Procoagulant phenotype of virus-infected pericytes is associated with portal thrombosis and intrapulmonary vascular dilations in fatal COVID-19.

BACKGROUND & AIMS: Mechanisms and clinical impact of portal microthrombosis featuring severe COVID-19 are unknown. Intrapulmonary vascular dilation (IPVD)-related hypoxia has been described in severe liver diseases. We hypothesized that portal microthrombosis is associated with IPVD and fatal respiratory failure in COVID-19. METHODS: Ninety-three patients who died from COVID-19, were analysed for portal microvascular damage (histology), IPVD (histology and chest-computed tomography, CT), and hypoxemia (arterial blood gas). Seventeen patients who died from COVID-19-unrelated pneumonia served as controls. Vascular lesions and microthrombi were phenotyped for endothelial (vWF) and pericyte (αSMA/PDGFR-ß) markers, tissue factor (TF), viral spike-protein and nucleoprotein (SP, NP), fibrinogen, platelets (CD41a). Viral particles in vascular cells were assessed by transmission electron microscopy (TEM). Cultured pericytes were infected with SARS-CoV-2 to measure TF expression and tubulisation of human pulmonary microvascular endothelial cells (HPMEC) was assessed upon vWF treatment. RESULTS: IPVD was present in 16/66 COVID-19 patients with both liver and lung histology, with a younger age (62 vs 78yo), longer illness (25 vs 14 days), worsening hypoxemia (PaO2/FiO2 from 209 to 89), and more ventilatory support (63 vs 22%) compared to COVID-19/Non-IPVD. IPVD, absent in controls, were confirmed by chest-CT. COVID-19/IPVD liver histology showed portal microthrombosis in >82.5% of portal areas, with a thicker wall of αSMA/PDGFR-ß+/ SP+/NP+ pericytes compared with COVID-19/Non-IPVD. Thrombosed portal venules correlated with αSMA+ area, whereas infected SP+/NP+ pericytes expressed TF. SARS-CoV-2 viral particles were observed in portal pericytes. In-vitro SARS-CoV-2 infection of pericytes up-regulated TF and induced endothelial cells to overexpress vWF, which expanded HPMEC tubules. CONCLUSIONS: SARS-CoV-2 infection of liver pericytes elicits a local procoagulant response associated with extensive portal microthrombosis, IPVD and worsening respiratory failure in fatal COVID-19. IMPACT AND IMPLICATIONS: Vascular involvement of the liver represents a serious complication of COVID-19 infection that must be considered in the work-up of patients with long-lasting and progressively worsening respiratory failure, as it may associate with the development of intrapulmonary vascular dilations. This clinical picture is associated with a pro-coagulant phenotype of portal venule pericytes, which is induced by SARS-CoV-2 infection of pericytes. Both observations provide a model that may apply, at least in part, to other vascular disorders of the liver, featuring obliterative portal venopathy, similarly characterized at the clinical level by development of hypoxemia and at the histological level, by phlebosclerosis and reduced caliber of the portal vein branches in the absence of cirrhosis. Moreover, our findings bring light to an as yet overlooked player of thrombosis pathophysiology, i.e. pericytes, which may provide novel therapeutic tools to halt prothrombotic mechanisms.

Profiling plasma alterations of extracellular vesicles in patients with acutely decompensated cirrhosis and bacterial infection.

BACKGROUND: Extracellular vesicles (EVs) modulate inflammation, coagulation and vascular homeostasis in decompensated cirrhosis. AIM: To characterize the profile of plasmatic EVs in patients with decompensated cirrhosis and bacterial infections and evaluate the association between EVs and the development of hemostatic complications. METHODS: We measured the levels of EVs using high-sensitivity flow cytometry and phospholipid-dependent clotting time (PPL) in a prospective cohort of hospitalized patients with acutely decompensated cirrhosis with versus without bacterial infections. A separate cohort of patients with bacterial infections without cirrhosis was also enrolled. We measured endothelium-, tissue factor (TF)-bearing, platelet- and leukocyte-derived EVs. In patients with infections, EVs were reassessed upon resolution of infection. Bleeding and thrombotic complications were recorded during 1-year follow-up. RESULTS: Eighty patients with decompensated cirrhosis were recruited (40 each with and without bacterial infections). Electron microscopy confirmed the presence of plasma EVs. Despite no difference in total EVs and PPL, patients with cirrhosis and infection had significantly higher TF+ EVs, P-Selectin+ EVs (activated platelet-derived), CD14+ EVs (monocyte/macrophages derived) and CD14+ TF+ EVs versus those with cirrhosis without infection. Upon infection resolution, levels of these EVs returned to those without infection. Patients with infections showed a significant association between reduced P-Selectin+ EVs and bleeding complications (HR 8.0 [95%CI 1.3-48.1]), whereas high levels of leukocyte-derived EVs (CD45+) and CD14+ EVs were significantly associated with thrombotic complications (HR 16.4 [95%CI 1.7-160] and 10.9 [95%CI 1.13-106], respectively). Results were confirmed in a validation cohort. CONCLUSION: Bacterial infections are associated with particular alterations of plasma EVs profile in decompensated cirrhosis. Bacterial infections trigger the release of EVs originating from various cell types, which may tip the precarious hemostatic balance of patients with acutely decompensated cirrhosis towards hyper- or hypocoagulability.

Cryptogenic ischemic stroke in cardiac transthyretin amyloidosis and sinus rhythm: a case report.

Cardiac amyloidosis is a group of diseases characterized by the deposition of amyloid fibers in cardiac tissue. Two forms are mainly reported: light chain (AL) and transthyretin (ATTR) amyloidosis. Among the complications of transthyretin amyloidosis there are thrombotic events and, to a lesser extent, hemorrhagic events. The latter are likely caused by perivascular amyloid deposition resulting in capillary fragility, in addition to INR lability during anticoagulant therapy. The onset of thrombotic events may be caused by the high prevalence of atrial fibrillation (AF), mechanical cardiac dysfunction and atrial myopathy observed in patients with transthyretin amyloidosis. It remains unclear why thromboembolic events occur even in patients with sinus rhythm or adequate anticoagulation, though a hypercoagulable state or underlying inflammation may be involved. We report a case of cryptogenic ischemic stroke in an 86-year-old woman with transthyretin amyloidosis and sinus rhythm. Traditional coagulation tests, whole blood rotational thromboelastometry and impedance aggregometry did not show a hypercoagulable state. The thrombin generation assay did not reveal a prothrombotic state. However, the study of extracellular vesicles highlighted underlying immune-mediated endothelial damage likely responsible for the thrombotic diathesis. It could be hypothesized that inflammation plays a role in the hypercoagulability of patients with transthyretin amyloidosis. Larger prospective studies are needed to validate our hypothesis.

Reticulated platelets are increased and hyper-activated in patients with cirrhosis, especially those with poor outcome.

BACKGROUND: Reticulated platelets (RePLT) are emergency circulating platelets released to contrast peripheral platelet destruction. AIM: We conducted a prospective study to [a] characterize RePLT in cirrhosis; [b] evaluate the association between RePLT and hepatic decompensation/death. METHODS: Cirrhosis patients without hepatocellular carcinoma were prospectively recruited and underwent assessment of RePLT and thrombopoietin (TPO). RePLT were evaluated by cytofluorimetry and immuno-fluorescence microscopy. Twenty healthy subjects were included as controls. Patients were followed for 6 months for hepatic decompensation and further decompensation/ACLF. RESULTS: Forty-five patients were included (Child-Pugh [CP] A/B/C 18/11/16). Compared to controls, RePLT in cirrhosis were significantly increased (0.82% vs. 0.05%; p < 0.001) and hyperactivated (4.35% vs. 0.17%; p = 0.004). No correlation was observed between RePLT and CP, platelet count, TPO, MELD score, and C-reactive protein. TPO was lower in cirrhosis than controls (28 pg/mL vs. 52 pg/mL; p = 0.005), decreasing significantly with CP stage. In CP B/C patients (n = 27), RePLT were significantly higher in those who progressed towards further decompensation/ACLF (2.11 [0.56-2.95] vs. 0.69 [0.02-1.22]; p < 0.01). A proportion of RePLT >2% accurately identified high-risk patients (AUROC 0.818; 95%CI: 0.639-0.997; sensitivity 94%, specificity 73%). CONCLUSION: RePLT in cirrhosis are increased and hyper-activated. In decompensated patients, higher RePLT appear to be associated with worse outcomes.

In vitro and ex vivo rescue of a nonsense mutation responsible for severe coagulation factor V deficiency.

BACKGROUND: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. OBJECTIVES: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. METHODS: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. RESULTS: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 µM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. CONCLUSION: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.

Whole blood thrombin generation shows a significant hypocoagulable state in patients with decompensated cirrhosis.

BACKGROUND: Patients with cirrhosis have a normal to increased thrombin generation (TG) capacity in platelet-poor plasma (PPP). By reflecting the contribution of all circulating blood cells, whole blood (WB) TG may allow a more physiological assessment of coagulation. OBJECTIVES: We compared WB-TG vs PPP-TG in patients with cirrhosis. METHODS: Assessment of coagulation included routine tests, factor VIII, natural anticoagulants, PPP-TG, and WB-TG. TG assays were performed with and without thrombomodulin. Twenty-five healthy subjects were included as controls. RESULTS: We included 108 patients (Child-Pugh A/B/C, 44/24/40). Compared with controls, patients had significantly lower platelet count, longer international normalized ratio, higher FVIII, and lower levels of protein C/S and antithrombin. Regarding thrombomodulin-modified TG assays, in compensated cirrhosis, both PPP-TG and WB-TG indicated an increased TG capacity, as reflected by an endogenous thrombin potential (ETP) significantly higher than controls. In contrast, in decompensated cirrhosis, PPP-TG indicated a hypercoagulable state with increased ETP, higher peak height, and shorter time-to-peak than controls, whereas WB-TG revealed a progressive impairment of TG kinetics and total capacity, ultimately resulting in a profound hypocoagulable state in patients with Child-Pugh C cirrhosis (ie, significant prolongation of lag time and time-to-peak with reduction of both ETP and peak height). In decompensated patients, bacterial infections and severity of anemia were associated with a further reduction of both ETP and peak height. CONCLUSION: Compensated cirrhosis is associated with an increased TG capacity. In decompensated cirrhosis, contrary to PPP-TG, which indicates hypercoagulability, WB-TG shows a significant hypocoagulable state. The clinical value of these findings deserves further investigation.

Crucial roles of red blood cells and platelets in whole blood thrombin generation.

Red blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI-induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.