Characterization of profunda femoris vein thrombosis.

OBJECTIVE: The incidence of and risk factors for profunda femoris vein (PFV) thrombosis are poorly characterized. We prospectively identified patients with PFV deep venous thrombosis (DVT) to characterize the demographics and anatomic distribution of proximal DVT in patients with PFV DVT. METHODS: A prospective study was conducted of patients at a tertiary care university hospital with DVT diagnosed by venous duplex ultrasound scanning between June 2014 and June 2015. DVT patients were categorized as having PFV involvement (yes or no), and the anatomic distribution of other sites of ipsilateral venous thrombi was further stratified to determine whether there was external iliac vein (EIV), common femoral vein (CFV), or femoropopliteal vein (FPV) DVT. Demographic characteristics of the patients were compared between groups, PFV DVT vs proximal DVT without PFV DVT. RESULTS: Of 4584 lower extremity venous duplex ultrasound studies performed, 398 (8.7%) scans were positive for proximal DVT from 260 patients; 23.1% of patients with DVT (60/260) had DVT involving the PFV. Of 112 patients who had CFV DVT, 55 (49.1%) also had ipsilateral involvement of the PFV. Of 60 patients with PFV DVT, 55 (91.7%) had involvement of the ipsilateral CFV. Patients in the PFV DVT group were more likely to have a history of a hypercoagulable disorder (26.7% vs 14.5%; P = .029) and a history of immobility (58.3% vs 42%; P = .026) compared with those with proximal DVT without PFV DVT. There were no differences in smoking, recent surgery, personal or family history of DVT, other medical comorbidities, inpatient status, or survival. There was no difference in laterality of DVT between the PFV DVT and proximal DVT without PFV DVT groups (35% vs 41.5% left, 35% vs 33.5% right, 30% vs 25% bilateral; P = .619). There was a higher proportion of PFV DVT with EIV involvement (21.7% vs 2.5%; P < .00001) and a higher proportion of PFV DVT with CFV + FPV involvement (65.0% vs 19%; P < .00001) compared with proximal DVT without PFV DVT. There was no difference in survival between the PFV DVT and proximal DVT without PFV DVT groups. CONCLUSIONS: Patients with PFV thrombosis tend to have more thrombus burden with more frequent concurrent DVT in the EIV and FPV. Patients with PFV DVT are also more likely to have a history of hypercoagulable disorder and immobility. Ultrasound protocols for assessment of DVT should include routine examination of the PFV as a potential marker of a more virulent prothrombotic state.

Determinants of pH-dependent modulation of translocation in dermonecrotic G-protein-deamidating toxins.

Cytotoxic necrotizing factors from E. coli (CNF1, CNF2) and Yersinia (CNFy)share N-terminal sequence similarity with Pasteurella multocida toxin (PMT). This common N-terminal region harbors the receptor-binding and translocation domains that mediate uptake and delivery of the C-terminal catalytic cargo domains into the host cytosol. Subtle variations in the N-terminal ~500 amino acids of CNFs and PMT could allow for selective recognition of cellular receptors and thus, selective target cell specificity. Through studies with cellular inhibitors, we have identified an additional novel function for this region in modulating responses of these toxin proteins to changes in pH during intoxication and delivery of the catalytic cargo domain into the cytosol.

Arf6-dependent intracellular trafficking of Pasteurella multocida toxin and pH-dependent translocation from late endosomes.

The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH(4)Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity.