Diagnosis of LVAD Thrombus using a High-Avidity Fibrin-Specific 99mTc Probe

Abstract

Treatment of advanced heart failure with implantable LVADs is increasing, driven by profound unmet patient need despite potential serious complications: bleeding, infection, and thrombus. The experimental objective was to develop a sensitive imaging approach to assess early thrombus accumulation in LVADs under operational high flow and high shear rates. Methods: A monomeric bifunctional ligand with a fibrin-specific peptide, a short spacer, and ^99mTc chelating amino acid sequence (F1A) was developed and compared to its tetrameric PEG analogue (F4A). Results:^99mTc attenuation by LVAD titanium (1 mm) was 23%. ^99mTc-F1A affinity to fibrin was K_d ~10 µM, whereas, the bound ^99mTc-F4A probe was not displaced by F1A (120,000:1). Human plasma interfered with ^99mTc-F1A binding to fibrin clot (p<0.05) in vitro, whereas, ^99mTc-F4A targeting was unaffected. The pharmacokinetic half-life of ^99mTc-F4A was 28% faster (124±41 min) than ^99mTc-F1A (176±26 min) with both being bioeliminated through the urinary system with negligible liver or spleen biodistribution. In mice with carotid thrombus, ^99mTc-F4A binding to the injured carotid was much greater (16.3±3.3 %ID/g, p=0.01) than that measured with an irrelevant negative control, ^99mTc-I4A (3.4±1.6 %ID/g). In an LVAD mock flow-loop (1:1, PBS:human plasma:heparin) operating at maximal flow rate, ^99mTc-F4A bound well to phantom clots in 2 min (p<0.05), whereas ^99mTc-F1A had negligible targeting. Excised LVADs from patients undergoing pump exchange or heart transplant were rewired, studied in the mock flow loop, and found to have spatially variable fibrin accumulations in the inlet and outlet cannulas and bearings. Conclusions:^99mTc-F4A is a high-avidity prototype probe for characterizing thrombus in LVADs that is anticipated to help optimize anticoagulation, reduce thromboembolic events, and minimize pump exchange.

Keywords: LVAD; fibrin; nuclear imaging; technetium; thrombosis.

Figure 1

A Diagrammatic representation of a HeartMate II (HMII) left ventricular assist device (LVAD). Blood (orange arrow) is drawn into the pump through a cannula from the ventricular apex, flows by the stator through the turbine and returns to systemic circulation through the outlet cannula into the aorta. The pump is powered through an externalized driveline cable on the outlet side of the LVAD. B Small residual LVAD thrombus on the inlet stator and around the bearing (C) from a patient admitted with elevated lactate dehydrogenase (LDH) (1300's) after symptomatic hemorrhagic/embolic stroke due to a suspected pump thrombus. White arrows point to residual thrombus. HMII image is copyright protected by Thoratec, Inc and used with permission.

Figure 2

A ^99mTc carbonyl aqua-ion and the complex formed with HH-peptide. B ^99mTc-F1A bi-functional monomer depicting the cyclic homing sequence, short spacer, and ^99mTc-chelating site depicting ^99mTc carbonyl coupling. C ^99mTc-F4A tetramer created by the balanced cross-linking of the four F1A monomers with tetrameric PEG arms (shown with one example of ^99mTc on one of the four arms for clarity)_. PEG: polyethylene glycol. Bold arrows point to ^99mTc carbonyl coupling in example illustrations.

Figure 3

A Titrated dosages of ^99mTc-F1A bound to uniform fibrin clot in PBS. The dissociation constant (K_d ~10.2 µM) was adjusted for decay and estimated using the Hill slope model, Y=Bmax*X^h / (Kd^h + X^h) three times independently (± s.e.m.). Total fibrin concentration was estimated from plasma fibrinogen (0.735 nmol per clot). B The percentage of ^99mTc-F4A bound to each clot in the presence of unlabeled F1A (monomer) at molar ratios up to 120,000 to 1. ^99mTc-F1A (C) and ^99mTc-F4A (D) bound to clots in the presence of PBS and in PBS with excess cysteine to demonstrate ^99mTc labelling stability. However, in a 50:50 PBS:human plasma mixture the binding of ^99mTc-F1A to clot was dramatically reduced (p<0.05) whereas ^99mTc-F4A was unaffected. E The attenuation of Na^99mTcO₄ from 20 µCi down to 0.5 µCi by titanium plate (1 mm), equivalent to the housing of HMII, was 23% ± 3% independent of radioactivity level (n=3/level). HMII: HeartMate II; PBS: phosphate buffered saline.

Figure 4

The pharmacokinetics (PK) and biodistribution (BD) of the reference radiolabeled ^99mTc-F1A compared to ^99mTc-F4A in mice (n=3/treatment). A Both agents, presented as percent injected radioactivity per gram blood (%ID/g), closely followed a two-compartment bi-exponential PK model. B The alpha distribution half-life of ^99mTc-F4A in mice (5.0 ± 1.9 min) was 41% faster (p=0.04) than that of ^99mTc-F1A (8.6 ± 1.9 min). Similarly, the beta elimination half-life of ^99mTc-F4A (124.7 ± 41.3 min) was 50 min shorter (28%) than that of ^99mTc-F1A (174.2 ± 26.2 min) (p= 0.08), which were likely related to minimal plasma interactions with ^99mTc-F4A. Mouse organ biodistribution including body remains (n=3/treatment) of ^99mTc-F1A (C) and ^99mTc-F4A (D) at the conclusion of the PK study (180 min) were similar. Neither probe accumulated substantially in the lung, liver or spleen. Virtually all of the ^99mTc activity was excreted through the urinary system (kidney, bladder and urine).

Figure 5

Targeted fibrin binding of ^99mTc-F4A to carotid thrombus was compared to an irrelevantly targeted ^99mTc-I4A probe (~75 µCi/animal). A The signal from the clot-bearing carotid (%ID/g) was compared to the clot-free contralateral vessel response. ^99mTc-F4A binding to carotid thrombus was much greater than uptake in the clot-free contralateral carotid vessel (p<0.05). ^99mTc-I4A bound poorly to carotid thrombus and nuclear probe uptake did not differ from the nonspecific signal obtained in the clot-free carotid. Binding of the fibrin-specific ^99mTc-F4A to carotid clot was markedly greater (p=0.01) than the nonspecific binding of ^99mTc-I4A to thrombus. *p<0.05. B Representative maximum intensity projection image with a color map (arbitrary units) from 1 of 5 mice administered ^99mTc-F4A (40 µCi) by tail vein injection then imaged 2 h later with a Multispectral FX multimodal imaging system (Bruker-Biospin, Billerica, MA). A Marked single source signal originating from the right carotid thrombus was noted. All animals (n=5) displayed a strong in vivo carotid nuclear signal following ^99mTc-F4A.

Figure 6

A HMII mock flow loop design: circulation media was 200 mL of heparinized 50:50 plasma:PBS. Activity injected for each probe was 0.5 mCi. B Average percent radioactivity (± s.e.m.) of ^99mTc-F4A versus ^99mTc-F1A bound to uniform fibrin clots after 2 min of circulation in HMII mock flow loop. *p<0.05, n=3/treatment group, HMII: HeartMate II

Figure 7

^99mTc-F4A signal was assessed for the inlet cannula (metallic and Dacron^® segments), the inlet stator/bearing, turbine, outlet bearing, and outlet cannula regions. Strong ^99mTc-F4A signals were noted in the metallic and Dacron^® inlet cannula segments in A and in the flexible, Dacron^® segment in panel B. Prominent ^99mTc-F4A signal was observed around the inlet bearing in C and both the inlet and outlet bearings/stator in D. Fibrin accumulation coating the walls of the outlet cannulas were frequently noted as in E. This LVAD was notable also for thrombus associated with the inlet and outlet bearings. Yellow arrows in each image point to regions of ^99mTc-F4A signal corresponding to fibrin deposition and accumulation. Abbreviated clinical histories: A Implanted 520 days. Early hemolysis after implantation. Peak LDH 1300 U/L. Resolved with eptifibatide and bivalrudin. B Implanted 830 days. No hemolysis. Routine transplant. C Implanted 146 days. Hemolysis noted with a peak LDH 800 U/L. Negative ramp study. LDH trended downward and the heart was transplanted soon thereafter. D Implanted 410 days. Sustained hemolysis with a LDH peak 3400 U/L. Pump exchanged for suspected pump thrombosis. E Ischemic stroke 7 days after implantation. No hemolysis. Pump exchanged. LVAD: left ventricular assist device; LDH: lactic dehydrogenase.