The reference interval for FXIII-A2B2 in plasma has been

established as 14–28 mg L)1 [11], corresponding to 67–133%.

Among the patients were 24 women with FXIII-A levels of

< 70% (minimum: 52.4%). Whether these low levels were

attributabletoconsumptionorgenetic influenceswasnotfurther

studied, and this might represent a limitation of our study.

However, among the controls were 22 women with FXIII-A

levelsof< 70%(minimum:51.9%).Wethereforeconcludethat

this mild FXIII-A deficiency that we observed in some patients

and controls is probably not related to pregnancy loss.

For the first time, we have compared FXIII-A and FXIII-B

plasma levels between women with unexplained recurrent

pregnancy loss and controls without a history of miscarriage

and with at least one successful pregnancy. Our results suggest

that recurrent pregnancy loss in the general population is not

associated with reduced FXIII plasma levels. Whether locally

reduced FXIII-A levels or impaired FXIII function in the

placenta may contribute to an increased risk of abortion

remains to be investigated.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

References

1 Muszbek L, Bereczky Z, Bagoly Z, Komaromi I, Katona E. Fac-

tor XIII: a coagulation factor with multiple plasmatic and cellular

functions. Physiol Rev 2011; 91: 931–72.

2 Ichinose A, Asahina T, Kobayashi T. Congenital blood coagulation

factor XIII deficiency and perinatal management. Curr Drug Targets

2005; 6: 541–9.

3 Inbal A, Muszbek L. Coagulation factor deficiencies and pregnancy

loss. Semin Thromb Hemost 2003; 29: 171–4.

4 Asahina T, Kobayashi T, Okada Y, Itoh M, Yamashita M, Inamato

Y, Terao T. Studies on the role of adhesive proteins in maintaining

pregnancy. Horm Res 1998; 50(Suppl. 2): 37–45.

5 Asahina T, Kobayashi T, Okada Y, Goto J, Terao T. Maternal blood

coagulation factor XIII is associated with the development of cyto-

trophoblastic shell. Placenta 2000; 21: 388–93.

6 van Wersch JW, Vooijs ME, Ubachs JM. Coagulation factor XIII in

pregnant smokers and non-smokers. Int J Clin Lab Res 1997; 27: 68–

71.

7 Ogasawara MS, Aoki K, Katano K, Ozaki Y, Suzumori K. Fac-

tor XII but not protein C, protein S, antithrombin III, or fac-

tor XIII is a predictor of recurrent miscarriage. Fertil Steril 2001; 75:

916–19.

8 Pasquier E, Bohec C, Mottier D, Jaffuel S, Mercier B, Ferec C, Collet

M, De Saint Martin L. Inherited thrombophilias and unexplained

pregnancy loss: an incident case–control study. J Thromb Haemost

2009; 7: 306–11.

9 De Saint Martin L, Duchemin J, Bohec C, Courturaud F, Mottier D,

Collet M, Blouch MT, Pasquier E. Increased thrombin generation

measured in the presence of thrombomodulin in women with early

pregnancy loss. Fertil Steril 2011; 95: 1813–15.

10 Ariens RA, Kohler HP, Mansfield MW, Grant PJ. Activity and sub-

unit antigen levels of blood coagulation factor XIII: relationship to

age, smoking and hypertension in healthy individuals. Arterioscler

Thromb Vasc Biol 1999; 19: 2012–16.

11 Katona E, Haramura G, Karpati L, Fachet J, Muszbek L. A simple,

quick one-step ELISA assay for the determination of complex plasma

factor XIII (A2B2). Thromb Haemost 2000; 83: 268–73.

Convalescent plasma levels of TAFI activation peptide predictdeath and recurrent vascular events in ischemic stroke survivors

K . JOOD,* P . REDFOR S ,* A . G ILS ,� C. BLOMSTRAND,* P . J . DECLERCK� and C . J ERN **Institute of Neuroscience and Physiology, the Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; and �Laboratory for

Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium

To cite this article: Jood K, Redfors P, Gils A, Blomstrand C, Declerck PJ, Jern C. Convalescent plasma levels of TAFI activation peptide predict death

and recurrent vascular events in ischemic stroke survivors. J Thromb Haemost 2012; 10: 725–7.

Thrombin activatable fibrinolysis inhibitor (TAFI) is a zymo-

gen present in human plasma. Upon activation by trypsin-like

enzymes such as thrombin, plasmin or the thrombin/throm-

bomodulin complex, the activation peptide (TAFI-AP) is

released from the catalytic domain (TAFIa). TAFIa operates

by continuously removing C-terminal lysine residues on

plasmin-modified partially degraded fibrin, thus attenuating

the rate of plasminogen activation and fibrinolysis [1].

Wehave investigatedplasma levelsof intactTAFIandTAFI-

AP, asmeasured by two genotype-independent ELISAs [2], in a

large case–control studyof ischemic stroke (IS), theSahlgrenska

AcademyStudyon Ischemic Stroke (SAHLSIS) [3]. BothTAFI

measures showed association with IS independently of vascular

risk factors. The associationwas stronger forTAFI-AP than for

intact TAFI, and was present in all major etiologic IS subtypes,

Correspondence: Katarina Jood, Department of Clinical Neuroscience

and Rehabilitation, Institute of Neuroscience and Physiology, the

Sahlgrenska Academy at University of Gothenburg, Per Dubbsg 14,

S-413 45 Gothenburg, Sweden.

Tel.: +46 31 3429052; fax: +46 31 842160.

E-mail: katarina.jood@neuro.gu.se

DOI: 10.1111/j.1538-7836.2012.04638.x

Received 21 November 2011, accepted 12 January 2012

Letters to the Editor 725

� 2012 International Society on Thrombosis and Haemostasis

indicating that TAFI activation may contribute to the develop-

ment of IS irrespective of the underlying etiology. In the present

study, we used data from a prospective follow-up of the

SAHLSIScohort to investigate thepossible associationbetween

TAFI levels and prognosis after stroke with respect to future

death and/or recurrent vascular events.

Details of SAHLSIS have been described elsewhere [4]. In

brief, 600 patients with acute ischemic stroke, aged 18–

69 years, were consecutively recruited at stroke units in western

Sweden. Each case was classified according tomodified Trial of

Org 10172 in Acute Stroke Treatment (TOAST) criteria [4].

Stroke severity was scored using the Scandinavian Stroke Scale.

Risk factors were defined as described [4]. All participants

provided written informed consent. This study was approved

by the Ethics Committee of the University of Gothenburg.

Blood was drawn under standardized conditions 3 months

after the index event (median 102 days, range 85–125 days) [3].

Plasma levels of intact TAFI and released AP were measured

by sandwich-type ELISAs as described [2,3]. High-sensitivity

C-reactive protein (hsCRP) was analyzed as described [5].

Two years after inclusion in the study, the survival rates of

patients were assessed using data from the Swedish population

register. Surviving patients were contacted for a telephone

interview that involved questions on recurrent stroke, transient

ischemic attacks (TIA) and coronary events (CE). If the patient

was unable to provide answers, a relative or caregiver was

interviewed. In addition, the Swedish Hospital Discharge

Register was screened for all patients. Reported vascular events

were confirmed by review of the medical records. Given that

some early recurrent events may have gone undetected using

this approach, all of the medical records from hospitals and

primary care units collated in the 3 months after the index

stroke were reviewed by two neurologists (P.R. and K.J.).

Recurrent stroke was defined as a new sudden neurologic

deficit or a deterioration of the previous deficit occurring after

> 24 h of stability, and not attributable to edema, hemor-

rhagic transformation or concomitant illness. For TIA, the

neurologic deficit had to have resolved completely within 24 h.

CE was defined as a myocardial infarction, hospitalization for

unstable angina, coronary-artery bypass grafting or percuta-

neous coronary intervention.

To avoid influence of an acute vascular event on convales-

cent plasma TAFI measures [6], patients suffering from a

recurrent vascular event before follow-up were excluded

(n = 42). Measurements of convalescent TAFI plasma levels

were missing in 38. Thus, the present analysis was performed

on 520 IS patients surviving free from recurrent vascular events

at follow-up after 3 months.

The small number of events precluded separate analyses of

the prespecified endpoints of death, recurrent stroke/TIA and

CE. Instead, associations with the composite endpoint of

death, recurrent stroke/TIA and/or CE were investigated.

Plasma TAFI measures and hsCRP were logarithmically

transformed. We compared baseline characteristics using

standard statistical tests. We used Cox regression to obtain

univariate and multivariable hazard ratios (HRs) for the

composite outcome. Multivariable analyses were performed

using the backward elimination procedure, including age, sex,

hypertension, smoking, diabetes, hyperlipidemia, stroke sever-

ity, TOAST subtype, anticoagulant treatment, intact TAFI,

TAFI-AP and hsCRP in the initial model. Plasma levels were

standardized so that reported HRs represented an increase by

one standard deviation (SD) (0.245 for logarithmically trans-

formed intact TAFI and 0.242 for TAFI-AP). Data were

analyzed using SPSS 18.0 (IBM, Armonk, NY, USA), and

statistical analyses were performed in a two-tailed fashion.

P < 0.05 was considered significant.

Two-year follow-up data were obtained for 517 (99.5%)

patients. During follow-up 12 deaths, 19 fatal or non-fatal

recurrent strokes, eight TIAs and three CEs occurred, resulting

in 37 composite endpoints. Patients who developed a compos-

ite endpoint during follow-up were older (P < 0.01), hadmore

severe index strokes (P < 0.01), and had higher convalescent

plasma concentrations of released TAFI-AP (P < 0.05) at

baseline as compared with patients surviving free from vascular

events (Table S1). The univariate HR of the composite

endpoint for one SD increase of TAFI-AP was 1.44 (95%

CI, 1.05–1.97) and this association remained after adjustment

for confounders (Table 1). Similar results were obtained when

excluding CE from the analysis (data not shown). In contrast

and interestingly, there was no significant association between

the composite endpoint and plasma concentration of intact

TAFI (Table 1). We also went on to investigate the possible

predictive value of the TAFI-AP/intact TAFI ratio. However,

no significant associations with the composite endpoint were

detected (univariate HR, 1.97; 95% CI, 0.83–4.14).

Prospective data from clinical studies using genotype-inde-

pendent assays of plasma TAFI are limited. However, in line

with results from our study, no association between total TAFI

and the risk of a future cardiovascular event in healthy

individuals was observed in the PRIME study [7]. Likewise, in

the AtheroGene study, a prospective study of patients with

coronary artery disease, no association betweenplasma levels of

total TAFI and future cardiovascular death was observed [8].

However, in that study, TAFIa/TAFIai, another measure of

Table 1 Hazard ratios and 95% confidence intervals of a composite

endpoint during follow-up for one SD increase in logarithmically trans-

formed convalescent plasma thrombin activatable fibrinolysis inhibitor

(TAFI) concentration

Univariate

hazard ratios

Multivariable

hazard ratios

Intact TAFI 1.20 (0.87–1.64) 0.87 (0.58–1.31)

TAFI-activation

peptide

1.44 (1.05–1.97) 1.51 (1.08–2.12)

Cox proportional hazards regression models. Multivariable analyses

were performed using the backward elimination procedure, including

age, sex, hypertension, smoking, diabetes, hyperlipidemia, stroke

severity, Trial of Org 10172 in Acute Stroke Treatment (TOAST)

subtype, anticoagulant treatment, intact TAFI, TAFI-activation pep-

tide (AP) and high-sensitivity C-reactive protein in the initial model.

Age, TAFI-AP, hyperlipidemia and stroke subtype were included in

the final model.

726 Letters to the Editor

� 2012 International Society on Thrombosis and Haemostasis

plasma-activated TAFI, but not TAFI-AP, predicted future

cardiovascular death. Whether the discrepant findings with

respect toTAFI-APin theAtheroGeneandour studyrepresents

biological differences between IS and coronary artery disease

remains to be elucidated, as the number of events in both studies

was small. Nevertheless, taken together, findings from clinical

prospective studies suggest anassociationbetweenplasma levels

of activated, but not intact, TAFI and arterial thrombotic

disease. This finding is biologically plausible as experimental

data show that it is the amount of activated TAFI, and not the

total amount, that plays a crucial role in retarding fibrinolysis

[9]. Alternatively, the association between markers for plasma-

activated TAFI and arterial thrombotic disease is mediated by

an increased thrombin generation, as TAFI activation is

dependent on the thrombin and thrombomodulin concentra-

tions.

The strengths of our study are a comprehensive follow-up

with few patients lost to follow-up, standardized blood

sampling, and genotype-independent assays. However, this

study also has important limitations, including limited statis-

tical power due to the small number of events. Moreover, it

would be of interest to investigate markers of thrombin

generation in SAHLSIS as well as in other longitudinal studies

of IS.

In conclusion, in this prospective study of IS survivors,

we found that convalescent plasma TAFI-AP, but not intact

TAFI, showed association with future death and/or recur-

rent vascular events in IS survivors, independently of

conventional vascular risk factors. Whether this finding

represents a causal role of plasma-activated TAFI in the

prognosis after IS cannot be concluded, and further studies

are thus warranted.

Acknowledgements

The authors thank collaborators within the SAHLSIS group.

The authors thank M. Peeters for excellent laboratory

assistance.

This study was supported by the Swedish Research Council

(K2011-65X-14605-09-6), the Swedish state (ALFBGB-

148861), the Swedish Heart and Lung Foundation

(20100256), the Swedish Stroke Association, the Yngve Land

Foundation for Neurological Research, the Rune and Ulla

Amlovs Foundation for Neurological Research, the John and

Brit Wennerstrom Foundation for Neurological Research, the

Per-Olof Ahl Foundation for Neurological Research, and the

Research Grant G.0676.10 of the Fund for Scientific Research-

Flanders.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

Supporting Information

Additional Supporting Informationmay be found in the online

version of this article:

Table S1. Baseline characteristics according to the occurrence

of a composite endpoint during follow-up.

Please note: Wiley-Blackwell is not responsible for the content

or functionality of any supporting materials supplied by the

authors. Any queries (other than missing material) should be

directed to the corresponding author for the article.

References

1 Boffa MB, Koschinsky ML. Curiouser and curiouser: recent advances

in measurement of thrombin-activatable fibrinolysis inhibitor (TAFI)

and in understanding its molecular genetics, gene regulation, and

biological roles. Clin Biochem 2007; 40: 431–42.

2 Ceresa E, Brouwers E, Peeters M, Jern C, Declerck PJ, Gils A.

Development of ELISAs measuring the extent of TAFI activation.

Arterioscler Thromb Vasc Biol 2006; 26: 423–8.

3 Ladenvall C, Gils A, Jood K, Blomstrand C, Declerck PJ, Jern C.

Thrombin activatable fibrinolysis inhibitor activation peptide shows

association with all major subtypes of ischemic stroke and with TAFI

gene variation. Arterioscler Thromb Vasc Biol 2007; 27: 955–62.

4 Jood K, Ladenvall C, Rosengren A, Blomstrand C, Jern C. Family

history in ischemic stroke before 70 years of age: the Sahlgrenska

Academy Study on Ischemic Stroke. Stroke 2005; 36: 1383–7.

5 Ladenvall C, Jood K, Blomstrand C, Nilsson S, Jern C, Ladenvall P.

Serum C-reactive protein concentration and genotype in relation to

ischemic stroke subtype. Stroke 2006; 37: 2018–23.

6 Brouns R, Heylen E,Willemse JL, Sheorajpanday R, De Surgeloose D,

Verkerk R, De Deyn PP, Hendriks DF. The decrease in procarboxy-

peptidase U (TAFI) concentration in acute ischemic stroke correlates

with stroke severity, evolution and outcome. J Thromb Haemost 2010;

8: 75–80.

7 Morange PE, Tregouet DA, Frere C, Luc G, Arveiler D, Ferrieres J,

Amouyel P, Evans A, Ducimetiere P, Cambien F, Tiret L, Juhan-

Vague I; Prime Study Group. TAFI gene haplotypes, TAFI plasma

levels and future risk of coronary heart disease: the PRIME Study. J

Thromb Haemost 2005; 3: 1503–10.

8 Tregouet DA, Schnabel R, Alessi MC, Godefroy T, Declerck PJ, Ni-

caud V,Munzel T, Bickel C, Rupprecht HJ, Lubos E, Zeller T, Juhan-

Vague I, Blankenberg S, Tiret L, Morange PE; AtheroGene Investi-

gators. Activated thrombin activatable fibrinolysis inhibitor levels are

associated with the risk of cardiovascular death in patients with cor-

onary artery disease: the AtheroGene study. J Thromb Haemost 2009;

7: 49–57.

9 Leurs J, Nerme V, Sim Y, Hendriks D. Carboxypeptidase U (TAFIa)

prevents lysis from proceeding into the propagation phase through a

threshold-dependent mechanism. J Thromb Haemost 2004; 2: 416–23.

Letters to the Editor 727

� 2012 International Society on Thrombosis and Haemostasis