Specific Enhanced Expression of Platelet-Derived Endothelial Cell Growth Factor in Submucosa of Human Colorectal Cancer

PURPOSE Platelet-derived endothelial cell growth factor, identified to be an angiogenic factor, has been implicated in metastases of colorectal cancer. This study aimed to clarify the role and localization of platelet-derived endothelial cell growth factor associated with human colorectal cancer invasion.METHODS Thirty-two patients with colorectal cancer who had undergone surgery were analyzed. Platelet-derived endothelial cell growth factor enzyme activities in the colorectal cancer specimens were measured. Cells that expressed platelet-derived endothelial cell growth factor were identified and localized by immunohistochemical analysis with anti-human platelet-derived endothelial cell growth factor antibody and by in situ hybridization with specific RNA probe.RESULTS Platelet-derived endothelial cell growth factor enzyme activity increased significantly in cancer tissues compared with normal colonic mucosa at various distances from the cancer. Immunohistochemical analysis and in situ hybridization demonstrated platelet-derived endothelial cell growth factor expression in stromal macrophages and fibroblasts located in cancer tissues and surrounding noncancerous tissues, although the tumor cells and normal colonic mucosa were negative. The value of platelet-derived endothelial cell growth factor expression was highest at the border of the colorectal cancer (35.3 ± 8.9 percent), followed by the cancer nest (15.2 ± 9.2 percent) and normal mucosa (7.7 ± 3.4 percent). In the border area, the highest value of platelet-derived endothelial cell growth factor expression was observed in the submucosa (35.3 ± 8.9 percent), followed by the muscular propria (21.9 ± 7.7 percent) and the subserosa (14.9 ± 5.5 percent).CONCLUSIONS Stromal macrophages and fibroblasts are responsible for elevated platelet-derived endothelial cell growth factor activity in colorectal cancer. The significance of enhanced expression of platelet-derived endothelial cell growth factor in the submucosa at the cancer border remains unclear. Cancer stroma may be an important factor for cancer angiogenesis and may serve as a treatment target through specific modulation of angiogenic factors.Presented at the meeting of the Japan Surgical Society, Sendai, Japan, April 11 to 13, 2001.     

Loss of membrane-dependent factor Va cleavage: a mechanistic interpretation of the pathology of protein CVermont

Clinical manifestations of arterial and venous thrombosis in a family with protein C deficiency was associated with two mutations in the light chain of protein C: Glu20-->Ala and Val34-->Met. Further studies showed that the mutation Glu20-->Ala which eliminated a gamma- carboxylation site was exclusively responsible for the anticoagulant defect of activated protein C (APC). Membrane-bound human factor Va is inactivated by APC after two sequential cleavages of the heavy chain at Arg506 and Arg306. Human factor Va inactivation by human recombinant APC (rAPC) and a mutant molecule with an alanine instead of a glutamic acid at position 20 (rAPC(gamma 20A)) was investigated in the presence and absence of phospholipid vesicles. During a 2-hour incubation period of the cofactor with either rAPC or rAPC(gamma 20A). In the absence of a membrane surface, factor Va is cleaved quantitatively at Arg506 and retains approximately 60% of its initial cofactor activity. After a 2- hour incubation period with rAPC membrane-bound factor Va has no cofactor activity, whereas in the presence of a membrane surface and rAPC(gamma 20A) factor Va retains 60% of its initial cofactor activity. The completed loss in factor Va cofactor activity upon incubation of the membrane-bound cofactor with phospholipid vesicles and rAPC is associated with cleavages at Arg506 and Arg306, whereas membrane-bound factor Va cleavage at Arg306 by rAPC(gamma 20A) is impaired, resulting in a cofactor that is cleaved at Arg506. Slow cleavage at Arg306 occurs when membrane-bound factor Va is incubated with rAPC(gamma 20A) and only small amounts of fragments of M(r) = 45,000 and 30,000 are noticed. Our data show that the genetic defect which leads to the absence of a gamma-carboxylation site at Glu20 impairs membrane binding of human APC, which in turn is required for cleavage of factor Va at Arg306 and inactivation of the cofactor. The consequence of impaired membrane-dependent cleavage at Arg306 is manifested in vivo by venous and arterial thrombosis.     

Characterization of Factors Affecting the Stability of Frozen Heparinized Plasma

The use of heparin rather than citrate as primary anticoagulant has been shown to significantly improve the initial activity, stability and recovery of factor VIII:C from human plasma, cryoprecipitates or factor VIII concentrates if the plasma was initially frozen at -80°C and subsequently stored at this temperature. If frozen and stored at progressively warmer temperatures however, increasing amounts of insoluble protein aggregates, termed storage precipitates (SPs), were recovered in the thawed plasma and cryoprecipitate fractions. Plasma recovery by centrifugation at 7,000 g for 7 min [Method I (MI)], 2 times 10 min (MII) or 15 min (MIII) had little effect on SP formation after 1 month at any storage temperature. After 4 months at -20°C, more SP was recovered from MIII plasma whereas at -40°C, more SP was recovered from MI plasma. Also, the preparation method had little or no effect on factor VIII:C activity at equivalent storage times or temperatures. A trend towards improved factor VIII recoveries was noted at lower freezing and storage temperatures however. SP formation was associated with reduced fibrinogen levels in the recovered plasma without loss of antithrombin-III or increased fibrinopeptide-A. Western blots showed polymerization of Aα or γ-chains of fibrinogen. SP formation was reduced or eliminated with factor XIII inhibitors, antibody to the active factor XIII a subunit or adjustment of heparinized plasma to 5-10mM sodium citrate before initial freezing and storage. Although plasma factor VIII:C recoveries were only slightly affected at these citrate concentrations under most conditions, its recovery in cryoprecipitates was substantially improved owing to the reduction or absence of SPs.     

Association Factor of Ribosomal Subunits from Bacillus stearothermophilus

The isolation of a new factor, which can cause the in vitro association of 30S and 50S ribosomal subunits at low Mg⁺⁺ concentration, is described. The association factor is eluted together with the dissociation protein when ribosomes of Bacillus stearothermophilus are washed with salt solutions of high concentration. The association activity is heat-stable, whereas dissociation factor is inactivated after 10 min at 80°C. This treatment allows the separation of both factors. Several properties rule out the possibility that uncharged, amino-acyl-, or peptidyl-tRNA are responsible for the association process described in this report. Digestion with trypsin shows that the association factor contains at least two components, one of which is a protein.     

Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay

Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg506-->Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 micromol/L), and APC. APC, at concentrations < or = 5.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a clot was observed in plasma from APC-resistant individuals. Gel electrophoresis analyses of factor V fragments showed that membrane-bound factor V is primarily cleaved at Arg306 in both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast degradation after cleavage at Arg506/Arg306, in the APC-resistant individuals' plasma, early generation and accumulation of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg306. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed in either plasma from normal or APC-resistant individuals. Our data show that resistance to APC in patients with the Arg506-->Gln mutation is due to the inefficient degradation (inactivation) of factor Va heavy chain by APC.     

No effect of a new second-generation B-domain-deleted recombinant product on lymphocyte transformation in vitro: a study of plasma-derived and recombinant products

Immunomodulatory effects of various factor VIII and factor IX clotting factor concentrates (CFCs) and of albumin were evaluated by a sensitive assay measuring the incorporation of ³H-thymidine in phytohaemagglutinin-stimulated lymphocytes in the presence of monodansylthiacadaverine.
In contrast to previous findings by others, we found lymphocyte transformation to be inhibited by all plasma-derived factor VIII concentrates at concentrations of 0.02, 0.2 and 2.0 IU/ml, including those purified by monoclonal antibodies ( P  < 0.05). Kryobulin TIM3 had the most pronounced effect. In addition, three plasma-derived human albumin preparations exerted a similar inhibitory effect as the factor VIII concentrates, whereas the corresponding plasma-derived factor IX concentrates only manifested minor immunomodulatory effects.
Of the recombinant preparations, only Recombinate exerted an inhibitory effect at 0.02 and 0.2 IU/ml, whereas both Kogenate and Recombinate decreased ³H-thymidine incorporation at 2.0 IU/ml ( P  = 0.01). No immunomodulatory effect at all was observed with r-VIII SQ, a new B-domain-deleted recombinant factor VIII preparation free from added albumin. The significance of this finding regarding immunological side-effects including inhibitor development remains to be evaluated, but this second-generation recombinant product opens up new and interesting perspectives yet to be explored.     

A Radioisotope Dilution Assay for Unlabelled Vitamin B12-Intrinsic Factor Complex Employing the Binding Intrinsic Factor Antibody: Probable Evidence for Two Types of Binding Antibody

A new radioisotope dilution assay for vitamin B₁₂-intrinsic factor complex is described. The method is based on the use of the binding type intrinsic factor antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B₁₂ complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel at pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B₁₂-intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for at least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B₁₂ complex and one which combines equally well with this complex or with free intrinsic factor.     

Stability of factor VIII concentrates after reconstitution

Adjusted-dose continuous infusion of factor VIII (FVIII) has recently been shown to reduce the doses of the factor in patients undergoing surgery by 50-75%. The main limitation of this method has been the instability of factor concentrates. All manufacturers are recommending infusion of the concentrate within hours after reconstitution. We studied the stability of 15 different lyophillzed F VIII products. Reconstituted samples were stored for periods of 4, 24, and 72 hr and 1, 2, 3, and 4 weeks at temperatures of WC, 20-23°C, and 37°C in their original glass containers and in plastic tubes and then frozen. Assays were performed in duplicate, using a one-stage clotting method and a chromoge nlc assay for F VIII, wlth all samples from a single concentrate In the same run. Activation of the coagulation factor occurred In some concentrates, more often at 44°C than at 20-23°C or 3PC. The stability of all products was substantially better than that declared by the manufacturers. Several concentrates maintained factor activities above 80% of baseline for the entlre period of 4 weeks at 44°C or at 20-23°C. The results demonstrate that many of the F VIII concentrates may be used for continuous infusion. © 1994 Wiley-Liss, Inc.     

Differential modulation of mitogenic and metabolic actions of insulin-like growth factor I in rat glomerular mesangial cells in high glucose culture

Summary In order to explore the possible contribution of insulin-like growth factor I to the development of diabetic nephropathy, the effect of glucose on the mitogenic and metabolic actions of insulin-like growth factor I in cultured rat glomerular mesangial cells was examined. The stimulation of [³H]-thymidine incorporation by insulin-like growth factor I in the cells exposed to high concentrations (55 mmol/l) of glucose (4.6±1.3 fold stimulation) was significantly suppressed as compared with that in the cells cultured in 11 mmol/l glucose (17.5±0.8 fold). In contrast, [³H]-aminoisobutylic acid uptake into the mesangial cells was significantly enhanced by glucose (2.03±0.03 nmol · mg protein^–1 · 15 min^–1 at 55 mmol/l glucose vs 0.59±0.01 at 11 mmol/l glucose), while 2-deoxyglucose uptake remained unchanged. [¹²⁵I]-insulin-like growth factor I binding was slightly but significantly increased in the cells exposed to high concentrations of glucose. Thus, glucose may modulate the mitogenic and metabolic actions of insulin-like growth factor I differently in cultured mesangial cells probably at the post-insulin-like growth factor I receptor level. These results may indicate that the differential modulation of the actions of insulin-like growth factor I by glucose could result in the increase in amino acid uptake and decrease in the cell proliferation in the mesangial cells, possibly leading to enhanced mesangial matrix synthesis with a relatively small increase in mesangial cell volume as seen in diabetic nephropathy.     

No inhibitor development after continuous infusion of factor concentrates in subjects with bleeding disorders undergoing surgery: a prospective study

Inhibitor development against von Willebrand factor, factor VIII or factor IX is one of the most severe complications of treating patients with von Willebrand's disease (VWD), haemophilia A or haemophilia B respectively. Continuous infusion of factor concentrate has been implicated as a risk factor for inhibitor development. This prospective study investigated inhibitor development after continuous infusion of factor concentrate for surgical procedures in subjects with VWD or a severe form of haemophilia (factor activity <1%). Observations were made on the occurrence of inhibitor formation, adverse events and virus seroconversions. Main inclusion criteria comprised a negative history of inhibitors to replacement factor concentrate, ≥50 exposure days to factor concentrate and anticipated surgery requiring replacement factor coverage for ≥3 days. Therapy began with a bolus dose of 30–50 IU kg^?1 body weight of factor concentrate followed by continuous infusion with 3–4 IU kg^?1 h^?1. Continuous infusion dose of factor concentrate was adjusted based on factor levels measured at least once daily. In 46 subjects included in the study to date, no inhibitors have been identified at discharge or follow‐up (3–4 weeks after surgery), and no thrombotic events or postoperative wound infections occurred. All subjects underwent surgery without major blood loss, and hemostatic efficacy was generally rated ‘excellent’. The results of the current study are promising, although the number of subjects is too small to make a definitive statement about the incidence of inhibitor development during continuous infusion of factor concentrate. Therefore, this study will be continued.     

Coagulation Factors in the Human Fetus of about 20 Weeks of Gestational Age

S ummary In plasma samples of 11 fetuses of about 20 weeks of gestational age the following coagulation factors have been determined (mean values found are given in parentheses): fibrinogen (0·30 U/ml), prothrombin (±0·17 U/ml), factor V (0·28 U/ml), factor VII (0·21 U/ml), factor VIII coagulant activity (factor VIII:C) (0·12 U/ml), factor VIII-related antigen (factor VIIIR:Ag) (1·04 U/ml), coagulant factor VIII-related antigen (factor VIII:CAg) (0·19 U/ml), factor IX coagulant activity (0·05 U/ml), factor IX antigen (≤0·03 U/ml), factor X (0·19 U/ml) and antithrombin III (AT-III) (0·23 U/ml).
Our data support the evidence that prenatal diagnosis of haemophilia A is at present possible; less optimism is warranted where haemophilia B is concerned. The number of samples is sufficient to establish normal values for the age group.
Means of quality control of the sample—which is often difficult to obtain—are discussed.     

Changes in the factor VIII complex in diabetic ketoacidosis: evidence of endothelial cell damage?

Summary Factor VIII-related antigen and von Willebrand factor are synthesised by and released from vascular endothelium. Acute increases in the plasma concentration of these proteins may reflect endothelial cell damage. We have thus measured the plasma concentration of factor VIII-related antigen and von Willebrand factor, together with procoagulant factor VIII, during the course of acute diabetic ketoacidosis in seven patients. In addition, evidence for qualitative changes in the factor VIII complex was sought. Plasma factor VIII-related antigen and von Willebrand factor were markedly increased (plasma factor VIII-related antigen at presentation, median 2.75 U/ml; von Willebrand factor 2.95 U/ml) and returned toward normal with clinical and biochemical resolution (plasma factor VIII-related antigen at clinical recovery, median 1.80 U/ml; von Willebrand factor 2.05 U/ml). Plasma procoagulant factor VIII followed a similar pattern, but levels were less elevated (plasma procoagulant factor VIII, at presentation, median 1.6U/ml; at clinical recovery, 1.2U/ml). Crossed immunoelectrophoresis and sodium dodecyl sulphate-acrylamide electrophoresis with autoradiographic identification of multimeric structure revealed no evidence of structurally abnormal factor VIII-related antigen in diabetic ketoacidosis. However, an extra peak on crossed immunoelectrophoresis (pre-peak) was a feature in the acute phase ketoacidotic plasma in six subjects, and may represent aggregated factor VIII. Changes in plasma factor VIII are a feature of diabetic ketoacidosis and, whilst not specific to this condition, may be the result of endothelial cell damage.     

Platelet activity of high-dose factor VIIa is independent of tissue factor

High-dose recombinant factor VIIa has been successfully used as therapy for haemophiliacs with inhibitors. The mechanism by which high-dose factor VIIa supports haemostasis is the subject of some controversy. Postulating a mechanism in which activity is dependent on tissue factor at the site of injury explains the localization of activity but not the requirement for high doses. Postulating a mechanism in which factor VIIa acts on available lipid independently of tissue factor explains the requirement for high doses but not the lack of systemic procoagulant activity. We report that factor VIIa bound weakly to activated platelets (K_d ～ 90 nm ). This factor VIIa was functionally active and could initiate thrombin generation in the presence of plasma concentrations of prothrombin, factor X, factor V, antithrombin III and tissue factor pathway inhibitor. The activity was not dependent on tissue factor. The concentration of factor VIIa required for detectable thrombin generation agreed well with the lowest concentration of factor VIIa required for efficacy in patients. High-dose factor VIIa may function on the activated platelets that form the initial haemostatic plug in haemophilic patients. These observations are in agreement with clinical trials which have shown that high-dose factor VIIa was haemostatically effective without causing systemic activation of coagulation.     

Cardio-renal-endocrine dynamics during stepwise infusion of physiologic and pharmacologic concentrations of atrial natriuretic factor in the dog

Infusion of alpha-human atrial natriuretic factor (alpha-h-ANF) into pentobarbital anesthetized dogs (n = 10) at 0.0025, 0.005, 0.01, and 0.3 micrograms/kg/min was performed to differentiate the physiologic actions of atrial natriuretic factor from its pharmacologic actions. The lowest doses of atrial natriuretic factor infusion resulted in circulating levels that were previously produced by 0-10% saline volume expansion. At the lowest infusion rate, circulating ANF increased 31 +/- 3 pg/ml, resulting in a significant increase in absolute sodium excretion, fractional excretion of sodium, and fractional excretion of lithium, and a significant decrease in urine osmolality. A greater change in circulating atrial natriuretic factor (96 +/- 12 pg/ml) was required to significantly decrease right atrial pressure, cardiac output, and plasma renin activity, and to increase systemic vascular resistance and total and fractional excretion of potassium. The highest dose of atrial natriuretic factor infused was required to decrease arterial pressure and renal vascular resistance. The present study demonstrates that atrial natriuretic factor is natriuretic and diuretic at physiologic concentrations; at low concentrations, atrial natriuretic factor appears to decrease the whole kidney proximal tubular reabsorption of sodium and does not affect glomerular filtration rate; a greater (but physiologic) change in circulating atrial natriuretic factor is required to significantly decrease cardiac output, cardiac filling pressure, and plasma renin activity than is required to significantly increase sodium excretion; and a decrease in systemic arterial pressure and vascular resistance does not occur at physiologic concentrations of atrial natriuretic factor.     

Regulation of factor VIIIa by human activated protein C and protein S: inactivation of cofactor in the intrinsic factor Xase

Factor VIIIa is a trimer of A1, A2, and A3-C1-C2 subunits. Inactivation of the cofactor by human activated protein C (APC) results from preferential cleavage at Arg336 within the A1 subunit, followed by cleavage at Arg562 bisecting the A2 subunit. In the presence of human protein S, the rate of APC-dependent factor VIIIa inactivation increased several-fold and correlated with an increased rate of cleavage at Arg562. (Active site-modified) factor IXa, blocked cleavage at the A2 site. However, APC-catalyzed inactivation of factor VIIIa proceeded at a similar rate independent of factor IXa, consistent with the location of the preferential cleavage site within the A1 subunit. Addition of protein S failed to increase the rate of cleavage at the A2 site when factor IXa was present. In the presence of factor X, cofactor inactivation was inhibited, due to a reduced rate of cleavage at Arg336. However, inclusion of protein S restored near original rates of factor VIIIa inactivation and cleavage at the A1 site, thus overcoming the factor X-dependent protective effect. These results suggest that in the human system, protein S stimulates APC-catalyzed factor VIIIa inactivation by facilitating cleavage of A2 subunit (an effect retarded in the presence of factor IXa), as well as abrogating protective interactions of the cofactor with factor X. (Blood. 2000;95:1714-1720)     

The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor.

Previous studies indicated that human and bovine factor VII exhibit 71% amino acid sequence identity. In the present study, competition binding experiments revealed that the interaction of human factor VII with cell-surface human tissue factor was not inhibited by 100-fold molar excess of bovine factor VII. This finding indicated that bovine and human factor VII are not structurally homologous in the region(s) where human factor VII interacts with human tissue factor. On this premise, we synthesized three peptides corresponding to regions of human factor VII that exhibited marked structural dissimilarity to bovine factor VII; these regions of dissimilarity included residues 195-206, 263-274, and 314-326. Peptide 195-206 inhibited the interaction of factor VII with cell-surface tissue factor and the activation of factor X by a complex of factor VIIa and tissue factor half-maximally at concentrations of 1-2 mM. A structurally rearranged form of peptide 195-206 containing an aspartimide residue inhibited these reactions half-maximally at concentrations of 250-300 microM. In contrast, neither peptide 263-274 nor peptide 314-326, at 2 mM concentration, significantly affected either factor VIIa interaction with tissue factor or factor VIIa-mediated activation of factor X. Our data provide presumptive evidence that residues 195-206 of human factor VII are involved in the interaction of human factor VII with the extracellular domain of human tissue factor apoprotein.     

Platelet activity of high-dose factor VIIa is independent of tissue factor

High-dose recombinant factor VIIa has been successfully used as therapy for haemophiliacs with inhibitors. The mechanism by which high-dose factor VIIa supports haemostasis is the subject of some controversy. Postulating a mechanism in which activity is dependent on tissue factor at the site of injury explains the localization of activity but not the requirement for high doses. Postulating a mechanism in which factor VIIa acts on available lipid independently of tissue factor explains the requirement for high doses but not the lack of systemic procoagulant activity. We report that factor VIIa bound weakly to activated platelets ( K _d ∼ 90 n m ). This factor VIIa was functionally active and could initiate thrombin generation in the presence of plasma concentrations of prothrombin, factor X, factor V, antithrombin III and tissue factor pathway inhibitor. The activity was not dependent on tissue factor. The concentration of factor VIIa required for detectable thrombin generation agreed well with the lowest concentration of factor VIIa required for efficacy in patients. High-dose factor VIIa may function on the activated platelets that form the initial haemostatic plug in haemophilic patients. These observations are in agreement with clinical trials which have shown that high-dose factor VIIa was haemostatically effective without causing systemic activation of coagulation.     

Levels of factor VIII and factor IX in fresh-frozen plasma produced from whole blood stored at 4 °C overnight in Turkey

Background

The aim of this study was to assess whether the quantities of factor VIII and factor IX in fresh-frozen plasma produced from whole blood stored at 4 °C for 24 hours are adequate for their intended purpose.

Materials and methods

Fresh-frozen plasma separated from whole blood after storage at 4 °C overnight (24 hours from donation) was compared with plasma prepared 8 hours after donation using a standard method. The amounts of factor VIII and factor IX obtained with the two methods were compared.

Results

Compared to the levels of factor VIII and factor IX in plasma prepared within 8 hours of blood collection, the levels in plasma prepared after 24 hours of storage at 4 °C were 25% and 9% lower, respectively. Ninety percent of the factor VIII and 100% of the factor IX levels were above 0.5 IU/mL (standard haematology reference range) after 24 hours of storage.

Discussion

These data suggest that there is good retention of coagulation factor activity in plasma produced from whole blood stored at 4 ºC for 24 hours and that such plasma would be an acceptable product for most patients requiring fresh-frozen plasma.     

Studies on urinary excretion of vitamin B12CO60 in pernicious anemia for determining effective dosage of intrinsic factor concentrates

Studies were made in pernicious anemia patients on the urinary excretion ofB₁₂Co⁶⁰ after a small oral dose followed by a large parenteral injection of nonradioactive vitamin B₁₂.

(1) Increasing doses of intrinsic factor concentrates give increasing excretionsof radioactivity at low doses; little additional increase at moderate doses; andat times a subsequent diminution at excessive doses. Data on 34 tests of aparticular intrinsic factor concentrate in 18 pernicious anemia patients tend tosupport an excretion proportional to the logarithm of intrinsic factor dosage atlow to moderate levels, but do not exclude the possibility of a linear approachto a plateau.

(2) Assay by hematologic response was compared with the urinary excretiontests in 13 pernicious anemia patients. This data shows a relation between thetwo tests though the correlation is far from complete.

(3) Methods are outlined for testing all intrinsic factor preparations with thesame amount of tracer vitamin B₁₂ as will be incorporated commercially. Moresensitive comparative tests of similar intrinsic factor preparations may be madeusing smaller amounts of B₁₂Co⁶⁰.

(4) The literature is reviewed to determine which variations in technic mightlead to the most reliable quantitation of intrinsic factor activity.

Submitted on August 8, 1955 Accepted on January 12, 1956     

Recurrence of inhibitor after orthotopic liver transplantation in severe haemophilia A

Objective: Orthotopic liver transplantation (OLT) treats both hepatitis C‐associated cirrhosis and haemophilia A with factor VIII activity increasing into the normal range in most patients. However, the results of OLT in haemophilia patients with inhibitors have been mixed. We report the follow‐up factor VIII values after OLT in an inhibitor patent. Methods: A middle‐aged white male with severe haemophilia A, who had been on immune tolerance for >10 years for a high‐responding factor VIII inhibitor underwent OLT. Factor VIII activity and inhibitor titres were followed over the next 9 months. Summary: The patient had received factor VIII 40 u kg⁻¹ three times/week for many years, which suppressed inhibitor titres to <2 BU. Higher doses of factor had been used to support him through surgery with good results. At the time of OLT, his inhibitor titre was 0.7BU. He was treated prior to transplant surgery with 116 u kg⁻¹ with smaller doses every 2–4 h with monitoring during the operation. In the next 24 h he required another 300 u kg⁻¹ of factor VIII to maintain activity between 61–122%. On postop days 1 and 2 he received 46 and 35 u kg⁻¹ to maintain mean activity of about 50%. Around Day 6 his requirement for factor VIII increased to 70 u kg⁻¹ daily to maintain the same levels. He was treated for possible acute liver rejection with high dose Solu–Medrol and his requirements for factor VIII decreased so that it was discontinued on day 13 postop. Over the next month, his factor VIII activity increased from 57–91% and he had no bleeding complications. Immune suppression was achieved with tacrolimus, mycophenolate and prednisone, the latter being discontinued about 1 month post OLT. After a peak of factor VIII activity at one month, his level decreased so that it was 52% and 37% at 4 and 6 months, respectively, despite the continuous use of immunosuppression for the OLT. Seven months after transplant, the patient underwent a procedure with a prefactor VIII infusion activity of 25% and a 2 h postinfusion activity of 71% after receiving 50 u kg⁻¹. The Bethesda titre for the inhibitor was 0.9 units at this time. The most recent factor activity has dropped to 19% at 9 months. The patient has not had any bleeds post‐transplantation, however. Conclusions: 1. An inhibitor patient on long‐term immune tolerance underwent successful orthotopic liver transplantation with initial normalization of factor VIII activity. 2. Beginning 2 months after OLT and continuing to the present, factor VIII activity gradually decreased associated with detection of a low titre recurrent factor VIII inhibitor. Synthesis of factor VIII by the donor liver is not sufficient for total suppression of the long‐standing inhibitor.