cathepsin-g and Thrombosis

Topics: Arachidonic Acid; Cathepsin G; Cathepsins; CD18 Antigens; Cell Adhesion; Cell Adhesion Molecules; Humans; Neutrophils; P-Selectin; Platelet Adhesiveness; Serine Endopeptidases; Thrombosis

Topics: Animals; Arachidonic Acid; Cathepsin G; Cathepsins; Cell Communication; Humans; Neutrophils; P-Selectin; Platelet Activation; Platelet Adhesiveness; Serine Endopeptidases; Signal Transduction; Thrombosis

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome characterized by platelet anti-PF4 (platelet-activating antiplatelet factor 4)-related thrombosis. Platelet-neutrophil interaction has been suggested to play a role, but the underlying mechanism has not been fully elucidated.. The study included 10 patients with VITT after ChAdOx1 (chimpanzee adenovirus Oxford 1) nCoV-19 (Oxford-AstraZeneca) vaccine administration, 10 patients with ischemic stroke (IS), 10 patients with acute deep vein thrombosis, and 10 control subjects in whom blood levels of neutrophil extracellular traps (NETs), soluble TF (tissue factor), and thrombin generation were examined. Furthermore, we performed in vitro studies comparing the effect of serum from patients and controls on NETs formation. Finally, immunohistochemistry was performed in cerebral thrombi retrieved from a patients with VITT and 3 patients with IS.. Compared with patients with IS, patients with deep vein thrombosis, controls, and patients with VITT had significantly higher blood values of CitH3 (citrullinated histone H3), soluble TF, D-dimer, and prothrombin fragment 1+2 (. Patients with VITT display enhanced thrombogenesis by PAD4-mediated NETs formation via cathepsin G-mediated platelet/neutrophil interaction.

Topics: Cathepsin G; Humans; Neutrophils; Thrombin; Thrombocytopenia; Thrombosis; Vaccines

Inhalation of particulate matter in polluted air causes direct, size-restricted passage in the circulation and pronounced lung inflammation, provoking platelet activation and (non)-fatal cardiovascular complications. To determine potency and mechanism of platelet sensitization via neutrophil enzymes, we performed in vitro aggregation studies in washed human platelets and in murine and human blood, in the presence of elastase, cathepsin G and regular platelet agonists, present in damaged arteries. The impact of both enzymes on in vivo thrombogenicity was studied in the same thrombosis mouse model, previously having demonstrated that neutrophil activation enhances peripheral thrombogenicity. At 0.05 U/mL, cathepsin G activated washed human platelets via PAR1, whereas at 0.35 U/mL, aggregation occurred via PAR4. In Swiss mouse platelet-rich plasma no aggregation occurred by cathepsin G at 0.4 U/mL. In human and murine blood, aggregations by 0.05-0.1 U/mL cathepsin G were similar and not PAR-mediated, but platelet aggregation was inhibited by ADP antagonists, advocating cathepsin G-released ADP in blood as the true agonist of sustained platelet activation. In the mouse thrombosis model, cathepsin G and elastase amplified mild thrombogenicity at blood concentrations that activated platelets in vitro. This study shows that cathepsin G and elastase secreted in the circulation during mild air pollution-induced lung inflammation lyse red blood cell membrane proteins, leading to ADP-leakage into plasma, sensitizing platelets and amplifying their contribution to cardiovascular complications of ambient particle inhalation.

Topics: Adenosine Diphosphate; Animals; Arteries; Biomarkers; Blood Platelets; Cathepsin G; Disease Susceptibility; Humans; Mice; Mice, Knockout; Neutrophil Activation; Neutrophils; Pancreatic Elastase; Platelet Activation; Platelet Aggregation; Protein Serine-Threonine Kinases; Thrombosis

Adequate cleavage of von Willebrand factor (VWF) prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM) was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3), cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

Topics: ADAMTS13 Protein; Adult; Blood Platelets; Cathepsin G; Endothelial Cells; Glomerular Basement Membrane; Humans; Immunoblotting; Kidney; Kidney Glomerulus; Leukocyte Elastase; Male; Matrix Metalloproteinase 9; Microscopy, Electron, Transmission; Myeloblastin; Neutrophils; Peptide Hydrolases; Proteinase Inhibitory Proteins, Secretory; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thrombosis; von Willebrand Factor

Von Willebrand factor (VWF) plays an important role in mediating platelet adhesion and aggregation under high shear rate conditions. Such platelet aggregates are strengthened by fibrin-network formation triggered by tissue factor (TF). However, little is known about the role of TF in VWF-dependent thrombus formation under blood flow conditions. We evaluated TF in thrombus formation on immobilized VWF under whole blood flow conditions in an in vitro perfusion chamber system. Surface-immobilized TF amplified intra-thrombus fibrin generation significantly under both low and high shear flow conditions, while TF in sample blood showed no appreciable effects. Furthermore, immobilized TF enhanced VWF-dependent platelet adhesion and aggregation significantly under high shear rates. Neutrophil cathepsin G and elastase increased significantly intra-thrombus fibrin deposition on immobilized VWF-TF complex, suggesting the involvement of leukocyte inflammatory responses in VWF/TF-dependent mural thrombogenesis under these flow conditions. These results reveal a functional link between VWF and TF under whole blood flow conditions, in which surface-immobilized TF and VWF mutually contribute to mural thrombus formation, which is essential for normal hemostasis. By contrast, TF circulating in blood may be involved in systemic hypercoagulability, as seen in sepsis caused by severe microbial infection, in which neutrophil inflammatory responses may be active.

Topics: Blood Flow Velocity; Blood Platelets; Cathepsin G; Fibrin; Humans; Neutrophils; Platelet Adhesiveness; Platelet Aggregation; Thromboplastin; Thrombosis; von Willebrand Factor

Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

Topics: Adult; Animals; Blood Platelets; Cathepsin G; Female; Hemostasis; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Platelet Aggregation; Thrombosis

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P- and L-selectin but not thrombin. Heparin prevents mucin binding to P- and L-selectin and mucin-induced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P- and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1-deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet P-selectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. Thus, carcinoma mucins promote thrombosis through adhesion-dependent, bidirectional signaling in neutrophils and platelets.

Topics: Adenocarcinoma, Mucinous; Animals; Antibodies, Neoplasm; Antibodies, Neutralizing; Blood Platelets; Cathepsin G; Cell Line, Tumor; Colonic Neoplasms; Disease Models, Animal; Humans; L-Selectin; Membrane Glycoproteins; Mice; Mice, Knockout; Mucins; Neoplasm Proteins; Neutrophil Activation; Neutrophils; P-Selectin; Platelet Activation; Receptors, Thrombin; Syndrome; Thrombosis

In human abdominal aortic aneurysm, the accumulation of blood-derived cells and proteases within the mural thrombus plays a pivotal role in the evolution toward vessel wall rupture. We sought to identify peptides released from abdominal aortic aneurysm specimens, characterized by an intraluminal thrombus.. Intraluminal thrombus samples were analyzed by differential proteomics, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. A 1309-Da peptide was detected in larger amounts in the newly formed luminal thrombus layer relative to older layers. It was identified as being LVVYPWTQRF (known as LVV-Hemorphin 7), a peptide generated from hemoglobin by cathepsin D. By immunohistochemical analysis, we showed that Hemorphin 7 (H7) colocalizes with cathepsin D and cathepsin G in the luminal layer of the intraluminal thrombus. In vitro, cathepsin G was able to generate H7 peptides at pH 7.4, whereas cathepsin D was only active in acidic conditions. Finally, H7 peptides were shown to be increased 3- to 4-fold in sera of abdominal aortic aneurysm patients relative to controls, and their levels were positively correlated with the volume of the thrombus.. Our results suggest that circulating H7 peptides may reflect proteolysis of hemoglobin in the aneurysmal intraluminal thrombus and may be used as a biological marker of pathological vascular remodeling.

Topics: Aortic Aneurysm, Abdominal; Biomarkers; Case-Control Studies; Cathepsin D; Cathepsin G; Hemoglobins; Humans; Hydrogen-Ion Concentration; Immunohistochemistry; Peptide Fragments; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thrombosis; Tissue Culture Techniques; Up-Regulation

Human neutrophil proteases cathepsin G and elastase can directly alter platelet function and/or participate in coagulation cascade reactions on the platelet or neutrophil surface to enhance fibrin formation. The clotting of recalcified platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (to shut down contact activation) was studied in well-plates or flow assays. Inhibitors of cathepsin G or elastase significantly delayed the burst time (t(50)) of thrombin generation in neutrophil-supplemented PRP from 49 min to 59 and 77 min, respectively, in well-plate assays as well as reduced neutrophil-promoted fibrin deposition on fibrinogen-adherent platelets under flow conditions. In flow assays, purified cathepsin G was a far more potent activator of platelet-dependent coagulation than elastase. Anti-tissue factor had no effect on neutrophil protease-enhanced thrombin formation in PRP. The addition of cathepsin G (425 nm) or convulxin (10 nm) to PRP dramatically reduced the t(50) of thrombin generation from 53 min to 17 or 23 min, respectively. In contrast, the addition of elastase to PRP left the t(50) unaltered. Whereas perfusion of PFP (gamma(w) = 62.5 s(-1)) over fibrinogen-adherent platelets did not result in fibrin formation until 50 min, massive fibrin could be observed on cathepsin G-treated platelets even at 35 min. Cathepsin G addition to corn trypsin inhibitor-treated PFP produced little thrombin unless anionic phospholipid was present. However, further activation inhibition studies indicated that cathepsin G enhances fibrin deposition under flow conditions by elevating the activation state of fibrinogen-adherent platelets rather than by cleaving coagulation factors.

Topics: Adult; Blood Platelets; Cathepsin G; Cathepsins; Cell Adhesion; Fibrin; Fibrinogen; Hemodynamics; Humans; Microscopy, Fluorescence; Microscopy, Video; Models, Biological; Neutrophils; Pancreatic Elastase; Perfusion; Phospholipids; Protein Structure, Tertiary; Serine Endopeptidases; Thrombin; Thromboplastin; Thrombosis; Time Factors

Many human thrombi lyse spontaneously without the administration of lytic drugs and cause no clinical symptoms. The mechanisms by which this occurs are incompletely understood. We found that model thrombi prepared from whole human blood in a Chandler loop also exhibited significant spontaneous lysis. Lysis was inhibited by chemical protease inhibitors, consistent with proteolysis resulting primarily from serine proteases, with a small contribution from matrix metalloproteinases. Whole blood was fractionated into platelet-rich plasma and cell populations. Significant spontaneous lysis was observed in platelet-rich thrombi enriched with polymorphonuclear leucocytes (PMNs), whereas mononuclear cells (MCs) and erythrocytes did not contribute to lysis. Incorporation of antibodies to urokinase (u-PA) and its receptor u-PAR neutralized a large proportion of the activity. Incubation of plasma with PMNs generated free u-PA activity, which was also detectable in model thrombi and in vivo human thrombi. Purified neutrophils, free of eosinophils, generated activity identical to PMNs. Smaller contributions to lysis by tissue-type plasminogen activator (t-PA), elastase and cathepsin G were also identified. These findings suggest a major role for circulating PMNs in endogenous thrombus lysis.

Topics: Antibodies, Monoclonal; Cathepsin G; Cathepsins; Electrophoresis, Polyacrylamide Gel; Humans; Immunohistochemistry; Models, Biological; Neutrophils; Pancreatic Elastase; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Serine Endopeptidases; Thrombosis; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

Activated neutrophils may promote thrombus formation by releasing proteases which may activate platelets, impair the fibrinolytic balance and injure the endothelial monolayer. We have investigated the morphological correlates of damage induced by activated neutrophils on the vascular wall, in particular the vascular injury induced by released cathepsin G in both static and dynamic conditions. Human umbilical vein endothelial cells were studied both in a cell culture system and in a model of perfused umbilical veins. At scanning electron microscopy, progressive alterations of the cell monolayer resulted in cell contraction, disruption of the intercellular contacts, formation of gaps and cell detachment. Contraction was associated with shape change of the endothelial cells, that appeared star-like, while the underlying extracellular matrix, a potentially thrombogenic surface, was exposed. Comparable cellular response was observed in an "in vivo" model of perfused rat arterial segment. Interestingly, cathepsin G was active at lower concentrations in perfused vessels than in culture systems. Restoration of blood flow in the arterial segment previously damaged by cathepsin G caused adhesion and spreading of platelets on the surface of the exposed extracellular matrix. The subsequent deposition of a fibrin network among adherent platelets, could be at least partially ascribed to the inhibition by cathepsin G of the vascular fibrinolytic potential. This study supports the suggestion that the release of cathepsin G by activated neutrophils, f.i. during inflammation, may contribute to thrombus formation by inducing extensive vascular damage.

Topics: Animals; Aorta; Cathepsin G; Cathepsins; Cells, Cultured; Endothelium, Vascular; Humans; In Vitro Techniques; Male; Microscopy, Electron, Scanning; Neutrophils; Perfusion; Rats; Serine Endopeptidases; Thrombosis; Umbilical Veins

Activated polymorphonuclear leukocytes (PMNs) may affect the integrity of blood vessels by endothelial cell injury. We investigated the effects of cathepsin G purified from human neutrophils on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVECs). Cathepsin G (5 and 10 micrograms/ml) induced marked intercellular gap formation after 1 hour of treatment, whereas 1 microgram/ml did not, even after 6 hours incubation. In contrast, plasminogen activator inhibitor-1 (PAI-1) antigen levels, measured by a double antibody enzyme-linked immunosorbent assay, were significantly increased in culture media (CM) on cathepsin G (1 microgram/ml) treatment after 15 minutes (5.1 +/- 1.2 ng/ml vs 2.6 +/- 0.6 ng/ml for controls, p < 0.01) and 6 hours of incubation (69.6 +/- 17.5 ng/ml vs 40.0 +/- 9.0 ng/ml for controls, p < 0.01). Likewise, PAI activity, measured by reverse fibrin autography, increased on cell treatment with cathepsin G. Preincubation of cathepsin G with eglin C (10 micrograms/ml) almost completely abolished the increase in both PAI antigen and activity levels induced by cathepsin G. Cycloheximide, a protein synthesis inhibitor, did not block cathepsin G-induced PAI-1 release. PAI-1 mRNA levels were not affected by HUVEC treatment with cathepsin G (1 microgram/ml for 15 minutes), even after 24 hours. In the extracellular matrix (ECM) PAI-1 antigen levels decreased to 77% and 40% of controls, respectively, after 15 minutes and 6 hours of cathepsin G (1 micrograms/ml) treatment. Reverse fibrin autography also demonstrated a dose-dependent reduction of PAI activity in the ECM on 6 hours of cell treatment with 1 or 5 micrograms/ml cathepsin G. Moreover, ECM prepared from confluent HUVECs released PAI-1 in supernatants on 1 micrograms/ml cathepsin G incubation in a cell-free system. Tissue-type plasminogen activator (t-PA) activity was strongly depressed on cathepsin G treatment, both in CM from HUVECs or in a cell-free system. Finally, PAI-1 was also released from cathepsin G-stimulated platelets in a dose-dependent manner. In summary, our results support a potentially thrombogenic role of cathepsin G, which could impair the fibrinolytic potential of the endothelium. These data give a new insight into the mechanisms by which activated PMNs may promote thrombus formation. On the other hand, the decrease of PAI-1 in ECM could favor penetration and migration of inflammatory or tumor cells through the subendothelial layers.

Topics: Cathepsin G; Cathepsins; Endothelium, Vascular; Extracellular Matrix; Gene Expression; Humans; In Vitro Techniques; Neutrophils; Plasminogen Activator Inhibitor 1; Platelet Aggregation; RNA, Messenger; Serine Endopeptidases; Thrombosis; Tissue Plasminogen Activator