In some tissues, FVIIa levels remained high (similar to that were found at 30 min) even at 24 h following rFVIIa administration, at which time rFVIIa in plasma was undetectable

In some tissues, FVIIa levels remained high (similar to that were found at 30 min) even at 24 h following rFVIIa administration, at which time rFVIIa in plasma was undetectable. administered rFVIIa may be simply supplanting the endogenous FVII/FVIIa bound to peri/extravascular tissues without increasing the net levels of FVIIa in tissues. Therefore, we thought it was important to evaluate FVIIa levels in tissues following its administration in order to assess the extent of rFVIIa transfer Anisindione from the blood stream into tissues, and the functional status of rFVIIa associated with extravascular tissues. First, to determine whether TF in perivascular tissue is saturated with endogenous FVII/FVIIa, we have examined the presence of Spry2 mouse FVII in adventitia of blood vessels from mice receiving saline by immunohistochemistry using polyclonal antibodies specific to mouse FVII/FVIIa. The antibodies were raised in rabbits using purified recombinant mouse rFVIIa as the antigen. The antibodies detect both mouse FVII and FVIIa. Out of 25 or more tissue sections of skin, liver and heart that we have examined, positive immunostaining of FVII was found only once around a single blood vessel in skin (data not shown). None of the other blood vessels or tissues stained positive for FVII/FVIIa. It is unclear why our immunohistochemistry data differs from that of Hoffman et al. [9]. It is possible that different mouse FVIIa antibodies used in these studies could have contributed to this difference. Although our present data suggest that it is unlikely that the peri or extravascular TF is saturated with endogenous FVII, we cannot rule out the possibility that mouse FVIIa Anisindione antibodies used in this study may not have sufficient sensitivity to detect traces of FVII/FVIIa in tissues. Therefore, in additional studies we have examined whether exogenously added FVIIa binds to tissue sections. Skin and other tissue sections were incubated with mFVIIa Anisindione (10 nM), CaCl2 (5 mM), or mFVIIa + CaCl2 for 1 h, washed and then stained with anti-mFVIIa. Only tissue sections incubated with mFVIIa in the presence of calcium ions stained positively, whereas other tissue sections stained negatively for FVIIa (data not shown). These data indicate that exogenously added FVIIa is capable of binding to extravascular TF, suggesting that TF sites are not saturated with endogenous FVII. To determine the extent of rFVIIa transported to tissues following its administration into the blood stream, mice were injected intravenously with saline or mFVIIa (120 g/kg body weight, n = 3 to 4 4) via tail vein. Thirty minutes, 6 h and 24 h post-administration of rFVIIa (and 30 min post-administration of saline), mice were exsanguinated by flushing 10 ml of ice-cold saline (supplemented with CaCl2, 5 mM) through the heart and draining the blood by severing the renal artery. Various tissues (e.g., lung, liver, kidney, brain, spleen, heart and skin) were collected and stored at ?80C until homogenization. The tissues were weighed and homogenized in ice-cold TBS (0.01M Tris-HCl, 0.15 M NaCl, pH 7.4, 0.5 ml/100 mg tissue). Initially, we measured FVII/FVIIa activity in total tissue homogenates as well as supernatants of tissue homogenates after the addition of EDTA (20 mM) in factor X activation assay using saturating Anisindione concentrations of relipidated TF (100 ng/ml). We obtained similar levels of FVII/FVIIa activity in both tissue homogenates and the supernatants. FVII/FVIIa activity levels were higher in tissues derived from mice receiving rFVIIa compared to mice receiving saline. These data suggest that rFVIIa administered to mice entered into tissues and remained functionally active. Based on this information, Anisindione we have used the EDTA supernatants for all of our subsequent assays. We have chosen the supernatant over the tissue homogenates for measuring FVIIa levels because varying levels of TF present in different tissue extracts may introduce error in determining FVIIa levels accurately. More importantly, presence of cell-derived full-length TF does not permit measurement of FVIIa activity in FVIIa-specific clotting assay using soluble TF [10]. Furthermore, it is not feasible to measure FVIIa antigen accurately in tissue homogenates if it is bound to TF or other cell-associated proteins. Measurement of FVIIa activity in tissues using FVIIa-specific clotting assay [10] from saline administered mice showed that all tissues contained.