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Schizophrenia Research 162 (2015) 175–182

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Schizophrenia Research

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Global hypercoagulability in patients with schizophrenia receiving long-term therapy

Vincent Chow a,b,c,CarolineReddela,b,c, Gabrielle Pennings a,b,c, Elizabeth Scott d,TundraPasqualone, Austin C.C. Ng b,c,ThomasYeohb, Jennifer Curnow a,c,f, Leonard Kritharides a,b,c,⁎ a ANZAC Research Institute, Sydney, Australia b Department of Cardiology, Concord Repatriation General Hospital, Sydney Local Health District, Sydney, Australia c University of Sydney, Australia d Brain & Mind Research Institute, University of Sydney, Australia e Department of Psychiatry, Croydon Health Centre, Sydney, Australia f Department of Haematology, Concord Repatriation General Hospital, Sydney Local Health District, Sydney, Australia article info abstract

Article history: Background: Patients with schizophrenia are at increased risk of venous thromboembolism. The mechanisms Received 21 January 2014 underlying this association are poorly understood. Received in revised form 4 December 2014 Aims: We investigated whether there is a global hypercoagulable state in patients with schizophrenia utilising the Accepted 6 December 2014 overall haemostatic potential (OHP) assay which assesses overall potential (OCP), haemostatic Available online 27 January 2015 potential (OHP) and fibrinolytic potential (OFP). Method: Citrated plasma was collected for OHP assays from patients with schizophrenia on long-term antipsy- Keywords: chotic treatment and compared with healthy age- and sex-matched controls. Time courses of fibrin formation Schizophrenia coagulation test and degradation were measured by spectrophotometry (absorption of 405 nm) after the addition of tissue factor Coagulation and tissue plasminogen activator to plasma. Thrombosis Results: Ninety patients with schizophrenia (antipsychotic treatment-15.9 ± 9.7 years) and 30 controls were Fibrinolysis recruited. Patients with schizophrenia had higher rates of smoking and levels of inflammatory markers (high- sensitivity C-reactive protein and neutrophil-to-lymphocyte ratio) than controls. Whilst D-dimer, fibrinogen and count did not differ between patients with schizophrenia and controls, the OCP (54.0 ± 12.6 vs 45.9 ± 9.1, p = 0.002) and OHP (12.6 ± 5.8 vs 7.2 ± 3.7, p b 0.001) were higher, and OFP was lower (76.6 ± 9.8% vs 84.9 ± 6.4%, p b 0.001) in patients with schizophrenia, implying both a hypercoagulable and hypofibrinolytic state in these patients. Importantly, abnormalities in overall coagulation were independently predicted by levels of plasminogen-activator-inhibitor-1, fibrinogen, platelet count, inflammatory markers and plasma , suggesting a multifactorial aetiology. Conclusion: Patients with schizophrenia have evidence of a global hypercoagulable and hypofibrinolytic state which may contribute to their increased risk of venous thromboembolism. © 2015 Elsevier B.V. All rights reserved.

1. Introduction reports of increased incidence of VTE amongst patients receiving con- ventional antipsychotic (Varia et al., 1983; Roche-Bayard Patients with schizophrenia have an increased risk of venous throm- et al., 1990)wereconfirmed by larger case–control studies which boembolic disease and other cardiovascular diseases. This increased reported a seven-fold increase in risk of VTE (Zornberg and Jick, 2000) risk is attributed to multiple factors including conventional risk factors and 13-fold increase risk of fatal pulmonary embolism (PE) (Parkin for such as smoking, obesity, the metabolic et al., 2003). There is some evidence of higher risk amongst new users syndrome and systemic inflammation. Other specific factors may and those receiving atypical antipsychotic medications (Walker et al., include immobility, catatonia during severe illness and the use of anti- 1997; Hagg et al., 2009; Parker et al., 2010). VTE has a reported early psychotic medications (both conventional and atypical). Early case case fatality rate of 7–11% (Stein et al., 2004), and a reported 5-year cumulative mortality rate of up to 32% (Ng et al., 2011). An increased risk of VTE occurs when at least one of Virchow's triad ⁎ Corresponding author at: Department of Cardiology, Concord Repatriation General is present (i.e. vascular endothelial damage, stasis of blood flow, and/or Hospital, Level 3 West, Hospital Road, Concord, NSW 2139, Australia. Tel.: +61 2 9767 7359; fax: +61 2 9767 6994. hypercoagulability of blood) (Anderson and Spencer, 2003). In the most E-mail address: [email protected] (L. Kritharides). recent VTE guidelines and current literature, hypercoagulability has

http://dx.doi.org/10.1016/j.schres.2014.12.042 0920-9964/© 2015 Elsevier B.V. All rights reserved. 176 V. Chow et al. / Schizophrenia Research 162 (2015) 175–182 been shown to increase the risk of VTE by 2–9 fold in the general popu- cardiac evaluation and screening of patients with schizophrenia receiv- lation (Rogers et al., 2012; Authors/Task Force et al., 2014). ing antipsychotic treatment were recruited. The control groups com- Appropriate risk stratification for VTE risk and development of prised of healthy volunteers of similar age and gender with no prior prevention strategies requires a clear understanding of the mechanisms history of schizophrenia or VTE or ischaemic heart disease and those involved, and very few studies have directly quantified or addressed the not receiving antipsychotic or long-term anticoagulation medications. mechanisms of increased thrombotic risk in patients with schizophrenia Amongst the 90 patients with schizophrenia, there were no patients receiving long-term antipsychotic treatment. Overall coagulation and receiving conventional neuroleptics. 68/90 (76%) of patients were fibrinolysis are determined by the interplay between pro-coagulant receiving clozapine treatment, and the remaining 22 patients received factors such as factor VIII (FVIII) and plasminogen activator inhibitor the following treatment: olanzapine (6), aripiprazole (4), quetiapine type-1 (PAI-1), and anti-coagulant factors such as protein C and protein (4), paliperidone (4), risperidone (2), amisulpride (1) and zuclopenthixol S. FVIII is an important component of the intrinsic coagulation pathway (1). Patients were excluded if they: 1) were prescribed an antipsychotic and is responsible for the amplification of the coagulation cascade after for less than one year; 2) deemed noncompliant with initial thrombin generation (Furie and Furie, 2008). PAI-1 is one of the antipsychotic medications based on psychiatry reviews and serum most important inhibitors of the plasma fibrinolytic activity and is clozapine levels for patients receiving this ; 3) had previous raised in inflammatory states, atherosclerosis and obesity (Cesari et al., history of VTE (PE and/or deep vein thrombosis [DVT]); 4) had symp- 2010). Elevated levels of FVIII and PAI-1 have been shown to increase toms suggestive of ischaemic heart disease and/or were found to the risk of VTE by 4.8-fold (Koster et al., 1995a; Jenkins et al., 2012b) have ischaemic heart disease on functional testing or 5) receiving and 1.6-fold (Meltzer et al., 2010) respectively. Proteins C and S exert long-term oral anticoagulation. All patients with schizophrenia met effects by inactivating coagulation factors V and VIII. DSM-IV diagnostic criteria for a schizophrenic disorder according Patients with protein C and S deficiencies have a 7.3–8.5 fold lifetime to the guidelines of the American Psychiatric Association (1994). increased risk of VTE (Koster et al., 1995b). This cross-sectional study was approved by the institutional Human Previous studies have found inconsistent associations between FVIII Ethics Committees. levels (Bank et al., 2007; Jenkins et al., 2012a), protein C and S levels (Mahmoodi et al., 2010; Bucciarelli et al., 2012), PAI-1 levels (Yukizawa et al., 2012), direct effects of (Carrizo et al., 2.2. Overall haemostatic potential (OHP) assay 2008) and VTE risk in these patients (Reitter-Pfoertner et al., 2013). Conventional testing for thrombophilia involves screening for specific Blood samples were collected by venipuncture from the cubital vein individual abnormalities in haemostasis which can be costly and with a 21 gauge butterfly needle. The tourniquet was removed and the prone to misinterpretation (Jennings et al., 2005). Consequently, cur- first 3 ml of blood was discarded. Blood was collected into BD rent guidelines do not recommend routine screening for heritable Vacutainer 0.109 M sodium citrate tubes (Becton-Dickinson, Franklin thrombophilia in asymptomatic individuals (Baglin et al., 2010; Lakes, NJ, USA) then centrifuged for 10 minutes (min) at 23 °C and Middeldorp, 2011a). 2500 g (Eppendorf 5804 R, USA). The plasma supernatant was centri- As the underlying biological mechanisms by which patients with fuged under the same conditions to produce platelet-poor plasma. We schizophrenia develop VTE are likely multifactorial, these may lend used a modified OHP assay based on that described by Blomback (He themselves to investigation by global coagulation assays rather et al., 1999, 2001; Curnow et al., 2007). Fibrin time curves were gener- than by measuring individual factors. The overall haemostatic ated in microtiter plate wells and plasmas were tested in duplicate. potential (OHP) assay is a simple, validated and inexpensive global The OHP assay blank buffer consisted of Tris (tris(hydroxymethyl) coagulation assay that detects both the dynamic coagulation and aminomethane), NaCl and CaCl2,andpurified water was used to make fibrinolytic potential of plasma samples. The underlying principle the buffer in the OHP assay. The concentrations of the reagents in the of the OHP assay determines whether the hypercoagulable state is buffer were: Tris—66 mM, NaCl—130 mM and CaCl2—35 mM. The buffer due to increased fibrin generation (hypercoagulability), reduced was adjusted to a pH of 7 and stored at 4 °C. fibrinolysis (hypofibrinolysis), or a combination of these. Fibrinolysis The overall coagulation potential (OCP) microtiter wells contained is the process of degradation of a fibrin blood clot and limits throm- 75 μl plasma and 75 μl OCP buffer, at pH 7.0 with final concentrations bus extension beyond the site of endothelial damage. The fibrinolytic of Tris 33 mmol/l, NaCl 65 mmol/l, CaCl2 16.5 mmol/l and tissue factor system is a complex cascade that controls the dissolution of the fibrin 0.85 pmol/l. OCP curves were generated from automated absorption blood clot into soluble fibrin degradation products and consists of measurements at 405 nm taken every minute for 100 min for all many factors including plasminogen, plasminogen activator, PAI-1 samples, an extension to the original 60 minute protocol to ensure the (Van De Craen et al., 2012). The fibrinolytic potential as measured fibrinolysis curves in all samples had returned to zero (baseline) to in the OHP assay measures the aggregate effect of these components. enable calculation of OHP assay parameters. OHP curves were gener- It has been shown to be useful in identifying hypercoagulable states ated using a similar method, except that the added buffer also in patients with acute coronary syndromes, autoimmune diseases contained rt-PA to give a final concentration of 300 ng/ml. Values and following cardiac bypass surgery (Vedin et al., 2005; Curnow for OCP and OHP represent the area under the relevant fibrin time et al., 2007; Reddel et al., 2013). curve calculated by summation of absorption values (He et al., The primary aim of this study was to investigate whether patients 1999, 2001). The overall fibrinolysis potential (OFP) value was calcu- with schizophrenia taking long-term antipsychotics demonstrated a lated by (OCP − OHP) / OCP × 100% and represents the area under global hypercoagulable state as determined by the OHP assay (He the fibrinolytic portion of the curve as a percentage of the total OCP et al., 1999, 2001; Curnow et al., 2007). The secondary aim of the value. Additional data derived from the fibrin time curve included study was to assess for clinical characteristics and laboratory markers maximum optical density (Max OD), maximum slope (Max slope) associated with alterations in global coagulation and fibrinolysis. and delay in onset of fibrin generation (Delay). Because all assays were performed in duplicate, Max OD is the mean of the maximum 2. Methods and materials OD reached in the two OCP curves. The slope was calculated progres- sively for each OD reading on the OCP curve, using a minimum of 2.1. Study population three time points. The greatest increase in OD for these points repre- sents the Max slope. Delay was defined as the time intercept From January 2010 to December 2012, 90 consecutive patients with between the line of maximum slope on the OCP curve and the line schizophrenia who attended a specialist clinic established for the of baseline absorbance at time zero. V. Chow et al. / Schizophrenia Research 162 (2015) 175–182 177

2.3. Biochemical analyses Table 1 Baseline characteristics.

Peripheral venous blood sample biochemical analyses included Patients with schizophrenia Controls fasting plasma lipid profile, electrolytes, and and thyroid function (n = 90) (n = 30) fl tests. Measurements of systemic in ammation included full blood Age (years) 40 ± 13 43 ± 12 count and its differentials (neutrophil, lymphocyte and eosinophil Gender counts) and high-sensitivity C-reactive protein (hsCRP). All measure- Male 61 (68) 17 (57) ments of hsCRP were performed using an immuno-turbidimetric Schizophrenia Duration of illness (years) 15.9 ± 9.7 – method, on an autoanalyser (IMMAGE CRPH; Beckman Coulter, Family history of mental illness 18 (20) 0 Villepinte, France). Glycosylated haemoglobin (HbA1c) was measured Clozapine therapy 68 (76) – by high-pressure liquid chromatography (BioRad, Hercules, CA). Body mass index (kg/m2) 29.1 ± 6.5 27.6 ± 4.4 The standard clotting assays prothrombin time, activated partial Comorbidities Alcohol excess 9 (10) 0 thromboplastin time, D-dimer and Clauss fibrinogen were performed Atrial fibrillation 0 0 on an automated coagulation analyser (STA-R, Diagnostica Stago). Heart failure Reagents were from Dade-Behring Diagnostics (NSW, Australia) Previous history of heart failure 1 (1) 0 and Diagnostica Stago. Platelet count was obtained by optical and NYHA class N 1 1 (1) 0 impedance methods (Abbott Diagnostics CELL-DYN Sapphire). Stroke 1 (1) 0 Thyroid dysfunction 1 (1) 0 Plasminogen-activator-inhibitor-1 (PAI-1) (TriniLIZE PAI-1 Antigen, Risk factors for ischaemic heart disease Trinity Biotech, Bray, Ireland) antigen levels were measured by ELISA Diabetes 21 (24) 3 (10) in accordance with the manufacturer's instructions (samples were 25 (28) 0 diluted 2 in 3 with sample buffer solution). therapy 19 (21) 0 11 (12) 5 (17) Smoking 35 (39) 2 (7) 2.4. Statistical analyses Cardiac observations Systolic blood pressure (mm Hg) 124 ± 11 119 ± 11 Diastolic blood pressure (mm Hg) 77 ± 9 73 ± 8 Data were summarized as frequencies and percentages for categori- Resting heart rate (bpm) 88 ± 13 70 ± 7 cal variables. Continuous variables were presented as mean ± standard Categorical variables are presented as numbers (%). Continuous values are presented as deviation. Comparison between two groups was based on unpaired t mean ± SD. test (parametric distribution) or Mann–Whitney test (non-parametric bpm, beats per minute; NYHA, New York Heart Association functional class. distribution) for continuous variables and χ2 tests or the Fisher exact test for dichotomous variables. A two-tailed p value of b0.05 was used as a cut-off for statistical significance. Amongst patients with schizo- 3.2. Biochemical and OHP assay parameters phrenia, multivariate backward selection linear regression analysis was performed to identify independent associations of the OHP assay Patients with schizophrenia had higher levels of inflammatory parameters (OCP, OHP and OFP). All variables with a univariate p markers including hsCRP (3.7 ± 5.3 vs 0.9 ± 0.8 mg/l, p b 0.001) value of b0.10 were included in the multivariate modelling to ensure and neutrophil-to-lymphocyte ratio (NLR) (2.5 ± 1.4 vs 1.7 ± 0.8, capture of all independent predictors of the above parameters. p = 0.03) compared to healthy controls. levels were Multicollinearity between variables was tested using Variance Inflation significantly higher (2.0 ± 1.4 vs 1.2 ± 0.6 mmol/l, p = 0.02) and Factor calculation. Statistical analysis was performed using GraphPad HDL levels were significantly lower (1.2 ± 0.4 vs 1.4 ± 0.3 mmol/l, Prism 6.01 (GraphPad Software, San Diego, CA) and SPSS (Version p = 0.01) in patients with schizophrenia. 16.0, SPSS Inc., Chicago, IL). Whereas D-dimer, fibrinogen and platelet count did not differ be- Based on previous published literature and in house data of OHP tween the two groups, the OCP (54.0 ± 12.6 vs 45.9 ± 9.1, p = 0.002) assay parameters and standard deviation values (Curnow et al., 2007; and OHP (12.6 ± 5.8 vs 7.2 ± 3.7, p b 0.001) were both significantly Edelman et al., 2013), a sample size of 30 subjects in each group could higher, and OFP (76.6 ± 9.8% vs 84.9 ± 6.4%, p b 0.001) significantly detect significance difference in OHP assay parameters between lower, in patients with schizophrenia, indicating both a hypercoagula- patients with schizophrenia and healthy controls with a two-sided ble and hypofibrinolytic state, compared to the controls. There were significance level of 0.05 and statistical power of at least 80%. no observable differences in Max OD and Max slope in the two groups. The delay in the onset of fibrin generation was significantly shorter in 3. Results healthy controls compared to patients with schizophrenia (13.5 ± 3.4 vs 16.0 ± 5.3 min, p = 0.02) (Fig. 1). The reduced OFP prompted inves- 3.1. Baseline characteristics tigation of PAI-1 levels in these patients. PAI-1 levels were significantly higher in patients with schizophrenia than in the control group (29.0 ± A total of 120 subjects participated in the study, 90 patients with 12.1 vs 21.8 ± 9.4, p = 0.006) (Table 2). schizophrenia and 30 healthy controls matched for age, gender and Although D-dimer, fibrinogen and platelet count did not differ body mass index (BMI). Table 1 summarizes the clinical characteristics significantly between controls and patients with schizophrenia, of the subjects. For patients with schizophrenia, the mean duration of their levels did correlate with OHP assay values in the patients antipsychotic treatment was 15.9 ± 9.7 years, with the majority with schizophrenia (Tables 3 and 4). In patients with schizophrenia, receiving clozapine treatment. They had higher rates of smoking, family OCP values demonstrated significant univariate associations with history of mental illness and hypercholesterolemia. No subjects had a raised D-dimer, fibrinogen, hsCRP, NLR and triglyceride levels. In history of PE or DVT prior to recruitment. Following the initial recruit- multivariate analysis, elevated fibrinogen (β = 0.39, p = 0.001) ment, one patient with schizophrenia was subsequently diagnosed and hsCRP (β = 0.26, p = 0.02) levels were independent predictors with DVT and PE, and there were two subsequent sudden deaths in of OCP level in these patients. the schizophrenia group. One of the deaths was presumed to be due Elevated OHP and decreased OFP levels indicate a hypofibrinolytic to drug overdose whilst the other was due to an acute myocardial state. Significant univariate associations with higher OHP included infarction (an incidental unilateral right lower limb DVT was found on higher BMI, fibrinogen, D-dimer, PAI-1, hsCRP levels and raised NLR. In autopsy). multivariate analysis, raised fibrinogen (β = 0.43, p b 0.001), platelet 178 V. Chow et al. / Schizophrenia Research 162 (2015) 175–182

Fig. 1. OHP assay parameters between patients with schizophrenia and healthy controls. A) Overall coagulation potential (OCP); B) overall haemostatic potential (OHP); C) overall fibrinolytic potential (OFP); D) maximum optical density (Max OD); E) Delay and F) Max slope. G) Reference sample of the OCP and OHP in a patient with schizophrenia and a healthy control. count (β = 0.25, p = 0.01) and PAI-1 levels (β = 0.23, p = 0.02) (Supplementary Fig. 1). However, there were no significant differences remained as significant independent predictors of a raised OHP. Signifi- in OHP, OCP and OFP values between patients receiving clozapine or cant univariate associations with lower OFP included raised white cell non-clozapine medications. There were 36 patients receiving adjunct count, BMI, fibrinogen, PAI-1, total and triglyceride levels therapy in additional to neuroleptic and these included: sertraline and lower high-density lipoprotein (HDL) levels. In multivariate analy- (8), sodium valproate (6), citalopram (5), escitalopram (4), fluoxetine sis, the independent predictors of a reduced OFP state included raised (4), lithium (3), lamotrigine (3), venlafaxine (2) and mirtazepine (1). triglyceride (β = −0.35, p = 0.001), PAI-1 (β = −0.29, p = 0.002) On exploratory analysis, we found no associations between adjunct and fibrinogen (β = −0.23, p = 0.03) levels. usage and OHP assay parameters (OCP: p = 0.55, OHP: p = 0.26, OFP: We investigated the influence of antipsychotic medications on the p=0.21). coagulation status of patients with schizophrenia, specifically investi- gating whether clozapine use affected these parameters given previous- 4. Discussion ly reported increased VTE risks in these patients (Walker et al., 1997). Patients with schizophrenia receiving clozapine or non-clozapine We have identified increased overall coagulation potential and antipsychotic medications were significantly more hypercoagulable impaired overall fibrinolysis potential in patients with schizophrenia (higher OHP, p = 0.006) and had impaired fibrinolysis (higher OCP, receiving long-term antipsychotics. These haemostatic abnormalities p b 0.001 and lower OFP, p = 0.002) than did healthy controls were independently associated with abnormalities in PAI-1, fibrinogen, V. Chow et al. / Schizophrenia Research 162 (2015) 175–182 179

Table 2 Biochemical parameters.

Patients with schizophrenia (n = 90) Controls (n = 30) p value

Biochemical parameters Haemoglobin (g/l) 143 ± 14 148 ± 10 0.19 9 ⁎ White cell count (×10 /l) 7.8 ± 2.5 6.5 ± 1.5 0.004 9 ⁎ Neutrophil count (×10 /l) 4.9 ± 2.1 3.6 ± 1.0 b0.001 ⁎ Neutrophil:lymphocyte ratio 2.5 ± 1.4 1.7 ± 0.8 0.03 Eosinophil count (×109/l) 0.17 ± 0.1 0.17 ± 0.1 0.82 ⁎ hsCRP (mg/l) 3.7 ± 5.3 0.9 ± 0.8 b0.001 HbA1c (%) 5.9 ± 1.1 5.6 ± 0.9 0.27 Total cholesterol (mmol/l) 5.0 ± 1.1 4.9 ± 0.7 0.63 ⁎ Triglycerides (mmol/l) 2.0 ± 1.4 1.2 ± 0.6 0.02 ⁎ HDL (mmol/l) 1.2 ± 0.4 1.4 ± 0.3 0.01 LDL (mmol/l) 3.0 ± 1.0 3.0 ± 0.7 0.95 Platelet count (×109/l) 261 ± 75 249 ± 53 0.48 Fibrinogen (g/l) 3.4 ± 0.7 3.1 ± 0.6 0.35 D-dimer (mg/l) 0.19 ± 0.11 0.22 ± 0.28 0.71 ⁎ PAI-1 (ng/ml) 29.0 ± 12.1 21.8 ± 9.4 0.006

Intra-assay CV (%) Inter-assay CV (%)

OHP assay ⁎ OHP 12.6 ± 5.8 7.2 ± 3.7 b0.001 1.73 3.78 ⁎ OCP 54.0 ± 12.6 45.9 ± 9.1 0.002 7.35 10.43 ⁎ OFP (%) 76.6 ± 9.8 84.9 ± 6.4 b0.001 2.61 1.90 Max OD (units) 0.7 ± 0.1 0.7 ± 0.2 0.52 0.81 4.28 Max slope (units) 222.3 ± 58.9 227 ± 43.5 0.69 7.37 13.36 ⁎ Delay (min) 16.0 ± 5.3 13.5 ± 3.4 0.02 3.37 8.18

Continuous values are presented as mean ± SD. hsCRP, high sensitivity C-reactive protein; HDL, high density lipoprotein; LDL, low density lipoprotein; HbA1c, glycosylated haemoglobin, PAI-1, plasminogen-activator-inhibitor-1; OHP, overall haemostatic potential; OCP, overall coagulation potential; OFP, overall fibrinolysis potential; OD, optical density; CV, coefficient of variation. ⁎ p b 0.05. platelet count, inflammatory markers and lipoprotein levels suggesting or venous thromboembolism, are often not detected by the traditional these as contributing factors to the risk of VTE in these patients. panel of tests as part of thrombophilia screening (including deficiencies Routine coagulation assays are based on the traditional cascade of antithrombin, proteins C and S, factor V Leiden and prothrombin model of coagulation and detect hypercoagulable states due to inherited G20210A mutations). A negative thrombophilia test may lead to false deficient or abnormal coagulation factors and acquired inhibitors. Unse- assurance as thrombophilia can only identify approximately 50% of all lected screening for inherited thrombophilic states in asymptomatic patients presenting with venous thrombosis by utilising the above patients is not recommended due to the low frequency, low penetrance tests (Whiteman and Hassouna, 2000; Middeldorp and van Hylckama of symptomatic VTE amongst carriers of the most common Vlieg, 2008; Middeldorp, 2011b). thrombophilic conditions and the high cost associated with testing The OHP assay is a simple and inexpensive global coagulation assay (Machin, 2003; Middeldorp, 2011a). Furthermore, hypercoagulable that detects both the dynamic coagulation and fibrinolytic potential of states, which are defined as those with the potential to develop arterial plasma samples. It has been shown to be useful in identifying

Table 3 Correlation analysis in patients with schizophrenia (n = 90).

OCP OHP OFP

Correlation coefficient p value Correlation coefficient p value Correlation coefficient p value

Clinical variables Age 0.08 0.45 −0.02 0.87 0.02 0.90 ⁎ ⁎ Body mass index 0.20 0.07 0.33 0.002 −0.22 0.04 Diabetes −0.07 0.51 0.02 0.85 −0.11 0.34 Hypercholesterolemia −0.07 0.53 0.09 0.41 −0.19 0.08 Smoking −0.03 0.77 0.04 0.74 −0.07 0.51 Clozapine treatment 0.07 0.56 −0.05 0.64 0.05 0.63

Laboratory markers ⁎ ⁎ Platelet 0.13 0.23 0.31 0.004 −0.26 0.02 ⁎ ⁎ D-dimer 0.35 0.001 0.23 0.04 −0.03 0.82 ⁎ ⁎ ⁎ Fibrinogen 0.61 b0.001 0.50 b0.001 −0.22 0.04 ⁎ ⁎ PAI-1 −0.02 0.83 0.30 0.008 −0.31 0.005 ⁎ ⁎ hsCRP 0.49 b0.001 0.35 0.001 −0.10 0.35 ⁎ ⁎ ⁎ White cell count 0.25 0.02 0.41 b0.001 −0.33 0.002 ⁎ ⁎ Neutro:lymph ratio 0.39 b0.001 0.23 0.03 −0.07 0.53 ⁎ Cholesterol −0.19 0.11 0.17 0.11 −0.41 b0.001 ⁎ HDL 0.03 0.81 −0.17 0.10 0.23 0.03 ⁎ ⁎ Triglyceride 0.34 0.002 0.12 0.28 −0.41 b0.001

PAI-1, plasminogen-activator-inhibitor-1; hsCRP, high sensitivity C-reactive protein; HDL, high density lipoprotein; OCP, overall coagulation potential; OHP, overall haemostatic potential; OFP, overall fibrinolysis potential. Pearson's and Spearman's correlations were performed for parametric and non-parametric variables respectively. Point biserial correlation was applied to dichotomous and continuous variables. ⁎ p b 0.05. 180 V. Chow et al. / Schizophrenia Research 162 (2015) 175–182

Table 4 medications will be required to establish differences between specific

Standardized coefficient β p value antipsychotic medications. The , which is associated with elevated inflam- Multivariate predictors of increased OCP in patients with schizophrenia (n = 90) matory markers and plasma triglycerides, is an independent risk factor Fibrinogen 0.39 0.001* hsCRP 0.26 0.02* for VTE (Ageno et al., 2006; Wang et al., 2010). Whether raised HDL is Backward selection regression model using a p value cut-off of b0.1, R2 = 0.36. protective against VTE remains unclear. Some cohort studies have dem- hsCRP, high sensitivity C-reactive protein. *p b 0.05. onstrated no clear association between HDL and risk of VTE, whilst Multivariate predictors of increased OHP in patients with schizophrenia (n = 90) others indicated that elevated HDL may increase risk of Fibrinogen 0.43 b0.001* unprovoked VTE in women (Chamberlain et al., 2008; Braekkan Platelet count 0.25 0.01* et al., 2009; Everett et al., 2009). A recent meta-analysis concluded PAI-1 0.23 0.02* that HDL was protective against VTE (Ageno et al., 2008). Reduced b 2 Backward selection regression model using a p value cut-off of 0.1, R = 0.33. fi PAI-1, plasminogen-activator-inhibitor-1. *p b 0.05. HDL was identi ed in patients presenting with massive PE (Wang et al., 2010)andwasassociatedwithVTErecurrence(Eichinger Multivariate predictors of reduced OFP in patients with schizophrenia (n = 90) et al., 2007). Possible HDL-related protective mechanisms include Triglycerides 0.35 0.001* PAI-1 0.29 0.002* reduced thrombin generation via promotion of the protein C path- Fibrinogen 0.23 0.03* way (Griffin et al., 1999), enhanced endothelial nitric oxide synthase WCC 0.19 0.07 activity and reduced leukocyte adhesion to endothelium (Mineo 2 Backward selection regression model using a p value cut-off of b0.1, R = 0.35. et al., 2006). In our patients with schizophrenia, raised hsCRP and PAI-1, plasminogen-activator-inhibitor-1; WCC, white cell count. *p b 0.05. triglycerides were independent predictors of impaired OCP and OHP supporting a role for inflammation and the metabolic syndrome. The independent associations identified on multivariate analysis hypercoagulable states in a number of patient groups (Vedin et al., highlight the potential for interaction between factors in determining 2005; Curnow et al., 2007; Reddel et al., 2013). The generation of a fibrin the overall hypercoagulable state in patients with schizophrenia. The time curve from serial spectrophotometric measurements in an ex vivo association between an inflammatory state (raised hsCRP) with hyper- environment represents the balance between fibrin generation and coagulability and the association between raised triglyceride levels with lysis. This underlying principle of the OHP assay determines whether hypofibrinolysis are interesting. Hypofibrinolysis has been shown to be the hypercoagulable state is due to increased fibrin generation, reduced a risk factor for a first venous and/or arterial thrombotic event in three fibrinolysis or a combination of these. Thus, the OHP assay is expected to large case–control studies (Lisman et al., 2005; Guimaraes et al., 2009; be dependent on levels of factors such as plasma fibrinogen (promotion Meltzer et al., 2009) and was an independent predictor for VTE in of coagulation) and PAI-1 inhibitor (inhibition of fibrinolysis). In our pa- another study (Cellai et al., 2013). Furthermore, the combination of tients with schizophrenia, plasma fibrinogen was an independent pre- hypercoagulability and hypofibrinolysis appears to synergistically dictor of raised OCP, OHP and OFP whilst PAI-1 was an independent increase the risk for VTE (Meltzer et al., 2008). Potential therapeutic predictor of OFP and OHP, even after adjusting for BMI and other clinical intervention with statin and/or fibrate therapy for these modifiable factors. Our OHP assay results indicate that patients with schizophrenia risk factors may reduce the overall hypercoagulable state and incidence have a hypercoagulable state due to a combination of increased fibrin of VTE in patients with schizophrenia. generation (higher OCP, p = 0.002) and reduced fibrinolysis (higher In addition to modifying the lipid profile, statin therapy has been OHP and lower OFP, both p b 0.001). proposed to have multiple pleiotropic effects (Waldman and The underlying biological mechanisms by which patients with Kritharides, 2003) with significant reductions in the occurrence of schizophrenia receiving antipsychotic medications develop VTE are symptomatic VTE (hazard ratio 0.57, p = 0.007) and hsCRP as reported complex and likely multifactorial. Several mechanisms have been in the JUPITER trial of healthy older adults treated with suggested, including enhanced platelet aggregation and adhesion (Glynn et al., 2009). These findings were reaffirmed in recent meta- (Boullin et al., 1978; Axelsson et al., 2007), the development of analyses showing significant reduction in VTE events amongst patients antiphospholipid antibodies (Davis et al., 1994) and raised homocyste- treated with statin therapy (Agarwal et al., 2010; Squizzato et al., 2010; ine levels (Hu et al., 2013). The increased risk may also be exacerbated Li et al., 2011; Pai et al., 2011; Rodriguez et al., 2012). may by antipsychotic-induced sedation resulting in venous stasis. Whilst increase nitric oxide bioavailability, regulate angiogenesis, reduce the obesity is an independent risk factor for VTE (Torbicki et al., 2008)and inflammatory response and down-regulate the blood coagulation is frequent in patients with schizophrenia, in particular those receiving cascade (Undas et al., 2005). In addition, statins have been shown to clozapine (Fitzsimons et al., 2005), previous studies have confirmed decrease platelet aggregation, tissue factor (Rosenson and Tangney, that the increased risk of VTE amongst patients with schizophrenia 1998; Undas et al., 2002), PAI-1 (Rosenson and Tangney, 1998; persists even after controlling for BMI and other clinical factors Nishino et al., 2008), interleukin-6 and interleukin-8 (Rezaie-Majd (Zornberg and Jick, 2000; Parkin et al., 2003). Furthermore, the et al., 2002) and hsCRP (Albert et al., 2001; Glynn et al., 2009) levels, schizophrenic illness in itself has been shown to have a propensity for and increase tPA and thrombomodulin expression (Rosenson and increasing the risk of thrombogenesis with raised markers for thrombo- Tangney, 1998; Perez and Bartholomew, 2010). Given these prior sis (D-dimer), thrombocyte activation (soluble P-selectin and L-selectin) results, future studies should explore the effects of statin therapy on and platelet dysfunction (increased platelet expression of integrins) OHP assay parameters in patients with schizophrenia. reported in antipsychotic drug-naive patients with schizophrenia We note several limitations to our study. First, the standard OHP (Walsh et al., 2002; Iwata et al., 2007; Masopust et al., 2011). assay uses platelet-poor plasma and may therefore underestimate the Our study cannot draw conclusions regarding a causal role for role of in global assays of coagulation. Second, our patients antipsychotic medications in mediating the altered coagulation status with schizophrenia had more cardiovascular risk factors than controls of these patients. All our patients had been stabilised on their current and this may contribute to their observed hypercoagulable state, antipsychotic regime prior to enrolment in this study and the effect of making the drawing of conclusions regarding thrombotic risk due to medication per se in our cohort is unknown. Amongst the patients psychosis itself less straightforward. However, this is clinically quite with schizophrenia, we did not find any association between the type important as the thrombosis risk in patients with schizophrenia is likely of medication used and OHP parameters. Larger patient numbers and contributed to many factors including co-existing cardiovascular risk the evaluation of patients before and after starting antipsychotic factors. V. Chow et al. / Schizophrenia Research 162 (2015) 175–182 181

In conclusion, we report for the first time that a hypercoagulable and Cellai, A.P., Lami, D., Antonucci, E., Fiorillo, C., Becatti, M., Olimpieri, B., Bani, D., Grifoni, E., fi Cenci, C., Marcucci, R., Mannini, L., Poli, D., Abbate, R., Prisco, D., 2013. Fibrinolytic hypo brinolytic state is present in patients with schizophrenia on long- inhibitors and fibrin characteristics determine a hypofibrinolytic state in patients term antipsychotic medications. These data may help our understand- with pulmonary embolism. Thromb. Haemost. 109 (3), 565–567. ing of the mechanisms underlying the increased risk for VTE in this Cesari, M., Pahor, M., Incalzi, R.A., 2010. Plasminogen activator inhibitor-1 (PAI-1): a key factor linking fibrinolysis and age-related subclinical and clinical conditions. patient population. Cardiovasc. Ther. 28 (5), e72–e91. Supplementary data to this article can be found online at http://dx. 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