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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. Copyright 2016 KEI Journals. All Rights Reserved Page │4 Medical Research Archives, Vol. 4, Issue 6, October 2016 Protein Z and thrombin generation *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).
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