Porcine Factor Research Articles

Syndrome hémorragique sévère chez un hémophile A bon répondeur. Intérêt du facteur VIII porcin

The case is reported of a high responder haemophiliac A, aged 37 yr, who had a severe bleeding episode after surgical treatment of a renal stone. Preoperative anti-factor VIII antibody concentration was 2 UB. Although the surgery was carried out with no real problem (partial nephrotomy), on the 5th postoperative day, there was a sudden increase in the concentration of anti-factor VIII antibodies (21 UB) with a concomitant drastic fall in Factor VIII C level, and important bleeding at the surgical site. The sequential administration of human Factor VIII, « activated prothrombin complex concentrates(FEIBA followed by AUTOPLEX), and total nephrectomy failed to stem the haemorrhage. Despite intravenous gammaglobulins, the anti-factor VIII titre reached 1300 UB. Bleeding could only be controlled by concentrated porcine Factor VIII (HYATE-C), 100 U · kg −1 8 h −1. This treatment was continued for 50 days, with no side-effects. An average dose of 130 U · kg −1 · 8 h −1 maintained a residual level of Factor VIII between 1 and 23 %. There was a simultaneous fall in the antibody-concentration to < 1 UB, due to steroid treatment.

Molecular characterization of commercial porcine factor VIII concentrate

Commercial porcine factor VIII concentrate (Hyate:C) is effective in treatment of patients with hemophilia A who have circulating antibodies to factor VIII. The molecular forms of factor VIII in the concentrate were identified and evaluated in light of the known properties of porcine and human factor VIII. The factor VIII in the concentrate was isolated by tandem chromatography on gelatin-Sepharose and monoclonal anti-factor VIII-Sepharose. The factor VIII was 1% of the protein mass of the concentrate when calculated by either quantity of protein recovered or by radioimmunoassay. Both functional assay and Western blotting of the crude concentrate indicated that maximum coagulant function was achieved by proteolytic activation of procofactor forms of factor VIII. The factor VIII can be fractionated by cation-exchange high-performance liquid chromatography (HPLC) into two or three species of heterodimers depending on the lot. The specific activity of the purified porcine factor VIII was 550 U/mg using pooled porcine plasma at 1 U/mL as a standard. From this value, a factor VIII concentration in normal pig plasma of 2 micrograms/mL was calculated. This agreed well with a value of 3 micrograms/mL obtained by radioimmunoassay (RIA) of factor VIII in porcine plasma. In contrast, reported values for human factor VIII average 5800 U/mg, resulting in a calculated concentration in plasma of 0.2 microgram/mL. The finding that porcine plasma contains a significantly higher circulating mass of factor VIII than human plasma appears to explain previous difficulties in comparing porcine and human factor VIII in standard assays.

Molecular Characterization of Commercial Porcine Factor VIII Concentrate

AbstractCommercial porcine factor VIII concentrate (Hyate:C) is effective in treatment of patients with hemophilia A who have circulating antibodies to factor VIII. The molecular forms of factor VIII in the concentrate were identified and evaluated in light of the known properties of porcine and human factor VIII. The factor VIII in the concentrate was isolated by tandem chromatography on gelatin-Sepharose and monoclonal anti-factor Vlll-Sepharose. The factor VIII was 1% of the protein mass of the concentrate when calculated by either quantity of protein recovered or by radioimmunoassay. Both functional assay and Western blotting of the crude concentrate indicated that maximum coagulant function was achieved by proteolytic activation of procofactor forms of factor VIII. The factor VIII can be fractionated by cation-exchange high-performance liquid chromatography (HPLC) into two or three species of hete-rodimers depending on the lot. The specific activity of the purified porcine factor VIII was 550 U/mg using pooled porcine plasma at 1 U/mL as a standard. From this value, a factor VIII concentration in normal pig plasma of 2 μg/mL was calculated. This agreed well with a value of 3 μg/mL obtained by radioimmunoassay (RIA) of factor VIII in porcine plasma. In contrast, reported values for human factor VIII average 5800 U/mg, resulting in a calculated concentration in plasma of 0.2 μg/mL. The finding that porcine plasma contains a significantly higher circulating mass of factor VIII than human plasma appears to explain previous difficulties in comparing porcine and human factor VIII in standard assays.

Stoichiometry of the porcine factor VIII-von Willebrand factor association.

Factor VIII and von Willebrand factor (vWF) are glycoproteins that form a tightly bound complex in plasma. The interaction of porcine factor VIII with porcine vWF was studied by analytical velocity sedimentation. A single approximately 240-kDa species of factor VIII was isolated for use in the analysis. In contrast, when analyzed by agarose/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, vWF consisted of a population of greater than 10 multimers derived from a 270-kDa monomer. A single boundary (So20,w = 7.2 S) was observed during velocity sedimentation of factor VIII at 260,000 x g. A single boundary also was observed for vWF (weight-average So20,w = 21 S) at 42,000 x g. Under condition of excess factor VIII, the weight-average So20,w of the factor VIII-vWF complex was 40 S at 42,000 x g. At 260,000 x g, the factor VIII-vWF complex had sedimented completely, leaving only free factor VIII. The height of the plateau region of the factor VIII sedimentation velocity curve at 260,000 x g was studied as a function of several starting concentrations of vWF. The experiments were done under conditions in which the effect of radial dilution was negligible so that the plateau height was a measure of the concentration of free factor VIII. The plateau height decreased linearly as the concentration of vWF was increased, indicating that the association was essentially irreversible under the conditions used. A stoichiometry of 1.2 vWF monomers/factor VIII molecule was calculated from the slope of the line. Assuming one factor VIII-binding site/vWF monomer, these results indicate that all factor VIII-binding sites are accessible in the vWF multimer.

Degradation of coagulation proteins by an enzyme from Malayan pit viper (Akistrodon rhodostoma) venom.

Three hydrolases from the crude venom of the Malayan pit viper (Akistrodon rhodostoma) can be differentiated. The first, which we designate ARH alpha, is the well-known fibrinogenolytic enzyme ancrod. The second, ARH beta, which has not been described previously, is identified by its electrophoretic mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by its ability to hydrolyze H-D-phenylalanyl-L-piperyl-L-arginyl-rho-nitroanilide, and by inhibition of its activity by diisopropyl phosphorofluoridate. The third, ARH gamma, also previously not described, has been purified by using gel permeation and ion-exchange chromatography and preparative PAGE. Chemical, electrophoretic, and hydrodynamic data indicate that it is a single-chain, nonglobular glycoprotein with a molecular weight of 25,600. ARH gamma catalyzes the degradation of several plasma vitamin K dependent coagulation factors, including factor IX, factor X, prothrombin, and protein C. The products are electrophoretically similar to factor IXa beta, factor Xa, thrombin, and activated protein C, respectively. However, these products contain little or no enzymatic activity. ARH gamma-degraded factor IX, factor X, prothrombin, and protein C can be subsequently activated by factor XIa, Russell's viper venom X coagulant protein, crude taipan snake venom, and thrombin, respectively. The N-terminal sequence of the peptides resulting from the ARH gamma digest of porcine factor IX shows that at least three bonds are hydrolyzed: (1) at position 152, seven residues from the Arg145-Ala146 factor XIa cleavage site; (2) at position 167 within the factor IX activation peptide; and (3) at position 177, three residues from the Arg180-Val181 factor XIa cleavage site. The degradation of factor IX by ARH gamma is not affected by several serine protease inhibitors. ARH gamma catalyzes the degradation of both the heavy and light chains of porcine factor VIII which results in the inability of thrombin to activate factor VIII. ARH gamma also catalyzes the degradation of porcine antithrombin III which abolishes its ability to inhibit thrombin. These findings may have relevance to studies of hemostatic derangements following envenomation by this snake. Additionally, several novel coagulation factor derivatives have been generated for structure-function studies.

Inhibitors of coagulation in children

Acquired inhibitors of coagulation factor interaction in nonhemophilic children are usually nonspecific, transient, and unassociated with clinical bleeding. They occur with some frequency and are the most common cause for a prolonged APTT found by routine testing. In children, some association with viral infections and treatment with penicillin has been noted, but their interrelationship with the development of antibodies remains unclear. The exact nature of these antibodies, usually directed against coagulant factor phospholipid, is not clear and multiple antibodies both specific as well as nonspecific may occur. No therapy is generally required. Rarely has the acute development of antibodies directed against specific coagulation factors occurred. The laboratory evaluation of the type of inhibitor is, therefore, most important as specific inhibitors may be associated with life-threatening bleeding situations. Their therapy should probably include attempts at eradication of the inhibitor by immunosuppressive agents or other newer modalities. Unfortunately, little information is available regarding the nature and outcome of specific inhibitors in children. Acquired inhibitors in hemophilic patients occur in about 6 to 10% of patients. Newer approaches to their therapy include activated PCC which have generally improved the outlook for such patients. Treatment regimens involve a knowledge of inhibitor response and the concomitant use of plasmapheresis, high-dose or continuous i.v. Factor VIII, and porcine Factor VIII, followed by activated PCC. The role of immunosuppressive agents and other newer modalities appears promising, however, prospective controlled studies are necessary to evaluate their role in the overall management of such patients.

Acquired Factor VIII Inhibitor Associated with Multiple Sclerosis, Successfully Treated with Porcine Factor VIII

Acquired Factor VIII Inhibitor Associated with Multiple Sclerosis, Successfully Treated with Porcine Factor VIII

A paradoxical effect of antibody concentration on vWF-dependent platelet agglutination distinguishes between botrocetin and ristocetin-induced agglutination.

Several heterologous antisera directed against either human or porcine von Willebrand factor (vWF), inhibited botrocetin-induced vWF-dependent agglutination at high concentrations but were found to enhance this reaction at low concentrations. Purified IgG from these immune sera also potentiated botrocetin-induced agglutination as did its F(ab)'2 fragments. However, monovalent Fab fragments of the purified IgG did not. The requirement for a divalent antigen combining region suggests that one possible mechanism of enhanced agglutination may involve intra or inter-molecular cross-linking of vWF multimers by the antibody. Another possible mechanism could be a conformational change in the vWF molecule induced by antibody binding. Such a conformational change may provide additional active sites on the vWF molecule that, in the presence of botrocetin, lead to enhancement of the agglutination reaction. Antisera to human vWF that showed this paradoxical inhibitory/enhancing effect on botrocetin-induced agglutination also inhibited ristocetin-induced platelet agglutination at high concentrations, but failed to enhance agglutination at any concentration. The different effects of these antisera on botrocetin and ristocetin-induced platelet agglutination suggest that no single mechanism can explain the action of both of these mediators.

A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity.

Factor VIII (antihemophilic factor) is a high molecular weight plasma glycoprotein that participates in the blood clotting cascade. The recent cloning and sequence analysis of the cDNA encoding human factor VIII revealed an obvious domain structure for the protein, which can be represented as A1-A2-B-A3-C1-C2. We now report the DNA sequence analysis of porcine exons encoding the entire B domain and part of the A2 and A3 domains. We found an unusually high degree of porcine-human amino acid sequence divergence in the B region compared with the limited sequence available for other regions of the porcine factor VIII molecule. In addition to sequence divergence, there are numerous gaps in the porcine B domain totalling over 200 amino acids. Recombinant DNA techniques were used to effect the removal of large segments of DNA encoding the B domain from the full-length human factor VIII cDNA. These constructs directed the synthesis of biologically active factor VIII when introduced into mammalian cells despite the deletion of up to 38% of the factor VIII molecule.

Von Willebrand factor contaminating porcine factor VIII concentrate (Hyate:C) causes platelet aggregation.

Severe thrombocytopenia has been reported to occur in some patients treated with a polyelectrolyte-fractionated porcine factor VIII concentrate (Hyate:C) (Kernoff et al, 1984; Gatti & Mannucci, 1984). We report here that Hyate:C induces aggregation of platelet-rich plasma in vitro accompanied by ATP release and thromboxane B2 formation, and is inhibited by EDTA and prostaglandin E1. Hyate:C induced platelet aggregation is affected by monoclonal antibodies anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa and suppressed by polyclonal anti-porcine von Willebrand factor antiserum. We conclude that the aggregating ability of Hyate:C is due to porcine von Willebrand factor present in the concentrate. The occasional thrombocytopenia described after Hyate:C infusions probably results from the interaction between platelets and the porcine von Willebrand factor contaminating the concentrate.

Relative Potencies of Human, Rat, Bovine/Caprine, Porcine and Ovine Hypothalamic Growth Hormone-Releasing Factors to Release Growth Hormone by the Rat Anterior Pituitary in vitro

Synthetic rat, human, bovine/caprine, porcine and ovine growth hormone-releasing factor (GRF) were tested for their capacity to release growth hormone (GH) by the rat anterior pituitary in vitro. All peptides elicited parallel and dose-dependent increases in GH release and produced similar maximal GH secretion. Rat GRF was 3-6 times more potent in stimulating the release of GH than all other GRFs, while human, bovine/caprine, porcine and ovine GRF had potencies that were not statistically different. The increased potency of both rat GRF(1-27)NH2 and rat GRF(1-23)NH2 when compared to human GRF(1-27)NH2 and human GRF(1-23)NH2, respectively, suggests that the increased potency of this molecule resides in structural differences in the amino terminal of native GRF. The results demonstrate increased sensitivity of rat pituitary cells for their homologous releasing factor.

Activation of porcine factor VIII:C by thrombin and factor Xa.

The activation of porcine factor VIII:C by thrombin and by factor Xa was studied by a chromogenic substrate assay and by sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of 125I-labeled factor VIII:C activation products. In the chromogenic assay, the kinetics of factor VIII:C dependent activation of factor X by factor IXa in the presence of calcium and phosphatidylserine/phosphatidylcholine vesicles were measured with N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine p-nitroanilide (S2222) as substrate. Substrate dependence of initial rates of the reaction at fixed factor IXa, factor VIII:C, lipid, and calcium obeyed Michaelis-Menten kinetics. At fixed factor IXa, factor X, lipid, and calcium the initial rates of the reaction varied linearly with lower factor VIII:C concentrations and plateaued at higher concentrations. The linear initial rate dependence formed the basis of a rapid, plasma-free assay of activated factor VIII:C. The activation of factor VIII:C by thrombin or factor Xa and the enzyme-independent rate of spontaneous inactivation were studied under conditions of excess enzyme. A model of the activation kinetics was developed and fit to the data by a nonlinear least-squares technique. From the model, the catalytic efficiencies (kcat/Km) of factor VIII:C activation by thrombin and factor Xa were 5.0 X 10(6) M-1 s-1 and 1.1 X 10(6) M-1 s-1, respectively. By comparison with published values of the catalytic efficiencies of several other coagulation enzymes for various substrates, both thrombin and factor Xa are efficient enzymes toward factor VIII:C.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor VIII Inhibitor

To the Editor.— Dr Waddell and colleagues 1 described the use of porcine factor VIII concentrate in a patient with factor VIII inhibitor. They gave in detail the disadvantages and complications of this material and prothrombin complex concentrates. I would like to mention that a blood fraction devoid of such drawbacks has recently been tested on hemophilic dogs. 2 This fraction, known as Bridge anticoagulant neutralizing agent (BANA), apart from increasing three-fold the level of injected factor VIII, acted directly on factor VIII inhibitor. 2,3 Prowse 4 calculated the amount of donor plasma required to produce sufficient material to neutralize 100 units of factor VIII bypassing activity in the case of two activated prothrombin complex concentrates and BANA. For FEIBA and Autoplex, 45 mL and 89 mL of plasma were needed, respectively. In the case of BANA, only 12.5 mL of plasma were required. In other words, the introduction of

Factor VIII Inhibitor-Reply

In Reply.— The material described by Dr Nour-Eldin is of great interest and certainly warrants further evaluation in patients with factor VIII inhibitors. However, clarification must be made regarding use of the porcine factor VIII concentrate. The patient did achieve a normal factor VIII level following administration of porcine factor VIII (Hyate) and no untoward side effects were attributed to this material.

Use of Porcine Factor VIII Concentrate

To the Editor.— Factor VIII inhibitors develop in patients with hemophilia A (factor VIII deficiency) in a reported incidence of 5% to 20% 1 and have also been described in a variety of immunologic disorders, in postpartum women, and in elderly patients with no apparent underlying disorders. Bleeding in the presence of factor VIII inhibitors may be severe and difficult to control. We describe a patient with ulcerative colitis and factor VIII inhibitor who developed substantial postoperative bleeding following emergency colectomy. Corticosteroids, human factor VIII concentrate, cyclophosphamide, and activated prothrombin complex concentrates were ineffective, but a highly purified porcine factor VIII concentrate did increase the factor VIII level. No adverse effects were attributed to the porcine factor VIII preparation. Report of a Case.— A 36-year-old man with ulcerative colitis developed persistent bleeding following colectomy. Partial thromboplastin time (PTT) was 58 s (control of 30 s). Factor VIII level was 12%.

Efficacy of porcine factor VIII in the management of haemophiliacs with inhibitors.

We report our experience with polyelectrolyte-fractionated highly purified porcine factor VIII concentrate (Hyate:C). Porcine factor VIII concentrate was used to treat 14 haemorrhagic episodes including seven severe haemarthroses, three severe haematomata, two episodes of oral bleeding, one traumatic and the other after multiple dental extractions and two life-threatening intestinal haemorrhages. Altogether 60 infusions of Hyate:C were given to five haemophiliacs with inhibitors. At the time of the first infusion with porcine factor VIII the anti-human inhibitor level ranged from 60 to 2.5 modified Bethesda units/ml (MBU 4 h incubation). The porcine cross-reactivity of these inhibitors was 25.6% and 5.4% of the human inhibitor level in two patients and scarcely measurable in the other three patients. We injected an amount of porcine factor VIII concentrate corresponding to the calculated porcine neutralizing units plus the units required for haemostasis. The clinical efficacy was excellent and no adverse effects were encountered, apart from two episodes of mild pyrogenic reactions well controlled by hydrocortisone. The anamnestic antibody response against human factor VIII was of slight degree in all cases, while the porcine cross-reactivity showed no significant variation. Our results emphasize the role of porcine factor VIII in the treatment of haemophiliacs with inhibitors.

The management of haemophiliacs who have antibodies to factor VIII.

The management of haemophiliacs with factor VIII antibodies is still one of the major problems in the treatment of haemophilia in spite of our greater understanding of the nature and mode of action of those antibodies.The approach to treatment varies from centre to centre and may consist of factor replacement with human or porcine factor VIII, administration of prothrombin complex concentrates, plasmapheresis, immunosuppressive drugs or different combinations of the above.At Oxford we have relied entirely on human factor VIII replacement for the management of acute bleeding. In twenty‐four patients receiving on demand therapy for 2–10 years we have details of amounts of factor VIII used, frequency of dosage and levels of factor VIII antibody. Eight patients with low levels of antibody have lost their antibody. In 7 patients with higher levels there has been a reduction in anamestic response and level of antibody in the blood. In the remaining patients some of whom are infrequently treated there has been little change in the antibody response.The above findings are described in the light of other regimens of therapy employing high or low dosage factor VIII replacement.

Innovative alternatives to human factor VIII.

Despite massive expenditure of effort and money in fractionation of human blood plasma, it is clear that the end‐product factor VIII is deficient in quantity and its quality seriously compromised by infectious agents both known and unknown. These problems may not be soluble by ever greater blood collection drives and ever more stringent process controls. The average fractionation puts only 20 per cent of donated factor into the bottle of concentrate. Are radical new methods of purification needed?Meanwhile, the vast majority of our potential resources of factor VIII and IX, free of all known human pathogens are thrown away. In the United Kingdom last year, over 600,000 million units of sheep, pig and cow factor VIII were washed down the abbatoir gutters or consumed as black pudding. Already, some haemophiliacs with inhibitors are successfully treated with highly purified porcine factor VIII. Could new purification methods and chemical modifications make this natural abundance of animal factor VIII available to treat all haemophiliacs?Genetically engineered factor VIII made by cultured cells, pure and in unlimited quantities is a theoretical possibility which stands as a ‐ perhaps the ‐ supreme challenge to biotechnology today. Undaunted, several groups have taken up this quest. Huge obstacles bar the way but like Everest it attracts our highest endeavours because it is there.Finally, what thoughts should we entertain of gene therapy? “Ah, could we not seize this sorry scheme of things entire and mould it closer to the heart's desire?” There have been false dawns but recent progress on gene insertion and control raise more than a glimmer of fresh hope.

Inhibition of activated porcine Factor IX by dansyl-glutamyl-glycyl-arginyl-chloromethylketone

Activated porcine Factor IX is irreversibly inhibited by an active site histidine-directed serine protease inhibitor, dansyl-glutamyl-glycyl-arginyl-chloromethylketone (DEGR-CK). The kinetics of inhibition are second order up to inhibitor concentrations of 10 −5 m. The apparent second-order rate constant (in 0.20 m NaCl, pH 8.0) is 1.7 × 10 4 m −1 min −1, which is considerably lower than values reported for Factor Xa, thrombin, plasmin, and kallikrein. Reaction of increasing concentrations of DEGR-CK with Factor IXa, followed by analysis of residual enzymatic activity, yields 1.2 mol DEGR-CK/mol protein, indicating 1:1 stoichiometry for the DEGR-CK/Factor IXa interaction. DEGR-Factor IXa is a potent anticoagulant in vitro. A concentration of 1 n m causes 50% inhibition of the ability of normal porcine-citrated plasma to correct either Factor VIII- or Factor IX-deficient plasmas (intrinsic pathway factors). In contrast, more than 100 n m DEGR-Factor IXa is required to cause 50% inhibition of Factor VII (extrinsic pathway) or Factor X (common pathway) assays. Activation of porcine Factor VIII:C by thrombin in the presence of DEGR-Factor IXa and phosphatidylcholine-phosphatidylserine vesicles reveals that DEGR-Factor IXa markedly stabilizes the spontaneous loss of Factor VIII:Ca activity as does unmodified Factor IXa [P. Lollar, G. J. Knutson, and D. N. Fass (1984) Blood 63, 1303–1308]. These results suggest that DEGR-Factor IXa incorporates into the intrinsic pathway Factor X-activator enzymatic complex, and also that stabilization of Factor VIII:Ca by this complex is independent of the active site of Factor IXa. Inhibition of Factor IXa by DEGR-CK results in the first reported irreversible active-site-modified derivative of this enzyme. DEGR-CK promises to be a useful reagent in the study of the Factor X activator complex. Conceivably, its specific anticoagulant properties could have future clinical benefit.

Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid

The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino- naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.