Thrombolysis vs. bleeding from hemostatic sites by a prourokinase mutant compared with tissue plasminogen activator

BACKGROUND: A single site mutant (M5) of prourokinase (proUK) was developed to make proUK less vulnerable to spontaneous activation in plasma. This was a problem that seriously compromised proUK in clinical trials, as it precluded proUK-mediated fibrinolysis at therapeutic concentrations. METHODS AND RESULTS: After completing dose-finding studies, 12 anesthetized dogs with femoral artery thrombosis were given either M5 (2.0 mg kg(-1)) or tissue plasminogen activator (t-PA) (1.4 mg kg(-1)) by i.v. infusion over 60 min (20% administered as a bolus). Two pairs of standardized injuries were inflicted at which hemostasis was completed prior to drug administration. Blood loss was quantified by measuring the hemoglobin in blood absorbed from these sites. Thrombolysis was evaluated at 90 min and was comparably effective by both activators. Rethrombosis developed in one t-PA dog. The principal difference found was that blood loss was 10-fold higher with t-PA (mean approximately 40 mL) than with M5 (mean approximately 4 mL) (P = 0.026) and occurred at more multiple sites (mean 2.7 vs. 1.2). This effect was postulated to be related to differences in the mechanism of plasminogen activation by t-PA and M5 in which the latter is promoted by degraded rather than intact (hemostatic) fibrin. In addition, two-chain M5 was efficiently inactivated by plasma C1 inactivator, an exceptional property which helped contain its non-specific proteolytic effect. CONCLUSIONS: Intravascular thrombolysis by M5 was accompanied by significantly less bleeding from hemostatic sites than by t-PA. This was attributed to the proUK paradigm of fibrinolysis being retained at therapeutic concentrations by the mutation.     

Hindered dissolution of fibrin formed under mechanical stress

See also Weisel JW. Stressed fibrin lysis. This issue, pp 977–8. Summary. Background: Recent data indicate that stretching forces cause a dramatic decrease in clot volume accompanied by gross conformational changes of fibrin structure. Objective: The present study attempts to characterize the lytic susceptibility of fibrin exposed to mechanical stress as a model for fibrin structures observed in vivo. Methods and results: The relevance of stretched fibrin models was substantiated by scanning electron microscopic (SEM) evaluation of human thrombi removed during surgery, where surface fibrin fibers were observed to be oriented in the direction of shear forces, whereas interior fibers formed a random spatial meshwork. These structural variations were modeled in vitro with fibrin exposed to adjustable mechanical stress. After two‐ and three‐fold longitudinal stretching (2 × S, 3 × S) the median fiber diameter and pore area in SEM images of fibrin decreased two‐ to three‐fold. Application of tissue plasminogen activator (tPA) to the surface of model clots, which contained plasminogen, resulted in plasmin generation which was measured in the fluid phase. After 30‐min activation 12.6 ± 0.46 pmol mm^?2 plasmin was released from the non‐stretched clot (NS), 5.5 ± 1.11 pmol mm^?2 from 2 × S and 2.3 ± 0.36 pmol mm^?2 from 3 × S clot and this hampered plasmin generation was accompanied by decreased release of fibrin degradation products from stretched fibrins. Confocal microscopic images showed that a green fluorescent protein‐fusion variant of tPA accumulated in the superficial layer of NS, but not in stretched fibrin. Conclusion: Mechanical stress confers proteolytic resistance to fibrin, which is a result of impaired plasminogen activation coupled to lower plasmin sensitivity of the denser fibrin network.     

Plasminogen on the surfaces of fibrin clots prevents adhesion of leukocytes and platelets

Summary. Background and Objectives: Although leukocytes and platelets adhere to fibrin with alacrity in vitro, these cells do not readily accumulate on the surfaces of fibrin clots in vivo. The difference in the capacity of blood cell integrins to adhere to fibrin in vivo and in vitro is striking and implies the existence of a physiologic antiadhesive mechanism. The surfaces of fibrin clots in the circulation are continually exposed to plasma proteins, several of which can bind fibrin and influence cell adhesion. Recently, we have demonstrated that adsorption of soluble fibrinogen on the surface of a fibrin clot results in its deposition as a soft multilayer matrix, which prevents attachment of blood cells. In the present study, we demonstrate that another plasma protein, plasminogen, which is known to accumulate in the superficial layer of fibrin, exerts an antiadhesive effect. Results: After being coated with plasminogen, the surfaces of fibrin clots became essentially non‐adhesive for U937 monocytic cells, blood monocytes, and platelets. The data revealed that activation of fibrin‐bound plasminogen by the plasminogen‐activating system assembled on adherent cells resulted in the generation of plasmin, which decomposed the superficial fibrin layer, resulting in cell detachment under flow. The surfaces generated after the initial cell adhesion remained non‐adhesive for subsequent attachment of leukocytes and platelets. Conclusion: We propose that the limited degradation of fibrin by plasmin generated by adherent cells loosens the fibers on the clot surface, producing a mechanically unstable substrate that is unable to support firm integrin‐mediated cell adhesion.     

Construction and characterization of a recombinant plasminogen activator composed of an anti-fibrin single-chain antibody and low-molecular-weight urokinase.

BACKGROUND: Targeting of plasminogen activators to the fibrin component of a thrombus by antibodies directed against human fibrin can enhance their thrombolytic potency and clot specificity. OBJECTIVES: To overcome the disadvantages of chemical conjugation, we investigated whether the recombinant fusion of a single-chain antibody and a plasminogen activator results in an active bifunctional molecule that might be useful as a therapeutic agent. METHODS: The cDNA of low-molecular-weight single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scuPA(LMW)), was cloned from human endothelial cells and fused to a single-chain antibody specific for the 7 N-terminal amino acids (beta(15-22)) in the beta-chain of human fibrin (scFv(59D8)). The fusion protein was purified using affinity chromatography with the beta(15-22)-peptide of human fibrin. RESULTS: Purified scFv(59D8)-scuPA(LMW) migrated as a 60-kDa band, which is consistent with a molecule composed of one scFv(59D8) and one scuPA(LMW) moiety. Both functions of the fusion molecule, fibrin-specific binding and plasminogen activation, were fully preserved. In human plasma clots, thrombolysis by scFv(59D8)-scuPA(LMW) is significantly faster and more potent compared with the clinically used urokinase. CONCLUSIONS: ScFv(59D8)-scuPA(LMW) constitutes a new recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency and relative fibrin selectivity, that can be produced with modern methods at low cost, large quantities and reproducible activity in Escherichia coli.     

Fibrinogen counteracts the antiadhesive effect of fibrin‐bound plasminogen by preventing its activation by adherent U937 monocytic cells

Summary. Background: Fibrinogen and plasminogen strongly reduce adhesion of leukocytes and platelets to fibrin clots, highlighting a possible role for these plasma proteins in surface‐mediated control of thrombus growth and stability. In particular, adsorption of fibrinogen on fibrin clots renders their surfaces non‐adhesive, while the conversion of surface‐bound plasminogen to plasmin by transiently adherent blood cells results in degradation of a superficial fibrin layer, leading to cell detachment. Although the mechanisms whereby these proteins exert their antiadhesive effects are different, the outcome is the same: the formation of a mechanically unstable surface that does not allow firm cell attachment. Objectives: Since fibrin clots in circulation are exposed to both fibrinogen and plasminogen, their combined effect on adhesion of monocytic cells was examined. Methods: Fibrin gels were coated with plasminogen and its activation by adherent U937 monocytic cells in the presence of increasing concentrations of fibrinogen was examined by either measuring ¹²⁵I‐labeled fibrin degradation products or plasmin amidolytic activity. Results: Unexpectedly, the antiadhesive effects of two fibrin binding proteins were not additive; in fact, in the presence of fibrinogen, the effect of plasminogen was strongly reduced. An investigation of the underlying mechanism revealed that fibrinogen prevented activation of fibrin‐bound plasminogen by cells. Confocal microscopy showed that fibrinogen accumulates in a thin superficial layer of a clot, where it exerts its blocking effect on activation of plasminogen. Conclusion: The results point to a complex interplay between the fibrinogen‐ and plasminogen‐dependent antiadhesive systems, which may contribute to the mechanisms that control the adhesiveness of a fibrin shell on the surface of hemostatic thrombi.     

Functions of the plasminogen receptor Plg‐RKT

Plg‐R_KT is a structurally unique transmembrane plasminogen receptor with both N‐ and C‐terminal domains exposed on the extracellular face of the cell. Its C‐terminal lysine functions to tether plasminogen to cell surfaces. Overexpression of Plg‐R_KT increases cell surface plasminogen binding capacity while genetic deletion of Plg‐R_KT decreases plasminogen binding. Plasminogen binding to Plg‐R_KT results in promotion of plasminogen activation to the broad spectrum serine protease plasmin. This function is promoted by the physical association of Plg‐R_KT with the urokinase receptor (uPAR). Plg‐R_KT is broadly expressed in cells and tissues throughout the organism and its sequence is remarkably conserved phylogenetically. Plg‐R_KT also is required for lactation and, thus, is necessary for survival of the species. This review provides an overview of established and emerging functions of Plg‐R_KT and highlights major roles for Plg‐R_KT in both the initiation and resolution of inflammation. While the roles for Plg‐R_KT in the inflammatory response are predominantly plasmin(ogen)‐dependent, its role in lactation requires both plasminogen‐dependent and plasminogen‐independent mechanisms. Furthermore, the functions of Plg‐R_KT are dependent on sex. In view of the broad tissue distribution of Plg‐R_KT, its role in a broad array of physiological and pathological processes should provide a fruitful area for future investigation.     

The utility of a single glucometer measurement of fasting capillary blood glucose in the prevalence determination of diabetes mellitus in an urban adult Palestinian population

This paper aims to evaluate the utility of a single glucometer fasting capillary blood glucose (FCBG) measurement in determining the prevalence of diabetes mellitus in a homogeneous adult population. FCBG measurements were compared with results of the oral glucose tolerance test (OGTT) in 445 subjects aged 30-65 years in an urban cross-sectional study in Old Ramallah. Prevalence of diabetes, sensitivity, specificity and predictive values were calculated at different cut-off levels of FCBG, using OGTT as the reference. The prevalence of OGTT-diagnosed diabetes was 2.7%, while it varied considerably using different cut-off levels of FCBG. The sensitivity of a single glucometer (Exac Tech II) measurement of FCBG at the cut-off level of 6.7 mmol l^-1 was 33.3%, with a specificity of 98.8%. Using the cut-off level of 6.1 mmol l^-1 as suggested by the 1998 provisional report of a WHO consultation, the sensitivity increased to 41.7%. At a cut-off level of 5.6 mmol l^-1, a sensitivity of 66.6% was reached, but the specificity decreased slightly. It can be concluded that a single glucometer measurement of FCBG in an adult population is not useful in determining the prevalence of diabetes mellitus.     

Activation of single‐chain urokinase‐type plasminogen activator by platelet‐associated plasminogen: a mechanism for stimulation of fibrinolysis by platelets

Summary. Background and Objective: Platelets are essential for hemostasis, and they cause resistance to fibrinolysis by tissue‐type plasminogen activator. In contrast, platelets enhance fibrinolysis mediated by single‐chain urokinase‐type plasminogen activator (scu‐PA). This study investigated the mechanism behind this profibrinolytic role of platelets. Methods and Results: Platelets enhanced scu‐PA activity, but not urokinase‐type plasminogen activator (u‐PA) activity, in plasma clot lysis and chromogenic assays. We established, using the non‐cleavable scu‐PA mutant (Lys158→Glu) and protease inhibitors, that platelets increased activation to u‐PA by a serine protease. Activation of scu‐PA was platelet‐dependent, even in plasma. It occurred in platelet‐rich but not in platelet‐poor plasma, as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis and zymography after addition of plasminogen activator inhibitor‐1. Candidate proteases that are known to activate scu‐PA and are present in platelet preparations were investigated. Factor VII activating protease was detected in platelet preparations by western blotting, but its inhibition by antibodies did not inhibit activation of scu‐PA by platelets. Plasmin and plasma kallikrein both mimicked the platelet effect, but were distinguished by their responses to a range of inhibitors. Analysis of platelet‐associated protease activity and the time course of scu‐PA activation pointed towards plasminogen, and the data were consistent with a mechanism of reciprocal activation. The essential role of plasminogen was revealed using platelets from plasminogen‐deficient mice, which could not activate scu‐PA. Local plasminogen on platelet membranes was markedly more effective than solution‐phase plasminogen in activation of scu‐PA. Conclusions: Platelets enhance fibrinolysis by scu‐PA through reciprocal activation of scu‐PA and platelet‐associated plasminogen, a system that is potentially important in the lysis of platelet‐rich thrombi.     

Increased zymogen activity of thrombin‐activatable fibrinolysis inhibitor prolongs clot lysis

Summary. Background and objectives: Thrombin‐activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated by proteolytic cleavage into the active enzyme TAFIa. Hydrolysis of the C‐terminal lysines on fibrin by TAFIa results in a down‐regulation of fibrinolysis. Recent studies demonstrated that the zymogen also exerts an intrinsic enzymatic activity. Our objective was to identify and characterize zymogen‐stimulatory nanobodies. Methods and results: The screening of 24 nanobodies against TAFI revealed that two nanobodies (i.e. Vhh‐TAFI‐a51 and Vhh‐TAFI‐i103) were able to stimulate the zymogen activity 10‐ to 21‐fold compared with the baseline zymogen activity of TAFI. The increase in catalytic efficiency can be attributed mainly to an increased catalytic rate, as no change in the K_M‐value was observed. The stability, the susceptibility towards PTCI and GEMSA and the kinetics of the stimulated zymogen activity differ significantly from those of TAFIa activity. Epitope mapping revealed that both Asp⁷⁵ and Thr³⁰¹ are major determinants in the binding of these nanobodies to TAFI. Localization of the epitope strongly suggests that this instability is as a result of a disruption of the stabilizing interactions between the activation peptide and the dynamic flap region (residues 296–350). In TAFI‐depleted plasma reconstituted with a non‐activatable variant of TAFI (TAFI‐R92A), clot lysis could be prolonged by nanobody‐induced stimulation of its zymogen activity as well as by increasing its concentration. Conclusions: Increasing the zymogen activity of TAFI results in an antifibrinolytic effect.     

Fibrinolytic abnormalities in acute respiratory distress syndrome (ARDS) and versatility of thrombolytic drugs to treat COVID‐19

The global pandemic of coronavirus disease 2019 (COVID‐19) is associated with the development of acute respiratory distress syndrome (ARDS), which requires ventilation in critically ill patients. The pathophysiology of ARDS results from acute inflammation within the alveolar space and prevention of normal gas exchange. The increase in proinflammatory cytokines within the lung leads to recruitment of leukocytes, further propagating the local inflammatory response. A consistent finding in ARDS is the deposition of fibrin in the air spaces and lung parenchyma. COVID‐19 patients show elevated D‐dimers and fibrinogen. Fibrin deposits are found in the lungs of patients due to the dysregulation of the coagulation and fibrinolytic systems. Tissue factor (TF) is exposed on damaged alveolar endothelial cells and on the surface of leukocytes promoting fibrin deposition, while significantly elevated levels of plasminogen activator inhibitor 1 (PAI‐1) from lung epithelium and endothelial cells create a hypofibrinolytic state. Prophylaxis treatment of COVID‐19 patients with low molecular weight heparin (LMWH) is important to limit coagulopathy. However, to degrade pre‐existing fibrin in the lung it is essential to promote local fibrinolysis. In this review, we discuss the repurposing of fibrinolytic drugs, namely tissue‐type plasminogen activator (tPA), to treat COVID‐19 associated ARDS. tPA is an approved intravenous thrombolytic treatment, and the nebulizer form has been shown to be effective in plastic bronchitis and is currently in Phase II clinical trial. Nebulizer plasminogen activators may provide a targeted approach in COVID‐19 patients to degrade fibrin and improving oxygenation in critically ill patients.     

An international collaborative study to establish the WHO 4th International Standard for Streptokinase: Communication from the SSC of the ISTH

Streptokinase is used worldwide as a cost‐effective treatment for acute myocardial infarction. Manufacturers use the World Health Organization (WHO) International Standard (IS) for Streptokinase to potency label their products, ensuring consistent, safe, and effective dosing. Stocks of the third IS for streptokinase (coded 00/464) are running low, and an international collaborative study was organized to calibrate a replacement. A total of 15 laboratories from nine countries took part, using chromogenic and/or fibrin clot lysis methods to determine the potency of two candidate preparations, coded 16/356 (sample B) and 16/358 (sample C), relative to the third IS (00/464). A third sample (88/824, sample A), which was used in the collaborative studies to establish the second and third IS, was also included. There was good agreement in potency estimates from different assay methods and low variability both within and between laboratories. Long‐term stability modeling indicated the candidates are very stable. Comparison of potency estimates for 88/824 (sample A) with potencies calculated in previous studies revealed a variability of only 1.9% over the course of three collaborative studies spanning 30 years and more than 50 years of streptokinase standardization. This indicates excellent continuity of the International Unit (IU) and assay methods. Following agreement by study participants and Scientific and Standardization Committee experts of the International Society on Thrombosis and Haemostasis, the WHO Expert Committee on Biological Standardization established 16/358 (sample C) as the fourth IS for Streptokinase with a potency of 1013 IU per ampoule in October 2019.     

Apolipoprotein(a) inhibits the conversion of Glu‐plasminogen to Lys‐plasminogen: a novel mechanism for lipoprotein(a)‐mediated inhibition of plasminogen activation

Summary. Background: Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for thrombotic disorders. Lp(a) is a unique lipoprotein consisting of a low‐density lipoprotein‐like moiety covalently linked to apolipoprotein(a) [apo(a)], a homologue of the fibrinolytic proenzyme plasminogen. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue‐type plasminogen activator‐mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin‐dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin‐mediated conversion of Glu‐plasminogen to Lys‐plasminogen. Objective: Using acid–urea gel electrophoresis to resolve the two forms of radiolabeled plasminogen, we determined whether apo(a) is able to inhibit Glu‐plasminogen to Lys‐plasminogen conversion. Methods: The assays were performed in the absence or presence of different recombinant apo(a) species, including point mutants, deletion mutants and variants that represent greater than 90% of the known apo(a) isoform sizes. Results: Apo(a) substantially suppressed Glu‐plasminogen conversion. Critical roles were identified for the kringle IV types 5–9 and kringle V; contributory roles for sequences within the amino‐terminal half of the molecule were also observed. Additionally, with the exception of the smallest naturally‐occurring isoform of apo(a), isoform size was found not to contribute to the inhibitory capacity of apo(a). Conclusion: These findings underscore a novel contribution to the understanding of Lp(a)/apo(a)‐mediated inhibition of plasminogen activation: the ability of the apo(a) component of Lp(a) to inhibit the key positive feedback step of plasmin‐mediated Glu‐plasminogen to Lys‐plasminogen conversion.     

Non‐invasive in vivo tracking of fibrin degradation by fluorescence imaging

Fibrin‐based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid‐phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non‐invasive molecular imaging systems enable real‐time tracking of processes in the living animal. In this study, a non‐invasive fluorescence based imaging system was applied to follow and quantify site‐specific degradation of fibrin sealant. To enable non‐invasive tracking of fibrin in vivo, fibrin‐matrix was labelled by incorporation of a fluorophore‐conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore‐labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16 days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site. Copyright © 2014 John Wiley & Sons, Ltd.