Appendix
| a Values expressed as mean (range). |
V617F JAK-2 mutation in patients with essential thrombocythemia: relation to platelet, granulocyte, and plasma hemostatic and inflammatory molecules
Anna Falanga
Marina Marchetti
Alfonso Vignoli
Donatella Balducci
Laura Russo
Vittoria Guerini
Tiziano Barbui
Department of Hematology-Oncology, Ospedali Riuniti, Bergamo, Italy
Offprint requests to: Anna Falanga, M.D., Department of Hematology, Ospedali Riuniti di Bergamo, Largo Barozzi 1-24128, Bergamo, Italy; E-mail: annafalanga@yahoo.com
Objective. This article evaluates patients with essential thrombocythemia (ET) to determine whether the V617F mutation in the JAK2 gene affects platelet hemostatic and adhesive molecules, platelet-polymorphonuclear leukocyte (PMN) interactions, and PMN-activation characteristics, as well as plasma hypercoagulation markers.
Patients and Methods. Thirty-seven ET patients with V617F JAK2 mutation and 38 wild-type, and 50 healthy controls were studied.
Results. Platelets from overall ET patients, compared to controls, expressed significantly higher membrane tissue factor (TF) and P-selectin (p < 0.01) and lower CD41 and CD42b (p < 0.01). TF appeared significantly higher in the V617F JAK2 carriers compared to wild-type, and total platelet TF antigen levels confirmed the same result. The presence of circulating platelet/PMN aggregates was significantly greater in the JAK2-mutation carriers than in the wild-type and controls (p < 0.05). PMN surface activation and inflammatory markers (i.e., CD14, TF, CD11b, and leukocyte alkaline phosphatase [LAP]) were all significantly higher in ET versus control subjects, with CD14 and LAP being the highest in the JAK2 mutation carriers. Finally, a significant increase in plasma hypercoagulation markers was found in ET patients, and the only difference for the V617F JAK2 carriers was higher plasma thrombomodulin levels (p < 0.01). Differences in white blood cell and PMN count, platelet TF, PMN CD14, and LAP, and plasma thrombomodulin remained significant after multivariate analysis.
Conclusions. These results show that a correlation exists between the presence of V617F JAK2 mutation and selected hemostatic activation variables.
Essential thrombocythemia (ET) is a myeloproliferative disorder (MPD) with a relatively long median survival [1]. However, the clinical course of the disease is complicated by a high incidence of thrombohemorrhagic episodes [2], and arterial and venous thromboses significantly contribute to the morbidity and mortality of ET patients [3]. The pathogenesis of these complications remains to be clarified and is under active investigation. High red blood cell, platelet, and leukocyte counts are all associated, to varying degrees, with thrombotic risk in MPD patients [4–6], but the role of qualitative abnormalities of circulating blood cells is also becoming increasingly important [7,8]. It was, therefore, of particular interest to find activated circulating platelets and polymorphonuclear leukocytes (PMN) in ET patients with parallel increases in circulating platelet/PMN aggregates and plasma thrombotic markers [9–12]. These findings suggest a central role for cellular activation in the thrombotic diathesis of MPD.
Recent investigations have demonstrated the presence of an acquired gain-of-function mutation in the tyrosine kinase JAK2 gene in neutrophils and platelets of patients with MPD [13–16]. JAK2 kinase is a tyrosine kinase associated with the cytoplasmic domains of cytokine and growth factor receptors. This mutation is present in the majority of patients with polycythemia vera (90%), and half of those with ET or myelofibrosis. Notably, clinical data indicate an association between the presence of this mutation and the severity of the disease [14,17,18]. It is unknown whether mutation status can specifically alter the hemostatic system, but it has recently been reported that the mutation is associated with increased expression of platelet surface P-selectin [19] and PMN surface activation markers [20]. This suggests that JAK2 kinase hyperactivity can affect cellular pathways potentially involved in the activation of the hemostatic system, which represents a relevant mechanism of morbidity in MPD. Therefore, this study was designed to investigate whether the presence of the V617F JAK2 mutation identifies subjects with specific hemostatic abnormalities. We studied the expression of procoagulant, adhesive, and inflammatory molecules by platelets and PMNs of ET patients, about half of whom expressed the V617F JAK2 mutation. Specifically, we evaluated: 1) platelet expression of tissue factor (TF), P-selectin, and adhesive glycoproteins; 2) levels of circulating platelet/PMN aggregates; and 3) PMN expression of TF and other activation/inflammatory molecules, such as CD14, (the receptor for bacterial endotoxin (lipopolysaccharide [LPS]) [21], CD11b, and leukocyte alkaline phosphatase (LAP) [8,17]. Finally, soluble plasma markers of activation of coagulation (i.e., prothrombin fragment 1+2 [F1+2], thrombin-antithrombin complex [TAT], and d-dimer), fibrinolysis (i.e., tissue plasminogen activator [t-PA] and plasminogen activator inhibitor [PAI-1]), endothelium (i.e., thrombomodulin [TM]), and PMN (i.e., elastase) were analyzed.
Patients and methods
Patients
Seventy-five consecutive ET outpatients (44 women, 31 men; age range, 23–86 years) admitted to our department were enrolled in the study after giving informed consent. ET was diagnosed according to the Polycythemia Vera Study Group criteria [3]. Thirty-seven patients were carriers of the V617F JAK2 mutation and 38 were carriers of the JAK2 wild-type gene. Fifty healthy subjects (28 women, 22 men; age range, 20–83 years) without a history of thrombohemorrhagic events acted as the control group, and none of these subjects had symptoms of active infection or inflammatory disease. Characteristics of patients and healthy controls are summarized in Table 1. Investigations were approved by the local ethics committee (Comitato di Bioetica, Ospedali Riuniti di Bergamo, Italy), procedures followed were in accordance with the Helsinki Declaration of 1975 as revised in 2000, and samples were obtained only after subjects provided informed consent. Median time interval between ET diagnosis and blood sampling was 72 months (range, 12-288 months) for V617F JAK2 carriers and 60 months (range, 13-216 months) for wild-type JAK2 patients (p = NS).
Blood samples
Blood samples were drawn early in the morning. Venous blood was drawn with a 21-gauge butterfly needle after applying a light tourniquet. After discarding the first 5 mL, blood was collected into sterile siliconized tubes containing trisodium citrate (0.129 mol/L, 9:1 v/v) and immediately processed. Sample preparation and cytofluorimetric analysis were accomplished within a maximum of 1 hour of sample collection.
Routine hematological assays
White blood cell differential count, hematocrit, hemoglobin, red blood cell, and platelet counts were determined by automated methods using a NE800 Analyzer (Dasit, Milan, Italy).
Study of platelet function by PFA100 analysis
PFA-100 simulates primary hemostasis by forcing whole blood to flow at a high shear rate through a hole (147-μm diameter) cut into a collagen-coated membrane coated with either adenosine diphosphate (ADP; 50 μg) or epinephrine (EPI; 10 μg). The blood comes into contact with the membrane surface and aggregates, and a platelet plug forms, occluding the hole and stopping the blood flow. Closure time reflects platelet function in the evaluated sample. The upper limits of the normal range of closure times were set at the 95th percentiles (mean ± 2 SD) of normal values (114 and 167 seconds for collagen/ADP and collagen/EPI cartridges, respectively).
Cytofluorimetric study of platelets
Flow cytometry analysis was used to study the platelets in whole-blood samples, both in basal conditions and after in vitro activation by the PFA-100 system (Dade Behring, Milan, Italy) [22]. The PFA-100 system was selected for the study of activated platelets because the PFA-100 system assay is performed in whole blood in the presence of other blood cells, the stimuli consist of combinations of EPI with collagen or ADP with collagen, and the conditions mimic the high physiological shear rate.
Both fresh and ADP/collagen-activated or EPI/collagen-activated blood samples were diluted 1:20 in phosphate-buffered saline (PBS) and incubated for 20 minutes at room temperature with the following fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb): anti-CD41, anti-CD42b (Serotec, Milan, Italy), anti-CD62P (BenderMedSystems, Vienna, Austria), and anti-TF (American Diagnostica, Stamford, CT, USA). After incubation, samples were analyzed by FACSCalibur cytofluorimeter (Becton Dickinson, Mountain View, CA, USA) [12]. Platelets were identified by their forward- and side-scatter properties, and this region was 100% positive for CD41 and CD42b antigen expression. Ten-thousand events were collected from each sample, and data acquisition and processing were performed with CellQuest software (Becton Dickinson). Results for CD41 and CD42b are expressed as mean fluorescence intensity (MFI) in arbitrary units, because these molecules are constitutively expressed by all platelets, while results for CD62P are expressed as the percentage of positive cells, because it is not expressed by all platelets in resting conditions. The MFI represents the mean level of each expression/cell marker.
Cytofluorimetric study of platelet/PMN aggregates
Whole-blood samples (50 μL) were incubated for 20 minutes with phycoerythin (PE)-conjugated mouse anti-human anti-CD11b mAb (Becton Dickinson) in combination with either FITC-conjugated anti-CD41 or FITC-conjugated anti-CD62P, or with isotype-identical negative control mAb (IgG2a-PE and IgG1-FITC; Becton Dickinson). After incubation, erithrocytes were lysed, samples centrifuged, resuspended in PBS, and immediately analyzed on the FACSCalibur cytofluorimeter. PMN were identified by their forward- and side-scatter properties and 100% CD11b-positivity. PMN-platelet aggregates were defined by double staining with both PMN (anti-CD11b mAb) and platelet markers (either anti-CD41 or anti-CD62P mAbs); 5000 events were collected from each sample [12].
Platelet TF: total antigen measurement
Immediately after venipuncture, platelet-rich plasma was prepared by centrifuging citrated venous blood at 180g for 20 minutes. The platelet-rich plasma was collected and washed three times in saline solution containing 5 mM ethylenediamine tetraacetic acid. The platelet pellet was then resuspended in PBS-Triton X100 buffer at the concentration of 109 platelets/mL and lysed by three cycles of freezing and thawing. Total platelet TF antigen was determined with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Imubind TF, American Diagnostica). Results are expressed as pg/109 platelets.
Cytofluorimetric study of PMN
PMN analysis was performed as described previously [11,12]. Briefly, 50 μL fresh whole blood was incubated for 20 minutes with the following mAb: anti–TF-FITC, anti–CD14-FITC, anti–CD11b-PE, anti–LAP-FITC, and irrelevant isotype-identical negative control mAbs (IgG2a-PE and IgG1-FITC). After incubation, the samples were lysed, centrifuged, resuspended in PBS, and immediately analyzed by FACSCalibur. PMN were selectively gated using their forward- and side-scatter properties and 5000 PMN-gated events were measured for each sample. Results are expressed as the percentage of positive cells and in MFI units.
Plasma hemostatic markers
Plasma was separated by centrifugation of venous blood at 3000g for 20 minutes and stored in aliquots at −80°C until the assay (<3 months). All parameters were measured using the following commercial ELISA kits: Enzygnost F1+2 (Dade Behring, Milan, Italy); Enzygnost TAT (Dade Behring); Zymutest d-dimer (Hyphen Biomed, Neuville-sur-Oise, France); Zymutest PAI-1 (Hyphen Biomed); Zymutest t-PA (Hyphen Biomed); Human PMN Elastase (BenderMedSystems); and Asserachrom Thrombomodulin (Roche Diagnostics, Monza, Italy). Assays were performed according to manufacturers' instructions.
Allele-specific polymerase chain reaction for V617F JAK2 mutation
The V617F mutation was tested for, according to published procedures [13]. Briefly, DNA was extracted from the blood samples using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA). Polymerase chain reaction was performed to amplify the portion of the JAK2 region that contains the JAK2 V617F mutation. Two amplicon products, 203-bp for mutant samples and 364-bp for mutant and wild-type alleles, were generated as described previously [6]. The heterozygous or homozygous status of the mutant exon 12 of JAK2 was analyzed by a restriction enzyme–based assay, as described previously [6].
Statistical analysis
All results are expressed as mean ± SD. The difference analysis was performed using the Student's t-test for unpaired and paired data. A p value < 0.05 was considered significant. To evaluate differences in categorical variables, Chi-square and Fisher's exact tests were used. Next, the parameters found to be statistically significant between the V617F JAK2-positive and V617F JAK2-negative patients, were included in a multivariate analysis adjusted for age, gender, and concomitant therapy (hydroxyurea [HU] or aspirin) performed with Stata/SE 8.0 software (Stata, College Station, TX, USA). All probability values are two-tailed (p < 0.05).
Results
Clinical and hematological characteristics
A total of 75 patients with ET entered the study; 37 patients were heterozygous carriers of the acquired V617F JAK2 mutation and 38 patients were carriers of the JAK2 wild-type gene. As shown in Table 1, the whole ET patient group has significantly greater white blood cell (WBC), PMN, and platelet counts compared to the control group. Furthermore, WBC and PMN counts are significantly higher in the V617F JAK2 mutation group compared with the JAK2 wild-type carriers. No differences between the two ET subgroups were found in platelet count, hemoglobin, or hematocrit levels. At the time of the study, patients were receiving the following treatments: 26 patients (11 V617F JAK2 carriers; 15 JAK2 wild-type) were not receiving antiplatelet or cytoreductive therapies; 27 patients (14 V617F JAK2 carriers; 13 JAK2 wild-type) were on aspirin; and 22 (12 V617F JAK2 carriers; 10 JAK2 wild-type) were on HU.
Sixteen patients (21.3%) had a previous history of thrombosis; 11 of these were V617F JAK2 carriers (29.7%) and 5 were wild-type subjects (13.15%; p = NS). Previous thrombotic episodes included venous events (deep vein thrombosis, pulmonary embolism, superficial thrombophlebitis) in five JAK2 carriers and three JAK2 wild-type subjects and arterial events (e.g., transient ischemic attack, acute myocardial infarction, angina) in six JAK2 carriers and two JAK2 wild-type subjects.
PFA100 study
In the ET patient group, the overall mean closure times with ADP/collagen (110 ± 51 seconds) and EPI/collagen (215 ± 76 seconds) are significantly (p < 0.01) higher than the controls (ADP/collagen: 84 ± 15 seconds; EPI/collagen: 114 ± 26 seconds). Specifically, the ADP/collagen closure time is prolonged in 23% and the EPI/collagen in 66% of ET subjects, in agreement with other published data [23]. The EPI/collagen closure time is significantly affected for the majority of patients receiving ASA. No significant differences were found between V617F JAK2 carriers (ADP/collagen: 104 ± 36 seconds; EPI/collagen: 207 ± 80 seconds) and wild-type subjects (ADP/collagen: 116 ± 73 seconds; EPI/collagen: 222 ± 73 seconds).
Platelet surface glycoproteins and activation marker
Baseline condition
Cytofluorimetric analysis of TF, CD62P, CD41, and CD42b expressed on the platelet surface was conducted before (baseline) and after ADP/collagen and EPI/collagen stimulation in the PFA100 system. As shown in Figure 1, the baseline percentage of platelets expressing TF is significantly higher in samples from ET patients (42.9% ± 2.57%) compared to controls (27.7% ± 3.48%; p = 0.0019). Within ET patients, the V617F JAK2 carriers have significantly (p < 0.01) higher levels of TF-positive platelets (47.8% ± 3.57%) than the wild-type subjects (37.7% ± 3.56%). This difference remains significant also when the results are expressed as MFI ratio (5.9 ± 0.41 vs 4.63 ± 0.39 MFI ratio; p < 0.01). Similarly, the percentage of platelets expressing P-selectin (CD62P) is significantly increased in ET vs control samples (p < 0.001). However, no statistically significant differences in CD62P expression were found between the JAK2 mutation carriers compared to the JAK2 wild-type subjects. Finally, the surface levels of the adhesion glycoproteins CD41 and CD42b are significantly lower on platelets from ET patients compared to controls (p = 0.0001 for both), but no differences were recorded between the V617F JAK2 and wild-type JAK2 ET subjects.
After ADP/collagen and EPI/collagen stimulation
As shown in Figure 2, EPI/collagen stimulation significantly increases the percentage of platelets expressing TF in both control (p < 0.001) and ET subjects (p < 0.001), whereas ADP/collagen significantly increases TF expression in the controls (p < 0.05) but not in ET patients. The percentage of TF positive platelets after stimulation is significantly higher (p = 0.01) in the V617F JAK2 carriers (ADP/collagen: 51.1% ± 3.41%; EPI/collagen: 59.4% ± 3.32%) compared to the JAK2 wild-type (ADP/collagen: 41.3% ± 3.52%; EPI/collagen: 42% ± 3.96%).
A significant increase in the percentage of platelets expressing CD62P was induced by either the ADP/collagen or EPI/collagen stimuli in both ET and controls, with significantly higher levels in the control group vs ET patients. No significant differences were found in the levels of platelet CD62P between the two JAK2 patient subgroups.
Concerning the two adhesive glycoproteins, a significant increase in platelet CD41 levels occurs after stimulation by either ADP/collagen or EPI/collagen in in both healthy controls and ET patients, with ADP/collagen being a more potent stimulus than EPI/collagen in both groups. The CD41 levels on stimulated ET platelets were confirmed to be lower compared to controls. Differently from CD41, the expression of CD42b was little or not at all affected by either ADP/collagen or EPI/collagen stimulation in both controls and ET patients. No significant differences were detected in the response levels of either CD41 or CD42 between the V617F JAK2 mutation carriers and JAK2 wild-type subjects.
Total platelet TF antigen
A significantly greater TF-antigen level is present in platelets isolated from ET patients (97 ± 16 pg/mL) compared to controls (57 ± 19 pg/mL; p < 0.05). Furthermore, in patients carrying the V617F JAK2 mutation, the platelet TF level (132 ± 22 pg/mL) is significantly higher than that of both JAK2 wild-type carriers (59 ± 17 ng/mL; p < 0.01) and healthy control subjects (p < 0.01). No statistically significant differences occurred in platelet TF levels between JAK2 wild-type ET patients and controls.
Platelet-PMN aggregates
Significantly higher levels of both CD11b/CD41 and CD11b/CD62P mixed aggregates were measured in ET patients compared to controls. In addition, the levels of mixed cell aggregates are significantly more elevated in JAK2 mutation carriers as measured by both CD41 (p < 0.05) and CD62P (p < 0.05) positive PMNs (Fig. 3). No significant correlations were found between the WBC and PMN counts and the level of platelet/PMN aggregates (data not shown).
Analysis of PMN surface activation markers
PMN expression of CD14 (15% ± 1.4%; p = 0.01), TF (20% ± 1.5%; p = 0.03), CD11b (154 ± 7.5 MFI; p = 0.0001), and LAP (63% ± 2.1%; p = 0.001) is significantly increased for the overall ET patient compared to the control group (CD14: 10% ± 1.5%; TF: 14.8% ± 1.2%; CD11b: 89 ± 5.4 MFI; LAP: 38% ± 5.3%) (Fig. 4). For V617F JAK2 mutation carriers, PMNs express statistically significant greater levels of CD14 (p = 0.02) and LAP (p = 0.003) compared to JAK2 wild-type. No statistically significant differences were found in PMN membrane TF and CD11b between the JAK2 mutational status subgroups.
Plasma markers
Plasma levels of hemostatic parameters were measured to assess the hypercoagulable state of ET subjects. As shown in Table 2, the levels of hypercoagulation markers (i.e., F1+2, TAT and d-dimer), fibrinolytic proteins (PAI-1 and t-PA), PMN azurophil degranulation markers (i.e., elastase) and endothelial shed molecules (i.e., TM) are all significantly greater in the plasma of ET patients compared to healthy controls. However, no statistically significant differences were detected between the V617F JAK2 carriers and wild-type carriers with the exception of TM, which is significantly higher (p = 0.02) in the V617F JAK2 carrier group.
Statistical multivariate analysis
A multivariate analysis was performed to evaluate the effect of age, gender, and different therapies (i.e., HU, aspirin) on the parameters found statistically different in univariate analysis between JAK2 V617F-positive and -negative patients. A logistic conversion model was applied. The results show that increased WBC (p = 0.004) and PMN counts (p = 0.049), platelet TF (p = 0.010), PMN CD14 (p = 0.009), and LAP (p = 0.012) levels, plasma TM (p = 0.004) were significant predictors for the presence of V617F JAK2 mutation. Differently, high levels of PMN-platelet aggregates did not confirm significant after correction in the multivariate model (p = NS).
Discussion
The present study, involving 37 V617F JAK2 gene mutation-carriers vs 38 wild-type subjects with ET, was initiated to investigate whether the presence of the mutation changes the expression of molecules relevant to the activation of the hemostatic system, particularly procoagulant, proadhesive, and inflammatory molecules by platelets and PMN.
The results show that platelets from all 75 ET patients compared to controls express significantly higher membrane TF and P-selectin and lower CD41 and CD42b levels. Interestingly, TF, but not the other three molecules, is significantly more elevated in the V617F JAK2 carriers compared to wild-type subjects, both before and after stimulation of platelets with EPI/collagen and ADP/collagen.
This is the first report on TF measurement in platelets from ET patients. TF is a transmembrane receptor for FVII and is the main initiator of blood coagulation, which leads to thrombin generation and fibrin deposition. TF is not normally exposed to circulating blood, but may be produced by the endothelium and monocytes under pathological conditions. Aberrant TF expression within the vasculature initiates thrombosis in various diseases including sepsis, atherosclerosis, and cancer [24]. Different studies show that TF is present in platelets [25–27]. However, whether platelets produce their own TF [26] or take up TF-positive microparticles from leukocytes is still matter of debate [27]. Under the conditions of this experiment, platelet surface TF antigen increased after a short stimulation in vitro by the PFA-100 system. These experiments were conducted in whole blood; therefore, platelets may have either acquired TF from contact with leukocytes or may have released TF from their intracellular stores [26]. The increase in platelet surface TF in the JAK2 mutation carriers remained significant after multivariate analysis taking age, sex and treatment (i.e., HU and aspirin) into account. The observed increase of TF on the platelet surface measured by cytofluorimetric analysis was associated with a concomitant increase in the total antigen content as measured by ELISA in washed isolated platelets.
P-selectin is also increased on the surface of platelets from ET patients compared to healthy controls, in agreement with previous findings from our [12] and other groups [9,10,28] showing that platelets circulate in an activated state in ET patients. P-selectin is a component of the alpha-granule membrane of resting platelets, which is only expressed on the platelet surface upon activation and alpha-granule secretion [29]. It mediates the adhesion of activated platelets to monocytes and PMN through binding to P-selectin glycoprotein ligand-1 (PSGL-1) expressed by leukocytes. Signaling by P-selectin through its receptor PSGL-1 on leukocytes induces the generation of TF-positive, highly procoagulant microparticles [30,31]. The data in this study show that while the percentage of platelets expressing P-selectin is higher in ET patients compared to controls in basal conditions, upon stimulation with ADP/collagen and EPI/collagen, ET platelets are less responsive and express lower P-selectin than stimulated platelets from healthy controls, which was more evident after ADP/collagen stimulation. A similar dysfunction of ET and polycythemia vera platelets was reported by Jensen et al. [28], who found less P-selectin exposure following platelet activation by ADP or thrombin receptor activating peptide in ET and polycythemia vera patients than in controls.
In this study, no difference was observed in platelet P-selectin expression between JAK2 mutation carriers and wild-type subjects, either before or after stimulation. These results differ from those of Arellano-Rodrigo et al. [19], who found a significant association between the JAK2 mutation and increased baseline or arachidonic acid–induced platelet P-selectin expression. We have no definite explanations for this discrepancy, although one reason may reside in the larger number of patients analyzed in this study (i.e., 37 JAK2 mutation carriers vs 21, and 38 wild-type vs 29). Another reason may be related to the differences between the two study patient populations. In Arellano's study, 50% of patients had had thrombotic events, while in this study population a lower percentage of ET patients had a positive history of thrombosis (21%). Furthermore, the patients' hematologic characteristics were also different in Arellano's study. In that study, the overall ET group compared to the control group had a lower hemoglobin level, lower leukocyte and neutrophil counts, and a very high percentage of subjects on cytoreductive therapy (89.7%); whereas in this study ET patients had hemoglobin levels not different from controls, higher leukocyte and neutrophil counts and a lower percentage of subjects on cytoreductive therapy (29.3%). Concerning the V617F JAK2 mutation, only the hemoglobin level of V617F JAK2 patients was described in Arellano's article, and it was higher than that of the wild-type subjects, while in this article there was no difference. Finally, the patients in Arellano's study were older (median age, 68 years). All these characteristics clearly define two very different populations, which makes the direct comparison of results difficult.
Regarding platelet membrane CD41 and CD42b adhesion glycoproteins, this study shows that at baseline, platelets from ET patients have lower CD41 and CD42b expression. This reduced expression was also evident after ADP/collagen and EPI/collagen stimulation. No differences were found between the V617F JAK2 mutation and wild-type carriers. Platelet membrane glycoproteins serve a variety of functions, including receptors necessary for platelet adhesion to leukocytes, fibrin, endothelial cells, and extracellular matrix. In MPD patients, several abnormalities have been described in membrane glycoprotein content [32,33], as well as in the pattern of redistribution upon agonist stimulation [28]. However, the observed reduction in these adhesion receptors seems not to be so relevant to affect the interaction of platelets with PMNs.
This study confirmed the previous finding of increased levels of platelet/PMN aggregates (both CD41- and CD62P-positive PMN) circulating in the blood of all ET patients [9,10,12]. Furthermore, these data demonstrate that these aggregates are significantly greater in the V617F JAK2 mutation carriers than in the wild-type and control groups. Another study reports that JAK2 mutation carriers tend to possess increased levels of platelet-PMN complexes (evaluated as CD42-positive PMN) versus their wild type counterparts, although this difference is not significant [19].
Platelet/PMN mixed aggregates are generated from the interactions between platelets and PMNs, and are increased in several pathological conditions associated with a propensity to thrombosis. The activation status of both cell types is crucial to allow the cell–cell interaction. The evidence that both platelet and PMNs from JAK2 mutation carriers expressed increased activation features is in good agreement with the findings of increased mixed cell aggregate formation in ET patients carrying the V617F JAK2 mutation. Interaction between platelets and leukocytes is gaining an increasingly relevant role, as it favors the cellular activation, metabolic changes and transfer of microvesicles between the two cell types. Most importantly, TF-bearing microvesicles are indeed transferred during the inter-cellular trafficking between platelets and leukocytes [34–37]. Of interest to this discussion is the finding of increased TF content in platelets from V617F JAK2 mutation carriers, who also had increased circulating mixed cell aggregates.
PMN surface activation/inflammatory markers (i.e., CD14, TF, CD11b, and LAP) are all significantly higher in the whole ET group vs controls, and in particular, CD14 and LAP were demonstrated to be more highly expressed in JAK2 mutation carriers. The increase in surface PMN CD14 and LAP in the JAK2 mutation carriers remained significant even after multivariate analysis. CD14 is a myeloid receptor for bacterial cell membrane/wall components, which is strongly induced on the PMN surface during sepsis. This marker is sensitive to proinflammatory stimuli, and an increase in this receptor in ET patients carrying the JAK2 mutation further supports the presence of highly activated PMNs. This is also confirmed by the parallel significant increase of surface LAP levels in the same JAK2 mutation samples, and further supports the hypothesis that patients with MPD may have patterns of granulocyte activation very similar to those of healthy stem cell donors given granulocyte colony-stimulating factor [20]. No difference was found in CD11b expression between carriers and wild-type ET patients, as observed by others [19].
These data show increased expression of surface TF on PMNs of ET patients compared to controls. However, differently from platelets, no significant difference was observed in the levels of this protein between the two subgroups of ET patients. It is recognized that among leukocytes, the monocytic fraction produces and expresses TF upon stimulation by bacterial endotoxin or cytokines, although whether PMN are able to produce and express TF as well is still controversial [38–42]. In our experimental conditions (i.e., whole blood) PMN surface TF might either come from interactions with platelets or platelet/monocyte microparticles or be produced by PMNs. This is the first report of PMN surface TF evaluation in ET subjects carrying the V617F JAK2 mutation vs wild-type subjects. Others have evaluated monocyte surface TF without finding significant differences between JAK2 mutation carriers and wild-type [19].
Finally, a significant increase in plasma hypercoagulation markers was found in ET patients. This is in agreement with our previous study that also showed a correlation between thrombotic markers and markers of activated PMNs [11]. In comparing the levels of hypercoagulability parameters of JAK2-mutation carriers and wild-type patient, only TM is different between the two subgroups of ET patients. TM is an indirect ex vivo parameter of endothelial dysfunction [43], and an increase of plasma soluble TM level is reported in several conditions, such as coronary artery disease, disseminated intravascular coagulation, and atheromatous arterial disease. Membrane-bound TM is likely shed and released into the circulation by the action of leukocyte-derived proteases (elastase, myeloperoxidase). In ET patients carrying the JAK2 mutation, more elevated levels of TM are present compared to the wild-type and control subjects. The greatest PMN activation, which occurs in V617F JAK2 mutation carriers, could render ET patients more susceptible to vascular damage. The importance of elevated circulating levels of soluble TM in hemostasis guarantees further studies in these patients.
In conclusion, these data show that the V617F JAK2 mutation in ET influences specific patterns of hemostasis and inflammation, with increases noted in platelet expression of TF, platelet/PMN interactions, PMN expression of CD14 and LAP and plasma TM levels. All but platelet-PMN mixed aggregates remained significant predictors for V617F JAK2 mutation even after correction for confounding variables, including HU treatment. These findings link the JAK2 mutation to alterations of cellular (i.e., leukocytes and platelets) as well as plasma (i.e., TM) compartments of blood coagulation, providing new insights into the mechanisms of thrombosis in MPD and favoring the hypothesis of an increased hypercoagulable condition in V617F JAK2 carriers. Prospective studies are warranted to define the role of these abnormalities in predicting thrombotic events in ET patients.
Acknowledgments
This work was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) to A.F. and from the Myeloproliferative Disorders Research Consortium.
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