No effect of fasting plasma total homocysteine on protein C activity in vitro

Although the association between the plasma levels of total homocysteine (tHcy) and the risk of atherosclerosis and thrombosis is well documented, the mechanism(s) by which hyperhomocysteinemia might contribute to atherogenesis and thrombogenesis are scarcely understood.1 In vitro studies showed that homocysteine, among other effects, inhibits both protein C activation and the activity of activated protein C (APC).1,2 The inhibition of APC activity in vitro apparently depends on the interaction of homocysteine with cysteine residues of factor V, which interferes with the proteolytic action of APC on factor Va, resulting in APC resistance, a very common and well-established risk factor for venous thromboembolism.2 However, several in vitro findings have not been confirmed in in vivo studies. For instance, in a study of healthy individuals and patients with previous thrombotic events, Cattaneo et al3 showed that neither the fasting plasma levels of tHcy nor their acute increase after an oral methionine load affects the plasma concentration of APC. These data, which suggest that hyperhomocysteinemia does not interfere with protein C activation in vivo, have recently been supported by the results of the study by Lentz et al,4 which showed that hyperhomocysteinemia did not interfere with protein C activation in vivo in cynomolgus monkeys to which an intravenous injection of thrombin had been administered. In the same study, Lentz et al4 showed that hyperhomocysteinemia did not interfere with the in vitro inactivation of factor V by APC in mice deficient in cystathionine-β-synthase nor in 10 human volunteers in whom hyperhomocysteinemia was acutely induced by an oral methionine load. Like Lentz and collaborators, we recently addressed the problem of whether or not hyperhomocysteinemia influences the anticoagulant response to APC. We studied subjects referred to our center between June 1991 and December 2001 to undergo screening for thrombophilic states, which included the measurement of fasting tHcy and the in vitro anticoagulant response to APC. The plasma levels of tHcy were measured by high-performance liquid chromatography (HPLC) and fluorometric detection5; APC resistance was measured using an activated partial thromboplastin time (APTT)–based clotting assay using undiluted patient plasma.

The results were expressed as normalized ratios.6Hyperhomocysteinemia was diagnosed when the plasma tHcy exceeded the 90th percentile of distribution of values obtained in a population of 553 healthy subjects (14.8 μM). We enrolled 1254 subjects (median age 43 years, range 9-83 years) who had normal APTT and absence of factor V Leiden (the most common congenital cause of APC resistance7). Of these subjects, 730 were women, of whom 108 (15%) used oral contraceptives; 434 (34.6%) had previous episodes of venous or arterial thrombosis; and 820 had a negative personal history for thrombosis. The plasma levels of tHcy ranged from 3.9 μM to 223 μM in the subjects studied. The mean APC normalized ratio of subjects with normal tHcy plasma levels (0.97 ± 0.13) was not different from that of subjects with hyperhomocysteinemia (0.96 ± 0.16, P = .73). There was no statistically significant correlation between the plasma levels of fasting tHcy and APC ratio (Table 1) whether considering the total number of subjects, those with hyperhomocysteinemia, or those with previous thrombotic events. In conclusion, in agreement with Lentz et al,4 our findings do not support the hypothesis that hyperhomocysteinemia negatively affects the in vitro anticoagulant response to APC.