Laurent Macchi

Anti‐platelet antibodies in patients with systemic lupus erythematosus and the primary antiphospholipid antibody syndrome: their relationship with the observed thrombocytopenia

The role of antiphospholipid antibodies in the pathogenesis of the thrombocytopenia observed during primary antiphospholipid antibody syndrome (APAS) and systemic lupus erythematosus (SLE) remains controversial. We have used the MAIPA test to examine the frequency and specificity of anti‐platelet antibodies directed against the major platelet membrane glycoproteins (GP IIb–IIIa, GP Ib–IX, GP Ia–IIa and GP IV) in patients where SLE and APAS were associated or not with thrombocytopenia. Results were compared with a series of 26 ITP patients, 46% of whom were shown to possess anti‐platelet antibodies directed against one or more of the platelet surface glycoproteins. When APAS was associated with thrombocytopenia, 7/10 patients possessed antibodies against GP IIb–IIIa and/or GP Ib–IX. For SLE patients with thrombocytopenia, 6/10 patients were shown to have antiplatelet antibodies against GP IIb–IIIa, GP Ib–IX or GP IV. In contrast, for APAS (n=11) and SLE patients (n=11) without thrombocytopenia, only one patient had an antibody directed against GP IIb–IIIa and one patient had an antibody to GP IV. Our results suggest that antibodies directed against major platelet membrane glycoproteins may play a role in the thrombocytopenia that is seen during SLE and APAS.

Markers of platelet activation in coronary heart disease patients.

Abstract. We have applied flow cytometry to the detection of activated platelets in patients with coronary heart disease. Paraformaldehyde‐fixed platelets were incubated with one of the following monoclonal antibodies (MAbs): Bx‐1 (anti‐GP Ib), AP‐2 (anti‐GP IIb‐IIIa complex), VH10 (anti‐GMP‐140, a glycoprotein of the α‐granule membrane), or PAC‐1 (directed against an activation‐dependent determinant on GP IIb‐IIIa complexes). Bound antibody was quantitated after the addition of FITC‐conjugated anti‐immunoglobulin. This report highlights studies on 16 unstable angina patients undergoing transluminal angioplasty. Blood samples were taken at different periods before and after the angioplasty. Levels of activated platelets were variable, remaining in the 2–4% range of control donors for some, but increasing to 10–30% postangioplasty for others (despite all patients receiving heparin and aspirin). Maximum numbers of activated platelets were detected at 24 or 48 h. Nonetheless, the amount of antibody bound to individual platelets rarely reached the levels seen when control platelets were stimulated with thrombin in vitro. Results with VH10 and PAC‐1 often, but not always, correlated suggesting different pathways of platelet activation.

Direct Thrombin Inhibitor Prevents Delayed Graft Function in a Porcine Model of Renal Transplantation

Background. Kidney transplantations from donors after cardiac arrest (DCA) are characterized by an increase in the occurrence of delayed graft function and primary nonfunction. In this study, Melagatran, a selective reversible direct thrombin inhibitor was used to limit renal injury in a DCA pig kidney transplantation model. Methods. We used a porcine model of DCA to study the effects of treatment with Melagatran in the peri-conservation period. Thromboelastography was used to check Melagatran antithrombin effect on in vitro clot formation. Reverse-transcriptase polymerase chain reaction was used to analyze the peripheral immune cells activation status. Renal function and morphologic study were performed at days 1 and 7. Finally, we analyzed the mechanisms of Melagatran protection on kidney microvasculature primary endothelial cells. Results. Prolongation of coagulation time (Ex-Tem) was observed 10 min after injection; however, Melagatran did not modulate increases of thrombin-antithrombin complexes following reperfusion. Melagatran significant treatment lowered the proinflammatory status of circulating immune cells. Animals survival was increased in Melagatran-treated groups (9 of 10 in Melagatran groups vs. 4 of 10 in controls at day 7). Renal injury and inflammation were also significantly reduced in treated groups. We also demonstrated a direct protective effect of Melagatran against endothelial cell activation and inflammation in vitro. Conclusion. Direct thrombin inhibitor administration in the periconservation period improved graft outcome and reduced renal injury in a model of DCA.

Major clinical vascular events and aspirin-resistance status as determined by the PFA-100 method among patients with stable coronary artery disease: a prospective study.

Aspirin inhibits platelet activation and reduces major vascular events in patients with stable coronary artery disease. The extent of platelet inhibition, denoted as aspirin resistance, however, is not always sufficient. A correlation between aspirin resistance as measured by aggregometry and adverse clinical events has been demonstrated. The point-of-care platelet function analyzer PFA-100 is usually used to detect aspirin resistance, but the relation between PFA-100 results and the vascular prognosis is not assessed. We prospectively enrolled 97 patients with stable coronary artery disease who were on aspirin (160 mg per day since 1 month or longer). Aspirin resistance was measured by the PFA-100 analyzer. Median follow-up was 2.5 years and the primary outcome was the composite of death, myocardial infarction, and ischemic cerebral infarction or acute limb ischemia. In our study, 29 patients (29.9%) showed resistance to aspirin, with a higher percentage of female patients (38 vs. 15%; P = 0.01). During the follow-up, aspirin resistance was not associated with an increased risk of death, myocardial infarction, or ischemic vascular event compared with the aspirin-sensitive patients (17 vs. 13%; P > 0.60). In this cohort of stable coronary artery disease, patients on aspirin dose of 160 mg per day, the aspirin-resistance status based on the PFA-100 results is not associated with a significant increase in major vascular clinical events.

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb–IIIa in a patient with an unusual platelet membrane glycoprotein composition

The subject (E.B.) is a 63‐year‐old woman with autoimmune thrombocytopenic purpura (AITP) who was first examined some 6 years ago with symptoms of epistaxis and gum bleeding, severe thrombocytopenia, and large platelets. Her serum tested positively with control platelets in the MAIPA assay performed using monoclonal antibodies (MoAb) to glycoprotein (GP) IIIa (XIIF9, Y2/51), yet was negative in the presence of MoAbs to GP IIb (SZ 22) or to the GP IIb‐IIIa complex (AP2, P2). The patients platelets failed to aggregate with all agonists tested except for ristocetin. IgG isolated from the patients serum inhibited ADP‐induced aggregation of control platelets. Unexpectedly, flow cytometry showed an altered expression of membrane glycoproteins on the patients platelets. Levels of GP Ib‐IX were much higher than previously located by us in platelets. In contrast, the expression of GP IIb‐IIIa was about half that seen with control subjects. When Western blotting was performed, a striking finding was a strong band of 250 kDa recognized by a series of MoAbs to GP Ibα in addition to the band in the normal position of GP Ibα. Finally, ADP‐stimulated (E.B.) platelets failed to express activation‐dependent epitopes on GP IIb‐IIIa as recognized by PAC‐1, AP6, or F26 and additionally gave a reduced P‐selectin expression after thrombin addition. In conclusion, we present a novel patient with a severely perturbed platelet function where an altered membrane GP profile is associated with the presence of an autoantibody recognizing a complex‐dependent determinant on GP IIb–IIIa and inhibitory of platelet aggregation. Am. J. Hematol. 57:164–175, 1998.

An ex vivo evaluation of blood coagulation and thromboresistance of two extracorporeal circuit coatings with reduced and full heparin dose

OBJECTIVES Bioactive Carmeda® heparin-coated extracorporeal circuits (ECCs) have been shown to reduce contact phase and coagulation activation during cardiopulmonary bypass (CPB). Heparin coating is therefore effective in safely reducing coagulation during routine CPB. Balance® Biosurface is a new, recently developed biopassive coating containing negatively charged sulphonated polymers. This study sought to compare the clotting activation and thromboresistance of the Balance® (B) circuit with that of the Carmeda® (C) with full-dose systemic heparin (FDH) and reduced-dose systemic heparin (RDH). METHODS This ex vivo study set-up comprising 40 experiments consisted of simplified ECC and circulation of freshly donated human blood. RDH and FDH regimens were obtained with 0.5 IU/ml and 1 IU/ml heparin administered to reach target activated clotting times (ACTs) of 250 and 500 s, respectively. The study design comprised four groups: FDH-C, FDH-B, RDH-C and RDH-B (all n = 10). Blood was sampled prior to and during the 2-h CPB. Coagulation activation was assessed (FXIIa, F1.2) and electron microscope scan imaging of oxygenators enabled determination of adhesion scores. RESULTS With a biopassive compared with bioactive surface, mean ACT was lower, regardless of the heparin regimen applied (P < 0.001), whereas the total heparin dose required to maintain ACT was above target level (P < 0.001). However, FXIIa and F1.2 values were similar in all groups throughout, as were pressure gradients among oxygenators. All groups demonstrated similar adhesion scores following ultrastructural oxygenator assessment. CONCLUSIONS In the absence of surgical-related haemostatic disturbances and based on target ACT levels under reduced- or full-dose heparin, the clotting process was similar to heparin-coated and new sulphonated polymer-coated ECC, both demonstrating similar thromboresistance.

A rapid and specific whole blood HPA-1 phenotyping by flow cytometry using two commercialized monoclonal antibodies directed against GP IIIa and GP IIb-IIIa complexes

The human platelet antigen 1 (HPA‐1) system has been implicated in rare but severe diseases, such as neonatal alloimmune thrombocytopenic purpura, post‐transfusion purpura and immune platelet refractoriness. We developed a flow cytometry assay for HPA‐1 phenotyping using two commercial monoclonal antibodies, P2 and SZ21, directed against glycoprotein (GP) IIb–IIIa and GP IIIa respectively. One hundred and twenty‐seven healthy controls were studied and ratios of mean fluorescence intensity for P2 and SZ21 discriminated between HPA‐1a homozygotes and heterozygotes. These two monoclonal antibodies, coupled with flow cytometry represent a rapid and reliable tool for platelet HPA‐1 typing to aid diagnosis.

Automated hematology analysers and spurious counts. Part 1. Platelets

Hematology analysers provide now quick, accurate, and reproducible cell blood counts. However, depending on detection methods, spurious counts may occur. If undetected, such spurious counts may lead to inappropriate medical care and to unneeded explorations. Focusing first on platelet counts, situations leading to spurious decrease include several preanalytical considerations, the major one corresponding to EDTA-induced platelet aggregation and to platelet satellitism around polymorphs. In other instances, related to the presence of small particles mimicking platelets, including fragmented red blood cells, lipids, cryoglobulins, fibrin strands, or cytoplasmic fragments of leukocytes, spurious elevation of platelet count may occur. According to the analyser and to the methods used for the determination of the cell blood count, flags or messages related to these spurious changes differ. For each spurious change, the authors describe the mechanism leading to the anomaly, the way the analysers generate flags, and what should be done to provide accurate results.

Anomalies et erreurs de détermination de l'hémogramme avec les automates d'hématologie cellulaire

Hematology analysers provide now quick, accurate, and reproducible cell blood counts. However, depending on detection methods, spurious counts may occur. If undetected, such spurious counts may lead to inappropriate medical care and to unneeded explorations. Focusing first on platelet counts, situations leading to spurious decrease include several preanalytical considerations, the major one corresponding to EDTA-induced platelet aggregation and to platelet satellitism around polymorphs. In other instances, related to the presence of small particles mimicking platelets, including fragmented red blood cells, lipids, cryoglobulins, fibrin strands, or cytoplasmic fragments of leukocytes, spurious elevation of platelet count may occur. According to the analyser and to the methods used for the determination of the cell blood count, flags or messages related to these spurious changes differ. For each spurious change, the authors describe the mechanism leading to the anomaly, the way the analysers generate flags, and what should be done to provide accurate results.

Automated hematology analysers and spurious counts

Hematology analysers provide now quick, accurate, and reproducible cell blood counts. However, depending on detection methods, spurious counts may occur. If undetected, such spurious counts may lead to inappropriate medical care and to unneeded explorations. Focusing first on platelet counts, situations leading to spurious decrease include several preanalytical considerations, the major one corresponding to EDTA-induced platelet aggregation and to platelet satellitism around polymorphs. In other instances, related to the presence of small particles mimicking platelets, including fragmented red blood cells, lipids, cryoglobulins, fibrin strands, or cytoplasmic fragments of leukocytes, spurious elevation of platelet count may occur. According to the analyser and to the methods used for the determination of the cell blood count, flags or messages related to these spurious changes differ. For each spurious change, the authors describe the mechanism leading to the anomaly, the way the analysers generate flags, and what should be done to provide accurate results.