Importance of monocyte deactivation in determining early outcome after ventricular assist device implantation.

BACKGROUND: Patients undergoing mechanical circulatory support using ventricular assist devices (VADs) experience a postoperative mixed antagonistic (proinflammatory and antiinflammatory) response syndrome. This response can result in immunoparalysis, exposing VAD recipients to infection and interfering with patient recovery despite adequate hemodynamic support. We undertook the present study to evaluate whether postoperative monocytic human leukocyte antigen-DR (mHLA-DR) expression is of prognostic value for mortality or infection of VAD recipients during their initial intensive care unit (ICU) stay after implantation. METHODS: Since 2004, we have monitored postoperative mHLA-DR expression in 50 VAD recipients using flow cytometry. RESULTS: Thirty-seven patients (74%) developed infection, and 22 patients (44%) died during their initial ICU stay. mHLA-DR expression was lowest in the immediate postoperative period (postoperative days [PODs] 1-3) but increased progressively thereafter. Multiple regression analysis showed that preoperative aspartate aminotransferase level was the only significant and independent predictor of the percentage of HLA-DR-positive monocytes on PODs 1-3 (ß = -0.726, p = 0.0001). ICU death and infection were associated with significantly lower percentages of HLA-DR-positive monocytes on PODs 1-3. ROC curve analysis revealed that the percentage of HLA-DR-positive monocytes on PODs 1-3 had significant discriminative power for ICU death (area under the curve = 0.73, 95% confidence interval, 0.545-0.912, p = 0.037), but not for infection. CONCLUSIONS: Postoperative mHLA-DR expression was closely related to preoperative hepatic cytolysis. It appeared to be the only early postoperative biological parameter that had some predictive power for death of VAD recipients in the ICU.

24-hour time-dependent aspirin efficacy in patients with stable coronary artery disease.

Aspirin-induced cyclooxygenase (COX)-1 acetylation is irreversible and it is assumed that the platelet thromboxane-A2 aggregation pathway is inhibited for at least 24 hours (h) after aspirin ingestion. However, time course of biological efficacy of daily low-dose aspirin has rarely been assessed in patients with coronary artery disease (CAD). We aimed to assess the 24-h biological efficacy of daily low-dose aspirin in CAD patients. The peak and trough (2 h-24 h) effect of a chronic treatment with once daily dose aspirin were studied in 150 consecutive stable CAD patients. The main outcome measure was light transmission aggregometry (LTA) triggered with 0.5 mg/ml arachidonic acid (AA). In the last 47 consecutive patients, additional tests were conducted at 6, 12, 16, 20 h after last aspirin administration. 4.7% of the patients had significant aggregation (>20% maximal intensity LTA-AA) 2 h after aspirin ingestion and 24.7% at 24 h (p<0.0001). The more precise assessments in the last 47 patients showed that significant platelet aggregation progressively reappeared with time after aspirin intake (2 h--4% of patients, 6 h-- 4%, 12 h--11%, 16 h--16%, 20 h--19% and 24 h--28%). Concordant results were observed using production of thromboxane-B2 and other techniques evaluating AA-induced platelet aggregation/activation. No significant differences were found between lower (75-100 mg/day) and higher (>100 mg/day) dose aspirin. Such aspirin «resistance¼ at 24 h after ingestion was related to biological inflammatory markers, current smoking and diabetes. In conclusion, once daily aspirin does not provide stable 24-h antiplatelet protection in a significant proportion of CAD patients. Any biological assessment of aspirin efficacy should take time since last aspirin intake into consideration.

Differential sensitivity and kinetics of response of different ex vivo tests monitoring functional variability of platelet response to clopidogrel.

We previously showed that variability of response to clopidogrel is linked to occupancy of the P2Y12 receptor by clopidogrel active-metabolite, and that maximal platelet aggregation intensity (MAI) measured by light transmission aggregometry (LTA) correlates with occupancy. The present study compared a range of ex vivo platelet tests at various levels of P2Y12 occupancy. After screening with clopidogrel 75 mg/day for seven days, subjects were selected to obtain 'low', 'average' and 'high' responders and randomised to clopidogrel (75 mg/day days 1-10; 300 mg day 11), or placebo. Assays were LTA in platelet-rich plasma using 2, 5 and 20 mM ADP, VerifyNow P2Y12, flow cytometric analysis of platelet activation markers and vasodilator-stimulated phosphoprotein (VASP) index, and a thromboelastographic test that is sensitive to clopidogrel. The reference test was P2Y12 receptor occupancy, measured by binding of 33P-2MeS-ADP to platelets. MAI showed the best correlation with P2Y12 occupancy. Similar results were seen with different ADP concentrations and when LTA data were expressed as inhibition of platelet aggregation. A plot of free receptors/cell versus VASP index was biphasic, with poor correlation for low-level P2Y12 occupancy. Sensitivity of the VerifyNow P2Y12 assay decreased at higher clopidogrel responses. Thromboelastography and P-selectin expression had poor correlation with receptor occupancy. In conclusion, LTA data correlate best with P2Y12 occupancy, the gold standard for detecting clopidogrel's effect at the receptor level. Our results highlight a limitation of the VASP index, which appears unable to distinguish low, average and high responders early after clopidogrel initiation when P2Y12 occupancy is still low.

Single-centre experience with the Thoratec paracorporeal ventricular assist device for patients with primary cardiac failure.

BACKGROUND: Temporary mechanical circulatory support may be indicated in some patients with cardiac failure refractory to conventional therapy, as a bridge to myocardial recovery or transplantation. AIMS: To evaluate outcomes in cardiogenic shock patients managed by the primary use of a paracorporeal ventricular assist device (p-VAD). METHODS: We did a retrospective analysis of demographics, clinical characteristics and survival of patients assisted with a Thoratec p-VAD. RESULTS: p-VADs were used in 84 patients with cardiogenic shock secondary to acute myocardial infarction (35%), idiopathic (31%) or ischaemic (12%) cardiomyopathy, myocarditis or other causes (23%). Before implantation, 23% had cardiac arrest, 38% were on a ventilator and 31% were on an intra-aortic balloon pump. Cardiac index was 1.6+/-0.5 L/min/m(2) and total bilirubin levels were 39+/-59 micromol/L. During support, 29 patients (35%) died in the intensive care unit and seven (10%) died after leaving. Forty-seven patients (56%) were weaned or transplanted, with one still under support. Despite significantly more advanced preoperative end-organ dysfunction, survival rates were similar in patients with biventricular devices (74%) and those undergoing isolated left ventricular support (24%) (63% versus 45%, respectively; p=0.2). Actuarial survival estimates after transplantation were 78.7+/-6.3%, 73.4+/-6.9% and 62.6+/-8.3% at 1, 3 and 5 years, respectively. CONCLUSIONS: Our experience validates the use of p-VAD as a primary device to support patients with cardiogenic shock. In contrast to short-term devices, p-VADs provide immediate ventricular unloading and pulsatile perfusion in a single procedure. Biventricular support should be used liberally in patients with end-organ dysfunction.

Monocytic HLA-DR expression in intensive care patients: interest for prognosis and secondary infection prediction.

OBJECTIVES: To test early measurement of human leukocyte antigen-DR expression on circulating monocytes (mHLA-DR) as prognostic marker, and the trend of mHLA-DR recovery for the prediction of late secondary infection risk in a large intensive care unit population. DESIGN: Prospective, observational study over 16 mos. SETTING: Intensive care unit in a tertiary teaching hospital. INCLUSION CRITERIA: Simplified Acute Physiology Score II >15, age >18 yrs. MEASUREMENTS AND MAIN RESULTS: The mHLA-DR was measured by flow cytometry within the first 3 days and twice a week until discharge. We used a logistic regression model for outcome prediction, and a competing risk approach to test the relationship between mHLA-DR recovery (log (mHLA-DR) slope) and incidence of secondary infection. A total of 283 consecutive patients suffering from various pathologies were monitored (Simplified Acute Physiology Score II = 39, Sepsis-related Organ Failure Assessment of 5 on day 0). Early mHLA-DR was decreased in the whole population, however, more deeply in sepsis (p < .0001). Low mHLA-DR was associated with mortality in the whole population (p = .003), as in subgroups (nonseptic, neurologic, and septic), but not when adjusted on Simplified Acute Physiology Score II. In patients with a length of stay of >7 days (n = 70), the lower the slope of mHLA-DR recovery, the higher the incidence of the first secondary infection (adjusted on early mHLA-DR, p = .04). CONCLUSIONS: For a given severity, mHLA-DR proved not to a predictive marker of outcome, but a weak trend of mHLA-DR recovery was associated with an increased risk of secondary infection. Monitoring immune functions through mHLA-DR in intensive care unit patients therefore could be useful to identify a high risk of secondary infection.

Functional variability of platelet response to clopidogrel correlates with P2Y(12) receptor occupancy.

Interindividual variability of response to clopidogrel is currently a subject of much interest. We tested the hypothesis that functional variability in the platelet response to clopidogrel correlates with occupancy of the platelet P2Y(12) receptor by clopidogrel active metabolite. Healthy subjects were screened after seven days' treatment with clopidogrel 75 mg/day to select three clopidogrel-response groups (n = 12/group), defined as 'average' (40-60% inhibition of platelet aggregation [IPA]), 'low' (<10% IPA) or 'high' (>80% IPA) responders. After a two- to six-week wash-out period, subjects were randomized (double-blind) to clopidogrel 75 mg/day (n = 10/group) for 10 days, followed by clopidogrel 300 mg on day 11, or placebo (n = 2/group). IPA induced by adenosine diphosphate (ADP), and P2Y(12) receptor occupancy were measured repeatedly. The incidence of low responders was 3.7%, and low responses to clopidogrel were maintained during the randomized evaluation phase. Treatment with clopidogrel for 10 days induced a significant increase in P2Y(12) receptor occupancy in each group of responders versus placebo; receptor affinity was unchanged. This reduction correlated with IPA response (r = 0.54). The additional 300 mg dose of clopidogrel on top of 75 mg chronic treatment increased IPA and P2Y(12) receptor occupancy in all groups, but relatively more in low responders. Variability in the response to clopidogrel appears to be linked to differences in P2Y(12) receptor occupancy. An additional 300 mg dose of clopidogrel improves both IPA and P2Y(12) receptor occupancy mostly in the subset of 'low' responders.

Management of intracranial hemorrhage in patients with mechanical circulatory support: a role for low-molecular-weight heparins?

Patients on mechanical circulatory support are at high risk of bleeding, particularly intracranial hemorrhage (ICH). The management of ICH in patients needing anticoagulation and antiplatelet therapies remains a challenge. We report our initial experience of ICH management with low-molecular-weight heparin in patients with mechanical circulatory support, without bleeding or thromboembolic complications.

Defective collagen-induced platelet activation in two patients with malignant haemopathies is related to a defect in the GPVI-coupled signalling pathway.

The occurrence of a thrombocytopathy concomitantly to the development of a malignant haemopathy has been reported for some time, but little is known about the mechanism(s) involved in the platelet dysfunction. Platelet glycoprotein VI (GPVI) has now been identified as a principal platelet receptor for collagen. In this paper, we report the cases of two patients with a myelodysplasia and a B lymphopathy, respectively, who presented with thrombocytopathy in relation to a defective GPVI-mediated platelet reactivity to collagen. Thus, with regard to the different steps of adhesion, activation secretion or aggregation, patients' platelet responses to collagen and to the GPVI specific agonists, collagen related peptide (CRP) or convulxin were null or dramatically impaired. Platelet responses to other agonists ADP, TRAP, Arachidonic acid were normal or showed only a moderate decrease. GPVI content was repeatedly normal, and binding of specific ligands, such as convulxin, satisfactory. Nevertheless, specific activating monoclonal antibodies and convulxin failed to induce platelet secretion; collagen, CRP or convulxin were unable to provoke calcium mobilisation. Furthermore, using a perfusion chamber model, we showed that ex vivo collagen-induced thrombi formation was very impaired. Taken together, these data provide evidence, for the first time, of an acquired defect in GPVI-mediated platelet reactivity to collagen, which reflects data observed in constitutional GPVI deficiencies, in two patients with malignant haemopathies.

Histocompatibility leukocyte antigen-D related expression is specifically altered and predicts mortality in septic shock but not in other causes of shock.

Although the expression of monocyte histocompatibility leukocyte antigen (HLA)-DR has been shown to be decreased during human sepsis, its level of expression in other nonseptic critical conditions is unclear. The aim of this study was to compare the level of HLA-DR expression on circulating monocytes among patients with septic, hemorrhagic, and cardiogenic shocks and severe sepsis without shock. At admission, HLA-DR expression was exclusively decreased in patients with septic shock (n = 30; P < 0.001), whereas the expression was similar between the other studied groups: cardiogenic shock (n = 16), hemorrhagic shock (n = 11), severe sepsis without shock (n = 18), and healthy volunteers (n = 8). HLA-DR expression was not predictive for overall mortality, but at day 1, an HLA-DR expression of less than 14 of mean fluorescence intensity (that corresponds to 40% labeled monocytes) was predictive of mortality exclusively in patients with septic shock (odds ratio, 11.4 and 95% confidence interval, 1.7; 78.4; P < 0.008). Catecholamine infusion, mechanical ventilation, positive blood culture, and number of units of blood or plasma transfused did not correlate with decreased HLA-DR expression. Thus, the decrease in HLA-DR expression is specific to septic shock and is associated, in septic shock patients, with increased mortality risk.

Platelet activation and aggregation profile in prolonged external ventricular support.

BACKGROUND: Platelet function plays a major role in the understanding of thromboembolic events in prolonged mechanical support. We studied the platelet activation, platelet aggregation profile, and efficacy of aspirin in patients in whom an external ventricular assist device had been implanted. PATIENTS AND METHODS: Fifteen patients were studied prospectively up to 6 weeks after implantation of the same type of ventricular assist device. Platelet function was studied weekly before daily aspirin administration. Aspirin efficacy was tested ex vivo by measuring platelet aggregation triggered by arachidonic acid. Flow cytometry was used to quantify the spontaneous and induced (adenosine diphosphate stimulation) expression of glycoproteins alphaIIbbeta3, Ibalpha, and CD62P on platelet membranes. The plasma levels of von Willebrand factor (von Willebrand factor activity and von Willebrand factor antigen) and fibrinogen were also determined. RESULTS: Six of the 15 patients (26%) maintained an arachidonic acid-induced platelet aggregation despite daily aspirin treatment (250 mg). CD62P values remained increased during a 5-week postoperative period. Spontaneous levels of glycoproteins alphaIIbbeta3 and Ibalpha on platelet membranes remained within a normal range with a preserved reactivity. The plasma levels of fibrinogen and von Willebrand factor remained increased during the entire study period. CONCLUSION: In patients with an implanted external ventricular assist device, the platelet activation profile displays a persistent activation with a preserved reactivity associated with a persistent high inflammatory state and endothelial activation.

Ex vivo differentiated endothelial and smooth muscle cells from human cord blood progenitors home to the angiogenic tumor vasculature.

OBJECTIVES: Recent studies have provided increasing evidence that postnatal neovascularization does not rely exclusively on sprouting of preexisting vessels, but also involves bone marrow-derived circulating endothelial precursors (BM-EPCs). Animal studies revealed that neovascularization of ischemic tissue can be enhanced by BM-EPCs transplantation. But a possible limitation to the use of vascular precursors for therapeutic angiogenesis is the relatively low number of these cells. In this study, we demonstrate that ex vivo expanded differentiated endothelial cells (ECs) and smooth muscle cells (SMCs), may home to the tumor vasculature allowing targeting of transgene expression to the neoangiogenic site. METHODS: Mononuclear cells (MNCs) or CD34+ -enriched cells were purified from cord blood. We have defined culture conditions in which we observed two types of clones easily differentiated according to their morphology: cobblestone or spindle-shaped. Phenotypic characterization was assessed by immunocytochemistry, flow cytometry analysis and polymerase reaction with reverse transcription. Formation of capillary-like network in vitro was studied in three-dimensional collagen culture. And recruitment of these cells to a tumoral neoangiogenic site was assessed into tumor-bearing Severe Combined Immunodeficient (SCID) mouse model. RESULTS: The cobblestone cells uniformly positive for CD31, VE-cadherin, vWF, VEGF R1 and R2, ecNOS and incorporating acetylated LDL were ECs. Spindle-shaped cells expressed alpha-smooth muscle actin (alpha-SMA), Smooth Muscle Heavy Chain (SMHC), SM22 and calponin. They also displayed a carbachol-induced contractility in a medium containing IGF1. So we concluded that spindle-shaped cells were SMCs. ECs and SMCs interacted with each other to form a capillary like network in three-dimensional type I collagen culture. Moreover, these ex vivo differentiated cells are able to home to the tumor vasculature. CONCLUSION: We provide evidence that progenitors for ECs and SMCs circulate in human cord blood and differentiate into functional ECs and SMCs. These differentiated cells could provide a biomaterial for vascular cell therapy, because of their homing capacity to the neovascularization site.

A Leu7Pro mutation in the signal peptide of platelet glycoprotein (GP)IX in a case of Bernard-Soulier syndrome abolishes surface expression of the GPIb-V-IX complex.

This paper describes the molecular defect of the second case of Bernard-Soulier syndrome, initially reported in 1957. Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib-V-IX complex and revealed small amounts of intracellular GPIbalpha, GPIbbeta and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard-Soulier syndrome. The change occurred in a prototypic alpha-helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co-transfection of GPIXPro7 with normal GPIbalpha and GPIbbeta into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbalpha and GPIbbeta, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb-IX complex.

AGEs bind to mesothelial cells via RAGE and stimulate VCAM-1 expression.

BACKGROUND: Excess advanced glycation end-products (AGEs) are formed during renal failure, and AGE formation also may be connected with the high glucose concentration of peritoneal dialysis (PD) fluids. To determine the effect of human peritoneal mesothelial cell (HPMC) exposure to glycated proteins, we studied the HPMC receptor of AGE expression (RAGE), and analyzed the results of AGE-RAGE interaction on adhesion molecule expression and leukocyte binding. METHODS: RAGE was detected by FACS analysis, and RAGE mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Vascular and intercellular cell adhesion molecule (VCAM-1 and ICAM-1) expression was measured by a known radiometric technique under basal conditions, after the addition of an AGE-specific compound, Nepsilon-carboxylmethyllysine (CML-albumin). Leukocyte adhesion on HPMC was analyzed by videomicroscopy after HPMC stimulation. RESULTS: RAGE protein was detected on HPMC, and RAGE mRNA was evidenced by RT-PCR. VCAM-1 expression was stimulated by CML-albumin (P < 0.01), while ICAM-1 was unchanged. By blocking the AGE-RAGE interaction, anti-RAGE antibodies or recombinant RAGE inhibited the increase in VCAM-1 expression. CML-albumin stimulation potentiated leukocyte adhesion to HPMC (P < 0.001). This effect was prevented by the incubation of leukocytes with recombinant VCAM-1 (P < 0.001). CONCLUSIONS: AGE binding to RAGE stimulated mesothelial cell activity, and resulted in the overexpression of VCAM-1, a structure for leukocyte adhesion. The AGE-RAGE interaction resulted in HPMC activation, which may promote local inflammation, and thus is implicated in the peritoneal injury found in long-term PD patients.