Direct oral anticoagulants in patients with antiphospholipid syndrome: a retrospective study in a real-life patient cohort.

The current mainstay for the treatment of thrombotic antiphospholipid syndrome (APS) is anticoagulation with vitamin K antagonists (VKAs). The use of direct oral anticoagulants (DOACs) is under debate. We aimed to assess whether DOACs would be safe in APS patients presenting to the thrombosis clinic. A retrospective cohort study was conducted. All patients presenting to our thrombosis clinic between 2010 and 2017 with a diagnosis of APS taking either VKAs or DOACs were included. APS diagnosis was based on the revised Sapporo criteria. Clinical and laboratory data were collected from the electronic and physical patient files. Out of 200 patients, 81 received VKAs, and 119 DOACs. The two cohorts did not differ with regard to their initial clinical manifestation or additional prothrombotic risk factors. Only a small minority of patients was antiphospholipid antibody triple positive (VKA, 7.0% vs. DOAC, 4.2%). Numberofon-treatment events was low (3 vs. 2). The hazard ratio for any thromboembolic event for patients taking DOACs was 0.78 (95% confidence interval, 0.12-5.19). Treatment with DOACs was not associated with an increased risk of recurrent thromboembolism in comparison with VKAs in this retrospective study. Our observation supports the assumption that in nontriple positive (low risk) APS patients, DOACs might be safe. Prospective data are urgently needed.

Impact of double heterozygosity for Factor V Leiden and Prothrombin G20210A on the thrombotic phenotype.

INTRODUCTION: Because of the rarity of double heterozygosity for Factor V Leiden (FVL) and Prothrombin (FII) G20210A, little is known about the thrombotic phenotype in double heterozygotes. MATERIAL AND METHODS: In a retrospective cohort study of patients referred for a thrombophilia work-up, we investigated whether double heterozygotes (n = 138) exhibit a more severe thrombotic phenotype compared with single FVL or FIIG20210A heterozygotes, single FVL homozygotes, or wildtype carriers. RESULTS: The risk of venous thromboembolism (VTE) was higher for female but not male double heterozygotes compared with single heterozygotes (FVL: 2.51, 95%CI 1.55-4.08, FIIG20210A: 1.75, 95%CI 1.14-2.68) and wildtype carriers (HR 2.53, 95%CI 1.58-4.05) but not compared with FVL homozygotes (HR 1.31, 95%CI 0.94-1.83). Female double heterozygotes developed VTE nearly a decade earlier than wildtype carriers and FVL heterozygotes (mean 44.2 vs. 52.6 and 52.2 years), most often in association with oral contraceptives. Spontaneous VTE and arterial thromboembolic events were not more frequent in double heterozygotes compared with the other genotype groups. Deep vein thrombosis (DVT) of the lower limb was the predominant VTE location in double heterozygotes, atypical vein thrombosis was rare. A phenomenon that has been described as the FVL paradox, a higher proportion of isolated DVT than pulmonary embolism, was also found for double heterozygotes. CONCLUSION: The thrombotic phenotype in double heterozygotes resembles the appearance of the thrombotic phenotype in FVL carriers but the thrombotic risk is aggravated by women-specific risk factors.

Familial Multiple Coagulation Factor Deficiencies (FMCFDs) in a Large Cohort of Patients-A Single-Center Experience in Genetic Diagnosis.

BACKGROUND: Familial multiple coagulation factor deficiencies (FMCFDs) are a group of inherited hemostatic disorders with the simultaneous reduction of plasma activity of at least two coagulation factors. As consequence, the type and severity of symptoms and the management of bleeding/thrombotic episodes vary among patients. The aim of this study was to identify the underlying genetic defect in patients with FMCFDs. METHODS: Activity levels were collected from the largest cohort of laboratory-diagnosed FMCFD patients described so far. Genetic analysis was performed using next-generation sequencing. RESULTS: In total, 52 FMCFDs resulted from coincidental co-inheritance of single-factor deficiencies. All coagulation factors (except factor XII (FXII)) were involved in different combinations. Factor VII (FVII) deficiency showed the highest prevalence. The second group summarized 21 patients with FMCFDs due to a single-gene defect resulting in combined FV/FVIII deficiency or vitamin K-dependent coagulation factor deficiency. In the third group, nine patients with a combined deficiency of FVII and FX caused by the partial deletion of chromosome 13 were identified. The majority of patients exhibited bleeding symptoms while thrombotic events were uncommon. CONCLUSIONS: FMCFDs are heritable abnormalities of hemostasis with a very low population frequency rendering them orphan diseases. A combination of comprehensive screening of residual activities and molecular genetic analysis could avoid under- and misdiagnosis.

Should emicizumab be used in patients with acquired hemophilia A?

Emicizumab is currently approved to prevent bleeding in patients with congenital hemophilia A with or without neutralizing antibodies (inhibitors) against factor VIII (FVIII). Here, we present a case-based discussion of its potential use in acquired hemophilia A (AHA), a severe bleeding disorder caused by autoantibodies against FVIII. State-of-the-art management is based on bypassing agents (recombinant factor VIIa, activated prothrombin complex concentrate) and recombinant porcine FVIII; immunosuppressive therapy (corticosteroids, rituximab, cyclophosphamide) is used to suppress autoantibody formation. Case reports and one series suggest that emicizumab can reduce the risk of bleeding and the requirement for hemostatic therapy until remission of AHA is achieved. Further, it may allow to postpone the start of immunosuppressive therapy or to use less intense regimens. However, the risk-benefit assessment of emicizumab in AHA is difficult because demographic and clinical characteristics are different compared with congenital hemophilia. Prospective clinical trials are needed before the use of emicizumab can be recommended in AHA.

Safety of direct oral anticoagulant exposure during pregnancy: a retrospective cohort study.

BACKGROUND: Direct oral anticoagulants (DOACs) have largely replaced vitamin K antagonists in many indications for anticoagulation. Prescribed to millions of patients, including women of reproductive age, exposure to DOACs in early pregnancy is not uncommon, but data on the embryotoxic risks are scarce. We aimed to assess the risk of DOAC embryotoxicity in a large sample of reported cases. METHODS: In this retrospective cohort study, we collected individual case reports of DOAC exposure in pregnancy from gynaecologists, haematologists, and vascular specialists starting from May, 2015. We obtained exports in April and October, 2017, August, 2018, and December, 2019, from the pharmacovigilance databases of the DOAC manufacturers, the European Medicines Agency (EMA), the German drug authority, and searched the homepage of the US Food and Drug Administration (FDA) for pregnancy exposure reports. Data from the International Society of Thrombosis and Haemostasis (ISTH) registry were obtained in August, 2018, and on July 21, 2020; data from the Teratology Information Service in Ulm, Germany, were received July 22, 2020. We also ran a systematic literature search on July 22, 2020, for cases of DOAC exposure. These data were compiled with those from our 2016 risk assessment and duplicate reports were excluded. Fetal or neonatal abnormalities were classified as a major birth defect according to the European Concerted Action on Congenital Anomalies and Twins (EUROCAT) classification and adjudicated into four categories: relation to DOAC exposure likely, possible, unlikely, or unrelated. FINDINGS: We identified 1193 reports of DOAC exposure during pregnancy: 49 from physicians, 48 from the ISTH registry, 29 from the Teratology Information Service, 62 from the German drug authority, 536 from Bayer (extracted from the Bayer pharmacovigilance system, the WHO VigiBase, and from the FDA Adverse Event Reporting System), 87 from Boehringer Ingelheim, 16 from Daiichi Sankyo, 98 from the literature search, two from the FDA homepage search, ten from the Risk Evaluation and Mitigation Strategy Review, and 256 from the EMA reports. After excluding potential duplicates, we identified 614 unique cases of DOAC exposure in pregnancy occurring between Feb 1, 2007, and July 9, 2020, that consisted of rivaroxaban in 505 (82%) pregnancies, dabigatran in 36 (6%), apixaban in 50 (8%), and edoxaban in 23 (4%). The median duration of DOAC exposure was 5·3 weeks (IQR 4·0-7·0) into pregnancy. Information on pregnancy outcome was available in 336 (55%) of 614 pregnancies: 188 (56%) livebirths, 74 (22%) miscarriages, and 74 (22%) elective pregnancy terminations. 21 (6%; 95% CI 4-9) of 336 showed fetal abnormalities, of which 12 (4%; 2-6) were adjudicated as major birth defects potentially related to DOAC exposure. INTERPRETATION: Although reports of pregnancy outcomes after DOAC exposure are missing important details and predominantly describe rivaroxaban exposures, the available data do not suggest that DOAC exposure in pregnancy carries a high risk of embryopathy. The 2016 ISTH guidance against elective pregnancy termination for fear of DOAC embryotoxicity and the recommendation in favour of close pregnancy surveillance is still valid. Pregnancy outcome data are inconsistently captured in pharmacovigilance databases, indicating a strong need for a more robust system of reporting. FUNDING: None.

Prevalence of hereditary antithrombin mutations is higher than estimated in patients with thrombotic events.

Antithrombin is the major inhibitor of blood coagulation, primarily inactivating thrombin and factor Xa. Inherited antithrombin deficiency is associated with an increased risk of thromboembolism. Its prevalence is estimated to be about 0.2% in the general healthy population, whereas it is 2% in patients with a history of thrombosis. We previously demonstrated thrombosis patients receiving therapeutic dosage of low-molecular-weight heparin having a lower antifactor Xa activity than originally estimated. In those patients, mutations in the AT gene (SERPINC1) were detected despite normal antithrombin activity. Thus we concluded that prevalence of antithrombin mutations might be higher than previously calculated. The aim of the present study was to investigate the prevalence of hereditary antithrombin mutations by analyzing the antithrombin gene in patients with a history of thromboembolism. Mutation analyses were performed by direct sequencing of SERPINC1 in 150 patients (aged <40 years) with thromboembolism and normal antithrombin levels. Patients with thrombophilic defects [factor V Leiden (G1691A), prothrombin (G20210T) mutation, protein C deficiency, protein S deficiency, and antiphospholipid antibodies] were excluded. The results were compared with those of 150 healthy controls without any personal history of thrombosis. Mutation analyses of all seven antithrombin exons revealed five (3.5%) patients with antithrombin mutations: three different heterozygous missense mutations were found in exon 2 and one (in two patients) in exon 7. Prevalence of inherited antithrombin mutations in thrombosis patients is higher than previously estimated. Functional antithrombin tests are unable to detect all clinically relevant antithrombin defects.

Deficiencies of antithrombin, protein C and protein S - practical experience in genetic analysis of a large patient cohort.

Deficiencies of natural anticoagulant proteins including antithrombin (AT), protein C (PC) and protein S (PS) are important causes of inherited thrombophilia. This study aimed to report on the practical experience gained in performing genetic analyses of a large cohort of patients with AT, PC and PS deficiencies and to relate this knowledge to clinical application. We genotyped a large cohort of 709 unrelated patients with AT (231), PC (234) and PS (244) deficiencies referred to us by physicians throughout Germany. Mutations were detected by direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The highest mutation detection rate (MDR) was found for the SERPINC1 gene (83.5%), followed by the PROC (69%) and PROS1 (43%) genes. Even at AT activities close to the normal range (75%), the MDR was 70%. Contrastingly, for PC and PS deficiencies, the MDR dropped significantly and mildly lowered to subnormal values. At PS activities >55% for PS no mutations were detected. Mutation profiles of all three genes were similar with the highest prevalence for missense mutations (63-78%), followed by nonsense (7-11%), splice-site mutations (7-13%), small deletions (1-8%), small insertions/duplications (1-4%) and large deletions (3-6%). In conclusion, genetic testing is a useful diagnostic tool for diagnosing thrombophilia. Based on our data, genetic analysis for patients with AT deficiency is indicated for all subnormal activities. In contrast, genotyping is not advisable for PC activities >70% and for PS activities >55%.

Low-molecular-weight heparin versus placebo for the prevention of venous thromboembolism in metastatic breast cancer or stage III/IV lung cancer.

In 2 double-blind studies, ambulatory patients with objectively proven, disseminated metastatic breast carcinoma (TOPIC-1) or stage III/IV non-small-cell lung carcinoma (TOPIC-2) were randomized to certoparin 3000 IU or placebo subcutaneously once daily, for 6 months. Primary efficacy outcome was objectively confirmed symptomatic or asymptomatic venous thromboembolism (VTE). Safety outcomes included bleeding (major and minor), and thrombocytopenia. TOPIC-1 was halted after an interim analysis. Venous thromboembolism occurrence was not different between treatment groups in TOPIC-1 (4% treated with certoparin, 7 of 174 vs 4% receiving placebo, 7 of 177, odds ratio [OR] 1.02; 95% confidence interval [CI] 0.30-3.48) and in TOPIC-2 (4.5%, 12 of 268) vs 8.3%, 22 of 264, respectively, OR 0.52; CI 0.23-1.12). Mortality was not different between groups. A post hoc analysis showed certoparin significantly reduced VTE in stage IV lung carcinoma (3.5% vs 10.2%; P = .032) without increased bleeding. In conclusion, thrombosis risk and prophylactic benefit was highest in stage IV lung carcinoma patients.

Increase in the plasma levels of protein Z-dependent protease inhibitor in normal pregnancies but not in non-pregnant patients with unexplained recurrent miscarriage.

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a serine protease inhibitor which efficiently inactivates activated factor X, when ZPI is complexed with PZ in plasma. Reduced plasma levels of ZPI and PZ have been reported in association with thrombosis. It has also been reported that PZ increases during pregnancy and that its partial deficiency is related to early pregnancy loss or recurrent miscarriage (RM). However, until now there has been no report on ZPI in pregnancy. To explore the possible role(s) of ZPI in the maintenance of pregnancy, we studied 42 non-pregnant normal women, 32 women with normal pregnancies, and 134 cases of unexplained RM in Japan, as well as 64 non-pregnant normal German females. Plasma ZPI was measured by in-house ELISA. There were significantly higher concentrations of plasma ZPI in normal pregnancies compared to non-pregnant women. The present study also confirmed that both factor X, the major target of ZPI, and protein Z increased during normal pregnancies. This increased ZPI and PZ may counteract the increased activated factor X, which may in turn contribute to the maintenance of normal placental circulation. Plasma ZPI levels were unchanged in non-pregnant RM women, while the plasma PZ level was slightly reduced, a finding consistent with existing reports. The exact relationship between RM and this unaltered ZPI with mild PZ reduction relative to normal pregnancies warrants further investigation.

Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin time, antithrombin and D-dimer.

This study evaluates the effect of whole blood storage on common coagulation parameters in order to confirm or revise acceptable storage limits as defined by current guidelines and diverse study reports. Aliquots were taken from the citrated whole blood of inpatients and outpatients (n = 147) within 4 h after blood withdrawal and after extended storage of whole blood for 8 and 24 h at ambient temperature. Aliquots were centrifuged and analyzed for prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fbg), antithrombin (AT), thrombin time (TT) and D-dimer. For each parameter, samples from 33-56 patients were investigated covering a wide range of normal and pathological values. Samples from patients receiving heparin were excluded from analyses of APTT and TT. All assays were performed using reagents and an analyzer from Siemens Healthcare Diagnostics Products GmbH. The mean percentage change after 8 and 24-h storage was below 10% for all parameters. Considering the changes in individual samples, all parameters can be reliably tested after 8-h storage, since less than 15% of the samples demonstrated individual changes of above 10%. The acceptable storage time can be extended to 24 h for PT, TT and D-dimer. Clinically relevant changes were detected after 24-h storage for APTT: 41% of the investigated samples demonstrated changes of above 10%. After 24-h storage, changes for Fbg and AT values were more than 15% in five out of 49 and in three out of 45 samples, respectively. This sporadic increase of values is clinically acceptable except for borderline samples.

Fibrinogen promotes early atherosclerotic changes of the carotid artery in young, healthy adults.

AIM: To determine whether high plasma levels or activities of different hemostatic proteins contribute to the development of early atherosclerotic vessel wall changes. Elevated levels of various hemostatic proteins and markers of inflammation have been linked to an increased risk of ischemic cardiovascular events; however, the mechanisms by which these molecules might contribute to this increased risk is not clear. METHODS: The intima-media thickness of the common carotid arteries (CCA-IMT) of 125 healthy young volunteers without known cardiovascular risk factors was measured by high-resolution ultrasound. Plasma concentrations of fibrinogen, thrombomodulin, protein Z and CRP were quantified, and the plasma activities of protein C, plasminogen and factor VIII were measured. Other established risk factors, such as body mass index (BMI) and plasma levels of cholesterol, triglycerides and homocysteine, were also determined. Furthermore, the carotid arteries were examined for the presence of plaques and stenoses. RESULTS: Univariate analysis showed a significant negative correlation between CCA-IMT and HDL cholesterol, and positive correlations with age, BMI, LDL cholesterol, triglycerides, homocysteine, fibrinogen and thrombomodulin, but not with total cholesterol, lipoprotein(a) and hsCRP. CCA-IMT was also statistically independent of the activities of protein C, factor VIII and plasminogen. Multivariate analysis revealed a significant correlation of CCA-IMT with age, BMI and fibrinogen. CONCLUSION: Our data suggest that fibrinogen levels correlate with early atherosclerotic changes of the carotid artery in a population with very low cardiovascular risk.

Factor VII-activating protease (FSAP): vascular functions and role in atherosclerosis.

FSAP is a plasma serine protease for which a potential role in the regulation of coagulation and fibrinolysis is postulated, based on its property to activate factor VII (FVII) as well as pro-urokinase (uPA). In clinical studies, the G534E single nucleotide polymorphism (Marburg I) of FSAP has been linked to late complications of atherothrombosis and is associated with a low proteolytic activity, particularly, towards pro-uPA. This has stimulated much interest in a search for additional functions of FSAP in the cardiovascular system. FSAP is a potent inhibitor of vascular smooth muscle cell proliferation and migration in vitro and local application of FSAP (but not Marburg I variant) in animal models reduces neointima formation. This is due to a reduced proteolytic activity of the variant isoform towards platelet derived growth factor-BB, a key mediator of neointima development. Moreover, appreciable quantities of FSAP are localized to unstable atherosclerotic plaques and may contribute to plaque instability. These data indicate that the cellular regulatory effects of FSAP may be more important than its influence on haemostasis. In this review the contribution of FSAP to vascular fibroproliferative inflammatory diseases in the context of pericellular proteolysis of the extracellular matrix, growth factor activity and haemostasis will be highlighted.

Risk stratification and heparin prophylaxis to prevent venous thromboembolism in pregnant women.

Women with a history of venous thromboembolism (VTE), thrombophilia or both may be at increased risk of thrombosis during pregnancy, but the optimal management strategy is not well defined in clinical guidelines because of limited trial data. A strategy of risk assessment and heparin prophylaxis was evaluated in pregnant women at increased risk of VTE. In a prospective trial (Efficacy of Thromboprophylaxis as an Intervention during. Gravidity [EThIG]), 810 pregnant women were assigned to one of three management strategies according to pre-defined risk factors related to history of VTE and thrombophilic profile. Low-risk women (group I), received 50-100 IU dalteparin/kg body weight/day for 14 days postpartum, or earlier when additional risk factors occurred. Women at high (group II) or very high risk (group III) received dalteparin from enrollment until six weeks postpartum (50-100 IU and 100-200 IU/kg/day, respectively). Objectively confirmed, symptomatic VTE occurred in 5/810 women (0.6%; 95% confidence interval [CI], 0.2 to 1.5%) (group I, 0 of 225; II, 3/469; III, 2/116). The rate of serious bleeding was 3.0% (95 % CI, 1.9 to 4.4%); 1.1% (95 % CI, 0.5 to 2.2%) was possibly dalteparin-related. There was no evidence of heparin-induced thrombocytopenia, one case of osteoporosis, and rates of miscarriage and stillbirth were similar to previous, retrospective studies. Risk-stratified heparin prophylaxis was associated with a low incidence of symptomaticVTE and few clinically important adverse events. Antepartum heparin prophylaxis is, therefore, warranted in pregnant women with idiopathic thrombosis or symptomatic thrombophilia.

The G534E polymorphism of the gene encoding the factor VII-activating protease is associated with cardiovascular risk due to increased neointima formation.

The G534E polymorphism (Marburg I [MI]) of factor VII-activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.

Value of soluble adhesion molecules and plasma coagulation markers in assessing transplant coronary artery disease in pediatric heart transplant recipients.

BACKGROUND: With an increasing number of heart transplantations (HTx) performed in children and an extended long-term survival of these patients, the importance of transplant coronary artery disease (TCAD) rises in this group of transplant recipients. Reliable serum markers for diagnosis or non-invasive monitoring of this disease in pediatric transplant recipients are still missing. We studied the systemic expression of adhesion molecules as well as plasma coagulation markers and the occurrence of TCAD and/or rejection in pediatric heart transplant recipients. METHODS AND RESULTS: The systemic plasma levels of soluble forms of sVCAM-1 and sICAM-1, d-dimer, tissue factor (TF), prothombin fragments F(1+2), and tissue factor pathway inhibitor (TFPI) were assessed in serial venous blood samples (2-4 per patient) in 50 pediatric transplant recipients children and 63 age- and sex-matched non-transplanted controls. TCAD and rejection were diagnosed angiographically or by combined histological, echocardiographic, or clinical signs, respectively. Plasma levels of sICAM-1 and sVCAM-1, d-dimers and prothrombin fragment F(1+2) but not TF and TFPI were significantly increased in children following HTx compared with non-transplanted controls (p<0.001). Among the transplanted patients, sICAM-1 levels were significantly higher in patients with angiographically detectable TCAD than in patients without evidence of TCAD (p<0.005). Plasma sICAM-1 levels above a cutoff value of 1500 ng/mL (95.5 percentile of control values) were indicative of the presence of TCAD (odds ratio 2.7; 95% confidence interval, 1.34-5.56, p = 0.022; Fisher's exact test). Only d-dimers were found to be significantly elevated in children with signs of myocardial rejection compared with those without rejection. CONCLUSIONS: Our results suggest that plasma sICAM-1 and d-dimer levels may be potentially useful to non-invasively assess TCAD and rejection, respectively, in pediatric heart transplant recipients.

Argatroban treatment for patients with intolerance to heparin and hirudin.

Argatroban is a synthetic thrombin inhibitor that acts independently of any cofactor. It inhibits free as well as fibrin- and clot-bound thrombin. The clinical history of two patients is presented, both with previous allergic reactions toward heparin and hirudin. In both patients, antibodies against hirudin were documented. Argatroban was successfully used in both patients without any side effects. We conclude argatroban to be the drug of choice in patients with intolerance to heparin and hirudin. Moreover, genetic variations affecting the hepatic catabolism of argatroban might exist, because very different dosages of argatroban were needed in the two patients.

R255h amino acid substitution of protein Z identified in patients with factor V Leiden mutation.

The clinical significance of diminished protein Z in plasma is controversial. Studies in mice demonstrated that deficiency of protein Z dramatically increases the prothrombotic tendency of factor V Leiden mutation. This finding was confirmed by initial results in humans, indicating that thromboembolism in factor V Leiden patients with lowered protein Z level occurs earlier than in patients with normal protein Z levels. Consequently, the aim of our present study was to find out whether genetic alterations of protein Z were demonstrated in patients with factor V Leiden mutation and early onset of thromboembolic disease. DNA-sequencing of the protein Z gene was performed in two patients with factor V Leiden mutation, early onset of thromboembolism, and lowered protein Z levels. In both patients, R255H substitution of the protein Z gene was identified. Subsequently, the R255H substitution was also found in 12 of 132 additional patients. Patients presenting with the R255H substitution in addition to factor V Leiden mutation showed thromboembolic events more frequently than factor V Leiden patients without R255H substitution of the protein Z gene. In conclusion, R255H substitution of the protein Z gene seems to influence clinical symptoms of thromboembolism in factor V Leiden patients.

A naturally occurring E30Q mutation in the Gla domain of protein Z causes its impaired secretion and subsequent deficiency.

Protein Z is a vitamin K-dependent glycoprotein that plays a role in the regulation of coagulation. A nucleotide substitution of G by C in exon II of the protein Z gene, resulting in the replacement of Glu-30 with Gln (E30Q), and a G to A transition at the 79th nucleotide in intron F (IntF79G/A) were heterozygously identified in a patient with a severe thrombotic tendency, whose plasma protein Z level was about 15% of normal. Other vitamin K-dependent coagulation factors were within normal ranges. Glu-30 is one of 13 gamma-carboxylation sites in protein Z and is well conserved among vitamin K-dependent proteins. Expression studies revealed that the E30Q mutant was not released from synthesizing cells, although wild-type protein Z was readily secreted in a vitamin K-dependent fashion. The E30Q mutant was N-glycosylated, gamma-carboxylated, and translocated from the endoplasmic reticulum (ER) to the Golgi in the presence of vitamin K, as was the wild type. Coexpression of E30Q with wild-type protein Z interfered with the secretion of the wild type, while only a minor or no effect was observed on the secretion of factor X and plasminogen. The IntF79A allele has been reported to be also associated with lowered protein Z levels.