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Matricryptins are collagen fragments proteolytically released from the extracellular matrix (ECM) with biological activity that can regulate several processes involved in ECM remodeling. Vessel wall matrix reorganization after lesion is important to the recovery of vascular function. This study aimed to analyze the effect of the peptide p1158/59 (Lindsey, 2015) on thrombosis, neointimal formation, and vascular remodeling of C57BL6 mice abdominal aorta. We used a FeCl 3 induced vascular injury mice model and analyzed thrombus size, neointima formation, gelatinase activities in situ, re-endothelization, and collagen fibers organization on the arterial wall using polarization microscopy. As result, we observed that 2 days after injury the treatment with p1158/59 increased thrombus size and gelatinase activity, vascular lesion and it did not recover the endothelium loss induced by the chemical injury. We also observed that the peptide increased neointima growth and collagen birefringence, indicating collagen fibers reorganization. It also promoted increased re-endothelization and decreased activity of gelatinases 14 days after injury. Thus, we conclude that the peptide p1158/59 impaired the initial thrombosis recovery 2 days after injury but was able to induce vascular ECM remodeling after 14 days, improving vessel re-endothelization, collagen fibers deposition, and organization., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Endothelial progenitor cells (EPCs) are circulating progenitor cells that can play an essential role in vascular remodelling. In this work, we compared the role of two EPCs cultivated with different mediums in the resolution of the arterial thrombus induced by FeCl 3 lesion and in vessel re-endothelization in the mouse carotid artery., Methods: Mice mononuclear cells were differentiated into EPCs using Dulbecco's Modified Eagle's Medium (DMEM) and vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and IGF (Insulin Growth Factor) called EPCs--M1) or with EGM2(endothelial growth medium) (media supplemented with growth factors from Lonza called (EPCs-M2) for 30days and characterized using flow cytometry. The animals received three EPC injections post-lesion, and we analyzed thrombosis time, vessel re-endothelization, metalloproteinases activities, eNOS (endothelial Nitric oxide synthase) presence and SDF-1(Stromal Derived Factor- 1) levels in circulation., Results: EPC-M1 presented a more immature progenitor profile than EPC-M2 cells. The injection of EPC-M1 prolonged the thrombosis time, and the treatment with the different EPCs increased eNOS expression and MMP2 (Metalloproteinase 2) activity and decreased SDF-1 in plasma. Only EPC-M1 treatment increased both MMP2 and MMP9 and reduced thrombus after 7days. Also, both EPCs decreased platelet aggregation in vitro., Conclusions: EPCs-M1 were more efficient in all of the analyzed assays. EPCsM2 may be a more mature EPC, proliferating less and promoting a less significant matrix remodelling. EPCs can promote vascular remodelling by inhibiting thrombosis and stimulating vascular wall remodelling and the treatment with a more immature progenitor may be more efficient in this process., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The practice of physical exercise is highly indicated to prevent cardiovascular diseases and is directly related to the improvement of endothelial function and the regulation of arterial blood pressure. The objective of this study was to analyze the effect of physical exercise in vascular remodeling after FeCl 3 chemically induced arterial injury on atherosclerotic mice. To analyze the effect of exercises on thrombus formation, LDL receptor-deficient mice were fed for 6 weeks with a high-fat diet and performed or not physical exercises for 2 weeks before the arterial injury. To verify endothelium recovery the animals were exercised or not 2 weeks before the injury, and 3 weeks after it, when the vessels were analyzed. In this work, we observed that physical exercises done only before arterial injury reduced thrombosis time, protected the endothelial layer, promoted the recruitment of CD34 positive progenitor cells, increased the level of eNOS and gelatinases activities and decreased the number of inflammatory cells in the vessel, but do not avoid the growth of neointima. Otherwise exercises done before and continued after injury, increased gelatinase activities, reduced lipid deposition in the aortic arch and prevented neointima formation. Thus, we could conclude that physical exercises are done before and continued after endothelial injury stimulate endothelial recovery by promoting endothelial cell growth, matrix remodeling and decreasing inflammation in the vessel wall., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment with recombinant human DNase 1 reversed the prothrombotic phenotype of tumor-bearing mice in both models. Remarkably, 4T1-derived exosomes induced NET formation in neutrophils from mice treated with granulocyte colony-stimulating factor (G-CSF). In addition, tumor-derived exosomes interacted with NETs under static conditions. Accordingly, the intravenous administration of 4T1-derived exosomes into G-CSF-treated mice significantly accelerated venous thrombosis in vivo. Taken together, our observations suggest that tumor-derived exosomes and neutrophils may act cooperatively in the establishment of cancer-associated thrombosis.

Introduction: MAGP1 is a glycoprotein present in the elastic fibers and is a part of the microfibrils components. MAGP1 interacts with von Willebrand factor and the active form of TGF-β and BMP. In mice lacking MAGP1, thrombus formation is delayed, increasing the occlusion time of carotid artery despite presenting normal blood coagulation in vitro. MAGP1-containing microfibrils may play a role in hemostasis and thrombosis. In this work, we evaluated the function of MAGP1 and its relation to TGF-β in the arterial thrombosis process., Methods and Results: We analyzed thrombus formation time in wild type and MAGP1-deficient mice comparing Rose Bengal and Ferric Chloride induced arterial lesion. The potential participation of TGF-β in this process was accessed when we treated both wild type and MAGP1-deficient mice with losartan (an antihypertensive drug that decreases TGF-β activity) or captopril (an angiotensin converting enzyme inhibitor that was used as a control antihypertensive drug). Besides, we evaluated thrombus embolization and the gelatinolytic activity in the arterial walls in vitro and ex vivo. Losartan and captopril were able to recover the thrombus formation time without changing blood pressure, activated partial thromboplastin time (aPTT), PT (prothrombin time), platelet aggregation and adhesion, but decreased gelatinase activity., Conclusions: Our results suggest that both treatments are effective in the prevention of the sub-endothelial ECM degradation, allowing the recovery of normal thrombus formation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background Aims: Dermatan sulfate (DS), an anticoagulant and antithrombotic glycosaminoglycan, also has anti-inflammatory activity. In this study, we investigated the effect of DS treatment in the presence or absence of bone marrow mononuclear cells (MNCs) or endothelial progenitor cells (EPCs) in the vascular response to carotid artery lesion in C57BL6 mice., Methods: Thrombus formation, the expression of adhesion molecules and factors involved in vascular remodeling, inflammation or vascular tone were analyzed by histologic examination, Western blotting and enzyme-linked immunoassay 1 and 3 days after vascular injury., Results: DS injections prevented thrombus formation and decreased P-selectin expression after 3 days of the injury. DS treatment also increased plasma SDF-1 levels but failed to rescue endothelial nitric oxide synthase (eNOS) expression, which is responsible for vascular tone. Treatment with MNCs alone failed to prevent thrombus formation 1 day after injury and increased intercellular adhesion molecule-1 expression, likely because of the inflammatory nature of these cells. Treatment with EPCs with DS was the most efficient among all therapies studied. Dual administration of EPCs and DS promoted an increase in the expression of adhesion molecules and, at the same time, induced a higher expression of eNOS at the injury site. Furthermore, it stimulated an elevated number of EPCs to migrate and adhere to the vascular wall., Discussion: Simultaneous treatment with EPCs and DS increased the expression of adhesion molecules, prevented thrombosis, rescued the expression of eNOS and increased migration of EPCs to the site of injury, thereby affecting thrombus remodeling and inflammation and can be involved in vessel hemostasis., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) is a Kunitz-type serine peptidase inhibitor of plant origin that has been shown to impair the viability of some tumor cells and to feature a potent inhibitory activity against human and rat plasma kallikrein (Kiapp 2.4 nmol/L and 5.2 nmol/L, respectively). This inhibitory activity is possibly responsible for an effect on hemostasis by prolonging activated partial thromboplastin time (aPTT). Because the association between cancer and thrombosis is well established, we evaluated the possible antithrombotic activity of this protein in venous and arterial thrombosis models. Vein thrombosis was studied in the vena cava ligature model in Wistar rats, and arterial thrombosis in the photochemical induced endothelium lesion model in the carotid artery of C57 black 6 mice. BbKI at a concentration of 2.0 mg/kg reduced the venous thrombus weight by 65% in treated rats in comparison to rats in the control group. The inhibitor prolonged the time for total artery occlusion in the carotid artery model mice indicating that this potent plasma kallikrein inhibitor prevented thrombosis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A basic phospholipase A₂ (LmrTX) isoform was isolated from Lachesis muta rhombeata snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-5 Discovery® Bio Wide column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of LmrTX was measured as 14.277.50 Da. The amino acid sequence showed a high degree of homology between PLA₂ LmrTX from L. muta rhombeata and other PLA₂ from snake venoms, like CB1 and CB2 from Crotalus durissus terrificus; LmTX-I and LmTX-II from Lachesis muta muta. LmrTX had PLA₂ activity in the presence of a synthetic substrate and alkylation of histidine residues significantly inhibited (P < 0.05) the enzymatic activity of LmrTX and its anticoagulant and antithrombotic activity. In this study, we examined the ability of the LmrTX in altering thrombus formation in living mouse, using a photochemically induced arterial thrombosis model. The control animals that did not receive protein injection showed a normal occlusion time, which was around 57 ± 7.8 min. LmrTX, the PLA₂ from L. muta rhombeata venom, caused a change in the occlusion time to 99 ± 10 min with doses of 7.5 μg/mice. Additionally, LmrTX showed the anticoagulant activity in vitro and ex vivo and prolonging the time aggregation in wash platelet induced by ADP and Thrombin., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mice lacking the extracellular matrix protein microfibril-associated glycoprotein-1 (MAGP1) display delayed thrombotic occlusion of the carotid artery following injury as well as prolonged bleeding from a tail vein incision. Normal occlusion times were restored when recombinant MAGP1 was infused into deficient animals prior to vessel wounding. Blood coagulation was normal in these animals as assessed by activated partial thromboplastin time and prothrombin time. Platelet number was lower in MAGP1-deficient mice, but the platelets showed normal aggregation properties in response to various agonists. MAGP1 was not found in normal platelets or in the plasma of wild-type mice. In ligand blot assays, MAGP1 bound to fibronectin, fibrinogen, and von Willebrand factor, but von Willebrand factor was the only protein of the 3 that bound to MAGP1 in surface plasmon resonance studies. These findings show that MAGP1, a component of microfibrils and vascular elastic fibers, plays a role in hemostasis and thrombosis.

The anionic phospholipid, phosphatidyl-L-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid beta-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor-initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents.

We compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate dermatan sulfate containing 2-O-sulfated alpha-L-iduronic acid and 4-O-sulfated N-acetyl-beta-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, another invertebrate dermatan sulfate, also enriched in 2-O-sulfated alpha-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-beta-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.