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Keywords:

  • endogenous thrombin potential;
  • genotype;
  • hemophilia A;
  • hemophilia B;
  • phenotype

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Summary. Background: Patients with severe hemophilia may show very varied bleeding tendencies, and the reasons for this heterogeneous clinical expression are unclear. The factor VIII/FIX genotype is the main determinant of the residual factor activity; however, different bleeding phenotypes have also been reported in patients sharing the same mutation. Such global coagulation tests as thrombin generation assays are tools with which to investigate different coagulation profiles among severe hemophiliacs. Objectives, patients and methods: This case–control study was aimed at comprehensively evaluating the role of genotype and endogenous thrombin potential (ETP) as predictors of the clinical phenotype in severe hemophiliacs with an extremely mild bleeding tendency (cases, n = 22), in comparison with those showing a typical bleeding tendency (controls, n = 50). Results: Cases were more frequently affected by hemophilia B than by hemophilia A, and showed a lower incidence of severe FVIII/FIX gene defects (referred to as null mutations), higher FVIII and FIX antigen levels and higher ETP values in platelet-rich plasma than controls (P < 0.05). By multivariate logistic regression, only non-null mutations were confirmed as an independent predictor of a mild clinical phenotype. Conclusions: These results indicate that non-null mutations represent the main determinant of the bleeding tendency, and that ETP measurement in platelet-rich plasma is able to identify severe hemophiliacs with a mild clinical phenotype.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

On the basis of the degree of the plasmatic deficiency of the coagulant activity of factor VIII or FIX (FVIII:C and FIX:C), hemophiliacs usually present with different bleeding tendencies. A typical patient with severe hemophilia (plasma factor < 1 IU dL−1) develops spontaneous hemorrhages into joints, muscles or soft tissues that, on average, require coagulation factor replacement two or more times per month [1,2]. However, the severity and frequency of bleeding may be different in hemophiliacs sharing the same factor activity, a mild bleeding tendency being reported in 10–15% of severe hemophiliacs [1,2]. The underlying gene mutation is responsible for the residual level of FVIII:C/FIX:C, so that molecular defects that totally prevent the synthesis of the protein (referred to as null mutations) are usually associated with undetectable factor activity, whereas non-null mutations account for variable factor levels [3,4]. Nevertheless, different clinical phenotypes have been reported in hemophiliacs with identical mutations in the FVIII or FIX genes (F8 and F9) [3,4]. The basis for this heterogeneity in the clinical expression of severe hemophilia is still poorly understood, and possible explanations include the effect of mutations or polymorphisms at other loci, or interlaboratory differences in FVIII:C and FIX:C assays. It has been suggested, for instance, that the mild hypercoagulable state associated with gain-of-function thrombophilic mutations such as FV Leiden or prothrombin G20210A may protect hemophiliacs from excessive bleeding; however, the clinical significance of these polymorphisms is still controversial [5–9]. Other coagulation abnormalities, pertaining to natural anticoagulants, platelets and fibrinolytic factors, have also been investigated but their role as modulators of disease severity seems negligible [10,11].

Global coagulation tests, which are able to reflect the ensemble of plasmatic procoagulant and anticoagulant activities, may be more sensitive than functional factor assays for the investigatation of hypocoagulable and hypercoagulable states [12,13]. In the hemophilia setting, thrombin generation measured as endogenous thrombin potential (ETP) is significantly correlated with FVIII:C levels [13–15]. However, a wide range of ETP values was reported in patients with undetectable FVIII:C [14], suggesting the potential of this test for distinguishing different bleeding tendencies among severe hemophiliacs.

The goal of this study was to evaluate, at the same time, the roles of F8/F9 mutations, thrombophilic polymorphisms and thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP) as determinants of the clinical phenotype in patients with severe hemophilia, by comparing those showing an extremely mild bleeding tendency with those with a typically severe phenotype.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Study design

This was a single-centre case–control study approved by the Institutional Review Board. Written informed consent for clinical data collection and laboratory measurements was obtained. Data on F8/F9 genotype, age at first bleed and at first joint bleed, annual bleeding frequency and concentrate consumption were collected from medical records and patient diaries. X-rays of elbows, knees and ankles obtained during the last 5 years prior to study entry were also evaluated by the same operator. Orthopedic examination and blood sampling were performed at enrollment. Radiologic and orthopedic joint scores were assigned according to the scoring system recommended by the Orthopaedic Advisory Committee of the World Federation of Hemophilia [16,17]. A score of zero reflects normal features in all six joints, the maximum radiologic score is 78 points (possible score per joint: 0–13) and the maximum orthopedic score is 90 points (possible score per joint: 0–15).

Patients

Adult patients (age ≥ 18 years) with severe hemophilia (FVIII:C/FIX:C of < 1 IU dL−1), with no history of inhibitors and treated exclusively on demand since diagnosis were considered to be eligible. Any detectable level of FVIII:C by chromogenic assay was considered to be an exclusion criterion. F8/F9 gene defects were classified as null mutations (large deletions, inversions and nonsense mutations) or non-null mutations (small deletions/insertions, and splice site and missense mutations).

Before the study was started, patients were defined as cases or controls on the basis of their bleeding frequency and concentrate consumption (excluding major injuries and surgical procedures). Cases (mild bleeders) were patients with an extremely mild bleeding tendency, defined by an annual frequency of two or fewer bleeding episodes and a median lifelong concentrate consumption lower than 500 IU kg−1 year−1. Controls were patients with an annual bleeding frequency of more than two episodes and/or a median concentrate consumption higher than 500 IU kg−1 year−1. The subgroup of controls characterized by a markedly severe bleeding tendency (severe bleeders) was defined on the basis of an annual bleeding frequency of 25 or more episodes requiring treatment in the last 5 years prior to enrollment plus a median concentrate consumption of > 2000 IU kg−1 year−1 during the same time period.

Laboratory measurements

Plasma preparation  Blood was drawn by clean venipuncture after a washout period of at least 5 days from the last concentrate infusion and collected in vacuum tubes (Becton Dickinson, Meylan, France) containing 109 mm trisodium citrate as anticoagulant in the ratio of one part anticoagulant to nine partsblood. Blood was centrifuged within 30 min at controlled room temperature with two different procedures.

Procedure A involved centrifugation for 20 min at 2880 × g (controlled room temperature) and separation of PPP, which was aliquoted, quick-frozen in liquid nitrogen and stored at − 70 °C until being tested for thrombin generation and used for conventional coagulation assays.

Procedure B involved centrifugation for 5 min at 150 × g (controlled room temperature) to obtain PRP. Supernatant plasma was harvested, and platelets were counted (ABX Micros 60; ABX International, Montpellier, France). Platelet numbers from each subject were then adjusted by appropriate dilutions of the autologous PRP into autologous PPP to a standard platelet count of 150 × 109 L−1. For each subject, platelet numbers were also adjusted to correspond to the whole blood count. PRP was tested for thrombin generation within 2 h of preparation.

FVIII:C and FIX:C were measured with one-stage coagulation bioassays, using the appropriate deficient plasmas as substrate and the activated partial thromboplastin time as reagent. Measurements were performed on multiple dilutions of reference and test plasmas, and the calculation of results was performed with parallel-lines bioassay. Reference plasmas were calibrated against the international standard for FVIII or FIX. The sensitivity threshold for both assays was 1 IU dL−1.

Chromogenic FVIII:C was measured with a two-stage amidolytic assay with commercially available kits (Coamatic Factor VIII; Chromogenix, Lexington, MA, USA). Reference plasma was the same as for the FVIII:C assay.

FVIII antigen (FVIII:Ag) and FIX antigen (FIX:Ag) were measured with appropriate enzyme-linked immunosorbent assay-based methods, using commercially available kits (Asserachrom VIII:Ag; Diagnostica Stago, Asnieres, France) (FIX-EIA; Affinity Biologicals Inc., Ancaster, ON, Canada). Reference plasmas were the same as for FVIII:C/FIX:C assays. The sensitivity threshold for both assays was 0.5 IU dL−1.

The thrombin generation assay (TGA) was performed according to Hemker et al. [18], as described in detail by Chantarangkul et al. [14]. The test is based on the activation of coagulation in PPP or PRP after addition of human relipidated recombinant tissue factor (Recombiplastin; Instrumentation Laboratory, Orangeburg, NY, USA), which acts as a coagulation trigger in the presence (only for the measurement in PPP) of the synthetic phospholipids 1,2-dioleoyl-sn-glycero-3-phosphoserine, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine and 1,2-dioleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids Inc., Alabaster, AL, USA), in a molar ratio of 20 : 20 : 60. The concentrations of tissue factor and phospholipids (in PPP only) in the test system were 2 pm and 1.0 μm, respectively. Continuous registration of the generated thrombin was achieved with a fluorogenic synthetic substrate (Z-Gly-Gly-Arg-AMC-HCl; Bachem, Bubendorf, Switzerland) added to the test system to a final concentration of 617 μm. The procedure was performed with an automated fluorometer (Fluoroskan Ascent; ThermoLabsystem, Helsinki, Finland). Readings from the fluorometer were automatically recorded and calculated with dedicated software (Thrombinoscope; Thrombinoscope BV, Maastricht, The Netherlands), which displays thrombin generation curves (time vs. generated thrombin) and calculates the area under the curve, defined as ETP and expressed as nm thrombin × minutes. Thrombin generation is measured as function of an internal calibrator for thrombin (Thrombin Calibrator; Thrombinoscope BV). ETP represents the balance between the actions of procoagulants and anticoagulants in plasma.

Molecular analysis  Intron 22 and intron 1 inversions were detected by polymerase chain reaction (PCR), and F8 mutational analysis was performed by direct gene sequencing [19]. F9 was studied by direct sequencing after PCR analysis of the relevant fragments [20]. FV G1691A (FV Leiden) and prothrombin G20210A mutations were detected by PCR amplification methods.

Statistical analysis

Continuous variables were expressed as median values and interquartile ranges (IQRs), and were compared by the Mann–Whitney U-test. Categorical variables were expressed as frequencies and percentage values, and compared by chi-square or Fisher’s exact test. Type of hemophilia [hemophilia A (HA) or hemophilia B (HB)], F8/F9 mutation, FVIII:Ag/FIX:Ag levels and ETP values were considered to be potential predictors of the bleeding tendency. Their association with the clinical phenotype was then evaluated by univariate and multivariate logistic regression, and expressed as crude and adjusted odds ratios with 95% confidence intervals. Continuous variables were transformed into dichotomous variables and, pertaining to ETP, the median value in controls was chosen as the cut-off. All P-values reported are two-sided, and P < 0.05 was considered to be statistically significant. All analyses were performed with spss software (release 17.0; SPSS Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

From March 2004 to November 2005, 266 adults with severe HA (n = 231) or HB (n = 35) were seen for their annual check-up visit at the Hemophilia Centre in Milan; 95, who had no history of inhibitors and had been treated exclusively on demand, gave their written consent. However, 23 of the HA patients were excluded because FVIII:C was found to be measurable by the chromogenic assay. Therefore, 72 patients (median age 36 years; IQR 29–44) were ultimately included in the analysis. Cases (n = 22) were compared with all controls (n = 50) and with the subgroup of controls fulfilling the severe bleeder definition (n = 22). The main characteristics of cases and controls are shown in Table 1. Cases had older ages at first bleed and at first joint bleed, lower orthopedic and radiologic scores, lower incidence of null mutations, higher FVIII:Ag/FIX:Ag levels and higher ETP values measured in PRP than controls (Table 1; Fig. 1). Cases and controls were similar with regard to ETP values measured in PPP and other parameters of the thrombin generation curve (i.e. lag time, peak, time to peak and start tail) assessed both in PRP and in PPP (data not shown).

Table 1.   Main characteristics of the 72 patients included in the study
 Cases, MB (n = 22)All controls (n = 50)Controls, SB (n = 22)P-value (cases vs. all controls)P-value (MB vs. SB)
  1. ETP, endogenous thrombin potential; IQR, interquartile range; MB, mild bleeders; NS, not significant; PRP, platelet-rich plasma; SB, severe bleeders.

Median age, years (IQR)32 (27–43)38 (30–44)38 (29–53)NSNS
Hemophilia B, no. (%)7 (32)4 (8)2 (9)0.03NS
Median age at first bleed, months (IQR)42 (12–75)12 (12–24)12 (12–15)< 0.01< 0.01
Median age at first joint bleed, months (IQR)84 (36–108)24 (21–48)24 (12–36)< 0.01< 0.01
Median number of bleeds per year (IQR)0 (0–1)20 (9.5–31.5)36 (25–39)< 0.01< 0.01
Median factor consumption, IU kg−1 year−1 (IQR)60 (37–158)1957 (862–2238)2207 (2113–2790)< 0.01< 0.01
Median orthopedic joint score (range)3 (0–7)15 (9–24)18 (13–28)< 0.01< 0.01
Median Pettersson score (range)18 (9–25)35 (25–46)44 (22–50)< 0.01< 0.01
Median FVIII antigen, IU dL−1 (IQR)1.4 (< 0.5–3.5)< 0.5 (< 0.5–0.7)< 0.5 (< 0.5–0.8)0.02NS
Median FIX antigen, IU dL−1 (IQR)9.9 (0.8–148.0)< 0.5 (< 0.5–0.8)< 0.5 (< 0.5)0.020.04
Median ETP in PRP, n× min (IQR)850 (476–1145)460 (137–830)414 (136–994)0.010.04
Null mutations, no. (%)2/20 (10)28/48 (58)13/21 (62)< 0.01< 0.01
PTG20210A, no. (%)1/21 (5)2 (4)0NSNS
FV Leiden, no. (%)03 (6)1 (4.5)NSNS
image

Figure 1.  Endogenous thrombin potential (ETP) measured in platelet-rich plasma (PRP) in 22 cases (mild bleeders) and 50 controls included in the study. Each box plot represents interquartile range with median value (black line in the middle) and 95% confidence intervals.

Download figure to PowerPoint

Four pairs of HA patients and two HB pairs were from the same family; all HA pairs were concordant with respect to clinical phenotype (two pairs were cases and two controls), one HB pair was also concordant (cases) and one HB pair was discordant. In order to evaluate the influence of kinship, the prevalence of null mutations in cases and controls was evaluated, including only one member of each family and assessing all the possible combinations of unrelated subjects: in each possible scenario, the incidence of null mutations remained significantly lower in cases (data not shown).

As a greater proportion of HB patients were cases, the features of HA and HB patients were compared (Table 2). HB patients were older at first joint bleed and had lower orthopedic joint scores. In these patients, null mutations were less common and FIX:Ag levels were more frequently detectable; ETP values measured in PRP were higher, but this difference was not statistically significant, perhaps owing to the small sample size (Table 2).

Table 2.   Main characteristics of the 72 patients according to the type of hemophilia
 Hemophilia A (n = 61)Hemophilia B (n = 11)P-value
  1. ETP, endogenous thrombin potential; IQR, interquartile range; MB, mild bleeders; NS, not significant; PRP, platelet-rich plasma. *Two patients never had any joint bleed.

MB cases, no. (%)15 (25)7 (64)0.03
Median age, years (IQR)37 (29–44)35 (30–41)NS
Median age at first bleed, months (IQR)12 (12–36)24 (12–48)NS
Median age at first joint bleed, months (IQR)24 (24–48)66 (51–102)*0.01
Median number of bleeds per year (IQR)11 (2–25)0 (0–12)0.03
Median factor consumption, IU kg−1 year−1 (IQR)1280 (369–2170)333 (38–487)0.01
Median orthopedic joint score (range)13 (6–20)5 (2–9)0.04
Median Pettersson score (range)28 (20–45)23 (9–31)NS
Median FVIII:Ag/FIX:Ag, IU dL−1 (IQR)< 0.5 (< 0.5–1.1)0.9 (0.5–144.0)0.006
Undetectable FVIII:Ag/FIX:Ag, no. (%)33/59 (56)2 (18)0.04
Median ETP in PRP, n× min (IQR)554 (264–949)840 (334–908)NS
Null mutations, no. (%)28/57 (49)2 (18)0.05

Levels of FVIII:Ag/FIX:Ag were associated with the type of F8/F9 mutation, being undetectable in 25 of 30 (83%) patients with null mutations and in eight of 37 (22%) with non-null mutations (P < 0.01); this association was confirmed by analyzing HA and HB patients separately (P < 0.05 for both). Similarly, FVIII:Ag/FIX:Ag levels were more frequently undetectable in patients with ETP ≤ 597 n× min (the median value as measured in PRP of the whole series) than in those with ETP > 597 n× min (24/35, 69% vs. 12/35, 34%; P < 0.01). This association was confirmed in HA but not in HB patients, because only two had undetectable FIX:Ag (Table 2). No association was found between ETP values and type of F8/F9 mutation.

The distribution of F8 mutations was as follows: intron 22 inversions, 36% (n = 22); missense mutations, 31% (n = 19); small deletions/insertions, 16% (n = 10); intron 1 inversions, 5% (n = 3); nonsense mutations, 3% (n = 2); and intron 24 inversions, 2% (n = 1). In four patients (7%), F8 mutation was not found. The missense mutation p.Arg1997Trp was the second most frequent F8 mutation after intron 22 inversions, being detected in nine patients (15%) originating from the same region: consanguinity was clear in two pairs (two brothers and two first cousins). The comparison between patients with p.Arg1997Trp and those with intron 22 inversions showed that cases were more frequent in the former (7/9, 78% vs. 1/22, 5%; P < 0.01), who had more frequently measurable FVIII:Ag (9/9, 100% vs. 5/22, 23%; P < 0.01) and higher ETP values in PRP (median: 939 vs. 486 n× min; P < 0.01).

The distribution of F9 mutations was as follows: small deletions, 36% (n = 4); missense mutations, 36% (n = 4); nonsense mutations, 18% (n = 2); and splice site mutations, 10% (n = 1). The types of gene mutation and laboratory findings in cases are summarized in Table 3. The same information concerning controls is provided in Table S1.

Table 3.   Genotype and laboratory findings in 22 mild bleeders included in the study
PatientType of hemophiliaF8/F9 mutationFVIII:Ag/IX:Ag (IU dL−1)ETP in PRP (n× min)
  1. ETP, endogenous thrombin potential; HA, hemophilia A; HB, hemophilia B; NA, not available; PRP, platelet-rich plasma.

 1HAp.Gly174Glu15.01214
 2HAp.Arg1997Trp4.0797
 3HAp.Arg1997Trp3.5777
 4HAp.Arg1997Trp3.5860
 5HAp.Arg1997Trp3.31501
 6HAp.Arg1997Trp2.61122
 7HAp.Arg1997Trp1.4614
 8HAp.Arg1997TrpNA1566
 9HANot found0.61526
10HANot foundNA1454
11HAIntron 22 inversion0.61111
12HAp.N963fsX8< 0.5590
13HAIntron 1 inversion< 0.5314
14HAp.Val84Glu< 0.5261
15HAp.P606fsX17< 0.5196
16HBp.Pro368Thr158.0947
17HBp.Pro368Thr148.0840
18HBp.Val181Phe144.0238
19HBp.Gly190Asp9.9906
20HBIVSD-12 C>G4.1523
21HBg.118_121delgttt0.8334
22HBg.118_121delgttt0.7889

The results of logistic regression are shown in Table 4. By univariate analysis, HB, non-null mutations, detectable FVIII:Ag/FIX:Ag levels and higher ETP values in PRP were determinants of a mild clinical phenotype. However, after adjustment for the other variables, only non-null mutations were confirmed as an independent predictor. Similar results were obtained when only mild and severe bleeders were included in the multivariate analysis (data not shown).

Table 4.   Univariate and multivariate analysis of factors associated with a mild clinical phenotype of severe hemophilia
 Cases, MB (n = 22)All controls (n = 50)Crude OR (95% CI)Adjusted OR (95% CI)
  1. CI, confidence interval; ETP, endogenous thrombin potential; HA, hemophilia A; HB, hemophilia B; MB, mild bleeders; OR, odds ratio; PRP, platelet-rich plasma.

Type of hemophilia
 HA1546Ref.Ref.
 HB745.4 (1.4–20.9)4.0 (0.8–19.6)
Type of F8/F9 defect
 Null mutations228Ref.Ref.
 Non-null mutations182012.6 (2.6–60.5)8.9 (1.3–60.6)
Levels of FVIII:Ag/FIX:Ag
 < 0.5 IU dL−1431Ref.Ref.
 ≥ 0.5 IU dL−116196.5 (1.9–22.4)1.1 (0.2–6.2)
Median ETP in PRP
 ≤ 460 n× min525Ref.Ref.
 > 460 n× min17253.4 (1.1–10.6)2.2 (0.5–8.7)

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

In the past, common indicators of the bleeding phenotype of patients with hemophilia included age at the first joint bleed, bleeding frequency, factor consumption and orthopedic scores [1,2,21]. However, the introduction of continuous primary prophylaxis has dramatically changed the natural history of the disease, reducing the bleeding frequency and allowing, if started early, maintenance of the pristine orthopedic status [22,23]. Therefore, new criteria are warranted to define the degree of clinical severity of hemophilia and to avoid the unnecessary burden and costs of prophylaxis in patients doomed to have a mild bleeding tendency. To tackle this issue, we performed this study only in adult patients treated lifelong on demand, for whom annual bleeding frequency and concentrate consumption represent reliable and accurate indicators of the clinical phenotype. Very low cut-off values for these parameters were chosen to define cases, in order to investigate a subgroup strictly composed of patients with an extremely mild bleeding tendency, who showed, as expected, older ages at bleeding onset and lower orthopedic scores than controls. On the other hand, the enrollment of patients exclusively treated on demand and able to remain untreated with factor concentrates for at least five days may have introduced a selection bias by excluding a relevant proportion of patients with an extremely severe bleeding diathesis from the control group.

This study provides evidence that the type of F8/F9 mutation is the main determinant of bleeding tendency in severe hemophilia, the presence of non-null mutations being a strong predictor of a mild clinical phenotype. In the literature, evidence of mitigation of the bleeding phenotype comes only from two case reports of severe patients with small deletion/insertions within polyA runs of exon 14 of F8 [24,25]. In our study, these mutations were not present; however, we found that a mild bleeding phenotype was associated with heterogeneous non-null F8/F9 defects. Among these non-null mutations, p.Arg1997Trp was the most frequent among cases with HA, as this is a common mutation that has already been reported in severe as well as in moderate–mild HA [3,19], often in association with measurable FVIII:Ag levels [3].

It is well established that less severe gene defects are more common [4,20], and antigen levels more often detectable [26], in HB than in HA. It was suggested that these features may help to explain the differences in clinical severity of HB shown in some recent studies, in which lower clinical severity scores [27], later onset of first bleed, reduced use of prophylaxis [28] and lower rate of joint replacement [29] than in HA were reported. The number of HB patients included in this study was small; however, the ratio between HB and HA patients was 1 : 6, as expected, and the distribution of F9 mutations was also comparable to that reported in the Italian mutation database [20]. Although the study design was not aimed at comparing HB with HA, we found that the odds of having a mild bleeding diathesis was five-fold higher in HB than in HA patients. This association was no longer statistically significant after adjustment for the other variables. However, an independent role of HB in mitigation of the clinical phenotype cannot be excluded, because the number of these patients analyzed was too small.

For the first time in this study, an association was found between the clinical phenotype of severe hemophilia and ETP as measured in PRP, ETP values being higher in patients with a mild bleeding tendency. In a previous study [12], all hemophiliacs with FVIII:C/FIX:C < 1 IU dL−1 and severe clinical phenotype (n = 20) showed ETP values in PPP lower than 50% of normal. However, no comparison was made with the counterpart of severe patients characterized by an extremely mild clinical phenotype. We observed no correlation between the bleeding tendency and thrombin generation in PPP, as already reported by Beltràn-Miranda et al. [15], so the results obtained in PRP seem to reflect in vivo conditions more accurately. The role of platelets in thrombin generation was investigated in patients with severe hemophilia by Siegemund et al. [30], who demonstrated a linear relationship between increasing platelet count and thrombin generation. Furthermore, the authors found that the influence of platelets on ETP was different between HA and HB, and that platelets increased thrombin generation significantly more in HB than in HA, owing to the role of platelets in the assembly of the tenase complex on their surface. The TGA in our study did include, as coagulation trigger, tissue factor at low concentrations, and this may variably influence thrombin generation when contact activation is not prevented by the addition of corn trypsin inhibitor [31]. This may be regarded as a limitation of the present and previous studies [12,30], because blood was not collected in the presence of corn trypsin inhibitor. However, it is unlikely that the effect (if any) of contact activation on thrombin generation in our experimental conditions was different in cases and controls to such an extent as to undermine the results and conclusions. Furthermore, a recent study showed that the purported influence of contact activation on thrombin generation is negligible when the concentration of tissue factor is higher than 0.5 pm [32]. This observation makes any effect unlikely, because the concentration of tissue factor in our experimental conditions was 2 pm.

In this study, higher ETP values in PRP were associated with detectable levels of FVIII:Ag/FIX:Ag. Perhaps the detection of measurable FVIII:Ag/FIX:Ag represents an indirect marker of the presence of very low plasma levels of FVIII:C/FIX:C (not detectable even by the most sensitive chromogenic assay), which may have contributed to the increased thrombin generation detected in these samples.

In conclusion, our data on ETP in PRP suggest that this measurement is able to identify severe hemophiliacs with very mild clinical expression. The practical application of this test in the clinical context is limited, because the method is time-consuming, and PRP can only be stored for a very short time period before testing. On the other hand, the evidence that non-null F8/F9 mutations are independent predictors of a mild bleeding pattern in severe hemophiliacs may have implications for patient management, because it would influence the choice and time of onset of prophylaxis and/or the adoption of individualized dose-escalating regimens. This piece of evidence is added to that of a lower risk of inhibitor development associated with less severe gene defects [33], and strengthens the recommendation to obtain an early molecular diagnosis to plan different therapeutic strategies in children.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

The study was funded by the Istituto Superiore di Sanità in the frame of the research progamme Italy – USA on rare diseases.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

This study was supported by an unrestricted educational grant received from Bayer HealthCare in the frame of the Bayer Haemophilia Awards Programme (Birmingham, UK, 2003) to E. Santagostino as a Special Project Award.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Table S1. Genotype and laboratory findings in 50 controls included in the study.

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JTH_3767_sm_TableS1.doc75KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.