Thrombotic Biomarker Profiling Of Plasma Samples From Patients Undergoing Bypass Surgery Using Protein Chip Array
Authors: Walenga Jeanine M. , Matsuo Takefumi , Wanaka Keiko , Cunanan Josephine , Hoppensteadt Debra , Fareed Jawed 
Published: October 2019

Abstract

Introduction
Cardiovascular bypass surgical procedures, such as the coronary bypass procedure (CAP), patients are exposed to large doses of heparin and protamine sulfate. There is a high prevalence of anti-heparin platelet factor-4 antibodies in these patients. Although a fraction of these patients develop HIT syndrome, the role of circulating anti-heparin platelet factor-4 antibodies is not clear. Some of these patients may be at a high risk for post-surgical thrombotic complications. Such thrombogenic mediators as the microparticle and tissue factor, may also be up regulated in these patients. Inflammatory processes may also contribute to the overall post-thrombotic complications. The relevance of thrombotic mediators and inflammatory processes remains to be further explored in these patients. Recently protein chip array approach using SELDI/TOF mass spectrometry methods have been employed to identify the unique biomarker in various diseases. The purpose of this study was to determine the protein chip array profile and quantification of various mediators of thrombotic activation in patients who have undergone bypass surgery.

Materials & Methods
Plasma samples from 79 patients were collected immediately prior to and two weeks after the CAP. Protein chip array profiling was carried out on a SELDI/TOF mass spectrometric method (PCS4000, BioRad, Richmond, CA) employing a gold chip array in the molecular weight range of 3000-150,000. The intensity of unique peaks was also calculated in terms of relative intensities. Microparticles were measured using a functional method (Hyphen Biomedical, France) and tissue factor antigen levels were measured using an ELISA method. The anti-heparin platelet factor-4 antibodies were also measured using a commercially available ELISA method (Genprobe, Wisconsin).

Results
Of the 79 patients, 20 showed a unique biomarker peak around 11-12kDa in the pre-op samples, which was absent from normal controls. 77 of the patients showed this unique biomarker peak at one week, whereas only 48 patients exhibited at two weeks after surgery. The relative intensity of the 11.6kDa biomarker was much higher at one week (6-fold) and was decreased at two weeks (3-fold). In addition to this unique peak, other biomarker peaks were noted at 15.1 and 15.8kDa. However, these peaks were not changed at different time points. In comparison to the normal, the microparticle levels were higher at the baseline sample (10.1±3.2nM) and increased to 19.3±6.1 and 24.5±8.1nM. Similarly, the tissue factor levels were increased at three weeks’ time period. The anti-heparin platelet factor-4 titer rose 28% from the baseline at week one and 33% at week two.

Conclusions
The results on the biomarker profile are consistent to the earlier finding, where the presence of a unique biomarker in the range of 11-12kDa have been reported in patients with high prevalence of anti-heparin platelet factor-4 antibody. The increased level of microparticles and tissue factor at post-surgical periods of one week and two weeks suggest endogenous activation of thrombogenic mechanisms, which appears proportional to the up regulation of the anti-heparin platelet factor-4 antibodies. Thus, this data indicates that the non-functional anti-heparin platelet factor-4 antibodies may result in the activation of cellular processes leading to thrombogenesis. Further characterization of the unique biomarkers identified in these patients may be useful in understanding of the pathogenesis of inflammatory processes and their relevance to thrombogenesis in CABG patients.

Clinical Implications
These results indicate that the nonfunctional anti-heparin platelet factor 4 antibodies are capable of mediating inflammatory and thrombotic responses without symptomatic thrombocytopenia. Therefore, the measurement of these antibodies along with inflammatory and thrombogenic mediators may be helpful in the diagnostic and prognostic management of these patients.

Disclosures:
No relevant conflicts of interest to declare.