TF Independent Potentiation of FVIIa Activity in CAD Plasma: An Assessment Using Two Chromogenic Assays.

Background. Tissue Factor (TF), the regulatory cofactor of FVII(a), triggers blood coagulation as it converts FX and FIX to their active forms FXa and FIXa, respectively. In 2003 we showed that level of decryptable circulating TF activity in diabetic, smoker, and hyperlipidemic populations is a functionally labile entity. Existence of such form of circulating TF has generated the concept of vulnerable or hyperreactive blood. Recently, heightened TF activity was noted by many groups in various types of clinical samples, including human plasma, via utilization of a commercial TF activity assay termed ACTICHROME® whose approach to measure plasma TF activity is direct and distinct from that developed by us.

Objective. To assess levels of plasma TF activity in patients with coronary artery disease (CAD), by the ACTICHROME® as well as our assay.

Results. Suitability of the ACTICHROME® assay for direct assessment of TF activity in plasma was compared with our technique of plasma TF immunocapture followed by TF relipidation. We assessed TF activity in plasma aliquots pre-incubated with FVIIai, an inactive form of FVIIa that retains binding affinity to TF yet lacks protease activity, thereby serving as a highly effective and specific TF inhibitor. The ACTICHROME® assay was not able to detect bona fide TF activity: the tested plasma exhibited activity that could not be inhibited by pre-incubation with FVIIai. Thus, under the conditions of the ACTICHROME® assay, FX to FXa conversion in human platelet rich plasma is achieved by FVIIa in a TF independent manner. In contrast to the ACTICHROME® assay, in our TF assay pre-incubation of samples with FVIIai fully precluded FX activation. We therefore refer to plasma TF activity levels obtained using our TF assay as tTFp (total TF potential), and to the FVIIa activity levels obtained using the ACTICHROME® assay – ACTI-FVIIa. Assessment of tTFp in CAD plasma samples (n=28) revealed that in a subset of patients, tTFp was lower than that found in normal plasma (58.5%-69.6%, p=0.0001 NL, n=6 vs CAD tTFp <70%, n=4), while another subset of patients presented with tTFp that was higher than that of normal plasma (134.2% – 233.6%, p=0.003 NL, n=6 vs CAD tTFp >130%, n=7). The tTFp values in age matched healthy subjects (n=6) were in the close range of 75.2%-109.5%. We also assessed each healthy control and CAD patient sample for ACTI-FVIIa. To our surprise, ACTI-FVIIa values in all CAD samples were high, ranging between 15.8–132.1 pM of the TF standard, whereas ACTI-FVIIa values in the healthy subject group were much lower, in the close range of 3.2–8.8 pM. Interestingly, ACTI-VIIa of the three CAD samples exhibiting the highest tTFp was significantly lower than that of the three CAD samples exhibiting the lowest tTFp (highest tTFp: ACTI-VIIa = 39.2 ± 17.3, lowest tTFp: ACTI-VIIa = 97.4 ± 30.1, p=0.043). Heightened ACTI-FVIIa values in CAD samples prompted us to ascertain whether circulating MP and/or platelets may account for this phenomenon. Conventional size MP and platelets were removed from plasma by ultracentrifugation. Approximately 50% of ACTI-FVIIa in normal plasma and ≥95% of ACTI-FVIIa in CAD plasma was attributable to conventional size MP; in contrast, tTFp values in plasma samples pre- and post-spin were fairly similar: ultracentrifugation of CAD plasma resulted in only ~24.5% reduction of TF activity whereas in normal plasma, it resulted in ~42% reduction in TF activity, indicating that similarly to CAD plasma, the majority of functional TF species in normal plasma are not physically associated with conventional size MP, yet a somewhat larger proportion of functional TF is indeed carried by them compared to CAD plasma.

Conclusions. We show that in CAD plasma, circulating MP of the conventional size do not carry most of functional TF yet, interestingly, CAD plasma strongly potentiates FVIIa activity in a TF-independent manner. We also show that the ACTICHROME® assay, when used to measure TF activity in human plasma, yields results that do not reflect bona fide TF activity. The majority of functional TF in human plasma appears to circulate in very small MP and in true solution; lastly, all examined plasma samples carried TF activity, underscoring the fact that potentially active TF protein is always present in circulation. These observations highlight the therapeutic possibilities of TF pathway inhibitors for treatment and prevention of atherothrombotic diseases.