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A 2.2-kilobase human antithrombin III (ATIII) "minigene" has been constructed which preserves many of the features of the 16-kilobase parental gene. These include approximately 300 base pairs of the 5'-flanking region containing the "TATA"-less ATIII promoter, the entire protein-coding sequence including the signal peptide, a single intervening sequence with intact donor and acceptor splice sites, and a polyadenylation signal. Unique PstI termini allow the minigene to be cloned in many commonly used plasmid vectors. The minigene has been reintroduced into heterologous cells by calcium phosphate precipitation. RNA is transcribed with heterogeneous 5'-ends and is processed by splicing either to the normal acceptor site or to an alternate site within the intervening sequence. The alternately spliced mRNA is also found in liver where it accounts for 20-40% of ATIII-specific transcripts. The alternate splicing event introduces a 42-nucleotide segment between codons -19 and -18 of the signal peptide. This segment contains an in-frame ochre termination codon such that the predicted protein product encoded by the alternately spliced mRNA would be only 19 amino acids long. The minigene is transcribed to high levels in mouse fibroblasts when present as an episome in a bovine papilloma virus-derived vector although no protein product is detected.

Animal mitochondrial DNA contains genes for 13 potential polypeptides of significant size. Five of these genes have been assigned to distinct proteins and eight remained unassigned reading frames (URFs). Short peptides corresponding to URF protein sequences were synthesized chemically. Antibodies raised to these synthetic peptides were used to establish the existence of all eight URF proteins in mouse tissues and cells by the complementary techniques of immunoperoxidase staining, protein blotting and immunoprecipitation. Immunoperoxidase staining of thin-sectioned, freeze-substituted tissue may prove generally useful for the identification of gene products for which no formal genetic data exist. Furthermore, the ability to determine the cellular and tissue distribution of such proteins may provide the first insight into their function.

A colorimetric assay for plasma antithrombin III (ATIII) has been developed, using a new synthetic peptide substrate, PS-915 (H-D-Phe-Pro-Arg-CHA). PS-915 is freely water-soluble and its aqueous solution is stable. It is highly specific for thrombin and its Km value is about the same as that of S-2238. By enzyme hydrolysis, the new substrate liberates 3-carboxy-4-hydroxyaniline (CHA), which turns blue in color due to the complex formation with added alkaline-pentacyanoammine ferroate. The assay is a three-stage kinetic, one-point procedure, based on the method of antithrombin (heparin cofactor activity) determination. Since absorption measurements of the CHA are usually obtained at 700 nm, no blank correction is necessary even when hyperbilirubinemic, lipemic and/or hemolyzed plasma are used. The calibration curve for ATIII is linear over the range of 0-175% of normal and has a high degree of reproducibility. The results obtained by this method are well correlated with data obtained by the commonly used chromogenic assay or by radial immunodiffusion.

We have studied vessel wall function in two groups of patients with chronic renal failure - 1) conservative treatment only and 2) maintenance hemodialysis. Three proteins synthesized by vascular endothelium-plasminogen activator (PA), factor VIII related antigen (VIII:RAg) and antithrombin III (ATIII) - were assayed before and after a fifteen minute period of venous occlusion. The release of PA was significantly reduced in patients on maintenance hemodialysis as compared to both undialyzed uremics and controls. Lesser amounts of VIII:RAg were also released by hemodialysis patients than by undialyzed uremics. These defects, which are suggestive of vessel wall dysfunction on maintenance hemodialysis, may contribute to the high incidence of arteriopathy and thrombotic disease observed in this group of patients.

Human alpha-thrombin was poorly immunogenic in Balb/c mice. Nevertheless, following fusion of spleen cells from a responding mouse with NS-1 cells, 8 mouse monoclonal antibodies against alpha-thrombin were isolated, and 6 were characterised. Five of these were isotype IgG2a, and one was IgG1. One, EST 1, bound thrombin only minimally, and was directed against a neoantigen on the thrombin-ATIII (T-AT) complex. This antibody also recognised a site on prothrombin, though with much lower affinity. Its binding was markedly temperature-dependent, indicating a requirement for molecular mobility. A second antibody, EST 4, would not bind the T-AT complex. It inhibited both the clotting and amidase activities of thrombin, and modification of the active site histidine, but not the active site serine, reduced the affinity constant of binding to EST 4. This antibody appears to be directed against an epitope in the vicinity of the enzyme active site. The epitopes for EST 1 and EST 4 were both remote from those of the other monoclonal antibodies, EST 2, 6, 7 and 8. These four competed with each other for binding to thrombin, and all inhibited clotting but not amidase activity. Thrombin binding was not affected by modification of the active site, though formation of the T-AT complex reduced the affinity of binding to EST 6 and EST 8. These monoclonals recognise epitopes in the region of the fibrinogen binding site.

The changes in antithrombin III in 31 patients who underwent open-heart surgery were measured during the period of extracorporeal circulation and afterwards over a total period of 24 hours by means of a chromogenic substrate, as well as by laser nephelometry. Both the actual changes in antithrombin III, as well as the theoretical changes calculated by eliminating the effect of haemodilution are discussed. The functional test indicates that the loss of antithrombin-III activity, in general, is due to the effect of haemodilution. However, marked individual differences were noted in the reaction pattern. The results of nephelometric measurements were inconsistent and seemed to indicate that routine procedures in laser nephelometry are hardly suitable for the purpose of comparison of samples with marked haematocrit differences.

We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the coagulation system at concentrations considerably less than 1.0 nM.