Dominique Baruch

Decreased ADAMTS-13 (A disintegrin-like and metalloprotease with thrombospondin type 1 repeats) is associated with a poor prognosis in sepsis-induced organ failure.

Objective:The inability to regulate the inflammatory response initiated upon infection leads to severe sepsis, characterized by widespread microvascular injury and thrombosis, organ ischemia, and dysfunction. A disintegrin-like and metalloprotease with thrombospondin type 1 repeats (ADAMTS)-13 regulates primary hemostasis by proteolyzing von Willebrand factor (VWF). Decreased ADAMTS-13 has been reported in disseminated intravascular coagulation due to severe sepsis. The present study investigates whether the sepsis-related dysregulation of endothelial activation leads to specific changes of ADAMTS-13. Design:Case-control study. Setting:Adult intensive care unit in a university hospital. Patients/Subjects:Three groups were studied: a case group of 30 patients with severe sepsis, a control group of 29 patients with comparable organ failure unrelated to sepsis, and 30 age- and gender-matched healthy subjects. Interventions:None. Measurements and Main Results:Significantly lower ADAMTS-13 activity was observed in patients with severe sepsis (43.2%; interquartile range, 32.7, 67.0) than in patients with organ failure unrelated to sepsis (67.8%; 57.4, 87.9; p < .05) and healthy subjects (105.6%; 87.2, 125.6; p < .001). Accordingly, ADAMTS-13 antigen was more decreased in patients with severe sepsis than in patients with organ failure unrelated to sepsis and healthy subjects. VWF antigen was higher in patients with severe sepsis than in patients with organ failure unrelated to sepsis and healthy subjects. We found strong negative correlations in severe sepsis but not in organ failure unrelated to sepsis, between ADAMTS-13 activity and 1) VWF antigen; 2) thrombomodulin; 3) interleukin-6; 4) Acute Physiology and Chronic Health Evaluation II score; 5) shock; 6) acute renal injury. Moreover, patients above the median of ADAMTS-13 activity presented a higher survival compared with those below the median in the patients with severe sepsis but not in the patients with organ failure unrelated to sepsis. In contrast, there was no significant association between VWF antigen and survival in either the severe sepsis group or the group with organ failure unrelated to sepsis. Conclusions:We observed low ADAMTS-13 activity and antigen in severe sepsis and in other conditions associated with organ dysfunction. ADAMTS-13 levels were significantly associated with differences in morbidity, mortality, and variables of inflammation and endothelial dysregulation only in severe sepsis patients. This suggests that ADAMTS-13 deficiency may have a pathophysiological relevance specific to severe sepsis.

Exposure of human megakaryocytes to high shear rates accelerates platelet production

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation.

Localization of von Willebrand factor binding domains to endothelial extracellular matrix and to type VI collagen.

We have recently shown that von Willebrand factor (vWF) binds to endothelial and fibroblastic extracellular matrixes (ECM) in a dose-dependent, specific, and saturable way. To localize the domain on the vWF subunit responsible for this interaction, purified proteolytic fragments of vWF were compared for their ability to inhibit 125I-vWF binding to ECM. A tryptic dimeric fragment of 116 kD (T116), extending from amino acid (aa) residues 449 to 728, produced a significant inhibition of 125I-vWF binding to the ECM. In contrast, P34 (aa 1-272), SpI (aa 911-1,365), and SpII (aa 1,366-2,050) had no significant effect on 125I-vWF binding to the ECM. Using an immunofluorescence technique, we identified type VI collagen and heparan sulfate in the endothelial ECM. 125I-vWF was found to bind specifically to purified type VI collagen. Unlabeled vWF and SpIII were able to completely inhibit 125I-vWF binding to type VI collagen. T116 and SpI appeared as competitors of this interaction, whereas P34 and SpII were not. Our data suggest that vWF binds to the endothelial ECM through the T116 fragment and that T116 and SpI each contain a binding site for type VI collagen. Heparin is known to be a vWF ligand, but did not appear as a competitor of vWF binding to the ECM, nor did heparan sulfate.

Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains

A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E. coli. This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter. On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE. It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb. Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin.

The Cleavage of Neutrophil Leukosialin (CD43) by Cathepsin G Releases Its Extracellular Domain and Triggers Its Intramembrane Proteolysis by Presenilin/γ-Secretase

The highly negatively charged membrane sialoglycoprotein leukosialin, CD43, is shed during neutrophil activation. This is generally thought to enhance cell adhesion. We here describe two novel consequences of this shedding, during neutrophil activation by phorbol esters or by chemoattractants after TNF-α priming. CD43 proteolysis was investigated by Western blotting, using a polyclonal antibody to CD43 intracellular domain. Our data emphasize the importance of a juxtamembranous cleavage of about 50% of membrane CD43 molecules by cathepsin G. Indeed, it is inhibited by α1-antichymotrypsin and cathepsin G inhibitor I and is reproduced by exogenous purified cathepsin G. The resulting membrane-anchored C-terminal fragment, CD43-CTF, becomes susceptible to presenilin/γ-secretase, which releases CD43 intracytoplasmic domain: preincubation with three different γ-secretase inhibitors, before PMN treatment by agonists or by purified cathepsin G, results in the accumulation of CD43-CTF. Because CD43 binds E-selectin, we also investigated the effect of the soluble extracellular domain CD43s, released by cathepsin G juxtamembranous cleavage, on neutrophil adhesion to endothelial cells. A recombinant CD43s-Fc fusion protein inhibited neutrophil E selectindependent adhesion to endothelial cells under flow conditions, while it had no effect on neutrophil static adhesion. We thus propose that, in addition to its potential pro-adhesive role, CD43 proteolysis results in: (i) the release, by cathepsin G, of CD43 extracellular domain, able to inhibit the adhesion of flowing neutrophils on endothelial cells and thus to participate to the natural control of inflammation; (ii) the release and/or the clearance, by presenilin/γ-secretase, of CD43 intracellular domain, thereby regulating CD43-mediated signaling.

von Willebrand factor is a major determinant of ADAMTS-13 decrease during mouse sepsis induced by cecum ligation and puncture

Summary.  Background: During sepsis, von Willebrand factor (VWF) is abundantly secreted; the main mechanism regulating its size involves specific proteolysis by the metalloprotease ADAMTS‐13. Objectives: To determine whether ADAMTS‐13 consumption due to its binding to, and/or cleavage, of VWF contributes to its decrease during sepsis and whether abrogating or enhancing ADAMTS‐13 activity influences sepsis outcome. Methods: ADAMTS‐13 activity was evaluated in a model of sepsis induced by cecum ligature and puncture (CLP) in wild‐type and Vwf−/− mice. Sepsis outcome was studied in those mice and in Adamts‐13−/− mice. Finally, survival was studied in wild‐type mice injected hydrodynamically with the human ADAMTS‐13 gene. Results: In wild‐type mice, CLP‐induced sepsis elicited a significant ADAMTS‐13 decrease, and a strong negative correlation existed between VWF and ADAMTS‐13. In Vwf−/− mice, CLP also induced severe sepsis, but ADAMTS‐13 was not significantly diminished. Notably, Vwf−/− mice lived significantly longer than wild‐type mice. In contrast, Adamts‐13−/− mice and wild‐type mice were comparable with regard to thrombocytopenia, VWF concentrations, absence of thrombi, and survival. Hydrodynamic hADAMTS‐13 gene transfer with the pLIVE expression vector resulted in high and stable ADAMTS13 activity in CLP mice; however, no impact on survival was observed. Conclusions: VWF secretion is a major determinant of ADAMTS‐13 decrease in the CLP model, and plays an important role in sepsis‐induced mortality, but the complete absence of its regulating protease, ADAMTS‐13, had no detectable impact in this sepsis model. Furthermore, increasing ADAMTS‐13 activity had no impact on survival.

Human neutrophil integrin α9β1: up-regulation by cell activation and synergy with β2 integrins during adhesion to endothelium under flow

Neutrophil β1 integrin expression and contribution to cell adhesion were revisited in this study. α9β1 and α5β1 appeared here as the main β1 integrins expressed on the membrane of resting platelet‐depleted neutrophils—α6β1 representing <15% and α2β1 undetectable. Neutrophil activation slightly enhanced α5 expression, did not change α6, but resulted in a two‐ to threefold increase of α9β1, which then became the major β1 integrin of the neutrophil membrane. α9β1 was the only β1 integrin to be up‐regulated after transendothelial migration across TNF‐α‐activated HUVECs. As α9β1 binds VCAM‐1, we analyzed its participation to neutrophil adhesion to TNF‐α‐activated endothelial cells. Blocking anti‐α9 mAb had little effect on neutrophil static adhesion, contrasting with the strong inhibition by anti‐β2 mAb. Under flow conditions, the anti‐α9 mAb had no effect by itself on neutrophil adhesion to activated HUVECs but enhanced the blocking effect of anti‐β2 antibodies significantly and further enhanced the velocity of β2–blocked rolling neutrophils. In conclusion, we describe here for the first time a nearly exclusive up‐regulation of α9β1 expression among all β1 integrins during neutrophil activation and transendothelial migration and a possibly important synergy between α9β1 and β2 integrins in stabilizing neutrophil adhesion to endothelium under flow conditions.

Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics.

We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.

6 Von Willebrand factor and platelet function

Summary vWF is an adhesive protein that binds to two distinct platelet glycoproteins, GP Ib and GP IIb-IIIa complex. Its interaction with GP Ib is primarily responsible for platelet adhesion to the subendothelium. The current model is that vWF binds to collagen and/or another component of the subendothelium, after which a conformational change in the vWF molecule exposes the GP Ib binding site. This interaction may not only promote the initial attachment of platelets to the subendothelium but also play a role in thrombus formation through exposure of GP IIb-IIIa to which vWF and fibrinogen can bind. The second important function of vWF is to be a carrier for F.VIII, protecting it from degradation and playing a role in its activation by thrombin. Circulating vWF has a complex multimeric structure that ranges in Mrs from 0.5 to 20 × 10 6 Daltons. The basic subunit has a Mr of 270 kDa. Amino acid sequencing of vWF demonstrated that the basic subunit or mature vWF is made up of 2050 amino acids. Molecular cloning of the vWF cDNA revealed that the primary transcript consists of 8900 base pairs that encode for 2813 amino acids, including a 22 amino acid signal peptide and a propolypeptide of 741 amino acids, called vWF antigen II. Recent studies on the expression of recombinant vWF molecules indicate that the propolypeptide is involved in the multimerization of vWF. The domains on the vWF molecule involved in the interactions of vWF with GP Ib, GP IIb-IIIa, collagen, F.VIII and heparin have been localized to varying extents. It is anticipated that peptide analysis and recombinant DNA techniques, such as in vitro mutagenesis, will further define the structural requirements of these binding domains. vWF is synthesized in a cell-specific manner by endothelial cells and megakaryocytes. It undergoes a complex intracellular biosynthesis involving transcription of a 200 kb gene, splicing out more than 42 introns, translation of a 8900 bp mRNA, glycosylation, disulphide bond formation, sulphatation, multimerization and proteolytic cleavage. The molecule can be secreted in a constitutive or regulated manner upon pertubation of the endothelial cells with physiological and non-physiological secretagogues. The mechanisms that control the synthesis of vWF should be an exciting area of further research. vWD is probably the most common of all congenital disorders of haemostasis. It is an extremely heterogeneous syndrome involving quantitative or qualitative disorders of vWF. All the variants described so far show an abnormal response to ristocetin and are thus likely to have a molecular abnormality that directly or indirectly involves the GP Ib binding domain of vWF. The gene defects associated with some types of vWD are just now being investigated and it can be expected that future studies will clarify the role of transcriptional, translational or post-translational mechanisms in the various forms of vWD.

Terminal platelet production is regulated by von Willebrand factor.

It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s−1) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf −/− mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf −/− and Vwf +/+ blood, exposed to VWF at 1800 s−1 in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf−/− mice than from Vwf+/+ mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf−/− mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s−1 on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.