Benoît Polack

Nanostructure of the Fibrin Clot

The nanostructure of the fibrin fibers in fibrin clots is investigated by using spectrometry and small angle x-ray scattering measurements. First, an autocoherent analysis of the visible light spectra transmitted through formed clots is demonstrated to provide robust measurements of both the radius and density of the fibrin fibers. This method is validated via comparison with existing small-angle and dynamic light-scattering data. The complementary use of small angle x-ray scattering spectra and light spectrometry unambiguously shows the disjointed nature of the fibrin fibers. Indeed, under quasiphysiological conditions, the fibers are approximately one-half as dense as their crystalline fiber counterparts. Further, although the fibers are locally crystalline, they appear to possess a lateral fractal structure.

Micro-macro link in rheology of erythrocyte and vesicle suspensions.

We report on the rheology of dilute suspensions of red blood cells (RBC) and vesicles. The viscosity of RBC suspensions reveals a previously unknown signature: it exhibits a pronounced minimum when the viscosity of the ambient medium is close to the value at which the transition from tank-treading to tumbling occurs. This bifurcation is triggered by varying the viscosity of the ambient fluid. It is found that the intrinsic viscosity of the suspension varies by about a factor of 4 in the explored parameter range. Surprisingly, this significant change of the intrinsic viscosity is revealed even at low hematocrit (5%). We suggest that this finding may be used to detect blood flow disorders linked to pathologies that affect RBC shape and mechanical properties. This opens future perspectives on setting up new diagnostic tools, with great efficiency even at very low hematocrit. Investigations are also performed on giant vesicle suspensions, and compared to RBCs.

Quantitative high D-dimer value is predictive of pulmonary embolism occurrence independently of clinical score in a well-defined low risk factor population

Summary.u2002 We performed a prospective study to assess whether positive quantitative D‐dimer (DD) levels could be integrated for a selected population in a defined strategy to accurately diagnose pulmonary embolism (PE). For this purpose, 1528 in‐ or outpatients with clinically suspected PE were investigated according to our prescription rules. Clinical probability was defined as low, intermediate or high. Patients in whom DD levels were measured met criteria defined by our previously described decision‐making algorithm: in‐ and outpatients, <u200380u2003years, without surgery in the previous 30u2003days or active cancer. Nine hundred and twenty‐three patients (60.4%) had quantitative DD measurement using automated latex DD assay (STA‐Liatest D‐Di®). According to our decision‐making algorithm, DD measurement was applied to 70.5% of out‐, and 55.7% of inpatients, and PE diagnosis was ruled out in 49.5% of the 923 patients. This allowed us to confirm prospectively that our specific rules greatly improve the DD testing efficiency. PE was diagnosed in 115 (12.5%) patients. For a 0.5u2003mgu2003L−1 cut‐off, the test sensitivity was 97.4%, but its specificity was only 56.7%. However, PE prevalence increased gradually with DD levels. The true observed PE prevalence, according to the quantitative assessment of DD levels, differed from that predicted with pretest clinical probability only. Moreover, in this well‐defined patient group, a quantitative DD level >u20032u2003mgu2003L−1 was predictive of PE occurrence independently of the clinical score (odds ratio 6.9, 95% confidence interval 3.7, 12.8). As part of a defined strategy, knowledge of positive DD quantitative value, together with the clinical probability score, improves the PE predictive model. A clinical validation of these results in a follow‐up study would now be necessary before considering the implementation of this strategy into clinical practice.

HDAC6 controls the kinetics of platelet activation

HDAC6, a major cytoplasmic deacetylase, is shown here to fine-tune the kinetics of platelet activation, a process that must be precisely regulated to ensure hemostasis after blood vessel injury while preventing pathologic thrombus formation. The discoid shape of resting platelets in the circulation is maintained by several highly acetylated microtubules organized in a marginal band. During platelet activation, microtubules undergo major reorganizations, which contribute to the shape change of activating platelets. We show that, during these activation-induced shape changes, a dramatic HDAC6-mediated tubulin deacetylation takes place, followed by microtubule reacetylation in spread platelets. In addition, although HDAC6-controlled tubulin deacetylation is not required for platelet activation, the capacity of HDAC6 to prevent tubulin hyperacetylation influences the speed of platelet spreading. These results are particularly important in view of HDAC6 inhibitors being currently used in clinical trials and represent the first example of cell signaling by lysine acetylation in platelet biology.

Effects of two sex steroids (17β estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937

We investigated the effects of two sex steroids (17beta estradiol and testosterone) on five human leukemia cell lines. We observed a statistically significant inhibition of proliferation, dose and time dependent, of the human monoblastic leukemia cell line U937. This inhibition was associated with a dose dependent decrease in the number of CFU-blasts in clonogenic cultures. Cytostatic effect was obtained with doses of 5 microM for estrogen and 10 microM for androgen and was not due to a non-specific cytotoxic effect, some cell viability remained high (> 90%) even after 6 days of incubation. More accurately, we demonstrated that growth inhibition was associated with a cell cycle arrest, U937 cells accumulating in G2/M phase. This blockade was dose related with a maximum number of cells accumulating at day 4. Sensitivity of these cells to an S-phase specific agent (hydroxyurea) was not increased, suggesting that these cells were blocked in G2/M and did not undergo mitosis. Expression in U937 cells of high affinity nuclear receptors for estrogen and androgen was negative which was in favour of a type II estrogen binding site, mediated mechanism. Moreover, a small fraction of these cells underwent apoptosis or differentiation with about 12% apoptotic cells and a significant increase (more than 30%) of two myelomonocytic markers (CD13 and CD64). These results demonstrate that the proliferation of some leukemic cells may be inhibited by micromolar concentrations of sex steroids, independently of nuclear receptor expression. The main mechanism seems to be a block in cell cycle associated with modulation of apoptosis and differentiation. It provided additional evidence for the potential value of sex steroids and their analogues in the treatment of leukemias.

Inversion of hematocrit partition at microfluidic bifurcations

Partitioning of red blood cells (RBCs) at the level of bifurcations in the microcirculatory system affects many physiological functions yet it remains poorly understood. We address this problem by using T-shaped microfluidic bifurcations as a model. Our computer simulations and in vitro experiments reveal that the hematocrit (ϕ0) partition depends strongly on RBC deformability, as long as ϕ0<20% (within the normal range in microcirculation), and can even lead to complete deprivation of RBCs in a child branch. Furthermore, we discover a deviation from the Zweifach-Fung effect which states that the child branch with lower flow rate recruits less RBCs than the higher flow rate child branch. At small enough ϕ0, we get the inverse scenario, and the hematocrit in the lower flow rate child branch is even higher than in the parent vessel. We explain this result by an intricate up-stream RBC organization and we highlight the extreme dependence of RBC transport on geometrical and cell mechanical properties. These parameters can lead to unexpected behaviors with consequences on the microcirculatory function and oxygen delivery in healthy and pathological conditions.

Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase activation.

Management of haemophilia A-inhibitor patients: clinical and regulatory perspectives.

Inhibitors to factor VIII (FVIII) are alloantibodies directed against epitopes able to neutralise FVIII procoagulant activity. They may render FVIII replacement therapy ineffective. They represent the most severe complication of haemophilia A. At least three mechanisms of FVIII neutralisation activity by anti-FVIII antibodies have been described: (1) steric hindrance; (2) recognition of neo-epitopes and (3) catalytic activity. The Nijmegen modification of the Bethesda is the recommended method for inhibitor surveillance. The occurrence of inhibitors is a relatively frequent and early event in previously untreated patients. Conversely, it is rare in previously treated patients. Therapeutic strategies for managing inhibitors include: inhibitor eradication, haemostatic management of bleeding episodes and/or surgery and supportive care. For high responding inhibitors, immune tolerance induction (ITI) is the strategy for achieving antigen-specific tolerance to FVIII. ITI success rate ranges commonly between 60% and 80%. For treatment of patients with high-titre, high-responding inhibitors, ‘by-pass’ therapy is generally recommended. Activated prothrombin complex concentrates represent the historically primary ‘by-pass’ treatment. Recombinant factor VIIa has also been widely used as a by-passing agent. Considering the small patient population, it has to be considered that full immunogenicity data cannot be collected premarketing authorisation. Thus, stringent follow-up of patients in the post-authorisation phase is required.

Is primary prophylaxis required in afibrinogenemia

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Should Plasma Homocysteine Be Used as a Biomarker of Venous Thromboembolism? A Case—Control Study

Mild or moderate hyperhomocysteinemia as a risk factor for venous thrombosis is still a matter of debate. The strength of this study is to bring a body of elements to evaluate whether hyperhomocysteinemia should be used as a biomarker for venous thromboembolism (VTE). These elements consist of a biological evaluation of several hematological risk factors, and an original control group made of patients with a negative Doppler ultrasonography. A total of 151 cases and 155 controls were included. Total plasma homocysteine level, MTHFR C677T polymorphism, inherited abnormalities of the natural anticoagulant system as well as plasma folate and cobalamin levels were determined. A total of 41 (27.2 %) of cases and only 9 (5.8%) of controls had at least one of the coagulation defects studied. No significant difference was observed for total homocysteine levels between the 2 groups: median (interquartile range) = 8.3 (7.2-10.8) μmol/L for cases and 8.4 (7-10.9) μmol/L for controls. We found significantly more plasma folates and/or cobalamin deficiencies in controls (18.3%) than in cases (8.6%). After adjustment for several variables significantly related to risk factors of VTE, hyperhomocysteinemia (> xbd=2200 xhg=2176 ybd=1302 yhg=1257/>13.2 μmol/L) was not found statistically associated with VTE: odds ratio 1.36 (95% confidence interval, 0.52-3.54). The prevalence of the homozygous 677TT polymorphism in the MTHFR gene was not increased in cases compared with controls. Mild or moderate hyperhomocysteinemia does not seem to be a strong determinant in VTE not only when the control group does not exclusively include healthy persons but also in investigated disease-free (thromboembolic disease) controls.