Original article 621

Induction therapy alters plasm

a fibrin clot properties inmultiple myeloma patients: association withthromboembolic complicationsAnetta Undasa, Lidia Zubkiewicz-Usnarskab, Grzegorz Helbigc,Dariusz Woszczykd, Justyna Kozinskae, Anna Dmoszynskae, Jakub Debskib,Maria Podolak-Dawidziakb and Kazimierz Kuliczkowskib

Induction therapy in patients with multiple myeloma

increases the risk of thromboembolism. We have recently

shown that multiple myeloma patients tend to form denser

fibrin clots displaying poor lysability. We investigated the

effect of induction therapy on fibrin clot properties in

multiple myeloma patients. Ex-vivo plasma fibrin clot

permeability, turbidity, susceptibility to lysis, thrombin

generation, factor VIII and fibrinolytic proteins were

compared in 48 multiple myeloma patients prior to and

following 3 months of induction therapy, mainly with

cyclophosphamide-thalidomide-dexamethasone regimen.

Patients on thromboprophylaxis with aspirin or heparins

were eligible. A 3-month induction therapy resulted in

improved clot properties, that is higher clot permeability,

compaction, shorter lag phase and higher final turbidity,

along with shorter clot lysis time and higher rate of D-dimer

release from fibrin clots than the baseline values. The

therapy also resulted in lower thrombin generation,

antiplasmin and thrombin-activatable fibrinolysis inhibitor

(TAFI), but elevated factor VIII. Progressive disease was

associated with lower posttreatment clot permeability and

lysability. Despite thromboprophylaxis, two patients

developed ischemic stroke and 10 had venous

thromboembolism. They were characterized by

pretreatment lower clot permeability, prolonged clot lysis

Copyright © 2015 Wolters Kluwer

0957-5235 Copyright � 2015 Wolters Kluwer Health, Inc. All rights reserved.

time, longer lag phase, higher peak thrombin generation,

TAFI and plasminogen activator inhibitor -1. Formation of

denser plasma fibrin clots with reduced lysability and

increased thrombin generation at baseline could

predispose to thrombotic complications during induction

treatment in multiple myeloma patients. We observed

improved fibrin clot properties and thrombin generation in

multiple myeloma patients except those with progressive

disease. Blood Coagul Fibrinolysis 26:621–627 Copyright �2015 Wolters Kluwer Health, Inc. All rights reserved.

Blood Coagulation and Fibrinolysis 2015, 26:621–627

Keywords: fibrin clot, fibrinolysis, induction treatment, multiple myeloma,thromboembolism

aInstitute of Cardiology, Jagiellonian University Medical College, and John Paul IIHospital, Krakow, bDepartment of Hematology, Blood Neoplasms and BoneMarrow Transplantation, Wrocław Medical University, Wroclaw, cDepartment ofHematology and Bone Marrow Transplantation, Silesian Medical University,Katowice, d1st Department of Hematology, City Hospital, Opole andeDepartment of Hematology, Medical University of Lublin, Lublin, Poland

Correspondence to Anetta Undas, MD, PhD, Institute of Cardiology JagiellonianUniversity Medical College, 80 Pradnicka St., 31202 Cracow, PolandTel: +48 12 6143004; fax: +48 12 4233900; e-mail: mmundas@cyf-kr.edu.pl

Received 30 July 2014 Revised 16 March 2015Accepted 25 March 2015

IntroductionMultiple myeloma is associated with an increased throm-

boembolic risk, particularly venous thromboembolism

(VTE) during therapy based on thalidomide combined

with chemotherapy [1,2]. Cerebrovascular ischemic

events and myocardial infarction (MI) also occur more

frequently in multiple myeloma patients [3]. Throm-

boembolism in multiple myeloma patients has been

demonstrated to be associated with a three-fold higher

risk of death during follow-up [4].

The mechanism underlying increased thromboembolic

risk in multiple myeloma patients is poorly understood.

Paraproteins have been shown to enhance platelet

adhesion and aggregation, increase blood viscosity, lead

to acquired activated protein C resistance and endothelial

damage, together with increased circulating factor (F)VIII

[5,6]. The immunomodulatory drugs have been shown to

enhance expression of tissue factor (TF) and vascular

endothelial growth factor, downregulate thrombospondin,

cause activated protein C resistance and increase FVIII

[7]. Genes and pathways associated with drug transport/

metabolism, DNA repair and cytokine balance may also

be involved in thalidomide-associated VTE [8]. Thalido-

mide given in combination with other chemotherapies or

dexamethasone, endothelial cell and monocyte-associated

procoagulant activity may be induced through phospha-

tidylserine exposure, or TF expression [9].

The final step of the blood coagulation is the formation of

clots relatively resistant to lysis [10]. Such clot phenotype

has been demonstrated in patients with MI [11,12],

cryptogenic ischemic stroke [13] and VTE [14], along

with advanced atherosclerotic vascular disease [15].

Recently, we have reported that unfavourable clot prop-

erties can be detected in multiple myeloma patients at

the time of diagnosis compared with the matched control

individuals [16].

Health, Inc. All rights reserved.

DOI:10.1097/MBC.0000000000000315

622 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 6

The current study was designed to evaluate the effect

of induction therapy on fibrin characteristics in associ-

ation with thromboembolic episodes in multiple myel-

oma patients.

Patients and methodsWe studied 48 adult patients with newly diagnosed

multiple myeloma out of the 106 patients recruited in

four Polish hematologic centres, in Wroclaw from

November 2010 to June 2012 and in Lublin, Katowice,

Opole, all three from November 2011 to July 2012. The

inclusion and exclusion criteria were described pre-

viously [16].

The International Staging System (ISS) for multiple

myeloma was used to stratify patients into three stages

[17]. The exclusion criteria were a recent (<6 months)

thrombotic event, liver cirrhosis, end-stage renal failure,

current anticoagulant therapy, severe thrombocytopenia

and hypofibrinogenemia (<1 g/l). Control individuals

recruited from outpatients and hospital staff were fre-

quency matched for age, sex, arterial hypertension, dia-

betes and previous thromboembolic events. The

University Ethical Committee approved the study, and

patients provided written, informed consent.

The patients received induction treatment, predominantly

using a cyclophosphamide-thalidomide-dexamethasone

regimen (CTD), cyclophosphamide 500 mg/m2 per day

intravenously (i.v.) on day 1, thalidomide 100 mg/d orally

(p.o.) each day, dexamethasone 20 mg/d p.o. on days 1–4

and 9–12. Individuals receiving thalidomide-dexametha-

sone regimen were also eligible. None of the patients

received lenalidomide-based regimens or erythropoietin.

Other therapies considered necessary by the physician for

the supportive care were allowed.

Response to therapy was categorized into four groups,

that is partial remission, very good partial response

(VGPR), complete remission (including stringent com-

plete response, CR) and progressive disease based on

the current criteria.

All patients received thromboprophylaxis either with

aspirin 75–150 mg/day or enoxaparin 40 mg/day during

induction treatment as recommended in 2008 [18].

Patients were assessed before the onset of immunomo-

dulatory therapy and after the first 90� 10 days since

enrolment. We analysed documented VTE episodes, MI,

ischemic stroke and death within the first 3 months of

therapy on the basis of medical records. The diagnosis of

deep-vein thrombosis (DVT) was established by a

positive finding of colour duplex sonography. The diag-

nosis of pulmonary embolism was based on the presence

of typical symptoms and positive results of high-resol-

ution spiral computed tomography. Ischemic stroke and

MI were diagnosed according to the WHO criteria.

Patients who developed a thromboembolic event and

Copyright © 2015 Wolters Kluwer

required administration of vitamin K antagonists (VKAs)

or therapeutic doses of low-molecular-weight heparins

(LMWHs) were excluded from the final analysis.

Laboratory investigationsBlood was drawn from an antecubital vein between 0800

and 1000 h. Patients receiving LMWH as thrombopro-

hylaxis were drawn at least 12 h since the last injection.

Blood cell count, glucose, total cholesterol, triglycerides,

activated partial thromboplastin time (aPTT), prothrom-

bin time, D-dimer, lactate dehydrogenase (LDH),

calcium, albumin, IgG, IgA, creatinine and b2-microglo-

bulin were assayed by routine laboratory techniques.

Fibrinogen was determined using the Clauss method.

Blood and urine immunofixation was performed to

confirm the diagnosis. The kappa and lambda free light

chain (FLC) concentrations were measured and the

kappa/lambda FLC ratios were calculated.

Plasminogen and antiplasmin were measured by chromo-

genic assays (STA Stachrom; Diagnostica Stago, Asnieres,

France). Plasma PAI-1 activity was determined using a

biofunctional immunosorbent assay (Chromolize PAI-1,

Biopool, Sweden). Plasma thrombin-activatable fibrinoly-

sis inhibitor (TAFI) activity was measured by

a chromogenic assay using the ACTICHROME Plasma

TAFI Activity Kit (American Diagnostica, Stamford, Con-

necticut, USA). Plasma factor VIII activity was determined

by the coagulometric assay (Siemens, Marburg, Germany).

Plasma thrombogenic potential was assessed using cali-

brated automated thrombography (CAT; Thrombino-

scope BV, Maastricht, Netherlands) at 378C according to

the manufacturer’s instructions. Eighty microlitres of pla-

telet poor plasma were diluted with 20 ml of a TF-based

activator (Diagnostica Stago, Asnieres, France) containing

5 pmol/l recombinant TF, 4 micromolar phosphatidylser-

ine/phosphatidylcholine/phosphatidylethanolamine

vesicles and 20 ml of FluCa solution (Hepes, pH 7.35,

100 nmol/l CaCl2, 60 mg/ml bovine albumin and

2.5 mmol/l Z-Gly-Gly-Arg-amidomethylcoumarin). The

peak thrombin level was analysed.

Plasma fibrin clot analysisPlasma fibrin clot variables were determined in duplicate

by technicians blinded to the origin of the samples

(intraassay and interassay coefficients of variation, 5–

8%, for all variables), as described [11,13,14].

(1) F

He

ibrin clot permeation using a pressure-driven

system, with calculation of a permeation coefficient

(Ks), which indicates the pore size. Lower Ks values

indicate reduced permeability.

(2) T

he lag phase of the turbidity curve, which reflects

the time required for initial protofibril formation and

maximum absorbance at 405 nm at the plateau phase

(DAbmax), indicating the number of protofibrils

per fibre.

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Thromboembolism in multiple myeloma Undas et al. 623

(3) F

Table

AgeMalePreviDiabeHypePreviHaemRBCWBCPlateIgG (IgA (LDHCalciAlbumb2 mCreaProthFibrinAPTTD-dimGlucTotalTrigly

Valuenons

ibrinolysis induced by 1 mg/ml recombinant tPA (rt-

PA; Boerhinger Ingelheim, Ingelheim, Germany)

added simultaneously with human thrombin to

citrated plasma. Clot lysis time was defined as the

time required for a 50% decrease in clot turbidity at

405 nm (t50%).

(4) F

ibrinolysis of the clots formed as described above

and perfused with the same buffer containing

0.2 mmol/l rtPA. The lysis rate was determined by

measuring D-dimer every 20 min in the effluent.

Maximum rates of increase in D-dimer levels (D-

Drate), and their maximum values (D-Dmax) detected

at 80 or 100 min were analysed.

(5) C

ompaction of a plasma fibrin clot formed in the

presence of 0.7 IU/ml thrombin and 20 mmol/l

calcium in tubes and then centrifuged at 6000g for

60 s. Compaction was expressed as the volume of the

supernatant evacuated from the tubes divided by the

initial plasma volume used to form the clot.

Scanning electron microscopyClots from patients and controls were fixed using 2.5% of

glutaraldehyde PBS solution for 2 h. Fixed clots were

gently removed and washed with distilled water, and

then dehydrated in graded water-ethanol solutions, dried

by the critical point procedure and sputter coated with

gold. Samples were scanned in six different areas (micro-

scope HITACHI S-4700).

Statistical analysisContinuous variables were expressed as mean�SD for

normally distributed variables and as median and inter-

quartile range for nonnormally distributed variables.

Copyright © 2015 Wolters Kluwe

1 Comparison of multiple myeloma patients at baseline and aft

MM patients at baseline (n¼48)

(years) 62 (56–71), n (%) 18 (37.5)ous VTE, n (%) 2 (4.2)tes, n (%) 9 (18.8)rtension, n (%) 14 (29.2)ous stroke, n (%) 1 (2.1)oglobin (g/dl) 10.2 [8.9–12.5](106/ml) 3.4 [2.9–4.1](103/ml) 6.2 [4.8–8.4]

lets (103/ml) 236 [176–282]g/l) 26.5 [6.8–54.0]g/l) 1.1 [0.3–31.1](IU/l) 195 [138–329]um (mmol/l) 3.3 [2.3–4.6]in (g/l) 34 [29–37]

icroglobulin (mg/l) 4.3 [2.5–6.6]tinine (mmol/l) 89.6 [67.2–167.1]rombin time (s) 13.4 [12.8–14.3]ogen (g/l) 3.4 [2.6–4.3](s) 31 [28–34]er (ng/ml) 1111 [584–2755]

ose (mmol/l) 5.5 [5–6.6]cholesterol (mmol/l) 4.7 [3.7–5.2]cerides (mmol/l) 1.2 [0.8–1.9]

s are given as number (percentage) or median (IQR). APTT, activated partial tignificant; RBC, red blood cells; VTE, venous thromboembolism; WBC, white bl

Continuous variables were checked for normal distri-

bution by the Shapiro–Wilk statistic. To analyse two

independent groups, we used the Kruskal–Wallis test.

To compare the patients before and after treatment,

Wilcoxon signed ranks test was used. Categorical vari-

ables were compared by x2 test or Fisher’s exact test as

appropriate. The Spearman rank correlation coefficients

were calculated to test the association between two

variables. Multivariate linear regression models was used

to choose variables that influence the fibrin indicators.

Only those with P values less than 0.1 in univariate

regression models were included in multivariable models.

Models were built in order to optimally explain the

variation in fibrin variables and checked for collinearity

between independent ones. All models were additionally

adjusted to age and fibrinogen. Logistic regression

analysis was used to show odds ratios (ORs) of throm-

boembolic events in the extreme quartiles of fibrin clot

variables at baseline. A two-sided P value less than 0.05

was considered statistically significant. All analyses were

performed using STATISTICA version 10.0 (StatSoft,

Inc., Tulsa, Oklahoma, USA).

ResultsPatient characteristics and therapy outcomeForty-eight multiple myeloma patients, including 24

(50%) with IgG multiple myeloma, 13 (27.1%) with IgA

multiple myeloma and 11 (22.9%) with light chain disease

(LCD) were studied (Table 1). The stage III ISS was

observed in 29 patients (60.4%), stage II in 10 patients

(20.8%) and stage I in nine patients (18.8%). Forty (83.3%)

patients received CTD treatment. Six (12.5%) patients

r Health, Inc. All rights reserved.

er 3 months’ induction therapy

MM patients after 3 months (n¼34) P

63 (57–70) NS12 (35.3) NS

1 (2.9) NS5 (14.7) NS

13 (38.2) NS0 –

11.7 [10.8–12.9] <0.0013.9 [3.7–4.0] <0.0015.2 [4.5–6.9] 0.003

251 [204–311] NS7.7 [4.9–20.2] <0.0011.2 [0.4–12.9] NS169 [124–301] 0.042.6 [2.2–4.3] 0.0139 [35–43] <0.001

2.7 [2.3–3.7] <0.00182.8 [63.3–120.1] 0.0213.1 [12.5–14.4] NS

3.3 [2.7–4.1] NS30 [27–33] NS

692 [450–1153] 0.025.4 [5.1–6.5] NS4.6 [3.7–5.6] NS1.5 [1.1–2] 0.05

hromboplastin time; LDH, lactate dehydrogenase; MI, myocardial infarction; NS,ood cells.

624 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 6

Fig. 1

(a)

(b)

Representative scanning electron microscopy images of plasmafibrin clots obtained for a male patient with IgG multiple myelomaprior to (a) and after the induction therapy with thalidomide-basedregimen (b) resulting in a complete remission associated with thereduction of serum IgG from 56 to 15 g/l. Magnification 5000x.A bar denotes 2 mm.

were treated with melphalan-prednisone-thalidomide

(MPT) and the remaining three patients with thalido-

mide-dexamethasone. Forty-two patients declared taking

aspirin 75–150 mg/day, and six patients (12.5%) received

LMWH.

After 3 months of therapy, we observed partial remission

in 23 (48.0%) patients, VGPR in six (12.5%), CR in nine

(18.8%) and progressive disease in the remaining seven

individuals (14.6%). Three patients (8.3%) died.

There were posttreatment increases in haemoglobin, red

blood cells, white blood cells, albumin and LDH, while

beta2-microglobulin, calcium, creatinine and D-dimer

decreased (Table 1). Fibrinogen remained unaltered.

During induction therapy, 10 VTE episodes, including

nine DVT and one pulmonary embolism, and two

ischemic strokes were recorded in 12 multiple myeloma

patients (25%), including 10 individuals in stage III ISS.

IgG multiple myeloma, IgA multiple myeloma and LCD

were equally represented (four individuals from each

group). Seven patients received CTD regimen. Of the

12 patients with thromboembolic complications, pro-

gression was observed in five patients, partial remission

in four, CR in two and one patient died during the 3

months’ follow-up. All episodes developed despite

thromboprophylaxis, that is nine patients were on low-

dose aspirin and three were on prophylactic enoxaparin at

the time of the event. The multiple myeloma patients

with thromboembolism (n¼ 12) did not differ from the

remainder with regard to demographic variables or rou-

tine laboratory parameters (data not shown).

Posttreatment changes in coagulation and fibrinvariablesAfter induction treatment, a slight, but significant,

improvement in all the variables describing fibrin clot

structure and function was observed in 34 available

multiple myeloma patients (70.8%). The analysis

excluded 14 individuals who died or developed throm-

boembolic episodes. Clot permeability and compaction

increased, indicating looser fibrin meshwork structure.

Fibrinolysis was accelerated in both assays used. The

DAbmax increased and the lag phase became slightly

shorter (all P< 0.05) after 3 months of therapy. In seven

patients with progressive disease, in whom none of the

fibrin parameters altered after therapy, as compared with

the individuals with CR, we observed lower posttreat-

ment Ks, longer lysis time and lower DAbmax.

Representative SEM images of a multiple myeloma

patient free of thromboembolic events were shown in

Fig. 1. Posttreatment images for the patients without

those with progressive disease were similar to those

typical of normal individuals [11,13,16].

Induction therapy was associated with the reduction in

peak thrombin generation, TAFI activity, and slightly,

Copyright © 2015 Wolters Kluwer

antiplasmin (Table 2). Importantly, FVIII rose at the

same time. In seven patients with progressive disease

versus the remaining 29 individuals, we observed higher

posttreatment peak thrombin generation (þ15%) and

TAFI activity (þ12%; both P< 0.05). Plasma levels of

PAI-1, antiplasmin, plasminogen and FVIII were similar

in the two subgroups (data not shown).

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Thromboembolism in multiple myeloma Undas et al. 625

Table 2 Comparison of fibrin clot variables and associated proteins in multiple myeloma patients

VariableMM patients at

baseline (n¼48)MM patients after3 months (n¼34) P

MM patients withthromboembolism

(n¼12)

MM patients withoutthromboembolism

(n¼36) P

Ks (10–9 cm2) 6 [5.7–6.6] 6.6 [5.9–7.2] 0.003 5.5 [5.1–5.9] 6.1 [5.6–6.8] 0.03t50% (min) 11 [10.4–11.9] 10.1 [9.6–11.3] <0.001 11.6 [11.2–11.9] 11.3 [10.3–11.8] NSD-Dmax (mg/l) 4.52 [4.4–5.1] 4.48 [4.3–4.8] NS 5.1 [4.9–5.2] 4.8 [4.6–5.1] NSD-Drate (mg/l/min) 0.067 [0.064–0.07] 0.072 [0.067–0.077] <0.001 0.061 [0.059–0.062] 0.067 [0.063–0.071] <0.001DAbmax (405 nm) 0.61 [0.52–0.7] 0.73 [0.63–0.75] <0.001 0.61 [0.5–0.67] 0.6 [0.51–0.71] NSLag phase (s) 51 [47–55] 48 [43–52] 0.02 60 [52–60.5] 50 [47–55] 0.01Compaction (%) 42 [37–44] 49 [40–51] 0.004 38.5 [36.5–49] 44 [40–47] NSPeak trombin (nmol/l) 344 [275–398] 284 [248–389] <0.001 503.5 [418–550] 344.8 [269–411] <0.001TAFI act (mg/ml) 36.4 [30.7–40.8] 30.9 [25.2–37.9] 0.005 45.3 [44.6–47.4] 38.9 [33.5–42.3] <0.001Antiplasmin (%) 102.2 [87.4–116.4] 96.3 [84.2–106.9] 0.04 101.5 [86.3–111.8] 104.1 [90.2–110.5] NSPlasminogen (%) 104.6 [93.2–111.8] 103.2 [93.5–110.8] NS 97.5 [87–103.8] 105.8 [97.4–113.1] 0.03PAI-1 act (IU/ml) 7.8 [6.2–10.0] 7.6 [6–10.8] NS 11 [9.9–12.8] 8.3 [6.4–10.5] 0.004Factor VIII (%) 196 [156–235] 219 [195–269] 0.002 192.5 [183.5–266.5] 188 [166.5–223] NS

n values are given as median [IQR]. DAbmax, maximum absorbance of fibrin gel determined by using turbidimetry; act, activity; D-Dmax, maximum D-dimer levels in the lysisassay; D-Drate, maximum rate of increase in D-dimer levels in the lysis assay; Ks, permeability coefficient; NS, nonsignificant; PAI-1, plasminogen activator inhibitor; t50%, half-lysis time; TAFI, thrombin-activatable fibrinolysis inhibitor.

Predictors of thromboembolic eventsPatients who experienced venous or arterial thromboem-

bolic complications had lower Ks, and D-Drate together

with longer lag phase measured at baseline than the

remaining patients (Table 2). Other fibrin variables were

similar in these two subgroups (Table 2).

Moreover, thromboembolism in multiple myeloma

patients was associated with higher baseline peak throm-

bin concentration, activated TAFI and PAI-1 activity as

well as lower plasminogen (Table 2).

Logistic regression analysis showed that two baseline

fibrin clot parameters could predict thromboembolism in

our group. The OR of thromboembolism in multiple

myeloma patients in the lowest quartile of baseline D-

Drate was 12.1 (3.6–59.1; P< 0.0001) relative to the

remaining patients, with the highest area under the curve

(AUC) of 0.78. Similar ORs were noted for the upper

quartile of the lag phase [OR 95% confidence interval

(95% CI), 7.5 (2.1–30.8), P< 0.001] with the AUC

of 0.72.

DiscussionThe current study shows that plasma fibrin clot phe-

notype is favourably altered after 3 months of induction

therapy largely based on thalidomide in multiple myel-

oma patients. We observed that no improvement in clot

phenotype occurs in individuals with progressive dis-

ease. Importantly, the multiple myeloma patients who

experienced thromboembolism during induction treat-

ment displayed lower clot permeability and tPA-

induced lysis at baseline. This suggests that unfavour-

able altered fibrin clot characteristics prior to therapy

may contribute to thrombotic complications observed

in multiple myeloma patients and cannot be normal-

ized by aspirin or LMWH prophylaxis.

Until now, there have been no data as to whether

specific immunomodulatory drugs produce specific

Copyright © 2015 Wolters Kluwe

fibrin-altering effects in multiple myeloma patients.

Several studies have shown that thrombosis occurs

mainly when thalidomide is used in combination with

high-dose dexamethasone, anthracyclines or any che-

motherapy in multiple myeloma patients [19]. Multiple

prothrombotic mechanisms by which thalidomide-

based therapy promotes thrombosis have been postu-

lated [1,19]. Posttreatment prothrombotic alterations

in multiple myeloma patients we observed here

include elevation of FVIII activity and lower antiplas-

min activity.

We found that induction therapy, when analysed in the

whole group, slightly improved plasma fibrin clot charac-

teristics and reduced thrombin formation. Changes in

clot structure were previously demonstrated by Carr

et al. [20] with a major contribution of paraprotein levels

in multiple myeloma patients. We found a significant

impact of thrombin formation on fibrin variables in

multiple myeloma patients as suggested in other clinical

situations [21], including cancer patients [22]. Stimu-

lation of prothrombinase by paraproteins has been

suggested in vitro [23]. A role of thrombin generation

in predicting thromboembolism in various malignant

diseases, including multiple myeloma, has been shown

by Ay et al. [24]. This study indicates that this role of

thrombin could be in part mediated by unfavourable

clot properties.

In terms of unfavourable changes in coagulation during

thalidomide-based therapy, our findings show that

there is no overall activation of blood coagulation,

higher thrombin generation and fibrin formation in

individuals treated with mostly thalidomide combined

with corticosteroids. However, the described favour-

able changes in coagulation and fibrinolysis are largely

driven by the effects observed in patients with partial

or complete remission, with no alterations to coagu-

lation parameters in individuals with progressive dis-

ease. Importantly, progression of multiple myeloma

r Health, Inc. All rights reserved.

626 Blood Coagulation and Fibrinolysis 2015, Vol 26 No 6

was associated with worse posttreatment fibrin para-

meters compared with those in individuals with CR. A

beneficial modulator of on-treatment fibrin clot pheno-

type in our study could be thromboprophylaxis. It has

been shown that aspirin, which acetylates fibrinogen

and other proteins, may accelerate plasma fibrin clot

lysis and make clots more permeable [25]. Similar

effects can induce LMWH [26]. Thromboprophy-

laxis-induced changes in fibrin structure were not

potent enough to abolish pretreatment differences

between individuals with thromboembolism during induc-

tion therapy versus the remainder. Our results suggest that

there is a subgroup of multiple myeloma patients ident-

ifiable prior to therapy, based on a prothrombotic plasma

fibrin clot phenotype, in whom there is a high risk for

thromboembolism. It is likely that a markedly higher

baseline thrombin generation observed in multiple myel-

oma patients prone to thromboembolism might contribute

to unfavourable clot properties.

Recently, we have reported hypofibrinolysis in

multiple myeloma patients [16], which is in line with

a study by Yagci et al. [27]. We found that thalidomide-

based therapy is associated with slightly faster clot lysis

using two assays with different final rtPA concen-

trations. Von Marion et al. [28] observed impaired

efficiency of lysis during induction therapy. Different

patient characteristics and methodology used could

explain in part this discrepancy. Moreover, our study

suggests that pretreatment higher TAFI and PAI-1

activity with lower plasminogen can distinguish the

multiple myeloma patients who suffered from throm-

boembolic events during induction therapy from those

free of these complications.

Several limitations of this study should be acknowledged.

A sample size was small but sufficiently powered to show

posttreatment differences. Thromboembolic episodes

that were asymptomatic or not clinically recognized

would not have been detected. Posttreatment fibrin clot

analysis was not performed in all the individuals studied

due to anticoagulant therapy initiated after the diagnosis

of VTE, which suppresses thrombin formation and alters

clot structure [10]. We cannot exclude an influence of

undetected confounding factors, for example cigarette

smoking adversely affecting clot properties [10,29].

Finally, effects of novel agents used in multiple myel-

oma, for example lenalidomide, on clot phenotype were

beyond the scope of this study.

In conclusion, we have shown that induction treatment

based on thalidomide was associated with improved fibrin

clot phenotype, lower thrombin generation and faster

fibrinolysis. Our findings suggest that more compact

fibrin clots with reduced lysability, as assessed prior to

therapy, characterize individuals who experienced

thrombotic complications during induction treatment

for multiple myeloma.

Copyright © 2015 Wolters Kluwer

AcknowledgementsThe study was funded by Jagiellonian University

Medical College (grant K/ZDS/002936 to A.U.).

Conflicts of interestThe authors declare no competing financial interests.

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