Despite advances in surgical technique and post-operative management, including routine administration of ASA, the incidence of early SVG occlusion after CABG surgery remains substantial in a contemporary patient population. The rate of SVG occlusion 6 months after CABG surgery observed in the RIGOR study is consistent with results from several recent studies, including the PREVENT 4 (Project of Ex-vivo Vein Graft Engineering via Transfection 4) trial which reported an overall SVG occlusion rate of 26% in 2400 patients 12–18 months after CABG surgery (19
). Among patient and graft-specific variables associated with SVG occlusion, bypass of small diameter (≤1.5 mm) target vessels has historically been considered one of the strongest (17
). In addition to this traditional risk factor, we identified shear-dependent platelet activation, measured by PFA-100 CADP CT, and ASA-insensitive TXA2
generation, measured by UTXB2
, as potent novel independent risk factors for early SVG thrombosis. The combination of PFA-100 CADP CT and UTXB2
was further able to stratify subjects into those at particularly high- and low-risk for SVG thrombosis.
In normal individuals, the overwhelming majority of TXA2
generated in the body is produced in platelets by metabolism of arachidonic acid by the COX-1 enzyme. Because ASA efficiently and irreversibly inhibits platelet COX-1 activity, it markedly suppresses TXA2
generation as measured by UTXB2
and blunts platelet reactivity to several agonists (15
). Growing evidence suggests that some patients with cardiovascular disease have persistent TXA2
generation in spite of ASA therapy and are consequently at increased risk for adverse clinical events. This association was first noted in a subgroup analysis from the HOPE (Heart Outcomes Prevention Evaluation) study and later confirmed in the larger CHARISMA (Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management and Avoidance) trial where subjects on ASA with the highest compared to the lowest quartile of UTXB2
at study entry had an approximate 1.7-fold increased risk (P ≤0.03 for both studies) of myocardial infarction, stroke or cardiovascular death over the subsequent 2–5 years (8
). Our findings add to the body of evidence suggesting that ASA-insensitive TXA2
generation is a significant cardiovascular risk factor by demonstrating that it is independently associated with early SVG occlusion after CABG surgery. Follow up data on RIGOR subjects is currently being collected to determine if ASA-insensitive TXA2
generation after CABG surgery also adversely affects long-term clinical outcome.
A fundamental question arising from the above data is whether ASA-insensitive TXA2
generation in patients with cardiovascular disease represents a failure of ASA to inhibit platelet COX-1-mediated TXA2
biosynthesis, the strictest and most accurate definition of ASA resistance (5
), or the presence of alternate TXA2
production pathways that are not effectively inhibited by ASA. Zimmerman found that platelets from patients taking 100 mg/day of ASA generate more TXA2
in the first week after CABG surgery than platelets from healthy ASA-treated controls (11
generation was only partially suppressed by the further exposure of platelets in vitro to high concentrations of ASA, suggesting both incomplete COX-1 inhibition in vivo by low-dose ASA, as well as the presence of COX-1-independent TXA2
). Others have subsequently confirmed that platelets from ASA-treated subjects are capable of generating TXA2
via non-COX-1-mediated pathways, though the amounts appear relatively small in comparison to that produced by platelets from ASA-naïve patients (21
). While we did not directly measure platelet TXA2
generation in our study, arachidonic acid-induced platelet aggregation is known to be inhibited when platelet TXA2
generation is suppressed by >90% (22
). The suppression of arachidonic acid-induced platelet aggregation in ≥95% of subjects both early and late after CABG surgery argues for a high degree of platelet COX-1 inhibition by ASA and therefore a very low prevalence of true ASA resistance in our study population.
Despite evidence for effective platelet COX-1 suppression by ASA, UTXB2
was markedly elevated in a substantial number of subjects both early and late after CABG surgery. As UTXB2
generation in the entire body, not only from platelets, our data suggest that significant TXA2
production may originate from non-platelet sources that are not effectively inhibited by ASA. Potential sources include inflammatory and endothelial cells that are capable of producing TXA2
via both COX-1 and inducible COX-2-dependent pathways. ASA is a relatively weak inhibitor of COX-2 at standard doses (20
) and, unlike platelets, both cell types have the capacity to regenerate COX-1 that is irreversibly inhibited by ASA. Another potential source is from non-enzymatic conversion of arachidonic acid to F2
-isoprostanes under conditions of oxidative stress. F2
-isoprostanes are capable of activating platelet and cellular TXA2
receptors and can also directly stimulate TXA2
production in endothelial cells (23
). Additional studies are needed to identify the sources of this ASA-insensitive TXA2
generation and the mechanism by which its production adversely affects clinical outcome.
Various aspects of platelet function, including ASA responsiveness, can be characterized by a wide array of ex vivo assays. One of the most physiologic is the PFA-100 device which measures agonist-induced platelet aggregation under conditions of high shear by quantifying the closure time (CT) of a membrane aperture by the formation of a platelet plug (25
). The PFA-100 was primarily designed to screen for defects in primary hemostasis with the CEPI agonist cartridge detecting the antiplatelet effects of ASA and the CADP agonist cartridge identifying patients with congenital and acquired platelet disorders (26
). Among patients with cardiovascular disease, a CEPI CT in the normal range (94–193 seconds) despite ASA therapy is associated with an increased risk of recurrent cardiovascular events (27
). However, the specificity and ultimate utility of the PFA-100 at detecting true aspirin resistance has been called into question by the observation that CEPI CTs are highly influenced by plasma levels of vWF (29
). Our results not only confirm the poor correlation between ASA responsiveness defined by CEPI CT compared to arachidonic acid-induced platelet aggregometry but also reveal no association between CEPI CT and SVG outcome.
A major finding of our study was the strong association between low CADP CT and early SVG occlusion. Unlike CEPI CT, CADP CT is not influenced by ASA or thienopyridine use and is therefore considered an indicator of global platelet reactivity. Similar to CEPI CT, we found a correlation between low CADP CT and high plasma vWF levels (13
) but did not find an independent association between vWF levels and early SVG occlusion (data not shown). A CADP CT below the normal range (71–118 seconds) suggests platelet hyper-reactivity and has been observed in patients with acute coronary syndromes where it correlates with the degree of myocardial necrosis and recurrent ischemic events (30
). In a recent study of 700 patients undergoing percutaneous coronary intervention, a CADP CT <65 seconds measured before the procedure was associated with a 3.5-fold increase risk (CI 1.2–10.4, p<0.03) of major adverse cardiac events at 24 months (21
). These studies along with our own suggest the potential of measuring shear-dependent platelet activation as a means to risk stratify patients with cardiovascular disease.
A limitation of the RIGOR study was that ASA responsiveness and platelet reactivity were not assessed preoperatively. This was by design as we anticipated not all eligible subjects would have been on chronic ASA therapy prior to CABG surgery and some would have been on concurrent non-ASA antiplatelet agents that would confound interpretation of many of the assays. It was reasoned that values obtained 6 months after CABG surgery, following resolution of the associated inflammatory response and hematologic changes, would be indicative of the subject’s baseline state. Now that ASA-insensitive thromboxane generation and shear-dependent platelet activation have been identified as important parameters, additional studies will be needed to determine if their preoperative measurement can identify patients at high risk for SVG thrombosis and what therapies might be useful to mitigate such risk.
In conclusion, we have identified ASA-insensitive TXA2 generation, measured by UTXB2, and shear-dependent platelet hyper-reactivity, measured by PFA-100 CADP CT, as novel independent risk factors for early SVG thrombosis. Both entities appear to represent pathways that are independent of platelet COX-1 activity and not effectively inhibited by ASA. We further found that the prevalence of ASA non-responsiveness varied widely by several commonly used assays and time relative to CABG surgery but was not generally associated with graft outcome. Using arachidonic acid-induced platelet aggregometry as a specific indicator of platelet TXA2 generation, however, the overwhelming majority of patients appear to have a high degree of platelet COX-1 inhibition by ASA and therefore a low incidence of ASA resistance both early and late after CABG surgery.