Disposition of tissue factor pathway inhibitor during cardiopulmonary bypass.

BACKGROUND: The tissue factor (TF) factor (F) VIIa complex activates coagulation FIX and FX to initiate coagulation, and also cleaves protease activated receptors (PARs) to initiate inflammatory processes in vascular cells. Tissue factor pathway inhibitor (TFPI) is the only specific inhibitor of the TF-FVIIa complex, regulating both its procoagulant and pro-inflammatory properties. Upon heparin infusion during cardiopulmonary bypass (CPB), a heparin releasable pool of endothelial associated TFPI circulates in plasma. Following protamine neutralization of heparin, the plasma TFPI level decreases, but does not return completely to baseline, suggesting that during CPB a fraction of the plasma TFPI becomes heparin-independent. We have investigated the structural and functional properties of plasma TFPI during CPB to further characterize how TFPI is altered during this procedure. METHODS: We enrolled 17 patients undergoing first-time cardiac surgery involving CPB. Plasma samples were obtained at baseline, 5 min and 1 h after start of CPB (receiving heparin), 10 min after protamine administration (off CPB) and 24 h following surgery. Samples were analyzed for full-length and free (non-lipoprotein bound) TFPI antigen by enzyme-linked immunosorbent assay (ELISA) and for TFPI anticoagulant activity using an amidolytic assay. Western blot analysis was used to identify TFPI species of varying molecular weights in three additional patients. Dunnett's test for post hoc comparisons was used for statistical analysis. RESULTS: The ELISA and Western blot data indicated that an increase in full-length TFPI accounted for most of the heparin releasable TFPI. Following heparin neutralization with protamine, the full-length TFPI antigen returned to baseline levels while the free TFPI antigen and the total plasma TFPI activity remained elevated. This was associated with the appearance of a new 38 kDa form of plasma TFPI identified by Western blot analysis. The 38 kDa form of TFPI did not react with an antibody directed against the C-terminal region of TFPI indicating it has undergone proteolysis within this region. All TFPI measurements returned to baseline 24 h following CPB. CONCLUSIONS: During CPB the full-length form of TFPI is the predominant form in plasma because of its prompt release from the endothelial surface following heparin administration. Upon heparin neutralization with protamine, full-length TFPI redistributes back to the endothelial surface. However, a new 38 kDa TFPI fragment is generated during CPB and remains circulating in plasma, indicating that TFPI undergoes proteolytic degradation during CPB. This degradation may result in a decrease in endothelium-associated TFPI immediately post-CPB, and may contribute to the procoagulant and proinflammatory state that often complicates CPB.

Mechanical evaluation of a soft tissue interference screw in free tendon anterior cruciate ligament graft fixation.

In this study of bioabsorbable screw fixation of free tendon grafts used in anterior cruciate ligament reconstruction, we performed load-to-failure and cyclic loading of tendon fixation in porcine bone. Bone density measurements from dual photon absorptometry scans were obtained to correlate bone density with fixation failure. The average density of porcine bone (1.42 g/cm2) was similar to that of young human bone (1.30 g/cm2) and significantly higher than that of elderly human cadaveric bone specimens (0.30 g/cm2). Cyclic loading was performed on free tendon grafts fixed with a bioabsorbable screw alone and on grafts fixed with a bioabsorbable screw and an anchor (polylactic acid ball or cortical bone disk). Stiffness of fixation increased substantially with the addition of a cortical bone disk anchor or polylactic acid ball compared with the interference screw alone. Tensile fixation strength of central quadriceps free tendon and hamstring tendon grafts were significantly superior in porcine bone of density similar to young human bone than in elderly human cadaveric bone. The bioabsorbable interference screw yielded loads at failure comparable with traditional bone-tendon-bone and hamstring tendon fixation when controlled for bone density. The addition of a cortical bone disk anchor provided the most optimal fixation of free tendon with the bioabsorbable screw and reduced slippage with cyclic loading to a very low level.

Growth hormone-dependent and -independent regulation of cytochrome P-450 isozyme expression in streptozotocin-diabetic rats.

The sexually dimorphic GH secretory pattern is thought to be the major factor regulating constitutive expression of hepatic P450IIC11 (P-450h) and P450IIC12 (P-450i). In this study we investigated whether factors other than the diabetes-induced decrease in GH secretion contribute to alterations in P-450 isozyme expression in streptozotocin (STZ)-diabetic rats. In male rats, hepatic P-450h apoprotein and mRNA decreased to 13% and 24% of control male levels, respectively, within 14 days of STZ injection. STZ-diabetes had little effect on expression of P-450i in females. Treatment of diabetic male rats with GH did not reverse the suppression of P-450h. STZ treatment also suppressed P-450h expression in GH-treated hypophysectomized (Hx) male rats, but incompletely. Thus, GH can partially reverse diabetic suppression of P-450h. However, in Hx male rats without GH supplementation, STZ treatment suppressed P-450h apoprotein and mRNA expression to 16% and 6% of nondiabetic Hx male levels, respectively, demonstrating the existence of GH-independent regulation of P-450h expression. In Hx female rats, P-450h apoprotein levels were 40% of those in intact control males and were not significantly decreased by STZ. Concomitantly, STZ produced a greater decrease in serum insulin levels and a greater increase in serum glucagon in Hx male rats than in Hx females. The results provide evidence for the existence of STZ-sensitive GH-independent expression of P-450h and further document the gender differences in STZ sensitivity.

Effects of vanadate on hepatic cytochrome P-450 expression in streptozotocin-diabetic rats.

The ability of sodium metavanadate to reverse the effects of streptozotocin-induced diabetes on hepatic cytochrome P-450 isozymes was examined in male rats. Streptozotocin caused P-450h levels to fall 95%, and P-450j and P-450b levels to rise 8- and 40-fold, respectively, after 1 week. When diabetic rats were administered metavanadate in the drinking water for 7 days, P-450h apoprotein and mRNA levels remained no different from those of untreated diabetic rats, whereas levels of P-450b and P-450j decreased toward those of control rats. Metavanadate also lowered serum triglyceride and 3-hydroxybutyrate levels without lowering serum glucose in the diabetic rats. Furthermore, P-450h mRNA levels correlated well with levels of P-450h apoprotein for all treatment groups, indicating that P-450h suppression in diabetic rats is under pretranslational control and is independent of the increased expressions of P-450j and P-450b, and of the hyperlipidemia and ketosis that occurs in diabetes. Vanadate is capable of separating the effects of diabetes on expression of individual P-450 isozymes.

Free diffusion coefficient of ionic calcium in cytoplasm.

The free diffusion coefficient of ionic Ca was measured in isolated samples of Myxicola axoplasm by following the migration of 45Ca. When precautions were taken to minimize the sequestration and chelation of 45Ca (i.e., using inhibitors, energy deprivation, and saturation of Ca chelation sites), a diffusion coefficient of 5.3 x 10(-6) cm2 s-1 was measured. The diffusion coefficient was not appreciably changed by lowering free calcium from 100 microM to approximately 10 microM or by increasing the diffusion time from ten to twenty minutes. In untreated cytoplasm taken directly from the giant axon of Myxicola, the migration of Ca was more complex and could not be described by a single diffusion coefficient. This result is interpreted to suggest that bulk movement of Ca-buffers may occur in untreated Myxicola axoplasm, a system that contains few microtubules.