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Soluble endothelial protein C receptor (sEPCR) levels and venous thromboembolism in carriers of two dysfunctional protein C variants
Thrombosis Research (2006) 117, 523 — 528
intl.elsevierhealth.com/journals/thre
BRIEF COMMUNICATION
Soluble endothelial protein C receptor (sEPCR) levels and venous thromboembolism in carriers of two dysfunctional protein C variants Paolo Simionia,*, Olivier Morboeuf b, Giulio Tognina, Sabrina Gavassoa, Daniela Tormenea, Barry Woodhamsc, Antonio Pagnana a
Department of Medical and Surgical Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy b Diagnostica Stago, Asnieres, France c Stago R&D, Gennevilliers, France Received 20 March 2005; received in revised form 17 April 2005; accepted 26 April 2005 Available online 25 May 2005
KEYWORDS sEPCR levels; A3 haplotype; EPCR gene; Dysfunctional PC; Thrombophilia
Abstract The role of the A3 haplotype and soluble endothelial protein C receptor (sEPCR) plasma levels in predisposing the carriers of two peculiar dysfunctional protein C (PC) variants (PC Arg-1YCys and PC Arg-1YLeu, also known as PC Padua2 and PC Padua3, respectively) to venous thromboembolism (VTE) has been evaluated. The levels of sEPCR have been assessed in family members during 6 years of followup and correlated to the presence of the A3 haplotype. Individuals who carried both a dysfunctional PC and the A3 haplotype and presenting with high levels of sEPCR experienced severe VTE at young age. The increased plasma levels of sEPCR were not related to excessive thrombin generation. Carriers of the A3 haplotype showed levels of sEPCR above 135 ng/ml (80th percentile of the distribution in healthy subjects), which remained elevated during the follow-up. In non-carriers of the A3 haplotype, sEPCR levels remained persistently around 100 ng/ml or lower. In conclusion, we have observed that elevated sEPCR plasma levels and the concomitant presence of the A3 haplotype of EPCR gene are associated with severe thrombotic manifestations in carriers of two dysfunctional PC variants. Whether high plasma levels of sEPCR and/or the presence of the A3 haplotype increase the risk of thrombosis in carriers of other PC defects or thrombophilic conditions remains to be clarified. D 2005 Elsevier Ltd. All rights reserved.
* Corresponding author. Department of Medical and Surgical Sciences, via Ospedale 105, 35100 Padua, Italy. Tel.: +39 49 8212667; fax: +39 49 8212661. E-mail address: [email protected] (P. Simioni). 0049-3848/$ - see front matter D 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2005.04.029
524
Introduction Inherited defects of the protein C (PC) system are associated with an increased tendency to venous thromboembolism (VTE) [1]. Recently, a so-called A3 haplotype in the gene encoding for the endothelial protein C receptor (EPCR) has been reported to be a candidate risk factor for VTE [2]. It has been shown that carriers of the A3 haplotype exhibit increased plasma levels of soluble EPCR (sEPCR) [2—4]. Since sEPCR inhibits the anticoagulant activity of APC by blocking its binding to phospholipids and abrogating its ability to inactivate factor Va [5], an increase in sEPCR circulating levels is likely to impair the PC system function. It has been shown that activation of protein C with the soluble thrombin—thrombomodulin complex was not altered by the presence of sEPCR [6]. Whether the presence of the A3 haplotype is associated with an increased risk of VTE is still controversial [2—4]. In a recent study, Uitte de Willige et al. [3] did not find a strong association between EPCR gene haplotypes and thrombosis risk. However, they found that low sEPCR levels appeared to reduce the risk of thrombosis [3]. We have evaluated the role of the A3 haplotype and sEPCR plasma levels in predisposing the carriers of dysfunctional PC variants belonging to two Italian families to thrombotic manifestations. The two peculiar naturally occurring PC variants (i.e., Arg-1YCys and Arg-1YLeu) are elongated at the NH2 terminal end of the light chains [7,8] and cannot bind to phospholipid and/or EPCR and/or sEPCR [9]. Since carriers of these PC variants were heterozygous, sEPCR was expected to bind and block the normal and functionally active component of PC/aPC present in patients’ plasma resulting in increased thrombotic tendency. Because many blood samples were collected during the follow-up at the Thrombosis Centre of Padua University-Hospital, we had the opportunity to determine the levels of sEPCR in several family members during a period of up to 6 years and correlate them to the presence of the A3 haplotype in those who were also carriers of this genotype.
Materials and methods Patients and sample collection Clinical and laboratory details of family members with Arg-1YCys or Arg-1YLeu have been previously reported [7,8]. Blood samples were drawn and
P. Simioni et al. clinical information were recorded at each visit. Blood was collected into plastic syringes that contained 0.129 M sodium citrate, in a ratio of 1 : 9 (vol / vol) anticoagulant to blood. Platelet poor plasma was obtained by centrifugation at 2000 g for 20 min and stored at 80 8C until it was analyzed. Pellet of leukocytes were stored for DNA extraction using standard methods. The study was approved by the Ethics Committee of the University of Padua Medical School, Padua.
Laboratory assays Routine laboratory tests as well as protein C antigen, chromogenic and coagulometric activity levels were performed as previously described [7,8]. Briefly, PC antigen was assessed by ELISA using anti-PC polyclonal antibody from DAKO (Glostrup, Denmark) as catching antibody and anti-PC polyclonal antibody conjugated with horseradish peroxidase (DAKO, Denmark) as second antibody [7,8]. Chromogenic and coagulometric activities were measured in plasma using the Behrichrom PC and the PC Reagent Kit (DadeBehring, Marburg, Germany), respectively. The affected family members typically presented with a heterozygous type IIb PC defect with normal antigen and chromogenic activity levels but the coagulometric activity reduced to half the normal level [7,8]. No other thrombophilic conditions such as antithrombin or protein S defects, factor V Leiden mutation or prothrombin variant G20210A, anticardiolipin antibodies or lupus anticoagulant were present in the family members investigated.
Prothrombin fragment1 + 2 (F1 + 2) ELISA F1 + 2 were measured using Enzignost F1 + 2 micro (Dade-Behring, Marburg, Germany) according to the manufacturers instructions.
sEPCR ELISA Plasma levels of sEPCR were measured with Asserachrom sEPCR kit (Diagnostica Stago, Asnieres, France) according to the manufacturer’s instructions. To obtain the normal ranges, plasma level of sEPCR was assessed in 105 healthy subjects of both sexes aged 20 to 70. The mean level was 92.5 F 48.3 ng/ml (mean F SD). A bimodal distribution of sEPCR plasma levels was observed in normal subjects as already reported [2]. A cut-off level of 135 ng/ml of sEPCR corresponding to the
Main characteristics of family members investigated
Family
Sex
Age (y)
Clinical symptoms
P P1*
M
48
P2 P3 P4 P5 P6 P7
F F M M F F
48 36 74 20 2 80
P8 P9 P10 B B1
F M F
8 25 9
Recurrent DVT + PE (1st episode at 37 y) No No No No No Recurrent DVT + PE (1st episode at 32 y) No No No
F
58
B2
F
74
DVT at 47 y/SVT at 52 y No
PC Antigen % (nv. 70—130)
PC Chromogenic activity % (nv. 70—130)
PC Coagulometric activity % (nv. 80—120)
F1 + 2 (nv. 0.4—1.1 nmol/l)
sEPCR1 (nv. 92.5 F 48.3 ng/ml)
A3 Haplotype
PC variant
52
33
8
0.47
162
Hetero
PC Arg-1YCys Hetero
121 122 130 130 92 140
111 106 83 70 101 90
63 20 45 30 44 38
0.85 0.75 1.35 0.8 1.0 1.1
74 85 197 250 63 220
No No Hetero Hetero No Hetero
Hetero Hetero Hetero Hetero Hetero Hetero
104 84 97
104 71 83
46 602 21
1.19 0.59 1.06
59 108 102
No No No
106
93
37
1.22
98
No
Hetero No Hetero PC Arg-1YLeu Hetero
110
121
52
1.32
94
No
Hetero
sEPCR, A3 haplotype and dysfunctional PC
Table 1
*Under oral anticoagulant treatment; DVT: deep vein thrombosis; SVT: superficial vein thrombosis; PE: pulmonary embolism; nv.: normal values; y: years. 1 Level of sEPCR at the first visit; 2level of PC at the first visit; in the following visits PC level was always found to be around 80%.
525
526
P. Simioni et al.
80th percentile of the distribution in healthy subjects was selected. This cut-off was similar to that previously reported by Saposnik et al. [2]. Thus, patients were considered to have high sEPCR if plasma levels were above 135 ng/ml.
presence of two fragments only of 251 and 42 bp, respectively.
Haplotype A3 identification
The main clinical and laboratory characteristics of the individuals investigated are gathered in Table 1. Plasma levels of sEPCR above the cut-off value (135 ng/ml) were found in 4 subjects (one patient under oral anticoagulant treatment [OAT]) out of 12 family members. All of these were double heterozygous carriers of both the PC gene lesion (Arg-1YCys) and the A3 haplotype of EPCR gene (Table 1 and Fig. 1A). Two out of the four (patients P1 and P7), developed severe VTE characterized by recurrent episodes of deep vein thrombosis (DVT) and pulmonary embolism. The first VTE episode developed at the age of 37 and 32 in patient P1 and P7, respectively. Seven family members presenting with the dysfunctional PC alone showed levels of sEPCR in the normal range (within mean F SD). Among them, four individuals were older than 15
For the detection of the A3 haplotype a new method was developed using the G at nucleotide 1651 (according to the Genebank sequence AF106202) as a marker, which was different from that originally used by Saposnik et al. [2]. The region surrounding nucleotide 1651 was amplified using oligonucleotide 5V-gCTgAAATTTTgTATTCTgTCC-3V as upstream primer and 5V-CCAgTATAATggCTACATTTTACC-3V as downstream primer. The annealing temperature was 55 8C. The 293 bp PCR product was digested by using BstE II restriction enzyme which recognizes two cleavage sites in the presence of the wild type allele leading to three fragments of 176, 75 and 42 bp, respectively. The presence of the C1651G polymorphism, abolishes one cleavage site resulting in the
Results and discussion
Figure 1 (A) A3 haplotype identification method. Two percent agarose gel electrophoresis of PCR amplification product surrounding the region of the polymorphism at nucleotide 1651 (C/G). Nucleotide number is according to Genbank sequence AF106202). The PCR product is digested by restriction enzyme BstE II. In the presence of A3 haplotype the PCR product is not cleaved at position 1651 and a band of 251 bp appears. Individuals P1, P4, P5 and P7 are heterozygous for A3 haplotype. (B) sEPCR plasma levels in family members. All six individuals were carriers of heterozygous PC Arg-1YCys and were followed up to 6 years. Individuals P1, P4 and P5 were also carriers of heterozygous A3 haplotype. Individual P1 is under oral anticoagulant treatment.
sEPCR, A3 haplotype and dysfunctional PC years, exhibited sEPCR levels ranging between 52 and 98 ng/ml and, only in one case, provoked DVT (after surgery at the age of 47) and superficial vein thrombosis (after hormonal replacement therapy at the age of 52) had occurred. Thus, individuals carrying both the dysfunctional PC and the A3 haplotype and presenting with higher levels of sEPCR, have experienced severe clinical manifestations. Even though the limited number of cases does not allow us to draw definite conclusions, it is interesting to observe that carriers of dysfunctional PC alone with sEPCR levels in the lower range were mildly symptomatic as compared to carriers of the double defect. Since sEPCR is not able to bind PC Arg-1YCys or PC Arg-1YLeu because of the severe de-arrangement of the Gla-domain of these two molecules [9], the markedly increased levels of sEPCR related to the presence of the A3 haplotype may act as an additional risk for thrombosis by inhibiting the normal component of PC/APC in patients’ plasma. On the other hand, low levels of sEPCR found in several heterozygous carriers of dysfunctional PC alone might contribute to reduce such a risk. It has already been shown, in fact, that reduced levels of plasma sEPCR are associated with a reduced risk of thrombosis [3]. Unfortunately, no family members with the A3 haplotype alone and only one with neither A3 haplotype nor PC defect were available for this study. Therefore no information is available on the risk of thrombosis in these two situations. F1 + 2 levels did not correlate to the severity of the underlying genetic defect predisposing to VTE in these patients. In fact, no significant difference in the mean levels of F1 + 2 was evident between carriers of single or double gene lesion (Table 1). In addition, we did not find a correlation between F1 + 2 and sEPCR levels, excluding excessive thrombin generation to be responsible for the increase in circulating sEPCR. Do plasma levels of sEPCR remain constant in time in carriers of the A3 haplotype and in noncarriers accounting for persistent risk of or protection from thrombosis in these subjects with dysfunctional PC variants? We have attempted to address this issue by determining the levels of sEPCR in consecutive samples from six family members who were followed for up to six years. Three out of the six were heterozygous carriers of the A3 haplotype, one of whom (individual P1) was under stable OAT throughout the follow-up. The remaining three were non-carriers of the A3 haplotype. The levels of plasma sEPCR in these individuals during the follow-up are shown in Fig. 1B. Plasma sEPCR levels appeared to remain constantly elevated in subjects with the A3 haplo-
527 type (individuals P4 and P5). In patient P1 (carrier of both dysfunctional PC and A3 haplotype), sEPCR levels were slightly lower as compared to those observed in the other carriers of the A3 haplotype at similar time points. This can be related to lifelong OAT given to this patient. The effect of OAT in reducing sEPCR plasma levels has been previously reported [10]. Interestingly, the sEPCR levels in the OAT patient remained constantly above those observed in non-carriers of the A3 haplotype. Although these findings suggest that the reduction of sEPCR levels during OAT can be modulated by the A3 haplotype no conclusion can be drawn after the observation of a single subject on OAT. Finally, in non-carriers of the A3 haplotype, sEPCR levels remained constantly around 100 ng/ ml (patient P10) or even persistently lower (individuals P2 and P3). Taken together these findings suggest that elevated sEPCR levels might increase the risk for VTE in carriers of peculiar dysfunctional PC molecules. We confirm previous observation that the A3 haplotype is a genetic determinant of the elevation of sEPCR in plasma [2—4] which is also evident during long term follow-up. In addition, constantly low levels of sEPCR in plasma, in the absence of A3 haplotype, might play a protective role against thrombosis in carriers of dysfunctional PC. We believe that larger studies of individuals with different PC defects (or other coagulation defects) and a prospective follow-up might be useful to verify whether genetically controlled sEPCR levels are likely to influence the clinical expression of inherited thrombophilic disorders.
Acknowledgements This work was supported by Grants n. 60A07-8834/ 02 from M.U.R.S.T. Italy (to PS). O.M. and B.W. are employed by Diagnostica Stago and Stago R and D whose product was studied in the present work.
References [1] Dahlback B. The protein C anticoagulant system: inherited defects as basis for venous thrombosis. Thromb Res 1995; 77:1 – 43. [2] Saposnik B, Reny JL, Gaussem P, Emmerich J, Aiach M, Gandrille S. A haplotype of the EPCR gene is associated with increase plasma levels of sEPCR and is a candidate risk factor for thrombosis. Blood 2004;103:1311 – 8. [3] Uitte de Willige S, van Marion V, Rosendaal FR, Vos HL, de Visser MCH, Bertina RM. Haplotypes of the EPCR gene,
528 plasma sEPCR levels and the risk of deep venous thrombosis. J Thromb Haemost 2004;2:1305 – 10. [4] Medina P, Navarro S, Estelle ´s A, Vaya ` A, Woodhams B, Mira Y, et al. Contribution of polymorphisms in the endothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk. Thromb Heamost 2004;91: 905 – 11. [5] Liaw PC, Neuenschwander PF, Smirnov MD, Esmon CT. Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function. J Biol Chem 2000;275:5447 – 52. [6] Regan LM, Stearns-Kurosawa DJ, Kurosawa S, Mollica J, Fukudome K, Esmon CT. The endothelial cell protein C receptor: inhibition of activated protein C anticoagulant function without modulation of reaction with proteinase inhibitors. J Biol Chem 1996;271:17499 – 503.
P. Simioni et al. [7] Wojcik EGC, Simioni P, van den Berg M, Girolami A, Bertina RM. Mutations which introduce free cysteine residues in the Gla-domain of vitamin K dependent proteins result in the formation of complexes with a-1-microglobulin. Thromb Haemost 1996;75:70 – 5. [8] Simioni P, Kalafatis M, Tormene D, Luni S, Zerbinati P, Barzon L, et al. Abnormal propeptide processing resulting in the presence of two abnormal species of protein C in plasma. Thromb Haemost 2001;86:1017 – 22. [9] Preston RJS, Villegas-Mendez A, Sun Y-H, Hermida J, Simioni H, Philippou H, et al. Selective modulation of protein C affinity for EPCR and phospholipids by Gla-domain mutation. FEBS J 2005;272:97 – 108. [10] Stearns-Kurosawa DJ, Swindle K, D’Angelo A, Della Valle P, Fattorini A, Caron N, et al. Plasma levels of endothelial protein C receptor respond to anticoagulant treatment. Blood 2002;99:526 – 30.
intl.elsevierhealth.com/journals/thre
BRIEF COMMUNICATION
Soluble endothelial protein C receptor (sEPCR) levels and venous thromboembolism in carriers of two dysfunctional protein C variants Paolo Simionia,*, Olivier Morboeuf b, Giulio Tognina, Sabrina Gavassoa, Daniela Tormenea, Barry Woodhamsc, Antonio Pagnana a
Department of Medical and Surgical Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy b Diagnostica Stago, Asnieres, France c Stago R&D, Gennevilliers, France Received 20 March 2005; received in revised form 17 April 2005; accepted 26 April 2005 Available online 25 May 2005
KEYWORDS sEPCR levels; A3 haplotype; EPCR gene; Dysfunctional PC; Thrombophilia
Abstract The role of the A3 haplotype and soluble endothelial protein C receptor (sEPCR) plasma levels in predisposing the carriers of two peculiar dysfunctional protein C (PC) variants (PC Arg-1YCys and PC Arg-1YLeu, also known as PC Padua2 and PC Padua3, respectively) to venous thromboembolism (VTE) has been evaluated. The levels of sEPCR have been assessed in family members during 6 years of followup and correlated to the presence of the A3 haplotype. Individuals who carried both a dysfunctional PC and the A3 haplotype and presenting with high levels of sEPCR experienced severe VTE at young age. The increased plasma levels of sEPCR were not related to excessive thrombin generation. Carriers of the A3 haplotype showed levels of sEPCR above 135 ng/ml (80th percentile of the distribution in healthy subjects), which remained elevated during the follow-up. In non-carriers of the A3 haplotype, sEPCR levels remained persistently around 100 ng/ml or lower. In conclusion, we have observed that elevated sEPCR plasma levels and the concomitant presence of the A3 haplotype of EPCR gene are associated with severe thrombotic manifestations in carriers of two dysfunctional PC variants. Whether high plasma levels of sEPCR and/or the presence of the A3 haplotype increase the risk of thrombosis in carriers of other PC defects or thrombophilic conditions remains to be clarified. D 2005 Elsevier Ltd. All rights reserved.
* Corresponding author. Department of Medical and Surgical Sciences, via Ospedale 105, 35100 Padua, Italy. Tel.: +39 49 8212667; fax: +39 49 8212661. E-mail address: [email protected] (P. Simioni). 0049-3848/$ - see front matter D 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2005.04.029
524
Introduction Inherited defects of the protein C (PC) system are associated with an increased tendency to venous thromboembolism (VTE) [1]. Recently, a so-called A3 haplotype in the gene encoding for the endothelial protein C receptor (EPCR) has been reported to be a candidate risk factor for VTE [2]. It has been shown that carriers of the A3 haplotype exhibit increased plasma levels of soluble EPCR (sEPCR) [2—4]. Since sEPCR inhibits the anticoagulant activity of APC by blocking its binding to phospholipids and abrogating its ability to inactivate factor Va [5], an increase in sEPCR circulating levels is likely to impair the PC system function. It has been shown that activation of protein C with the soluble thrombin—thrombomodulin complex was not altered by the presence of sEPCR [6]. Whether the presence of the A3 haplotype is associated with an increased risk of VTE is still controversial [2—4]. In a recent study, Uitte de Willige et al. [3] did not find a strong association between EPCR gene haplotypes and thrombosis risk. However, they found that low sEPCR levels appeared to reduce the risk of thrombosis [3]. We have evaluated the role of the A3 haplotype and sEPCR plasma levels in predisposing the carriers of dysfunctional PC variants belonging to two Italian families to thrombotic manifestations. The two peculiar naturally occurring PC variants (i.e., Arg-1YCys and Arg-1YLeu) are elongated at the NH2 terminal end of the light chains [7,8] and cannot bind to phospholipid and/or EPCR and/or sEPCR [9]. Since carriers of these PC variants were heterozygous, sEPCR was expected to bind and block the normal and functionally active component of PC/aPC present in patients’ plasma resulting in increased thrombotic tendency. Because many blood samples were collected during the follow-up at the Thrombosis Centre of Padua University-Hospital, we had the opportunity to determine the levels of sEPCR in several family members during a period of up to 6 years and correlate them to the presence of the A3 haplotype in those who were also carriers of this genotype.
Materials and methods Patients and sample collection Clinical and laboratory details of family members with Arg-1YCys or Arg-1YLeu have been previously reported [7,8]. Blood samples were drawn and
P. Simioni et al. clinical information were recorded at each visit. Blood was collected into plastic syringes that contained 0.129 M sodium citrate, in a ratio of 1 : 9 (vol / vol) anticoagulant to blood. Platelet poor plasma was obtained by centrifugation at 2000 g for 20 min and stored at 80 8C until it was analyzed. Pellet of leukocytes were stored for DNA extraction using standard methods. The study was approved by the Ethics Committee of the University of Padua Medical School, Padua.
Laboratory assays Routine laboratory tests as well as protein C antigen, chromogenic and coagulometric activity levels were performed as previously described [7,8]. Briefly, PC antigen was assessed by ELISA using anti-PC polyclonal antibody from DAKO (Glostrup, Denmark) as catching antibody and anti-PC polyclonal antibody conjugated with horseradish peroxidase (DAKO, Denmark) as second antibody [7,8]. Chromogenic and coagulometric activities were measured in plasma using the Behrichrom PC and the PC Reagent Kit (DadeBehring, Marburg, Germany), respectively. The affected family members typically presented with a heterozygous type IIb PC defect with normal antigen and chromogenic activity levels but the coagulometric activity reduced to half the normal level [7,8]. No other thrombophilic conditions such as antithrombin or protein S defects, factor V Leiden mutation or prothrombin variant G20210A, anticardiolipin antibodies or lupus anticoagulant were present in the family members investigated.
Prothrombin fragment1 + 2 (F1 + 2) ELISA F1 + 2 were measured using Enzignost F1 + 2 micro (Dade-Behring, Marburg, Germany) according to the manufacturers instructions.
sEPCR ELISA Plasma levels of sEPCR were measured with Asserachrom sEPCR kit (Diagnostica Stago, Asnieres, France) according to the manufacturer’s instructions. To obtain the normal ranges, plasma level of sEPCR was assessed in 105 healthy subjects of both sexes aged 20 to 70. The mean level was 92.5 F 48.3 ng/ml (mean F SD). A bimodal distribution of sEPCR plasma levels was observed in normal subjects as already reported [2]. A cut-off level of 135 ng/ml of sEPCR corresponding to the
Main characteristics of family members investigated
Family
Sex
Age (y)
Clinical symptoms
P P1*
M
48
P2 P3 P4 P5 P6 P7
F F M M F F
48 36 74 20 2 80
P8 P9 P10 B B1
F M F
8 25 9
Recurrent DVT + PE (1st episode at 37 y) No No No No No Recurrent DVT + PE (1st episode at 32 y) No No No
F
58
B2
F
74
DVT at 47 y/SVT at 52 y No
PC Antigen % (nv. 70—130)
PC Chromogenic activity % (nv. 70—130)
PC Coagulometric activity % (nv. 80—120)
F1 + 2 (nv. 0.4—1.1 nmol/l)
sEPCR1 (nv. 92.5 F 48.3 ng/ml)
A3 Haplotype
PC variant
52
33
8
0.47
162
Hetero
PC Arg-1YCys Hetero
121 122 130 130 92 140
111 106 83 70 101 90
63 20 45 30 44 38
0.85 0.75 1.35 0.8 1.0 1.1
74 85 197 250 63 220
No No Hetero Hetero No Hetero
Hetero Hetero Hetero Hetero Hetero Hetero
104 84 97
104 71 83
46 602 21
1.19 0.59 1.06
59 108 102
No No No
106
93
37
1.22
98
No
Hetero No Hetero PC Arg-1YLeu Hetero
110
121
52
1.32
94
No
Hetero
sEPCR, A3 haplotype and dysfunctional PC
Table 1
*Under oral anticoagulant treatment; DVT: deep vein thrombosis; SVT: superficial vein thrombosis; PE: pulmonary embolism; nv.: normal values; y: years. 1 Level of sEPCR at the first visit; 2level of PC at the first visit; in the following visits PC level was always found to be around 80%.
525
526
P. Simioni et al.
80th percentile of the distribution in healthy subjects was selected. This cut-off was similar to that previously reported by Saposnik et al. [2]. Thus, patients were considered to have high sEPCR if plasma levels were above 135 ng/ml.
presence of two fragments only of 251 and 42 bp, respectively.
Haplotype A3 identification
The main clinical and laboratory characteristics of the individuals investigated are gathered in Table 1. Plasma levels of sEPCR above the cut-off value (135 ng/ml) were found in 4 subjects (one patient under oral anticoagulant treatment [OAT]) out of 12 family members. All of these were double heterozygous carriers of both the PC gene lesion (Arg-1YCys) and the A3 haplotype of EPCR gene (Table 1 and Fig. 1A). Two out of the four (patients P1 and P7), developed severe VTE characterized by recurrent episodes of deep vein thrombosis (DVT) and pulmonary embolism. The first VTE episode developed at the age of 37 and 32 in patient P1 and P7, respectively. Seven family members presenting with the dysfunctional PC alone showed levels of sEPCR in the normal range (within mean F SD). Among them, four individuals were older than 15
For the detection of the A3 haplotype a new method was developed using the G at nucleotide 1651 (according to the Genebank sequence AF106202) as a marker, which was different from that originally used by Saposnik et al. [2]. The region surrounding nucleotide 1651 was amplified using oligonucleotide 5V-gCTgAAATTTTgTATTCTgTCC-3V as upstream primer and 5V-CCAgTATAATggCTACATTTTACC-3V as downstream primer. The annealing temperature was 55 8C. The 293 bp PCR product was digested by using BstE II restriction enzyme which recognizes two cleavage sites in the presence of the wild type allele leading to three fragments of 176, 75 and 42 bp, respectively. The presence of the C1651G polymorphism, abolishes one cleavage site resulting in the
Results and discussion
Figure 1 (A) A3 haplotype identification method. Two percent agarose gel electrophoresis of PCR amplification product surrounding the region of the polymorphism at nucleotide 1651 (C/G). Nucleotide number is according to Genbank sequence AF106202). The PCR product is digested by restriction enzyme BstE II. In the presence of A3 haplotype the PCR product is not cleaved at position 1651 and a band of 251 bp appears. Individuals P1, P4, P5 and P7 are heterozygous for A3 haplotype. (B) sEPCR plasma levels in family members. All six individuals were carriers of heterozygous PC Arg-1YCys and were followed up to 6 years. Individuals P1, P4 and P5 were also carriers of heterozygous A3 haplotype. Individual P1 is under oral anticoagulant treatment.
sEPCR, A3 haplotype and dysfunctional PC years, exhibited sEPCR levels ranging between 52 and 98 ng/ml and, only in one case, provoked DVT (after surgery at the age of 47) and superficial vein thrombosis (after hormonal replacement therapy at the age of 52) had occurred. Thus, individuals carrying both the dysfunctional PC and the A3 haplotype and presenting with higher levels of sEPCR, have experienced severe clinical manifestations. Even though the limited number of cases does not allow us to draw definite conclusions, it is interesting to observe that carriers of dysfunctional PC alone with sEPCR levels in the lower range were mildly symptomatic as compared to carriers of the double defect. Since sEPCR is not able to bind PC Arg-1YCys or PC Arg-1YLeu because of the severe de-arrangement of the Gla-domain of these two molecules [9], the markedly increased levels of sEPCR related to the presence of the A3 haplotype may act as an additional risk for thrombosis by inhibiting the normal component of PC/APC in patients’ plasma. On the other hand, low levels of sEPCR found in several heterozygous carriers of dysfunctional PC alone might contribute to reduce such a risk. It has already been shown, in fact, that reduced levels of plasma sEPCR are associated with a reduced risk of thrombosis [3]. Unfortunately, no family members with the A3 haplotype alone and only one with neither A3 haplotype nor PC defect were available for this study. Therefore no information is available on the risk of thrombosis in these two situations. F1 + 2 levels did not correlate to the severity of the underlying genetic defect predisposing to VTE in these patients. In fact, no significant difference in the mean levels of F1 + 2 was evident between carriers of single or double gene lesion (Table 1). In addition, we did not find a correlation between F1 + 2 and sEPCR levels, excluding excessive thrombin generation to be responsible for the increase in circulating sEPCR. Do plasma levels of sEPCR remain constant in time in carriers of the A3 haplotype and in noncarriers accounting for persistent risk of or protection from thrombosis in these subjects with dysfunctional PC variants? We have attempted to address this issue by determining the levels of sEPCR in consecutive samples from six family members who were followed for up to six years. Three out of the six were heterozygous carriers of the A3 haplotype, one of whom (individual P1) was under stable OAT throughout the follow-up. The remaining three were non-carriers of the A3 haplotype. The levels of plasma sEPCR in these individuals during the follow-up are shown in Fig. 1B. Plasma sEPCR levels appeared to remain constantly elevated in subjects with the A3 haplo-
527 type (individuals P4 and P5). In patient P1 (carrier of both dysfunctional PC and A3 haplotype), sEPCR levels were slightly lower as compared to those observed in the other carriers of the A3 haplotype at similar time points. This can be related to lifelong OAT given to this patient. The effect of OAT in reducing sEPCR plasma levels has been previously reported [10]. Interestingly, the sEPCR levels in the OAT patient remained constantly above those observed in non-carriers of the A3 haplotype. Although these findings suggest that the reduction of sEPCR levels during OAT can be modulated by the A3 haplotype no conclusion can be drawn after the observation of a single subject on OAT. Finally, in non-carriers of the A3 haplotype, sEPCR levels remained constantly around 100 ng/ ml (patient P10) or even persistently lower (individuals P2 and P3). Taken together these findings suggest that elevated sEPCR levels might increase the risk for VTE in carriers of peculiar dysfunctional PC molecules. We confirm previous observation that the A3 haplotype is a genetic determinant of the elevation of sEPCR in plasma [2—4] which is also evident during long term follow-up. In addition, constantly low levels of sEPCR in plasma, in the absence of A3 haplotype, might play a protective role against thrombosis in carriers of dysfunctional PC. We believe that larger studies of individuals with different PC defects (or other coagulation defects) and a prospective follow-up might be useful to verify whether genetically controlled sEPCR levels are likely to influence the clinical expression of inherited thrombophilic disorders.
Acknowledgements This work was supported by Grants n. 60A07-8834/ 02 from M.U.R.S.T. Italy (to PS). O.M. and B.W. are employed by Diagnostica Stago and Stago R and D whose product was studied in the present work.
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