Blood coagulation at the site of microvascular injury in healthy and coumadin-treated subjects heterogenous for factor V Leiden mutation.

There is little knowledge regarding the in-vivo course of coagulation reactions in subjects with factor (F)V Leiden (G1691A, R506Q). The aim of the current study was to evaluate the effect of FV Leiden on coagulant reactions triggered by vascular injury. At the site of microvascular injury, prothrombin activation and FVa formation and inactivation have been evaluated in 16 apparently healthy subjects and 16 patients on long-term anticoagulation with acenocoumarol, with eight heterogenous carriers of FV Leiden in the healthy and in the anticoagulated group. Thrombin formation, measured by levels of thrombin-antithrombin complexes (TAT) and thrombin B-chain, proceeded at similar rates in the bleeding-time blood in carriers and non-carriers of FV Leiden. The onset and rate of FV activation did not differ significantly among the four groups. When healthy carriers of FV Leiden were compared to healthy non-carriers, the onset of FVa inactivation by activated protein C (APC) was delayed by 60 to 90 seconds (p=0.01). Anticoagulated individuals also showed the same pattern of FV Leiden associated differences in the time of occurrence of the 30 kD FVa heavy-chain fragment (p=0.021). Our results indicate that when blood clotting is triggered by microvascular injury, the heterozygous expression of FV Leiden has no effect on thrombin generation in healthy or coumadin-treated subjects. Inactivation of FVa by the APC mechanism is attenuated significantly in carriers of FV Leiden but the magnitude of this effect is smaller than that observed in most purified systems.

Mechanism of cyst specific protein 21 mRNA induction during Acanthamoeba differentiation.

The Acanthamoeba cyst specific protein 21 (CSP21) gene is tightly repressed in growing cells and highly induced early during differentiation into a dormant cyst. This increase is mediated by the rate of transcription of the CSP21 gene as determined by nuclear run-on assays. The promoter region of the CSP21 gene was analyzed by transcript start site mapping and in vitro transcription of wild-type or mutant templates, using extracts from growing cells. A sequence located 3' to a modified TATA box completely inhibits transcription and removal of this region permits robust transcription utilizing a start site approximately 35 base pairs downstream of the TATA box. Sequences 5' to the TATA box had no effect on transcription, suggesting that anti-repression is the only mechanism required for CSP21 induction. Fractionation of nuclear extracts yielded a fraction capable of transcription from the CSP21 promoter, and a fraction containing a promoter-specific repressing activity. Anti-repression may thus be a major mechanism regulating differentiation or maintenance of the proliferative cycle in Acanthamoeba.