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Microvesicles (MVs) are key markers in human body fluids that reflect cellular activation related to diseases as thrombosis. These MVs display phosphatidylserine at the outer leaflet of their plasma membrane as specific recognition moieties. The work reported in this manuscript focuses on the development of an original method where MVs are captured by bimetallic zinc complexes. A set of ligands have been synthetized based on a phenol spacer bearing in para position an amine group appended to a short or a longer alkyl chain (for grafting on surface) and bis(dipicolylamine) arms in ortho position (for zinc coordination). The corresponding dibridged zinc phenoxido and hydroxido complexes have been prepared in acetronitrile in presence of triethylamine and characterized by several spectroscopic techniques. The pH-driven interconversion studies for both complexes in H 2 O:DMSO (70:30) evidence that at physiologic pH the main species are mono-bridged by the phenoxido spacer. An X-Ray structure obtained from complex 2 (based on the ligand with the amine group on the short chain) in aqueous medium confirms the presence of a mono-bridged complex. Then, the complexes have been used for interaction studies with short-chain phospholipids. Both have established the selective recognition of the anionic phosphatidylserine model versus zwitterionic phospholipids (in solution by 31 P NMR and after immobilization on solid support by surface plasmon resonance (SPR)). Moreover, both complexes have also demonstrated their ability to capture MVs isolated from human plasma. These complexes are thus promising candidates for MVs probing by a new approach based on coordination chemistry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The regulation of plasmin generation on cell surfaces is of critical importance in the control of vascular homeostasis. Cell-derived microparticles participate in the dissemination of biological activities. However, their capacity to promote plasmin generation has not been documented. In this study, we show that endothelial microparticles (EMPs) from tumor necrosis factor alpha (TNFalpha)-stimulated endothelial cells served as a surface for the generation of plasmin. The generation of plasmin involved expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) at the surface of EMPs and was further increased by their ability to bind exogenous uPA on uPAR. Plasminogen was activated at the surface of EMPs in a dose-dependent, saturable, and specific manner as indicated by the inhibition of plasmin formation by epsilon-amino-caproic acid (epsilon-ACA) and carboxypeptidase B. EMP-induced plasmin generation affects tube formation mediated by endothelial progenitor cells. However, low amounts of EMPs increased tube formation, whereas higher concentrations inhibited it. Prevention of these effects by inhibitors of either uPA or plasmin underscore the key role of EMP-induced plasmin generation. In conclusion, we demonstrated that EMPs act as vectors supporting efficient plasmin generation and dissemination, a new pathway in the regulation of endothelial proteolytic activities with potential involvement in inflammation, angiogenesis, and atherosclerosis.

Acquired abdominal aortic aneurysms are usually associated with a mural thrombus through which blood continues to flow. Some early data suggest that aneurysmal evolution correlates with the biological activity of the thrombus. Our hypothesis was therefore that the thrombus could adsorb blood components and store, release, and participate in the activation of proteases involved in aneurysmal evolution. For this purpose, we have explored both the metalloproteinase and fibrinolytic systems in the thrombus and the wall of human aneurysms. We have first investigated blood clot formation and lysis in vitro. Spontaneous clotting induces a release of promatrix metalloproteinase (pro-MMP)-9 into the serum that was fourfold higher than in paired control plasma (P < 0.001). Fibrinolysis progressively released more MMP-9 in a time-dependent manner (P < 0.01). After selective isolation, we demonstrated that polymorphonuclear leukocytes are the main source of MMP-9 release during clot formation. Protease content was then analyzed in 35 mural thrombi and walls of human abdominal aortic aneurysms sampled during surgical repair. In 15 aneurysms, the liquid phase at the interface between the thrombus and the wall was sampled separately. Both thrombus and wall contained MMP-2 and MMP-9 but the ratio MMP-9/MMP-2 was higher in the thrombus than in the wall. The liquid interface also contained active MMP-9. Immunohistochemistry of the thrombus confirmed these findings, showing the presence of polymorphonuclear leukocytes at the luminal pole of the thrombus, co-localizing with MMP-9 storage. In contrast, MMP-3 and MMP-7 were only present in the aneurysmal wall. Plasminogen was present in the mural thrombus but plasmin activity was present in both thrombus and wall. In the liquid interface, plasmin-alpha(2)-anti-plasmin complexes were detected demonstrating in vivo the activation of plasminogen. In contrast, u-PA and t-PA were detectable only in the wall, suggesting that plasminogen present in the thrombus could be activated by factors secreted by the arterial wall. This was demonstrated in vitro, in which co-incubation of thrombus and wall extracts generated plasmin in the presence of a fibrin matrix and activated MMPs. In conclusion, our study strongly suggests that the mural thrombus, by trapping polymorphonuclear leukocytes and adsorbing plasma components could act as a source of proteases in aneurysms that may play a critical role in enlargement and rupture.

High levels of histidine-rich glycoprotein (HRGP) and plasminogen activator inhibitor-1 (PAI-1) have been claimed to contribute to the hypofibrinolytic state observed in patients with venous thrombosis. These abnormalities were detected, respectively, in eight and 10 members of a family from which four of seven members with both abnormalities had venous thromboembolism. Binding of tissue plasminogen activator (t-PA) by PAI-1 may induce hypofibrinolysis. To determine whether plasminogen binding by HRGP may influence plasminogen activation, we studied the fibrinolytic activity of members of this family cohort with a system that detects modifications in plasmin generation by proteins interfering with the binding of plasminogen to fibrin. Plasminogen activation was performed by adding plasma to fibrin surfaces to which t-PA had been previously bound in the presence of 40 mg/ml bovine serum albumin and 20 mumol/L of the lysine analog trans-4-(aminomethyl)-cyclohexane carboxylic acid to prevent nonspecific binding and thereby the inhibitory effect of elevated PAI-1 levels. The generation of plasmin as a function of time was detected (1) by photometric analysis with a chromogenic substrate highly selective for plasmin and (2) by measuring the binding and activation of plasminogen at the fibrin surface with radiolabeled plasminogen. The amount of plasmin generated by plasma from patients having high levels of HRGP (160% to 280%) was similar to that of a control group having normal levels of HRGP (100% +/- 22%). Similar results were obtained with a plasma artificially depleted in HRGP and supplemented with various amounts of this protein. No correlation between HRGP level and t-PA-mediated plasminogen activation was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied 28 patients with SLE, five of whom had had thrombotic episodes. Platelet function and coagulation and fibrinolytic studies were performed to determine whether any of these factors may predispose to thrombosis in SLE. An inhibitor of blood coagulation was detected in 12 patients, and von Willebrand's syndrome was observed in two others. The most striking findings which could be correlated with thromboembolic phenomena were the increase in VII R:WF and the absence of plasminogen activator release after venous occlusion. Both proteins are synthesized by the endothelial cell, and the abnormalities observed are possibly related to damage of the vascular endothelium by immune complexes observed in SLE.

We have studied the coagulation status of eight patients with the Chediak-Higashi syndrome (CHS), both in the chronic and the accelerated phase of the disease. It has been shown that during the accelerated phase there are coagulation abnormalities. These abnormalities include a peripheral thrombocytopenia, minor alterations of liver clotting factors, and mainly a profound hypofibrinogenemia and hypoplasminogenemia, which cause life-threatening bleedings. These disorders are of complex origin, but a fibrinolytic process, possibly primary, appears to play a significant role, since the present evidence for intravascular coagulation is not definitive. The accelerated phase of the CHS is characterized by a visceral infiltration by macrophages and lymphocytes. Therefore, we have investigated the possible role of the macrophages in the fibrinolytic process. We have found an excessive plasminogen activator (PA) production by CHS mononuclear cells in the accelerated phase and to a lesser extent in the chronic phase, except in one patient in whom no anomaly was found. Single-cell studies revealed an increased number of PA-producing cells among the monocyte-macrophage lineage rather than a higher level of production per cell. Polymorphonuclear cells (PMN) from patients with CHS were also shown to contain more PA. Slight but significant abnormalities in PA production were observed in obligatory heterozygotes (five out of nine), indicating the inherited nature of the excessive PA production. Finally, an enhanced PA production was similarly demonstrated using beige mice macrophages. The exacerbated production of PA by macrophages in the accelerated phase of the CHS can account to some extent for the coagulation abnormalities that have been observed.

The release of plasminogen activators (PA) from human isolated glomeruli has been studied by a sensitive radioenzymatic assay using 125I-fibrin coated tubes and plasminogen. The glomerular fibrinolytic activity (GFA) was detectable after 15 minutes of incubation. Then it increased with time, the glomerular protein concentration, and with the plasminogen concentration (P less than 0.001 for all). CaCl2 (1 mM) increased the GFA (9.7 +/- 0.9 versus 4.9 +/- 0.4 micrograms fibrin/mg/30 min, P less than 0.05). The GFA was also enhanced when pH increased. Arachidonic acid (AA, 1 to 20 micrograms/ml) increased the GFA in a saturable manner. Inhibitors of cyclooxygenase (aspirin) or of lipoxygenase (nordihydroguaiaretic acid) did not modify the basal and AA-stimulated GFA. Other polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), eicosatrienoic acid (ETA), eicosatetraynoic acid (ETYA), or dihomo-gamma-linoleic acid (DHL), also stimulated the GFA whereas linoleic acid and oleic acid did not. Polyunsaturated fatty acids also stimulated the fibrinolytic activity of glomerular supernatants. Specific antibodies to t-PA, and to a lesser extent to u-PA, decreased this fibrinolytic activity whether or not AA was added. Furthermore, AA and EPA were found to increase the activity of purified u-PA and t-PA. We conclude that human glomeruli release both t-PA and u-PA, and that this release is increased by calcium and alkaline pH. The polyunsaturated fatty acids enhanced the GFA, mainly by a stimulatory effect of PA activity rather than an increased release of PA from glomerular cells.

A new spectrophotometric solid-phase fibrin-tissue plasminogen activator activity assay (SOFIA-tPA), specific for the quantitation of tissue plasminogen activators, is described. The method is based on (1) the high-affinity binding (Kp = 1.4 +/- 2 nM) of tPA to a solid-phase fibrin network constructed by thrombin proteolysis of fibrinogen covalently coupled to polyglutaraldehyde-activated polyvinyl chloride microtiter plates, and (2) the subsequent development of PA activity by the fibrin-tPA complex and its measurement with a coupled assay using a chromogenic substrate highly selective for plasmin. Conditions were chosen such that the rate of para-nitroaniline release from the substrate is directly proportional to the concentration of tPA. The support is able to isolate tPA from the bulk of proteins present in any biological fluid allowing the assay to specifically detect tPA activity (range: 0.01 to 50 IU/ml) even in the presence of other activators, proteases, and inhibitors. Since the assay is done in a well-defined reaction mixture (the fibrin-tPA complex, plasminogen, and the synthetic substrate), kinetics studies using pure or crude tPA can be performed. Standard curves (rate measurement and endpoint methods) were made using the international standard (preparation 83/517) for tPA.

Monoclonal antibodies (MAbs) to vascular plasminogen activator (vPA), the tissue-type plasminogen activator (tPA) in human plasma, were produced to be used as probes for immunochemical analysis. Human tissue sections and one of these MAbs were used to demonstrate the endothelial origin of plasma-tPA by immunohistochemistry. To produce MAbs, mice were immunized with semipurified vPA isolated from postocclusion human venous blood. Primed spleen cells were fused with the mouse myeloma cell line NS-1. Screening for MAb-producing hybridomas was performed with postocclusion euglobulins as a source of antigen by means of a solid-phase fibrin-vPA immunoassay. The selective and high-affinity binding of vPA for fibrin ensures the specificity and sensitivity of this test. Thus, eight hybridomas secreting MAbs to vPA were selected, cloned, and established as permanent hybridoma cell lines. Immunohistochemical analysis of cryostat sections of human tissues was performed with EA-delta 12D, a MAb having no inhibitory effect against vPA activity but binding to vPA with a high affinity. Thus, the only structures immunostained were endothelial cells of venules, capillaries, and arterioles. The EA-delta 12D monoclonal localization of plasma vPA in the endothelial lining of blood vessels provides evidence that tPA in plasma originates from the vascular wall and validates its designation as vascular plasminogen activator, ie, vPA. Also, our results are consistent with the fact that vPA in blood and tPA in tissues are immunologically identical and have a common endothelial origin.

The selective detection of hybridomas producing monoclonal antibodies against tissue-plasminogen activators (t-PA) by means of a solid-phase fibrin immunoassay (SOFIA) is described. The screening method is based on the specific and high affinity binding of t-PA for fibrin solid phase. This, as in the physiological fibrin-t-PA interaction, provides a molecular structure conserving its antigenic and functional properties. The solid-fibrin-state support is prepared by thrombin proteolysis of fibrinogen coupled to a poly-glutaraldehyde activated polyvinylchloride plate. This procedure avoids fibrinogen desorption and provides a uniform fibrin network onto which t-PA from crude extracts or purified preparations, can be specifically absorbed. The fibrin-t-PA solid-phase support is able to fix anti-t-PA antibodies which can be subsequently revealed by a second enzyme- or radiolabelled anti-mouse Ig antibody. The results demonstrate the effectiveness of the SOFIA method for the selection of hybridomas producing anti-t-PA antibodies against different epitopic determinants of t-PAs.