Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor.factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-3H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.

No abstract

Tissue factor pathway inhibitor (TFPI) is a vascular anticoagulant that regulates the tissue (TF)-dependent pathway of coagulation. The majority of intravascular TFPI is thought to be noncovalently bound to the vessel wall. Our immunolocalization studies in cultures of human umbilical vein endothelial cells (HUVEC) and immortalized EA.hy926 cells that TFPI is located in well-defined granules evenly spread over the cell surface and with apical polarization within the cytoplasm. These granules are smaller than and distinct from Weibel-Palade bodies. Upon treatment of cultured cells with low concentrations of thrombin (0.01 to 1 NIH U/mL), a marked redistribution of TFPI, occurred with patching in focal points and increased exposure of both TFPI antigen and anticoagulant activity on the surface of the stimulated endothelial cells. This redistribution was paralleled by an acute release of TFPI in the cell medium. EA.hy926 cells responded more readily to thrombin stimulation than HUVECs. The process was inhibited by both hirudin and anti-thrombin receptor antibody. Our findings demonstrate a novel mechanism by which thrombin may exert a negative feedback control on blood coagulation. Therefore, this pathway can be physiological importance in controlling TF-mediated thrombin generation.

IMPORTANCEThe efficacy of antiplatelet therapy in critically ill patients with COVID-19 is uncertain.OBJECTIVE To determine whether antiplatelet therapy improves outcomes for critically ill adults with COVID-19. DESIGN, SETTING, AND PARTICIPANTSIn an ongoing adaptive platform trial (REMAP-CAP) testing multiple interventions within multiple therapeutic domains, 1557 critically ill adult patients with COVID-19 were enrolled between October 30, 2020, and June 23, 2021, from 105 sites in 8 countries and followed up for 90 days (final follow-up date: July 26, 2021).INTERVENTIONS Patients were randomized to receive either open-label aspirin (n = 565), a P2Y12 inhibitor (n = 455), or no antiplatelet therapy (control; n = 529). Interventions were continued in the hospital for a maximum of 14 days and were in addition to anticoagulation thromboprophylaxis. MAIN OUTCOMES AND MEASURESThe primary end point was organ support-free days (days alive and free of intensive care unit-based respiratory or cardiovascular organ support) within 21 days, ranging from −1 for any death in hospital (censored at 90 days) to 22 for survivors with no organ support. There were 13 secondary outcomes, including survival to discharge and major bleeding to 14 days. The primary analysis was a bayesian cumulative logistic model. An odds ratio (OR) greater than 1 represented improved survival, more organ support-free days, or both. Efficacy was defined as greater than 99% posterior probability of an OR greater than 1. Futility was defined as greater than 95% posterior probability of an OR less than 1.2 vs control. Intervention equivalence was defined as greater than 90% probability that the OR (compared with each other) was between 1/1.2 and 1.2 for 2 noncontrol interventions. RESULTSThe aspirin and P2Y12 inhibitor groups met the predefined criteria for equivalence at an adaptive analysis and were statistically pooled for further analysis. Enrollment was discontinued after the prespecified criterion for futility was met for the pooled antiplatelet group compared with control. Among the 1557 critically ill patients randomized, 8 patients withdrew consent and 1549 completed the trial (median age, 57 years; 521 [33.6%] female). The median for organ support-free days was 7 (IQR, −1 to 16) in both the antiplatelet and control groups (median-adjusted OR, 1.02 [95% credible interval {CrI}, 0.86-1.23]; 95.7% posterior probability of futility). The proportions of patients surviving to hospital discharge were 71.5% (723/1011) and 67.9% (354/521) in the antiplatelet and control groups, respectively (median-adjusted OR, 1.27 [95% CrI, 0.99-1.62]; adjusted absolute difference, 5% [95% CrI, −0.2% to 9.5%]; 97% posterior probability of efficacy). Among survivors, the median for organ support-free days was 14 in both groups. Major bleeding occurred in 2.1% and 0.4% of patients in the antiplatelet and control groups (adjusted OR, 2.97 [95% CrI,; adjusted absolute risk increase, 0.8% [95% CrI, 0.1%-2.7%]; 99.4% probability of harm).CONCLUSIONS AND RELEVANCE Among crit...

No abstract