Molecular Biomarkers for In Vitro Thrombogenicity Assessment of Medical Device Materials

Abstract

To develop standardized in vitro thrombogenicity test methods for evaluating medical device materials, three platelet activation biomarkers, beta-thromboglobulin (β-TG), platelet factor 4 (PF4), soluble p-selectin (CD62P), and a plasma coagulation marker, thrombin-antithrombin complex (TAT), were investigated. Whole blood, drawn from six healthy human volunteers into Anticoagulant Citrate Dextrose Solution A was recalcified and heparinized over a concentration range of 0.5-1.5 U/mL. The blood was incubated with test materials with different thrombogenic potentials for 60 min at 37°C, using a 6 cm²/mL material surface area to blood volume ratio. After incubation, the blood platelet count was measured before centrifuging the blood to prepare platelet-poor plasma (PPP) and platelet-free plasma (PFP) for enzyme-linked immunosorbent assay analysis of the biomarkers. The results show that all four markers effectively differentiated the materials with different thrombogenic potentials at heparin concentrations from 1.0 to 1.5 U/mL. When a donor-specific heparin concentration (determined by activated clotting time) was used, the markers were able to differentiate materials consistently for blood from all the donors. Additionally, using PFP instead of PPP further improved the test method's ability to differentiate the thrombogenic materials from the negative control for β-TG and TAT. Moreover, the platelet activation markers were able to detect reversible platelet activation induced by adenosine diphosphate (ADP). In summary, all three platelet activation markers (β-TG, PF4, and CD62P) can distinguish thrombogenic potentials of different materials and detect ADP-induced reversible platelet activation. Test consistency and sensitivity can be enhanced by using a donor-specific heparin concentration and PFP. The same test conditions are applicable to the measurement of coagulation marker TAT.

Keywords: biomaterials; coagulation; hemocompatibility; molecular markers; platelet activation; thrombogenicity.

Figure 1.

Summary of the major experimental steps. Test materials with different thrombogenic potentials (Low: Polytetrafluoroethylene (PTFE), High-Density Polyethylene (HDPE), Intermediate: Silicone (Si), Stainless Steel (SS), and High: Buna-N Rubber (BUNA), glass beads (Glass) were tested. For positive control (PC): Test was run with 20 μM ADP in recalcified blood. For negative control (NC): Test was run without any test material. PPP: Platelet-Poor Plasma, PFP: Platelet-Free Plasma

Figure 2.

Mean concentrations of β-TG (A), PF4 (B), and CD62P (C) after 1 hour incubation of test materials with blood at various heparin concentrations. Data are shown as mean ± SE (n=6). PC: positive control, NC: negative control

Figure 3.

(A) ACT values of heparinized blood (0 to 1.5 U/mL) from different blood donors. (B) Number of donors (from a total of n=6 donors) that enabled differentiation (>= 10% difference in the concentration of a marker) of the materials in different thrombogenic potential groups (Low- PTFE, HDPE; intermediate- Si, SS; high- Glass, BUNA) at different heparin concentrations. ACT-Hep is the minimum heparin concentration that achieved the targeted ACT value between 190–240s for blood from each donor.

Figure 4.

Comparison of platelet activation marker concentration normalized to the baseline and negative controls for the (A) positive control, and thrombogenic materials (B) Glass and (C) BUNA at different test heparin concentrations (1.0 U/mL, 1.25 U/mL, and ACT-Hep). (D) Statistical significance comparison of platelet activation markers, determined by Fishers LSD post hoc test. Data are shown as mean ± SE (n=6). PC: positive control, NC: negative control

Figure 5.

Comparison of using PPP and PFP on the test sensitivity for different markers. The marker concentrations of (A) β-TG, (B) PF4, and (C) CD62P in PPP and PFP were normalized to those of the negative control. All test samples were exposed to whole blood heparinized with ACT-Hep concentration at 37 °C for 1 hr. Data are shown as mean ± SE (n=6). *p<0.05 shows a statistical significance comparison between PPP and PFP for each material. NC: negative control

Figure 6.

(A) Mean TAT concentration after 1 hour incubation of test materials in whole blood at different heparin concentrations. (B) number of donors (out of total of n=6 donors) that enabled differentiation (≥10% difference in the concentration of a marker) of the materials in different thrombogenicity groups (Low - PTFE, HDPE; intermediate - Si, SS; high - Glass, BUNA) at different heparin concentrations. (C) Comparison of TAT concentrations (normalized to NC) in PPP and PFP after the test samples were incubated with whole blood, heparinized to the ACT-Hep concentration, at 37 °C for 1 hr. Data is shown as mean ± SE (n=6). *p <0.05, ** 0.05

Figure 7.

Detection of Reversible activation of Platelets. The effect of ADP on the platelet activation markers (β-TG, PF4, CD62P), coagulation marker (TAT), and platelet count reduction in the blood after 1 hr of incubation without and with different heparin concentrations. Data are shown as mean ± SE (n=6). (F) Statistical significance comparison of different materials. Green color indicates a statistically significant difference between compared groups (p<0.05). PC: positive control, NC: negative control