Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase.

hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln-. A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases. ch-hK4 was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified with an overall yield of 25%. The zymogen was readily self-activated during the refolding process to generate an active form (21 kDa) of hK4 (rhK4). rhK4 cleaved the chromogenic substrates Val-Leu-Arg-pNA (S-2266), Pro-Phe-Arg-pNA (S-2302), Ile-Glu-Gly-Arg-pNA (S-2222), and Val-Leu-Lys-pNA (S-2251), indicating that rhK4 has a trypsin-type substrate specificity. The rhK4 was inhibited by aprotinin (6 kDa), forming an equimolar 27 kDa complex. rhK4 readily activated both the precursor of PSA (pro-PSA) and single chain urokinase-type plasminogen activator (scuPA, pro-uPA). rhK4 also completely degraded prostatic acid phosphatase but failed to cleave serum albumin, another protein purified from human seminal plasma. These results indicate that hK4 may have a role in the physiologic processing of seminal plasma proteins such as pro-PSA, as well as in the pathogenesis of prostate cancer through its activation of pro-uPA.

The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites.

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.

Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving protease (VWFCP). Based on partial amino acid sequence, VWFCP was identified recently as a new member of the ADAMTS family of metalloproteases and designated ADAMTS13. The 4.6-kilobase pair cDNA sequence for VWFCP has now been determined. By Northern blotting, full-length VWFCP mRNA was detected only in liver. VWFCP consists of 1427 amino acid residues and has a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat, a Cys-rich domain, an ADAMTS spacer, seven additional thrombospondin-1 repeats, and two CUB domains. VWFCP apparently is made as a zymogen that requires proteolytic activation, possibly by furin intracellularly. Sites for Zn(2+) and Ca(2+) ions are conserved in the protease domain. The Cys-rich domain contains an RGDS sequence that could mediate integrin-dependent binding to platelets or other cells. Alternative splicing gives rise to at least seven potential variants that truncate the protein at different positions after the protease domain. Alternative splicing may have functional significance, producing proteins with distinct abilities to interact with cofactors, connective tissue, platelets, and von Willebrand factor.

Activation of prostate-specific antigen precursor (pro-PSA) by prostin, a novel human prostatic serine protease identified by degenerate PCR.

A novel serine protease was found in human prostate by degenerate oligonucleotide PCR amplification and cloned. The zymogen form of this enzyme, named prostinogen, is composed of 240 amino acid residues with an amino-terminal propiece of 5 residues and a 235-residue mature enzyme. The transcript has a signal peptide of 15 amino acid residues. The mature enzyme has 41% sequence identity with prostate specific antigen (PSA). Prostinogen was expressed in Escherichia coli and refolded from inclusion bodies. The zymogen, with a molecular mass of 28 kDa, was readily activated by agarose-immobilized trypsin to generate prostin, a serine protease, which cleaves the chromogenic substrate (N-benzoyl-L-Ile-L-Glu-L-Gly-L-Arg-p-nitroaniline hydrochloride) (S-2222). Recombinant prostin readily activates the precursor of PSA (pro-PSA) by cleavage of the amino terminal Arg(7)-Ile(8) peptide bond. These results indicate that prostin may be a physiological activator of pro-PSA following its own proteolytic activation, as part of a cascade system involving a series of serine protease precursor proteins in the prostate.

Traumatic posterior urethral injury and early realignment using magnetic urethral catheters.

PURPOSE: We determined the success of early urethral realignment using magnetic urethral catheters. MATERIALS AND METHODS: We retrospectively reviewed the records of 13 patients with complete urethral disruption treated with endourological realignment 0 to 11 days after injury using coaxial magnetic urethral catheters. RESULTS: Urethral realignment was established in 11 of the 13 patients (85%) using magnetic urethral catheters. Of the 10 patients for whom followup was available urethral strictures developed in 5 (50%) a mean of 6.1 months after realignment, necessitating a mean of 1.4 corrective procedures per patient. Impotence was noted in 1 of 7 patients (14%) and no urinary incontinence developed after realignment. CONCLUSIONS: Urethral realignment within 2 weeks of injury using magnetic urethral catheters is a safe and simple technique with minimal morbidity. The stricture formation, impotence and incontinence rates of this technique are comparable to those reported for delayed urethroplasty. We advocate early realignment using magnetic urethral sounds as an alternative treatment for traumatic urethral disruption.

Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein.

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.

Radiation therapy for local recurrence of prostate cancer after radical prostatectomy.

A definitive conclusion about the value of ART is not possible from the data available: Both the methods of radiation therapy and the techniques in the diagnosis of locally persistent disease have evolved over the years. Currently, the data lead to the conclusion that ART decreases local recurrence but does not improve overall survival. Yet the PSA data strongly suggest that only locally persistent disease is a common event after radical prostatectomy (particularly in margin-positive disease only) and that current ART techniques are inadequate in many but not all of these patients. Certainly some men seem to have their local disease eliminated by ART to remain NED, but it is unclear exactly how to select them. Many experts also believe that keeping the PSA as low as possible for as long as possible, with sequential applications of ART and then androgen ablation as necessary, is a good emotional if not medical strategy. For example, all patients now wish to know their PSA level and worry about it. Also, potency can be maintained or regained after ART but becomes improbable after androgen ablation because of loss of libido. Clearly, a study randomizing high-risk postoperative patients into observation versus ART is needed and indeed such a study is under way in the Southwestern and Eastern Cooperative oncology groups, but to date accrual is inadequate (Ian Thompson, Jr, MD, personal communication, May 1994). This study must be supported. When participation in the randomized study is not possible, we believe four tentative recommendations about the application of ART can be made based on the available data (Fig. 1): (1) For high-risk patients (e.g., high Gleason score and/or high pathologic stage) with initially undetectable PSA levels, we recommend instituting ART before any rise in postoperative PSA levels because low-volume disease may best respond to this therapy. (2) For patients with rapidly rising or initially detectable postoperative PSA levels (especially if NBA is negative), we believe that the disease has most likely already spread to distant sites and would initiate therapy aimed at systemic disease. (3) For those patients with rapidly rising postoperative PSA but with positive NBA, we recommend local irradiation. (4) if the postoperative PSAlevels rise gradually, we would initiate ART regardless of the needle biopsy result because of the possibility of NBA sampling error and the fact that the gradual increase in PSA suggests that the disease is still local.(ABSTRACT TRUNCATED AT 400 WORDS)

Newer applications of serum prostate-specific antigen in the management of prostate cancer.

The information contained in this article indicates that PSA will have an increasing role in the management of prostate cancer. For example, it is now essential for optimal diagnosis if prostate cancer detection is the goal. Prospects are high that more information about PSA density relationships, PSA velocity phenomenologies, and possible PSA isoforms will increase diagnostic accuracy. It would also seem that PSA will improve staging accuracy not only by better manipulation of multiple preoperative parameters (eg, cancer grade, volume, PSA, etc) but possibly by the molecular detection of minute amounts of occult prostate cancer cells in bone, blood or lymph nodes, or by improved use of immune scanning. Finally, the use of these more sophisticated staging approaches together with increasingly sensitive PSA assays and possibly androgen provocative testing might allow the prospect that the potentially curative therapies can be almost immediately assessed for efficacy, thereby increasing prospects for therapeutic progress. Finally, PSA may become even more important for manipulating hormone therapies (eg, IAS therapy) or it could form a basis for new treatments such as immune or gene therapy.

Urinary prostate specific antigen levels after radical prostatectomy.

It was recently demonstrated that urinary prostate specific antigen (PSA) is discordant with serum PSA in many patients after radical prostatectomy. This observation led to the speculation that elevated urinary PSA in the face of undetectable serum PSA may indicate early disease recurrence. We measured urinary PSA levels in 30 patients who had undergone radical prostatectomy for prostate carcinoma and 7 patients who had undergone cystoprostatectomy for bladder cancer. PSA levels of randomly collected urine samples ranged from 0.00 to 22.9 ng./ml. and 0.01 to 8.37 ng./ml., respectively. There was no correlation among urinary and serum PSA levels, pathological stage or type of operation. In 14 patients who had undergone radical prostatectomy and who had measurable levels of urinary PSA voided specimens were divided into initial stream and end stream voided samples. The PSA levels in the end stream voided samples were significantly less than the initial stream sample in 12 of the 14 patients. In men who had undergone radical prostatectomy urethral swab samples were analyzed for PSA. Of 26 patients 24 had detectable levels of urethral swab PSA (range 0.01 to 39.04 ng./ml., median 0.93 ng./ml.). Urethral swab PSA levels did not correlate with serum PSA values or pathological stage of disease. Of 7 patients who had defunctionalized urethras after radical cystoprostatectomy 5 had significantly elevated PSA in the urethral wash or swab samples (range 4.3 to 24.5 ng./ml.). Immunohistochemical analysis of urethrectomy specimens demonstrated positive staining for PSA in 3 of 4 specimens. We conclude that the major source of urinary PSA following total prostatectomy is the urethra itself rather than residual prostate tissue. Measuring serial urinary PSA appears to have limited value in monitoring patients after radical prostatectomy. Whether this urethral PSA can ever contaminate the serum levels of PSA after radical prostatectomy is currently under investigation.

The enhanced detection of persistent disease after prostatectomy with a new prostate specific antigen immunoassay.

Prostate specific antigen (PSA) determinations after radical prostatectomy are valuable in detecting persistent disease. Previously, we determined that 0.4 ng./ml. PSA was a reliable clinical threshold using the Hybritech Tandem-R PSA assay. Recently, we reported that a new PSA immunoassay (Abbott IMx PSA) correlated well with results of the Tandem-R immunoradiometric PSA assay and had a lower threshold. Using a conservative threshold of 0.1 ng./ml. PSA for the IMx PSA assay, we analyzed IMx PSA values in serial postoperative serum from 72 radical prostatectomy patients whose initial Tandem-R levels were less than 0.4 ng./ml. PSA. The lower detection limits of the IMx PSA assay allowed approximately a third (15 of 42) more detection of persistent disease within 8 months of surgery. When the PSA level remained undetectable for more than 8 months but the disease eventually recurred the lead times averaged 9 to 12 months when 0.1 ng./ml. PSA was used to signify persistent disease. All patients whose PSA levels reached 0.1 ng./ml. PSA and were subsequently followed for more than 3 months continued to have increasing levels. Also, every man who eventually had recurrence also had a PSA serum level of at least 0.1 ng./ml. PSA within 28 months postoperatively, although the subsequent increase from 0.1 to 0.4 ng./ml. PSA sometimes took several years. Although the clinical impact of these findings is yet unknown, new or altered PSA assays with lower detection limits can provide unique information that may offer opportunities for improved clinical investigation and possibly patient management.

A brief review of ultrasensitive prostate-specific antigen assays for the evaluation of patients after radical prostatectomy.

The development of immunoassays for prostate-specific antigen (PSA) and their clinical utility are summarized. Because of the complexity of the PSA molecule and anti-PSA antibodies, there is currently no standard in PSA measurement. Evaluating various immunoassays requires the knowledge of the lower limit of detection as well as the biological and clinical thresholds of a given assay. There have been recent reports demonstrating earlier detection of residual prostate cancer after radical prostatectomy by ultrasensitive assays for PSA. Because of the recent evidence for non-prostatic sources of PSA such as the male urethra, the possibility of their contaminating PSA levels must be evaluated when more sensitive assays for PSA are under consideration.

Recurrent prostate cancer despite undetectable prostate specific antigen.

Serum prostate specific antigen (PSA) is a sensitive tumor marker following definitive therapy for adenocarcinoma of the prostate. Detectable levels of PSA after radical prostatectomy indicate residual and/or recurrent cancer. Therefore, we were surprised to document recurrent cancer following radical retropubic prostatectomy in a man who had undetectable serum PSA levels. This observation suggests that careful clinical monitoring remains essential after definitive therapy, since rare patients may have recurrent and/or residual carcinoma despite undetectable levels of PSA.

Elevated tumor necrosis factor alpha production concomitant to elevated prostaglandin E2 production by trauma patients' monocytes.

The level of tumor necrosis factor alpha (TNF alpha), a monokine implicated in mediating septic shock, is elevated in the blood of some patients with sepsis. Monocytes from 11 trauma patients and 11 burn patients were suboptimally stimulated with interferon gamma and muramyl dipeptide, an analogue of bacterial wall products. The patients with sepsis showed significantly greater total TNF alpha levels (secreted in combination with cell-associated) 3 days before septic episodes, as compared with normal controls (32.38 to 2231.76 ng/10(6) monocytes per milliliter, median = 121.03 ng/10(6) monocytes per milliliter; normal control: 0.00 to 18.20 ng/10(6) monocytes per milliliter, median = 5.93 ng/10(6) monocytes per milliliter). Increases in patients' total monocyte TNF alpha levels greater than 30 ng/10(6) monocytes per milliliter correlated with septic episodes. In patients with sepsis, the total monocyte TNF alpha levels were increased despite a concomitant increase in their prostaglandin E2 levels in both stimulated (interferon gamma plus muramyl dipeptide) and unstimulated in vitro assays (9 patients: stimulated prostaglandin E2 range, 30.1 to 123.6 ng/10(6) monocytes per milliliter). Massively elevated monocyte TNF alpha and prostaglandin E2 production occurred simultaneously in patients with sepsis.