Axel Matzdorff

Markers of platelet activation and platelet-leukocyte interaction in patients with myeloproliferative syndromes.

INTRODUCTION Changes in platelet count and function contribute to thrombo-hemorrhagic episodes in chronic myeloproliferative syndromes (MPS). We used flow cytometry to study platelet-leukocyte conjugates and markers of platelet activation in patients with MPS. METHODS Whole blood from patients with chronic myelogenous leukemia (CML), polycythemia vera (PV), chronic myelofibrosis (MF), and essential thrombocythemia (ET) and from healthy volunteers was prepared for flow cytometry. Platelet microparticles and platelet microaggregates were identified with anti-CD42b and forward scatter, activated platelets with anti-CD62p. Anti-CD42b, anti-CD14, and anti-CD45 were used to study platelet-leukocyte conjugates. RESULTS The percentage of CD62p-positive platelets was elevated in all myeloproliferate syndrome subtypes. The median percentage of platelet microparticles was 5.2% in controls and significantly higher in PV (12.0%), MF (11.0%), and ET (11.0%, all p<0.05). There was an increased percentage of platelet-neutrophil conjugates in patients with PV (8.3%) and ET (10.4%) compared to normal controls (6.8%, all p<0.05). Platelet-monocyte conjugates were 8.0% in controls and elevated in PV (15.4%) and ET (15.0%, all p<0.05). Patients with a history of venous or arterial thrombotic events had slightly less platelet-leukocyte conjugates and slightly more microparticles than patients without thrombosis; however, this difference was not statistically significant. CONCLUSIONS These findings suggest that platelet-leukocyte conjugate formation occurs in myeloproliferative syndromes and indicates platelet activation. Also, platelet microparticles are elevated and might provide a catalytic surface for thrombin generation. This could explain the clinical observation that patients with myeloproliferative syndromes have an increased risk to experience arterial or venous thrombotic events.

Protein Z influences the prothrombotic phenotype in Factor V Leiden patients

Protein Z enhances the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI). Thus, diminution of protein Z should induce prothrombotic tendency due to lowered cofactor activity for ZPI. In Factor V Leiden mice, prothrombotic tendency of severe diminution or lack of protein Z was demonstrated. We here present first studies in humans, indicating that diminution of protein Z in factor V Leiden patients aggravates thromboembolic risk.

Comparison of β-Thromboglobulin, Flow Cytometry, and Platelet Aggregometry to Study Platelet Activation

This study compares granule membrane protein (GMP)-140 expression measured by flow cytometry, release of beta-thromboglobulin (beta-TG), and platelet aggregometry as markers of platelet activation in vitro. Whole blood was activated with different concentrations of thrombin. There was a significant increase in beta-TG plasma levels after stimulation with 0.01 and 0.04 U thrombin/ml. There was also an increase in GMP-140 expression, but interindividual variability was high. Aggregometry of platelet-rich plasma did not detect platelet activation and formation of platelet aggregates with 0.05 and 0.1 U thrombin/ml, while flow cytometry showed an early and significant increase of GMP-140 expression with these doses. Beta-TG release is a more sensitive marker of platelet activation than GMP-140 while flow cytometry is easier to perform and less susceptible to artifacts.

Comparison of GP IIB/IIIA inhibitors and their activity as measured by aggregometry, flow cytometry, single platelet counting, and the rapid platelet function analyzer.

AbstractBackground: GP IIb/IIIa inhibitors have primarily been used short-term e.g., during PTCA. They failed to show clinical benefit during long-term therapy. One reason might be the absence of a method to monitor inhibitor activity. This study compared platelet aggregometry, the rapid platelet function analyzer (RPFA) test, single platelet counting, and flow cytometric determination of receptor occupancy to measure GP IIb/IIIa-receptor inhibitor activity. Methods: Increasing doses of abciximab, tirofiban, and eptifibatide were added to whole blood in vitro. Whole blood was used for the RPFA, for single platelet counting and flow cytometry. Platelet rich plasma was prepared for aggregometry. Results: The correlation between aggregometry and RPFA results was linear for abciximab and eptifibatide. Tirofiban was a stronger inhibitor with the RPFA (IC50 7.7[emsp4 ]nM) than with aggregometry (IC50 19.6[emsp4 ]nM). The single platelet counting technique showed that even supratherapeutic concentrations of all three inhibitors could not completely suppress microaggregation. Abciximab concentrations that were equipotent to tirofiban with aggregometry were less potent with regards to the inhibition of microaggregation. This difference was more pronounced with TRAP induced microaggregation than with ADP. The flow cytometric receptor occupancy test showed that occupancy was 95[emsp4 ]% with 5[emsp4 ]μg/ml abciximab and almost 97[emsp4 ]% with 10[emsp4 ]μg/ml. Tirofiban reached a maximum receptor occupancy of 56[emsp4 ]%, eptifibatide 64[emsp4 ]%. Conclusions: While aggregometry is time consuming the RPFA provides results fast and with little variability. There is still a discrepancy between aggregometry and RPFA results for tirofiban. The single platelet counting technique detects the inhibition of microaggregation the relevance of which for the clinical outcome is not known. The flow cytometric receptor occupancy assay is best suited for abciximab.

Relative and Absolute Changes of Activated Platelets, Microparticles and Platelet Aggregates after Activation in vitro

Standard flow cytometers provide relative numbers of activated platelets, microparticles, and platelet aggregates. With fluorescent beads it is now possible to determine absolute numbers. Whole blood and platelet-rich plasma were incubated with agonists (ADP, collagen, thrombin). CD62p expression, microparticle and platelet aggregate formation were measured. Flow-Count Fluorospheres® were added to calculate absolute concentrations. After activation there was an increase in the percentage of CD62p-positive platelets. However, the total number of platelets decreased and therefore the absolute number of CD62p-positive platelets did not increase but decreased. The number of CD62p-positive platelets decreased not as much as the number of CD62p-negative platelets, which explains why the relative percentage of CD62p-positive platelets increased. A similar increase in percent and decrease in absolute counts was found for microparticles. Platelet aggregates increased both in relative and absolute numbers. These results suggest that the detection of activated platelets by flow cytometry has to be complemented by the determination of the absolute concentrations to avoid misinterpretation.

Reactivity of Platelets from Healthy Individuals and from Patients with Hematologic Diseases following Incubation with Pegylated RecombinantHuman Megakaryocyte Gr owth and Development Factor in vitro

Platelets express the receptor for thrombopoietin. It is possible that thrombopoietin modulates platelet reactivity. We examined the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet activation in vitro using flow cytometry. We compared samples from healthy individuals and from patients with various hematologic diseases (AML, myeloma, postchemotherapy). Citrated whole blood was incubated with PEG-rHuMGDF (10 or 100 ng/ml), then a mild stimulus was added (0.1 U thrombin/ml). Blood from healthy individuals showed a significantly higher degree of platelet activation (CD62p expression), microparticle generation, and aggregate formation after incubation with PEG-rHuMGDF+thrombin versus thrombin alone (p < 0.05). However, this difference could not be shown for platelets from patients with thrombocytopenia or other hematologic diseases. The use of PEG-rHuMGDF should be safe and not cause an additional risk of thromboocclusive disease in these patients.