Yale S. Arkel

Protein Z, protein S levels are lower in patients with thrombophilia and subsequent pregnancy complications

Summary.  Objective: We posit that low levels of protein S (PS) and protein Z (PZ) contribute to adverse pregnancy outcome (APO). Patients: We evaluated 103 women with subsequent normal pregnancy outcome (NPO), 106 women with APO, and 20 women with thrombophilia (TP). Methods: We compared first trimester (1st TRI) PZ levels in 103 women with NPO, 106 women with APO, and in 20 women with TP. We compared plasma levels of PZ and free PS antigen during the second (2nd TRI) and third trimesters (3rd TRI) of pregnancy in 51 women with APO and 51 matched women with NPO. Results: The mean 1st TRI PZ level was significantly lower among patients with APO, compared to pregnant controls (1.81 ± 0.7 vs. 2.21 ± 0.8 µg mL−1, respectively, P < 0.001). Of patients with known TP, those with APO had a tendency for lower mean PZ levels compared to those TP women with NPO (1.5 ± 0.6 vs. 2.3 ± 0.9 µg mL−1, respectively, P < 0.0631). There was a significant decrease in the PZ levels in patients with APO compared to NPO (2nd TRI 1.5 ± 0.4 vs. 2.0 ± 0.5 µg mL−1, P < 0.0001; and 3rd TRI 1.6 ± 0.5 vs. 1.9 ± 0.5 µg mL−1, P < 0.0002). Protein S levels were significantly lower in the 2nd and 3rd TRIs among patients with APO compared to patients with NPO (2nd TRI 34.4 ± 11.8% vs. 38.9 ± 10.3%, P < 0.05, respectively; and 3rd TRI 27.5 ± 8.4 vs. 31.2 ± 7.4, P < 0.025, respectively). Conclusions: We posit that decreased PZ and PS levels are additional risk factors for APO.

Circulating levels of inflammatory cytokines (IL-1β and TNF-α ), resistance to activated protein C, thrombin and fibrin generation in uncomplicated pregnancies

We studied 33 women during normal uneventful pregnancies and with no history of previous adverse pregnancy events for markers of activated coagulation and thrombin activity including prothrombin fragment 1.2(PF1.2), thrombin- antithrombin (TAT), and soluble fibrin polymer (SFP). In addition, we measured potential thrombin generation through the addition of thromboplastin to patient plasma in the presence of a thrombin-specific chromogenic substrate determined serially over a period of time--Endogenous Thrombin Potential assay (ETP). This assay was performed with plasma treated and untreated with activated protein C (APC). The fibrinolytic system was assessed by measurement of thrombin activatable fibrinolysis inhibitor (TAFI). These findings were correlated with the levels of pro-inflammatory cytokines, interleukine-1 beta and tumor necrosis factor-alpha. Our data supports previous reports that indicate that resistance to activated protein C and coagulation activation markers are commonly increased in the later 2/3rds of pregnancy. There are no differences in thrombin generation potential, as determined by the ETP assay without the addition of APC, in the three trimesters. However, the thrombin reserve (TR), the ETP result without APC divided by the ETP result with the addition of APC, is increased above the reference range in the 2nd and 3rd trimesters. Patients with increased TR and resistance to APC had increased levels of TNF-alpha. Increased proinflammatory cytokines are reportedly associated with changes in the APC system with a decrease in the ability to generate APC. A sub-group of pregnancies with APC resistance had increased levels of TNF-alpha and may be important in the risk for adverse pregnancy outcomes.

Does heparin therapy improve pregnancy outcome in patients with thrombophilias

Bernstein M, Brisson L, Cairney B, DeSai D, Grant R, Israel S, Jardine L, Luke B, Massicotte P, Silva M. Venous thromboembolic complications (VTE) in children: first analyses of the Canadian Registry of VTE. Blood 1994; 83: 1251–7. 3 Ehrenforth S, Junker R, Koch HG, Kreuz W, Munchow N, Scharrer I, Nowak-Gottl U. Multicenter evaluation of combined prothrombotic defects associated with thrombophilia in childhood. Eur J Pediatr 1999; 158 (Suppl. 3): S97–104. 4 Kosch A, von Kries R, Nowak-Gottl U. Thrombosen im kindersalter. Monatsschr Kinderheilkd 2000; 148: 387–97. 5 Revel-Vilk S, Chan A, Bauman M, Massicotte P. Prothrombotic conditions in an unselected cohort of children with venous thromboembolic disease. J Thromb Haemost 2003; 1: 915–21. 6 Young G, Manco-Johnson M, Gill JC, Dimichele DM, Tarantino MD, Abshire T, Nugent DJ. Clinical manifestations of the prothrombin G20210A mutation in children: a paediatric coagulation consortium study. J Thromb Haemost 2003; 1: 958–62. 7 Nowak-Gottl U, Strater R, Heinecke A, Junker R, Koch HG, Schuierer G, von Eckardstein A. Lipoprotein(a) and genetic polymorphism of clotting factor V, prothrombin, and methylenetetrahydrofolate reductase are risk factors of spontaneous ischemic stroke in childhood. Blood 1999; 94: 3678–82. 8 Strater R, Becker S, von Eckardstein A, Heinecke A, Gutsche S, Junker R, Kurnik K, Schobess R, Nowak-Gottl U. Prospective assessment of risk factors for recurrent stroke during childhood—a 5years follow-up study. Lancet 2002; 360: 1526–7. 9 Tormene D, Simioni P, Prandoni P, Franz F, Zerbinati P, Tognin G, Girolami A. The incidence of venous thromboembolism in thrombophilic children: a prospective cohort study. Blood 2002; 100: 2403–5. 10 Sutor AH. Screening children with thrombosis for thrombophilic proteins. Cui bono? J Thromb Haemost 2003; 1: 886–8. 11 Van Ommen CH, Heijboer H, Buller HR, Hirasing RA, Heijmans HS, Peters M. Venous thromboembolism in childhood: a prospective twoyear registry in the Netherlands. J Pediatr 2001; 139: 676–81. 12 Kosch A, Junker R, Kurnik K, Schobess R, Gunther G, Koch H, Nowak-Gottl U. Prothrombotic risk factors in children with spontaneous venous thrombosis and their asymptomatic parents. A family study. Thromb Res 2000; 99: 531–7.

The use of coagulation activation markers (soluble fibrin polymer, TpP, prothrombin fragment 1.2, thrombin-antithrombin, and D-dimer) in the assessment of hypercoagulability in patients with inherited and acquired prothrombotic disorders.

A total of 260 consecutive patients, referred for hypercoagulable assessment, was included in this study. Four coagulation activation markers were utilized to assess these patients [enzyme-linked immunosorbent assays for soluble fibrin polymer (TpP), prothrombin fragment 1.2, thrombin–antithrombin complex, and D-dimer]. The mean levels of the activation markers directly correlated with the number of hypercoagulable abnormalities. The percentage of patients with increased TpP levels for each group was lower than the other activation markers. The findings indicate that activation markers reflect the number of underlying thrombophilic abnormalities. Our data suggest that there is a utility in performing a panel of coagulation activation markers to assess the thrombotic risk. The measurement of soluble fibrin polymer may be more reflective of an impending vascular event.

The use of thrombus precursor protein, D-dimer, prothrombin fragment 1.2, and thrombin antithrombin in the exclusion of proximal deep vein thrombosis and pulmonary embolism.

We examined various nonSTAT commercially available coagulation activation markers in an attempt to help diagnose or exclude the often subtle clinical presentations of proximal deep vein thrombosis (PDVT) and pulmonary embolism (PE). Fifty-five patients presenting to the Emergency Department were completely assessed. Eleven patients were diagnosed with PDVT, six patients were diagnosed with PE, and three patients were diagnosed with both PDVT and PE. Thrombus precursor protein (TpP) excluded the diagnosis in 19 of the 35 patients negative for PDVT and/or PE, D-Dimer in 15 patients, prothrombin fragment 1.2 in 17 patients, and thrombin–antithrombin (TAT) in 14 patients. Both the TpP and TAT enzyme-linked immunosorbent assay (ELISA) tests had 100% sensitivity and negative predictive value for evaluating PDVT and/or PE. The TpP ELISA had the highest specificity (54%) of all four markers studied.

Soluble monocyte cluster domain 163, a new global marker of anti‐inflammatory response, is elevated in the first trimester of pregnancy

1 Ferrari E, Chevallier T, Chapelier A, Baudouy M. Travel as a risk factor for venous thromboembolic disease: a case control study. Chest 1999; 115: 440–4. 2 Lapostolle F, Surget V, Borron SW, Desmaizières M, Sordelet D, Lapandry C, Cupa M, Adnet F. Severe pulmonary embolism associated with air travel. N Engl J Med 2001; 345: 779–83. 3 Rosendaal FR. Venous thrombosis: amulticausal disease.Lancet 1999; 353: 1167–73. 4 Geroulakos G. The risk of venous thromboembolism from air travel. Br Med J 2001; 322: 188. 5 Virchow R. Phlogose und Thrombose im Gefässsystem In: Gesammelte Abhandlungen Zur Wissenschaftlichen Medizin. Frankfurt: Meidinger Sohn & Co., 1856: 458–612. 6 Landgraf H, Vanselow B, Schulte-Huermann D, Mulmann MV, Bergau L. Economy class syndrome: rheology, fluid balance, and lower leg edema during a simulated 12-hour long distance flight. Aviat Space Environ Med 1994; 65: 930–5. 7 Stricker H, Colucci G, GodioM,Mossi G, Mombelli G. The influence of a prolonged sitting position on the biochemical markers of coagulation activation in healthy subjects: evidence of reduced thrombin generation. J Thromb Haemost 2003; 1: 380–1. 8 Hodkinson PD, Hunt BJ, Parmar K, Ernstin J. Is mild normobaric hypoxia a risk factor for venous thromboembolism? J Thromb Haemost 2003; 1: 2131–3. 9 Dahlbäck B. Blood coagulation. Lancet 2000; 355: 1627–32. 10 Bouma BN, Meijers JCM. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidaseU). J Thromb Haemost 2003; 1: 1566–74.

Pregnant patients with thrombophilia and subsequent adverse pregnancy outcomes have a decreased first trimester response to thrombomodulin in an activated partial thromboplastin time (APTT) system

Pregnancy is associated with changes in the hemostatic and fibrinolytic systems that predispose to thrombosis [1]. Inherited and acquired thrombophilic conditions have been associated with adverse pregnancy outcomes linked to uteroplacental thrombosis including fetal loss after 10 weeks, intrauterine growth restriction, abruption with or without premature rupture of membranes, and severe, early onset pre-eclampsia [2–5]. The protein C anticoagulant system, which includes protein C, protein S, activated protein C (APC), thrombomodulin (TM) and endothelial cell protein C receptor (EPCR), is the major natural inhibitor system for thrombin regulation [6–8]. This inhibition is accomplished by the interaction of thrombin with TM to decrease both thrombin activity and activation of protein C to APC by the resultant complex. In turn, APC with its cofactor protein S degrades factors Va and VIIIa [9]. In this study, we utilized an assay that measures the effect of TM on the activated partial thromboplastin time (APTT) clotting system (TACT) [10] to determine whether the maternal plasma response to TM in patients with adverse pregnancy outcomes differs from those with normal pregnancy. In addition, we examined whether pregnant patients with established thrombophilic disorders demonstrated a decreased response to TM, favoring a prothrombotic tendency. Group 1: normal pregnancy (N 1⁄4 59) Patients with normal pregnancies and subsequent normal pregnancy outcome served as controls. These patients were identified retrospectively following an uncomplicated delivery. Exclusion criteria for normal pregnancy include complications such as intrauterine fetal demise, intrauterine growth restriction <10th percentile, pre-eclampsia, abruption, gestational hypertension, preterm labor, and medical disorders such as diabetes, stroke, and thromboembolic disease. Group 2: adverse pregnancy outcomes (N 1⁄4 96) Pregnant patients whose pregnancies resulted in pre-eclampsia defined by established criteria [11], birth weight below the < 10 percentile [12,13], preterm premature rupture of membranes, preterm delivery (delivery less than 37 weeks), and abruption defined as recurrent bleeding in more than one trimester [14,15]. Group 3: thrombophila (N 1⁄4 26) Pregnant patients with acquired and/or inherited thrombophilic conditions. These include the presence of the factor V Leiden or prothrombin 20210A gene mutations, as well as protein S, protein C or anti-thrombin deficiencies, hyperhomocysteinemia, anti-phospholipid antibodies or the lupus inhibitor. We have modified the TACT assay based on the method described by Suehisa et al [10]. The amount of rabbit TM (American Diagnostica Company, Greenwich, CT, USA) added to the test was determined by preliminary experiments to cause a 2-fold prolongation in the APTT using pooled normal plasma in 1 : 2.5 dilution. Twenty-five microliters of diluted TM (0.4 U mL) or a buffer solution (0.15 mol L NaCl, 0.5% Triton X-100, 50 mmol L Tris-HCl pH 7.4) was mixed with 50 lL of tested plasma. After incubation at 37 C for 5 min, the coagulation reaction was started by adding 85 lL of 25 mM CaCl2. The clotting time was measured with an STA Compact Coagulation Analyzer (Diagnostica Stago, Parsippany, NJ, USA). The TACT ratio was calculated by dividing clotting time with TM by the clotting time with buffer solution. Data were analyzed by the t-test. The mean gestational age at delivery and infant birth weight were significantly lower in adverse pregnancy outcomes and thrombophilia groups compared to controls (t-test, P < 0.05). The thrombophilia and adverse pregnancy outcomes groups were noted to have significantly lower TACT ratios compared to normal pregnancy outcome patients (mean 1.88 ± 0.32 and 1.79 ± 0.44 vs. 2.14 ± 0.53; P < 0.02 and < 0.0001, respectively). Stratifying by race among patients in the normal pregnancy outcome group, Asians were noted to have Correspondence: M. J. Paidas, The Center for Thrombosis and Hemostasis in Women’s Health, Department of Obstetrics and Gynecology, Yale University, New Haven, CT 06520-8063, USA. Tel.: +203 7857894; fax: +203 7856885; e-mail: michael.paidas@ yale.edu

Ischemic stroke in a young patient with protein C deficiency and prothrombin gene mutation G20210A.

We report a patient who had an ischemic stroke aged 22 years, an inherited type I protein C deficiency and a heterozygous genotype of prothrombin gene 20210A. In view of recent reports of an increased risk for ischemic cerebral vascular disease in patients with the prothrombin 20210A mutation, we suggest that many of the reported cases of ischemic stroke and protein C deficiency may have had additional prothrombotic disorders such as the prothrombin mutation. The current data concerning the magnified risk for stroke in patients with the prothrombin 20210A mutation suggests the need to study all patients with premature stroke for this mutation and the other risk factors for thrombosis. This would include homocysteine, lupus inhibitor, anticardiolipin antibodies, and possibly the natural inhibitors of coagulation. It is possible that patients with the prothrombin 20210A mutation and ischemic cerebral vascular disease would benefit from long-term anticoagulation therapy in a similar way to patients with the antiphospholipid syndrome.

Inherited Thrombophilias and Early Pregnancy Loss

Inherited thrombophilias are a heterogeneous group of conditions which have been associated with a variety of pregnancy complications, including early and late fetal loss, intrauterine growth restriction, abruptio placentae, and preeclampsia.1 As the functional significance of the burgeoning list of thrombophilic conditions is better understood, more rational and thoughtful approach to their detection and usefulness in clinical practice will likely emerge. While dominant conditions, such as antithrombin deficiency are rarely present without clinical manifestations, other less thrombogenic mutations, such as factor V Leiden, often are not associated with obvious pregnancy complications, as noted by the finding that the presence of heterozygous factor V Leiden is associated with a 0.2% risk of maternal thromboembolism. Emerging data suggests the quality and quantity of thrombophilic conditions, in addition to genetic and environmental influences create a ‘threshold milieu’ for the clinical manifestation of these heterogeneous prothrombotic conditions. This review will summarize the current knowledge of thrombophilic conditions and their association with first trimester pregnancy outcome.

Elevated first trimester soluble fibrin polymer is associated with adverse pregnancy outcome in thrombophilic patients

Inherited and acquired thrombophilic conditions are associated with maternal thromboembolic events and a variety of adverse perinatal outcomes, including, preeclampsia, intrauterine growth restriction (<10th percentile), second and third trimester fetal loss, and abruptio placentae [1,2]. Currently, there is a paucity of functional or global tests that will provide insight as to the prediction of adverse pregnancy outcome [3,4]. Activation markers are of limited predictive value for adverse outcome [5]. Bombeli et al. [6] found that thrombin–antithrombin complexes (TAT) or D-dimers did not correlate with risk stratification in pregnant women based upon personal or family history of thrombosis and the presence of a thrombophilic condition. However, these authors did find that women with ongoing thrombosis during pregnancy had significantly elevated TAT and D-dimers with or without anticoagulant therapy. Coagulation activation markers such as prothrombin fragment 1.2 and TAT are increased with the progression of pregnancy to levels as seen in those patients with active thrombosis [4]. Bremme et al. [7] found that normal pregnancy (n=26 women) was associated with both increased thrombin activity, increasedsoluble fibrin levels (9.2–13.4 nmol/l), as well as fibrinolysis, as evidenced by increased levels of fibrin D-dimer (91–198 µg/l). Soluble fibrin polymer (SFP) has been shown to be a specific and sensitive marker of active clotting and thrombin generation in a group of nonpregnant individuals at high risk for thromboembolic event [8]. The purpose of this study was to determine if elevated SFP is an additional risk for adverse pregnancy outcome in thrombophilic patients. We conducted a retrospective cohort study among 39 patients with inherited and acquired thrombophilia (41 pregnancies) and 50 uncomplicated gestations. Thrombophilic women previously underwent a uniform evaluation for inherited and acquired thrombophilic conditions. A reference laboratory was used to evaluate the relevant thrombophilic conditions. Factor V Leiden, methylenetetrahydrofolate reductase gene mutation, and prothrombin gene mutation 20210 assay were performed by using multiplex allele-specific primers’ PCR amplification. We defined protein S as deficient if protein levels were below 40%, as determined by functional assay. Protein C and antithrombin were measured by chromogenic and antigenic assays, and respective levels below the laboratory’s normal reference range were considered abnormal. Homocysteine testing was evaluated by high performance liquid chromatography (HPLC), and values above the laboratory’s normal reference range were considered abnormal. Antiphospholipid antibody syndrome was diagnosed by established criteria [9]. Anticardiolipin and anti-beta 2 glycoprotein I antibodies were performed by enzyme-linked immunosorbent assay (ELISA), and values above the laboratory’s reference range were considered abnormal. The presence of the lupus anticoagulant was detected by screening with a sensitive activated partial thromboplastin time (APTT) and confirmed by the dilute Russel viper venom time with correction by adding the high concentration of phospholipids. This is felt to be a reliable confirmatory test for the lupus inhibitor. PAI-1 antigen and activity were measured by ELISA and chromogenic assay respectively, and levels three-fold above the upper limit of the reference range were considered abnormal. Thrombocytosis, as part of the myeloproliferative syndrome, was determined by the presence of an elevated platelet count, associated with abnormal platelet function studies, platelet flow cytometry, and compatible bone marrow findings, clinical presentation, such as splenomegaly, and other white cell or red cell abnormalities. Following informed consent and institutional review board approval, pregnant patients enrolled in the maternal fetal medicine faculty practice of New York University School of Medicine who underwent phlebotomy for other routine laboratory tests as part of their obstetrical care were asked to donate an additional aliquot of blood for SFP evaluation. Specimens were collected at routine blood draw and the plasma samples were stored at −80°C until analysis. Maternal plasma SFP concentrations were measured with a commercial ELISA [thrombus precursor protein (TpP); American Biogenetic Sciences, Inc., Copiague, New York, USA], as previously described [8]. The standard curve consistently had a R value of more than 0.98, the interassay and intraassay errors were less than 7%. Using a computerized obstetrical database, all thrombophilic patients, with an available blood sample obtained during their first prenatal visit and who met the criteria for thrombophilia, were identified and recruited. All charts were reviewed in detail to confirm the accuracy of the diagnosis. To establish a reference range for SFP in pregnancy, blood specimens from the first, second, and third trimesters were obtained from a group of healthy pregnant women with normal pregnancy outcome. Patients with thrombophilia were identified as having the following conditions: protein S deficiency (N = 8), factor V Leiden mutation R506Q (N=12), prothrombin gene mutation 20210A (N=6), antiphospholipid antibody syndrome (N=17), hyperhomocysteinemia (N=1), and thrombocythemia and myeloproliferative disorder with predominant manifestation of thrombocytosis (N=3). Five patients had two defects. Preeclampsia was defined by American College of Obstetricians and Gynecologist’s criteria [10]. Severe preeclampsia was defined when the following were present: systolic blood pressure (SBP) of at least 160mmHg or diastolic blood pressure (DBP) of at least 110mmHg and plus two or more protein on urinary dipstick. Severe preeclampsia was considered present if one of the blood pressure or proteinuria requirements was met along with either thrombocytopenia (platelet count<100 000/ml) or elevated liver enzymes (serum glutamic oxalo-acetic transaminase or serum glutamic pyruvic transaminase). The data were analyzed by Wilcoxon rank sum, t-test (DF, 1), and x2 (DF, 1) where appropriate. Level of significance was set at a P value less than 0.05. Receiver operator characteristic (ROC) curve analysis was performed to determine the optimum cutoff for SFP, to predict adverse pregnancy outcome. Thrombophilic patients had significantly lower birth weights and delivered earlier than controls, respectively: 2943 g ± 829 g vs. 3498 g ± 527 g and 37.0 weeks ± 4.5 weeks vs. 39.4 weeks ± 1.5 weeks (P<0.01). First trimester mean SFP maternal levels (mg/ml) were significantly higher among thrombophilic patients compared with controls (21.5 ± 3.7 vs. 3.7 ± 0.8mg/ml; P<0.02). The ROC curve of SFP to predict adverse pregnancy outcomes by using 3 mg/ml in the first trimester as cutoff (P = 0.03) produced a sensitivity of 83%, specificity 64%, positive predictive value of 57%, and negative predictive value of 97% for adverse pregnancy outcome. Five thrombophilic patients had at least two thrombophilic conditions, and their SFP values were higher than cases with one (26.4 ± 16.0; P<0.01). Even with heparin therapy, cases also had higher SFP values than controls in the second trimester (31.7 ± 8 vs. 10.9 ± 2; P<0.01) and third trimester (35.1 ± 16.1 vs. 12.9 ± 3; P<0.01). Table 1 shows the distribution of the adverse pregnancy outcomes in the thrombophilia group. Table 1 Distribution of adverse pregnancy outcome There were no significant differences between the thrombophilic patients (n=39) and controls (n=50) regarding mean maternal age, years_SD (32.6 ± 5.8 vs. 33.1 ± 5.6), median gravidity and range [3 (1–11) vs. 2 (0–9)], median parity and range [1 (0–4) vs. 0 (0–5)], nulliparity (17 vs. 16%), multiparity (83 vs. 84%), race expressed as percent Caucasian (80 vs. 83%), respectively. There was no significant difference in gestational age at blood draw [median (range) between the thrombophilic patients (n=41, 40, 39) and controls (n=50, 50, 50)] in the first trimester [10.2 (4.0–14.0) vs. 9.0 (4.6–13.0)], second trimester [21.3 (15.0–27.4) vs. 17.6 (15.3–26.9)], or the third trimester [33.5 (27.3– 40.4) vs. 29.7 (27.7–40.0)], respectively. Our study confirms the progressive increase of thrombin generation as pregnancy proceeds with a markedly accelerated thrombin generation in thrombophilic patients. Higher first-trimester SFP increases the risk for adverse pregnancy outcomes. Even some normal pregnant patients with uneventful pregnancies have elevated SFP values. The significance of this is not known at this time and may be reflective of normal biologic variability or indicative of indolent processes. SFP levels are inversely related to gestational age at delivery and birth weight confirming our previous data regarding multiple gestations [11]. Testing for SFP may be useful in predicting patients at risk for thrombophilia-induced poor pregnancy outcome and monitoring anticoagulation efficacy. After the completion of this study, the ELISA test for SFP became unavailable commercially. It employs a very specific antibody to SFP. Perhaps, other tests may be developed that will exhibit similar properties. Our data indicate that reintroduction of SFP measurement may have clinical utility, particularly if confirmed in a larger prospective study.