Selective disruption of heparin and antithrombin-mediated regulation of human factor IX.

BACKGROUND: Interaction with antithrombin and heparin regulates distribution, activity, and clearance of factor IXa (FIXa). Hemophilia B prophylaxis targets plasma FIX levels > 1% but neglects extravascular FIX, which colocalizes with antithrombin-heparan sulfate. OBJECTIVE: Combined mutagenesis of FIX was undertaken to selectively disrupt heparin- and antithrombin-mediated regulation of the protease. METHODS: Human FIX alanine substitutions in the heparin (K126A and K132A) and antithrombin (R150A) exosites were characterized with regard to coagulant activity, plasma thrombin generation, antithrombin inhibition, and plasma half-life. RESULTS: Single or combined (K126A/R150A or K132A/R150A) exosite mutations variably reduced coagulant activity relative to wild-type (WT) for FIX (27-91%) and FIXa (25-91%). Double mutation in the heparin exosite (K126A/K132A and K126A/K132A/R150A) markedly reduced coagulant activity (7-21%) and plasma TG. In contrast to coagulant activity, FIX K126A (1.8-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased tissue factor-initiated plasma TG, while FIX K132A and K126A/R150A were similar to WT. FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased FIXa-initiated TG, while FIXa K132A, R150A, and K126A (0.8-0.9-fold) were similar to WT. Dual mutations in the heparin exosite or combined mutations in both exosites synergistically reduced the inhibition rate for antithrombin-heparin. The half-life of FIXa WT in FIX-deficient plasma was remarkably lengthy (40.9 ±1.4 min) and further prolonged for FIXa R150A, K126A/R150A, and K132A/R150A (> 2 h). CONCLUSION: Selective disruption of exosite-mediated regulation by heparin and antithrombin can be achieved with preserved or enhanced thrombin generation capacity. These proteins should demonstrate enhanced therapeutic efficacy for hemophilia B.

Factor XI anion-binding sites are required for productive interactions with polyphosphate.

BACKGROUND: Conversion of factor XI (FXI) to FXIa is enhanced by polymers of inorganic phosphate (polyP). This process requires FXI to bind to polyP. Each FXIa subunit contains anion-binding sites (ABSs) on the apple 3 (A3) and catalytic domains that are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin. AIMS: To determine the importance of FXI ABSs to polyP enhancement of FXI activation. METHODS: Recombinant FXI variants lacking one or both ABSs were tested in polyP-dependent purified protein systems, plasma clotting assays, and a murine thrombosis model. RESULTS: In the presence of polyP, activation rates for FXI lacking either ABS were reduced compared with wild-type FXI, and FXI lacking both sites had an even greater defect. In contrast to heparin, polyP binding to FXIa did not enhance inhibition by antithrombin and did not interfere with FXIa activation of FIX. FXI lacking one or both ABSs does not reconstitute FXI-deficient plasma as well as wild-type FXI when polyP was used to initiate coagulation. In FXI-deficient mice, FXI lacking one or more ABSs was inferior to wild-type FXI in supporting arterial thrombus formation. CONCLUSIONS: The ABSs on FXIa that are required for expression of heparin's cofactor activity during protease inhibition by antithrombin are also required for expression of polyP cofactor activity during FXI activation. These sites may contribute to FXI-dependent thrombotic processes.

Kinetic theory of plasma sheaths surrounding electron-emitting surfaces.

A one-dimensional kinetic theory of sheaths surrounding planar, electron-emitting surfaces is presented which accounts for plasma electrons lost to the surface and the temperature of the emitted electrons. It is shown that ratio of plasma electron temperature to emitted electron temperature significantly affects the sheath potential when the plasma electron temperature is within an order of magnitude of the emitted electron temperature. The sheath potential goes to zero as the plasma electron temperature equals the emitted electron temperature, which can occur in the afterglow of an rf plasma and some low-temperature plasma sources. These results were validated by particle in cell simulations. The theory was tested by making measurements of the sheath surrounding a thermionically emitting cathode in the afterglow of an rf plasma. The measured sheath potential shrunk to zero as the plasma electron temperature cooled to the emitted electron temperature, as predicted by the theory.

Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin-independent mechanism.

BACKGROUND: Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined. OBJECTIVES: To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (FIXa-FVIIIa) activity. PATIENTS/METHODS: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC(50) s) of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX-dependent and FIX-independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock-depleted or antithrombin/FIX-depleted plasma supplemented with recombinant FIX. RESULTS: UFH and fondaparinux demonstrated similar potency under FIX-dependent and FIX-independent conditions, whereas LMWH (2.9-fold) and ssLMWH (5.1-fold) demonstrated increased potency with limiting TF. UFH (62-fold) and fondaparinux (42-fold) demonstrated markedly increased EC(50) values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (two-fold) were less affected, with an EC(50) within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin-depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin-binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition. CONCLUSIONS: Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin-independent interaction with the FIXa heparin-binding exosite.

DDAVP use in patients undergoing transsphenoidal surgery for pituitary adenomas.

BACKGROUND: Disorders of fluid and sodium regulation, often termed "diabetes insipidus," are a frequent occurrence following surgery for pituitary adenomas. The present study was undertaken to identify the incidence of diabetes insipidus after pituitary surgery and its associated factors. METHODS: A retrospective review of the medical records 300 patients who underwent transsphenoidal surgery for pituitary adenoma was undertaken. Information regarding patient gender, perioperative serum sodium levels and urinary output volumes, tumor size, previous pituitary surgery, tumor subtype, and the use of DDAVP was gathered. A multivariate statistical analysis was performed. FINDINGS: Follow-up data were available on 288 patients. During the inpatient postoperative hospital stay, DDAVP was administered to 19% of all patients and 16% of patients not taking DDAVP preoperatively. Of patients with normal fluid/sodium regulation preoperatively, DDAVP was prescribed for 9% at discharge and 4% at 6 weeks postoperatively. Only 1.4% of patients were taking vasopressing replacement at the time of last follow-up. Significant correlations were found between gender, previous surgery, serum sodium levels, and urine volumes at various time points. Immunohistochemical type of tumor and tumor size were not related to DDAVP requirement. CONCLUSIONS: Transient hypotonic polyuria is frequently encountered after pituitary surgery. However, only a small number of patients will develop a long-term requiring for ongoing medical treatment. Previous surgery, female gender, and elevated serum sodium and urine volumes in perioperative period were associated with DDAVP requirement.

A horseradish peroxidase-light and electron microscopic study of immunoliposomes utilized for intracellular delivery to the rat striatum.

Liposomes can deliver plasmid DNA, viruses, antisense oligonucleotides, and pharmacological agents to the central nervous system. Conjugation of antibodies to liposomes increases delivery specificity. Immunoliposomes created with Thy 1.1 antibody have previously been shown to be effective for neuronal delivery. The intracellular delivery of these immunoliposomes is evaluated by light and electron microscopy. Thy 1.1 conjugated liposomes were loaded with horseradish peroxidase and stereotactically injected into rat striatum. On light microscopy, immunoliposomes were concentrated within 0.2 mm of the injection site 8 h following delivery but, 24 h post-operatively, had diffused more than 0.5 mm from the injection site. With transmission electron microscopy, immunoliposomes were observed entering numerous neurons and some astrocytes in a process distinct from the clathrin-coated pit mechanism. These findings suggest that Thy 1.1 immunoliposomes are effective for intracellular delivery in vivo and their endocytosis occurs independently of a coated pit process. The research has helped to elucidate alternative mechanisms for immunoliposomal delivery. A more fundamental understanding of these attributes is needed to achieve the therapeutic potential of immunoliposomes.

Transsphenoidal approaches for the extracapsular resection of midline suprasellar and anterior cranial base lesions.

OBJECTIVE: The transsphenoidal approach is an effective method for treating tumors contained within the sella or extending into the suprasellar cistern. The technique of tumor dissection is predicated on preservation of the integrity of the diaphragma, i.e., intracapsular removal. Gross total extracapsular dissection may, however, be accomplished either by using a standard approach to the pituitary fossa or by extending the exposure to include removal of a portion of the planum sphenoidale and division of the superior intercavernous sinus. METHODS: Included in this series were 14 patients with parasellar or sellar tumors with extension into the anterior fossa and/or suprasellar cistern. For 4 of 14 patients (29%), extracapsular access was gained by broaching the tumor capsule from within the pituitary fossa. For the remaining 10 of 14 patients (71%), the dura of the floor of the sella and the planum sphenoidale was exposed, using neuronavigation to verify the limits of bony dissection; extracapsular tumor resection was performed using the operating microscope and endoscopy as indicated. The dural defect was repaired with abdominal fat, the sellar floor and planum sphenoidale were reconstructed, and in selected cases a lumbar drain was placed. RESULTS: Seven of 14 tumors (50%) were craniopharyngiomas, 3 of 14 (21%) were pituitary adenomas, and 2 of 14 (14%) were meningiomas. There was one case of lymphocytic hypophysitis and one yolk sac tumor. Gross total resection was possible in 11 of 14 cases (79%). Immediate postoperative visual function worsened in 2 of 14 cases (14%), improved in 3 of 14 cases (21%), and was stable in the remainder of cases. Postoperatively, 2 of 14 patients (14%) developed bacterial meningitis. Overt postoperative cerebrospinal fluid rhinorrhea was not observed. CONCLUSION: Gross total extracapsular resection of midline suprasellar tumors via a transsphenoidal approach is possible but is associated with a higher risk of complications than is standard transsphenoidal surgery.

Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism.

Phosphorothioate oligonucleotides (PS ODNs) prolong the activated partial thromboplastin time in human plasma by inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity. This inhibition was characterized using ISIS 2302, a 20-mer antisense PS ODN. ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of factor X activation by the intrinsic tenase complex. The decrease in V(max(app)) was analyzed by examining complex assembly, cofactor stability, and protease catalysis. ISIS 2302 did not inhibit factor X activation by the factor IXa-phospholipid complex, or significantly affect factor VIII-phospholipid affinity. Inhibitory concentrations of ISIS 2302 modestly decreased the affinity of factor IXa-factor VIIIa binding in the presence of phospholipid (K(D) = 11.5 vs 4.8 nM). This effect was insufficient to explain the reduction in V(max(app)). ISIS 2302 did not affect the in vitro half-life of factor VIIIa, suggesting it did not destabilize cofactor activity. In the presence of 30% ethylene glycol, the level of factor X activation by the factor IXa-phospholipid complex increased 3-fold, and the level of chromogenic substrate cleavage by factor IXa increased more than 50-fold. ISIS 2302 demonstrated partial inhibition of factor X activation by the factor IXa-phospholipid complex, and chromogenic substrate cleavage by factor IXa, only in the presence of ethylene glycol. Like the intact enzyme complex, ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of chromogenic substrate cleavage by factor IXa (K(I) = 88 nM). Equilibrium binding studies with fluorescein-labeled ISIS 2302 demonstrated a similar affinity (K(D) = 92 nM) for the PS ODN-factor IX interaction. These results suggest that PS ODNs bind to an exosite on factor IXa, modulating catalytic activity of the intrinsic tenase complex.

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Radiosurgery for Cushing's disease after failed transsphenoidal surgery.

OBJECT: Although transsphenoidal surgery has become the standard of care for Cushing's disease, it is often unsuccessful in normalizing cortisol production. In this study the authors investigate the safety and efficacy of gamma knife radiosurgery (GKRS) for Cushing's disease after failed transsphenoidal surgery. METHODS: The records of all patients who underwent GKRS at the authors' institution after unsuccessful transsphenoidal surgery for Cushing's disease were retrospectively reviewed. Successful treatment was considered a normal or below-normal 24-hour urinary free cortisol (UFC) level. Records were also evaluated for relapse, new-onset endocrine deficiencies, interval change in tumor size, and visual complications. Forty-three patients underwent 44 gamma knife procedures with follow up ranging from 18 to 113 months (mean 39.1, median 44 months). Normal 24-hour UFC levels were achieved in 27 patients (63%) at an average time from treatment of 12.1 months (range 3-48 months). Three patients had a recurrence of Cushing's disease at 19, 37, and 38 months, respectively, after radiosurgery. New endocrine deficiencies were noted in seven patients (16%). Follow-up magnetic resonance images obtained in 33 patients revealed a decrease in tumor size in 24, no change in nine, and an increase in size in none of the patients. One patient developed a quadrantanopsia 14 months after radiosurgery despite having received a dose of only 0.7 Gy to the optic tract. CONCLUSIONS: Gamma knife radiosurgery appears to be safe and effective for the treatment of Cushing's disease refractory to pituitary surgery. Delayed recurrences and new hormone deficiencies may occur, indicating the necessity for regular long-term follow up.

Chemotherapy for esthesioneuroblastomas.

Esthesioneuroblastoma can be regarded as a chemosensitive tumor based on multiple reports of response to treatment. Neoadjuvant therapy is seldom curative, however, and may be of no benefit in some patients. Individuals who respond to preoperative therapy have a greater chance of long-term disease-free survival. Platinum-based therapy has been the mainstay of treatment. Toxicity has been mild, and treatment failures have been treated with postoperative chemotherapy with or without bone marrow transplantation. Given the small number of cases of esthesioneuroblastoma diagnosed annually, it is unlikely that any consensus on this issue is forthcoming. Either post- or presurgical treatment of advanced stage or recurrent disease has become the standard of care, however.

Results of transsphenoidal surgery for Cushing's disease in patients with no histologically confirmed tumor.

OBJECTIVE: Pathological confirmation of surgical resection of an adenoma for Cushing's disease is not always achieved. We reviewed our experience to determine the prognostic significance of this lack of confirmation regarding outcome, and we evaluate explanations for this situation. METHODS: The records of all patients undergoing transsphenoidal surgery for Cushing's disease from 1992 to 1998 were reviewed, and those with no histological confirmation of tumor were identified. Information regarding preoperative and postoperative hormonal levels and clinical symptoms, preoperative magnetic resonance imaging data, intraoperative findings, and the number of reoperations were recorded. RESULTS: There were 29 patients with no confirmation of tumor. Nineteen (66%) of these patients were cured with surgery and only one had a recurrence of disease, with an average follow-up of 38 months. An abnormality thought to represent an adenoma at the time of surgery was removed in 26 patients (90%). Preoperative magnetic resonance imaging suggested a discrete lesion in 21 patients (72%). Neither intraoperative impression nor magnetic resonance imaging appearance was correlated with outcome. CONCLUSION: Patients with no histological confirmation of tumor after transsphenoidal surgery for Cushing's disease are likely to have a good outcome. The results do not differ significantly from reported cure rates in patients with confirmed adenomas. Possible explanations for this situation are discussed.

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Bone morphogenetic proteins and bone morphogenetic protein gene therapy in neurological surgery: a review.

OBJECTIVE: To review the uses of bone morphogenetic proteins (BMPs) and BMP gene therapy for the treatment of neurosurgical disorders. METHODS: Literature review. RESULTS: BMPs are members of the transforming growth factor beta superfamily, and they play an important role in the growth and development of numerous tissues, including bone, brain, and spinal cord. Although the majority of previous studies have focused on the regulatory functions of BMPs in the normal growth and differentiation of the skeletal system, BMPs also seem to be exquisitely involved in the regulation of cellular proliferation, survival, differentiation, apoptosis, and lineage commitment in the central nervous system. When specific BMPs are delivered on biological matrices, they have the capacity to induce bone, cartilage, ligament, and tendon at both heterotopic and orthotopic sites, suggesting that they may play a major role in the future treatment of spinal and craniofacial pathology. For example, recent studies have clearly demonstrated the usefulness of BMPs and BMP gene therapy for the induction of spinal arthrodesis in several animal models. In addition, several BMPs have been shown to have a neuroprotective effect in animal models of head injury, cerebral ischemia, and Parkinson's disease and may therefore have direct clinical applications for the treatment of central nervous system disorders. CONCLUSION: As the physiological activity of BMPs in the development and pathology of the central nervous system and spine are more fully elucidated, BMP therapeutics and gene therapy will probably have numerous applications in neurological surgery.