cause of mortality/morbidity. Thus, there remains an unmet clinical need for effective antiplatelet/anticoagulant drugs that do not affect hemostasis. We developed an scFv (single-chain antibody), which specifically binds to activated GP (glycoprotein) IIb/IIIa (αIIbß3), which mediates platelet cross-linking/aggregation and thrombus formation. Our scFvTarg binds specifically to the fibrinogen-binding pocket, which is only exposed on activated GPIIb/IIIa, and thereby inhibits platelet aggregation but not adhesion.1–3 We have genetically fused scFvTarg to TAP (tick anticoagulant peptide), an FXa (Factor Xa) inhibitor, generating a unique dual-function antiplatelet/anticoagulant drug termed as Targ-Tap. Being targeted to activated platelets, this fusion protein provides enrichment of anticoagulant activity at the site of activated platelet accumulation and thereby allows application at low systemic doses. We have recently demonstrated its antithrombotic potency while maintaining hemostasis.2

Here, we investigate whether Targ-TAP can prevent cardiac I/R injury. Inducing transient ischemia by occlusion of the left anterior descending artery of mice for 60 minutes,1 we recapitulated the clinical scenario of patients with myocardial infarction undergoing recanalization of the occluded coronary artery. Our drug was administered at the time of reperfusion to mimic its potential clinical application. Male C57Bl/6J mice (20–25 g), sourced from AMREP Animal Services, were randomized to receive control (PBS or non–Targ-TAP, a nontargeting-TAP mutant without binding to GPIIb/IIIa), or Targ-TAP (both at 0.03 μg/g body weight, intravenously). All animal procedures were performed in accordance with institutional guidelines. Echocardiography was performed at baseline and 4 weeks post-I/R.1 Imaging and all analyses were performed blinded. Targ-TAP protected against loss of cardiac function, whereas a significant decline in ejection fraction and fractional shortening was observed in control animals (Figure [A]). Significant increases in left ventricular volume-at-diastole and volume-at-systole were noted in control groups, whereas Targ-TAP–treated mice were protected from dilatation (Figure [A]). Control animals had significantly more regional abnormalities as compared with Targ-TAP–treated mice; reflected in the significant reduction in peak radial strain for both the infarcted area and global readouts, as well as increased time in maximum opposite-wall delay (Figure [B]). Importantly, this protection of cardiac function by Targ-TAP correlated with a significantly reduced infarct size in comparison with controls, as assessed by Evans Blue/triphenyltetrazolium chloride staining 4 weeks post-I/R (Figure [C]). Confirming that Targ-TAP does not exhibit systemic effects on hemostasis, mice treated with Targ-TAP did not display prolonged tail bleeding times or increased blood loss (Figure [D]). With the caveat that tail clip experiments have limitations in predicting bleeding in patients, we additionally measured the activated partial thromboplastin time and prothrombin time in mice. No prolongation was observed with Targ-TAP (Figure [E]).

Figure.

Figure. Targ-TAP (dual-function antiplatelet/anticoagulant drug) preserves myocardial function and reduces infarct size after ischemia/reperfusion (I/R).A, Four-week post-I/R, ejection fraction is preserved in Targ-TAP-treated mice as compared with baseline, non–Targ-TAP, or control-treated mice. Targ-TAP also improved fractional shortening and prevented volume-at-diastole (V;d) and volume-at-systole (V;s) left ventricular (LV) dilatation. B, Representative radial strain curves. Colored lines represent the six myocardial regions, black lines represent the average (global) strain. Control mice exhibit a marked decrease in % peak (PK) radial strain, both in the infarcted area and globally compared with Targ-TAP. Control mice show significant increases in time for maximum opposite-wall delay as compared to Targ-TAP. C, Representative images of Evans Blue/TTC (triphenyltetrazolium chloride)- stained hearts 4 wk post-I/R. Targ-TAP-treated hearts show a reduced I/AaR (infarct to area at risk ratio) and infarct size compared with controls. D, No difference is observed for bleeding time and blood loss in mice injected with Targ-TAP, compared with control; both are significantly increased in mice injected with Eptifibatide. E, Targ-TAP did not show a difference in aPTT (activated partial thromboplasin time) or PT (prothrombin) compared with controls. After normality was confirmed (Anderson-Darling and Shapiro-Wilk tests), 1-way or 2-way ANOVA with Tukey post hoc test were applied. PK indicates peak.

To date, there is no clinically available therapeutic to reduce the deleterious consequences of cardiac I/R injury. Targeted drug delivery provides an attractive platform to deliver potent, site-directed antithrombotic drugs, without untoward systemic effects on hemostasis. Targeting activated platelets, which express an activation-specific molecular epitope and accumulate early, and in abundance, in ischemic/reperfused myocardium, provide an ideal cellular target for site-directed drug delivery to the area at risk for I/R injury. No other such cellular or molecular target has yet been described. Targ-TAP possesses this targeting capability and thereby allows local delivery/enrichment of both antiplatelet potency, by blocking fibrinogen binding to GPIIb/IIIa, and anticoagulant effects, by TAP’s factor anti-Xa effects. Targ-TAP can be applied intravenously as an acute application in the catheterization laboratory or subcutaneously for chronic application. The recombinant antibody-drug format allows optimization in size and consequently in pharmacokinetic/dynamic characteristics.

In summary, we describe Targ-TAP as a highly effective antithrombotic drug uniquely combining localized antiplatelet and targeted anticoagulant effects while preserving hemostasis. Its flexible drug format supports broad application and includes prophylaxis and treatment of arterial and venous thrombosis and, importantly, prevention of cardiac I/R injury. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Nonstandard Abbreviations and Acronyms

GP

glycoprotein

I/R

ischemia/reperfusion

scFv

single-chain antibody

TAP

tick anticoagulant peptide

Acknowledgments

We acknowledge the help of Dr Erica Malan.

Disclosures

C. Haller, E.L. Chaikof, K. Peter, and X. Wang are inventors on patents describing recombinant antibodies. The other authors report no conflicts.

Footnotes

*K.P. and X.W. are equally contributing senior authors.

For Sources of Funding and Disclosures, see page 1212.

Correspondence to: Karlheinz Peter, MD, PhD, Baker Heart and Diabetes Institute, 75 Commercial Rd, Melbourne, 3004 Victoria, Australia. Email
Xiaowei Wang, PhD, Baker Heart and Diabetes Institute, 75 Commercial Rd, Melbourne 3004, Victoria, Australia. Email

References

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  • 2. Hanjaya-Putra D, Haller C, Wang X, Dai E, Lim B, Liu L, Jaminet P, Yao J, Searle A, Bonnard T, et al. Platelet-targeted dual pathway antithrombotic inhibits thrombosis with preserved hemostasis.JCI Insight. 2018; 3;e99329. doi: 10.1172/jci.insight.99329CrossrefMedlineGoogle Scholar
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