Concentrations Of Tissue Plasminogen Activator Research Articles

Assessment of Fibrinolytic Activity by Measuring the Lysis Time of a Tissue Factor-induced Clot: A Feasibility Evaluation

A clot lysis time assay in which a tissue factor-induced fibrin clot is lysed by exogenously added tissue plasminogen activator has been recently reported. We evaluated the feasibility of clot lysis time in a routine hemostasis laboratory, and its correlation with thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 levels and changes with aging in 185 healthy participants. Clot lysis time was assessed by monitoring changes in turbidity during clot formation and subsequent lysis using a computerized kinetic spectrophotometric microtiter plate. After preliminary experiments, 100 and 160 ng/mL tissue plasminogen activator concentrations were chosen for the study. Clot lysis time was calculated by a new mathematical analysis of the lysis curve based on discrete derivative. Clot lysis time, thrombin activatable fibrinolysis inhibitor, and plasminogen activator inhibitor-1 plasma levels showed a normal distribution. For both concentrations of tissue plasminogen activator, clot lysis time progressively increased with increase in age (P < .0001) and was significantly correlated with thrombin activatable fibrinolysis inhibitor antigen, thrombin activatable fibrinolysis inhibitor activity, and plasminogen activator inhibitor-1 antigen (at least P < .01). During linear regression analysis, thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 antigen were found to significantly influence clot lysis time (at least P < .01). Clot lysis time determination has a good laboratory performance. Our new method of calculation is independent of the time of reading and allows a more accurate and consistent detection of both short and prolonged lysis times. Our data suggest the feasibility of the use of this test in the work of routine hemostasis laboratory.

Banking of Biological Fluids for Studies of Disease-associated Protein Biomarkers

With the increasing demand of providing personalized medicine the need for biobanking of biological material from individual patients has increased. Such samples are essential for molecular research aimed at characterizing diseases at several levels ranging from epidemiology and diagnostic and prognostic classification to prediction of response to therapy. Clinically validated biomarkers may provide information to be used for diagnosis, screening, evaluation of risk/predisposition, assessment of prognosis, monitoring (recurrence of disease), and prediction of response to treatment and as a surrogate response marker. Many types of biological fluids or tissues can be collected and stored in biorepositories. Samples of blood can be further processed into plasma and serum, and tissue pieces can be either frozen or fixed in formalin and then embedded into paraffin. The present review focuses on biological fluids, especially serum and plasma, intended for study of protein biomarkers. In biomarker studies the process from the decision to take a sample from an individual to the moment the sample is safely placed in the biobank consists of several phases including collection of samples, transport of the samples, and handling and storage of samples. Critical points in each step important for high quality biomarker studies are described in this review. Failure to develop and adhere to robust standardized protocols may have significant consequences as the quality of the material stored in the biobank as well as conclusions and clinical recommendations based on analysis of such material may be severely affected.

Effects of omentectomy on the peritoneal fibrinolytic system

Decreased fibrinolytic activity in the serosal surfaces of peritoneal tissue appears to be a major factor in the development of peritoneal fibrous adhesions. The omentum reduces peritoneal adhesion by creating a mechanical barrier and producing fibrinolytic factors. This experimental study was designed to investigate the effects of omentectomy on the peritoneal fibrinolytic system. Thirty animals were assigned randomly to a control group or an omentectomy group. On postoperative day 10, peritoneal and blood samples were collected and adhesions were graded qualitatively. We measured the concentrations of serum and peritoneal tissue plasminogen activator, peritoneal plasminogen activator inhibitor-1, tissue plasminogen activator/plasminogen activator inhibitor complex, and hydroxyproline. Adhesions were significantly increased after omentectomy. Omentectomy also resulted in a reduction of both serum and tissue "tissue plasminogen activator" levels. On the other hand, an increment in "plasminogen activator inhibitor-1" levels was observed after omentectomy. There were no differences in "tissue plasminogen activator/plasminogen activator inhibitor" complex or "hydroxyproline" levels. Omentectomy reduced peritoneal fibrinolytic activity significantly and the peritoneal plasminogen activator system showed corruption that did not resolve with the rest of the peritoneal system after omentectomy.

An Evaluation of Normal Saline and Taurolidine on Intra-Abdominal Adhesion Formation and Peritoneal Fibrinolysis

Intraoperative lavage with normal saline or taurolidine solutions is commonly used in abdominal surgery. For this purpose, normal saline and taurolidine, which may modify the intrinsic fibrinolytic activity of the peritoneum by breaking the peritoneal adhesions, are frequently used. We aimed to evaluate how normal saline and taurolidine affect peritoneal fibrinolysis and adhesion formation. A rat model of peritoneal adhesion was used. Control animals received no medication, while normal saline or taurolidine solutions were administered intraperitoneally to the remaining two groups (n = 20 for each group). At postoperative day 10 adhesions were graded, and cardinal parameters of peritoneal fibrinolysis (activities and concentrations of tissue plasminogen activator [tPA], plasminogen activator inhibitor type 1 [PAI-1], and tPA/PAI-1 complex), and hydroxyproline contents were determined in peritoneal tissue samples. Total adhesion scores were decreased in both of the treated groups. Median tissue levels of tPA and tPA activity were higher in the treated groups versus controls. The PAI-1 levels were similar among the three groups. tPA/PAI-1 complex levels were higher in animals that received normal saline and in those treated with taurolidine solution compared with control animals. Peritoneal hydroxyproline levels were similar across the three groups. Our results suggest that normal saline and taurolidine solution administrations might reduce peritoneal adhesion formation, probably by altering peritoneal fibrinolytic activity.

Oxidative stress and endothelium influenced by metformin in type 2 diabetes mellitus

Metformin may influence atherogenesis but the mechanisms are not well understood. A pilot study was undertaken to determine whether metformin administration is associated with changes in oxidative stress and endothelial function. Fifteen type 2 diabetic patients were treated for 3 months with metformin (1,700 mg daily) or with a placebo in a crossover study. Laboratory parameters of oxidative stress, fibrinolysis and endothelial function were evaluated both prior to and following the respective treatments. In addition, laser Doppler was used to determine microcirculation changes in the skin. Increases in serum N-acetyl-beta-glucosaminidase activity (p < 0.05) and plasma malondialdehyde concentration were found following 1 month of metformin administration. Three months of treatment was accompanied by significantly increased plasma malondialdehyde (p < 0.001) and ascorbic acid (p < 0.01) concentrations as well as the alpha-tocopherol/(cholesterol + triglyceride) ratio (p < 0.001). The concentration of tissue plasminogen activator (tPA), vascular cell-adhesion molecules (VCAM) and intercellular cell-adhesion molecules (ICAM) were significantly decreased (p < 0.01) compared with placebo. Microcirculation measured by laser Doppler flowmetry was not significantly changed. We conclude that initiation of metformin treatment in type 2 diabetic patients is associated with improved diabetes control as well as with activation of oxidative stress together with antioxidant system. The atherogenic process measured by biochemical indicators is diminished in parallel. Our results show that in short-term metformin administration in type 2 diabetes promotes endothelium effects associated with a complex of metabolic changes.

Fibrinolytic responses of human peritoneal fluid in laparoscopic cholecystectomy: a prospective clinical study

The reduction in peritoneal fibrinolysis is believed to be the pathogenetic mechanism of adhesion formation. The general conclusion based on previous clinical and experimental studies is that laparoscopic procedures produce less adhesion formation. The association between this beneficial effect of laparoscopic cholecystectomy and peritoneal fibrinolytic changes is not clear. Therefore, the authors aimed to compare the effects of open and laparoscopic cholecystectomy on peritoneal fibrinolysis. For this purpose, fibrinolytic parameters in peritoneal fluid were investigated 24 h after laparoscopic and open cholecystectomies. In a prospective clinical study, peritoneal fluid was sampled via a drain 24 h after laparoscopic (n = 10) and open (n = 9) cholecystectomies. Activities and concentrations of tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and tPA/PAI-1 complex were determined by enzyme-linked immunosorbent assay (ELISA) kits. In peritoneal fluids, tPA and tPA/PAI-1 complex concentrations were higher in the open cholecystectomy group (p = 0.009 and p < 0.001, respectively), but tPA activity and PAI-1 concentrations did not differ between the groups (p = 0.514 and p = 0.716, respectively). Fibrinolytic changes in peritoneal fluid have several similarities in open and laparoscopic cholecystectomies with regard to tPA activity and PAI-1 levels. However, higher tPA levels after the open procedure probably are secondary to more intense tissue handling leading to mesothelial release of tPA.

Elevated tissue plasminogen activator in patients with screening-detected abdominal aortic aneurysm

A population-based case-control study with historical and current data was conducted in a population with a high prevalence of disease to explore the hypothesis that the fibrinolytic system may be involved in the early pathogenesis of abdominal aortic aneurysm (AAA). Forty-two patients found to have AAA at population-based screening were compared with 100 controls matched for age and sex. Mass concentration of tissue plasminogen activator (tPA mass) and tissue plasminogen activator/plasminogen activator inhibitor-1 complex (tPA/PAI-1 complex mass) were analyzed in blood samples obtained at the screening (current), and in blood samples obtained from a study conducted 12 years previously on the same population (historical). Current tPA mass levels were significantly higher in AAA patients compared with controls (13.6 vs 11.4 microg/L, P=.016). A similar trend was observed in historical tPA mass levels (9.8 vs 8.2 microg/L, P=.062). Current and historical mass concentrations of tPA/PAI complex in AAA patients were similar to those in controls. Current tPA mass levels retained the associations with AAA in a logistic regression model after adjustment for history of atherosclerosis (odds ratio [OR], 1.1 per microg/L, P=.039) and current smoking (OR 1.1 per microg/L, P=.039). When family history of AAA was added in a logistic regression model, the OR for current tPA mass was 1.1 per microg/L (P=.056) and 1.1 per microg/L (P=.070) when treated hypertension was added. The finding of elevated tPA mass, in contrast to tPA/PAI-1 complex, in plasma among patients with screening-detected AAA supports the hypothesis that the fibrinolytic system may be important in the early pathogenesis of AAA.

Microvascular Function, Metabolic Syndrome, and Novel Risk Factor Status in Women With Cardiac Syndrome X

To characterize microvascular function, candidate risk pathways, and metabolic syndrome prevalence in women with cardiac syndrome X, 52 nondiabetic women with angiographically normal epicardial arteries but >1 mm of planar ST depression during exercise testing (patients) and 24 healthy controls of similar age were recruited. In addition to fasting blood samples and anthropometric measurements, forearm cutaneous microvascular function after iontophoresis of acetylcholine and sodium nitroprusside was assessed by laser Doppler imaging. Despite body mass index correction and a larger proportion on statin therapy, patients had high levels of insulin (p=0.016), triglycerides (p=0.018), intercellular adhesion molecule-1 (p=0.021), von Willebrand factor (p=0.005), and leptin (p=0.005) and lower levels of high-density lipoprotein cholesterol (p=0.042) compared with controls. Consistent with these data, 30% of patients but only 8% of controls fulfilled criteria for the metabolic syndrome as defined by the National Cholesterol Education Program (p=0.015). Endothelium-dependent and -independent microvascular functions were markedly impaired in patients (p<0.001), and the odds ratio for cardiac syndrome X was 7.38 (95% confidence interval 2.2 to 24.7) if the acetylcholine response was <8,710 flux units. In conclusion, women with cardiac syndrome X more commonly have metabolic syndrome and related adiposity, metabolic, and inflammatory derangements. They also have significantly impaired skin microvascular function as assessed by laser Doppler imaging, consistent with generalized vascular dysfunction, a finding with potential diagnostic implications.

Pentoxifylline, a Methyl Xanthine Derivative, Reduces Peritoneal Adhesions and Increases Peritoneal Fibrinolysis in Rats

Peritoneum has an intrinsic fibrinolytic activity that breaks the peritoneal adhesions. Peritoneal injuries with ischemia interfere this fibrinolytic activity and cause adhesions. Pentoxifylline, a methyl xanthine derivative, improves blood flow by decreasing its viscosity and also increases fibrinolytic activity in plasma. We hypothesized that pentoxifylline would increase peritoneal fibrinolysis and ameliorate adhesions. A rat model of peritoneal adhesion (cecal abrasion with gauze, n = 15 for each group) was used to test this hypothesis and cardinal parameters of peritoneal fibrinolysis were measured in peritoneal samples. No medication was given in control animals, while pentoxifylline was administered intraperitonealy (IP) (25 mg/kg, before abdominal closure to whole abdomen) or intravenously (IV) (25 mg/kg, for 9 days after operation) in the experimental groups. At postoperative day 10, peritoneal biopsies were obtained and adhesions were graded qualitatively. Activities and concentrations of tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), tPA/PAI-1 complex and hydroxyproline contents were determined. Total adhesion scores were decreased in both treated groups. Mean levels of tPA concentration and tPA activity were increased in the treated groups compared to controls (p < 0.001 and p = 0.001, respectively). The tPA/PAI-1 complex levels were similar among the three groups. PAI-1 levels were lower in animals receiving IP pentoxifylline compared to control animals and those treated with IV pentoxifylline (p = 0.048, p = 0.015, respectively). Peritoneal hydroxyproline levels were similar among the three groups. Our results suggest that pentoxifylline administration either through IV or IP may reduce peritoneal adhesion formation probably by altering peritoneal fibrinolytic activity.

Tissue plasminogen activator in chronic subdural hematomas as a predictor of recurrence

Chronic subdural hematomas (CSDHs) recur in 7 to 18% of cases. The present study was conducted to determine whether serum or lesion concentrations of coagulofibrinolytic and angiogenic factors, which have been reported to be potential markers of CSDH development, might predict such recurrences. Sixty consecutive patients (mean age 71.5 years) with CSDHs (74 affected sides) were studied. Samples of serum in preoperative peripheral venous blood and of hematomas (obtained during surgery) were collected and analyzed. The CSDH recurred in six (8.1%) of the 74 affected sides in six patients. None of the values of the coagulative factors or tests in serum showed significant variation between cases with and those without recurrence. Among coagulofibrinolytic factors, tissue plasminogen activator (TPA) in hematomas demonstrated significantly greater levels in recurrent than in nonrecurrent cases; a similar tendency was noted for alpha2-plasmin inhibitor-plasmin complex in hematomas. Both factors were greater in the lesions than in the serum. Among the angiogenic factors, levels of hepatic growth factor (HGF) and vascular endothelial growth factor (VEGF) in hematomas were significantly greater than in serum, whereas those of basic fibroblast growth factor were rather lower. Note that comparisons between recurrent and nonrecurrent cases revealed no significant difference. Patients harboring CSDHs with high TPA concentrations on sampling at the initial surgery have a relatively high probability of recurrence and require follow up with computerized tomography scanning. Angiogenic factors, such as HGF and VEGF, might be candidate markers of CSDH enlargement but are not useful as predictors of recurrence.

Human intraperitoneal fibrinolytic response to elective surgery.

Intra-abdominal adhesions develop in over 90 per cent of patients undergoing laparotomy. Peritoneal fibrinolysis is believed to be important in the pathophysiology of adhesion formation. This study investigated the fibrinolytic response of postoperative peritoneal fluid in 12 patients undergoing elective laparotomy. There was a significant reduction in the plasminogen activating activity to undetectable levels at 24 h, which was sustained at 48 h (P < 0.05). While there was an early reduction in the concentration of tissue plasminogen activator (median 40.0, 28.2, 16.3 and 31.9 ng/ml at 2, 6, 24 and 48 h respectively; P < 0.05), the abolition of functional fibrinolytic activity appeared to be secondary to a marked increase in the concentration of plasminogen activator inhibitor (PAI) 1 (median 86, 196, 800 and 730 ng/ml at 2, 6, 24 and 48 h respectively; P < 0.05) and PAI-2 (median less than 6, 12, 155 and 245 ng/ml at 2, 6, 24 and 48 h respectively; P < 0.05). This reduction in the plasminogen activating activity of peritoneal fluid may favour the formation of permanent fibrous adhesions following surgery.

Coagulation and fibrinolytic responses of human peritoneal fluid and plasma to bacterial peritonitis

Significantly higher (P < 0.05) thrombin-antithrombin III complex levels were found in the abdominal exudate of patients with peritonitis (median 5500 ng/ml) than in that of controls (median 89 ng/ml). In patients, peritoneal fluid concentrations of tissue and urokinase-type plasminogen activator were increased by factors of 65 and 10 respectively (P < 0.05). The concentration of plasminogen activator inhibitor (PAI) 1 was increased by a factor of about 800 (median 395 versus 0.5 ng/ml, P < 0.05). Despite markedly raised concentrations of PAI, peritoneal fluid displayed fibrinolytic activity as demonstrated by significantly increased (P < 0.05) concentrations of plasmin-alpha 2-antiplasmin complex (median 10,952 versus 57 ng/ml) and fibrin degradation products (median 40,360 versus 126 ng/ml). There was no correlation between plasma and peritoneal fluid concentrations. Intraabdominal coagulation and fibrinolysis are stimulated in the abdominal cavity of patients with bacterial peritonitis.

Stearic, Oleic, and Linoleic Acids Have Comparable Effects on Markers of Thrombotic Tendency in Healthy Human Subjects

Because human studies concerning the effects of stearic acid on thrombotic tendency are inconsistent, we compared the effects of stearic acid with those of its unsaturated derivatives, oleic acid and linoleic acid. In this randomized, crossover study, 45 subjects (27 women and 18 men) consumed, in random order, 3 experimental diets, each for 5 wk. Diets contained approximately 38% of energy as fat. Dietary compositions were the same except for 7% of energy from stearic, oleic, or linoleic acids. At the end of each period, ex vivo and in vitro platelet aggregation, and variables of coagulation, fibrinolysis, and hematology were evaluated. In men, ex vivo platelet aggregation time as measured by filtragometry (P = 0.036 for diet effects) was favorably prolonged during consumption of the linoleic acid diet compared with the stearic acid diet (P = 0.040), but there was no difference with consumption of the oleic acid diet (P = 0.198). In vitro platelet aggregation induced by collagen and ADP, and variables of coagulation (factor VII amidolytic activity and concentrations of fibrinogen and prothrombin fragment 1 and 2) and fibrinolysis [plasminogen activator inhibitor (PAI) activity and concentrations of tissue plasminogen activator (tPA)/PAI-1 complexes] did not differ among the 3 diets. The mean platelet volume of the subjects decreased during consumption of the stearic acid diet by 0.32 fL compared with the oleic acid diet (P < 0.001) and by 0.35 fL compared with the linoleic acid diet (P < 0.001). In conclusion, our results do not suggest that stearic acid is highly thrombogenic compared with oleic and linoleic acids.

Fibrinolytic capacity in peritoneal fluid after laparoscopic and conventional colorectal resection: data from a randomized controlled trial

A reduced peritoneal fibrinolytic capacity after surgery is currently accepted to be the main cause for postoperative adhesions. The aim of this prospective randomized trial was to determine the fibrinolytic activity in peritoneal fluid after laparoscopic as compared to conventional colorectal resection. A randomized controlled trial in parallel with the multicenter trial Lapkon II was conducted. Peritoneal fluid was sampled via drain at 2, 8, and 24 h after elective laparoscopic (n=14; LAP) and conventional (n=16; CON) colorectal resections. Activities and concentrations of tissue plasminogen activator (t-PA), plasminogen activator inhibitor type-1 (PAI-1) and t-PA/PAI complex were determined in all specimen by ELISA kits. There was no difference in age, sex or body mass index between both groups. Postoperatively, t-PA activity decreased in both groups and was lower 2 h after closing the abdomen in the laparoscopic group (p<0.05). PAI-1 activity and concentration increased in both groups. Difference between the groups was measured for PAI-1 concentration after 24 h (p<0.05). There were no differences between the groups regarding t-PA concentrations, PAI-1 activity and t-PA/PAI complex. After closing the abdominal cavity, postoperative changes in fibrinolytic capacity of peritoneal fluid can be determined in samples collected by a drain. However, there were no major differences in the postoperative course of fibrinolytic capacity in peritoneal fluid after laparoscopic and conventional colorectal resections.

In Vitro Models for Assessing Transcranial Ultrasound-Enhanced Thrombolysis

To the Editor: Contrary to the promising results of recent clinical studies, Pfaffenberger et al conclude from in vitro data that diagnostic ultrasound (US) is not suitable for transcranial enhancement of tissue plasminogen activator (tPA) thrombolysis.1 Their contention is based on surprising control data showing significant clot lysis without tPA and plasminogen and relatively modest increases in clot dissolution with tPA. Interestingly, their reported clot weight loss of 21.8% after 1 hour of incubation in saline alone is much higher than that which they recently reported in another journal using the same methods (2.8%±1.8%2 and 11.0%±3.8%3). Likewise, the effect of tPA alone (27.4%) also differs with earlier results of 19.9%±4.3%3 and 22.7%±9.0%.2 These discrepancies seem important, because a 19.9% effect of tPA alone would have been lower than combined US and recombinant tPA through the skull (26.2%), thus encouraging the authors to positively assess transcranial US thrombolysis. Because clot dissolution is dependent on the amount of plasminogen present and on the amount of tPA available for activation of the plasminogen, little clot lysis should occur during the first 60 minutes of incubation in saline, with or without tPA. To study our hypothesis, we used experimental conditions similar to those of Pfaffenberger et al. 1.5 mL of citrated …

Relationship Between Myocardial Injury and Soluble P-Selectin in Non-ST Elevation Acute Coronary Syndromes

The relationships among troponin concentration, early phase coagulation activation and soluble P-selectin concentration was evaluated in this study. Troponin-l, soluble P-selectin, von Willebrand factor (vWF), fibrinogen, plasminogen, plasminogen activator inhibitor 1 (PAI-1) and tissue plasminogen activator (t-PA) concentrations of 202 consecutive patients with non-ST elevation acute coronary syndrome (NSTE-ACS) were measured at the time of admission. Patients were classified into 2 groups as troponin-negative (<0.2 ng/ml, n=129) or positive (>or=0.2 ng/ml, n=73). Soluble P-selectin concentrations were found to be higher (p<0.001) and correlated with troponin concentrations (r=0.313, p<0.005) in troponin-positive patients with NSTE-ACS. In these patients fibrinogen (p<0.001), plasminogen (p<0.001) and PAI-1 (p<0.026) concentrations were higher and t-PA concentrations were lower (p<0.013) and all these parameters correlated with P-selectin concentrations (p<0.001). There was no difference between the groups according to vWF concentrations (p=0.379). Soluble P-selectin concentrations were found to be an independent predictor of troponin positivity (beta=0.295, odds ratio =1.05, p<0.001). Analysis of regression revealed a significant effect of troponin on soluble P-selectin concentrations (r=0.52, r2=0.27, p<0.001). The results suggest that higher soluble P-selectin concentrations might be involved in increased coagulation activation and myocardial injury development in patients with NSTE-ACS.

Sesamol regulates plasminogen activator gene expression in cultured endothelial cells: a potential effect on the fibrinolytic system

Sesamol is a component in the nutritional makeup of sesame that was identified as an antioxidant. In recent years, the importance of the plasminogen activator (PA) and its adjustment factor, plasminogen activator inhibitor-1 (PAI-1), in the prevention of atherosclerosis has gradually received recognition. The objective of this in vitro study was to demonstrate the effects of sesamol on PA and PAI-1. We also compared the effects of sesamol with two well-known antioxidants, vitamins C and E, by using human umbilical vein endothelial cells as an experimental model and by treating them with the above-mentioned three nutrients with doses up to 100 μmol/L. After 24 h, cells and cultural medium were collected for analysis. The concentrations of tissue PA (tPA), urokinase PA (uPA) and PAI-1 were measured by an enzymatic immunity method. Northern blot method was used to analyze the expression of mRNA of these three types of proteins. The results showed that sesamol increased the production of uPA and tPA significantly and also up-regulated the mRNA expressions of these proteins. On the other hand, vitamins C and E could induce tPA but not uPA. As for PAI-1, none of the nutrients induced any evident response. These findings suggest that the overall vascular fibrinolytic capacity may be enhanced by using sesamol to regulate PA gene expression.

Several Mechanisms Contribute to the Basic Carboxypeptidase Mediated Downregulation of Fibrinolysis.

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is an intrinsically unstable basic carboxypeptidase which removes C-terminal lysine residues from plasmin-degraded fibrin. This modification of the fibrin surface attenuates fibrinolysis via several mechanisms. First, plasminogen activation is downregulated due to the loss of high affinity plasminogen binding. Second, the rate of plasmin inhibition is increased due to the loss of the protection from inhibition that degraded fibrin confers on plasmin. Finally, fibrin degradation by plasmin becomes less efficient due to the loss of cleavage cooperativity. Although carboxypeptidase-mediated downregulation of the fibrinolytic process has been studied, the relative contribution of each mechanism to the aggregate antifibrinolytic process remains unclear. We, therefore, undertook the following study to measure the contribution of plasmin protection and fibrin cleavage cooperativity to the overall antifibrinolytic effect. To this end, we studied the lysis of clots formed from purified fibrinogen, thrombin and plasmin in the presence and absence of various concentrations of aprotinin, a tight-binding but reversible plasmin inhibitor, and pancreatic carboxypeptidase B (CPB), a stable carboxypeptidase highly homologous to TAFIa. To determine the overall antifibrinolytic effect, we studied the lysis of clots formed from the same components, but using plasminogen with various concentrations of tissue plasminogen activator (tPA) in place of plasmin. In the absence of the inhibitor, CPB saturably increased the plasmin-mediated lysis time by 1.2-fold of that seen in the absence of CPB, suggesting that the loss of cleavage cooperativity increases lysis time by ~20%. The effect of plasmin protection from aprotinin, mediated by recruitment of plasmin to the degraded fibrin surface, was demonstrated using a fixed concentration of plasmin (20nM) and various concentrations of aprotinin (5–20nM). When plasmin was equimolar with or in excess of aprotinin, CPB saturably prolonged lysis by a factor which increased as the concentration of aprotinin increased: saturating CPB (50nM) increased the lysis times by 1.3, 1.5, 1.9 and 2.3-fold at 5, 10, 15 and 20nM aprotinin. When an excess of aprotinin was included (30nM), CPB saturably increased the plasmin-mediated lysis time identically over a range of plasmin concentrations: saturating CPB (50nM) increased the lysis times 2.4, 2.4, 2.3 and 2.3-fold at plasmin concentrations of 10, 15, 20 and 25nM, respectively. Since the total increase (2.4-fold) results from the loss of plasmin protection in addition to the loss of cleavage cooperativity (1.2-fold), we conclude that the loss of plasmin protection increases lysis time ~ 2.4/1.2 = 2-fold. When plasminogen (200nM) and tPA (25–200pM) were used in place of plasmin, CPB also saturably increased lysis time: saturating CPB (50nM) increased lysis times by ~ 15-fold at 150nM aprotinin and by ~ 17-fold at 300nM aprotinin for 25, 50, 100 and 200pM tPA. Since the total increase in lysis time (~ 16-fold) in these experiments results from the downregulation of plasminogen activation in addition to the loss of cleavage cooperativity (1.2-fold) and the loss of plasmin protection (2-fold), we estimate that the downregulation of plasminogen activation resulting from sustained removal of C-terminal lysine residues by a basic carboxypeptidase during fibrinolysis increases clot lysis time by 16/(1.2 x 2) ~ 6.7-fold.

Tissue plasminogen activator (t-PA) and placental plasminogen activator inhibitor (PAI-2) in gingival crevicular fluid from patients with Papillon-Lefèvre syndrome.

Numerous patients with Papillon-Lefèvre syndrome (PLS) express a severe periodontal inflammation that results in premature loss of deciduous and permanent teeth. The plasminogen activating (PA) system is involved in physiological and pathological processes including epithelial healing, extracellular proteolysis and local inflammatory reactions. The aim of the study was to explore a possible role of the PA system in patients with PLS. Samples of gingival crevicular fluid (GCF) were collected from areas with gingival infection in 20 patients with PLS and in 20 healthy controls. The concentration of tissue plasminogen activator (t-PA) and inhibitor (PAI-2) was measured with ELISA. The median level of PAI-2 was significantly higher (p < 0.01) in PLS patients than in the controls, while the median value of t-PA did not differ between the groups. No difference in t-PA or PAI-2 levels was found regarding age, gender or presence of active periodontal disease. The findings indicate an atypical activity of the PA system with a disturbed epithelial function in PLS patients, suggesting that the periodontal destruction seen in patients with PLS is secondary to a hereditary defect in the defense system.

Hemostatic factors as risk markers for intracerebral hemorrhage: a prospective incident case-referent study.

Abnormalities in the hemostatic system may cause hemorrhagic complications. The aim of the present study was to examine whether total concentrations of tissue plasminogen activator (tPA), plasminogen inhibitor-1 (PAI-1), tPA/PAI-1 complex, von Willebrand factor (VWF), and soluble thrombomodulin were associated with a first-ever intracerebral hemorrhage (ICH). This prospective study was an incident case-referent study nested within the Västerbotten Intervention Program and the Northern Sweden Monitoring Trends and Determinants in Cardiovascular Disease (MONICA) cohorts. By 2000, approximately 74 000 subjects had been screened, and 39 ICH cases were defined according to the World Health Organization MONICA criteria. A total of 78 matched controls were selected from the same cohort. The average time from screening to the ICH event was 5.1 years. tPA/PAI-1 complex, systolic and diastolic blood pressures, and hypertension were associated with ICH in the univariate analysis. In the multivariate model, only hypertension (odds ratio [OR], 3.96; 95% confidence interval [CI], 1.27 to 12.36) and the tertile with the highest level of VWF compared with the lowest tertile (OR, 0.27; 95% CI, 0.08 to 0.90) were independently associated with ICH. The OR for the combined exposure to hypertension and low levels of VWF was 8.95, indicating a possible synergistic interaction. No associations were observed for smoking, cholesterol, body mass index, PAI-1, tPA, and soluble thrombomodulin. This study showed that hypertension and low concentrations of VWF were independently associated with ICH. Furthermore, we observed a possible synergistic interaction between low levels of VWF and hypertension, suggesting 2 different pathways in the development of ICH.