Delphine Borgel

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.

Elevated growth-arrest-specific protein 6 plasma levels in patients with severe sepsis.

Objective:Growth-arrest-specific protein 6 (Gas6), an intracellular protein released by apoptotic cells, has been detected in normal plasma. As the Gas6 system has been implicated in mouse susceptibility to sepsis, and as leukocyte apoptosis is thought to play a major role in the physiopathology of human severe sepsis, we studied Gas6 plasma levels and possibly related variables in patients with severe sepsis. Design:Matched case-control study. Setting:Adult intensive care unit in a university hospital. Patients:Thirty patients with severe sepsis, 30 patients with organ failure not related to infection, and 30 healthy subjects matched for age and gender. Interventions:Blood draw. Measurements and Main Results:Gas6 plasma levels were quantified using enzyme-linked immunosorbent assay. Whole-blood gas6 messenger RNA levels were measured by quantitative real-time polymerase chain reaction. Gas6 plasma levels were elevated (110 ng/mL [75, 139]; median values [interquartile range]) in severe sepsis patients compared with organ failure patients (85 ng/mL [56, 101]) and healthy subjects (54 ng/mL [49, 68]). In patients with severe sepsis, this increase correlated with the Acute Physiology and Chronic Health Evaluation II severity score, the organ failure Organ Dysfunction and Infection (ODIN) score, and the existence of a septic shock. Gas6 messenger RNA levels were increased in patients with severe sepsis and correlated specifically with the monocyte count. Conclusions:In severe sepsis, the recently described anti-apoptotic protein Gas6 was found at high levels in plasma and correlated well with the degree of organ dysfunction.

Molecular basis for protein S hereditary deficiency: Genetic defects observed in 118 patients with type I and type IIa deficiencies

Circulating protein S (PS) is partly bound to C4b-binding protein, and only free PS can act as a cofactor for protein C (PC), a natural anticoagulant. Two types of PS deficiencies are commonly observed in patients with unexplained thrombosis, and they are characterized by having both a low total PS level and a low free PS level (type I) or by having only a low free PS level (type IIa). To elucidate the genetic mechanisms responsible for these two plasma phenotypes, we screened 118 symptomatic patients with type I or type IIa PS deficiency for a PS gene coding sequence variation. A total of 34 mutations, 17 of which were novel, were identified in 65 propositi (70% in type I and 44% in type IIa). In type I deficiency, 29 different mutations were distributed throughout the coding sequence. In type IIa deficiency, five different missense mutations were clustered in exons XII and XIII, with a Ser 460 to Pro mutation accounting for most cases (82%). This points to a role of the domain encoded by exons XII and XIII in the distribution between bound and free PS. The Ser 460 to Pro mutation was associated with the factor V Arg 506 to Gin mutation or a PC gene mutation in about half the patients, suggesting a cooperative effect on clinical expression.

Connexin 37 Limits Thrombus Propensity by Downregulating Platelet Reactivity

Background— Formation of platelet plug initiates hemostasis after vascular injury and triggers thrombosis in ischemic disease. However, the mechanisms leading to the formation of a stable thrombus are poorly understood. Connexins comprise a family of proteins that form gap junctions enabling intercellular coordination of tissue activity, a process termed gap junctional intercellular communication. Methods and Results— In the present study, we show that megakaryocytes and platelets express connexin 37 (Cx37). Deletion of the Cx37 gene in mice shortens bleeding time and increases thrombus propensity. Aggregation is increased in murine Cx37−/− platelets or in murine Cx37+/+ and human platelets treated with gap junction blockers. Intracellular microinjection of neurobiotin, a Cx37-permeant tracer, revealed gap junctional intercellular communication in platelet aggregates, which was impaired in Cx37−/− platelets and in human platelets exposed to gap junction blockers. Finally, healthy subjects homozygous for Cx37–1019C, a prognostic marker for atherosclerosis, display increased platelet responses compared with subjects carrying the Cx37–1019T allele. Expression of these polymorphic channels in communication-deficient cells revealed a decreased permeability of Cx37–1019C channels for neurobiotin. Conclusions— We propose that the establishment of gap junctional communication between Cx37-expressing platelets provides a mechanism to limit thrombus propensity. To our knowledge, these data provide the first evidence incriminating gap junctions in the pathogenesis of thrombosis.

Gross deletions/duplications in PROS1 are relatively common in point mutation-negative hereditary protein S deficiency

Hereditary protein S (PS) deficiency is an autosomal disorder caused by mutations in the PS gene (PROS1). Conventional PCR-based mutation detection identifies PROS1 point mutations in approximately 50% of the cases. To verify if gross copy number variations (CNVs) are often present in point mutation-negative hereditary PS deficiency we used multiplex ligation-dependent probe amplification (MLPA) as a detection tool in samples from individuals with a high probability of having true PS deficiency. To this end, DNA samples from nine PS deficient probands with family members (seven type I and two type III) and nine isolated probands (three type I and six type III), in whom PROS1 mutations were not found by DNA sequencing, were evaluated. An independent quantitative PCR (qPCR) was performed to confirm the findings of the MLPA assay. Family members were also tested when DNA was available. Gross abnormalities of PROS1 were found in six out of eighteen probands. In three probands complete deletion of the gene was detected. Two probands had a partial deletion involving different parts of the gene (one from exon 4 through 9 and another from exon 9 through 11). One family showed a duplication of part of PROS1. qPCR analysis was in accordance with these results. In conclusion, this study substantiates that gross gene abnormalities in PROS1 are relatively common in hereditary PS deficient patients and that MLPA is a useful tool for direct screening of CNVs in PROS1 point mutation-negative individuals.

Increased secretion of Gas6 by smooth muscle cells in human atherosclerotic carotid plaques

Vitamin K-dependent protein Gas6 (growth-arrest specific gene 6) plays a role in vascular smooth muscle cell (VSMC) survival and migration, as well as in endothelium and leukocyte activation, and could therefore be involved in atherosclerosis. However, the study of mouse models has led to contradictory results regarding the pro- or anti-atherogenic properties of Gas6, and relatively few data are available in human pathophysiology. To better understand the implication of Gas6 in human atherosclerosis, we studied Gas6 expression and secretion in vitro in human VSMC, and analysed the effect of Gas6 on inflammatory gene expression in these cells. We show that Gas6 secretion in VSMC is strongly induced by the anti-inflammatory cytokine transforming growth factor (TGF)β, and that VSMC stimulation by recombinant Gas6 decreases the expression of inflammatory genes tumour necrosis factor (TNF)α and intracellular adhesion molecule (ICAM)-1. The study of Gas6 expression in human carotid endarterectomy samples revealed that Gas6 is mainly expressed by VSMC at all stages of human atherosclerosis, but is not detected in normal vessel wall. Analysis of plaque secretomes showed that Gas6 secretion is markedly higher in non-complicated plaques than in complicated plaques, and that TGFβ secretion pattern mirrors that of Gas6. We conclude that Gas6 is secreted in human atherosclerotic plaques by VSMC following stimulation by TGFβ, and that Gas6 secretion decreases with plaque complication. Therefore, we propose that Gas6 acts as a protective factor, in part by reducing the pro-inflammatory phenotype of VSMC.

Plasma levels of the growth arrest-specific gene 6 product (Gas6) and antiplatelet drug responsiveness in healthy subjects

Aspirin targets the thromboxane (Tx) A2 pathway and clopidogrel the adenosine diphosphate (ADP) pathway of platelet activation process. The biological effects of aspirin and clopidogrel vary from one subject to another and this variability has been implicated in the risk of recurrent ischemic events (1). We recently examined aspirin and clopidogrel responsiveness in 96 healthy subjects (2) and found aspirin pseudo-resistance (APR) in 30% of cases. As in the other subjects, the production of TxB2 (the stable breakdown product of TxA2) was abolished by aspirin in these pseudo-resistant subjects, but their platelet closure times (CT) determined with the PFA-100 � device (collagen/epinephrine cartridge) were normal, despite aspirin intake (100 mg day )1 ) for 7 days. This suggested that platelets continued to aggregate despite adequate inhibition of TxA2 production by aspirin, and that additional platelet amplification pathways therefore compensated for the lack of TxA2. Several studies have linked low PFA-100 � (Dade Behring, Dudingen, Switzerland) CT values to vascular events in aspirin-treated patients (3-5), suggesting that APR may be a risk factor for recurrent cardiovascular events. The importance of the growth arrest-specific gene 6 product (Gas6) in platelet activation and thrombus stabilization has been demonstrated in mice (6-8), but the expression of Gas6 and its receptors in human platelets is controversial (6,9). We and others have detected Gas6 protein with an ELISA method in human and mouse plasma (9-11). We recently showed that plasma Gas6 levels in healthy subjects do not influence platelet aggregation ex vivo in response to selected platelet agonists (11), but the role of Gas6 in the variability of biological responses to antiplatelet drugs has not been investigated. Plasma Gas6 levels were determined in the 96 healthy volunteers enrolled in the above-mentioned study (2) with an ELISA method (10) in baseline samples, and were expressed as a percentage of the Gas6 level in a normal plasma pool (11). Statistical analysis used the Kruskall-Wallis test and a logistic regression model to test the relationship between antiplatelet drug responsiveness and quartiles of Gas6 levels, after adjustment for potentially confounding variables. Plasma Gas6 levels ranged from 47.7% to 167.1% of the value obtained with the normal plasma pool (median 85%, interquartile range (IR): 68-103%). These values are compat- ible with those found in another population of healthy male volunteers (11). Subjects in the first and second quartiles of plasma Gas6 levels had median values of 64.3% and 79.2%, respectively, while subjects in the third and fourth quartiles had values of 88.9% and 115.7% (P < 0.001). Subjects with APR had a median Gas6 level of 88% (IR: 78-133%), while aspirin- sensitive subjects had a median Gas6 level of 79% (IR: 57- 132%, P ¼ 0.033). The proportion of APR subjects increased from 8.0% in quartile 1 of plasma Gas6 levels to 21.7%, 45.8% and 43.4% in quartiles 2-4, respectively (P ¼ 0.009). Table 1 shows the results of univariate and multivariate logistic regression analysis testing the association of APR and quartiles

Implication of protein S thrombin-sensitive region with membrane binding via conformational changes in the gamma-carboxyglutamic acid-rich domain.

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the gamma-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the gamma-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.

Cleaved Protein S (PS), Total PS, Free PS, and Activated Protein C Cofactor Activity as Risk Factors for Venous Thromboembolism

BACKGROUND Although hereditary protein S (PS) deficiency is clearly associated with venous thromboembolism (VTE), the importance of low PS concentrations as a risk factor for VTE in other patients is still a matter of debate. To clarify this issue, we designed a case-control study to evaluate the role of different molecular forms of plasma PS. METHODS We quantified plasma cleaved, total, and free PS and activated protein C (APC) cofactor activity in 87 VTE patients and 174 controls matched for age, sex, and hormonal treatment. Free PS was measured by ELISA or by enzyme-linked ligand sorbent assay (ELSA). Cleaved and total PS were measured by ELISA. RESULTS In controls, the mean (SD) concentration of circulating cleaved PS was 39 (14) nmol/L, corresponding to 10% (3.5%) of total PS. Concentrations of cleaved PS and total PS were not significantly different in patients with VTE compared with controls. However, in our population, low free PS measured by ELISA or ELSA, as well as APC cofactor activity values were significantly associated with VTE with odds ratios (95% confidence intervals) of 2.9 (1.3-6.3), 2.5 (1.1-5.6), and 2.9 (1.3-6.4), respectively, in multivariate analyses. CONCLUSION Phenotypic low PS detected by APC cofactor activity assay or by an assay specific for free PS should be considered a risk factor for VTE.

A chemically‐modified inactive antithrombin as a potent antagonist of fondaparinux and heparin anticoagulant activity

Heparin and its analogs, mediating their anticoagulant activity through antithrombin (AT) activation, remain largely used for the preventive and curative treatment of thrombosis. The major adverse reaction of these drugs is the bleeding risk associated with overdose. Unfractionnated heparin (UFH) can be efficiently and rapidly neutralized by protamine sulfate, but this reversal partially neutralizes low‐molecular‐weight heparin (LMWH) and is inefficient in reversing fondaparinux. To secure administration of AT‐mediated anticoagulants and counteract bleeding disorders, we previously designed a recombinant inactive AT as an antidote to heparin derivatives.