Severe ischemia of the hand following intra-arterial promazine injection: effects of vasodilation, anticoagulation, and local thrombolysis with tissue-type plasminogen activator.

Promazine hydrochloride was injected accidentally in the antecubital artery of a 42-year-old woman, resulting in severe ischemia of the second and third fingers of her right hand which lasted for four days before she was hospitalized. Vasodilation by combining axillary plexus block and intravenous sodium nitroprusside did not improve ischemia and local thrombolysis was performed using recombinant tissue-type plasminogen activator (50 mg over 8 hours), resulting in normalization of digital pressure in one of the two affected fingers. The outcome was favourable and amputation could be avoided.

The hepatic clearance of recombinant tissue-type plasminogen activator decreases after an inflammatory stimulus

We have shown that tissue-type plasminogen activator (tPA) and plasma kallikrein share a common pathway for liver clearance and that the hepatic clearance rate of plasma kallikrein increases during the acute-phase (AP) response. We now report the clearance of tPA from the circulation and by the isolated, exsanguinated and in situ perfused rat liver during the AP response (48-h ex-turpentine treatment). For the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of 125I-tPA from the circulation of turpentine-treated rats (2.2 ± 0.2 ml/min, N = 7) decreases significantly (P = 0.016) when compared to normal rats (3.2 ± 0.3 ml/min, N = 6). The AP response does not modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80%) cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of turpentine-treated rats (15.5 ± 1.3 µg/min, N = 4) was slower (P = 0.003) than the clearance rate by the liver of normal rats (22.5 ± 0.7 µg/min, N = 10). After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment), tPA was cleared by the perfused liver at 16.2 ± 2.4 µg/min (N = 5), suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kallikrein were not altered during the AP response. These results contribute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered.

Time- and dose-related regional fluxes of tissue-type plasminogen activator in anesthetized endotoxemic pigs

BACKGROUND: Acute endotoxinemia elicits an early fibrinolytic response. This study analyzes the effects of the dose and duration of endotoxin infusion on arterial levels of tissue-type plasminogen activator (tPA) and pulmonary, mesenteric and hepatic plasma tPA fluxes. METHODS: Pigs were randomized to receive an acute, high-dose (for 6 h, n=13, high ETX) or a prolonged, low-dose (for 18 h, n=18, low ETX) infusion of endotoxin or saline vehicle alone (for 18 h, n=14, control). All animals were fluid resuscitated to maintain a normodynamic circulation. Systemic and regional blood flows were measured and arterial, pulmonary arterial, portal and hepatic venous blood samples were analyzed to calculate regional net fluxes of tPA. Plasma tumor necrosis factor (TNF-alpha) levels were analyzed. RESULTS: Mesenteric tPA release and hepatic uptake increased maximally at 1.5 h in ETX groups related to dose. Maximal mesenteric tPA release [high ETX 612 (138-1185) microg/min/kg, low ETX 72 (32-94) microg/min/kg, median+/-interquartile range] and hepatic tPA uptake [high ETX -1549 (-1134 to -2194) microg/min/kg, low ETX -153 (-105 to -307) microg/min/kg] correlated to TNF-alpha levels. Regional tPA fluxes returned to baseline levels at 6 h in both ETX groups and also remained low during sustained low ETX. No changes were observed in control animals. CONCLUSIONS: Endotoxemia induces an early increase in mesenteric tPA release and hepatic tPA uptake related to the severity of endotoxemia. The time patterns of changes in mesenteric and hepatic tPA fluxes are similar in acute high-dose endotoxemia and sustained low-dose endotoxemia.

Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with late-phase long-term potentiation in both Schaffer collateral and mossy fiber pathways.

The gene encoding tissue-type plasminogen activator (t-PA) is an immediate response gene, downstream from CREB-1 and other constitutively expressed transcription factors, which is induced in the hippocampus during the late phase of long-term potentiation (L-LTP). Mice in which the t-PA gene has been ablated (t-PA-/-) showed no gross anatomical, electrophysiological, sensory, or motor abnormalities but manifest a selective reduction in L-LTP in hippocampal slices in both the Schaffer collateral-CA1 and mossy fiber-CA3 pathways. t-PA-/- mice also exhibit reduced potentiation by cAMP analogs and D1/D5 agonists. By contrast, hippocampal-dependent learning and memory were not affected in these mice, whereas performance was impaired on two-way active avoidance, a striatum-dependent task. These results provide genetic evidence that t-PA is a downstream effector gene important for L-LTP and show that modest impairment of L-LTP in CA1 and CA3 does not result in hippocampus-dependent behavioral phenotypes.

Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator.

The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.

Origins of the specificity of tissue-type plasminogen activator.

The role of subsite interactions in defining the stringent substrate specificity of tissue-type plasminogen activator (t-PA) has been examined by using an fd phage library that displayed random hexapeptide sequences and contained 2 x 10(8) independent recombinants. Forty-four individual hexapeptides were isolated and identified as improved substrates for t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide that contained the primary sequence of the actual cleavage site in plasminogen. These results suggest that small peptides can mimic determinants that mediate specific proteolysis, emphasize the importance of subsite interactions in determining protease specificity, and have important implications for the evolution of protease cascades.

Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients

Background: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue-type plasminogen activator (t-PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1 beta (IL-1 beta) can up regulate the level of t-PA and plasminogen activator inhibitor-2 (PAI-2) synthesis by human gingival fibroblasts. Method: In the present study, the levels of t-PA and PAI-2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from 106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post-treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA and PAI-2. Results: The results showed that significantly high levels of t-PA and PAI-2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI-2, but not t-PA, after 14 days. A significant positive linear correlation was found between t-PA and PAI-2 in GCF (r=0.80, p

Plasminogen activator system in osteoclasts

To determine which genes of the plasminogen activator (PA) system were expressed in osteoclasts, RNA extracted from microisolated mouse osteoclasts was used as template for reverse transcribed polymerase chain reaction (RT-PCR) with gene-specific primer pairs, Using this approach, the expression of RNAs for tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, protease nexin, and urokinase receptor isoform 1 (uPAR1) were detected in mouse osteoclasts. The expression of uPAR RNA in osteoclasts was confirmed by in situ hybridization with a uPAR1 probe, RNA encoding the uPAR isoform 2 was not detected in mouse osteoclasts, but a novel unspliced uPAR RNA variant was detected in these cells, The novel uPAR variant and uPAR1 RNA were also detected in mouse calvarial osteoblasts, kidney, muscle, and the mouse macrophage cell line J774A.1 by RT-PCR The presence of RNAs for most of the components of the PA system in osteoclasts suggests that it may have a functional role in this cell type.

The expression of plasminogen activator system in a rat model of periodontal wound healing

Background: The plasminogen activator system has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling, including wound healing. The aim of this study was to elucidate the presence of components of the plasminogen activator system during different stages of periodontal wound healing. Methods: Periodontal wounds were created around the molars of adult rats and healing was followed for 28 days. Immunohistochemical analyses of the healing tissues and an analysis of the periodontal wound healing fluid by ELISA were carried out for the detection of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and 2 plasminogen activator inhibitors (PAI-1 and PAI-2). Results: During the early stages (days 1 to 3) of periodontal wound healing, PAI-1 and PAI-2 were found to be closely associated with the deposition of a fibrin clot in the gingival sulcus. These components were strongly associated with the infiltrating inflammatory cells around the fibrin clot. During days 3 to 7, u-PA, PAI-1, and PAI-2 were associated with cells (particularly monocytes/macrophages, fibroblasts, and endothelial cells) in the newly formed granulation tissue. During days 7 to 14, a new attachment apparatus was formed during which PAI-1, PAI-2, and u-PA were localized in both periodontal ligament fibroblasts (PDL) and epithelial cells at sites where these cells were attaching to the root surface. In the periodontal wound healing fluid, the concentration for t-PA increased and peaked during the first week. PAI-2 had a similar expression to t-PA, but at a lower level over the entire wound-healing period. Conclusions: These findings indicate that the plasminogen activator system is involved in the entire process of periodontal wound healing, in particular with the formation of fibrin matrix on the root surface and its replacement by granulation tissue, as well as the subsequent formation of the attachment of soft tissue to the root surface during the later stages of wound repair.

Increased expression of urokinase-type plasminogen activator mRNA determines adverse prognosis in ErbB2-positive primary breast cancer.

PURPOSE: To evaluate and validate mRNA expression markers capable of identifying patients with ErbB2-positive breast cancer associated with distant metastasis and reduced survival. PATIENTS AND METHODS: Expression of 60 genes involved in breast cancer biology was assessed by quantitative real-time PCR (qrt-PCR) in 317 primary breast cancer patients and correlated with clinical outcome data. Results were validated subsequently using two previously published and publicly available microarray data sets with different patient populations comprising 295 and 286 breast cancer samples, respectively. RESULTS: Of the 60 genes measured by qrt-PCR, urokinase-type plasminogen activator (uPA or PLAU) mRNA expression was the most significant marker associated with distant metastasis-free survival (MFS) by univariate Cox analysis in patients with ErbB2-positive tumors and an independent factor in multivariate analysis. Subsequent validation in two microarray data sets confirmed the prognostic value of uPA in ErbB2-positive tumors by both univariate and multivariate analysis. uPA mRNA expression was not significantly associated with MFS in ErbB2-negative tumors. Kaplan-Meier analysis showed in all three study populations that patients with ErbB2-positive/uPA-positive tumors exhibited significantly reduced MFS (hazard ratios [HR], 4.3; 95% CI, 1.6 to 11.8; HR, 2.7; 95% CI, 1.2 to 6.2; and, HR, 2.8; 95% CI, 1.1 to 7.1; all P < .02) as compared with the group with ErbB2-positive/uPA-negative tumors who exhibited similar outcome to those with ErbB2-negative tumors, irrespective of uPA status. CONCLUSION: After evaluation of 898 breast cancer patients, uPA mRNA expression emerged as a powerful prognostic indicator in ErbB2-positive tumors. These results were consistent among three independent study populations assayed by different techniques, including qrt-PCR and two microarray platforms.

Fibrinolysis in pregnancy: a study of plasminogen activator inhibitors.

During pregnancy the plasma concentration of two different inhibitors of plasminogen activators (PAIs) increases. The only one found in the plasma of nonpregnant women (PAI1) is immunologically related to a PAI of endothelial cells; its plasma activity, as deduced from the inhibition of single-chain tissue-type plasminogen activator (t-PA), increased from 3.4 +/- 2.3 U/mL (mean +/- 95% confidence limits) in the plasma of nonpregnant women to 29 +/- 7 U/mL at term, and its antigen level, measured by a radioimmunoassay, increased from 54 +/- 17 ng/mL to 144 +/- 25 ng/mL. In pregnancy plasma a second PAI (PAI 2) related to a PAI found in placenta extracts was observed. Its level, quantified with a radioimmunoassay, increased from below the detection limit (approximately 10 ng/mL) in normal plasma to 260 ng/mL at term. One hour after delivery, PAI 1 activities and antigen decreased sharply, but the PAI 2 antigen levels remained constant. Three days later, the PAI 1 antigen levels had fallen to normal levels, but the PAI 2 antigen levels were still at least eightfold above the nonpregnant values. During pregnancy, the t-PA and prourokinase (u-PA) antigen concentrations increased 50% and 200%, respectively, whereas the plasminogen and alpha 2-antiplasmin levels remained constant. Despite the large variations in the levels of PAs and PAIs, the overall fibrinolytic activity as measured in diluted plasma by a radioiodinated fibrin plate assay did not change significantly. Just after delivery, a great increase in the t-PA antigen levels was observed. Three to five days after delivery most parameters of the fibrinolytic system were normal again. Our results demonstrate that during pregnancy and in the puerperium profound alterations of the fibrinolytic system occur that are characterized by increases in PAs and their inhibitors, but these alterations do not affect the overall fibrinolytic activity.

Ex vivo Pulsatile Perfusion of Human Saphenous Veins Induces Intimal Hyperplasia and Increased Levels of the Plasminogen Activator Inhibitor 1.

Vessel wall trauma induces vascular remodeling processes including the development of intimal hyperplasia (IH). To assess the development of IH in human veins, we have used an ex vivo vein support system (EVVSS) allowing the perfusion of freshly isolated segments of saphenous veins in the presence of a pulsatile flow which reproduced arterial conditions regarding shear stress, flow rate and pressure during a period of 7 and 14 days. Compared to the corresponding freshly harvested human veins, histomorphometric analysis showed a significant increase in the intimal thickness which was already maximal after 7 days of perfusion. Expression of the endothelial marker CD31 demonstrated the presence of endothelium up to 14 days of perfusion. In our EVVSS model, the activity as well as the mRNA and protein expression levels of plasminogen activator inhibitor 1, the inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), were increased after 7 days of perfusion, whereas the expression levels of tPA and uPA were not altered. No major change was observed between 7 and 14 days of perfusion. These data show that our newly developed EVVSS is a valuable setting to study ex vivo remodeling of human veins submitted to a pulsatile flow.