Assessment Of a Novel Synthetic Collagen Reagent By Platelet Aggregometry
Authors: Jeske Walter , Escalante Vicki , Walenga Jeanine M. , Bakhos Mamdouh 
Published: October 2019

Platelets play a critical role in hemostasis and depending on the physiological balance can have either decreased (bleeding) or increased (thrombosis) activity. The monitoring of platelet function is important to diagnose congenital and acquired platelet dysfunctions, predict surgical bleeding, identify the need for antiplatelet therapy and to monitor anti-platelet therapy efficacy. A growing concern today is that many anti-platelet drugs used with a one dose fits all protocol, may not achieve optimal clinical efficacy and safety in all patients. It may be desirable to dose to a targeted level of platelet function inhibition. Additionally, a subpopulation of patients treated with anti-platelet drugs may exhibit ‘anti-platelet drug resistance’ due to non-compliance, biochemical factors, genetic polymorphisms, concomitant therapies or patient-specific factors. Because platelets can be activated through a number of pathways, the sensitivity of current platelet agonists is not always optimal. This study evaluated the ability of a novel synthetic collagen reagent to detect the anti-platelet activity of ticagrelor, cilostazol and abciximab in normal and aspirinized human platelets.

Whole blood was drawn from 6 healthy volunteers from the antecubital vein using a double-syringe technique and was anticoagulated by the addition of 1 part 3.2% sodium citrate to 9 parts blood. Citrated blood was centrifuged for platelet rich plasma (PRP) and platelet poor plasma (PPP). The platelet count of the PRP was measured using an ICHOR II Analyzer (Helena, Beaumont, TX) and adjusted to 250,000-300,000/µl by the addition of homologous PPP. Each donor was drawn on separate days for the ‘non-aspirinized’ and ‘aspirinized’ portions of the protocol. For the ‘aspirinized’ portion of the protocol, three donors had ingested aspirin (325 mg)< 48 hours prior to blood draw. For the other three donors, solubilized aspirin was supplemented to the PRP in vitro at a final concentration of 100 µM. All PRPs were supplemented with abciximab (1.25-5 µg/ml; Eli-Lilly, Indianapolis, IN), ticagrelor (5-50 µg/ml; AstraZeneca, London, UK) and cilostazol (5-25 µM; Otsuka, Tokushima, Japan). Platelet aggregation was measured using a PAP-8 platelet aggregometer (BioData, Horsham, PA) in response to ADP (10 µM; BioData), arachidonic acid (AA) (500 µg/ml; BioData), biologic collagens (0.19 mg/ml; BioData or 10 µg/ml; Chronolog) and synthetic collagen (8-64 ng/ml; JNC, Tokyo, Japan).

In non-aspirinized PRP, ticagrelor strongly inhibited ADP- and AA-induced aggregation. Whereas the two biologic collagens were not able to identify the anti-platelet effect of ticagrelor, a clear anti-platelet effect was seen with the synthetic collagen at concentrations ≤ 32 ng/ml. In the presence of aspirin, the anti-platelet effect of ticagrelor was no longer observed with AA or 8 ng/ml synthetic collagen. Although the effect with 16 and 32 ng/ml synthetic collagen was reduced, ticagrelor could still be detected. In non-aspirinized PRP, the most marked effect of cilostazol was on AA-induced aggregation, where aggregation levels of ∼20% were observed at drug concentrations ≥ 12.5 µM (vs. 95% in the absence of cilostazol). Cilostazol at concentrations up to 25 µM did not inhibit aggregation induced by either biologic collagen, but at lower concentrations of synthetic collagen, the anti-platelet effect of higher concentrations of cilostazol could be observed. Addition of aspirin to the PRP negated the ability to detect cilostazol. Arachidonic acid and synthetic collagen (8 and 16 ng/ml) were the most sensitive for detecting abciximab. Inhibition of biologic collagen-induced aggregation was only observed at the 5 µg/ml drug concentration. Although the synthetic collagen reagent produced lower levels of aggregation in aspirinized plasma than in non-aspirinized plasma, abciximab was readily detectable in the presence of aspirin using the synthetic collagen reagent.

Low concentrations of the synthetic collagen reagent induced platelet aggregation that is inhibited by several classes of anti-platelet drugs (ADP receptor antagonists, GPIIb/IIIa antagonists, PDE inhibitors), and in addition combined anti-platelet effects with aspirin could be measured. Because such effects are not observed with standard biologic collagens, the new synthetic collagen may be a useful addition to clinical laboratory testing for platelet function.

Disclosures:

Jeske: BioData Corporation: Research Funding.