No abstract

Matrix metalloproteinase 2 (MMP2) has been reported to be secreted by collagen-stimulated platelets, and active MMP2 has been shown to play a role in platelet aggregation. It has been demonstrated that MMP2 activation is dependent on the complex (membrane type 1 [MT1]-MMP/tissue inhibitor of MMP2 [TIMP2]) receptor and MMP2. We have investigated human platelets as a possible source of MT1-MMP, and we have studied its role in MMP2 activation and in platelet aggregation. Gelatin zymograms showed the existence of MMP2 at proforms (68 kd) and activated-enzyme forms (62-59 kd) in supernatants of resting and activated platelets, respectively. No gelatinolytic activity was associated with the platelet pellet after aggregation, suggesting a total release of MMP2 during cell activation. By Western blot analysis in nonreduced conditions, MT1-MMP was found on resting platelet membranes in 2 forms–the inactive 45-kd form and an apparent 89-kd form, which totally disappeared under reduced conditions. After platelet degranulation, only the 45-kd form was detected. Reverse transcription–polymerase chain reaction experiments showed the expression in platelets of messenger RNA encoding for MMP2, MT1-MMP, and TIMP2. Flow cytometry analysis showed that MT1-MMP, MMP2, and TIMP2 expressions were enhanced at the activated platelet surface. MMP inhibitors, recombinant TIMP2, and synthetic BB94 inhibited collagen-induced platelet aggregation in a concentration-dependent manner, indicating the role of activated MT1-MMP in the modulation of platelet function. In conclusion, our results demonstrate the expression of the trimolecular complex components (MT1-MMP/TIMP2/MMP2) by blood platelets as well as the ability of MMP inhibitors to modulate the aggregating response.

SummaryThe in vitro closure time (CT), determined by the Platelet Function Analyzer (PFA-100 TM ), is used to monitor patients treated with aspirin. A relatively high percentage of in vitro aspirin resistance was reported despite an adequate inhibition of platelet response to arachidonic acid and we investigated whether high plasma levels of von Willebrand factor ristocetin cofactor activity (vWF:RCo) may contribute to this profile. Platelet aggregation test, CT [collagen adrenaline (CEPI-CT) and collagen adenosine 5¢-diphosphate (ADP) (CADP-CT)], and vWF:RCo levels were evaluated in 55 consecutive patients receiving aspirin (75-250 mg/d) versus 32 untreated control subjects. All the aspirin-treated patients showed platelet aggregation responses that reflected the aspirin intake. However, CT data analysis enabled aspirin good-responder (GR) and aspirin bad-responder (BR) patients to be identified. All GR group subjects (n ¼ 27), had a CEPI-CT and a CADP-CT longer than 300 s and 96 s respectively. The BR group (n ¼ 28) had CEPI-CT values below 200 s and all CADP-CT were in the normal range (77 ± 19 s). Interestingly, the BR plasma vWF:RCo levels were significantly higher (159 ± 43%) than those of the GR group (121 ± 34%) (P < 0AE01), which were similar to control values (114 ± 31%). A negative correlation between vWF:RCo and CT values was established. We demonstrate that in vitro aspirin-resistance, revealed by PFA-100 TM CT prolongation failure, is correlated to increased plasmatic vWF:RCo levels, reinforcing its particular importance in PFA-100 TM cartridges performance.

No abstract

SummarySickle cell disease (SCD) is linked to hypercoagulability and is characterised by high concentrations of erythrocyte-derived microparticles (Ed-MPs). However, the impact of procoagulant cell-derived microparticles on the thrombin generation process remains unclear. We analysed the alterations of each phase of thrombin generation (TG) in relation to the concentration of erythrocyte- or platelet-derived microparticles (Ed-MPs and Pd-MPs) in a cohort of patients with steady-state SCD. We studied 92 steady-state SCD patients, 19 of which were under treatment with hydroxyurea, and 30 healthy age- and sex-matched individuals. TG was assessed by calibrated automated thrombogram. Ed-MP and Pd-MP expressing or not phosphatidylserine (PS) were determined by means of flow cytometry. Procoagulant phospholipid-dependent activity in the plasma was evaluated by the Procoag-PPL assay. Levels of thrombomodulin and haemoglobin in the plasma as well as red blood cell and reticulocyte counts were measured. SCD patients, independently of the administration of hydroxyurea, were marked by a significant acceleration in the propagation phase of TG which correlated with the Ed-MP/PS+ concentration. TG was significantly attenuated in hydroxyurea-treated patients. In conclusion, the acceleration of the propagation phase of TG, driven by Ed-MP/PS+, is a major functional alteration in blood coagulation in patients with steady-state SCD. Treatment with hydroxyurea, in addition to the regulation of haemolysis, lowers Ed-MPs and attenuates thrombin generation. The thrombogram could be a useful tool for the diagnosis of hypercoagulability and optimisation of the treatment in patients with SCD.

No abstract

Heparin-induced thrombocytopenia (HIT) is a potentially lethal adverse effect of heparin therapy. Accurate and rapid HIT laboratory diagnosis when HIT is suspected is crucial. The combination of an immunological assay with a functional test improves the accuracy of HIT, but functional assays are currently limited to a few laboratories. Multiplate® analyzer (Dynabyte, Munich, Germany) is a practical, semi-automated and easy-to-perform platelet aggregation assay. The aim of this study is to explore whether heparin-induced platelet aggregation in whole blood assessed by Multiplate® (Heparin-induced multiple electrode aggregometry, HIMEA) can replace platelet aggregation test (PAT) in platelet-rich plasma. For this purpose, HIMEA performance in HIT diagnosis was prospectively evaluated. HIMEA and PAT were compared to serotonin-release assay (SRA) in 200 well-characterized consecutive patients suspected for HIT. HIMEA was found to be more sensitive (81% vs. 76%) and more specific (99% vs. 96%) than PAT compared to SRA. Both tests showed a high negative predictive value while HIMEA had a better positive predictive value. HIMEA has overall better performance characteristics than PAT for the detection of HIT platelet-activating antibodies. The combination of an immunological assay with HIMEA could be a feasible option in non-specialized laboratories for HIT diagnosis optimization.

It has been shown [Touqui, Jacquemin & Vargaftig (1983) Thromb. Haemostasis 50, 163; Touqui, Jacquemin & Vargaftig (1983) Biochem. Biophys. Res. Commun. 110, 890-893; Alam, Smith & Melvin (1983) Lipids 18, 534-538; Pieroni & Hanahan (1983) Arch. Biochem. Biophys. 224, 485-493] that rabbit platelets inactivate exogenous PAF (platelet-activating factor, PAF-acether) by a deacetylation-reacylation mechanism. The deacetylation step is catalysed by an acetyl hydrolase sensitive to the serine-hydrolase inhibitor PMSF (phenylmethanesulphonyl fluoride) [Touqui, Jacquemin, Dumarey & Vargaftig (1985) Biochim. Biophys. Acta 833, 111-118]. We report here that human platelets can produce PAF on thrombin stimulation. This production is marginal and transient, reaching a maximum at 10 min and decreasing thereafter. In contrast, 10-12 times more PAF is produced when platelets are treated with PMSF and stimulated with thrombin. Under these conditions, the maximum formation is observed at 30 min and no decline occurs for up to 60 min after stimulation. In addition, these platelets (treated with PMSF and stimulated with thrombin) incorporate exogenous labelled acetate in the 2-position of PAF, probably by an acetyltransferase-dependent mechanism. Production of PAF by human platelets during physiological stimulation can be demonstrated when PAF degradation is suppressed by the acetyl-hydrolase inhibitor PMSF.