Histopathological concordance between prostate biopsies and radical prostatectomy specimens-implications of transrectal and transperineal biopsy approaches.

BACKGROUND: This study aimed to evaluate the histopathological concordance rates between prostate biopsies and radical prostatectomy specimens according to the applied biopsy approach (transrectal or transperineal). METHODS: We studied patients who had been newly diagnosed with clinically significant prostate cancer and who underwent a radical prostatectomy between 2018 and 2022. Patients were included if they underwent a prebiopsy magnetic resonance imaging and if they had not been previously treated for prostate cancer. Histopathological grading on prostate biopsies was compared with that on radical prostatectomy specimens. Univariable and multivariable logistic regression analyses were performed to assess the effect of the applied biopsy approach on histopathological concordance. Additional analyses were performed to assess the effect of the applied biopsy approach on American Urological Association risk group migration, defined as any change in risk group after radical prostatectomy. RESULTS: In total, 1058 men were studied, of whom 49.3% (522/1058) and 50.7% (536/1058) underwent transrectal and transperineal prostate biopsies, respectively. Histopathological disconcordance was observed in 37.8% (400/1058) of men while American Urological Association risk group migration was observed in 30.2% (320/1058) of men. A transperineal biopsy approach was found to be independently associated with higher histopathological concordance rates (OR 1.33 [95% CI 1.01-1.75], p = 0.04) and less American Urological Association risk group migration (OR 0.70 [95% CI 0.52-0.93], p = 0.01). CONCLUSIONS: The use of a transperineal biopsy approach improved histopathological concordance rates compared to the use of a transrectal biopsy approach. A transperineal biopsy approach may provide more accurate risk stratification for clinical decision-making. Despite recent improvements, histopathologic concordance remains suboptimal and should be considered before initiating management.

Patient-reported outcome measures compared to clinician reported outcomes regarding incontinence and erectile dysfunction in localized prostate carcinoma after robot assisted radical prostatectomy: Impact on management.

PURPOSE/ BACKGROUND: Patient-reported outcome measures (PROMs) are widely used after robot assisted radical prostatectomy (RARP) in order to evaluate the impact/burden of the treatment. The most bothersome side effects of RARP are urine incontinence (UI) and erectile dysfunction (ED). During the follow up consultations, clinicians report these side effects in interviewing patients. Our study examined the discrepancy between the PROMs and clinician report outcomes (CROs) and hypothesized that the disagreement could have an impact on the management of UI and ED. METHODS: Up to 1 year after RARP, UI and ED recovery of 312 men with localized and locally advanced prostate cancer were assessed using the International Consultation Incontinence Questionnaire Short-Form (ICIQ-SF) and the International Index of Erectile Function (IIEF-EF) and CROs by interview. Discrepancies between PROs and CROs were studied in light of treatment offered and management. RESULTS: The ICIQ-SF Score matched with CROs in all sum score categories except in ICIQ sum score 6 to 12; here the UI was underreported by clinicians in 58% and 59% of patients at 8 and 12 months (P < 0.001). Furthermore, at 8 and 12 months postoperatively, clinicians underreported UI in 29% and 23% of patients with ICIQ score 13-18 (P < 0.001). The clinician significantly over-reported the recovery of erectile function ("normal erection") (P < 0.001), especially in men with IIEF-EF sum score 6 to 16. Independently of ICIQ-SF/IIEF-EF scores, discrepancy between PROs and CROs did not affect rate of health care offered to patients. CONCLUSIONS: This is to our knowledge the first study that compared the PROs with clinician reported functional outcomes and the impact of discrepancies on the management of side effects of RARP in prostate cancer. Observed discrepancies between the PROs and CROs did not affect offered management and counseling of UI and ED.

Functional outcomes rather than complications predict poor health-related quality of life at 6 months after robot-assisted radical prostatectomy.

The objective is to evaluate the effect of robot-assisted radical prostatectomy (RARP)-related postoperative complications on the 6-month postoperative health-related quality of life (HRQoL). A total of 1008 patients underwent a RARP with or without pelvic lymph node dissection (PLND) between 2012 and 2020 and were invited to complete questionnaires about HRQoL and functional outcomes (urinary incontinence (UI), erectile dysfunction (ED) and urinary complaints (UC)) before and 6 months after RARP. Patient characteristics and postoperative complications up to 90 days after surgery were prospectively recorded. Associations between complications and HRQoL/functional outcomes were assessed by multivariate linear regression analyses. In total, 528 patients (52.4%) were included in the analyses. Complications occurred in 165/528 (31.3%) patients, of which 30/165 (18.2%) had a Clavien-Dindo ≥ III complication. In multivariate regression analyses, postoperative complications were not significantly associated with postoperative HRQoL, UI and ED (p = 0.73, p = 0.72 and p = 0.95, respectively), but were significantly associated with a minor increase in UC (ß = 1.7, p < 0.001). More specifically, infectious and urological complications were significantly associated with an increase in UC (ß = 1.9, p < 0.001 and ß = 0.9, p = 0.004, respectively). The presence of UTI, in particular, was significantly associated with this minor increase (ß = 1.5, p = 0.002). Functional outcomes were all significantly associated with the HRQoL at 6 months postoperatively. No significant associations were found between postoperative complications and HRQoL at 6 months after RARP. However, worse functional outcomes were associated with a worse HRQoL at 6 months postoperatively. In addition, postoperative infectious and urological complications were significantly associated with a minor increase in UC.

Functional Outcomes and Urological Complications after Genital Gender Affirming Surgery with Urethral Lengthening in Transgender Men.

PURPOSE: We determined the urological complications and lower urinary tract function after genital gender affirming surgery with urethral lengthening in transgender men. MATERIALS AND METHODS: A single center, retrospective cohort study was performed from January 2013 to January 2018. Patient demographics, medical history, perioperative data, surgical and urological complications, and preoperative and postoperative urological outcomes were obtained. RESULTS: Of the 63 patients included in the study 8 (13%) underwent metoidioplasty and 55 (87%) phalloplasty, comprised of 27 (43%) free radial forearm flap, 19 (30%) anterolateral thigh flap and 9 (14%) superficial circumflex iliac artery perforator flap surgeries. In phalloplasty the types of urethral lengthening were tube-in-tube free radial forearm flap in 27 (49%), free radial forearm flap (second fasciocutaneous flap) in 18 (33%), superficial circumflex iliac artery perforator flap in 5 (9%) or labial in 5 (9%). Mean followup was 23 months (range 12 to 71). Stricture formation occurred in 35 (63%) phalloplasty and 5 (63%) metoidioplasty cases. Urethral fistula formation occurred in 15 (27%) phalloplasty and 4 (50%) metoidioplasty cases. Mean time to strictures and fistulas was approximately 3 months. Overall 46 (73%) patients needed revision surgery because of fistulas/strictures. After treatment 44 (70%) patients were able to void from the tip of the phallus. No clinically relevant differences in International Prostate Symptom Scores, frequency volume charts and uroflowmetry were found preoperatively vs postoperatively. CONCLUSIONS: Genital gender affirming surgery with urethral lengthening is a complex procedure with a high complication rate. After treating complications no clinically relevant differences in urological functioning were recorded. The majority of transgender men could void from the tip of the penis and showed favorable urological outcomes.

Analysis of current perioperative management with Haemate® P/Humate P® in von Willebrand disease: Identifying the need for personalized treatment.

INTRODUCTION: Patients with Von Willebrand disease (VWD) are regularly treated with VWF-containing concentrates in case of acute bleeding, trauma and dental or surgical procedures. AIM: In this multicentre retrospective study, current perioperative management with a von Willebrand factor (VWF)/Factor VIII (FVIII) concentrate (Haemate® P) in patients with VWD was evaluated. PATIENTS/METHODS: Patients with VWD undergoing minor or major surgery between 2000 and 2015, requiring treatment with a VWF/FVIII concentrate (Haemate® P), were included. Achieved VWF activity (VWF:Act) and FVIII during FVIII-based treatment regimens were compared to predefined target levels in national guidelines. RESULTS: In total, 103 patients with VWD (148 surgeries) were included: 54 type 1 (73 surgeries), 43 type 2 (67 surgeries) and 6 type 3 (8 surgeries). Overall, treatment resulted in high VWF:Act and FVIII levels, defined as ≥0.20 IU/mL above predefined levels. In patients with type 1 VWD, respectively, 65% and 91% of trough VWF:Act and FVIII levels were higher than target levels. In patients with type 2 and type 3 VWD, respectively, 53% and 57% of trough VWF:Act and 72% and 73% of trough FVIII levels were higher than target level. Furthermore, FVIII accumulation over time was observed, while VWF:Act showed a declining trend, leading to significantly higher levels of FVIII than VWF:Act. CONCLUSION: High VWF:Act and accumulation of FVIII were observed after perioperative FVIII-based replacement therapy in patients with VWD, both underlining the necessity of personalization of dosing regimens to optimize perioperative treatment.

Psychological and speech studies in Rubinstein-Taybi syndrome.

Forty individuals with Rubinstein-Taybi syndrome were tested using an extensive test battery to obtain more insight about their intelligence level, social competency, temperament, and behavior as well as articulation and receptive and expressive language level. Examination of individuals in the Netherlands who have this rare syndrome has been extensive, allowing some generalizations to be made. The intelligence level of affected individuals is usually low, although some persons have much higher scores than do others. The tested individuals were remarkably consistent in their social competency, temperament, and behavior. They were able to make good use of their limited verbal abilities. Comparable studies of other groups of persons with a specific syndrome are needed to determine whether the present findings are specific for Rubinstein-Taybi syndrome or may be found among persons with other syndromes.

Kinetic characterization of tissue-type plasminogen activator (t-PA) and t-PA deletion mutants.

The binding of t-PA to fibrin is mediated both by its "finger" (F) and its "kringle 2" (K2) domain. In addition, these domains are involved in the stimulation of t-PA activity by fibrin. We analyzed the kinetic characteristics of Glu-plasminogen activation by t-PA and a set of t-PA deletion mutants in the absence and the presence of desA-fibrin. In the absence of desA-fibrin, the activity of t-PA (variants) is determined by the presence of the protease domain, irrespective of the composition of the amino-terminal heavy chain. In the presence of the cofactor desA-fibrin, the activity of t-PA (variants) is dependent on the domain composition of the heavy chain. The activity of t-PA is stimulated 2,400 fold by desA-fibrin, whereas the activity of the mutant lacking the K1 domain (del. K1) increases 936 fold in the presence of this cofactor. Mutants lacking either the K2 domain (del. K2) or the F domain (del. F) exhibit an enhanced activity upon desA-fibrin addition of 200 and 210 fold, respectively. DesA-fibrin has no stimulatory effect on the activity of the mutant containing only the serine-protease domain (del.FE K1 K2) nor on the activity of the variant containing only the K1 domain and the serine-protease domain (del. FE K2). Furthermore, we determined the relative fibrin affinity of each t-PA variant, which is similarly dictated by the composition of the heavy chain.

Tissue-type plasminogen activator and its substrate Glu-plasminogen share common binding sites in limited plasmin-digested fibrin.

The enzyme tissue-type plasminogen activator (t-PA) and its substrate Glu-plasminogen can both bind to fibrin. The assembly of these three components results in about a 1000-fold acceleration of the conversion of Glu-plasminogen into plasmin. Fibrin binding of t-PA is mediated both by its finger (F) domain and its kringle-2 domain. Fibrin binding of Glu-plasminogen involves its kringle structures (K1-K5). It has been suggested that particular kringles contain lysine-binding sites and/or aminohexyl-binding sites, exhibiting affinity for specific carboxyl-terminal lysines and intrachain lysines, respectively. We investigated the possibility that t-PA and Glu-plasminogen kringles share common binding sites in fibrin, limitedly digested with plasmin. For that purpose we performed competition experiments, using conditions that exclude plasmin formation, with Glu-plasminogen and either t-PA or two deletion mutants, lacking the F domain (t-PA del.F) or lacking the K2 domain (t-PA del.K2). Our data show that fibrin binding of t-PA, mediated by the F domain, is independent of Glu-plasminogen binding. In contrast, partial inhibition by Glu-plasminogen of t-PA K2 domain-mediated fibrin binding is observed that is dependent on carboxyl-terminal lysines, exposed in fibrin upon limited plasmin digestion. Half-maximal competition of fibrin binding of both t-PA and t-PA del.F is obtained at 3.3 microM Glu-plasminogen. The difference between this value and the apparent dissociation constant of Glu-plasminogen binding to limitedly digested fibrin (12.1 microM) under these conditions is attributed to multiple, simultaneous interactions, each having a separate affinity. It is concluded that t-PA and Glu-plasminogen can bind to the same carboxyl-terminal lysines in limitedly digested fibrin, whereas binding sites composed of intrachain lysines are unique both for the K2 domain of t-PA and the Glu-plasminogen kringles.

Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA.

Plasminogen activation is catalyzed both by tissue-type-(t-PA) and by urokinase-type plasminogen activator (u-PA). This reaction is controlled by plasminogen activator inhibitor type 1 (PAI-1) that is either present in plasma or bound to fibrin, present in a thrombus. We studied the mechanism of in vitro inhibition of both t-PA and u-PA activity by PAI-1 bound to fibrin. It is shown that activation of latent PAI-1 unmasks a specific fibrin-binding site that is distinct from its reactive site. This reactive site of activated PAI-1 bound to fibrin is fully exposed to form complexes with t-PA and u-PA, that are unable to activate plasminogen. Upon complex formation with either one of the plasminogen activators, PAI-1 apparently undergoes a conformational change and loses its affinity for fibrin. Consequently, complexes of u-PA and PAI-1 dissociate from the fibrin matrix and are encountered in the fluid phase. In contrast, t-PA/PAI-1 complexes remain bound to fibrin. By employing recombinant t-PA deletion-mutant proteins, that precisely lack domains involved in fibrin binding, we demonstrate that binding of t-PA/PAI-1 complexes is mediated by both the "finger" (F) and the "kringle-2" (K2) domain of t-PA. A model is proposed that explains inhibition of the fibrinolytic process, at the level of plasminogen activation by t-PA, directed by PAI-1 bound to fibrin. An implication of the proposed model is that t-PA/PAI-1 complexes and free t-PA compete for the same binding sites on fibrin.

Identification of the domains of tissue-type plasminogen activator involved in the augmented binding to fibrin after limited digestion with plasmin.

The binding of recombinant tissue-type plasminogen activator (rt-PA) to fibrin increases upon digestion of fibrin with plasmin. Optimal binding is observed following a limited plasmin digestion of fibrin, coinciding with the generation of fibrin fragment X polymers. We studied the involvement of the separate domains of the amino-terminal "heavy" (H) chain of rt-PA in this augmentation of fibrin binding. The fibrin-binding characteristics of a set of rt-PA deletion mutants, lacking either one or more of the structural domains of the H chain, were determined on intact fibrin matrices and on fibrin matrices that were subjected to limited digestion with plasmin. The augmented fibrin binding of rt-PA is partially abolished when the plasmin-degraded fibrin matrices are subsequently treated with carboxypeptidase B, demonstrating that this increased binding is dependent on the generation of carboxyl-terminal lysine residues in the fibrin matrix. Evidence is provided that this increase of fibrin binding is mediated by the kringle 2 (K2) domain that contains a lysine-binding site. Further increase of the fibrin binding of rt-PA is independent of the presence of carboxyl-terminal lysines. It is shown that the latter increase is not mediated by the K2 domain. Based on our data, we propose that the increase in fibrin binding, unrelated to the presence of carboxyl-terminal lysine residues, is mediated by the finger (F) domain, provided that this domain is correctly exposed in the remainder of the protein.

Artificial exon shuffling between tissue-type plasminogen activator (t-PA) and urokinase (u-PA): a comparative study on the fibrinolytic properties of t-PA/u-PA hybrid proteins.

We constructed two human tissue-type plasminogen activator/urokinase (t-PA/u-PA) hybrid cDNAs which were expressed by transfection of mouse Ltk- cells. The properties of the secreted proteins were compared with those of recombinant t-PA (rt-PA) and high molecular weight (HMW) u-PA. The hybrid proteins each contain the amino-terminal fibrin-binding chain of t-PA fused to the carboxy-terminal serine protease moiety of u-PA but differ by a stretch of 13 amino acid residues between kringle 2 of t-PA and the plasmin cleavage site of u-PA. Hybrid protein rt-PA/u-PA I contains amino acids 1-262 of t-PA connected with amino acids 147-411 of u-PA, whereas hybrid protein rt-PA/u-PA II consists of the same t-PA segment and residues 134-411 of u-PA. We demonstrated fibrin binding for rt-PA, whereas the hybrid proteins bind to a lesser extent and HMW u-PA has no affinity for fibrin. Plasminogen activation by either one of the hybrid proteins in the absence of a fibrin substitute was similar to that by HMW u-PA, while rt-PA was much less active. The catalytic efficiency, in the presence of a fibrin substitute, increases more than 2000-fold for rt-PA, about 250-fold for hybrid proteins I and II, and 12-fold for HMW u-PA, respectively. Under these conditions the hybrid proteins are more efficient plasminogen activators than the parental ones. The hybrid molecules form a 1:1 molar complex with the human endothelial plasminogen activator inhibitor (PAI-1), analogous to that formed by rt-PA and HMW u-PA. The relative affinity of rt-PA for PAI-1 is 4.6-fold higher than that of HMW u-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the human plasminogen activator inhibitor 1 gene: nonrandom distribution of introns.

Plasminogen activator inhibitor 1 (PAI-1) is the primary inhibitor of tissue-type plasminogen activator and thus performs an essential role in the regulation of the fibrinolytic process. It is a member of a large family of serine protease inhibitors (serpins). We determined the structure of the PAI-1 gene in order to more completely investigate the relationship of PAI-1 to other serpins and, at the same time, to begin to delineate structure-function relations in PAI-1 itself. A human genomic cosmid DNA library was screened and found to contain two independent clones, each harboring the entire PAI-1 gene. Restriction site mapping, electron microscopic inspection of heteroduplexes, and nucleotide sequence analysis all demonstrate that the PAI-1 gene is approximately 12.2 kilobase pairs in length and consists of nine exons and eight introns. All intron-exon boundaries are in accord with the "GT-AG" rule, including a cryptic acceptor splice site found in intron 7. The intron-exon pattern of the PAI-1 gene is distinct from that of most other serpins except that intron 3 of PAI-1 occupies an identical position as intron E of ovalbumin. Comparison of our data with the proposed subdomain structure of serpins suggests that seven of the eight introns may occupy a nonrandom position in the gene. These introns either delineate boundaries of individual structural subdomains or are located in random coil regions of the protein. Transcription of the PAI-1 gene in cultured vascular endothelial cells results in two distinct mRNA species. Our data suggest that these two transcripts arise by alternative polyadenylation.

Mapping of epitopes on human tissue-type plasminogen activator with recombinant deletion mutant proteins.

An antigen assay based on a monoclonal antibody directed against the light chain of tissue-type plasminogen activator (t-PA) was developed to quantify seven recombinant (r) t-PA deletion mutant proteins. These recombinant proteins were then employed to map different epitopes on t-PA which interact with a panel of twenty-three monoclonal anti-t-PA antibodies. Twenty were directed against domains on the heavy chain, two against the "finger" domain, three against the "epidermal growth factor-like" domain, five against the kringle 1 domain, and ten against the kringle 2 domain. Only three monoclonal anti-t-PA antibodies interact with the light chain. The finding that the epitopes of each of the monoclonals could be determined with the deletion mutant proteins supports the hypothesis of autonomous folding of structural domains and emphasizes the validity of the use of the recombinant t-PA-deletion mutant proteins for structure-function studies.

On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid.

The binding of tissue-type plasminogen activator (t-PA) to fibrin is mediated both by its finger domain and by its kringle-2 domain. In this report, we investigate the relative affinities of these domains for lysine. Human recombinant t-PA deletion-mutant proteins were prepared and their ability to bind to lysine-Sepharose was investigated. Mutants containing the kringle-2 domain bound to lysine-Sepharose, whereas mutants lacking this domain but containing the finger domain, the epidermal growth factor domain or the kringle-1 domain did not bind to lysine-Sepharose. Mutant proteins containing the kringle-2 domain could be specifically eluted from lysine-Sepharose with epsilon-amino caproic acid. This lysine derivative also abolished fibrin binding by the kringle-2 domain but had no effect on the fibrin-binding property of the finger domain. Thus, a lysine-binding site is involved in the interaction of the kringle-2 domain with fibrin but not in the interaction of the finger domain with fibrin. The implications of the nature of these two distinct interactions of t-PA with fibrin on plasminogen activation by t-PA will be discussed.

Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)

Autonomous functions of structural domains on human tissue-type plasminogen activator.

Transfected mouse Ltk- cells were employed for transient expression of recombinant human tissue-type plasminogen activator (t-PA; EC 3.4.21.31) or of recombinant-t-PA deletion proteins, encoded by SV40-pBR322-derived t-PA cDNA plasmids. The t-PA cDNA deletion mutants have two features in common, i.e., cDNA programming the signal peptide and the coding region for the light chain. Consequently, recombinant t-PA mutant proteins are efficiently secreted and display plasminogen activator activity. The gene encoding the amino-terminal heavy chain [an array of structural domains homologous to other plasma proteins (finger, epidermal growth factor, and kringle domains)] was mutated using restriction endonucleases to delete one or more structural domains. The stimulatory effect of fibrinogen fragments on the plasminogen activator activity of t-PA was demonstrated to be mediated by the kringle K2 domain and to a lesser extent by the finger/epidermal growth factor region but not by the kringle K1 domain. These data correlate well with the fibrin-binding properties of the recombinant t-PA deletion proteins, indicating that the stimulation of the activity by fibrinogen fragments is based on aligning the substrate plasminogen and t-PA on the fibrin matrix. Our results support the evolutionary concept of exon shuffling, arranging structural domains that constitute autonomous functions of the protein.

Structure and function of human tissue-type plasminogen activator (t-PA).

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.

Comparison of the genes coding for the common 5' terminal sequence of messenger RNAs in three trypanosome species.

Messenger RNAs of Trypanosoma brucei share a common 5' terminal sequence of 35 nucleotides, encoded by a mini-exon located in 1.35-kb tandemly linked repeats. We show here that sequences, almost identical to the mini-exon of T. brucei, are present in mRNAs from members of two other kinetoplastid subgenera: Trypanosoma vivax and Trypanosoma cruzi. As in T. brucei, these mini-exons are encoded by small tandemly linked repeat elements. We have determined and compared the nucleotide sequences of the mini-exon repeats from T. brucei, T. vivax and T. cruzi. This analysis shows that the mini-exon, its immediate flanking sequences and a T-rich stretch downstream are conserved, but little else. Our data establish the generality of the novel transcription system, that was first found in T. brucei and that yields mRNAs with common, repeat-encoded, 5' termini.

Many trypanosome messenger RNAs share a common 5' terminal sequence.

The mRNAs for different variant surface antigens of Trypanosoma brucei start with the same 35 nucleotides. This sequence is encoded by a separate mini-exon, located in a 1.35-kb repetitive element. We have reported that trypanosomes contain many transcripts that hybridize to mini-exon probes, even if they do not make the surface antigens. We show here that these transcripts have the mini-exon sequence at their 5' end; they do not contain other sequences from the mini-exon repeat element and are polyadenylated. We have cloned DNA complementary to trypanosome mRNAs and randomly selected 17 clones containing mini-exon sequences. Thirteen of these are derived from different genes that do not code for surface antigens. We conclude that the mini-exon sequence is a common element at the 5' end of many trypanosome mRNAs. As the 200 genes for mini-exons are highly clustered, linkage of the mini-exon sequence to the remainder of most mRNAs may require discontinuous transcription.