RISK FACTORS IN THE DEVELOPMENT OF THROMBO-HEMORRHAGIC COMPLICATIONS IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS

DOI: 10.32471/oncology.2663-7928.t-22-1-2020-g.8672

Myeloproliferative Ph-negative neoplasia (MPN) — polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) are validated mainly at the age of 50–70 years with a benign and prolonged course of this pathology.

Due to excessive synthesis of cytokines and proteolytic enzymes with activation of IL6–JAK2–APRF–STAT signal cascade, regulating proliferation and differentiation of cells in increasing hypoxia, a specific metabolic microenvironment is formed.

A cell that has entered the path of differentiation inevitab­ly dies. Subsequent generations of cells synthesize their own proteins, some of which enter in the intracellular matrix. The glycan components of proteins are the most sensitive to changes in the composition of the cellular genome [1]. The specificity of the structure of glycans is determined by the microheterogeneity of proteins and is due to the presence of several molecular forms that differ in the composition of carbohydrate chains and functional activity. The appearance of atypical branched glycans is considered to be early specific biochemical markers of development of malignant diseases [2].

The intercellular matrix is a complicated complex of macromolecules, collagen proteins, proteoglycans and glycoproteins. In the biochemical aspect, a high degree of organization and ordering of the intercellular matrix is expressed by specific quantitative ratios of the biopolymers forming it. Any deviations from these specific ratios cannot but entail disorders in the structure and functions of the connective tissue. A highly ordered three-dimensional structure of the matrix, which includes type IV collagen — a protein from the group of glycoproteins is formed by the interaction of glycoprotein binding centers with adhesion proteins — laminin, fibronectin (FN) and integrins. The nature of protein-carbohydrate bonds determines the timing of cell attachment to the intracellular matrix [3].

Collagen is synthesized by fibroblasts: translation and post-translational modification of polypeptide chains occur inside the cell, and extracellularly collagen «matures» — protein modification is completed with the participation of FN with the formation of collagen fibers [4].

Collagen type IV contains 1α₁ (IV) and 2α₂ (IV) chains. After secretion by cells, they do not undergo proteolytic modification and therefore retain the structure of the N- and C-terminal globular domains (NC₁, 7S and NC₂). If intracellular protein synthesis is disturbed, oligosaccharide part is absent in them, this leads to increase in the activity of proteolysis processes — the leading post-translational mechanism for monitoring the state of extracellular matrix (ECM) [5].

FN is formed in hepatocytes, fibroblasts and neutrophilic granulocytes already at the stage of early myeloid maturation [6]. Increase in the adhesive ability of leukocytes in MPN patients due to a significant stiffness and impairment of the elasticity of the inner membrane [7] probably results from structural-functional distur­bances of glycane components of anomalous cells.

In the process of adhesion a FN molecule with one domain binds to the membrane receptor in with an­other one — with the corresponding centers on collagen molecules in the ECM. In ECM, multimers «fibronectin + platelet receptor GPIc-IIa + collagen» are located around collagen fibers and endothelial cells [8]. The carbohydrate component of FN makes up 25% of the whole molecule. In a state of physiological norm, the structure of glycans of cellular (leukocyte) FN is represented by O-glycans and bi- or tri-antenna N-glycans (asparagine N-residues containing core fucose.

Protease-resistant domains of FN ligands have sites of binding with heparin, fibrinogen, collagen, cell surface receptors. In the plasma FN is present in several forms: native — in the form of dimers, aggregated — consisting of multimers and complexes, degraded — represented by fragments formed during proteolysis [9, 10].

In MPN patients a significant decrease in the functional activity of FN with a decrease of its content in blood plasma and an increase in the level and activity of matrix metalloproteinase (MMP)-9 was established, which is interrelated — in the process of degradation of  FN under the action of trypsin and chymotrypsin, the amount of fFN with m.m. 165 kDa and 58 kDa in­creases, namely they activate the synthesis of MMP-9 in neutrophils [11]. A disorder in the synthesis of fibronectin, especially in the progression of PMF occurs due to a defect structure of hepatocytes, the main source of plasma FN synthesis; at a leukocyte level of more than 20•10⁹/l, functional activity of FN decrease significantly and the processes of degradation are enhanced due to the impairment of the structural features of its glycans (increased core fucosylation rate in their significant branching) and a significant increase in MMP activity, which proves the role of neutrophils of varying degree of maturity, including granulocytes of a pathological clone, in the synthesis of FN with an anomalous structure of glycans (branched hypersialized) and hyperproduction of MMP-9. It is the specific features of FN neutrophils (including hydrolyzed), the degree of their activation, migratory abilities and stimulation of adhesion of thrombocytes to collagen is determined [12]. Activation of proteolysis processes leads to disorder of adhesion processes — «cell-matrix».

The participation of C-reactive protein (CRP) in the expression of adhesive molecules, including FN, enhances the migration of leukocytes from the vasculature to the tissue and changes the transmission route of signals from receptors in the sequence: IL-1/TNFα/IL-6 → Ras → MAP kinase → NF/IL-6. Catabolism of cellular proteins and intracellular matrix is performed with participation of MMP. Expression of MMP is similar to the expression of acute phase proteins.

Under physiological conditions MMP are synthesized episodically in the form of non-glycosylated proenzymes on a specific area of the cellular membrane of fibroblasts, phagocytes, epithelial cells and lymphocytes. The source of MMP-9 synthesis is tertiary peroxidase-negative granules of activated myeloid cells — stab and segmentonuclear neutrophils in the form of pro­MMP­-9 with m.m. 78.4 kDa. During subsequent glycosylation in the Golgi apparatus, a proenzyme with m.m. 92 kDa — MMP-9 is formed and secreted. Due to the presence of an additional site identical to fibronectin in the catalytic domain of the proenzyme, MMP is ensured with a high affinity to membrane components [10]. In tumor cells in a state of hypoxia, MMP are continuously synthesized by the entire surface of the membrane with the involvement of Src tyrosine kinase in the process [13].

Variations concomitant to JAK2 mutations in 16 chromosome which encodes synthesis of metalloproteinases, bring epigenetic abnormalities in transcriptional regulation [14] and change ECM structure [15].

With the simultaneous exposure to several proteinases synthesized by tumor and/or stromal cells, disorganization­ of the ECM progresses [10, 16]. Activation of enzymes also occurs in response to changes in the state of components of the ECM itself [17–19]. Strengthening of synthesis and increase in activity of MMP, especially MMP-9 [20] contributes to the progression of stromal-vascular dystrophy with the formation of fibrosis in patients.

Elevated level and high activity of MMP and especially of MMP-9 contributes to progression of stromal-vascular dystrophy with formation of fibrotic changes in the bone marrow.

The impact of cytokine, MMP-2 and MMP-9 on FN molecule containing atypical glycans leads to their enhanced cascade degradation. FN and MMP are components of the ECM: FN provides cell adhesion to the substrate and opsonization of foreign material [21, 22], and MMP by means of proteolysis changes the structure of microenvironment proteins (collagen, fibronectin, laminin, and glycoproteins of the basement membrane) with matrix remodeling [23].

Therefore, presence of defective glycans in the composition of FN molecule as the most sensitive markers for myelofibrosis development and in the following of myeloproliferative syndrome, as well as products of their degradation, as markers of thrombo-hemorrhagic complications (THC), may be the earliest criteria when making a diagnosis and predicting the development of the leukemic process and defining risks of THC [24, 25].

In MPN patients an increased content of cellular elements with the impairment of the rheological properties of blood is followed by impairment of thomboresistance of capillaries. Rheologic disorders are caused by and enhanced spontaneous platelet aggregation and adhesion of leukocytes with formation of cellular conglomerates — phenomenon of leukocyte–platelet aggregation is realized. In disorders of endothelial-leukocyte interaction synthesis of MMP-2 and MMP-9 is enhanced with the increase in their activity with involvement of «acute phase» proteins (CRP), adhesive proteins (FN), and highly glycosylated alpha-acid glycoprotein (AAGP).

AAGP consists of five complex-type N-glycans­ with a different number of antennas; the biological activity of glycan «antennas» is determined by negatively charged sialic acids (12% of the molecule) and fucose components [26]. In conditions of physiological norm, the carbohydrate determinants of AAGP contain bi- and triantenic­ glycans of a complex type [27]. Cytokines (TNFα and IL-6) initiate the synthesis of AAGP with m.m. 42 kDa by hepatocytes. The source of alternative synthesis of AGP are polymorphonuclear neutrophils; its m.m. 50–64 kDa, and the structure is represented by a hypersalinated peptide glycoform with fucosylated glycans and polylactamase complexes.

Membrane glycoproteins of platelets, leukocytes, and endothelium belong to the products of gene families of receptors and have a similar structure. They determine the processes of proliferation, differentiation, intercellular contacts, and cell migration.

Taking into account specific features of formation of the stromal microenvironment of the bone marrow in the process of myelofibrosis development with subsequent myeloid metaplasia, complicated with vascular events, we judge it expedient to study the problem of THC in MPN in the aspect of membrane components of cellular elements at the level of glycans as the earliest markers of pathologic processe, including leukemic ones.

Aim: to determine the state of disintegration processes in the system of hemostasis in the formation of thrombotic complications in CNP patients, the development of new algorithms for the diagnosis of thrombotic conditions and antithrombotic prevention.

MATERIALS AND METHODS

Our sample included 120 patients with MPN (men — 66, women — 54) aged 22–75 years (average 60.1 ± 1.1 years), with established diagnoses: in 33 (27.5%) — PV; 78 (65.0%) — PMF; 9 (7.5%) — ET; 38 (31.7%) patients developed thrombohemorrhagic complications in the form of acute cerebrovascular accidents, myocardial infarction, episodes of thrombosis of arteries of the lower extremities. In the comparison group there were 95 patients with manifestations of lower limb ischemia of atherosclerotic origin (men — 70, women — 25) aged 55–75 years (average 63.4 ± 0.88 years), in 38 (40.0%) atherothrombosis has developed.

The examination of patients was carried out using modern methods: general-clinical, morphological, cytological, biochemical, hemostasiological in compliance with international standards according to the recommendations of [28], as well as specific methods for determining the level, structure and functional activity of AAGP, FN, fibronectin fragmentation (fFN), MMP and their glycan component.

The concentration of AAGP in plasma was determined by the method of rocket immunoelectrophoresis (RIEP), the lectin-binding ability of AAGP — by cross affinity immunoelectrophoresis (CAIEP) using lectins of different specificity [2].

Isolation of AAGP from human blood serum was performed in two ways: by hydrophobic extraction with phenol and by immunoaffinity chromatography on agarose with immobilized antibodies to this glycoprotein. Fractions enriched with AAGP were tested using immunodot and immunoblot methods. The concentration of FN was determined by immunodot analysis using polyclonal rabbit antibodies to FN and secondary antibodies conjugated with horseradish peroxidase (Bio-Rad, USA). GelProAnalyser 32 software was used to quantify the results of the analysis [29].

The functional activity of FN was determined according to the degree of its binding to heparin by the method of cold heparin precipitation. Proteolytic cleavage of FN in vitro was performed by incubating the commercial preparation of FN (Sigma, USA) with trypsin (3.4.21.4) (Sigma, USA), chymotrypsin (3.4.21.1) (Sigma, USA), collagenase (3.4.24.3) (Boehringer Mannheim, Nimechchina), MMP-2 (3.4.24.24) (Sigma, USA), MMP-9 (3.4.24.35) (Sigma, USA), thrombin (3.4.21.5) («Renam», Russia).

FN fragmentation was studied using electrophoresis in density gradient of 5–17.5% of polyacrylamide gel, using Laemmli method [30] and western blot analysis [31].

The microheterogeneity of AAGP and FN was determined by lectin-enzyme analysis using de-glycosylated with N-Glycosidase F (US Biological, USA) antibodies to this protein [32].

The conjugation of lectins with horseradish root peroxidase and lectin blot analysis was performed according to the recommendations of [33].

Determination of gelatinase activity (MMP-2 and MMP-9) in blood plasma was performed by gelatin-zymography with preliminary vertical electrophoresis of samples in 7.5% PAAG in the presence of 0.1% SDS and 1.0% gelatin (Sigma, USA).

Statistical processing of the research results was carried out using biostatistics methods, STATISTICA v.6.1 (license number AJAR909E415822FA). To build mathematical models for assessing the likelihood of developing thrombotic complications, we used the logistic function: P(y) = 1/(1+exp{-­­ y})­, (y) is a linear combination of prognostically significant thrombus formation factors.

RESULTS AND DISCUSSION

The analysis of hemograms during the initial examination of patients showed that in MPN the level of cellular elements (erythrocytes, leukocytes and platelets) in relation to the group of patients with atherosclerotic lesions of the vessels of the lower extremities was significantly higher: in ET there was noted a significant increase in platelet levels (by 3.9 times; p < 0.001), in PMF — leukocytes and granulocytes (by 2.3 and 1.4 times, p < 0.001, respectively), in PV — the level of red blood cells and hemoglobin — by 1.6 and 1.3 times, p < 0.001 (Table 1).

Note: ¹р < 0.05; ²р < 0.001 relative the to group of comparison.

In trepanobioptates and aspirates of the bone marrow, exemplified by patients with PV, there was revealed an increased content of megakaryocytes and myeloid cells of various maturity and sizes with single atypical forms. The adipose tissue content was reduced; in the perivascular zones of the bone marrow there was determined the proliferation of fibroreticular elements with structures of framework spaces replaced by them.

Clinical evaluation of MPN patients revealed manifestations of plethoric syndrome in 59 (49.2%) patients, including 42.3% of patients with PMF and 78.8% of patients with PV. Splenomegaly was noted­ in 51 (42.5%) cases, of which 53.8% with PMF, 15.2% with PV and 4 of 9 (44.4%) with ET. Manifestations of hepatomegaly were noted in every third patient with MPN (38 patients — 31.7%), mainly in PMF (34 patients — 43.6%).

The trigger mechanism in the processes of thrombus formation is an increase in platelet activity [1]. In our studies, indicators of spontaneous platelet aggregation (39.96 ± 0.95%) and ADP-induced aggregation (66.12 ± 1.43%) in PV; ristomycin-induced platelet aggregation (77.46 ± 2.32% and 77.21 ± 2.03%), blood clot retraction (61.22 ± 1.17% and 58.41 ± 1.33%) in patients with PMF and ET significantly exceeded the corresponding findings of patients with atherosclerotic process (Table 2).

Note: ¹p < 0.05; ²p < 0.01; ³p < 0.001 relative to the comparison group; «+» — degree of reaction.

According to the results obtained, findings of spontaneous platelet aggregation (39.96 ± 0.95%) and ADP-induced aggregation (66.12 ± 1.43%) in PV; ristomycin-induced platelet aggregation (77.46 ± 2.32% and 77.21 ± 2.03%) in patients with PMF and ET significantly differed from the corresponding findings in patients with the atherosclerotic process, which indicates severe microcirculatory disorders due to the features of cell composition, especially with the level of leukocytes being more than 20 •10⁹/l.

In the study of routine laboratory findings, mathematical models for assessing the likelihood of developing thrombotic complications in MPN patients are proposed, with the inclusion of a set of hemogram and hemostasiogram findings in the diagnostic algorithm.

In patients with MPN and atherosclerotic vascular lesions, fragments with a small and medium molecular weight were included in the wide range of fFN. Herewith, in patients of the main groups with decrease in the content of FN fragments with m.m. 175–160 and 155–150 kDa by 50% or more, the content of fFN with m.m. 98–90 (from 60% in PV to 87% in PMF) was significantly increased, which correlates with a high risk of thrombotic complications development.

Quantitative and qualitative composition of α-acid glycoprotein in MPN. In MPN patients in composition of AAGP, protein with m.m. 42 kDa (plasma) synthesized by hepatocytes prevailed; in patients with atherothrombosis and with vascular complications in patients with PV, peptides with m.m. 42 and 68 kDa were revealed; in PMF AAGP with m.m. 68, 84 and 126 kDa, due to the existence of additional sources of its synthesis, including leukemic clone cells was revealed.

The structure of glycans of AAGP of neutrophilic origin­ is different from those synthesized by hepatocytes. In MPN patients as part of AAGP with m.m. 84 and 126 kDa glycans contain residues of N-acetylneuraminic acid located in positions 2 → 6 and 1 → 3, in atherothrombosis, sialic acid was located in glycan in position 2 → 6. Activated neutrophils were the source of AAGP synthesis in both cases, but in MPN neutrophils were of varying maturity, which may be an additional criterion for the diagnosis of myeloproliferative neoplasms.

The lectin-binding activity of FN indicates that the glycans consisted of branched structural components, but the functional activity, which depends on the degree of glycosylation, is significantly different (Table 3).

Note: ¹p < 0.05; ²p < 0.01; ³p < 0.001 relative to the norm; ↓ — decrease; ↑ — increase; ↑↑↑ — significant increase; N — normal.

LАВА — laburnum anagyroides agglutinin; ААL — aleuria aurantia lectin; LСА — lentil lectin; WGА — wheat germ agglutinin; SNА — black elderberry; МАА — mannose binding lectin.

In MPN patients low plasma FN with a decrease in FA is due to differences in its glycan component (the presence of terminal fucose as a part of O-glycans and core fucose as a part of F-N-glycans) with increased degradation with the formation of fFN in the range of m.m. from 200–19 kDa.

Incubation of FN with MMP-2 and MMP-9 showed a significant increase in the activity of gelatinases in MPN patients compared with the norm, especially in the complicated course of the disease (Table 4), which also indicates the involvement of activated leukocytes of varying maturity in the proteolysis processes, including neutrophils of a pathological clone. MMP-2 was determined in active form, and MMP-9 was also present in the form of zymogen (pro-MMP-9).

Note: ¹p < 0.05; ²p < 0.01; ³p < 0.001 relative to the control.

In the serum of all patients which have undergone thrombotic complications, there was a significant increase in the content of fFN with m.m. 98–90 kDa ≥500% with a high total activity of MMP-2 and MMP-9 ≥200%, which should be considered as markers of vascular catastrophes.

Mutation V617F of JAK2 gene was detected in 16 of 24 patients with MPN (66.7%), of which 11 cases (68.8%) had a history of vascular complications — in 3 out of 4 patients with ET, in 5 out of 7 patients with PMF, in 3 out of 5 patients with PV. In 6 cases, episodes of vascular catastrophes repeated. In ailments in chronic kidney disease in the presence of a JAK2 mutation, a significant increase in the number of red blood cells, white blood cells and platelets in peripheral blood was established, as well as a significant increase in the level of LDH and MMP-9 with a decrease in the functional activity of FN (Table 5).

Note: ¹p < 0.05 in comparison with PMF/ET patients with JAK2 mutation; ²p < 0.05 in comparison with PV patients with JAK2 mutation.

It is advisable for patients with an increased risk of developing thrombohemorrhagic complications in MPN to undergo elective courses of inpatient treatment to correct overall health against the background of cytoreductive therapy.

According to the results of the correlation analysis, it has been established that a direct correlation between the increased number of platelets and leukocytes in MPN patients (r = 0.476; p < 0.001) is a negative prognostic factor in the progression of myeloproliferative syndrome.

Thus, we monitored the mechanisms of the formation and realization of THC based on the assessment of the cellular and coagulation components of the blood composition of MPN patients and atherosclerotic vascular lesions (as a comparison group) with an in-depth study of the composition of glycans in patients’ blood serum samples.

Thus, informative criteria for the likelihood of developing thrombotic and hemorrhagic events were established in MPN patients, and groups of increased risk of their development were formed, a mathematical model  was compiled taking into account the levels of hemoglobin, leukocytes, spontaneous and ristomycin-induced platelet aggregation.

Additional, earlier, criteria specifying the mechanisms of the pathogenesis of the development of THC are: increased activity of MMP-2 and MMP-9 with enhanced fibronectin degradation in the presence of fFN with m.m. 90–98 kDa ≥ 50% of the total amount of fFN; a high level of AAGP, due to an increase in the level of biennial glycans, the appearance of AAGP components with m.m. 126 and 84 kDa.

In the comparison group, in ischemic damage to the main vessels of the lower extremities of atherosclerotic genesis, significant risk criteria for the development of vascular events were high cholesterol, indicators of spontaneous and AAGP-induced platelet aggregation. The above changes were due to increased levels of AAGP, FN, increased functional activity of fibronectin, MMP-2 and MMP-9, a high degree of degradation of FN under the influence of MMP-2 and MMP-9 with a content of ffn with m.m. 90–98 kDa ≥ 50% of the total amount of fFN.

Considering the gender factor in comorbidity with cardiovascular pathology (metabolic syndrome, hypertension, diabetes mellitus, ischemic heart disease), the risk of developing fatal outcomes is significantly increased. Taking into account the above factors, considering the individual characteristics of patients, will allow for an adequate assessment of risk factors in the development of THC in patients in cases of comorbid conditions, and therefore, to prevent them.

Since the mathematical model of the risk of developing THC in MPN patients and atherosclerotic vascular damage is compiled on the basis of routine findings of a general blood test and a hemostasiogram, it is possible to use it by family practitioners at the primary care level in order to early identify the high-risk population for possible THC and timely the administration of adequate special and preventive therapy.

An in-depth study of the glycan components of the blood serum of patients made it possible to clarify the sources of synthesis of the main components that affect the mechanisms of development of THC and to determine the leading role and sequence of action of activated cells — neutrophils and platelets in the initiation of thrombosis mechanisms in MPN and atherosclerotic­ process.

The regularity and sequence of pathological changes in MPN patients allows timely and purposeful correction of possible disorders in the hemostasis system by the integrated administration of hydroxyurea, interferon, disaggregants (ASA, clopidogrel, curantyl) in combination with drugs that improve the rheological and metabolic characteristics of the blood.

CONCLUSIONS

1. Identification of the probable risk factors for the development of vascular complications in patients of the study groups using a mathematical model with a set of laboratory screening indicators (in MPN patients a high level of platelets, hemoglobin with increased spontaneous platelet aggregation was determined) may precede clinical manifestations of the disease and its complications.

2. The practical use of the laboratory diagnostic algorithm as an early marker for development of possible vascular complications will allow rationally and timely (in the early stages) to identify patient population that requiresthe treatment in specialized outpatient oncohematology offices and the administration of hydroxyurea and/or interferon agents in, and administration of disaggregants and agents for microcirculation improvement.

3. A study of the composition of glycans in the blood serum of patients made it possible to clarify the sources­ of synthesis of the main components that affect the mechanisms of development of THC in MPN and to determine the leading role and sequence of action of activated cells-neutrophils and platelets in the initiation of blood clotting mechanisms.

4. Progressing fibrosis formation in patients with MPN in the presence of concominant genetic deviations (mutation V617F in the JAK2 gene) contributes to the initiation of «defective» clones of white blood cells, platelets, red blood cells, which ultimately may transform into a leukemic process.

5. Patients with manifestations of microcirculatory disorders and corresponding changes in laboratory parameters require a more thorough prognostic assessment, constant monitoring of clinical and laboratory status with correction of administrations.

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Correspondence:
V.G. Bebeshko
SI «National Research Center for Radiation Medicine
of National Academy of Medical Sciences of Ukraine»
04050, Kyiv, Yu Illienka St., 53
E-mail: vg.bebeshko@gmail.com

Submitted: 07.05.2020