Tissue-Factor Bearing Microparticles and Thrombotic Risk in the Myeloproliferative Neoplasms

Thrombosis is a well recognized complication in the myeloproliferative neoplasms (MPN), essential thrombocytosis (ET), polycythemia vera (PV), and myelofibrosis (MF). Advanced age and prior thrombosis increase the risk of this complication; leukocytosis and the JAK2 V617F mutation or its allelic burden may also increase this risk. Yet mechanisms for thrombosis are not well-established, and the role of novel biomarkers to predict risk and/or recurrence is to be determined. Circulating cellular microparticles (MP) containing procoagulant tissue factor (TF) have been shown to correlate with thrombotic risk in many forms of cancer and cardiovascular diseases. To investigate the role of MP in the MPN, we studied 47 patients (ET=13; PV=18; MF=12; and MPN unclassifiable=4) and compared results to healthy subjects.

Citrated blood samples were collected from patients and controls. Platelet-poor plasma (PPP) was obtained by centrifuging at 1,500 G for 20 minutes. 100 μL of PPP was added to 1ml PBS (without Mg/Ca) and centrifuged at 20,000 G for 15 minutes. The sediment containing MP was resuspended in 100μL of buffer for labeling with TF, CD41a (platelets), CD14 (monocytes), PAI-1 and Annexin V. Following incubation, labeling buffer was added to the suspension to a volume of 1ml for flow cytometric analysis (LSR Fortessa, FlowJo software). Electronic triggering was done on side-scatter, and acquisition regions were defined based on sizing beads (0.3 to 1.0 micron) along with annexin A5 positivity. D-dimers were compared between patient and controls. Using MP sediment, thrombin generation (TGA) was assayed (Technothrombin TGA, DiaPharma), with results expressed as lag phase, velocity-index, peak thrombin, and area under the curve (AUC).

Among the MPN patients, 19 (40%) were male, the mean age was 60.5 years and the mean disease duration was 5.3 years. 34 (72%) patients were JAK2 V617F-positive, and 9 (19%) had a prior history of thrombosis. At the time of sample collection, 41 (87%) patients were on aspirin, 2 (4%) were on Coumadin, and 15 (32%) were on hydroxyurea. The median percentage of TF-bearing MP's was statistically similar in MPN patients compared to controls (N=15) (22.7% vs. 14.7%; p=0.16). When comparing MPN patients to controls, these TF-bearing MP were derived from CD41a (median 7.0% vs. 2.0%; p=0.009), reflecting platelet origins of the MPs in MPN patients. The median percentage of PAI-1 bearing MPs was statistically similar in MPN patients and controls (4.0% vs. 2.0%; p=0.074). The median d-dimer was statistically similar between MPN patients and controls (0.48 vs. 0.22 mcg/ml, p=0.089). Thrombin generation assays were performed in 46 MPN patients and 7 controls, with a shorter median lag phase (15.1 vs. 34.1 minutes; p=0.002), higher median velocity index (6.7 vs. 1.7; p=0.004), and greater median peak thrombin generation (63.2 nM vs. 23.2 nM; p=0.0050) in MPN patients compared to controls. The median AUC (1985.1 vs. 1419.2 p=0.16) was not statistically different. There were no significant differences in TF or PAI-bearing MPs, d-dimer, or TGA by JAK2 status nor were there significant differences when comparing within the MPN subgroups.

In this study, the proportion of TF-bearing MPs of platelet origin was higher in MPN patients, suggesting their possible role in thrombotic complications. However, we suspect that monocyte and myeloid lineage derived MPs may also play a role in thrombosis, but a lack of power prevented recognition of this finding. There were no differences in the proportion of PAI-1 bearing MPs or significant differences in the d-dimer between MPN patients and controls. The TF-bearing MPs in MPN patients appear functional, based on a shorter lag phase, higher velocity index, and greater peak thrombin generation. Future studies with prospective follow-up are indicated to determine the role of MPs and thrombin generation assays in predicting incident or recurrent thrombosis in the MPN.

No relevant conflicts of interest to declare.