Paroxysmal Nocturnal Hemoglobinuria Platelets Research Articles

Analysis of platelets by flow cytometry in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

The marked pro-thrombotic tendency in PNH is likely to be at least partly due to the population of platelets derived from the abnormal stem cell clone. However, identification of GPI (−) platelets by flow cytometry can be technically difficult. Here we describe a technique that involves the addition of aspirin immediately after the separation of platelet rich plasma and the use of gel filtration to isolate platelets away from plasma proteins and other blood cells. In a study of 92 analyses of samples from 50 patients, we have demonstrated that the percentage of PNH platelets correlates well with the percentage of PNH granulocytes. We also provide data on several cases where there was an extreme discrepancy between the proportion of PNH granulocytes and red cells; in these cases, the demonstration of abnormal platelets suggests that the patient is likely to be at risk of thrombosis. We believe this test will be potentially useful in the evaluation of samples from such patients and may serve as a tool to investigate the causes of hypercoagulability in PNH.

Analysis of Platelets By Flow Cytometry in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by a clonal population of hematopoietic stem cells with an acquired somatic mutation in the PIG-A gene, giving rise to populations of circulating mature cells that are unable to synthesize glycosylphosphatidylinositol (GPI). The disease is most readily diagnosed by flow cytometry analysis of red blood cells, using antibodies specific for the GPI-linked protein CD59, or analysis of granulocytes, using antibodies specific for the GPI-linked protein CD24, along with the FLAER reagent, a fluorescent protein that binds to the GPI structure and which is detected only on the surface of GPI (+) cells. However, other mature blood lineages can be derived from the PNH clone. Notably, thrombosis is a major life threatening complication of PNH and may be triggered by complement activation on platelets that belong to the GPI-negative stem cell clone. The PNH clone size generally predicts thrombosis, but sometimes the proportion of PNH red cells and granulocytes are highly discordant, in which case there might be a role for the determination of the proportion of PNH platelets. Historically, flow cytometry analysis of platelets in patients with PNH has been technically difficult. Here is described a method to do this that avoids technical challenges by using aspirin and sepharose gel filtration of platelets to prevent their activation as well as simultaneous determination of CD59 expression and uptake of the FLAER reagent. Red cells were analyzed based on CD59 expression and granulocytes based on CD24 and FLAER. We analyzed blood samples from 48 patients with PNH and or AA/PNH who provided informed consent, 16 of whom had a prior history of thrombosis. To separate platelet rich plasma (PRP), whole blood collected in EDTA tubes was centrifuged at 200g for 7 minutes at room temperature with the brake turned off. After this step, there was no further centrifugation or vortexing of the platelets. A solution of aspirin was made up immediately prior to use and was added to the PRP at a final concentration of 0.5mMolar. Aspirinated PRP was then loaded on top of a sepharose-2B column using Tyrode's buffer. The platelet-rich turbid drops were collected, to isolate platelets from red cells and coagulation proteins. 50 ul of platelet rich buffer was then incubated with FLAER-Alexa-488 (Pinewood, 1:20 dilution) and CD59-PE (Serotec, 1:10 dilution) in the dark for 30' at room temperature. To prevent doublet events from confounding the analysis, the platelet suspension was diluted 1:200 in Hanks with 0.1% BSA. The sample was passed through a 35 uM Falcon cell strainer, and platelets were identified by forward/side scatter acquired on a log-log scale on a BD Facscan. The median proportion of PNH red cells, granulocytes and platelets was 24%, 86%, 76% respectively in the group without a history of thrombosis and 23% ,82%, and 65% in the group with a history of thrombosis. The proportion of PNH platelets was highly correlated with the proportion of PNH granulocytes (r=0.84). In two patients with almost undetectable PNH red cells and over 90% PNH granulocytes, the proportion of PNH platelets was over 90%; both were on prophylaxis and neither had thrombosis. It is predicted that this technique may be useful for determining thrombosis risk, particularly when the results from the analysis of rbc's and granulocytes are discordant. DisclosuresNo relevant conflicts of interest to declare.

Terminal Complement Inhibitor Eculizumab Improves Complement-Mediated Platelet Consumption and Thrombocytopenia in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).

Abstract 4030Poster Board III-966PNH is a hematopoietic stem cell disorder in which unregulated activation of the complement system leads to significant morbidities with shortened lifespan. Life threatening thromboembolism (TE) is the most feared complication of PNH, accounting for 45% of patient deaths. Approximately 40% of PNH patients experience a clinically evident TE and 60% of patients without clinically diagnosed TE demonstrate TE by high-sensitivity MRI, indicating the ongoing thrombotic risk in PNH patients. Thrombocytopenia is found in 25-50% of PNH patients at presentation. PNH platelets are extremely sensitive to terminal complement activation, and circulating platelets are activated in thrombocytopenic PNH patients without clinically diagnosed TE. Eculizumab, a monoclonal antibody targeted to complement C5, blocks the production of both C5a and C5b-9, and has been demonstrated to reduce TE events by up to 92% in PNH patients. Eculizumab has also been shown to beneficially impact several prothrombotic processes in PNH patients including hemolysis, nitric oxide consumption, thrombin generation, tissue factor pathways, and inflammation. We hypothesized that terminal complement activation of PNH platelets contributes to ongoing platelet activation, which is reflected by platelet consumption, contributing to chronic thrombocytopenia defined as platelets <100×109/L. This hypothesis was tested by examining whether chronic inhibition of terminal complement activation with eculizumab increases platelet counts in thrombocytopenic PNH patients. The study population was comprised of the 49 thrombocytopenic PNH patients identified from the total 195 patients in the SHEPHERD, TRIUMPH, and Phase 2 eculizumab trials. Platelet counts were measured at baseline, 26 and 52 weeks of eculizumab treatment. At baseline the median platelet count in the thrombocytopenic group was 68×109/L (range 23 to 97). Patients with thrombocytopenia were more likely to have a history of TE than patients with normal platelet counts (45% vs 27%; P<0.022). Treatment with eculizumab markedly inhibited terminal complement activity in all thrombocytopenic patients, as measured by a significant reduction in LDH at 26 and 52 weeks (median values at baseline, 26 weeks, and 52 weeks: 1746 IU/L, 290 IU/L, 286 IU/L, respectively, UNL 220 U/L); P<0.00001 for each time point vs. baseline). Chronic terminal complement inhibition with eculizumab significantly increased platelet counts to a non-thrombocytopenic level in 33% (95% CI: 20-48%) and 36% (95% CI: 22-51%) of thrombocytopenic patients at 26 and 52 weeks, respectively, of treatment. By inhibiting C5 activity, the median platelet counts significantly increased in thrombocytopenic patients from 68×109/L at baseline to 80 and 85×109/L (P<0.001) at 26 and 52 weeks, respectively. Eculizumab treatment significantly increased platelet counts in both thrombocytopenic patients with a history of TE and no history of TE (P<0.05 vs baseline in both groups). Treatment with eculizumab significantly increased platelet counts at 52 weeks in this thrombocytopenic patient population irrespective of a history of bone marrow failure (P<0.05 vs baseline for each group). As expected, there was no apparent impact of terminal complement inhibition on marrow blood cell production as measured by no change in absolute neutrophil count at 26 and 52 weeks. This suggests that the mechanism of platelet increase may be due to inhibition of terminal complement-mediated consumption of platelets in thrombocytopenic patients with PNH. The present study indicates that thrombosis in PNH patients is more frequent in patients with thrombocytopenia. Terminal complement deposition on PNH platelets results in platelet activation which in turn would be expected to result in platelet consumption, suggestive of a chronic prothrombotic state, in PNH patients. Earlier studies have shown that chronic terminal complement inhibition with eculizumab reduces thrombotic risk in patients with PNH. The current study shows that chronic terminal complement inhibition with eculizumab treatment improves pre-existing thrombocytopenia in a significant proportion of patients with PNH. These clinical findings warrant further investigation and suggest that terminal complement C5 activity contributes to platelet activation and consumption with subsequent thrombocytopenia and an increased thrombotic risk in patients with PNH. Disclosures:Socie:Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees. Muus:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Schrezenmeier:Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Höchsmann:Alexion: Honoraria. Hill:Alexion: Honoraria. Bessler:Alexion: Membership on an entity's Board of Directors or advisory committees. Risitano:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients' plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.

High Definition Contrast-Enhanced MR Imaging in Paroxysmal Nocturnal Hemoglobinuria (PNH) Suggests a High Frequency of Subclinical Thrombosis.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by intravascular hemolysis and venous thrombosis. Thrombosis is the most feared complication in PNH and is reported to occur in >40% of patients. Proposed mechanisms of thrombosis include depletion of the coagulation regulator nitric oxide (NO) by intravascular hemolysis and increased sensitivity of PNH platelets to activation. The occurrence of subclinical thrombosis in PNH patients has not been previously studied using modern imaging techniques. In order to evaluate for subclincal thrombosis we evaluated PNH patients with a comprehensive state-of-the-art MRI protocol (which included the use of both blood pool and conventional Gadolinium based contrast agents) for the detection of subclinical thromboses and its sequelae. The detailed protocol consisted of:lung perfusion and pulmonary MRA,cardiac MR - including quantitative studies of both ventricles, right heart flow dynamics and delayed enhancement for the detection of left ventricular damage, andabdominal MR for the assessment of hepatic and portal venous systems and kidneys.10 PNH patients (median age 31.5 yrs) with large PNH clones but without previous clinical evidence of venous or arterial thrombosis underwent imaging. Five (50%) of the patients were on primary anticoagulant prophylaxis with warfarin. There was evidence of significant renal hemosiderosis, which was distributed throughout the cortices, in 8/10 patients. Two patients had small myocardial scars suggestive of previous unsuspected ischemic damage. Six patients had sub-segmental perfusion defects mainly distributed in the peripheries of the lung fields indicative of previous small pulmonary emboli. No such subclinical thromboses would be anticipated in an age-matched control population. 8 patients had mildly reduced right ventricular ejection fractions (mean 42.2±1.8%; normal range 48–63%). The plasma B-type natriuretic peptide (BNP) level was high in all 10 patients (median 29.4pmol/l; range 18.7–373.90; normal subjects 4.89±1.00pmol/l). BNP has been shown to increase in proportion to right ventricular dysfunction in pulmonary hypertenstion. No intraabdominal defects were identified with the current protocol. In summary, we identified abnormalities suggestive of previous subclinical thromboses in 6 of 10 hemolytic PNH patients by high-resolution MR imaging, including in patients on primary prophylaxis with warfarin. Effective prevention of thrombosis is an important aspect of the therapy in PNH.

Complement-Induced Vesiculation and Exposure of Membrane Prothrombinase Sites in Platelets of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane-binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b-9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9-treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane- binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b- 9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9- treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

Paroxysmal nocturnal hemoglobinuria. Enhanced stimulation of platelets by the terminal complement components is related to the lack of C8bp in the membrane.

Recently, a protein isolated from the membrane of human E, the so-called C8 binding protein (C8bp), has been described. C8bp is characterized as a 65-kDa protein that binds to C8 and inhibits the C5b-9-mediated lysis in a homologous system. In the present study, membranes of peripheral blood cells were tested for the presence of C8bp by SDS-PAGE and immunoblotting. In all cells a protein band reacting with anti-C8bp was seen, the Mr, however, was only about 50 kDa. To further analyze the 50-kDa protein, we isolated the protein by phenol-water extraction and isoelectric focusing from papain-treated platelets. The isolated protein behaved similar to the E-derived C8bp: it inhibited the lysis of model target cells by C5b-9. To examine the function of C8bp in platelets, we tested platelets from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). These platelets were deficient in C8bp, being in accordance with their higher lytic susceptibility in vitro. In response to sublytic C5b-9 doses, the PNH platelets released considerably more serotonin and thromboxane B2 than normal platelets. By addition of purified C8bp, the thromboxane B2 release was suppressed, indicating that C8bp not only restricts the lytic complement attack, but also regulates the C5b-9-mediated stimulation of target cells. Thus, lack of C8bp might not only result in enhanced hemolysis, but also in enhanced stimulation of platelets, which in turn might contribute to the thrombotic complications seen in some PNH-type III patients.

Interactions of the platelets in paroxysmal nocturnal hemoglobinuria with complement. Relationship to defects in the regulation of complement and to platelet survival in vivo.

The blood cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) have abnormal interactions with complement. The activity of the alternative pathway C3 convertase on the platelets of 9 out of 19 patients with PNH was elevated. 10 patients had C3 convertase activity within the normal range even though 80-95% of their platelets lacked the complement regulatory protein decay accelerating factor (DAF) that is absent from the affected blood cells in PNH. PNH and normal platelets released factor H when C3 was bound to their surfaces. This may account for the apparent regulation of C3 convertase activity on platelets that lack DAF. The abnormal uptake of the membrane attack complex of complement by PNH III erythrocytes was not seen in PNH platelets. 111Indium-labeled platelet survival times were normal in five of eight patients, which suggests that the lack of the membrane attack complex defect results in normal platelet survival in PNH.

AET-Treated Platelets: Their Usefulness for Platelet Antibody Detection and an Examination of Their Altered Sensitivity to Immune Lysis

Abstract2-Aminoethylisothiouronium bromide (AET) increases the sensitivity of blood cells to complement-mediated immune lysis. We compared the sensitivities of untreated or AET-treated platelets to immune lysis induced by different types of platelet antibody in the 51Cr platelet lysis test. AET platelets were 8–16 times more sensitive to autoantibody and alloantibody, but 8–16 times less sensitive to drug- dependent antibody. AET-platelets bound similar amounts of alloantibody but less drug-dependent antibody, and they lysed at higher complement dilutions than did untreated platelets. AET-platelets detected 10 of 25 autoantibodies, 9 of 9 alloantibodies, and 5 of 8 drug-dependent antibodies. Untreated platelets detected 1 of 25, 6 of 9, and 7 of 8 of these respective platelet antibodies. The use of AET-platelets in the 51Cr platelet lysis test increases its sensitivity for detecting non- drug-dependent platelet antibodies. AET-platelets resemble paroxysmal nocturnal hemoglobinuria (PNH) platelets in their enhanced sensitivity to complement-mediated lysis. They differ from PNH platelets in their insensitivity to immune lysis induced by drug-dependent antibodies and, in this respect, are similar to Bernard-Soulier syndrome platelets.

AET-treated platelets: their usefulness for platelet antibody detection and an examination of their altered sensitivity to immune lysis

2-Aminoethylisothiouronium bromide (AET) increases the sensitivity of blood cells to complement-mediated immune lysis. We compared the sensitivities of untreated or AET-treated platelets to immune lysis induced by different types of platelet antibody in the 51Cr platelet lysis test. AET platelets were 8–16 times more sensitive to autoantibody and alloantibody, but 8–16 times less sensitive to drug- dependent antibody. AET-platelets bound similar amounts of alloantibody but less drug-dependent antibody, and they lysed at higher complement dilutions than did untreated platelets. AET-platelets detected 10 of 25 autoantibodies, 9 of 9 alloantibodies, and 5 of 8 drug-dependent antibodies. Untreated platelets detected 1 of 25, 6 of 9, and 7 of 8 of these respective platelet antibodies. The use of AET-platelets in the 51Cr platelet lysis test increases its sensitivity for detecting non- drug-dependent platelet antibodies. AET-platelets resemble paroxysmal nocturnal hemoglobinuria (PNH) platelets in their enhanced sensitivity to complement-mediated lysis. They differ from PNH platelets in their insensitivity to immune lysis induced by drug-dependent antibodies and, in this respect, are similar to Bernard-Soulier syndrome platelets.

Mechanism of complement-mediated activation of human blood platelets in vitro: comparison of normal and paroxysmal nocturnal hemoglobinuria platelets.

The paroxysmal nocturnal hemoglobinuria (PNH) platelet differs from the normal human platelet in its interaction with activated complement components: (a) when complement is activated by the alternative pathway, greater amounts of C3 are fixed to the PNH platelet than to the normal platelet; (b) the platelet-release reaction, as measured by serotonin release, occurs after C3 fixation to the PNH platelet. This reaction does not occur with normal platelets; (c) although serotonin release mediated by antibody alone was the same for normal and PNH platelets, antibody-initiated complement activation resulted in the fixation of greater amounts of C3 to PNH platelets and greater consequent serotonin release; and (d) nearly maximal serotonin release; and (d) nearly maximal serotonin release from PNH platelets occurs after the fixation of C3 (or perhaps C5) to the membrane without completion of the terminal sequence. In contrast, completion of the terminal complement sequence beyond C5 is required for maximal serotonin release from normal platelets. These abnormalities of interaction of complement components and PNH platelets may explain the occurrence of thromboses in this disease.

A platelet and granulocyte membrane defect in paroxysmal nocturnal hemoglobinuria: usefulness for the detection of platelet antibodies.

The tendency of platelets and leukocytes to lyse after their interaction with antibody and complement was studied by measuring the release of (51)Cr from cells labeled with this isotope. Platelets from six patients with paroxysmal nocturnal hemoglobinuria (PNH) were 15-230 times more sensitive to antibodies and 10-32 times more sensitive to complement than normal platelets or platelets from patients with other types of thrombocytopenic or hemolytic disorders. Mixed white blood cell (WBC) preparations from patients with PNH were 3-20 times more sensitive to anti-WBC antibodies and 5-10 times more sensitive to C' than were WBC preparations from normal subjects, but PNH lymphocytes showed normal immunologic reactivity. PNH platelets, like PNH erythrocytes, lysed more readily than normal platelets in acidified serum and in media of reduced ionic strength, but these characteristics were not demonstrable with PNH WBC's under the conditions of study. In PNH, platelets appear to comprise a single population with respect to their sensitivity to immune lysis, yet their survival time as measured with (51)Cr falls within normal limits. PNH granulocytes likewise appear to consist of a single, uniformly sensitive population. It is concluded that, in PNH, platelets and granulocytes share the membrane defect characteristic of erythrocytes in this disorder. These observations support the concept that PNH arises as the result of a somatic mutation in a primitive cell capable of differentiating into erythroblast, myeloblast, and megakaryoblast lines. PNH platelets or enzymatically treated normal platelets permit the detection of some types of platelet antibodies in dilutions up to 2000-fold greater than is possible with currently available methods, a finding suggesting that the immune lysis technique will prove useful for the study of platelet immunology.