Development of a new test for the global fibrinolytic capacity in whole blood.

BACKGROUND: The development of global tests for the fibrinolytic capacity in blood is hampered by the low base-line fibrinolytic activity in blood, by the involvement of both plasmatic components and blood cells in the fibrinolytic system and by the loss of fibrinolytic activity as a result of the action of plasminogen activator inhibitor-1 (PAI-1). OBJECTIVE: To develop a new test for the global fibrinolytic capacity (GFC) of whole blood samples. METHODS AND RESULTS: Collection of blood in thrombin increased the subsequent generation of fibrin degradation products. This was ascribed to rapid clot formation and concomitant reduction of in vitro neutralization of tissue-type plasminogen activator (tPA) by PAI-1. On the basis of this observation, the following test was designed: blood samples were collected in thrombin with and without aprotinin and clots were incubated for 3 h at 37 degrees C. The GFC was assessed from the difference between the fibrin degradation products in the two sera. The assay was applied to blood samples from patients and healthy subjects. Other hemostasis parameters were determined in plasma samples taken simultaneously. The GFC varied considerably (normal range 0.13-13.6 microg mL(-1)); physical exercise strongly increased the GFC. Statistically significant correlations were found with tPA activity, PAI-1 activity and fibrinogen level. A mixture of antibodies against tPA and urokinase-type plasminogen activator (uPA) completely inhibited the GFC. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFI) accelerated fibrinolysis 8-fold. CONCLUSION: The new test represents a global assessment of the main fibrinolytic factors in plasma and potentially those associated with blood cells.

Clot penetration and fibrin binding of amediplase,a chimeric plasminogen activator (K2 tu-PA).

Amediplase (K(2) tu-PA) is a hybrid plasminogen activator, consisting of the kringle 2 domain of alteplase and the protease domain of urokinase. The objective of this study was to determine the in vitro clot penetration of amediplase in relation to its fibrin binding and to compare the properties with those of alteplase. The clot lysis activity of amediplase in internal clot lysis models (both purified system and plasma system) was about 10 times less than that of alteplase. The clot lysis activity of amediplase in an external clot lysis model (plasma system) was similar to that of alteplase at therapeutic concentrations around 1 micro g/ml. The fibrin-clot binding properties of amediplase and alteplase were studied in a purified system as well as in a plasma system. In both systems amediplase bound to fibrin although to a significantly lower extent than alteplase. The binding of amediplase or alteplase did not increase during plasmin-mediated degradation of fibrin. The binding of amediplase was fully inhibited by epsilon-aminocaproic acid, indicating that the observed binding was specific and occurred via the lysine binding site in the kringle of amediplase. Clot penetration was studied during pressure-driven fluid permeation using syringes containing plasma clots. Amediplase was able to enter the clot without significant hindrance, while alteplase was concentrated on the top of the plasma clot and hardly entered into the inner parts of the clot. Diffusion-driven clot penetration was studied during clot lysis using confocal microscopy. Alteplase was detected on or close to the clot surface, while two-chain urokinase, which has no affinity to fibrin, was also detected deep inside the clot. Amediplase showed a penetration behaviour, which was distinct from that of alteplase and similar to that of two-chain urokinase. We concluded that the fibrin binding of amediplase is moderate and does not hinder clot penetration under permeation-driven or diffusion-driven transport conditions. Enhanced clot penetration, especially in large clots, could allow a more efficient lysis during thrombolytic therapy.

Binding and retention of polycationic peptides and dendrimers in the vascular wall.

Extracellular matrix (ECM) of tissues, vascular tissue in particular, contains a high concentration of negatively charged glycosaminoglycans (GAGs), which are involved in the regulation of cell motility, cell proliferation and the regulation of enzyme activities. Previously, we have shown that the vascular ECM is capable of binding an extremely high concentration of positively charged molecules, such as polylysine. Vascular ECM can be used therefore as a substrate for binding and retention of drugs delivered intravascularly, if these drugs are endowed with an ability to bind to the vascular ECM. In this study, we evaluated a number of positively charged molecules as potential affinity vehicles for delivery of drugs to the vascular ECM. We labelled the molecules of interest with fluorescence and compared them ex vivo in terms of binding and retention in the de-endothelialised rat carotid artery after intravascular delivery under pressure. High molecular weight polylysine (84 kDa) and polyamidoamine (PAMAM) dendrimers accumulated in the wall of the artery up to a concentration of 10 mg/ml and were not washed away significantly after 4 h of perfusion of the artery. A 24-mer peptide containing a consensus sequence for binding to GAGs (ARRRAARA)(3), 2.7 kDa, was comparable to high molecular weight polylysine and dendrimers in terms of binding and retention. A 14-mer GAG-binding peptide from vitronectin and low molecular weight polylysine, 3 kDa, accumulated in the vascular wall up to about 3 mg/ml and was washed away after 30 min of perfusion. A 10-mer consensus GAG-binding peptide did not bind significantly to the vascular tissue. We conclude that the consensus 24-mer GAG-binding peptide is by far superior to polylysine of a similar molecular weight in terms of binding to vascular tissue, and can provide high accumulation and long-term retention of a low molecular weight compound (fluorescein, as a model molecule) in the vascular wall. Rationally designed GAG-binding peptides can be useful as affinity vehicles for targeting drugs to the vascular ECM.

Polylysine as a vehicle for extracellular matrix-targeted local drug delivery, providing high accumulation and long-term retention within the vascular wall.

We present the first steps in the elaboration of an approach of extracellular matrix-targeted local drug delivery (ECM-LDD), designed to provide a high concentration, ubiquitous distribution, and long-term retention of a drug within the vessel wall after local intravascular delivery. The approach is based on the concept of a bifunctional drug comprising a "therapeutic effector" and an "affinity vehicle," which should bind to an abundant component of the vessel wall. The aim of the present study was to select molecules suitable for the role of affinity vehicles for ECM-LDD and to study their intravascular delivery and retention ex vivo and in an animal model. By use of fluorescence microscopy, the following molecules were selected on the basis of strong binding to cross sections of human vessels: protamine, polylysine, polyarginine, a glycosaminoglycan-binding peptide from vitronectin, and a synthetic dendrimer. With polylysine as a prototypic affinity vehicle, we showed that after intravascular delivery, polylysine was concentrated throughout a luminal layer of the vascular wall to an extremely high concentration of 20 g/L and was retained therein for at least 72 hours of perfusion without noticeable losses. Low molecular weight (fluorescein) and high molecular weight (hirudin) compounds could be chemically conjugated to polylysine and were retained in the vessel wall after intravascular delivery of the conjugates. In conclusion, by use of the ECM-LDD method, an extremely high concentration and long-term retention of locally delivered drug can be reached. Polycationic molecules can be considered as potential affinity vehicles for ECM-LDD.

The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells.

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cell-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA/T that was generated by thrombin was not affected by the monoclonal antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracellular proteolysis during processes in which thrombin is also involved.

Urokinase-mediated fibrinolysis in the synovial fluid of rheumatoid arthritis patients may be affected by the inactivation of single chain urokinase type plasminogen activator by thrombin.

BACKGROUND: Excessive fibrin deposition within the inflamed joints of rheumatoid arthritis (RA) patients suggests that local fibrinolysis is inefficient, which seems to be in contrast with the observed increased levels of urokinase type plasminogen activator (u-PA). Thrombin-mediated inactivation of single chain u-PA (scu-PA) into an inactive form called thrombin-cleaved two chain u-PA (tcu-PA/T) may provide a possible explanation for this contradiction. AIM: To assess the occurrence of tcu-PA/T in the synovial fluid of patients with RA and with osteoarthritis (OA), and in the synovial fluid of controls to find support for thrombin-mediated inactivation of scu-PA in RA. METHODS: Levels of scu-PA and tcu-PA/T were measured in the synovial fluid of 20 RA patients, nine OA patients and 14 controls using sensitive bioimmunoassays. Total urokinase antigen was quantified by a urokinase ELISA. RESULTS: tcu-PA/T was found in the synovial fluid of all RA and OA patients. Only in seven of 14 control samples, levels of tcu-PA/T could be measured above the detection limit of the assay (0.2 ng/ml). The concentrations of tcu-PA/T, scu-PA and u-PA:Ag were significantly higher in the synovial fluid of the RA and OA patients as compared with the controls, while the RA patients had significantly higher levels of tcu-PA/T and u-PA:Ag than the OA patients. In RA, tcu-PA/T seemed to account for more than 40% of total urokinase antigen, while the contribution of tcu-PA/T to total urokinase antigen was only minor in OA and the controls (9.0% and 6.6%, respectively). CONCLUSION: A significant part of the high total urokinase antigen in the synovial fluid of RA patients can be attributed to tcu-PA/T, implying that a large amount of scu-PA is not available for fibrinolysis because of its inactivation by thrombin. Thus, thrombin may promote the inflammation process in RA by inhibiting the fibrinolytic system and preventing the removal of fibrin.

Interaction of reteplase with heparin. A comparison between reteplase and alteplase.

Reteplase is a protein consisting of the kringle-2 and protease domains of tissue-type plasminogen activator (t-PA). Because intravenous heparin will be used as an adjunct to thrombolytic therapy with reteplase, we investigated the interactions in vitro between heparin and reteplase as well as between heparin and recombinant t-PA (alteplase) as a control. Reteplase and alteplase bound completely to a heparin-agarose column and eluted respectively at 0.39 M and 0.60 M in a NaCl gradient. Two-chain derivatives of reteplase and alteplase eluted at 0.31 M and 0.52 M NaCl respectively. Plasminogen activation by the two-chain derivatives of reteplase and alteplase in a purified system at low ionic strength were stimulated by heparin up to 13- and 22-fold, respectively. However, reteplase required five times more heparin for maximal stimulation than alteplase. In addition, the heparin stimulation of reteplase was more salt sensitive than that of alteplase. In conclusion, both heparin binding and heparin stimulation experiments showed that heparin interacts with reteplase, but the interaction is weaker than with alteplase.

Interaction of plasminogen activators and plasminogen with heparin: effect of ionic strength.

In order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparin-agarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

Functional analogy between lipoprotein(a) and plasminogen in the binding to the kringle 4 binding protein, tetranectin.

Apolipoprotein(a), apo(a), contains 37 repeats structurally homologous to kringle 4 structures of the fibrinolysis zymogen plasminogen. The aim of the study was to explore the functional analogy between apolipoprotein(a) and plasminogen in the binding to the kringle-4-binding plasma protein, tetranectin. With a modified crossed immunoelectrophoresis technique, reversible binding between lipoprotein(a) and tetranectin could be demonstrated with an apparent Kd of 0.013 muMol/l. Lys- and Glu-plasminogen showed an apparent Kd of 0.5 muMol/l. Binding of lipoprotein(a) to fibrin and to fibrin-bound tetranectin was found to be negligible. The absence of fibrin binding of lipoprotein(a) excludes a potential mechanism of coexistence of fibrin and lipid deposits in arterial diseases and does not provide for a link between lipoprotein and the clotting system. Plasminogen and lipoprotein(a) show functional analogy in their binding to tetranectin, but tetranectin primarily targets at lipoprotein(a).

Acid treatment of plasma for the inactivation of plasminogen activator inhibitor-1 (PAI-1).

The present study was initiated to assess the effectiveness of various acid treatments of blood or plasma in the inactivation of PAI-1. It was shown that a frequently used treatment of blood or plasma with 1 M acetate buffer, pH 3.9, only partially inactivated PAI-1. The inactivation of PAI-1 in plasma was found to depend upon pH and temperature, showing an optimal inactivation at a pH less than or equal to 3, at 37 degrees C.

Haemostasis contact system and fibrinolysis in hereditary angioedema (C1-inhibitor deficiency).

Factors of the classical complement pathway, the contact system and fibrinolysis were evaluated both with functional and immunochemical methods, in patients with inherited deficiency of C1-inhibitor. Evaluations were performed under basal conditions, during acute attacks and during prophylaxis with low doses of anabolic steroids. Patients in the basal state showed no significant abnormalities of any of the parameters that we investigated. During acute attacks a slightly reduced prekallikrein concentration was registered. During treatment with low doses of danazol and stanozolol, protein C and plasminogen were found to be increased. Our data suggest that C1-inhibitor deficiency per se does not lead to a derangement of the fibrinolysis and coagulation contact system, and that the kinin system may be involved during acute attacks of angioedema.

Daytime fluctuations in blood of tissue-type plasminogen activator (t-PA) and its fast-acting inhibitor (PAI-1).

Circadian fluctuation in blood fibrinolytic activity was studied in 10 volunteers in the day-time period at 09.00, 12.00 and 15.00 h. Activity of tissue-type plasminogen activator (t-PA) was found to increase from 09.00 to 15.00 h in accordance with known results with global assays of blood activity. Also, activity and antigen of plasminogen activator inhibitor 1 (PAI-1) and antigen of t-PA showed fluctuations but with an opposite pattern to that of t-PA activity, with highest levels in the morning and lowest ones in the afternoon. Activity of a reversible t-PA inhibitor was constant. The discordance between fluctuations in antigen and in activity of t-PA is attributed to the inhibitory effect of PAI-1 on t-PA activity. It is concluded that the appearance into the blood of t-PA and PAI-1 shows a daytime fluctuation with a similar pattern, suggesting a co-ordinated circadian fluctuation in production of both proteins by endothelial cells.

The mutual relationship between the two molecular forms of the major fibrinolysis inhibitor alpha-2-antiplasmin in blood.

Alpha-2-antiplasmin, a major inhibitor of fibrinolysis, is synthesized in the liver and occurs in blood in two molecular forms: a very active plasminogen-binding (PB) form and a less active nonplasminogen-binding (NPB) form. This study investigates the origin and mutual relationship of these two forms in vivo and in vitro. Despite wide variation in plasma concentration of the inhibitor (16% to 138%), the ratio between the two forms in vivo was found to be, in the main, constant among healthy volunteers, heterozygotes for a congenital deficiency of alpha-2-antiplasmin, and patients with a stable liver cirrhosis: PB/NPB = 2.41 +/- 0.34 (SD). Resynthesis after depletion or increased synthesis in the acute-phase reaction showed a specific increase of the PB form of the molecule in blood after discontinuation of L-asparaginase or streptokinase therapy and after myocardial infarction. In vitro studies demonstrated that only the PB form was present after one day in the culture medium of the human cell line Hep G2, while the NPB form appeared after 11 days. Clearance after inhibition of synthesis by L-asparaginase therapy revealed a more rapid decrease in the PB form relative to the NPB form in blood, demonstrated by a change in the PB-NPB ratio from 2.86 +/- 0.55 to 1.74 +/- 0.24 (mean of 6, SD). An apparently spontaneous first order conversion from the PB to NPB form, with an apparent half-life of about eight days, was demonstrated at 37 degrees C in plasma and serum in vitro. The conversion was found to be temperature dependent and uninfluenced by the fibrinolytic components fibrinogen, fibrin, and plasminogen. Additions of a variety of enzymes or inhibitors did not interfere with the process. These results demonstrate that the PB form of alpha-2-antiplasmin is produced by the liver and that the NPB form is formed in the circulation.

The postoperative fibrinolytic shutdown: a rapidly reverting acute phase pattern for the fast-acting inhibitor of tissue-type plasminogen activator after trauma.

The plasma activity level of the recently discovered fast-acting inhibitor of tissue-type plasminogen activator (t-PA) was found to be temporarily increased after surgery, myocardial infarction and severe trauma. Detailed analysis of the postoperative period revealed simultaneously increased t-PA antigen and inhibition and decreased t-PA activity only on the first postoperative day. These changes were more rapid than those in fibrinogen and C-reactive protein. It is concluded that t-PA inhibition shows the most rapidly changing pattern observed so far in response to trauma. The postoperative fibrinolytic shutdown in blood fibrinolytic activity can be ascribed to a primary increase in t-PA inhibitor levels.

Identification of a reversible inhibitor of plasminogen activators in blood plasma.

Inhibition of tissue-type plasminogen activator (t-PA) by pooled plasma could be ascribed for only 60% to the endothelial cell type PA inhibitor. The residual inhibition is ascribed to a so-far undescribed plasma component present at 0.2 nmol/l. This component shows reversible binding to t-PA with an apparent Ki of 10 pmol/l (does not hinder t-PA binding to fibrin); also reacts with urokinase, but not with DIP-t-PA; is stable at 37 degrees C and does not occur in media of endothelial cells, hepatocytes and fibroblasts. This PA binding component in plasma adds to the regulation of plasminogen activator activities.

Evaluation of euglobulin methods for the study of blood fibrinolytic activity: results for patients with rheumatoid arthritis and in the postoperative period.

Euglobulin fractionation is a frequently employed pretreatment of plasma for the determination of fibrinolytic activity. The fractionation procedure suffers from possible in vitro artifacts, e.g., variable precipitation of C1-inactivator. This is illustrated by the following two situations. It is shown that increased amounts of C1-inactivator not related to an increased plasma concentration are present in euglobulin fractions in cases of classic rheumatoid arthritis. Similarly, postoperatively, a disproportional increase in C1-inactivator in euglobulin fractions occurs. In both cases, an artificially reduced fibrinolytic activity is recorded due to increased inhibition by C1-inactivator. This is circumvented and recognized by adding sodium flufenamate or C1s-esterase to euglobulin fractions to uniformly eliminate C1-inactivator. Two specific assays for tissue-type plasminogen activator activity in euglobulin fractions (as C1-inactivator-resistant activator activity and a parabolic rate assay on a synthetic substrate) correlate excellently (r = 0.8728; p less than 0.001; n = 108). The first mentioned is corrected for variable endogenous C1-inactivator; the latter assay is found to be insensitive to inhibition by C1-inactivator. It is concluded that with euglobulin methods a misinterpretation of blood fibrinolytic activity is possible in rheumatoid arthritis patients. In the postoperative period, the fibrinolytic shutdown concerns tissue-type plasminogen activator activity; the pattern of the shutdown can be misjudged in using traditional euglobulin methods.

Appropriate milieu for the assay of alpha-2-antiplasmin activity with chromogenic substrates.

Inhibition reference curves for alpha 2-antiplasmin showed a deviation from linearity at low inhibitor concentrations using the chromogenic substrate S-2251. Extrapolation of these curves to zero inhibition gave higher amidolytic activities than actually recorded with the free enzyme plasmin. It was further found that comparison of purified alpha 2-antiplasmin and that in plasma was prevented by the deviation. The difference between calculated and measured plasmin activity could not be attributed to instability of plasmin. It was established that the observations (1) were specific for high concentrations of S-2251, (2) were not the same with another plasmin substrate, chromozym PL, and (3) were related to the addition of plasma proteins. Apparently, the problem was related to the solubility state of S-2251. A solution to this problem is the addition of nonionic detergents, notably Tween 80 (0.01%) or Triton X-100 (0.03%), which prevent all deviations.

Intrinsic plasma fibrinolysis: involvement of urokinase-related activity in the factor XII-independent plasminogen proactivator pathway.

A portion of human blood fibrinolytic activity can be quenched by antibodies to urokinase. We attempted to identify this portion and its position in the three pathways (extrinsic, intrinsic factor XII dependent, and intrinsic factor XII independent) of plasminogen activator activity that have been defined in the DEFs of plasma. Activity quenching in various plasmas (including plasma adsorbed by agarose-bound AUK) demonstrated the involvement of a discrete activator activity of 40 to 50 BAU/ml with little variation among individuals (43 +/- 6 (SD) BAU/ml, n = 13). The quenching did not involve, quantitatively or qualitatively, the extrinsic (tissue-type) plasminogen activator activity varying between 1 and 146 BAU/ml in baseline plasma and in plasma obtained after stimulation by venous occlusion, exercise, or DDAVP administration. In confirmation, extrinsic activator activity was recovered in plasma adsorbed by agarose-bound AUK. The quenching also did not involve the factor XII-dependent activities; it was quantitatively normal in plasma with Hageman (n = 3) and Fletcher (n = 2) traits, and AUK did not interfere with the generation of factor XII-dependent fibrinolytic activity by addition of purified activated factor XII in plasma with Hageman trait. There was no effect of AUK on kallikrein generation, kallikrein activities, or the APPT, nor were these aspects altered in depleted plasma. In conclusion, the quenching involved the factor XII-independent system of intrinsic fibrinolysis. Addition of purified urinary urokinase did not result in restoration of missing activity in plasma adsorbed by AUK-agarose. The quenching, therefore, probably involved an apparent urokinase-related activator component in plasma.

The molecular form of alpha 2-antiplasmin with affinity for plasminogen is selectively bound to fibrin by factor XIII.

Two molecular forms of alpha 2-antiplasmin (PB = plasminogen-binding; NPB = non-PB) exist. We have studied the extent of binding of these two forms to fibrin by factor XIII. A modified crossed immunoelectrophoresis technique which separately determines both forms showed that, compared with plasma, the PB-form is reduced by 31 +/- 11% (SD) in serum of twelve individuals and the NPB form by only 6%. Laurell assay showed a reduction of 18 +/- 9% (n = 12) in total alpha 2-antiplasmin antigen (PB + NPB) in serum; the immediate plasmin inhibition test (mainly recording the PB-form) revealed 35 +/- 6% (n = 12) reduction in inhibition. Coagulation of blood, platelet-rich and platelet-poor plasma yielded comparable results. No decrease in alpha 2-antiplasmin or change in its composition was observed when factor XIII-deficient plasma was clotted. Fibrin binding was not significantly different from normal in either plasminogen-depleted or plasminogen-enriched plasma. It is concluded that the PB-form of alpha 2-antiplasmin becomes selectively bound to fibrin.