Identification & Characterization of Novel Hemostatic Biomarkers of Adverse Clinical Events in Patients with Continuous Flow Left Ventricular Assist Device Implants
Authors: McClane Nathan , Jeske Walter , Walenga Jeanine M. , Escalante Vicki , Coglianese Erin , Hoppensteadt Debra , Schwartz Jeffrey , Bakhos Mamdouh 
Published: October 2019

Abstract
Introduction: Heart failure affects over 5 million people in the US. Its rising prevalence coupled with a lack of donor hearts has led to an increase in the use of continuous flow left ventricular assist devices (CF-VAD). However, patients with CF-VAD implants are at risk for complications such as non-surgical bleeding and thrombosis, which significantly increase the risk of patient morbidity and mortality. Identification of novel biomarkers for these events could improve current risk assessment models, treatment, and thus, quality of life for VAD implanted patients.
Materials and Methods: 71 blood samples were collected peri-operatively and at varying time points post-operatively from 16 patients implanted with a Thoratec HeartMate II CF-VAD. Plasma was prepared and stored at -70°C until analysis. Plasma samples collected from normal individuals were used as controls. Samples were analyzed for annexin V-positive microparticles and microparticles exposing tissue factor, as well as plasma levels of annexin V and C-reactive protein (CRP) by ELISA. Additionally, surface-enhanced laser desorption ionization - mass spectrometry (SELDI-MS) analysis was used to identify changes in biomarker expression. The occurrence of adverse clinical events was determined using an internal RedCap database. CF-VAD thrombosis was characterized as: cerebrovascular accident/transient ischemic attack diagnosed by a neurologist, rise in LDH or plasma free hemoglobin, hemolysis, evidence of pump dysfunction consistent with thrombus identified by pump parameters, echocardiographic or computed tomographic evidence of clot, or surgical pump exchange for thrombus. CF-VAD bleeding events were characterized as: anemia and bleeding determined by a cardiologist.
Results: Annexin V plasma levels were increased approximately 3-fold compared to normal plasma (9.23 ±0.85 vs. 2.76 ±0.2 nM). CRP plasma levels were significantly elevated in CF-VAD patients compared to normals (14.5 ±2.7 vs. 2.2 ± 0.6 µg/ml; p=0.034). Although the level of annexin V-positive microparticles were the same in CF-VAD patients and normals, the amount of microparticles exposing tissue factor was significantly higher in CF-VAD patients (3.7 ± 1.1 vs. 0.5 ± 0.1 pg/ml; p=0.038). SELDI-MS analysis indicated three distinct peaks that were present in CF-VAD patients, but absent from normals (8.1, 11.7 and 15.2/16.2 kDa). During the follow-up period, patients experienced 12 CF-VAD-related thrombotic events, 8 hemorrhagic events and 2 septic events. The 8.1 kDa biomarker was present in 10/12 patients with a thrombotic event, 2/6 patients with a hemorrhagic event, 0/2 patients experiencing sepsis and 0/2 patients without an adverse event. Positive correlations with the SELDI-MS peaks and measured parameters were identified: the 8.1 kDa peak was associated with elevated plasma levels of annexin V (p=0.01) and the presence of annexin V-positive microparticles (p=0.005); the 11.7 kDa peak was associated with elevated plasma levels of CRP (p=0.01); the 15.2/16.2 kDa peaks were associated with the presence of microparticle exposing tissue factor (p=0.002), annexin V-positive microparticles (p<0.001) and elevated plasma levels of annexin V (p=0.03).
Conclusions: Despite treatment with low-dose aspirin and warfarin, CF-VAD patients exhibit signs of hemostatic activation. SELDI-MS may be useful in the identification of novel biomarkers of CF-VAD-associated adverse events. This data justifies further evaluation of the utility of the SELDI-MS to readily identify biomarkers related to adverse clinical events in a larger CF-VAD population.

Disclosures
No relevant conflicts of interest to declare.