Detailed mechanisms of the inactivation of factor VIIIa by activated protein C in the presence of its cofactors, protein S and factor V.

Factor VIIIa is inactivated by a combination of two mechanisms. Activation of factor VIII by thrombin results in a heterotrimeric factor VIIIa that spontaneously inactivates due to dissociation of the A2 subunit. Additionally, factor VIIIa is cleaved by the anticoagulant serine protease, activated protein C, at two cleavage sites, Arg(336) in the A1 subunit and Arg(562) in the A2 subunit. We previously characterized an engineered variant of factor VIII which contains a disulfide bond between the A2 and the A3 subunits that prevents the spontaneous dissociation of the A2 subunit following thrombin activation. Thus, in the absence of activated protein C, this variant has stable activity following activation by thrombin. To isolate the effects of the individual activated protein C cleavage sites on factor VIIIa, we engineered mutations of the activated protein C cleavage sites into the disulfide bond-cross-linked factor VIII variant. Arg(336) cleavage is 6-fold faster than Arg(562) cleavage, and the Arg(336) cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg(562) cleavage more than Arg(336) cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg(562) cleavage more than Arg(336) cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg(336) cleavage is only twice as fast as Arg(562) cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation.

Cathepsin G, a leukocyte protease, activates coagulation factor VIII.

Neutrophils and monocytes express cathepsin G and can also bind to activated platelets, thus they can be localized to the site of active coagulation. Previous studies have suggested that cathepsin G inactivated coagulation factor VIII (FVIII) and was thus anticoagulant. But other studies have indicated procoagulant functions for cathepsin G in activation of coagulation factor V or activation of platelets among other possible mechanisms. Therefore, it remains unclear if cathepsin G is anticoagulant or procoagulant. We investigated the effects of human neutrophil cathepsin G on FVIII/VIIIa. Cathepsin G activates FVIII to a partially active form while having only a minor inactivating effect on thrombin-activated FVIIIa. This inactivation is mostly due to decreased stability of FVIIIa since a disulfide bond that prevents A2 subunit dissociation from FVIIIa prevents any loss of activity due to cathepsin G proteolysis. FVIII that has been cleaved by cathepsin G can still be activated by thrombin if A2 subunit dissociation is prevented. Cathepsin G cleavages of FVIII are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. Therefore, it is possible that cathepsin G from neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII.

Subchronic studies in Sprague-Dawley rats to investigate mechanisms of MTBE-induced Leydig cell cancer.

High MTBE exposures caused rat Leydig cell (LC) tumors in inhalation and gavage cancer bioassays. Investigating early endocrine changes consistent with known mechanisms of LC carcinogenesis, we gavaged adult male Sprague-Dawley rats with MTBE in five different subchronic experiments and studied testosterone biosynthesis in isolated rat LCs exposed in vitro to MTBE or a major metabolite, t-butanol. In vitro LC testosterone production declined 29-50% following 3-h exposures to 50-100 mM MTBE or t-butanol. Within hours after gavaging with 1,000 or 1,500 mg/kg MTBE, circulating testosterone declined to 38-49% of control (p < 0.05). If sampled longer after treatment or with lower doses, testosterone reductions were less dramatic or nondetectable even after 28 days of treatment. Accessory organ:brain weight ratios decreased only slightly although showing dose response with 40-800 mg/kg/day after 28 days. High MTBE doses caused slight liver weight and total P450 increases. Reduced aromatase activity in liver and testis microsomes predicted low serum estradiol, but estradiol was 19% higher than corn oil controls concurrent with testosterone reduction 1 h after the last of 14 daily 1,200-mg/kg doses (p < 0.05). Pituitary luteinizing hormone (LH) and prolactin measured in both intact and orchiectomized rats, with testosterone implants in some castrated rats providing stable levels of testosterone, revealed no consistent direct effect on hypothalamic-pituitary function. MTBE-treated rat livers showed no evidence of peroxisome proliferation, a characteristic of some LC carcinogens. Considering recognized mechanisms of Leydig cell cancer in rats, collectively these results suggested reduced LC steroidogenesis enzyme activity as a possible mechanism underlying MTBE LC carcinogenesis.