Activated protein C (APC) has potent anticoagulant and antiinflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/ D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.

Retinoic acids are morphogenic signaling molecules that are derived from vitamin A and involved in a variety of tissue functions. Two groups of their nuclear receptors have been identified: retinoic acid receptors (RARs) and retinoic acid X receptors (RXRs). All-trans retinoic acid is the high affinity ligand for RARs, and 9-cis retinoic acid also binds to RXRs with high affinity. In cells at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid via unknown mechanisms. It was previously shown that retinoic acids prevents activation-induced death of thymocytes. Here, we report that both all-trans and 9-cis retinoic acid induce apoptosis of mouse thymocytes and purified CD4+CD8+ cells in ex vivo cultures, with 9-cis retinoic acid being 50 times more effective. The induction of apoptosis by retinoic acids is mediated by RARgamma because (a) the phenomenon can be reproduced only by RARgamma-selective retinoic acid analogs, (b) the cell death induced by either retinoic acids or RARgamma analogs can be inhibited by RARgamma-specific antagonists, and (c) CD4+CD8+ thymocytes express RARgamma. In vivo administration of an RARgamma analog resulted in thymus involution with the concomitant activation of the apoptosis-related endonuclease and induction of tissue transglutaminase. The RARgamma pathway of apoptosis is RNA and protein synthesis dependent, affects the CD4+CD8+ double positive thymocytes, and can be inhibited by the addition of either Ca2+ chelators or protease inhibitors. Using various RAR- and RXR-specific analogs and antagonists, it was demonstrated that stimulation of RAR alpha inhibits the RARgamma-specific death pathway (which explains the lack of apoptosis stimulatory effects of all-trans retinoic acid at physiological concentrations) and that costimulation of the RXR receptors (in the case of 9-cis retinoic acid) can neutralize this inhibitory effect. It is suggested that formation of 9-cis retinoic acid may be a critical element in regulating both the positive selection and the "default cell death pathway" of thymocytes.

Acquired factor XIII (FXIII) deficiency due to autoantibody against FXIII is a very rare severe hemorrhagic diathesis. Antibodies directed against the A subunit of FXIII, which interfere with different functions of FXIII, have been described. Here, for the first time, we report an autoantibody against the B subunit of FXIII (FXIII-B) that caused lifethreatening bleeding in a patient with systemic lupus erythematosus. FXIII activity, FXIII-A 2 B 2 complex, and individual FXIII subunits were undetectable in the plasma, whereas platelet FXIII activity and antigen were normal. Neither FXIII activation nor its activity was inhibited by the antibody, which bound to structural epitope(s) on both free and complexed FXIII-B. The autoantibody highly accelerated the elimination of FXIII from the circulation. FXIII supplementation combined with immunosuppressive therapy, plasmapheresis, immunoglobulin, and anti-CD20 treatment resulted in the patient's recovery. FXIII levels returned to around 20% at discharge and after gradual increase the levels stabilized above 50%. IntroductionBlood coagulation factor XIII (FXIII) is a protransglutaminase of tetrameric structure (FXIII-A 2 B 2 ). 1,2 Its potentially active A subunit (FXIII-A) is synthesized in cells of bone marrow origin; it is also present in platelets and monocytes/macrophages in dimeric form (FXIII-A 2 ). The noncatalytic B subunit (FXIII-B) is in excess and it is essential for the stabilization of FXIII-A 2 in plasmatic conditions. FXIII is converted into an active transglutaminase (FXIIIa) by limited proteolysis of FXIII-A and by Ca 2ϩ -induced dissociation of FXIII-B. Cross-linking of fibrin ␣-and ␥-chains and ␣ 2 -plasmin inhibitor to fibrin by FXIIIa stabilizes fibrin and protects it from prompt elimination by plasmin. 3 Inherited FXIII-A deficiency is a severe bleeding diathesis with the high risk of intracranial bleeding in nonsupplemented patients. 4 Only 5 cases of inherited FXIII-B deficiency with mild-to-moderate bleeding tendency have been reported. [5][6][7][8] In the absence of FXIII-B, plasma FXIII activity and FXIII-A concentration were considerably decreased, whereas in platelets a normal amount of FXIII-A was measured. 9 Thirty-six cases of severe FXIII deficiency due to an autoantibody against FXIII-A have been reported. In a few cases, the autoantibody was characterized and classified into subgroups according to its inhibition of FXIII activation, FXIIIa activity, or binding to fibrin. [10][11][12][13][14][15] In about one third of the cases the autoantibody was associated with systemic lupus erythematosus (SLE). No report on an autoantibody directed against FXIII-B has been published so far. MethodsA 28-year-old woman suffering from SLE with end-stage kidney disease was on hemodialysis. For preparation of cubital fistula she was admitted to a county hospital. In the proximity of the surgical wound, hematomas developed that were explored. The wounds showed no tendency of healing and remained open. A few days later extensive intramuscular hematoma ...

Endothelial cells express surface angiotensin-converting enzyme 2 (ACE2), the main receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that promotes the infection of endothelial cells showing activation and damage. Bronchoalveolar lavage fluid from coronavirus disease-2019 (COVID-19) subjects showed a critical imbalance in the renin-angiotensin-aldosterone system with the upregulated expression of ACE2. Recently, intravenous recombinant ACE2 was reported as an effective therapy in severe COVID-19 by blocking the viral entry to target cells. Here, we present a case of a critically ill COVID-19 patient with acute respiratory distress syndrome where circulating ACE2 was first measured to monitor disease prognosis. ACE2 activity increased about 40-fold over the normal range and showed a distinct time course as compared to 2-3-fold higher levels of endothelium biomarkers. Although the level of soluble E-selectin followed the clinical status of our patient similar to ferritin and IL-6 levels, the dramatic rise in serum ACE2 activity may act as an endogenous nonspecific protective mechanism against SARS-CoV-2 infection that preceded the recovery of our patient.

In the present study, humoral and T cell-mediated immune responses elicited by BBIBP-CorV (inactivated virus) and BNT162b2 (mRNA-based) vaccines against SARS-CoV-2 virus were compared. Convalescent volunteers were also investigated to evaluate adaptive immunity induced by live virus. Although both vaccines induced antibody- and T cell-mediated immune responses, our analysis revealed significant quantitative and qualitative differences between the two types of challenges. The BBIBP-CorV vaccine elicited antireceptor-binding domain (RBD) IgG, as well as anti-spike protein (S) IgG and IgA antibodies in healthy individuals, the levels of which were much lower than after BNT162b2 vaccination but still higher than in the convalescent patients. The cumulative IFNγ-positive T cell response, however, was only twofold higher in participants injected with BNT162b2 compared to those who were primed and boosted with BBIBP-CorV vaccine. Moreover, the inactivated virus vaccine induced T cell response that targets not only the S but also the nucleocapsid (N) and membrane (M) proteins, whereas the mRNA vaccine was able to elicit a much narrower response that targets the S protein epitopes only. Thus, the pattern of BBIBP-CorV-induced T cell response in virus-naive participants was similar to the cell-mediated anti-SARS-CoV-2 response observed in convalescent patients. Based on these data, we can conclude that the BBIBP-CorV inactivated virus vaccine is immunologically effective. However, the duration of BBIBP-CorV-induced integrated, antibody, and T cell-mediated, immune responses needs further investigation.