ALBERT

Description: Junctional adhesion molecule A (JAM-A) is a transmembrane adhesive glycoprotein that participates in the organization of endothelial tight junctions and contributes to leukocyte transendothelial migration. We demonstrate here that cultured endothelial cells not only express a cellular 43-kDa variant of JAM-A but also release considerable amounts of a 33-kDa soluble JAM-A variant. This release is enhanced by treatment with proinflammatory cytokines and is associated with the down-regulation of surface JAM-A. Inhibition experiments, loss/gain-of-function experiments, and cleavage experiments with recombinant proteases indicated that cleavage of JAM-A is mediated predominantly by the disintegrin and metalloproteinase (ADAM) 17 and, to a lesser extent, by ADAM10. Cytokine treatment of mice increased JAM-A serum level and in excised murine aortas increased ADAM10/17 activity correlated with enhanced JAM-A release. Functionally, soluble JAM-A blocked migration of cultured endothelial cells, reduced transendothelial migration of isolated neutrophils in vitro, and decreased neutrophil infiltration in a murine air pouch model by LFA-1– and JAM-A–dependent mechanisms. Therefore, shedding of JAM-A by inflamed vascular endothelium via ADAM17 and ADAM10 may not only generate a biomarker for vascular inflammation but could also be instrumental in controlling JAM-A functions in the molecular zipper guiding transendothelial diapedesis of leukocytes.

Description: Background: The Kunitz type tissue factor pathway inhibitor (TFPI) is an important regulator in hemostasis. Decreased concentrations are a risk factor for thrombosis and complete TFPI deficiency is associated with lethality. TFPI is also reported to be an important link between inflammation and thrombosis. Neutrophil extracellular traps (NETs) formed by NETosis can bind TFPI which then can be cleaved and inactivated by neutrophil elastase (NE) during thrombotic events. In neutrophils, the enzyme peptide arginine deiminase 4 (PAD4) is central in the citrullination of histones prior to the externalization of DNA during NETosis. In this study, we provide evidence that PAD4 also might regulate the activity of TFPI by posttranslational modification of its functional arginines into citrulline. Aim: To study the effect of citrullination of TFPI and various TFPI constructs on their functional activity on FXa or thrombin generation. Methods: Citrullination of TFPI by the neutrophil protein PAD4 was studied in a model system (FXa inhibition) and in plasma system (thrombin generation). Various TFPI constructs, Kunitz (K) domains K1K2, K2, and TFPI1-161 were used to study the effects of citrullination on inhibition of FXa. LC-MS was used to locate the specific sites of citrullination. Results: This study shows that PAD4 very efficiently citrullinates full lenght TFPI. Very low concentrations of PAD4 were sufficient (Ki 0.4 nM) to abolish FXa inhibition by TFPI. Citrullination is calcium-ion, time- and concentration-dependent. The truncated variants K1K2 and TFPI 1-161 and the isolated K2 domain were citrullinated less efficiently by PAD4 than TFPI, implying the presence of specific binding sites for PAD4 at the C-terminus of TFPI. The presence of phospholipids inhibited the citrullination reaction, an effect only seen for TFPI and not for all the other TFPI variants. Thus, the presence of the C-terminus in TFPI appears to be favourable for citrullination by PAD4. Thrombin generation in TFPI-deficient plasma triggered with TF or Russell's viper venom (RVV)-X showed almost complete absence of anticoagulant activity of citrullinated TFPI Conclusions: To conclude, only TFPI is very sensitive to citrullination by PAD4. Citrullinated TFPI has lost its ability to inhibit FXa. This process might play a role in the increased thrombosis risk with inflammation. Further experiments are needed to determine the physiologic or pathologic relevance of this process. Disclosures No relevant conflicts of interest to declare.

Description: The chemokines platelet factor 4 (PF4) and RANTES (regulated on activation normal T cell expressed and secreted) are secreted by activated platelets and influence multiple cell types and biologic processes. For instance, PF4 inhibits progenitor cell proliferation and angiogenesis, while platelet-derived RANTES is involved in vascular recruitment of monocytes. However, little is known about functional interactions of PF4 and RANTES. Here we show that the presence of PF4 enhanced the arrest of RANTES-stimulated monocytes and monocytic cells on activated endothelial cells under flow conditions, while binding of PF4 to the monocyte surface was increased by RANTES. Both RANTES-triggered arrest and PF4 binding involved monocytic chondroitin sulfate. Ligand blots and surface plasmon resonance revealed a robust heterophilic interaction of PF4 with RANTES but not with RANTES variants defective in higher order oligomerization. The tetrameric mutant E26A bound to the monocyte surface without increasing PF4 binding, and monocyte arrest induced by E26A-RANTES was not enhanced by PF4. Stimulation of monocytes with supernatants of activated platelets triggered arrest involving RANTES and PF4, as shown by inhibition studies. Our results suggest that heterophilic interactions with PF4 require structural motifs important in RANTES oligomerization and amplify RANTES-triggered effects on monocyte adhesion. This may have implications for the modulation of inflammatory recruitment by platelet-derived chemokines.

Description: Protein S exhibits anticoagulant activity independent of activated protein C (APC). An automated factor Xa–based one-stage clotting assay was developed that enables quantification of the APC-independent activity of protein S in plasma from the ratio of clotting times (protein S ratio [pSR]) determined in the absence and presence of neutralizing antibodies against protein S. The pSR was 1.62 ± 0.16 (mean ± SD) in a healthy population (n = 60), independent of plasma levels of factors V, VIII, IX, and X; protein C; and antithrombin, and not affected by the presence of factor V Leiden. The pSR strongly correlates with the plasma level of protein S and is modulated by the plasma prothrombin concentration. In a group of 16 heterozygous protein S–deficient patients, the observed mean pSR (1.31 ± 0.09) was significantly lower than the mean pSR of the healthy population, as was the pSR of plasma from carriers of the prothrombin G20210A mutation (1.47 ± 0.21; n = 46). We propose that the decreased APC-independent anticoagulant activity of protein S in plasma with elevated prothrombin levels may contribute to the thrombotic risk associated with the prothrombin G20210A mutation.