David L. Enfield

Clinical evaluation of the BTA trak assay and comparison to voided urine cytology and the bard BTA test in patients with recurrent bladder tumors

OBJECTIVES To assess the clinical performance of the BTA TRAK assay and to compare it with that of voided urine cytology (VUC) and the Bard BTA test (BTA) in the detection of recurrent bladder cancer (BC). METHODS The study was performed on randomly selected archival voided urine samples for many of which VUC and/or BTA information was available. Sensitivity was determined in samples from patients with histologically confirmed recurrent BC. Specificity was determined in samples from healthy volunteers, patients with three categories of current medical conditions, and patients with a history of BC but no current evidence of disease. RESULTS The TRAK assay was positive in 156 of 216 samples for patients diagnosed with BC, for an overall sensitivity of 72%. Mean values increased with progressing grade and stage of disease. In the comparison between TRAK and VUC, the overall sensitivities were 68% and 25%, respectively (P < 0.001). For Stages Ta and T1 and for all tumor grades, the sensitivity of the TRAK assay was significantly greater than that of VUC (P < 0.001). TRAK sensitivity was also significantly better than that of BTA (73% versus 58%, P = 0.005). The specificity of the TRAK assay ranged from 75% in samples from patients with genitourinary disease to 97% in healthy volunteers. CONCLUSIONS The TRAK assay is superior to VUC and the original BTA test in the detection of BC. The results of the study indicate that the TRAK assay may be a useful adjunct to cystoscopy in the management of patients with recurrent BC.

Bovine factor IX (christmas factor). Further evidence of homology with factor X (Stuart factor) and prothrombin

Factor IX is a single-chain glycoprotein involved in the intrinsic pathway of blood coagulation. The molecular characteristics of factor IX and the mechanism of its activation by factor XI, (activated factor XI) have recently been described [ 1,2] . In the presence of factor VIII, calcium, and phospholipid, factor IX, catalyzes the conversion of factor X to X,. Factor X, in the presence of factor V, calcium, and phospholipid in turn converts prothrombin to thrombin. Factor X, and thrombin are both inactivated by diisopropylphosphorofluoridate (DFP), but the same has not been established for factor IX,. Factor X, and thrombin are homologous with other mammalian serine proteases [3]. The amino terminal regions of factor IX, prothrombin, and the light chain of factor X are also homologous, but factor IX is immunologically distinct [4]. Nevertheless, evidence is presented here showing that considerable homology exists between bovine factors IX and X and prothrombin, suggesting that factor IX, has evolved from the same precursor and therefore is probably also a serine protease.

Cleavage and inactivation of Factor IX by granulocyte elastase.

Radioiodinated Factor IX was cleaved by a crude sonicate from leukocytes. In the absence of calcium, fragments of less than 15,000 mol wt were seen from reduced samples on gel electrophoresis. After digestion in 2 mM calcium, however, electrophoresis of reduced samples showed, in addition to low molecular weight fragments, protein bands corresponding in size to heavy and light chains of Factor XIa-activated Factor IX. The cleaving activity in leukocyte sonicates was inhibited by soybean trypsin inhibitor, but only to a small extent by aprotinin. Granulocyte elastase was isolated from purified polymorphonuclear leukocyte granules by affinity chromatography on soybean trypsin inhibitor-agarose and further chromatography on carboxymethyl cellulose. The purified fraction contained two isozymes on acidic gels which cleaved both an ester sensitive to elastase and radiolabeled Factor IX. These two activities were inhibited by elastase-specific chloromethyl ketone. The isolated protease fraction rapidly inactivated apparent Factor IX activity in a coagulant assay system. The degree of inactivation correlated with the amount of intact, radiolabeled Factor IX cleaved. As with the crude sonicate, generation of the larger heavy and light chain-sized fragments was dependent upon calcium. To assess directly the effect of elastase on Factor IX, an immunospecific, active site-directed assay was developed. In this assay, the sample was incubated with solid-phase antibody to Factor IX and the amount of activated product was detected as that which had complexed with radioiodinated antithrombin III. In this system, exposure of Factor IX to Factor XIa showed progressive increase in the ability to bind antithrombin III, whereas after elastase, Factor XIa was unable to generate antithrombin III binding. The elastase-degraded Factor IX did not inhibit activation of additional Factor IX in clotting assays. When Factor IXa was incubated with elastase, binding of antithrombin III was decreased, corresponding to appearance of low molecular weight fragments on parallel samples that were reduced and electrophoresed. These data are consistent with elastase inactivating Factor IX by cleaving bonds near, but distinct from, bonds cleaved by Factor XIa.

The amino-terminal sequence of an invertebrate trypsin (crayfish Astacus leptodactylus): Homology with other serine proteases

The serine proteases represent one of the best characterized families of proteins which have evolved from a common ancestor [ 11. They include such functionally diverse and distinct enzymes as the digestive proteases of the pancreas, trypsin, chymotrypsin and elastase, and the hepatic proteases of the blood coagulation system, thrombin, factor X, and factor IX, [2]. Plasmin [ 1) and kallikrein [3] also belong to this family. Among these enzymes, the trypsins, defined by common features of their active site, seem to have been preserved during the whole span of evolution ranging from bacteria to mammals. The complete covalent structures of three vertebrate trypsins, i.e., bovine [4], porcine [S] and dogfish [6] have been reported and structural details of several other vertebrate trypsins are known in part [4]. In contrast, little information is available on trypsins of invertebrate animals despite the fact that nine of ten phyla of the animal kingdom are represented by invertebrates. A proteolytic enzyme which exhibits tryptic specificity toward peptides and synthetic substrates [7,8] was found in the invertebrate crayfish Astacus leptoductylus and was later identified as a serine protease [9]. In contrast to bovine trypsin, the crayfish trypsin is a rather acidic protein which is irreversibly inactivated at low pH [8]. The molecular weight, calculated from the amino acid composition is 24 000 [lo] and in contrast to the vertebrate trypsins crayfish trypsin contains 6 rather than 12 half-cystine residues. The en-