D.L. Aronson

Turnover of human tissue plasminogen activator (tpa) in rabbits

The turnover of purified tissue plasminogen activator (tPA) from two different manufacturers was compared in rabbits. The first was melanoma derived one-chain tPA and the second was recombinant two-chain tPA. No differences were noted between the two products. A biphasic disappearance curve was observed for the protein (125Iodine labelled). The first phase was extremely rapid with a T1/2 of 0.59-0.89 min; the secondary phase had a T1/2 of 10-12 min. tPA accumulated rapidly in the liver (44% at twenty min) and appeared to be degraded as demonstrated by the increase in plasma of low molecular weight material which was also TCA soluble. Fractionation of purified recombinant two-chain tPA on a Dupont GF-250 column yielded two peaks of protein (Peak 1 and Peak 2) and the turnover of each in rabbits was compared.

Detection and measurement of low levels of prothrombin. Use of a procoagulant from Echis carinatus venom

Abstract The ability of a procoagulant fraction from the venom of the saw-scaled viper ( Echis carinatus sochureki ) to convert prothrombin to thrombin in the absence of Ca2+ or other cofactors was used as the basis of a sensitive test method for prothrombin. Incubation of a test sample with prothrombin-free human fibrinogen and the procoagulant fraction resulted in clotting if prothrombin was present in the test sample; clotting time was a function of the amount of prothrombin over the range 0.02 – 1.0 unit. The test method appears valuable for detecting and quantitating low levels of prothrombin contamination in therapeutic products and in laboratory preparations of coagulation factors.

Human prothrombin fragments FI(αβ) and F2: Preparation and characterization of structural and biological properties

Abstract Human prothrombin fragments F1(αβ) and F2 were isolated as by-products of α-thrombin preparations. Prothrombin concentrates were activated to > 90% completion with thromboplastin (30–60 min, 23° C). The generated thrombin was quantitatively removed from clarified activation mixtures with Amberlite CG-50 resin (polymethylacrylic acid). The nonadsorbed fraction, containing the activation fragments, was desalted, freeze-dried, and chromatographed on DE-23 cellulose (diethylaminoethyl cellulose) employing an increasing salt gradient. Two pools containing F1(αβ) and F2 were obtained and further purified by gel filtration through Sephadex G-100. Fragment identity and purity were determined by electrophoresis in sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. The two isolated fractions migrated electrophoretically in SDS-containing gels as distinct nonreduced proteins ( M r 27,600 ± 1,300 and 15,800 ± 700 for F1(αβ) and F2, respectively). Following reduction, F2 remained electrophoretically homogeneous ( M r 15,800 ± 500). The reduced F1(αβ) gave rise to predominantly an M r 22,800 component and low M r materials (intact F1 was detected in some F1(αβ) preparations). Homogeneity of F2 was confirmed by detecting only an NH2-terminal serine and a CO2H-terminal arginine (1.1 mol. Arg/mol. F2 released by carboxypeptidase B digestion). Secondary cleavages within F1(αβ) were demonstrated by identifying threonine (0.44 mol. Thr/mol. Ala), and aspartic acid (0.15 mol. Asp/mol. Ala) as NH2 terminal residues, and by obtaining arginine (0.6 mol. Arg/mol. FL(αβ) and trace amounts of lysine as the CO2H-terminal residues). Sequential NH2-terminal degradations (8 cycles) confirmed the deleted glycine at position-4 in human prothrombin and permitted identification of the internal F1 cleavage sites at Arg-52/Thr-53 and secondarily at Arg-55/Asp-56 (bovine prothrombin numbering). Since both cleavage sites are located within the same disulfide loops (Cys-48 to Cys-61), they account for the electrophoretic behavior of nonreduced versus reduced F1(αβ). Neither F1 (αβ) nor F2 exhibited biological activities similar to complement C3 or C5 fragments when examined in guinea pig ileal, skin permeability, or chemotaxis assays. The prothrombin fragments further did not promote cellular proliferation of cultured embryo fibroblasts. These findings suggest that the prothrombin fragments F1(αβ) and F2 are biologically inactive peptides except for their possible role of interfering with prothrombin activation.

The use of prothrombin activating snake venoms to measure human prethrombin 2: Absence of prethrombin 2 in serum

The activation of the prothrombin intermediate, Prethrombin 2, has been studied in order to establish test systems that would enable identification of Prethrombin 2 in serum and Factor IX concentrates. While activation of Prethrombin 2 by Taipan Snake Venom (TSV) was slow and incomplete, inclusion of approximately molar amounts of prothrombin fragments F1 or F1.2 markedly enhanced the amount of thrombin formed by TSV. This effect could also be obtained by the inclusion of serum. Neither normal serum nor Factor V deficient serum contain any identifiable Prethrombin 2. On the other hand substantial amounts of Prethrombin 2 are present in Factor IX concentrates used for the treatment of Christmas Disease (Hemophilia B).

The purification of human acarboxy prothrombin. Characterization of its derivatives after thrombin cleavage

Abstract After administration of vitamin K antagonists, the liver is able to give rise to a prothrombin molecule which is different from normal prothrombin. This abnormal protein has been isolated using plasma derived from patients under either nicoumalone or ethylbiscoumacetate therapy, and designated human acarboxy prothrombin. After purification, the electrophoretic and immunological characteristics of this protein were studied before and after cleavage by human purified thrombin. The methods used were sodium dodecyl sulfate gel electrophoresis, disc gel electrophoresis at alkaline pH and Ouchterlony double diffusion technique. The results indicate that human acarboxy prothrombin has apparently the same molecular weight as normal prothrombin but has a slower electrophoretic mobility at alkaline pH. Analysis of the fragments obtained by thrombin cleavage shows that Prethrombin 1 derived from normal or acarboxy prothrombin have the same molecular weight and the same electric charge. Fragment 1 derived from acarboxy prothrombin has the same molecular weight as normal Fragment 1 but differs by its slower mobility at alkaline pH and by the lack of some antigenic determinants as demonstrated by the use of both an antiserum specific for normal prothrombin and an antiserum specific for normal Fragment 1.

Heterogeneity of factor IX in therapeutic factor IX concentrates

Abstract Rabbit antibody to human factor IX was used to investigate the factor IX antigenic content and electrophoretic mobility of commercial products as well as experimental “activated products”. Rocket immunoelectrophoresis of all concentrates showed a 1.2–3 fold increased antigenic content/unit factor IX clotting activity when compared to plasma. Two dimensional crossed immunoelectrophoresis of standard Factor IX concentrates produced a single sharp peak whether electrophoresed in Ca2+ or EDTA containing buffer. “Activated” concentrates produced a dome shaped precipitin arc. The addition of plasma to standard factor IX concentrates yielded a marked shoulder only when the electrophoresis was run in EDTA. This effect could not be reproduced by the addition of antithrombin III (AT-III). The addition of plasma to some “activated” products revealed an even more pronounced heterogeneity whether in Ca2+ or EDTA and the addition of AT-III in the same products produced a second precipitin peak. These results indicate that at least three forms of factor IX exist in Factor IX concentrates. The absence of detectable AT-III reacting material in the standard concentrates is a priori evidence of the absence of major amounts of IXa, whereas the presence of AT-III reacting material in the “activated” concentrates is evidence of biologically active material.