Influence of Protein Z Plasma Level on Thrombin Generation Assay

Influence of protein Z plasma level on thrombin generation assay

Authors : Abstract Bérangère S. Joly Introduction: Protein Z (PZ) is a vitamin K- dependent factor, involved as a cofactor of PZ- Bénédicte Sudrié-Arnaud dependent protease inhibitor (ZPI) that inhibits the Jeanne-Yvonne Borg activated factor X on phospholipid surface. A severe Véronique Le Cam Duchez PZ deficiency could be associated with ischemic arterial diseases. The aim of this study was to Author affiliations: evaluate the influence of PZ plasma levels on thrombin generation (TG) and to detect a Bérangère S. Joly hypercoagulable state in patients with PZ deficiency. Centre Hospitalier Universitaire de Rouen, Patients and Methods: Young patients with Hématologie biologique, Unité Hémostase, personal history of arterial thrombosis and PZ Rouen, France; Hôpital Lariboisière, deficiency were included. PZ concentrations were APHP, Université Paris Diderot, assessed using ELISA method. TG assay was Hématologie biologique, Paris, France. performed using platelet-free plasma (PPP) E-mail: [email protected] containing 5 pM or 1 pM tissue factor and 4 μM phospholipids with and without thrombomodulin. Bénédicte Sudrié-Arnaud Then, we focused on the influence of successive Centre Hospitalier Universitaire de Rouen, overloads with purified PZ in two patients with PZ Hématologie biologique, Unité Hémostase, deficiency (Pat1PZdef and Pat2PZdef) and industrial Rouen, France. PZ deficiency (IndPZdef). E-mail: [email protected] Results: First, in 13 patients with PZ deficiency, Jeanne-Yvonne Borg none influence of PZ level was reported on TG assay Centre Hospitalier Universitaire de Rouen, parameters. Second, TG profiles performed on Pat1PZdef, Pat2PZdef and IndPZdef were close to Hématologie biologique, Unité Hémostase, the reference TG profile. In presence of TM, there Rouen, France. was no difference in TG parameters according to PZ E-mail: [email protected] plasma levels. After each overload, real PZ plasma levels were underestimated; differences in standards Corresponding author: of purified PZ between the two manufacturers might Véronique Le Cam Duchez, Centre explain such results. Hospitalier Universitaire de Rouen, Conclusion: The absence of influence of PZ Hématologie biologique, Unité Hémostase, concentration on TG assay is reliable to the absence INSERM U1096, Rouen, France. of well-demonstrated clinical consequences of PZ E-mail: deficiency in arterial thrombosis. Clinical [email protected] consequences of protein Z deficiency warranted further studies.

Keywords

Protein Z, thrombin generation assay, protein Z

deficiency Medical Research Archives, Vol. 4, Issue 6, October 2016 Protein Z and thrombin generation

1. Introduction due to a difference in aa sequence

Protein Z (PZ), a vitamin K- between bovine and human PZ. The dependent protein, was identified in complete aa sequence of human PZ was bovine plasma in 1977 [1] and in human previously described by Sejima and plasma in 1984 [2]. The amino acid (aa) Ichinose [4, 7], who reported the lack of sequence of bovine PZ (396 aa) was 36 aa C-terminal enhancing thrombin described in 1985 [3] and reported binding [6]. extensive homology to the other vitamin In 1997, Mc Donald et al reported

K-dependent coagulation factors: 13 Gla that PZ binds PL surface thanks to residues within the NH2 -terminal 40 gamma-carboxyglutamic acid-rich (GLA) residues, 2 “EGF-like” domains and 1 domain, such as vitamin K-dependent

Beta-hydroxyaspartic acid at position 64. factors, but PZ presents slower

However, the absence of active serine site kinetics [8]. The inhibition of activated and active histidine site in the homologous factor X (FXa) mediated by PZ underlines region with the family of serine proteases the need for another plasma protein for enhanced the lack of catalytic triad. This inhibition: PZ-dependent protease is consistent with the inability to activate inhibitor (ZPI) was isolated and

PZ into a serine protease by limited characterized by Han et al [9, 10]. ZPI hydrolysis [2, 3]. Moreover, PZ only has a belongs to the serpin family, producing cofactor function, and not a proteolytic rapid inhibition of procoagulant PL and function [4], like protein S (PS). In 1991, Ca2+ and FXa within PZ. Two possible

Hogg and Stenflo reported that bovine PZ pathways for PZ mediated inhibition of interacts with thrombin [5], and suggested FXa by ZPI were evoked [11] and Tabatai that bovine PZ mediates binding between et al described a plasma circulating thrombin and phospholipid (PL) surfaces. complex between PZ and ZPI [12].

In contrast, human PZ binds thrombin Moreover, Yin et al demonstrated that in poorly resulting in a minimal impact on presence of PZ, thrombin generation (TG) thrombin association with PL [6]. This is was significantly delayed and peak

Copyright 2016 KEI Journals. All Rights Reserved Page │2 Medical Research Archives, Vol. 4, Issue 6, October 2016 Protein Z and thrombin generation thrombin concentration was reduced by 2. Patients and Methods more than 50% [13]. 2.1. Patients

Therefore, reduced PZ blood In a first part, fifteen young patients concentrations might be expected to (age <56 years old) with personal history reduce inhibition of blood coagulation of arterial thrombosis (stroke or predisposing to thrombosis. There have myocardial infarction) and PZ deficiency been several clinical studies on PZ but no other coagulation abnormalities or deficiency in many different clinical anticoagulant therapy, were included in contexts but all with inconclusive and this study conducted at Rouen University controversial results. Indeed, PZ Hospital, France. Blood samples were deficiency has been described as a risk collected from tubes containing 0.109 M factor for bleeding [14] but these data of trisodium citrate (1:10) (Venosafe were not confirmed in other Plastic tubes, Terumo, Japan) and double studies [15, 16]. Other studies assessed PZ centrifuged (2250g-15min-20°C). Platelet- plasma level in several thrombotic events free plasmas were kept frozen at -80°C including stroke, myocardial infarction, until the assays. cerebral or deep venous thrombosis and In a second part, we focused on two pregnancy complications. However, many other patients with PZ deficiency and discrepancies were observed in the results without anticoagulant therapy (Pat1PZdef of these numerous studies reported in and Pat2PZdef). Pat1PZdef was a 52-year- several reviews [17, 18]. old woman admitted for stroke to the

The aim of this present study was to Neurology Department of Rouen evaluate the influence of PZ plasma levels University Hospital, France, with a PZ on coagulation activation and to detect a level of 0.43 mg/L (normal range from 0.9 hypercoagulable state in patients with PZ to 2.7 mg/L) and no other coagulation deficiency using TG assay. abnormalities. Pat2PZdef was a 33-year-

old man, admitted for myocardial

Copyright 2016 KEI Journals. All Rights Reserved Page │3 Medical Research Archives, Vol. 4, Issue 6, October 2016 Protein Z and thrombin generation infarction to the Cardiology Department coagulation factors. This industrial of Rouen University Hospital, France, deficient PZ plasma was overloaded too. with a PZ level of 0.31 mg/L controlled to For each spiked plasma, real PZ

0.48 mg/L. In this latter patient we concentration was assessed. evidenced isolated and persistent TG was measured according to the antibodies IgG antiphospholipid (Phopho- method previously described by Hemker LISA IgG/IgM, Theradiag, Marne la et al [19]. TG assay was performed in a Vallée, France) but no anticardiolipin, no Fluoroscan Ascent® fluorometer anti-beta2 GPI antibodies and no lupus (Thermoscientific Labsystems, Helsinki, anticoagulant. Finland) and Thrombinoscope™ software

2.2. Methods (Thrombinoscope BV, Maastricht, The

PZ assays were performed on Netherlands) [20-22], in three different plasma samples in our homeostasis unit at conditions (all reagents from Diagnostica

Rouen University Hospital, France using Stago, Asnières, France):

ELISA method (Asserachrom PZ, *condition 1: using PPP normal

Diagnostica Stago, Asnières, France). reagent containing final concentrations of

For both selected deficient PZ 5 pM tissue factor (TF) and 4 μM PL, plasmas, progressive PZ overloads were *condition 2: using PPP low performed using purified PZ (Hyphen reagent, containing final concentrations of

Biomed, Neuville/Oise, France). 1 pM tissue factor (TF) and 4 μM PL,

Moreover, we used an industrial PZ [23]. deficient plasma (Protein Z deficient For both these conditions, plasma, Hyphen Biomed, Neuville sur endogenous thrombin potential (ETP),

Oise France), (IndPZdef), restored with lagtime, peak concentration and time to distilled water and containing less than peak [21], were analyzed by

1% of PZ and normal range for other Thrombinoscope™ software, and velocity

was calculated for each sample.

*the third condition used software (Statistical Solutions, Cork, recombinant human thrombomodulin Ireland). We studied correlation between

(TM) (Diagnostica Stago, Asnières, PZ final concentration and respectively

France,) obtained to boost protein C lag time, peak, ETP and time to peak. pathway. Using our standardized 3. Results procedure, TG was measured with and without TM, and ratio of ETP was 3.1. TG assay performances calculated with and without TM as The intra-assay CV was calculated

ETP(TM+)/ETP(TM-) [19, 24]. from 8 measurements of IQC in the same

In each set of TG experiment, we set of experiments. The CV for each performed an assay on our in-house parameter were respectively 2% for lag internal quality control (IQC) (normal time, 4% for ETP and 2% for both the pooled plasmas) in order to validate the peak and time to peak. Forty-four assay set of experiment. runs were performed for IQC; inter-assay

reproducibility was correct for both TG Parameters of TG assay were parameters (mean CV of 11% for lag time, normalized against the IQC of the same 9% for ETP, 11.9% for peak and 9.3% for set of experiment or against the mean of time to peak). IQC assays. Finally, we standardized our set of experiments using a protocol 3.2. Patients with PZ deficiency described by Dargaud et al [25-27]. First, we performed PZ assays on

2.3. Statistical analysis plasma collected from 13 patients with PZ deficiency. PZ level did not influence the Data are expressed as mean ± parameters of TG assay (Fig. 1). standard deviation (SD). The intra-assay Nevertheless, this comparison was coefficient of variation (CV) for TG assay performed in patients with likely was calculated from consecutive differences in coagulation state. measurements of IQC. Statistical analysis was performed on NCSS 2007 statistical

Figure1: Parameters of thrombin generation assay for 13 patients with protein Z deficiency and the internal quality control (protein Z plasma concentration: 2.61 mg/L).

3.3. Overloading of PZ deficient PZ. We compared the TG profile of these plasmas 3 PZ deficient plasmas with the mean

Second, two other patients with PZ profile of IQC (44 sets) (Fig. 2), deficiency (Pat1PZdef and Pat2PZdef) considering the IQC profile as the and industrial PZ deficiency (IndPZdef) reference in standardized procedure were chosen for overload with purified (condition 1: TF 5 pM, PL 4 µM).

Figure 2: Comparison of thrombin generation assay before overload with purified protein Z for both patient with protein Z deficiency, the industrial protein Z deficient and the internal quality control. Thrombin generation assay was assessed with tissue factor 5 pM, phospholipids 4 µM; 44 measurements of internal quality control were assessed.

Pat1PZdef plasma had increased IndPZdef were close to the reference TG hypercoagulability (lagtime: 2 min; peak: profile, while the lagtime of Pat2PZdef

327 nM; ETP: 1816 nM.min; time to was shorter than the reference profile peak: 4.17 min) compared to normal (2.17 min versus 2.58 min). After each plasma (lagtime: 2.58 min; peak: 239 nM; overload, real PZ plasma levels were

ETP: 1409 nM.min; time to peak: 5.54 determined. The real concentration was min). TG profile of Pat2PZdef and under the theoretical level (Fig. 3).

Figure 3: Comparison of theoretical and measured protein Z concentrations according to theoretical overload. Lines illustrate theoretical protein Z concentrations (black lines), and measured protein Z concentrations (dotted lines).

Nevertheless, the PZ plasma profile of Pat1PZdef, Pat2PZdef or concentrations did not influence the TG IndPZdef (Fig. 4).

Figure 4: Influence of purified human protein Z overloads in thrombin generation profile of protein Z deficient plasmas. Protein Z concentrations measured by ELISA are indicated at the end of each term of legend.

A B

These data were the same after between measured PZ levels and the normalization against daily IQC or against parameters of TG assay were non- the mean of the 44 IQC. Regressions significant (Table 1).

Table 1: Coefficients of regression for thrombin generation parameters, according to the measured protein Z plasma concentrations in plasma protein Z deficient. Each TG assay was normalized against the daily internal quality control or against the mean of 44 internal quality control.

Normalization against daily IQC p(regression) Lagtime ETP Peak TTP

Pat1PZdef 0.264 0.808 0.831 0.264

Pat2PZdef 0.255 0.235 0.659 0.570

IndPZdef 0.513 0.535 0.316 0.630

Normalization against 44 IQC mean p(regression) Lagtime ETP Peak TTP

Pat1PZdef 0.267 0.810 0.829 0.264

Pat2PZdef 0.256 0.249 0.672 0.570

IndPZdef 0.05 0.390 0.902 0.252

In condition 2, (TF 1 pM), the pure Pat1PZdef and IndPZdef with one results were the same when comparing point of overload for each (Fig. 5).

Figure 5: Comparison of thrombin generation assay for patient with protein Z deficiency and industrial protein Z deficient before and after overload with purified protein Z. Thrombin generation assay was assessed with tissue factor 1 pM, phospholipids 4 µM; protein Z concentrations measured by ELISA are indicated at the end of each term of legend.

Finally, in presence of TM inhibition of ETP or peak according to PZ

(condition3), there was no difference in plasma levels (Fig. 6).

Figure 6: Comparison of thrombin generation assay for patient with protein Z deficiency (A), industrial protein Z deficient (B) before and after overload with purified protein Z, and internal quality control (C). Thrombin generation assay was assessed with tissue factor 5 pM, phospholipids 4 µM, with and without thrombomodulin; protein Z concentrations measured by ELISA are indicated at the end of each term of legend. The ratio between ETP with TM and ETP without TM are indicated in the table (D).

D A

ETP TM+ / ETP TM-

IQC 0.49

Pat1PZdef 0.5 0.61

Pat1PZdef 1,3 0.72

IndPZdef 0.1 0.48

IndPZdef 3.3 0.62

B C

4. Discussion contributes to FXa inhibition [11, 12] and

To our knowledge, this is the first thus should influence TG profile. study to observe the effect of plasma PZ Moreover in 2000, Yin et al demonstrated level on TG profile. As previously that PZ significantly delayed TG [13]. The described, PZ, the cofactor of ZPI, authors analyzed the effect of PZ on FXa

Copyright 2016 KEI Journals. All Rights Reserved Page │10 Medical Research Archives, Vol. 4, Issue 6, October 2016 Protein Z and thrombin generation activity and TG during in vitro studies. expected concentrations. Nevertheless, we Their results suggested that PZ decreased obtained concentrations of up to 6 mg/l coagulation response in human plasma. which is high compared to normal value First, the comparison between 13 different (0.9 to 2.7 mg/l) but without consequence PZ deficient patients did not reach on TG assay. difference in TG assay profile in our No proteolytic functions are conditions (5 pM FT, 4 µM PL). supported by PZ or PS: PS is the cofactor

However, TG assay is a global of protein C inhibiting activated factor V coagulation test influenced by several and activated factor VIII in presence of coagulation factors [21]. PL and calcium, and PZ is the cofactor of Hence, it could be possible to ZPI inhibiting FXa in presence of PL and demonstrate an influence by successive calcium [9, 10]. Several studies have been overloads of PZ deficient plasma with a published on the influence of PS on TG small quantity of purified PZ. In our assay. As described by Hezard et al, PS study, PZ plasma levels did not influence deficiency generates hypercoagulability in TG profile either for Pat1PZdef or TG assay, in platelet rich plasma Pat2PZdef. conditions, with and without activated

Second, concerning PZ overloads protein C [28]. Duchemin et al, in 1994, (Fig. 3), the measurement of PZ demonstrated that PS plasma level did not concentration was always lower than influence TG profile in absence of TM expected. Differences in standards of [29]; while inhibition of ETP was purified PZ between the two correlated with PS levels in presence of manufacturers (Hyphen Biomed for TM: lower PS level was correlated with IndPZdef and Diagnostica Stago for the lower ETP inhibition. We did not observe purified PZ calibrator Asserachrom PZ) these results with PZ plasma level: in our might explain such results. Differences in study, lower PZ level was correlated with concentrations are constant and a real higher ETP inhibition for IndPZdef as correlation exists between observed and well as for Pat1PZ def.

Later, Duchemin et al reported that involvement of PZ deficiency in human PS concentration was positively correlated pathologies was evaluated with opposite with lagtime especially with low TF results: PZ deficiency and risk factor of concentration. In contrast, PS venous thrombosis [31, 32], stroke [33, concentration did not influence ETP or 34], and myocardial infarction [35, 36]. peak regardless of TF concentration. The discrepancies are the same Dielis et al observed an influence of free concerning all thrombotic syndromes, as

PS level on lagtime in different conditions described in literature reviews [17, 18]. for TG assay [30]. In our study, PZ plasma concentration did not influence 5. Conclusion lagtime, peak or ETP irrespective of TF We were unable to demonstrate the concentration. influence of PZ plasma level on thrombin

Thus, the mechanisms of action for generation assay test according to PS and PZ are similar but the targets of Hemker's method. Our results likely serpin PC and ZPI are different: PC explain the absence of studies in the directly inactivates the boosters of TG literature on the clinical consequences of (activated factors V and VIII) while ZPI protein Z deficiency warranting further inactivates only FXa. studies.

Finally, the absence of influence of Acknowledgements

PZ concentration on TG assay could We thank Agnès Deshayes for her explain the absence of well demonstrated technical help. We are grateful to Nikki clinical consequences of PZ deficiency in Sabourin-Gibbs, Rouen University venous thrombosis and arterial Hospital, for her help in editing the thrombosis. In 2000 and 2001, two studies manuscript. demonstrated that PZ deficiency increased Conflict of interest. thrombotic risk in mice carrying factor V Leiden mutation [11, 13]. Later, the None declared.

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