Living Kidney Donor Transplantation in a Resource-limited Country: The Ivory Coast Experience.

Renal transplantation that offers a good quality of life still is not performed by the majority of countries of black Africa. We started a pilot project of renal transplantation in Ivory Coast 2 years ago. The present paper reports the preliminary results, difficulties related to the program, and perspectives regarding its expansion. Ten living related kidney transplantations have been performed over a 2-year period. Recipients and their respective donors were male. The mean age of the recipients was 42.8 years (22-57), and the mean age of the donors was 29.4 years (22-43). The mean number of mismatches was 3.2 (0-6). None was immunized. Recipients and donors were all EBV IgG positive and CMV IgG positive. All but 1 case were induced with basiliximab. The mean graft and patient survival time was 16.6 months (6-26). The mean cold ischemic time was 2.27 hours (1-3.32). The mean serum creatinine at discharge was 241.87 µmol/L (115.18-1063.2), at 6 months was 117.20 µmol/l (95.6-139.9), at 12 months was 104.55 µmol/L (62.02-132.9), and at 24 months was 104.55 µmol/L (62.02-132.9). The mean cyclosporine through level (C0) at 6 months was 137.57 ng/mL (70-366), at 12 months was 117.33 ng/mL (62-197), and at 24 months was 78 ng/mL. The mean cyclosporine 2-hour post-administration concentration levels (C2) at 6 months was 764.9 ng/mL (430-1421), at 12 months was 937.17 ng/mL (483-1292), and at 24 months was 690.66 ng/mL (488-853). Main complications were sepsis, adenovirus hemorrhagic cystitis, new-onset diabetes after transplantation, delayed graft function, polycythemia, and cytomegalovirus infection. No clinical rejection was diagnosed over the 2-year period. Patient and graft survival was 100% at a mean post-transplantation time of approximately 16.6 months.

A new HLA allocation procedure of kidneys from deceased donors in the current era of immunosuppression.

INTRODUCTION: It has recently been proposed to replace the current Eurotransplant kidney allocation based primarily on mismatches (MM) at the 3 HLA loci by a simpler system based on full HLA-DR compatibility. The present study analyzes this system in the current era of immunosuppression. METHODS: From 1999 to 2012, 723 renal grafts were performed on 586 patients who were treated with a calcineurin inhibitor, mycophenolate mofetil, and in most cases antilymphocyte globulins. Four groups of HLA MM were compared: (A) A+B 2-4/DR 1-2 MM (n = 397), (B) A+B 2-4 MM/DR 0 MM (n = 106), (C) A+B 0-1 MM/DR 1-2 MM (n = 138), and (D) A+B 0-1/DR 0 MM (n = 82). RESULTS: Acute rejection episodes were less frequent during the first post-transplantation year in group D than in the other groups (P = .018). Patient survival was lower in group A than in the other groups (P = .008). Immunologic graft survival was higher in group D than in the other groups in univariate (P = .015) and multivariate analyses (P = .033; 96.4% vs 90.1% at 10 years). CONCLUSIONS: In the current era of immunosuppression, allocation of kidneys from deceased donors could be performed primarily according to full DR compatibility then to the best A+B matching, affording excellent graft outcome to most recipients.

Patient and graft outcome in current era of immunosuppression: a single centre pilot study.

OBJECTIVES: The present single centre study aims at analyzing the impact on renal allograft outcome of the important changes which occurred in the transplant population and immunosuppressive therapy during the last two decades. METHODS: From 2000 to 2013, 779 single kidney transplantations were performed on 635 patients who all received on an intent-to-treat basis steroids, a calcineurin inhibitor, mycophenolate mofetil and an induction therapy with either antithymocyte globulin or an antagonist directed to the interleukin (IL)-2 receptor. Uni- and multivariate analyses of patient and immunologic graft survival were conducted. RESULTS: The sole factor predicting patient survival is recipient's age: 10-year survival rates are 94·7, 81·6 and 57·9% for the <45, 45-60 and >60 years age groups, respectively (P<0·001). Peak (>50% panel reactive antibodies) anti-human leucocyte antigens (HLA) sensitization, cold ischaemia time and HLA-B and -DR mismatches (MM) influence graft outcome: at 10 years, the difference in 10-year survival rates is 5·9% between grafts from sensitized and not sensitized patients (90·9 vs 96·8%, P = 0·002), 3·8% between grafts with <18 and ≧18 hours cold ischaemia (96·6 vs 92·8%, P = 0·003), 7·3% between grafts with no MM and either B or DR MM versus those with B and DR MM (96·8 vs 89·5%, P = 0·002). CONCLUSION: In our single centre experience, graft survival was most strongly determined by HLA matching, offering excellent long term graft outcome to most patients.

Thrombophilic factors do not predict outcomes in renal transplant recipients under prophylactic acetylsalicylic acid.

A cohort of recipients of renal transplant after 2000 (N=310) was prospectively screened on the day of transplantation and 1 month later for a panel of 11 thrombophilic factors to assess their effect on posttransplant outcomes. All patients received prophylactic acetylsalicylic acid, started before transplantation. The rate of thromboembolic events or acute rejection episodes during the first posttransplant year (primary composite endpoint) was 16.7% among patients free of thrombophilic factor (N=60) and 17.2% in those with >or=1 thrombophilic factor (N=250) (p>0.99). The incidence of the primary endpoint was similar among patients free of thrombophilic factors and those with >or=2 (N=135), or >or=3 (N=53) factors (16.3% and 15.1% respectively; p=1) and in patients who remained thrombophilic at 1 month (15.7%; p=0.84). None of the individual thrombophilic factor present at the day of transplantation was associated with the primary endpoint. The incidence of cardiovascular events at 1-year, serum creatinine at 1-year, 4-year actuarial graft and patient survival were not influenced by the presence of >or=1 thrombophilic factor at baseline (p=NS). In conclusion, the presence of thrombophilic factors does not influence thromboembolic events, acute rejection, graft or patient survival in patients transplanted after 2000 and receiving prophylactic acetylsalicylic acid.

Use of marginal donors for liver transplantation: a single-center experience within the Eurotransplant patient-driven allocation system.

BACKGROUND: Due to the organ shortage, marginal donors are increasingly used in liver transplantation (OLT). These grafts may be safely used in less critical recipients but, the real influence of extended donor criteria (EDC) remains uncertain when graft-recipient matching is not applied. Our study analyzed the impact of EDC on initial graft function within the Eurotransplant patient-driven allocation system. PATIENTS AND METHODS: We reviewed 70 OLT performed between 2004 and 2006. The impact of the following EDC were analyzed: age > 60; intensive care unit (ICU) stay > 4 days; peak serum Na(+) > 160 mEq/L; body mass index (BMI) > 30; cardiac arrest with cardiopulmonary resuscitation, and high doses of vasopressors. Early graft function, as defined according to peak transaminase level and spontaneous prothrombin time within the first 5 posttransplant days, was compared between the donors with none or one criterion (group A = 39) and those with >1 criterion (group B = 31). RESULTS: The most frequent EDC were high vasopressor use, ICU stay > 4 days and BMI > 30, were present in respectively 44%, 27%, and 16% of the donors. No EDC were present in 13 donors, one in 26, three in eight, and four in three. Demographics and origin and severity of the liver disease were similar in both groups. We failed to observe significant differences in initial graft function. CONCLUSION: The presence of EDC did not significantly affect early graft function in a population where donor and recipient were not matched. While this observation must be confirmed in a multicenter analysis, it tends to support the use of marginal liver grafts, even in patient-driven allocation systems.

Gonadal vein reconstruction for extension of the renal vein in living renal transplantation: two case reports.

BACKGROUND: When the left kidney is harvested for living donor transplantation (LDKT), the short length of the left renal vein may eventually created a technical problem for reimplantation of the graft. We report an original technique, using the donor gonadal vein to extend the left renal vein and facilitate graft implantation. CASE REPORT: In the first case, the native graft gonadal vein was successfully used to extent a short renal vein after laparoscopic donor nephrectomy. The graft gonadal vein was utilized for the creation of graft venous return in the second case. Good graft function was observed in both cases. DISCUSSION: This original technique could lead to a functionally acceptable anastomosis without use of supplementary donor or recipient vascular tissue and ultimately to good organ function without increased peri- or postoperative morbidity during LDKT. A precise preoperative assessment of donor vascular anatomy is a key factor for donor safety and successful LDKT.

Conversion from tacrolimus to cyclosporin is associated with a significant improvement of glucose metabolism in patients with new-onset diabetes mellitus after renal transplantation.

BACKGROUND: The incidence of new-onset posttransplant diabetes mellitus (PTDM) is increased in renal transplant patients treated with tacrolimus. METHODS: We retrospectively analyzed fasting plasma glucose and HbA1c levels as well as the dose of glucose-lowering agents in 34 renal transplant patients converted from tacrolimus to cyclosporine (CsA) for PTDM. Diabetes was defined according to current guidelines as repeated fasting plasma glucose (FPG) levels > or =126 mg/dL. RESULTS: At conversion, 11 patients received insulin, 5 received oral agents, and 18 had no glucose-lowering therapy. Fasting plasma glucose levels decreased from 146 +/- 64 mg/dL at conversion to 111 +/- 26 mg/dL at 3 months and 104 +/- 21 mg/dL at 12 months (P < .001). HbA1c levels decreased from 6.8 +/- 0.8% at conversion to 6.0 +/- 0.6% at 12 months (P = .001). Insulin was stopped in 3, the dose reduced in 7, and remained stable in 1 of the patients. The average daily insulin dose among these patients was reduced from 31 +/- 17 units at conversion to 13 +/- 12 units at 12 months (P < .05). There was no significant change in the number of patients treated with oral glucose-lowering agents. Diabetes reversed (fasting plasma glucose < or = 125 mg/dL without glucose-lowering therapy) in 44% (95% confidence interval, 23% to 64%) of patients during the first year after conversion (P < .001). Graft function, blood pressure, and lipid levels remained stable after conversion but the proportion of patients receiving lipid-lowering therapy increased from 18% to 49% (P < .01). CONCLUSIONS: Conversion from tacrolimus to CsA for PTDM was associated with a marked improvement in glucose metabolism and frequent reversal of diabetes.

Undiagnosed rupture of right hemidiaphragm--hepatothorax: a case report.

A case of late diagnosis of right diaphragmatic rupture due to blunt trauma is presented. Traumatic diaphragmatic rupture is an uncommon but severe problem in a patient with multiple injuries. One third of left-sided ruptures and half of right-sided ruptures have been undetected for several days. Traumatic diaphragmatic rupture should be suspected on the basis of an abnormal chest X-ray in patients with multiple injuries. Imaging studies like computed tomography, scan and magnetic resonance imaging can be helpful for differential diagnosis. Right-sided injuries occur more commonly than previously thought and often require thoracotomy.

Pharmacodynamics of trovafloxacin and levofloxacin against Bacteroides fragilis in an in vitro pharmacodynamic model.

An in vitro pharmacodynamic investigation was conducted to explore whether the area under the concentration time curve from 0 to 24 h (AUC(0-24))/MIC ratio could predict fluoroquinolone performance against Bacteroides fragilis. An in vitro model was used to generate kill curves for trovafloxacin (TVA) and levofloxacin (LVX) at AUC(0-24)/MIC ratios of 1 to 406 against three strains of B. fragilis (ATCC 25285, ATCC 23745, and clinical isolate M97-117). TVA and LVX were bolused prior to the start of experiments to achieve the corresponding AUC(0-24)/MIC ratio. Experiments were performed in duplicate over 24 h and in an anaerobic environment. Analyses of antimicrobial performance were conducted by comparing the rates of bacterial kill (K) using nonlinear regression analysis with 95% confidence intervals. Statistical significance was defined as a lack of overlap in the 95% confidence limits generated from the slope of each kill curve. For both TVA and LVX, K was maximized once an AUC(0-24)/MIC ratio of > or =40 was achieved and was not further increased despite a 10-fold increase in AUC(0-24)/MIC from approximately 40 to 400 against all three strains of B. fragilis. No significant differences were found in K between AUC(0-24)/MIC ratios of approximately 40 to 200. In experiments where AUC(0-24)/MIC ratios that were > or = 5 and < or = 44 were conducted, 64% demonstrated regrowth at 24 h. Resistant strains were selected in 50% of those experiments, demonstrating regrowth, which resulted in increased MICs of two- to 16-fold for both TVA and LVX. Regrowth did not occur, nor were resistant strains selected in any studies with an AUC/MIC that was > 44. Our findings suggest that fluoroquinolones provide antibacterial effects against B. fragilis in a concentration-independent manner associated with an AUC(0-24)/MIC ratio of > or =40. Also, the potential for the selection of resistant strains of B. fragilis may increase with an AUC(0-24)/MIC ratio of < or =44.

Abdominal aortic coarctation with splanchnic arterial occlusion.

Abdominal aortic coarctation is found in only 2% of aortic coarctation and is usually manifested by renovascular hypertension. Splanchnic arterial occlusive lesions occur in 22% of these patients and are exceptionally symptomatic. We present a case report of a young patient with abdominal aortic coarctation causing hypertension and visceral angina. The aetiopathogeny and treatment are discussed.

Fluoroquinolone resistance in Bacteroides fragilis following sparfloxacin exposure.

In vitro pharmacodynamic studies investigating the antimicrobial properties of five fluoroquinolones, (trovafloxacin, sparfloxacin, clinafloxacin, levofloxacin, and ciprofloxacin) against Bacteroides fragilis ATCC 23745 were conducted. The times required to reduce the viable counts by 3 log units were as follows: clinafloxacin, 2.9 h; levofloxacin, 4.6 h; trovafloxacin, 6 h; and sparfloxacin, 10 h. Exposure to ciprofloxacin did not achieve a 3-log decrease in viable counts. The susceptibility of B. fragilis was determined both prior to exposure and following 24 h of exposure to each of the five fluoroquinolones tested. The MICs of clinafloxacin, levofloxacin, trovafloxacin, sparfloxacin, ciprofloxacin, metronidazole, cefoxitin, chloramphenicol, and clindamycin were determined by the broth microdilution method. The MICs for B. fragilis preexposure were as follows: clinafloxacin, 0.25 microg/ml; trovafloxacin, 0.5 microg/ml; sparfloxacin, 2 microg /ml; levofloxacin, 2 microg/ml; and ciprofloxacin, 8 microg/ml. Similar pre- and postexposure MICs were obtained for cultures exposed to trovafloxacin, clinafloxacin, levofloxacin, and ciprofloxacin. However, following 24 h of exposure to sparfloxacin, a fluoroquinolone-resistant strain emerged. The MICs for this strain were as follows: clinafloxacin, 1 microg/ml; trovafloxacin, 4 microg/ml; sparfloxacin, 16 microg/ml; levofloxacin, 16 microg/ml; and ciprofloxacin, 32 microg/ml. No changes in the susceptibility of B. fragilis pre- and postexposure to sparfloxacin were noted for metronidazole (MIC, 1 microg/ml), cefoxitin (MIC, 4 microg /ml), chloramphenicol (MIC, 4 microg/ml), and clindamycin (MIC, 0.06 microg/ml). Resistance remained stable as the organism was passaged on antibiotic-free agar for 10 consecutive days. Mutant B. fragilis strains with decreased susceptibility to clinafloxacin, trovafloxacin, sparfloxacin, levofloxacin, and ciprofloxacin were selected on brucella blood agar containing 8x the MIC of levofloxacin at a frequencies of 6.4 x 10(-9), 4x the MICs of trovafloxacin and sparfloxacin at frequencies of 2.2 x 10(-9) and 3. 3 x 10(-10), respectively, and 2x the MIC of clinafloxacin at a frequency of 5.5 x 10(-11); no mutants were selected with ciprofloxacin. The susceptibilities of strains to trovafloxacin, levofloxacin, clinafloxacin, sparfloxacin, and ciprofloxacin before and after exposure to sparfloxacin were modestly affected by the presence of reserpine (20 microg/ml), an inhibitor of antibiotic efflux. The mechanism of fluoroquinolone resistance is being explored, but it is unlikely to be efflux due to a lack of cross-resistance to unrelated antimicrobial agents and to the fact that the MICs for strains before and after exposure to sparfloxacin are minimally affected by reserpine.

Occluding aortic endoluminal stent graft combined with extra-anatomic axillofemoral bypass as alternative management of abdominal aortic aneurysms for patients at high risk with complex anatomic features: a preliminary report.

PURPOSE: To describe an exclusion endoluminal technique for management of abdominal aortic aneurysms among high-risk patients with complex anatomic features. METHODS: From January 1995 to December 1996, among 143 patients with infrarenal abdominal aortic aneurysm treated by means of endograft placement, 9 (6.3%) had complex aortic or aortoiliac morphologic features. For these patients, the endograft was delivered through a femoral cutdown in an occluding aortoiliac configuration. The contralateral iliac artery was occluded with an iliac endograft. Axillofemoral bypass grafting was performed. Computed tomographic scans were obtained regularly. RESULTS: There was 1 postoperative death of severe arrhythmia. All aneurysms were found to be affected by thrombosis on immediately postoperative computed tomographic scans, except in 1 patient with a proximal leak, which was managed successfully with angiographic embolization. The mean follow-up time was 12 months. Aortic aneurysm diameter decreased from 2 mm at 6 months (2 patients) to 6 mm at 12 months (6 patients). All axillofemoral bypass grafts are patent. CONCLUSIONS: Placement of an occluding endograft associated with axillofemoral bypass grafting is a good alternative for patients at high risk with complex anatomic features. Longer-term follow-up study is needed to evaluate this endoluminal technique.

Adventitial cystic disease of the popliteal artery communicating with the knee joint. A case report.

We describe the case of a patient with adventitial cystic disease of the popliteal artery in which a direct anatomic communication between the cysts and the nearby knee joint was demonstrated by magnetic resonance imaging and confirmed by surgery. This unusual observation could shed some light on the much debated question of the cause, the pathogenesis, and the management of the affection. Moreover, it emphasizes the importance and the role of magnetic resonance imaging in the diagnosis of adventitial cystic disease of the popliteal artery.

Mechanism and effects of the binding of lupus anticoagulant IgG and prothrombin to surface phospholipid.

We report here experiments on how lupus anticoagulant antibodies (LA IgG) that react with prothrombin bind to surface phospholipid and affect prothrombin's affinity for surface phospholipid and activation to thrombin. LA IgG was purified by protein A chromatography from the plasma of 16 patients of whom four had associated hypoprothrombinemia and 10 had experienced thrombosis. Many LA IgG bound, in the absence of phospholipid and calcium, not only to immobilized prothrombin but to both prothrombin 1 and fragment 1, which established at least an oligoclonal origin of LA IgG. No LA IgG bound to thrombin. Although prothrombin and Ca2+ were required to support binding of LA IgG to immobilized phosphatidylserine (PS), prothrombin at higher concentrations inhibited binding, presumably by competing with prothrombin/LA IgG complexes for PS binding sites. Prothrombin 1, which cannot bind to PS, also inhibited binding of many LA IgG to PS, presumably by forming competing soluble prothrombin 1/LA IgG complexes. Despite their ability to react with prothrombin independent of phospholipid, LA IgG enhanced binding of prothrombin to immobilized phospholipid and to cultured human umbilical vein endothelial cells. Prothrombin bound with LA IgG to the surface of endothelial cell monolayers could be activated to thrombin after supernatant prothrombin and LA IgG were washed away. The relation is discussed of these observations to a hypothesis that LA IgG mediated concentration of prothrombin on cell surface phospholipid represents a mechanism by which LA IgG could increase thrombotic risk.

Differences in the interactions of lupus anticoagulant IgG with human prothrombin and bovine prothrombin.

Lupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin that the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell's viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing > 50% of inhibition. In contrast, only one LA IgG markedly inhibited (> 50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity.

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.

Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa.

Because free factor VIIa is inactivated only very slowly by a plasma concentration of antithrombin III (AT III) even in the presence of heparin, it has been assumed that AT III plays no significant role in regulating the initiation of tissue factor-dependent blood coagulation. However, in the present study, we present evidence that factor VIIa bound to tissue factor, unlike free factor VIIa, is readily inactivated by AT III in the presence of heparin. In a reaction mixture containing calcium ions and approximately equimolar concentrations of relipidated tissue factor (8.9 nmol/L) and factor VIIa (10 nmol/L), AT III (100 micrograms/mL) plus heparin (1 U/mL) inhibited 50% of the factor VIIa coagulant activity of the reaction mixture within 5 minutes. AT III/heparin was also shown to inhibit the catalytic activity towards factor X of factor VIIa/tissue factor complexes formed on monolayers of an ovarian carcinoma cell line (OC-2008) that constitutively expresses surface membrane tissue factor. AT III, even in the absence of exogenously added heparin, substantially inhibited the functional activity of factor VIIa/cell surface tissue factor complexes on intact monolayers. AT III alone and AT III/heparin, to a greater extent, also inhibited factor VIIa on "nonfunctional" factor VIIa/tissue factor complexes on intact monolayers, with resultant inhibition of their expression of factor VIIa/tissue factor catalytic activity toward factor X after cell lysis. The potential physiologic significance of these findings is discussed.

Binding of annexin V to a human ovarian carcinoma cell line (OC-2008). Contrasting effects on cell surface factor VIIa/tissue factor activity and prothrombinase activity.

Proteins of the annexin/lipocortin family bind tightly to anionic phospholipids and platelets and act as in vitro anticoagulants. Annexins may be useful as tools to study the availability of anionic phospholipids on cell surfaces and their role in the regulation of blood coagulation. In the present study, we investigated the binding of annexin V (placental anticoagulant protein I) to a human ovarian carcinoma cell line, OC-2008, that constitutively expresses surface membrane tissue factor activity. Binding of annexin V to cell monolayers was calcium-dependent, specific, saturable and reversible; Scatchard analysis indicated a single class of binding sites with an apparent Kd of 9.4 +/- 3.1 nM and 5.2 +/- 1 x 10(6) sites per cell. Binding was completely inhibited by phospholipid vesicles containing phosphatidylserine, but was not inhibited by vesicles containing phosphatidylcholine. Annexin V inhibited the cell surface-dependent activity of prothrombinase complex, but did not inhibit the activity of the factor VIIa/tissue factor complex. In conclusion, these results suggest that anionic phospholipid is present on the extracellular face of OC-2008 cells; this anionic phospholipid is functionally important for the activity of the prothrombinase complex, but the importance of anionic phospholipid for the cell surface factor VIIa/tissue factor functional activity is unclear.

Factor VIIa/tissue factor-catalyzed activation of factors IX and X on a cell surface and in suspension: a kinetic study.

The kinetics of activation of factor IX and factor X by factor VIIa was studied in the presence of various sources of tissue factor: (1) a surface membrane of human ovarian carcinoma cell line, OC-2008 (2) the cell lysate (of OC-2008) and (3) reconstituted purified human tissue factor. The rates of activation of factors IX and X were monitored in activation peptide release assays using tritiated substrates. The results indicate that the apparent Km values for factor IX and factor X were similar for a given tissue factor, but varied with tissue factor source. The source of tissue factor greatly influenced the apparent differences in Vmax for factors IX and X. When a surface of monolayer provided tissue factor, the Vmax of factor IX was only 2-3 fold lower than factor X, but when either a cell lysate or purified tissue factor was the source of cofactor activity, the difference in Vmax rose to about 8-10 fold. Although, the tissue factor apoprotein in the cells was expressed entirely on the outer surface membrane, the activity of tissue factor on the intact cell surface was 50 to 100-fold lower than in the lysed cell preparation.

Purification and characterization of rabbit factor IX and its existence as a two-chain factor IX alpha in circulating plasma.

The long term objective of this study is to immunodeplete rabbits of factor IX as a means of developing a rabbit model for hemophilia B for use in studies of tissue factor-dependent blood coagulation. As a first step, we have purified rabbit factor IX by basically two different methods: (1) conventional chromatography utilizing DEAE-Sephadex and heparin-agarose column chromatography (2) immunoaffinity chromatography on monoclonal anti-rabbit factor IX IgG column. Purified rabbit factor IX migrated as a single band with an apparent molecular mass of 76 kD on nonreduced SDS-PAGE. On reduced SDS-PAGE the majority of factor IX migrated as a two-chain molecule (molecular masses 51 and 28 kD) and a faint band corresponding to 78 kD. We have shown that the purification of rabbit factor IX as a two-chain molecule is not due to the partial proteolysis of factor IX during its purification from commercially obtained rabbit plasma. Analysis of 3H-labelled rabbit factor IXa on SDS-PAGE revealed that, in contrast to human factor IXa, carbohydrate was found associated with the heavy chain of activated factor IX (H beta) after release of the activation peptide. Further understanding of the molecular properties of rabbit factor IX and the generation of neutralizing monoclonal and polyclonal antibodies will facilitate the development of a rabbit model for hemophilia B.