Abstract. Studies were performed on a FrenchCanadian family afflicted with a bleeding disorder exhibiting an autosomal dominant inheritance pattern and a severe bleeding diathesis after trauma. Clinical laboratory coagulation tests were unimpressive; the only persistent abnormalities include mild thrombocytopenia and moderately reduced Factor V clotting activities. Some individuals had prolonged Stypven times when platelet-rich plasma was used, suggesting that their platelets could not support functional prothrombinase complex assembly. Detailed studies were performed by use of plasma and isolated, washed platelets from a sister and brother. Bioassay data indicate that both individuals had Factor V activities of -40 and 36% of normal, respectively. A comparison of the Factor V radioimmunoassay and bioassay data on the brother's plasma indicated that the circulating amount of Factor V functional activity was low relative to Factor V antigen concentration (-65-75%). In both individuals, the platelet Factor V functional activities were extremely low (2-4%) relative to antigen levels present as determined by radioimmunoassay. These discrepancies between Factor V activities and antigen concentration do not appear to be due to an unstable Factor V molecule or to the presence of a Factor V or Factor Va inhibitor or inactivator. Kinetics of prothrombin activation by use of purified clotting factors indicated that thrombinactivated platelets from both individuals supported prothrombinase complex assembly identical to controls in the presence of added purified Factor Va. Consequently, their bleeding diathesis appears to reflect their platelet, rather than their plasma, Factor V activity. These results suggest that platelet Factor V is an essential component in maintaining stable and prolonged hemostasis after trauma.

Phosphatidylinositol 3-kinases (PI3Ks) are key elements in the signaling cascades that lie downstream of many cellular receptors. In particular, PI3K ␦ and ␥ isoforms contribute to inflammatory cell recruitment and subsequent activation. For this reason, in a series of preclinical studies, we tested the potential of a recently developed small-molecule inhibitor of these two isoforms, pteridin-7-yl]phenol], as a form of anti-inflammatory therapy for respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). To determine pharmacokinetic profiles, aerosolized formulations of the drug were delivered to mice by a nose-only inhalation route, yielding high pulmonary TG100-115 levels with minimal systemic exposure. Safety assessments were favorable, with no clinical or histological changes noted after 21 days of daily dosing. In a murine asthma model, aerosolized TG100-115 markedly reduced the pulmonary eosinophilia and the concomitant interleukin-13 and mucin accumulation characteristic of this disease. As a functional benefit, interventional dosing schedules of this inhibitor also reduced airway hyper-responsiveness. To model the pulmonary neutrophilia characteristic of COPD, mice were exposed to either intranasal lipopolysaccharide or inhaled smoke. Aerosolized TG100-115 again inhibited these inflammatory patterns, most notably in the smoke model, where interventional therapy overcame the steroid-resistant nature of the pulmonary inflammation. In conclusion, aerosolized TG100-115 displays pharmacokinetic, safety, and biological activity profiles favorable for further development as a therapy for both asthma and COPD. Furthermore, these studies support the hypothesis that PI3K ␦ and ␥ are suitable molecular targets for these diseases.

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Rationale: The γ and δ isoforms of phosphoinositide 3‐kinase (PI3K) regulate immune/inflammatory responses by multiple cell types including Th2 T cells, mast cells, and eosinophils. We explored whether a dual PI3K γ/δ inhibitor could impact airway hyperresponsiveness (AHR) and inflammation.Methods: Mice were sensitized with ovalbumin (OVA) on Day 0 and boosted on Day 5, followed by inhalation challenges on Days 12–13. TG100–115, a PI3K γ/δ inhibitor (IC50 = 83 and 235 nM, respectively), was delivered by inhalation on Days 11–14 for total daily doses of 100 mg/kg; dexamethasone (DEX) was delivered at 3 mg/kg i.p on Days 8–14. On Day 15, AHR was determined following aerosolized methacholine (MC) challenge, polymorphonuclear (PMN) cell counts were determined from bronchoalveolar lavage fluid (BALF), and lungs were processed for histology.Results: Exposure to aerosolized TG100–115 resulted in sustained compound levels within the lung and airways with minimal plasma exposure. TG100–115 reduced both AHR and PMN accumulation vs. vehicle‐treated animals, to near maximal response of systemic DEX. Histology showed that both treatments reduced inflammation and mucin accumulation to near background levels.Conclusion: An aerosolized PI3K γ/δ inhibitor reduces asthma symptoms in a standard mouse model, and may represent a new anti‐inflammatory therapy for future clinical exploration.

Homogeneous, single-chain human factor V was used to develop a double antibody competition radioimmunoassay to measure factor V concentrations in plasma and platelets. Standard curves were constructed that allow for the detection of as little as 20 ng factor V/ml of plasma. Normal factor V concentrations range from 4 to 14 micrograms/ml of plasma with an average value of 7.0 +/- 2.0 micrograms/ml (n = 64). No correlation was observed between antigen levels and age or sex. The radioimmunoassay data are consistent with factor V clotting assays, providing freshly drawn plasma is used in the bioassay. Radioimmunoassay of washed platelets indicate that 0.63–1.93 microgram of factor V is present per 2.5 X 10(8) platelets (4612–14128 molecules of the factor V platelet). When normalized to individual hematocrits and platelet count, the data indicated that platelets contribute approximately 18%-25% of the factor V found in whole blood. In addition, two individuals with functionally deficient factor V were examined and found to be deficient in both antigen and activity.