Characterization of des-(741-1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa.

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

Function of isolated rabbit hearts perfused with erythrocyte suspension is not stable but improvement may be feasible with hemoglobin solution.

In the present study isolated rabbit hearts were perfused with erythrocyte suspensions (hematocrit 21.5 +/- 0.5%) or hemoglobin solutions according to Langendorff with a constant flow at 37 degrees C. In preliminary experiments three types of stroma-free hemoglobin were used: unmodified, but carefully purified, stroma-free hemoglobin (SFHb), HbNFPLP which is a chemically modified Hb molecule and polyHbNFPLP which is a polymer of HbNFPLP. In hearts perfused with erythrocyte suspensions left ventricular developed pressure and oxygen consumption decreased and perfusion pressure increased steadily from the beginning of the perfusion. Dark spots appeared on the surfaces of these hearts, which were the result of extravasation of erythrocytes. As a consequence capillaries probably became obstructed, leading to reduced cardiac function. Hearts perfused with stroma-free hemoglobin solutions showed an initial increase in left ventricular developed pressure after switching from Tyrode perfusion to perfusion with hemoglobin solutions. Left ventricular developed pressure and perfusion pressure were stable for about 2 hours in hearts perfused with SFHb and were reasonable for 2 hours when the heart was perfused with HbNFPLP or more than 4 hours with polyHbNFPLP. More extensive experiments with stroma-free hemoglobin solutions when these become available in sufficient quantities have, according to the results from preliminary experiments, the potential of showing good oxygen supply resulting in reasonable cardiac function.

Prevention of side effects by hemoglobin solutions; the selection of optimal test models, especially concerning thrombogenicity.

Modification of hemoglobin (Hb) by crosslinking and polymerization results in an improved oxygen release capacity and a prolonged vascular retention time. Modification improves the efficacy and prevents certain side effects. It eliminates leakage of Hb through the kidneys and accumulation in the tubuli. Another important issue is the degree of purification of Hb solutions. Traces of membrane fragments may cause immunogenic and thrombogenic side effects. To determine the contamination with erythrocyte membrane fragments, we developed assays for glycophorin-alpha and phospholipids. Special models were evaluated for testing the maximum allowable level of membrane contamination. As an in vitro model for thrombogenicity we used confluent monolayers of human umbilical vein endothelial cells. These cells were incubated with Hb solutions and subsequently tested on tissue factor (TF) procoagulant activity. TF was tested by the factor VII-catalyzed activation of factor X. The lower detection limit of this assay for endotoxin was 0.5 ng/ml. Hb did not cause any tissue factor expression even after prolonged incubation. No cooperation was found within endotoxin. As an in vivo test on thrombogenicity we developed a guinea pig model in which we can follow the generation of fibrinopeptide A (FPA). This is one of the most sensitive markers for thrombin activation in vivo. When slightly contaminated Hb solutions (phospholipid content 2 nmol/ml) were infused in the presence of factor Xa at a dose (9 micrograms/kg) which in itself did not induce FPA generation, we observed an increase in FPA levels in the plasma from 1.2 +/- 0.4 ng/ml to 5.2 +/- 0.7 ng/ml. Factor Xa is used to mimic a stressed clinical condition with activated coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of interleukin-6 production by monocytes for in vitro safety testing of hemoglobin solutions.

Induction of interleukin-6 (IL-6) production by isolated human mononuclear blood cells was taken as in vitro model for the induction of inflammatory reactions. The model was very sensitive to bacterial endotoxin (detection limit < 10pg/ml). Hemoglobin (Hb) solutions, prepared under non-sterile conditions also induced IL-6 production, which correlated with a positive reaction in the Limulus assay. Purification of the Hb solutions with a detergent prevented IL-6 production, showing that pure Hb itself does not activate the monocytes. We conclude that this assay is a useful and sensitive test of contamination with components that can induce inflammatory reactions, especially microbial products.

Detection of erythrocyte membrane components in hemoglobin-based blood substitutes.

Two methods for the detection of membrane components in human stroma-free hemoglobin solutions are described. The first is a phospholipid assay with a detection limit of 0.5-1 nmol phospholipid/ml hemoglobin-solution. For the detection of membrane proteins an immunoassay with a monoclonal antibody against glycophorin alpha was developed (detection limit 0.01% of the original amount). These methods were used to determine the purity of Hb solutions prepared in two different ways. Hb solutions prepared by filtration of red blood cells, gradually swollen in hypotonic buffer, contained 0.25% of the original amount of phospholipid and no detectable glycophorin alpha. For Hb solutions prepared in a similar way from red blood cells lysed in water, the values for phospholipid and glycophorin alpha were 2.5% and 0.06%, respectively. The determination of both glycophorin alpha and phospholipid gives a useful indication of the purity of Hb solutions.

Preparation and characterization of crosslinked and polymerized hemoglobin solutions.

In 1982 we synthesized 2-Nor-2-formylpyridoxal 5'-phosphate (NFPLP) and subsequently showed that coupling of the beta chains of hemoglobin (Hb) by this organic phosphate compound according to Benesch et al. (1) lowers the oxygen affinity and prolongs the retention time in the circulation of rats and rabbits with a factor 3 by prevention of excretion via the kidneys. Optimal conditions for the purification of HbNFPLP either by ion-exchange chromatography or by heat treatment were established with recoveries of 70% and 85%, respectively. By extrapolation from the data in rats and rabbits a half life of about 8 hours can be expected in the circulation of humans. However, under some conditions a further prolongation is required. The aim of further modification of HbNFPLP was to achieve a retention time of about 24 hours. Polymerization with glutaraldehyde to polyHbNFPLP resulted in a mixture of polymers of different size. We determined the optimal degree of polymerization with respect to the effects on vascular retention time, oncotic activity, viscosity and oxygen affinity. Depending on the degree of polymerization we found in rats a 5- to 7- fold increase in vascular half-life compared to native Hb. The change in oxygen affinity was found to be independent of the polymer size (P50 = 18-22 mmHg). A limiting factor for polymerization is the increase in viscosity, which was dramatic when large polymers (greater than 300 kD) were present in the preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of membrane fragments in hemoglobin solutions.

Two methods for the detection of membrane components in human stroma-free hemoglobin (SFHb) solutions are described. The first method is a phospholipid assay with a detection limit of 0.5-1 nmol phospholipid/ml SFHb. For the detection of membrane proteins an immunoassay with a monoclonal antibody against glycophorin alpha was developed (detection limit 0.01% of the original amount). The determination of both glycophorin alpha and phospholipid yields useful information on the purity of SFHb solutions, as was shown by determination of the purity of two SFHb solutions prepared in different ways.

Effects of modified hemoglobin solutions on the isolated rabbit heart.

The effects of modified hemoglobin (Hb) solutions on the coronary vasculature were studied. Hearts were perfused according to Langendorff with constant flow of Tyrode solution. The solutions studied were stroma- free Hb, prepared by lysis of red blood cells in water (SFHb-lys), or prepared by swelling of red blood cells in hypotonic phosphate buffer (SFHb). The increase in coronary vascular resistance at a dose of 200 mg Hb/dl was 68% for SFHb-lys and 13% for SFHb, respectively. Addition of the modified Hb solutions HbNFPLP and polyHbNFPLP produced an increase in coronary resistance of 11% and 8%, respectively. The left ventricular developed pressure (LVDP) (control value 72 +/- 12 mm Hg) increased by 18 and 12 mm Hg, respectively, for a dose of 250 mg Hb/dl. When HbNFPLP was converted to its met-Hb form the increase in LVDP was reduced to 3 mmHg and the increase in perfusion pressure to 6 mm Hg. We conclude that elimination of stromal contamination from Hb solutions can diminish vasoconstrictor effects. The increase in cardiac pressure development and in coronary vascular resistance found for dilute modified Hb solutions is partly due to an improved oxygen transport to the heart.

Interaction of 2,4-diaminopyridine with cholinesterase inhibitors.

The interaction between 2,4 diaminopyridine (2,4-DAP) and neostigmine or edrophonium was studied in vivo in the rat sciatic nerve-tibialis anterior muscle preparation using the constant infusion technique of pancuronium. The ED50 value (dose of drug which produces 50% antagonism) decreased from 17 to 7 micrograms/kg with neostigmine and from 70 to 20 micrograms/kg with edrofonium, when these 2 compounds were co-administered with 40 micrograms/kg 2,4-DAP. The ED50 value of 2,4-DAP decreased from 160 to 90 micrograms/kg when 3.5 micrograms/kg neostigmine was added. From these experiments we conclude that 2,4 DAP acts synergistically with both neostigmine and edrophonium. In experiments using the train of four (T4) stimulation, we compared the antagonistic potencies of the drugs alone and in combination with each other. Neostigmine proved to be the most potent. 2,4-DAP given alone was not able to restore the muscle contraction completely (T4 ratio greater than 70%) up to a dose of 400 micrograms/kg. The doses of neostigmine and edrophonium could be halved, when they were combined with a small amount of 2,4-DAP without apparent lose of antagonistic potency.

Pharmacokinetics and pharmacodynamics of 2,4-diaminopyridine in the cat.

The pharmacokinetics, antagonistic effects, and cardiovascular effects of 2,4-diaminopyridine (2,4-DAP) were studied in 7 anaesthetized cats. Cats received a pancuronium infusion at a constant rate to cause a 90% block of contraction of the anterior tibialis muscle, stimulated through the sciatic nerve. After steady state was reached, 2,4-DAP (750 micrograms/kg IV) was administered. Plasma, urine, and bile were collected over 8 h and analyzed by means of an HPLC assay. Plasma concentrations decreased biexponentially with half-lives of 9.0 +/- 5.7 min and 140 +/- 36 min, respectively. The volume of the central compartment was 0.85 +/- 0.27 L/kg, and the volume of distribution in the steady state was 3.1 +/- 1.1 L/kg. Total plasma clearance was 18 +/- 5 ml/kg/min. Ninety percent of the administered dose was recovered in the urine and 0.1 percent in the bile in 8 h. The antagonism of the pancuronium-induced steady-state block was 98% +/- 5%, with onset and duration of 3.5 +/- 2 min and 165 +/- 40 min, respectively.

Differential effects of 4-aminopyridine and 2,4-diaminopyridine on the in vivo release of acetylcholine and dopamine in freely moving rats measured by intrastriatal dialysis.

The central effects of 4-aminopyridine (4-AP), a blocking agent of voltage-dependent potassium channels, and its more polar analogue, 2,4-diaminopyridine (2,4-DAP), were studied after i.p. injection and direct intrastriatal administration in rats. The effects of the drugs on the release of acetylcholine (ACh) and dopamine (DA) were quantified by means of an in vivo microdialysis sampling technique. Both neurotransmitters were determined by on-line HPLC analysis. Both aminopyridines increased the release of ACh dose dependently when administered intrastriatally. After peripheral administration, however, 4-AP but not 2,4-DAP induced an increase in the release of ACh. These results are interpreted as being due to the greater lipid solubility of 4-AP compared to 2,4-DAP and hence its better penetration through the blood-brain barrier. Intrastriatal administration of 4-AP induced a much lower increase in the release of DA compared to ACh, whereas there was no change in the release of DA after peripheral administration. These results indicate that the sensitivity of excitable membranes to the releasing effects of 4-AP is not the same for DA- and ACh-containing neurotransmitter systems.

A comparison of the pharmacological actions of 4-aminopyridine and two of its derivatives in the monkey.

The neuromuscular, cardiovascular and central nervous system stimulating effects of 4-aminopyridine (4-AP), 2,4-diaminopyridine (2,4-DAP) and LF-14 were investigated in the monkey. All these compounds were shown to reverse the stable neuromuscular blockade produced by the intravenous infusion of pancuronium bromide. The doses producing 50% antagonism (ED50) of the pancuronium-induced neuromuscular block were 0.50, 0.54 and 0.71 mg/kg for LF-14, 2,4-DAP and 4-AP respectively. The compounds had only slight cardiovascular effects. In contrast to 4-AP, LF-14 and 2,4-DAP did not reduce the duration of ketamine/diazepam-induced anesthesia, suggesting minimal if any central nervous system effects of these two compounds.

Comparison of the pharmacological actions of some new 4-aminopyridine derivatives.

The pharmacological actions of 2,4-diaminopyridine (2,4-DAP) and 3-[(dimethylamino)-carbonyl] amino 4-aminopyridine (LF-14) were examined and compared with those of 4-aminopyridine (4-AP) in anaesthetized rats and on isolated rat and guinea-pig tissues. Both compounds were more potent than 4-AP in reversing the neuromuscular block caused by pancuronium bromide. The ED50S of LF-14, 2,4-DAP and 4-AP were 100 micrograms/kg, 140 micrograms/kg and 450 micrograms/kg, respectively. LF-14 and 2,4-DAP were also more potent in their in vitro actions on the neuroeffector junctions in the ileum and the isolated heart. 2,4-DAP and LF-14 either did not facilitate or only slightly facilitated the recovery time from xylazine/ketamine anaesthesia which was used as a test for their central action; 4-AP significantly reduced the recovery time. We therefore conclude that both 2,4-DAP and LF-14 are stronger peripherally acting compounds with less central action, and that they may be possible replacements for 4-AP as antagonists of non-depolarizing muscle relaxants.