Plasminogen activator inhibitor type I contributes to protective immunity during experimental Gram‐negative sepsis (melioidosis)

Summary. Background: Melioidosis is a frequent cause of sepsis in Southeast Asia caused by the Gram‐negative bacterium Burkholderia pseudomallei. Patients with melioidosis have elevated circulating levels of plasminogen activator inhibitor type 1 (PAI‐1), an important regulator of inflammation and fibrinolysis. Objectives: In this study, we aimed to investigate the role of PAI‐1 during melioidosis. Methods: Wild‐type (WT) and PAI‐1‐deficient (PAI‐1–/1^?/?) mice were intranasally infected with B. pseudomallei. Mice were killed after 24, 48 or 72 h. Lungs, liver and blood were harvested for measurement of bacterial loads, cytokines, clinical chemistry, histopathology, and coagulation parameters. Additionally, survival studies were performed. Results: PAI‐1^?/? mice demonstrated enhanced susceptibility to B. pseudomallei infection, as shown by a strongly increased mortality rate (100% vs. 58% among WT mice, P < 0.001), associated with enhanced bacterial loads in lungs, liver, and blood. Additionally, PAI‐1^?/? mice showed elevated levels of proinflammatory cytokines in lungs and plasma, accompanied by enhanced local and systemic coagulation activation (thrombin–antithrombin complexes and D‐dimer), increased hepatocellular injury (plasma aspartate aminotransferase and alanine aminotransferase), and renal failure (plasma creatinine and urea). Conclusions: PAI‐1 has a protective role during severe Gram‐negative sepsis caused by B. pseudomallei by limiting bacterial growth, inflammation, and coagulation, and probably, as a consequence thereof, distant organ injury.     

The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet‐mediated fibrinolysis resistance: a thromboelastographic study in whole blood

Summary. Background: The resistance of platelet‐rich thrombi to fibrinolysis is generally attributed to clot retraction and platelet PAI‐1 release. The role of TAFI in platelet‐mediated resistance to lysis is unclear. Objective: We investigated the contribution of TAFI to the antifibrinolytic effect of platelets in whole blood by thromboelastography. Methods: Platelet‐poor (PP‐WB, < 40 × 10³ μL^?1) and platelet‐rich (PR‐WB, > 400 × 10³ μL^?1) blood samples were obtained from normal human blood (N‐WB, 150–220 × 10³ μL^?1). Clot lysis time was measured by thromboelastography in recalcified blood supplemented with t‐PA (100 ng mL^?1) and tissue factor (1:1000 Recombiplastin). Results: t‐PA‐induced lysis time increased in parallel with platelet concentration (up to 3‐fold). Neutralization of TAFI, but not of PAI‐1, shortened the lysis time by ～ 50% in PR‐WB and by < 10% in PP‐WB. Accordingly, prothrombin F1+2 and TAFIa accumulation was greater in PR‐WB than in PP‐WB. A similar TAFI‐dependent inhibition of fibrinolysis was observed when clot retraction was prevented by cytochalasin D or abciximab, or when platelet membranes were tested. Moreover, in blood with an intact contact system, platelet‐mediated fibrinolysis resistance was attenuated by an anti‐FXI but not by an anti F‐XII antibody. Finally, platelets made the clots resistant to the profibrinolytic effect of heparin concentrations displaying a strong anticoagulant activity. Conclusions: Our data indicate that TAFI activation is one major mechanism whereby platelets make clots resistant to fibrinolysis and underscore the importance of TAFI inhibitors as new antithrombotic agents.     

Thrombin induces GPIb-IX-mediated fibrin binding to αIIbβ3 in a reconstituted Chinese hamster ovary cell model

BACKGROUND: The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. OBJECTIVES: In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX. METHODS: We used Chinese hamster ovary (CHO) cells stably expressing human alpha(IIb)beta(3) and/or GPIb-IX complexes (CHO-alpha(IIb)beta(3)-IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor. RESULTS: Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO-alpha(IIb)beta(3)-IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against alpha(IIb)beta(3) and GPIbalpha. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3zeta adaptor and calcium-calmodulin-dependent events are involved. CONCLUSIONS: The present data support a significant role of GPIb-IX and alpha(IIb)beta(3) receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin.     

EMMPRIN (CD147/basigin) mediates platelet–monocyte interactions in vivo and augments monocyte recruitment to the vascular wall

Summary. Background: Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. Objectives: To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet–monocyte interactions. Methods and Results: Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet–monocyte interactions under flow conditions. Monocytes readily bound to surface‐adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA‐1 were significantly decreased when ANA‐1 cells were pretreated with EMMPRIN‐silencing small interfering RNA prior to injection into wild‐type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN‐silenced ANA‐1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA‐1–platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN‐mediated platelet–monocyte aggregate formation in vivo. Conclusions: EMMPRIN supports platelet–monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development.     

Thrombin generation‐based assays to measure the activity of the TFPI–protein S pathway in plasma from normal and protein S‐deficient individuals

Summary. Background: Protein S acts as a cofactor for full‐length tissue factor pathway inhibitor (TFPI) in the downregulation of thrombin formation. Objective: To develop a functional test to measure the activity of the TFPI–protein S system in plasma. Methods/Patients: Using calibrated automated thrombography, we quantified the activity of the TFPI–protein S system in plasma by measuring thrombin generation in the absence and presence of neutralizing antibodies against protein S or TFPI. Moreover, we designed an enzyme‐linked immunosorbent assay (ELISA) to determine the level of full‐length TFPI in plasma. The performance of these assays was examined in plasma from 85 normal individuals and from 35 members of protein S‐deficient families. Results: The ratio of thrombin peaks determined in the absence and presence of anti‐protein S antibodies (protein S ratio = 0.5 in normal plasma) is a measure of the TFPI cofactor activity of protein S, whereas the ratio of thrombin peaks determined in the absence and presence of anti‐TFPI antibodies (TFPI ratio = 0.25 in normal plasma) is a measure of the overall activity of the TFPI–protein S system. Protein S and TFPI ratios were elevated in protein S‐deficient individuals, indicating an impairment of the TFPI–protein S system. Both ratios correlated well with full‐length TFPI levels, which were significantly lower in protein S‐deficient patients than in normal family members. Conclusions: Functional assays for the TFPI–protein S system and an ELISA for full‐length TFPI were developed. These assays show that the activity of the TFPI–protein S anticoagulant pathway is impaired in individuals with congenital protein S deficiency.     

Cholestatic liver injury as a side-effect of dabigatran and the use of coagulation tests in dabigatran intoxication and after reversal by idarucizumab in bleeding and sepsis

Idarucizumab, an antidote specific for dabigatran, became available recently. Dabigatran is not associated with increased risk of hepatotoxicity in comparison with warfarin, but it is seen as a rare side-effect. Cases of cholestatic liver injury due to dabigatran have not been reported previously. We present a case of severe gastro-intestinal bleeding with underlying dabigatran intoxication in a patient with renal failure and the effect of reversal of dabigatran using idaruzicumab on coagulation assays. International normalized ratio (INR) and activated partial thromboplastin time (APTT) results were elevated in a setting of sepsis, possibly due to liver failure. INR and APTT can be elevated if sepsis is complicated by disseminated intravascular coagulation (DIC) or liver failure, making it challenging to determine dabigatrans contribution to their prolongation. A rebound effect after administration of idarucizumab and slow elimination of dabigatran due to reduced kidney function could be detected using the Hemoclot^® diluted thrombin time (dTT) in this situation, in contrast to with non-dilutional assays. Before admission, cholestatic liver injury started shortly after initiation of dabigatran etexilate therapy. As no other cause was found, this liver injury was likely to be drug-induced. Bleeding cessated promptly after administration of idarucizumab in dabigatran intoxication. In conclusion, the anticoagulant effect of dabigatran can be measured by Hemoclot^® dTT in sepsis and cholestatic liver injury was seen as a possible rare side-effect of dabigatran treatment.     

Three‐dimensional culture of single embryonic stem‐derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling

In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES‐NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES‐NSPCs retained high cell viability and proliferated within small‐sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII‐tubulin and NF200 over time. At day 14, cell‐matrix constructs mainly comprised NSPCs and neurons (46.5% βIII‐tubulin⁺ cells). Gamma‐aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES‐NSPCs expressed matriptase and secreted MMP‐2/9, with MMP‐2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES‐NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three‐dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES‐NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin‐based hydrogels for ES‐NSPC delivery into the injured central nervous system. Copyright © 2016 John Wiley & Sons, Ltd.