Purpose: Little evidence of increased thrombotic risk is available in COVID-19 patients. Our purpose was to assess thrombotic risk in severe forms of SARS-CoV-2 infection. Methods:All patients referred to 4 intensive care units (ICUs) from two centers of a French tertiary hospital for acute respiratory distress syndrome (ARDS) due to COVID-19 between March 3rd and 31st 2020 were included. Medical history, symptoms, biological data and imaging were prospectively collected. Propensity score matching was performed to analyze the occurrence of thromboembolic events between non-COVID-19 ARDS and COVID-19 ARDS patients.Results: 150 COVID-19 patients were included (122 men, median age 63 [53; 71] years, SAPSII 49 [37; 64] points). Sixty-four clinically relevant thrombotic complications were diagnosed in 150 patients, mainly pulmonary embolisms (16.7%). 28/29 patients (96.6%) receiving continuous renal replacement therapy experienced circuit clotting. Three thrombotic occlusions (in 2 patients) of centrifugal pump occurred in 12 patients (8%) supported by ECMO. Most patients (> 95%) had elevated D-dimer and fibrinogen. No patient developed disseminated intravascular coagulation. Von Willebrand (vWF) activity, vWF antigen and FVIII were considerably increased, and 50/57 tested patients (87.7%) had positive lupus anticoagulant. Comparison with non-COVID-19 ARDS patients (n = 145) confirmed that COVID-19 ARDS patients (n = 77) developed significantly more thrombotic complications, mainly pulmonary embolisms (11.7 vs. 2.1%, p < 0.008). Coagulation parameters significantly differed between the two groups. Conclusion:Despite anticoagulation, a high number of patients with ARDS secondary to COVID-19 developed life-threatening thrombotic complications. Higher anticoagulation targets than in usual critically ill patients should therefore probably be suggested.

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.

Background and Purpose-Early reperfusion using tissue-type plasminogen activator is the only therapeutic agent to treat focal cerebral ischemia with proven efficacy in patients. Nevertheless, novel insights into the pathophysiology of neurons, glial cells, and the fate of the endothelium after stroke call for the use of new strategies to improve stroke treatment alone or in combination with tissue-type plasminogen activator-induced thrombolysis. Unfortunately, despite the plethora of drugs that display clear beneficial effects in animal models of experimental ischemia, their subsequent use in clinical trials has proven disappointing. As such, one is forced to consider that new animal models of focal cerebral ischemia may be required before clinical evaluation of a new molecule. Methods-In situ microinjection of purified murine thrombin was used to trigger a local clot formation in anesthetized mice. Cerebral blood velocity was measured continuously throughout the duration of the study. The efficiency of recombinant tissue-type plasminogen activator to induce thrombolysis and its subsequent effect on infarct volume were then measured. Results-In situ thrombin injection leads to a reproducible clot formation and cortical brain injury. Recombinant tissue-type plasminogen activator-induced thrombolysis reduced infarct volume by 36.8% when compared with untreated control mice. Conclusions-We describe an original and reproducible mouse model of in situ clot formation and reperfusion, which could be used to investigate new therapeutic strategies to improve stroke treatment.

The regulation of plasmin generation on cell surfaces is of critical importance in the control of vascular homeostasis. Cellderived 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 ␣ (TNF␣) - IntroductionMicroparticles (MPs) are vesicles resulting from the blebbing of the cellular membrane of most activated or apoptotic cells. 1 These microvesicles have been described in various cellular models and in different pathological conditions as reliable hallmarks of cell damage. 2 Because they convey various bioactive effectors originating from the parent cells, MPs may exhibit a spectrum of biological activities: they regulate endothelial or blood cell functions, participate in inflammatory responses or angiogenesis, and propagate biological responses involved in hemostatic balance. 3 We previously reported the capacity of endothelial cells to release microparticles after inflammatory stimulation and the presence of increased levels of circulating endothelial microparticles (EMPs) in patients with thrombotic disorders. 4 Since this initial report, elevated levels of EMPs have been documented in various pathological conditions including coronary syndromes, 5 renal failure, 6 diabetes, 7 antiphospholipid syndrome, 8 thrombotic thrombocytopenic purpura, 9 and sickle cell disease, 10 in which they reflect endothelial dysfunction and are associated with a poor clinical outcome.EMPs provide procoagulant phospholipid surfaces for the assembly and activation of coagulation factors, mainly through phosphatidylserine translocation to the exoplasmic leaflet as a result of membrane remodeling. Their involvement in thrombin generation also results from their capacity to harbor, deliver, or induce tissue factor activity. 11-13 However, a more complex contribution to the hemostatic balance is suggested by their expression of thrombomodulin, tissue factor pathway inhibitor, and endothelial protein C receptor, thus providing a possible antithrombotic counterbalance. 14,15 Another key regulator of the vascular homeostasis is the plasminogen activation system. Plasminogen activation is mediated by 2 serine proteases: tissue-type plasminogen activator (tPA), which is mainly implicated in fibrinolysis, and urokinase-type plasminogen activator (uPA), which is critically involved in pericellular proteolysis due to its high affinity cell-surface receptor uPAR. 16 Plasmin generation induced by uPA and subsequent activation of matrix metalloproteinases (MMPs) promote cell migration through interstitial matrix and participate in processes such as tissue remodeling, cancer invasion, and angiogenesis. [17][18][19] Importantly, we have shown that uncontrolled plasminogen activation can have deleterious consequences by inducing cell detachment and apoptosis. 20,21 The regulation of plasmin generation at the endothelial surface is therefore of critical importa...

In the present study we have quantitatively characterized the interaction of purified human Glu- and Lys-plasminogen with intact and degraded fibrin by ligand-binding experiments using a radioisotopic dilution method and antibodies against human plasminogen. A fibrinogen monolayer was covalently linked to a solid support with polyglutaraldehyde and was treated with thrombin or with thrombin and then plasmin to respectively obtain intact and degraded fibrin surfaces. Under these conditions, a well-defined surface of fibrin is obtained (410 +/- 4 fmol/cm2) and, except for a 39-kDa fragment, most of the fibrin degradation products remain bound to the support. New binding sites for plasminogen were detected on the degraded surface of fibrin. These sites were identified as carboxy-terminal lysine residues both by inhibition of the binding by the lysine analogue 6-aminohexanoic acid and by carboxy-terminal end-group digestion with carboxypeptidase B. The binding curves exhibited a characteristic Langmuir adsorption isotherm saturation profile. The data were therefore analyzed accordingly, assuming a single-site binding model to simplify the analysis. Equilibrium dissociation constants (Kd) and the maximum number of binding sites (Bmax) were derived from linearized expression of the Langmuir isotherm equation. The Kd for the binding of Glu-plasminogen to intact fibrin was 0.99 +/- 0.17 microM and for degraded fibrin was 0.66 +/- 0.22 microM. The Kd for the binding of Lys-plasminogen to intact fibrin was 0.41 +/- 0.22 microM and for degraded fibrin was 0.51 +/- 0.12 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Smooth muscle cell (SMC) rarefaction is involved in the development of several vascular pathologies. We suggest that the plasminogen activation system is a potential extracellular signal that can induce pericellular proteolysis and apoptosis of vascular SMCs. Using primary cultures of arterial SMCs, we show that plasmin generated from plasminogen on the cell surface induces cell retraction and fibronectin fragmentation, leading to detachment and morphological/biochemical changes characteristic of apoptosis (also called anoikis). The generation of cell-bound plasmin mediated by tissue-type plasminogen activator (t-PA), constitutively expressed by VSMCs, requires binding of plasminogen to the cell surface and is inhibited by epsilon-aminocaproic acid (IC50=0.9+/-0.2 mM), a competitor of plasminogen binding to membrane glycoproteins. Conversely, addition of alpha2-antiplasmin, which blocks free plasmin in the cell supernatant, could not fully prevent anoikis. Finally, an MMP inhibitor failed to prevent VSMC anoikis, arguing for a direct involvement of plasmin in this phenomenon. Indeed, similar changes are induced by plasmin directly added to VSMCs or to arterial rings, ex-vivo. We show for the first time that pathological anoikis can be triggered by a process that requires functional assembly of the plasminogen activation system on the surface of VSMCs.

The online version of this article has a Supplementary Appendix. BackgroundWe recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and MethodsCirculating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. ResultsCirculating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. ConclusionsEndothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium.Key words: fibrinolytic microparticles, plasmin, plasminogen, uPA; tPA. Plawinski L, Robert S, Doeuvre L, Sabatier F, Martinez de Lizarrondo S, Mezzapesa A, Anfosso F, Leroyer AS, Poullin P, Jourde N, Njock M-S, Boulanger CM, Anglés-Cano E, and Dignat-George F. Leukocyte-and endothelial-derived microparticles: a circulating source for fibrinolysis. Haematologica 2012;97(12):1864-1872. doi:10.3324/haematol.2012 This is an open-access paper. Citation: Lacroix R, Leukocyte-and endothelial-derived microparticles: a circulating source for fibrinolysis ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

Accurate sizing of nanoparticles in biological media is important for drug delivery and biomedical imaging applications since size directly influences the nanoparticle processing and nanotoxicity in vivo. Using fluorescence single particle tracking we have succeeded for the first time in following the aggregation of drug delivery nanoparticles in real time in undiluted whole blood. We demonstrate that, by using a suitable surface functionalization, nanoparticle aggregation in the blood circulation is prevented to a large extent.