Christilla Bachelot-Loza

The effectiveness of platelet supplementation for the reversal of ticagrelor-induced inhibition of platelet aggregation: An in-vitro study.

BACKGROUND Management of ticagrelor-induced bleeding is challenging, as no antidote is currently available. Platelet transfusion, usually proposed to reverse antiplatelet drugs, has been suggested to be ineffective but few data are available. OBJECTIVE To assess the efficacy of platelet supplementation to restore platelet aggregation inhibited by ticagrelor. DESIGN In vitro study. SETTING Blood samples were obtained from the French Blood Bank Institute. PARTICIPANTS Healthy blood donors. INTERVENTIONS Whole blood from healthy donors was spiked with ticagrelor or aspirin (used as a positive control). MAIN OUTCOME MEASURES Platelet aggregation was investigated with impedance aggregometry on whole blood [expressed in ohms (V)] and light transmission aggregometry (expressed in %) on platelet-rich plasma using ADP or arachidonic acid as agonists for ticagrelor or aspirin, respectively. Platelet supplementation was defined as the addition of washed platelet suspension increasing at least 60% of whole blood platelet count. RESULTS Ticagrelor (3.25u200amM) inhibited ADP-induced platelet aggregation compared with control either in whole blood (2 vs. 13u200aV, Pu200a<u200a0.05) or in platelet-rich plasma (15 vs. 75% Pu200a<u200a0.05). Aspirin (25u200amM) inhibited arachidonic acid-induced aggregation (1 vs. 7.5u200aV, Pu200a<u200a0.05 in whole blood and 5 vs. 77.5%, Pu200a=u200a0.01 in platelet-rich plasma). Platelet supplementation completely restored arachidonic acid-induced platelet aggregation in whole blood (10 vs. 1u200aV, Pu200a=u200a0.008) and platelet-rich plasma (73 vs. 5%, Pu200a<u200a0.01) in aspirin-treated samples, whereas it failed to correct ADP-induced aggregation (2 vs. 2u200aV in whole blood and 13.5 vs. 15% in platelet-rich plasma, Pu200a>u200a0.05) in ticagrelor-treated samples. We also report a case of a ticagrelor-treated patient in whom platelet transfusion failed to restore ADP-induced platelet aggregation. CONCLUSION Platelet supplementation restored platelet aggregation in aspirin-spiked but not in ticagrelor-spiked samples. These results do not support the use of platelet transfusion to reverse the effects of ticagrelor.

Polymorphonuclear leukocyte and monocyte activation induced by plasma from patients with heparin-induced thrombocytopenia in whole blood

Heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy, results from platelet activation by heparin-dependent antibodies. Previously, we have shown that plasma from patients with HIT (HIT plasma) induces leukocyteplatelet aggregation in blood. In this report, we examined leukocyte activation by HIT plasma and the contribution of heparin and platelets to this activation, in whole blood. Degranulation of leukocytes from HIT patients was evaluated as a leukocyte activation marker. We showed that polymorphonuclear leukocytes (PMN) and monocytes were the leukocyte subpopulations involved in platelet-leukocyte aggregation induced by HIT plasma in healthy donor blood. PMN and monocyte activation, reflected by increased surface expression of the CD11b adhesion molecule, was induced by HIT plasma in a heparin-dependent manner. The CD11b increase induced by HIT plasma was observed on PMN only when they were associated with platelets. Moreover, the increased CD11b expression on monocytes and PMN correlated strongly with the degree of platelet adhesion to these cells. Degranulation of leukocytes from HIT patients and control subjects (non-HIT heparin-treated patients and healthy subjects) was evaluated in vivo by measuring the plasma myeloperoxidase concentration. HIT plasma contained higher myeloperoxidase concentrations than control plasma, suggesting leukocyte degranulation during HIT. In conclusion, this study provides the first evidence that PMN activation is induced by HIT plasma. HIT plasma induced PMN and monocyte activation in a heparin-dependent manner. In whole blood, platelet association with monocytes and PMN, and the activation of these leukocytes by HIT plasma were interrelated. Finally, leukocyte degranulation could be involved in HIT physiopathology.

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

Molecules that reduce the level of cyclic adenosine 5-monophosphate (cAMP) in the platelet cytosol, such as adenosine 5-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4(-/-) platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets. Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin αIIbβ3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4-activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP-protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents.

Differential effect of the inhibition of Grb2-SH3 interactions in platelet activation induced by thrombin and by Fc receptor engagement

The adaptor protein Grb2 (growth factor receptor-bound protein 2) is involved in cell proliferation via the Ras signalling pathway. In order to study the role of Grb2 in blood platelet responses, we used a peptide containing two proline-rich sequences derived from Sos (peptidimer), which binds to Grb2-Src homology 3 domain (SH3) with a high affinity, and hence inhibits Grb2-SH3-mediated protein interactions. Platelet aggregation and 5-hydroxytryptamine (serotonin) release measured in the presence of the peptidimer were: (i) significantly decreased when induced by thrombin; and (ii) potentiated when induced by the engagement of the Fc receptor. In thrombin-activated platelets, the Grb2-SH2 domain formed an association with the beta3 subunit of the alphaIIb-beta3 integrin (GPIIb-IIIa), Shc, Syk, Src and SHP1 (SH2-containing phosphotyrosine phosphatase 1), whereas these associations did not occur after the engagement of the receptor for the Fc domain of IgG (FcgammaRIIa) or in resting platelets. Grb2-SH3 domains formed an association with the proline-rich sequences of Sos and Cbl in both resting and activated platelets, since the peptidimer abolished these associations. Inhibition of both fibrinogen binding and platelet aggregation by the peptide RGDS (Arg-Gly-Asp-Ser) had no effect on thrombin-induced Grb2-SH2 domain association with the aforementioned signalling molecules, indicating that these associations occurred during thrombin-induced inside-out signalling. Platelet aggregation induced by direct activation via alphaIIb-beta3 (outside-in signalling) was potentiated by the peptidimer. The results show that inhibition of Grb2-SH3 interactions with signal-transduction proteins down-regulates thrombin-induced platelet activation, but also potentiates Fc receptor- and alphaIIb-beta3-mediated platelet activation.

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.

Bleeding disorders in Lowe syndrome patients: evidence for a link between OCRL mutations and primary haemostasis disorders

Lowe syndrome (LS) is a rare X‐linked disorder caused by mutations in the oculocerebrorenal gene (OCRL), encoding OCRL, a phosphatidylinositol 5–phosphatase with a RhoGAP domain. An abnormal rate of haemorrhagic events was found in a retrospective clinical survey. Herein, we report the results of exploration of haemostasis in six LS patients. All patients had normal coagulation tests but prolonged closure times (CTs) in the PFA–100 system. Healthy donors’ blood samples incubated with a RhoA kinase inhibitor had prolonged CTs. This suggests that an aberrant RhoA pathway in platelets contributes to CT prolongation and primary haemostasis disorders in LS.

Tyrosine phosphorylation and association of FcγRII and p72Syk are not limited to the FcγRII signalling pathway

Abstract The tyrosine kinase p72Syk plays a critical role in platelet signal transduction. It associates with the platelet receptor for the Fc domain of IgGs, FcγRII, following stimulation by FcγRII cross-linking. Here, we show that p72Syk and FcγRII tyrosine phosphorylation and association occured following platelet stimulation by: (1) two monoclonal antibodies, which form a bridge between a target antigen and FcγRII, and (2) the G-protein-coupled receptor agonist thrombin. The kinetics of the p72Syk/FcγRII association depended on the signalling pathway (i.e., the antigen targeted or the thrombin receptor). We established a direct relationship between the level of FcγRII phosphorylation and the detection of its association with p72Syk. Inhibition of p72Syk by piceatannol resulted in partial or total inhibition of FcγRII phosphorylation, after immunological activation or addition of thrombin, respectively, suggesting that p72Syk participates in FcγRII phosphorylation. The results provide evidence that p72Syk/FcγRII association is not restricted to immunological activation.

MRP4 (ABCC4) as a potential pharmacologic target for cardiovascular disease

This review focuses on multidrug resistance protein 4 (MRP4 or ABCC4) that has recently been shown to play a role in cAMP homeostasis, a key-pathway in vascular biology and in platelet functions. In vascular system, recent data provide evidence that inhibition of MRP4 prevents human coronary artery smooth muscle cell proliferation in vitro and in vivo, as well as human pulmonary artery smooth muscle cell proliferation in vitro and pulmonary hypertension in mice in vivo. In the heart, MRP4 silencing in adult rat ventricular myocytes results in an increase in intracellular cAMP levels leading to enhanced cardiomyocyte contractility. However, a prolonged inhibition of MRP4 can promote cardiac hypertrophy. In addition, secreted cAMP, through its metabolite adenosine, prevents adrenergically induced cardiac hypertrophy and fibrosis. Finally, MRP4 inhibition in platelets induces a moderate thrombopathy. The localization of MRP4 underlines the emerging concept of cAMP compartmentalization in platelets, which is a major regulatory mechanism in other cells. cAMP storage in platelet dense granules might limit the cAMP cytosolic concentration upon adenylate cyclase activation, a necessary step to induce platelet activation. In this review, we discuss the therapeutic potential of direct pharmacological inhibition of MRP4 in atherothrombotic disease, via its vasodilating and antiplatelet effects.

Impact of aspirin and clopidogrel interruption on platelet function in patients undergoing major vascular surgery.

Aims To investigate functional platelet recovery after preoperative withdrawal of aspirin and clopidogrel and platelet function 5 days after treatment resumption. Methods/Results We conducted an observational study, which prospectively included consecutive patients taking aspirin, taking clopidogrel, and untreated controls (15 patients in each group). The antiplatelet drugs were withdrawn five days before surgery (baseline) and were reintroduced two days after surgery. Platelet function was evaluated by optical aggregation in the presence of collagen, arachidonic acid (aspirin) and ADP (clopidogrel) and by VASP assay (clopidogrel). Platelet-leukocyte complex (PLC) level was quantified at each time-point. At baseline, platelet function was efficiently inhibited by aspirin and had recovered fully in most patients 5 days after drug withdrawal. PLC levels five days after aspirin reintroduction were similar to baseline (+4±10%; pu200a=u200a0.16), in line with an effective platelet inhibition. Chronic clopidogrel treatment was associated with variable platelet inhibition and its withdrawal led to variable functional recovery. PLC levels were significantly increased five days after clopidogrel reintroduction (+10±15%; pu200a=u200a0.02), compared to baseline. Conclusions Aspirin withdrawal 5 days before high-bleeding-risk procedures was associated with functional platelet recovery, and its reintroduction two days after surgery restored antiplaletet efficacy five days later. This was not the case of clopidogrel, and further work is therefore needed to define its optimal perioperative management.

Recombinant activated factor VII does not reduce bleeding in rabbits treated with aspirin and clopidogrel

Combined antiplatelet agents (cAPA), aspirin plus clopidogrel, increase the risk of bleeding. We hypothesised that recombinant activated FVIIa (rFVIIa), which normalises thrombin generation in platelet-rich plasma from patients treated with cAPA, could limit this bleeding risk. It was the objective of this study to investigate the efficacy and safety of rFVIIa compared to placebo, in a bleeding and thrombosis model in rabbits treated with aspirin and clopidogrel. New-Zealand rabbits, randomised into two groups (Placebo1, n=36 ; cAPA, n=34), were anaesthetised, ventilated and monitored for blood pressure, temperature and carotid blood flow. The Folts model was applied to a carotid artery. Cyclic flow reductions (CFR) were recorded over a first 20-min period (Obs1). Each rabbit was then randomly assigned into one of three subgroups (Placebo2, 40μg/kg rFVIIa, 160 μg/kg rFVIIa) and CFR were monitored for a second 20-min period (Obs2). Ear bleeding time (BT) was measured at the end of each period. Hepatosplenic (HS) section was performed at the end of the experiment and HS blood loss defines the primary endpoint. Secondary endpoints were thrombosis (CFR), prothrombin time, platelet aggregation, and thrombin generation. Non- parametric statistical tests were used (p<0.05). cAPA significantly increased HS blood loss, BT and suppressed CFR compared to Placebo1 (p<0.05). rFVIIa injection did not modify HS blood loss, BT or CFR rate in Placebo1 rabbits nor in cAPA animals. These effects were unaffected by either rFVIIa dose. rFVIIa accelerated thrombin generation but had no effect on platelet aggregation in citrated platelet-rich plasma. rFVIIa did not modify HS blood loss associated with cAPA in rabbits.