Vampire bat salivary plasminogen activator is quiescent in human plasma in the absence of fibrin unlike human tissue plasminogen activator

The vampire bat salivary plasminogen activator (Bat-PA) is a potent PA that exhibits remarkable selectivity toward fibrin-bound plasminogen (Gardell et al, J Biol Chem 256: 3568, 1989). Herein, we describe the activity of recombinant DNA-derived Bat-PA (rBat-PA) in a human plasma milieu. rBat-PA and recombinant human single-chain tissue plasminogen activator (rt-PA) are similarly efficacious at lysing plasma clots. In stark contrast to rt-PA, the addition of 250 nmol/L rBat-PA to plasma in the absence of a clot failed to deplete plasminogen, alpha 2-antiplasmin and fibrinogen. The lytic activities exhibited by finger-domain minus Bat-PA (F- rBat-PA) and finger and epidermal growth factor-like domains minus Bat-PA (FG- rBat-PA) were less than rBat-PA, especially at low concentrations of PA; nevertheless, these truncated forms also possessed a strict requirement for a fibrin cofactor. The loss of PA activity following the addition of rBat-PA to plasma was slower than that observed when either rt-PA or two-chain rt-PA was added. The efficacy, fibrin selectivity, and decreased susceptibility to inactivation exhibited by rBat-PA in vitro in a human plasma milieu suggests that rBat-PA may be superior to rt-PA for the treatment of thrombotic complications.     

Bradykinin stimulates tissue plasminogen activator release in human vasculature.

Bradykinin stimulates tissue plasminogen activator (tPA) release in isolated perfused animal tissues. The present study tests the hypothesis that bradykinin increases tPA release in humans through local effects on the vasculature. Graded doses of sodium nitroprusside (0.8 to 3.2 micrograms/min), acetylcholine (ACh) (7.5 to 60 micrograms/min), and bradykinin (100 to 400 ng/min) were administered intra-arterially in random order in 10 salt-depleted (10 mmol/d of Na) normotensive volunteers. None of the drugs altered mean arterial pressure or heart rate. Forearm blood flow (FBF) was measured by strain-gauge plethysmography. All 3 drugs caused a dose-dependent increase in FBF, although ACh was less potent than either nitroprusside or bradykinin (maximum FBF 7.5+/-2.4 versus 10.0+/-1.5 and 11.9+/-2.1 mL. 100 mL-1. min-1, respectively). Bradykinin caused a significant, dose-dependent increase in venous (effect of dose F=9. 9, P=0.028 by ANOVA), but not arterial (F=0.154, P=0.92) tPA antigen in the infused arm. Thus, net tPA release increased significantly in response to bradykinin (50.6+/-13.3 at the highest dose versus 0. 9+/-0.4 ng. 100 mL-1. min -1 at baseline, P=0.014). In contrast, bradykinin did not affect plasminogen activator inhibitor antigen. Neither nitroprusside nor ACh altered plasma levels of tPA or plasminogen activator inhibitor antigen. Bradykinin increased tPA release across the forearm in the absence of systemic effects. This effect could not be attributed to changes in blood flow because doses of equivalent potency of the vasodilator nitroprusside did not increase tPA. These data demonstrate that bradykinin stimulates tPA release in the human vasculature.     

Activity of tissue plasminogen activator and plasminogen activator inhibitor in noninsulin-dependent diabetes mellitus

The activity of free tissue plasminogen activator (f-tPA) and plasminogen activator inhibitor (PAI) in the plasma of 82 noninsulin-dependent diabetics (NIDDM) was measured by bioimmunoassay of the euglobulin fraction obtained from the plasma, and the levels were compared with those of age- and gender-matched normal subjects. Comparison of these levels in both groups revealed that the f-tPA activity tended to be lower in NIDDM than in the controls, although the differences were not significant. Normal activity of PAI was seen, but f-tPA in NIDDM, when accompanied by macroangiopathy such as ischemic heart disease, was significantly depressed. When glycosylated hemoglobin levels were in excess of 10%, the f-tPA activity was significantly decreased, but no reduction was found in PAI activity as compared with controls. When NIDDM is associated with either macroangiopathy or high glycosylated hemoglobin levels, a decreased f-tPA activity, rather than an increased PAI activity, may contribute to the development of a defective fibrinolytic state.     

Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene

Neuronal migration is a critical phase of brain development, where defects can lead to severe ataxia, mental retardation, and seizures. In the developing cerebellum, granule neurons turn on the gene for tissue plasminogen activator (tPA) as they begin their migration into the cerebellar molecular layer. Granule neurons both secrete tPA, an extracellular serine protease that converts the proenzyme plasminogen into the active protease plasmin, and bind tPA to their cell surface. In the nervous system, tPA activity is correlated with neurite outgrowth, neuronal migration, learning, and excitotoxic death. Here we show that compared with their normal counterparts, mice lacking the tPA gene (tPA(-/-)) have greater than 2-fold more migrating granule neurons in the cerebellar molecular layer during the most active phase of granule cell migration. A real-time analysis of granule cell migration in cerebellar slices of tPA(-/-) mice shows that granule neurons are migrating 51% as fast as granule neurons in slices from wild-type mice. These findings establish a direct role for tPA in facilitating neuronal migration, and they raise the possibility that late arriving neurons may have altered synaptic interactions.     

组织型纤溶酶原激活剂及其抑制剂与肺血栓栓塞症

肺血栓栓塞症（PTE）的发病与机体的纤溶和凝血系统功能密切相关。组织型纤溶酶原激活物（t-PA）及其抑制物（PAI-1）因调节机体的纤溶系统而在静脉血栓形成及栓塞性疾病的发病机制中发挥重要作用,因此,本文对t—PA和PAI-1与PTE的关系作如下综述。     

Binding of tissue plasminogen activator to human monocytes and monocytoid cells

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 mumol/L) and high capacity (0.71 to 3.3 x 10(6) receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 mumol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 10(6) sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 mumol/L and 1.1 x 10(6) sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inactivated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50 (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125I-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.     

Thrombin regulation of mRNA levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in cultured human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells release tissue plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) in response to alpha thrombin stimulation. In order to study the mechanisms of thrombin stimulation, we measured changes in levels of mRNA for t-PA and PAI-1 following exposure of endothelial cells to 3 U/mL alpha thrombin. Alpha thrombin causes a significant and time- dependent increase in the mRNA levels of both t-PA and PAI-1. Catalytically inactivated diisofluorophosphate (DIP) treated thrombin and alpha thrombin pretreated with hirudin do not alter t-PA and PAI-1 mRNA levels. We conclude that the increased secretion of t-PA and PAI-1 by human umbilical vein endothelial cells in response to alpha thrombin is mediated at least partially through an increase in mRNA levels. In addition, an active thrombin catalytic site is required for these increases in mRNA to occur.     

Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties

Native tissue plasminogen activator (ntPA) has a variable glycosylation site on its kringle-2 domain. We have examined the effects of kringle glycosylation on functional properties by studying the simplified tPA molecule, tPA-6. tPA-6 is composed of kringle-2 and the serine protease domains and, like ntPA, cells expressing tPA-6 process it into two glycoforms: the monoglycosylated tPA-6-primary (tPA-6P, type II) with N- linked glycosylation at Asn-448 in the serine protease domain and diglycosylated tPA-6-variant (tPA-6V, type I) with glycosylation at Asn- 448 and at Asn-184 in kringle-2. When the two glycoforms were separated, we found that purified tPA-6V had reduced fibrin-stimulated plasminogenolytic activity toward Glu-plasminogen when compared to purified tPA-6P. However, in the presence of fibrin, tPA-6V unexpectedly exhibited a sixfold increase in selectivity toward Lys- plasminogen. In addition, tPA-6V was less susceptible than tPA-6P to plasmin-mediated conversion to the two-chain form. By site-directed mutagenesis of tPA-6, we eliminated variable glycosylation at Asn-184 and engineered a new glycosylation signal at a remnant site in the kringle. This derivative, designated tPA-6D, was secreted with complete kringle glycosylation. Like the naturally occurring tPA-6V, tPA-6D had lower rates of fibrin-stimulated Glu-plasminogen activation, increased specificity toward Lys-plasminogen, and greater resistance to plasmin digestion. Although the activity of tPA-6D could be stimulated by fibrin, its activity was not stimulated significantly by fibrinogen, and in human plasma the rate of fibrinogen depletion was reduced threefold. Although fibrin binding to kringle-2 of tPA-6D was slightly improved, there was a substantial increase in the dissociation constant (kd) for lysine binding, demonstrating a lack of correlation between these ligand-binding sites. Overall, our data demonstrate the marked effect of kringle glycosylation on functional properties. In addition, we have generated a derivative with properties that potentially improve clot specificity and single-chain half-life and reduce the potential for plasminogen activation in the plasma.     

Inhibition of tissue plasminogen activator activity by aspirin in vivo and its relationship to levels of tissue plasminogen activator inhibitor antigen, plasminogen activator and their complexes

The observation that aspirin inhibits the increment in tissue plasminogen activator (t-PA) activity induced by venous occlusion of the forearm became controversial with the publication of several nonconfirmatory studies. The current study was performed to confirm the original observation and determine the mechanism by which aspirin suppresses the incremental t-PA activity induced by venous occlusion. Aspirin (650 mg/d X 2) caused no change in resting levels of t-PA antigen (t-PA:Ag) or activity, plasminogen activator inhibitor 1 antigen (PAI-1:Ag), or activity or t-PA-PAI-1 complexes. In contrast, aspirin reduced the increments induced by venous occlusion as follows: t-PA:Ag by 45% (P = .001); t-PA activity (euglobulin lysis time, ELT) by 43% (P = .006); and t-PA activity (alpha 2-plasmin inhibitor-plasmin complexes, PIPC) by 41% (P = .003). The inhibition of incremental t-PA activity measured as ELT or PIPC was linearly correlated with the inhibition of incremental t-PA:Ag (respectively, r = .75, P less than .02; r = .67, P less than .05). Aspirin had no effect on the increment in PAI-1:Ag induced by venous occlusion, but similar to the effect on t- PA:Ag, aspirin induced a 51% inhibition of the increment in t-PA-PAI-1 complex formation. Aspirin did not alter the ability of alpha 2-plasmin inhibitor to bind plasmin, nor the ability of plasma to support the fibrin-catalyzed generation of plasmin by t-PA, nor the subsequent formation of PIPC. Aspirin inhibits the t-PA activity induced by venous occlusion primarily by inhibiting the release of t-PA antigen.     

Kinetic studies of plasminogen activation by epithelial tissue plasminogen activator

Initial-rate kinetic studies of the activation of plasminogen by epithelial activator were performed in the absence and in the presence of fibrinogen and CNBr-digested fibrinogen, under assay conditions similar to those described by Hoylaerts et al. (J Biol Chem, 257, 2912, 1982). In the purified system, and in the absence of any stimulator, Lys-plasminogen is more readily activated than Glu-plasminogen, with catalytic rate constants of 0.01 s-1 and 0.0034 s-1 and Michaelis constants of 1.2 microM respectively. With Glu-plasminogen, double reciprocal plots deviated from linearity at low concentrations of plasminogen, in agreement with the findings reported for melanoma activator. In the presence of fibrinogen, activation rates for both Lys and Glu-plasminogen were increased. (kcat = 0.017 and 0.041 s-1 and km = 1.4 and 41 microM, respectively). In the presence of CNBr-fragments of fibrinogen, the Michaelis constant is lowered for both forms of plasminogen, (km = 0.3 microM) thus indicating high affinity and efficient activation of plasminogen on fibrin clot. Comparison of the kinetic data with those reported for melanoma activator suggest that even though the values of the kinetic constants are different, epithelial activator has a similar mechanism of action for the activation of plasminogen as the melanoma enzyme.     

Gene polymorphisms of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with antiphospholipid antibodies

OBJECTIVE: Impaired fibrinolytical outcomes may be one of the pathogenic factors for thrombotic events in patients with antiphospholipid antibodies (aPL). We investigated the consequences of the gene polymorphisms of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) in patients positive for aPL. METHODS: Seventy-seven Japanese and 82 British patients with aPL were examined for Alu-repeat insertion (I)/deletion (D) polymorphism of the tPA gene by polymerase chain reaction (PCR), and 4G/5G polymorphism in the PAI-1 promoter gene by site-directed mutagenesis-PCR and restriction fragment length polymorphism analysis. Correlations between these polymorphisms and clinical symptoms of antiphospholipid syndrome (APS) (arterial thrombosis, venous thrombosis, miscarriage) were analyzed. RESULTS: Significant differences in the allele frequencies of these genes did not exist between patients and controls. There was no significant correlation between these gene polymorphisms and clinical symptoms of APS in patients with aPL. CONCLUSION: Polymorphisms of the tPA or PAI-1 genes probably do not significantly influence the risk of anerial thrombosis, venous thrombosis, or pregnancy morbidity in patients with aPL.     

Determination of plasma tissue type plasminogen activator and plasminogen activator inhibitor activity in patients with ischemic stroke

Plasma tissue-type plasminogen activator and plasminogen activator inhibitor were determined during the acute, recovery and sequelae stages of patients with ischemic stroke by chromophoric substrate assay. The result showed that t-PA activity was elevated during the acute phase, remained elevated during the recovery stage and declined during the sequelae stage. Lowering of PAI activity was found during acute phase, which reversed during recovery phase and remained significantly elevated during sequelae stage. As a result, the ratio of PAI/t-PA fluctuated during different stages of the disease. Significant elevation of PAI and PAI/t-PA ratio during sequelae stage may be one of the risk factors of further thrombosis and contribute partly to the high relapsing rate of the disease. In addition, a positive correlation was found between PAI and serum cholesterol content.     

Circadian fluctuations of tissue plasminogen activator antigen and plasminogen activator inhibitor-1 antigens in vasospastic angina.

To elucidate the circadian variation of fibrinolytic components in vasospastic angina, plasma levels of tissue plasminogen activator antigen (t-PA), free plasminogen activator inhibitor antigen (free PAI-1), t-PA/PAI-1 complex, and total PAI-1 were measured in venous plasma samples. Samples were taken every 6 hours (6:00 AM, noon, 6:00 PM, and midnight) for 24 hours in 14 patients with vasospastic angina, in 9 patients with exertional angina, and in 19 normal subjects. Twenty-four-hour Holter monitoring (Holter monitor, Del Mar Avionics, Irvine, Calif.) was also carried out in all subjects. All of the fibrinolytic components showed circadian variation, with a peak level at 6:00 AM in every study group except for the t-PA/PAI-1 complex in the group of patients with exertional angina. The values for all or the fibrinolytic components at each sampling time were higher in patients with coronary artery disease than in normal subjects. In particular, the mean value of free PAI-1 at 6:00 AM in patients with vasospastic angina was significantly higher than that in normal subjects and that in patients with exertional angina. This value of free PAI-1 in patients with vasospastic angina was closely associated with the duration of ischemic attacks. These results suggested that the circadian fluctuation of fibrinolytic components may be an important factor that leads to coronary thrombosis at the time of coronary spasm, especially in the early morning.     

Key role of tissue plasminogen activator in neurovascular coupling

The increase in blood flow evoked by synaptic activity is essential for normal brain function and underlies functional brain imaging signals. Nitric oxide, a vasodilator released by NMDA receptor activation, is critical for the flow increase, but the factors linking NMDA receptor activity to nitric oxide-dependent hyperemia are poorly understood. Here, we show that tissue plasminogen activator (tPA), a serine protease implicated in NMDA receptor signaling, is required for the flow increase evoked by somatosensory stimulation. tPA acts by facilitating neuronal nitric oxide release, but this effect does not involve enhancement of NMDA currents or the associated intracellular Ca(2+) rise. Rather, the evidence suggests that tPA controls NMDA-dependent nitric oxide synthesis by influencing the phosphorylation state of neuronal nitric oxide synthase. These findings unveil a previously unrecognized role of tPA in vital homeostatic mechanisms coupling NMDA receptor signaling with nitric oxide synthesis and local cerebral perfusion.     

Regulation of tissue plasminogen activator in sickle cell anemia

Evidence of activation of the clotting system in individuals with sickle cell anemia (SCA) has been observed by several investigators. It has been suggested that the clotting and fibrinolytic systems may play a role in the pathophysiology of vaso-occlusion in SCA. We reported previously evidence of abnormal fibrinolytic activity as reflected in decreased releasable tissue plasminogen activator (t-PA) using a functional assay. We have examined the mechanism of the decreased functional releasable t-PA in individuals with SCA. We studied 12 patients with respect to releasable t-PA, fast acting inhibitor to t-PA (or PAI-1), and immunoreactive or antigenic t-PA. These SCA individuals were at their baseline states and not taking medications known to interfere with the fibrinolytic or clotting systems. We found that the mean releasable t-PA for the SCA individuals was 0.01 IU/ml of plasma with a standard error of mean (SEM) of 0.01. The mean releasable t-PA of 118 healthy normal controls was 0.70 IU/ml with SEM 0.10 (P less than .001). The mean level of fast-acting inhibitor to t-PA in unoccluded circulation of the SCA patients' plasma was 16.5 IU/ml with SEM of 3.54. The mean plasma levels of fast-acting inhibitor to t-PA in 56 healthy controls was 2.56 IU/ml with SEM of 0.29 (P less than .0001). The SCA patients had a mean baseline t-PA antigen level of 5.98 ng/ml with SEM of 1.72. The mean level of t-PA antigen of 78 healthy controls using the same technique was 4.3 ng/ml with SEM of 2.7 (not significant). The mean baseline functional t-PA for SCA individuals was 0.15 IU/ml with SEM 0.01 and the mean baseline functional t-PA for 118 controls was 0.17 IU/ml with SEM 0.10. These data suggest that the mechanism of decreased releasable t-PA in sickle cell anemia is related to an elevation of fast-acting inhibitor to t-PA and that antigenically t-PA is present in normal quantities in the baseline plasma in this population.