Cell cultures established from cerebrum of 1-day-old rats were used to investigate hormonal regulation of the development of oligodendrocytes, which synthesize myelin in the central nervous system. The number of oligodendrocytes that developed was preferentially increased by insulin, or by insulin-like growth factor I (IGF-I), also known as somatomedin C. High concentrations (5 Pg/ml) of insulin were required for substantial induction of oligodendrocyte development, whereas only 3.3 ng of IGF-I per ml was needed for a 2-fold increase in oligodendrocyte numbers. At an IGF-I concentration of 100 ng/ml, oligodendrocyte numbers were increased 6-fold in cultures grown in the presence of 10% fetal bovine serum, or up to 60-fold in cultures maintained in serum-free medium. IGF-I produced less than a 2-fold increase in the number of nonoligodendroglial cells in the same cultures. Type I IGF receptors were identified on oligodendrocytes and on a putative oligodendrocyte precursor cell population identified by using mouse monoclonal antibody A2B5. These results indicate that IGF-I is a potent inducer of oligodendrocyte development and suggest a possible mechanism based on IGF deficiency for the hypomyelination that results from early postnatal malnutrition.Oligodendrocytes play a critical role in nervous system function by synthesizing and maintaining myelin in the central nervous system (CNS) (1). Very little is known about the stimuli or regulatory signals that promote the development of oligodendrocytes, but such information might provide the basis for approaches to promote repair in demyelinating disorders.We studied tissue cultures established from cerebrum of MATERIALS AND METHODS Cell Explantation and Culture. Cells were explanted by mechanical dissociation of cerebrum of 1-day-old strain LEC rats as described (3, 4) and were inoculated into polystyrene tissue culture dishes, glass microscope slide chambers, or dishes containing glass coverslips. Glass surfaces were previously treated with D-polylysine (10-50 pug/ml) or polylysine followed by fibronectin (1 Atg/cm2; purified by method B of ref. 9). Cells were maintained as mixed cultures without separation of individual cell types. Unless otherwise noted, culture medium was Eagle's minimum essential medium supplemented with glucose to a total of 6 g/liter, 0.1 mM of each nonessential amino acid, antibiotics, and 10% fetal bovine serum. For serum-free culture, cells were maintained in the presence of 10%6 fetal bovine serum for the first 16-24 hr of culture to allow the cells to attach (10, 11), and the cells were then rinsed and refed with serum-free culture medium. Serum-free medium (modified from ref. 10) consisted ofequal parts of Dulbecco's modified Eagle's medium (high-glucose formula) and Ham's F-12 medium, supplemented with glucose to a total of 6 g/liter/15 nM triiodothyronine/30 nM sodium selenite/transferrin (50 pug/ml). Insulin was absent except where noted. In all cases, medium was changed on the fourth day after explantation and every second da...

Human activated protein C (APC) is an antithrombotic, antiinflammatory serine protease that plays a central role in vascular homeostasis, and activated recombinant protein C, drotrecogin alfa (activated), has been shown to reduce mortality in patients with severe sepsis. Similar to other serine proteases, functional APC levels are regulated by the serine protease inhibitor family of proteins including ␣1-antitrypsin and protein C inhibitor. Using APC-substrate modeling, we designed and produced a number of derivatives with the goal of altering the proteolytic specificity of APC such that the variants exhibited resistance to inactivation by protein C inhibitor and ␣1-antitrypsin yet maintained their primary anticoagulant activity. Substitutions at Leu-194 were of particular interest, because they exhibited 4-to 6-fold reductions in the rate of inactivation in human plasma and substantially increased pharmacokinetic profiles compared with wild-type APC. This was achieved with minimal impairment of the anticoagulant͞ antithrombotic activity of APC. These data demonstrate the ability to selectively modulate substrate specificity and subsequently affect in vivo performance and suggest therapeutic opportunities for the use of protein C derivatives in disease states with elevated serine protease inhibitor levels.

To enhance the potency of 1,2-dibenzamidobenzene-derived inhibitors of factor Xa (fXa), an amidine substituent was incorporated on one of the benzoyl side chains to interact with Asp189 in the S1 specificity pocket. Lead molecule 1 was docked into the active site of fXa to facilitate inhibitor design. Subsequently, iterative SAR studies and molecular modeling led to a 1000-fold increase in fXa affinity and a refined model of the new inhibitors in the fXa active site. Strong support for the computational model was achieved through the acquisition of an X-ray crystal structure using thrombin as a surrogate protein. The amidines in this series show high levels of selectivity for the inhibition of fXa relative to other trypsin-like serine proteases. Furthermore, the fXa affinity of compounds in this series (K(ass) = 50-500 x 10(6) L/mol) translates effectively into both anticoagulant activity in vitro and antithrombotic activity in vivo.

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Conversion by α-thrombin of the zymogen human protein C (HPC) to activated protein C (aPC) is an important physiologic feedback control mechanism for the coagulation cascade. Although activation of HPC by thrombomodulin-bound thrombin is relatively rapid, activation by free thrombin occurs at a significantly slower rate. Previously, we generated a “hyper-activatable” derivative of HPC (FLIN-Q3) with an increased activation rate by free α-thrombin in vitro. In this study, the antithrombotic efficacy of FLIN-Q3 was compared with both native zymogen and aPC in an arteriovenous shunt model of thrombosis in the guinea pig. Recombinant proteins were infused 15 minutes before and throughout a 15-minute period while blood was circulated from carotid to jugular through tubing that enclosed a thread on which fibrin was deposited. Parallel dose-dependent antithrombotic responses were observed. Under these non–steady-state conditions, the calculated infusion doses associated with a 50% reduction of thrombus mass were 2.7, 24, and 250 mg/kg/h for aPC, FLIN-Q3, and HPC, respectively. Thrombus weight correlated inversely with plasma concentration of aPC, measured amidolytically, from either direct infusion of aPC or that generated from the zymogens in the animal, and similarly correlated inversely with anticoagulant activity measured by whole blood aPTT. Neither zymogen form showed significant aPC activity before shunt circulation, suggesting a requirement for exposure to thrombin. After the infusion was discontinued for 15 minutes, a second period of thrombus formation in the shunt demonstrated the ability of zymogen forms of PC, unlike aPC, to provide “on-demand” anticoagulant responses to repeated thrombotic stimuli. Thus, a “hyper-activatable” PC molecule such as FLIN-Q3 may represent a superior form of anticoagulant therapy than either the native zymogen or aPC.