The study included 20 newly diagnosed patients with MM (range 31–78, 45% males) according to the standards of the International Myeloma Working Group who were admitted to the First Affiliated Hospital of Harbin Medical University between October 2016 and February 2017, and 15 healthy volunteers (range 28–75, 46.67% males) (22). The exclusion criteria were a recent (<6 months) thrombotic event, current anticoagulant therapy, and associated disease, including antiphospholipid syndrome, chronic renal disease, heart disease, malignant or systemic disease, diabetes, acute infection, immobilization, surgery, hereditary thrombophilia and hyperviscosity, among other conditions. Harbin Medical University Ethical Committee (Harbin, China) approved the study, and patients provided written, informed consent.

Thalidomide (Tha), lenalidomide (Len) and Dex were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Human umbilical vein cells (HUVECs), ECs medium, and poly-L-lysine were from ScienCell Research Laboratories, Inc. (San Diego, CA, USA). RPMI-1640 medium, fetal bovine serum (FBS) and bovine serum albumin (BSA) were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Alexa Fluor 488 or Alexa Fluor 647-conjugated lactadherin were prepared in our laboratory. Human factors (F) Va, VIIa, VIII, IXa, X, Xa, prothrombin, thrombin, fluorescein EGR-chloromethylketone and biotinylated EGR-chloromethylketone were all from Haematologic Technologies, Inc. (Burlington, VT, USA). Mouse anti-fibrin II chain (cat. no. NYBT2G1) was from Accurate Chemical & Scientific Corporation (Westbury, NY, USA). Isotype control antibody (cat. no. X0931) was from Dako (Agilent Technologies, Inc., Santa Clara, CA, USA). Fluorescein-maleimide, fluorescein isothiocyanatephalloidin, DAPI, Alexa 647-labeled isotype-matched control antibody (cat. no. MA5-18168) were from Molecular Probes (Invitrogen; Thermo Fisher Scientific, Inc.). Propidium iodide (PI) was obtained from Shanghai Dobio Biotech Co., Ltd. (Shanghai, China). Chromogenic substrates S-2765 and S-2238 were from Diapharma Group, Inc. (West Chester, OH, USA). Percoll was from GE Healthcare Life Sciences (Uppsala, Sweden). Tyrode’s buffer containing 1 mM HEPES was prepared in our laboratory and filtered through a 0.22-mm syringe filter from EMD Millipore (Billerica, MA, USA).

Lactadherin was purified from bovine milk and labeled with Alexa Fluor 647 or Alexa Fluor 488 as described previously (12). The ratio of fluorescein to lactadherin was 1.2/1 or 1.1/1.

Blood was drawn prior to therapy with a 21-gauge needle and was collected into an anticoagulant tube containing 3.2% citrate (5 ml; BD Biosciences, San Jose, CA, USA). Blood cell isolation was performed ≤30 min post-blood collection. Following centrifuging at 200 × g for 15 min at 20°C, platelet-rich plasma (PRP) was aspirated carefully from the upper layer and erythrocytes were collected from the bottom layer of blood samples. Mixed peripheral blood leukocytes were isolated from the blood of study subjects using gradient centrifugation with 30% percoll and 68% percoll, according to the manufacturer’s protocol. Erythrocytes, PRP, and leukocytes were analyzed immediately by flow cytometry and confocal microscopy following isolation. To prepare platelet-free plasma (PFP), PRP was centrifuged for 20 min at 1,500 × g at room temperature. For microparticle-depleted plasma (MDP) preparation, 600 μl of PFP was centrifuged for 30 min at 20,000 × g at 20°C. The supernatant (400 μl) was collected, snap-frozen in liquid nitrogen, and then stored at −80°C prior to use.

Tha and Len were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) and Dex was resuspended in 1X PBS. The drugs were stored at −20°C prior to use. Tha or Len and Dex were diluted in culture medium to reach the final concentrations of 1.0 and 10 μM, respectively. The final concentration of DMSO was 0.01% in all experiments as preliminary experiments had demonstrated that DMSO concentrations of 0.01% had no significant effect on PS exposure on cells (data not shown).

HUVECs were maintained in EC medium in poly-L-lysine-coated cell culture flasks at 37°C and 5% CO₂ in a humid environment. HUVECs were incubated in growth media containing 20% pooled serum obtained from patients with MM or healthy subjects at room temperature for 24 h. Serum-cultured ECs were then treated with DMSO (0.01%), Tal (1.0 μM), Len (1.0 μM), Tal (1.0 μM)/Dex (10 μM), or Len (1.0 μM)/Dex (10 μM) at room temperature for 24 h. Cells in the logarithmic growth phase were used for all experiments. All cell culture results presented are based on at least three individual experiments.

Erythrocytes, platelets and leukocytes from patients with MM were cultured separately with DMSO (0.01%), Tal (1.0 μM), Len (1.0 μM), Tal (1.0 μM)/Dex (10 μM), or Len (1.0 μM)/Dex (10 μM) in a 5% CO₂ atmosphere at 37°C. Erythrocytes (10⁶/ml) were incubated in vitro for 24 h at a hematocrit of 0.4% in Ringer’s solution (23). Platelets (10⁶/ml) were suspended in Tyrode’s buffer for 1 h (24). Leukocytes (10⁶/ml) were incubated for 24 h in RPMI-1640 medium containing 10% FBS (25). Erythrocytes, platelets and leukocytes were washed twice before detection.

To quantify PS exposure on blood cells and ECs, 5 μl of each cell suspension (0.5–1×10⁶/ml) in Tyrode’s buffer was incubated with 5 μl Alexa Fluor 488-conjugated lactadherin for 15 min at room temperature in the dark. A total of 10,000 events per sample were acquired by flow cytometry and analyzed with BD FACSDiva software 6.0 (BD Biosciences).

PCA of various cell types was evaluated using a one-stage recalcification time assay in a KC4A-coagulometer (Amelung; Labcon GmbH, Heppenheim, Germany). Cell suspensions [100 μl of erythrocyte (1×10⁸), platelet (1×10⁷), leukocyte (1×10⁶), or EC (1×10⁶)] were incubated with 100 μl MDP from healthy volunteers at 37°C. After 3 min, 100 μl of warmed 25 mM CaCl₂ was added to start the reaction, and the time to subsequent fibrin strand formation was recorded. All clotting assays were performed in triplicate. For inhibition assays, cells were preincubated with lactadherin (final concentration 128 nM) prior to the assay.

For the intrinsic Xa formation, a total of 10⁴ platelets/leukocytes/ECs or 10⁵ erythrocytes were incubated with 1 nM factor IXa, 5 nM factor VIII, 0.2 nM thrombin, 130 nM factor X, and 5 mM CaCl₂ in factor Xa buffer (TBS with 0.2% BSA) for 5 min at 20°C. The reaction was stopped by the addition of EDTA (7 mM final concentration). Immediately following the addition of 10 μl S-2765 (0.8 mM) chromogenic substrate, factor Xa generation was determined using a kinetic absorbance reading at 405 nm on a SpectraMax M5 Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The assay to measure formation of extrinsic factor Xa was analogous to that for intrinsic factor Xa except that cells were mixed with 1 nM factor VIIa, 130 nM factor X and 5 mM CaCl₂. For the prothrombinase assay, the samples were incubated with 1 nM factor Va, 2 nM factor Xa, 1 μM prothrombin and 5 mM CaCl₂ in prothrombinase buffer (TBS with 0.05% BSA) for 5 min at 25°C. Thrombin production was evaluated after the addition of EDTA using the chromogenic substrate S-2238 (0.8 mM). For the inhibition assays of intrinsic/extrinsic factor Xa and prothrombinase formation, cells suspensions were pre-incubated with lactadherin (final concentration 128 nM) for 10 min at 25°C in Tyrode’s buffer. The mixture was then incubated with the specified clotting factors according to the above protocols. The quantity of thrombin or factor Xa formation was assessed as previously described (12).

Fibrin formation was quantified by turbidity as described (26). For assays in normal human MDP, blood cells and cultured ECs were added to recalcified (10 mM, final) normal human MDP (88% MDP, final) in the absence or presence of lactadherin (final concentration 128 nM). Fibrin production was measured by turbidity at 405 nm in a SpectraMax 340PC plate reader (Molecular Devices).

To locate PS, erythrocytes, platelets or leukocytes were incubated with Alexa Fluor 488-lactadherin (final concentration 4 nM) or PI, and ECs were incubated with the final concentrations of 4 nM Alexa Fluor 488-lactadherin and Alexa Fluor 647-CD31 [purified CD31, (clone L133.1), BD Biosciences)] for 10 min at room temperature in the dark. Then, cells were washed to remove unbound dye and imaged immediately. Observation of PS exposure on cells by confocal microscopy was performed as previously described (12). For fibrin generation experiments in vitro, ECs were cultured on 1% gelatin-coated coverslips in media containing 20% normal or patient serum for 24 h. Following rinsing with Tyrode’s buffer, the ECs were then overlaid with prewarmed MDP (15%) in the presence of 3 mM calcium. Fibrin networks were imaged using laser confocal microscopy in the presence of Alexa Fluor 647-conjugated anti-fibrin (final concentration, 1 μg/ml; prepared in our laboratory) at 37°C for 15 min. Background signal was calculated using a similarly labeled isotype matched control antibody (final 1 μg/ml) at 37°C for 15 min. To observe the location of coagulation factor binding sites, the ECs were co-stained with factor Va-fluorescein-maleimide (final concentration, 2 nM) and factor Xa-EGRck-biotin (final concentration, 2 nM, complexed to Alexa Fluor 647-streptavidin) at 37°C for 10 min. Samples were excited with 488 or 568 nm emission lines of a krypton-argon laser and narrow bandpass filters were used for restricting emission wavelength overlap. Images were obtained using the LSM 510 system (Carl Zeiss AG, Oberkochen, Germany).

Results are presented as mean ± standard deviation of at least triplicate measurements. Statistical analysis was performed with Student’s t-test or one-way analysis of variance followed by Fisher’s least significant difference post hoc tests for multiple comparisons as appropriate. P<0.05 was considered to indicate a statistically significant difference.

The characteristics of the study participants (20 patients with MM and 15 healthy subjects) are detailed in Table I. Patients with MM had a high serum concentration of M-component. Compared with healthy subjects, patients with MM had significantly higher levels of β2-microglobulin, fibrinogen, D-dimer, von Willebrand factor and factor VIII and shortened prothrombin time. However, patients with MM had lower levels of erythrocytes and hemoglobin than healthy subjects.

As lactadherin binds to PS with high affinity (21), fluorescence-labeled was used lactadherin to detect the level of PS exposure on various blood cells using a flow cytometer. Patients with MM had a significantly higher percentage of PS⁺ blood cells than healthy subjects (RBC, 1.12±0.58 vs. 0.54±0.29%; PLT, 10.24±3.37 vs. 2.35±1.10%; WBC, 19.26±2.55 vs. 5.46±1.41%; P<0.05 for all cell types; Fig. 1A). The percentage of PS⁺ ECs after treatment with patient serum was 4.30-fold higher than after treatment with healthy plasma (P<0.05; Fig. 1B). The absolute number of PS⁺ blood cells was also significantly higher in patients with MM compared with healthy subjects (P<0.05, for all; Table II). To observe PS binding on the outer membrane of cells, erythrocytes, platelets, or leukocytes were incubated with Alexa Fluor 488-lactadherin and PI, and imaged using confocal microscopy. Alexa Fluor 488-lactadherin staining was not observed on the membranes of platelets, erythrocytes and leukocytes from healthy subjects (Fig. 1C), whereas light green fluorescence was observed on platelets, leukocytes and erythrocytes from patients with MM (Fig. 1D). These results further confirmed that there is increased PS exposure on blood cells in patients with MM compared with healthy subjects.

ECs incubated for 24 h with serum from patients with MM or healthy control were stained with CD31-Alexa Fluor 647 and PS exposure was detected using lactadherin-Alexa Fluor 488. Confocal laser-scanning microscopy demonstrated that there was limited lactadherin staining on ECs cultured with normal serum (Fig. 1C), while large amounts of lactadherin, exhibited by green fluorescence, was observed on ECs treated with patient serum (Fig. 1D). Notably, the percentage of PS⁺ ECs was higher than that of erythrocytes, platelets and leukocytes in patients with MM.

To examine whether the increase in PS⁺ cells contributes to enhanced PCA, recalcification-time assays were performed using a KC4A-coagulometer. Compared with samples from healthy subjects, patient suspensions of erythrocytes, platelets and leukocytes (cell number controlled) exhibited significantly shorter coagulation time (P<0.05; Fig. 2A). ECs cultured in serum from patients with MM induced a shorter coagulation time when cultured in serum from healthy subjects (P<0.05; Fig. 2B). To confirm whether this increased PCA was attributed to membrane PS exposure, inhibition assays were performed where cells were incubated with 128 nM lactadherin prior to coagulation testing. Lactadherin prolonged the coagulation time of PS⁺ cells to similar values as healthy subjects (P<0.05; Fig. 2C).

In order to further evaluate the capacity of blood cells and ECs to support the formation of procoagulant enzyme complexes, intrinsic/extrinsic factor Xa and thrombin formation assays were performed using purified coagulation factors. The production of all three procoagulant enzyme complexes was increased in MM groups compared with healthy subjects (P<0.05; Fig. 3A–D). These findings suggest that PS⁺ blood cells contribute to coagulation, and that the relative number of these blood cells may increase thrombosis risk. In inhibition assays for all cell types, lactadherin (at 128 nM) blocked production of the three procoagulant enzyme complexes by ~75% (Fig. 3E). PS blockade almost entirely inhibited the formation of these complexes, suggesting that PS independently increases PCA in patients with MM.

Fibrin constitutes the primary structural protein of blood clots and intravascular thrombi and its formation involves the concerted action of coagulation factors and blood cells. Therefore, fibrin formation was evaluated in normal MDP. More fibrin was deposited on erythrocytes, platelets and leukocytes from patients with MM than those from healthy subjects (P<0.05; Fig. 4). The inhibition assays demonstrated that lactadherin reduced fibrin formation of all cells to a level lower than healthy subjects.

Confocal microscopy provided more evidence that PS on ECs has an important role in fibrin clot formation. Limited factor Va (red) or factor Xa (green) binding was observed on ECs treated with serum from healthy subjects (Fig. 5A, arrow). When ECs were cultured with the serum from patients with MM, factor Va and factor Xa were observed to be bound to the PS-enriched areas of the cell membrane (Fig. 5B, arrow). ECs were incubated with plasma in the presence of calcium and Alexa 647-conjugated anti-fibrin. Considerable fibrin fibrils (red) were spread along the filopodia of ECs visualized using actin and DAPI staining (Fig. 5C, arrow). The patient serum resulted in a 1.48-fold higher fibrin generation in ECs than the serum from healthy subjects (P<0.05; Fig. 5D).

The effects of the IMiDs (Tha and Len, with or without Dex treatment) on PS exposure of blood cells in vitro were determined. Erythrocytes (24 h), platelets (1 h), and leukocytes (24 h) from patient with MM were cultured with Tha (1.0 μM), Len (1.0 μM), Tha (1.0 μM)/Dex (10 μM), or Len (1.0 μM)/Dex (10 μM) at 37°C. Flow cytometry analysis demonstrated that, compared with control or either IMiD alone, Tha or Len plus Dex increased the level of PS exposure on erythrocytes (P<0.05; Fig. 6A). IMiD treatment alone did not significantly increase PS exposure on erythrocytes compared with controls. However, Tha and Len significantly increased PS exposure on platelets and leukocytes compared with controls (P<0.05; Fig. 6B and C). Treatment of platelets and leukocytes with an IMiD in combination with Dex resulted in higher PS exposure compared with IMiD treatment alone. The effects of IMiDs and Dex, either alone or in combination, on PS exposure on ECs were determined by flow cytometry. Incubation of ECs with an IMiD alone resulted in increased PS exposure compared with controls (P<0.05; Fig. 6D). Furthermore, an increased percentage of PS⁺ ECs was induced by the combination of an IMiD and Dex, as compared with Tha or Len alone. The results indicate that IMiDs and Dex used in combination can further increase PS exposure on blood cells from MM patients and ECs in vitro.