Heparin-protamine balance after neonatal cardiopulmonary bypass surgery.

Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB. SUMMARY: Background Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.

New insights into the biology of tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) dampens the initiation of blood coagulation by inhibiting two potent procoagulant complexes, tissue factor-factor VIIa (TF-FVIIa) and early forms of prothrombinase. TFPI isoforms, TFPIα and TFPIß, result from alternative splicing of mRNA, producing distinct C-terminal ends of the two proteins. Both isoforms inhibit TF-FVIIa, but only TFPIα can inhibit early forms of prothrombinase by binding of its positively charged C-terminus with high affinity to the acidic B-domain exosite of FVa, which is generated upon activation by FXa. TFPIα and TFPIß are produced in cultured human endothelial cells, while platelets contain only TFPIα. Knowledge of the anticoagulant mechanisms and tissue expression patterns of TFPIα and TFPIß have improved our understanding of the phenotypes observed in different mouse models of TFPI deficiency, the east Texas bleeding disorder, and the development of pharmaceutical agents that block TFPI function to treat hemophilia.

Comparison of the inhibitory activities of human tissue factor pathway inhibitor (TFPI)α and TFPIß.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an alternatively spliced protein with two isoforms, TFPIα and TFPIß, which differ in their C-terminal structure and cellular localization. Detailed characterization of their inhibitory activity is needed to define potentially unique inhibitory roles in tissue factor (TF)-mediated thrombotic and inflammatory disease, and to understand how pharmaceuticals targeted to different structural regions of the TFPI isoforms alter hemostasis in hemophilia patients. METHODS: The TF inhibitory activity of TFPIß localized to the surface of CHO cells was compared with that of soluble TFPIα by the use of in vitro and in vivo assays. RESULTS: In TF-factor VIIa-mediated FXa generation assays, TFPIß was a slightly better inhibitor than TFPIα, which was approximately three-fold better than TFPI-160, a soluble, altered form of TFPI similar to TFPIß. In direct FXa inhibitory assays, TFPIß had an IC50 2.5-fold lower than that of TFPIα and 56-fold lower than that of TFPI-160. TFPIß inhibited TF-mediated CHO cell migration though Matrigel, whereas TFPIα and TFPI-160 were poor inhibitors, demonstrating that TFPIß effectively blocks TF-initiated signaling events during cellular migration through matrices that are not permeable to soluble forms of TFPI. Furthermore, TFPIß inhibited TF-dependent CHO cell infiltration into lung tissue following tail vein injection into SCID mice, and blocked the development of consumptive coagulopathy. CONCLUSIONS: TFPIß is a slightly better inhibitor of TF procoagulant activity than TFPIα. As a surface-associated protein, TFPIß is a much better inhibitor of TF-mediated cellular migration than soluble TFPIα, and may specifically act in the inhibition of TF-mediated signaling events on inflamed endothelium and/or monocytes.

Tissue factor pathway inhibitor and bacterial infection.

See also van den Boogaard FE, Brands X, Schultz MJ, Levi M, Roelofs JJTH, van 't Veer C, van der Poll T. Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antimicrobial effects in murine pneumococcal pneumonia. This issue, pp 122-32.

Temporal expression of alternatively spliced forms of tissue factor pathway inhibitor in mice.

BACKGROUND: Mouse tissue factor pathway inhibitor (TFPI) is produced in three alternatively spliced isoforms that differ in domain structure and mechanism for cell surface binding. Tissue expression of TFPI isoforms in mice was characterized as an initial step for identification of their physiological functions. METHODS AND RESULTS: Sequence homology demonstrates that TFPIalpha existed over 430 Ma while TFPIbeta and TFPIgamma evolved more recently. In situ hybridization studies of heart and lung did not reveal any cells exclusively expressing a single isoform. Although our previous studies have demonstrated that TFPIalpha mRNA is more prevalent than TFPIbeta or TFPIgamma mRNA in mouse tissues, western blot studies demonstrated that TFPIbeta is the primary protein isoform produced in adult tissues, while TFPIalpha is expressed during embryonic development and in placenta. Consistent with TFPIbeta as the primary isoform produced within adult vascular beds, the TFPI isoform in mouse plasma migrates like TFPIbeta in SDS-PAGE and mice have a much smaller heparin-releasable pool of plasma TFPIalpha than humans. CONCLUSIONS: The data demonstrate that alternatively spliced isoforms of TFPI are temporally expressed in mouse tissues at the level of protein production. TFPIalpha and TFPIbeta are produced in embryonic tissues and in placenta while adult tissues produce almost exclusively TFPIbeta.

Tissue factor pathway inhibitor-gamma is an active alternatively spliced form of tissue factor pathway inhibitor present in mice but not in humans.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor procoagulant activity produced as two alternatively spliced isoforms, TFPIalpha and TFPIbeta, which differ in domain structure and mechanism for cell surface association. 3' Rapid amplification of cDNA ends was used to search for new TFPI isoforms. TFPIgamma, a new alternatively spliced form of TFPI, was identified and characterized. METHODS: The tissue expression, cell surface association and anticoagulant activity of TFPIgamma were characterized and compared to those of TFPIalpha and TFPIbeta through studies of mouse and human tissues and expression of recombinant proteins in Chinese hamster ovary (CHO) cells. RESULTS: TFPIgamma is produced by alternative splicing using the same 5'-splice donor site as TFPIbeta and a 3'-splice acceptor site 276 nucleotides beyond the stop codon of TFPIbeta in exon 8. The resulting protein has the first two Kunitz domains connected to an 18 amino acid C-terminal region specific to TFPIgamma. TFPIgamma mRNA is differentially produced in mouse tissues but is not encoded within the human TFPI gene. When expressed in CHO cells, TFPIgamma is secreted into conditioned media and effectively inhibits tissue factor procoagulant activity. CONCLUSIONS: TFPIgamma is a third alternatively spliced form of TFPI that is widely expressed in mouse tissues but not made by human tissues. It contains the first two Kunitz domains and is a secreted, rather than a cell surface-associated, protein. It is a functional anticoagulant and may partially explain the resistance of mice to coagulopathy in tissue factor-mediated models of disease.

Combined tissue factor pathway inhibitor and thrombomodulin deficiency produces an augmented hypercoagulable state with tissue-specific fibrin deposition.

BACKGROUND AND OBJECTIVE: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are endothelial-associated anticoagulant proteins thought to control hemostasis in specific vascular beds. Here, we have examined the consequences of TFPI deficiency in the presence of a compounding procoagulant state caused by reduced TM function. METHODS AND RESULTS: TFPI(+/-)/TM(pro/pro) mice are born at less than expected frequency in either TFPI(+/-)/TM(pro/+) or TM(pro/pro) mothers but are born at near the expected frequency in TM(pro/+) mothers. Adult TFPI(+/-)/TM(pro/pro) mice have elevated thrombin-antithrombin complex and increased thrombus volume in an electrical injury model of venous thrombosis. In striking contrast to mice with single deficiency of TFPI or TM, TFPI(+/-)/TM(pro/pro) mice exhibit augmented fibrin deposition not only in the liver, but also in the cerebral microvasculature. CONCLUSIONS: TFPI(+/-)/TM(pro/pro) mice exhibit partial intrauterine lethality when carried by mothers with an underlying prothrombotic state, providing the first experimental evidence in an animal model that TFPI-dependent control of hemostasis in the vascular bed of the placenta fulfills a critical role for successful pregnancy outcome. In addition to the placenta, partial TFPI deficiency interacts with decreased TM function in an organ selective manner to produce fibrin deposition in other specific vascular beds, the liver and brain.

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored co-receptor. OBJECTIVES/METHODS: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. RESULTS AND CONCLUSIONS: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

The family pet as an unlikely source of group A beta-hemolytic streptococcal infection in humans.

This study examines the possibility of the family pet serving as a reservoir for group A beta-hemolytic streptococcal infections in humans. We obtained oropharyngeal cultures from children with acute pharyngitis and concurrent oropharyngeal cultures from their household pets. Children with culture-proved group A beta-hemolytic streptococcal pharyngitis were detected in 26 of 42 households surveyed. Oropharyngeal cultures were also collected from a group of children without pharyngitis and their pets. Additionally 149 dogs and cats from a local veterinary hospital were cultured from 371 body sites including the oropharynx, axilla and vagina. All beta-hemolytic bacterial isolates were identified by colonial and microscopic morphology, catalase and pyrrolidonylarylamidase production, bacitracin susceptibility and serogrouping. No group A beta-hemolytic streptococci were recovered from any of the body sites surveyed from a total of 230 animals. Based on these findings, the family pet seems to be an unlikely reservoir for group A beta-hemolytic streptococci.

Space Station Freedom crew training.

The nature of the Space Station Freedom Program presents an array of new and enhanced challenges which need to be addressed en route to developing an effective and affordable infrastructure for crew training. Such an infrastructure is essential for the safety and success of the program. The three major challenges that affect crew training are the long lifetime of the program (thirty years), the interdependence of successive increments, and the participation of the three International Partners (Canada, European Space Agency, and Japan) and a myriad of experimenters. This paper addresses these major challenges as they drive the development of a crew training capability and the actual conduct of crew training.