- Email: [email protected]
Activated protein C resistance and Factor V Leiden in patients with hemolysis, elevated liver enzymes, low platelets syndrome
Activated Protein C Resistance and Factor V Leiden in Patients With Hemolysis, Elevated Liver Enzymes, Low Platelets Syndrome THOMAS KRAUSS, MD, HELLMUT G. AUGUSTIN, DVM, PhD, ¨ DIGER OSMERS, MD, HARALD MEDEN, MD, MICHAEL UNTERHALT, MD, AND RU WALTHER KUHN, MD
Objective: Hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is characterized by a distinct activation of the coagulation system. A mutation of the gene coding for coagulation Factor V (Factor V Leiden) has been identified as the most frequent risk factor for thrombosis. To identify risk factors for HELLP syndrome, we determined coagulation parameters and the Factor V Leiden mutation in women who previously had developed HELLP syndrome. Methods: Coagulation parameters (activated protein C resistance, antithrombin, protein C, protein S) were determined in 21 women 6 months to 9 years after they had developed HELLP syndrome in the third trimester. In addition, these women were analyzed for the presence of the Factor V Leiden mutation. Results: Of these analyzed women, 33% (seven of 21) had an activated protein C resistance (activated protein C ratio less than 2.0). Another 38% of the women had subnormal activated protein C ratios (2.0 –2.3). Only 57% of the women with an activated protein C resistance were identified as heterozygous carriers of the Factor V Leiden mutation (four of seven). Conclusion: Women with HELLP syndrome have a higher incidence of Factor V Leiden mutations. This increased incidence does not, however, account fully for the increased frequency of activated protein C resistance in these patients. (Obstet Gynecol 1998;92:457– 60. © 1998 by The American College of Obstetricians and Gynecologists.)
Hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is a severe, life-threatening disorder of pregnancy with a perinatal maternal mortality of 0 –24%1,2 and a perinatal infant mortality of 7.7– 60%.1 It From the Departments of Gynecology and Obstetrics and Hematology and Oncology, University of Go¨ttingen, Go¨ttingen, Germany. Supported by a grant from the Deutsche Forschungsgemeinschaft Kr1571/1-2.
VOL. 92, NO. 3, SEPTEMBER 1998
is a severe manifestation of preeclampsia with a typical constellation of laboratory parameters (hemolysis, elevated liver enzymes, low platelet count).3– 6 Both etiology and pathogenesis of HELLP syndrome remain unknown. The clinical manifestation of HELLP syndrome involves an activation of the coagulation system that may lead to microthrombi and consequently to a compensated disseminated intravascular coagulopathy.7 A number of genetic defects have been identified in patients with hereditary thrombophilia. These include a deficiency of protein S, protein C, and antithrombin. Changes of these parameters also have been reported to be involved in preeclampsia.8,9 In 1993, Dahlba¨ck et al10 first described the phenomenon known as activated protein C resistance. Activated protein C resistance is defined by an insufficient prolongation of partial thromboplastin time (PTT) after addition of activated protein C compared with basal PTT. Activated protein C resistance or a deficiency of protein C, protein S, or antithrombin accounts for 60 –70% of all cases of hereditary thrombophilia.11 Genetic analysis of a family with activated protein C resistance identified a point mutation within the Factor V gene.12 This mutation is characterized by an exchange of bp1691 from guanine to adenine, resulting in an arginine 3 glutamine replacement of aa506. As a consequence of this mutation, Factor Va becomes resistant to activated protein C. On the basis of the characteristic disturbance of the coagulation system in women with HELLP syndrome involving intravascular fibrin deposition7 and the identification of the Factor V Leiden mutation as the most frequent risk factor for thrombosis, we hypothesized
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Table 1. Clinical and Laboratory Findings of 21 Patients 6 Months to 9 Years After Development of Hemolysis, Elevated Liver Enzymes, Low Platelets Syndrome
Mean Range
Syst. blood pressure (mmHg)
Diast. blood pressure (mmHg)
Thrombocyte count* (cells/mL)
ASAT† (U/L)
ALAT† (U/L)
LDH‡ (U/L)
Haptoglobin§ (g/L)
147 110 –260
96 80 –130
83,500 18,000 –140,000
89 28 –192
98 24 –236
455 293–999
0.27 0.00 –1.09
Syst. 5 systolic; Diast. 5 diastolic; ASAT 5 asparate aminotransferase; ALAT 5 alanine aminotransferase; LDH 5 lactate dehydrogenase. Normal reference values *150 – 400 3 103 cells/mL, † , 17 U/L, ‡ 90 –200 U/L, § 0.45–2.05 g/L.
that women who are heterozygous carriers of the Factor V Leiden mutation may have an increased risk of developing HELLP syndrome during pregnancy.
Materials and Methods A total of 80 patients with HELLP syndrome were diagnosed and treated at the University of Go¨ttingen Medical School between 1987 and 1996. Of these, 21 women could be identified 6 months to 9 years after parturition who still lived in the Go¨ttingen area, were willing to participate in the study, and were not users of oral contraceptives at the time of the study. Because none of these selection criteria had a direct relationship to any of the experimental parameters of the study, this selection was considered random. The age range of the 21 women who participated in this study was 26 – 45 years (median 31) at the time of sample collection. Two independent plasma samples from each woman were collected and analyzed, and the minimum time between sampling was 2 weeks. The age range of the women at parturition was 18 –36 years (median 28). Median gestational age of these women at the time of developing clinical symptoms of HELLP syndrome was 33 weeks (range 26 – 41). Of the 21 women, 18 were primigravidas. The laboratory parameters of the patients that had led to the diagnosis of HELLP syndrome are summarized in Table 1. All patients with HELLP syndrome had significant proteinuria (more than 0.3 g/L). Given the diagnosis HELLP syndrome, all newborns were delivered by cesarean delivery. To determine normal values, we determined activated protein C ratios in 70 female healthy, nonpregnant blood donors and 70 male healthy blood donors. The age range of the blood donors was 20 – 60 years (median 32). Protein C, protein S, and antithrombin were determined in samples of citrate plasma with the use of commercially available test systems (activated protein C resistance: Chromogenics, Mo¨lndal, Sweden; protein C and protein S: Immuno AG, Vienna, Austria; assays performed using a BCS analyzer: Behring, Marburg, Germany). All measurements were performed using
458 Krauss et al
Factor V Leiden in HELLP Syndrome
Factor V– deficient plasma. Prothrombin time, coagulation Factors II and V, and fibrinogen also were determined, to exclude hepatic changes and as control parameters for the assessment of sample quality (Behring). The presence of an activated protein C resistance was defined as an activated protein C ratio of less than 2 in repeated measurements of two independent samples. Likewise, a deficiency of protein C or protein S was defined as a measurement of less than 70% in repeated measurements or 60% in a single measurement (duplicate determinations). The normal reference levels in our laboratory for protein C and protein S are 110 6 36% and 97 6 16%, respectively. Normal values for antithrombin vary between 80% and 130%. For the detection of the Factor V Leiden mutation, a 287-bp fragment of the Factor V gene flanking the mutation site at nucleotide 1691 was amplified by polymerase chain reaction (PCR) essentially as described previously.11 The PCR product was digested with the restriction enzyme Mnl I, and the resulting DNA fragments were resolved on a 3% agarose gel. When the Factor V gene is mutated, one Mnl I site is lost, resulting in fragments of 157 bp and 130 bp. Differences in the activated protein C ratios of normal control women and women who previously had developed HELLP syndrome were analyzed by Wilcoxon rank-sum test.
Results Activated protein C resistance determinations now are employed widely. Nevertheless, results are known to vary significantly and the analytic systems are not standardized. We consequently determined the frequency distribution of activated protein C ratios in randomly selected plasma sample donors in the Go¨ttingen (central Germany) area. As shown in Figure 1, a subgroup of 90% of these values were normally distributed, varying around a mean of 2.65. There was no difference between male and female donors. When a cutoff value of 2.0 was applied, approximately 5% of plasma donors had an activated protein C resistance, corresponding to the reported values in the central European population.13 Another subpopulation of sam-
Obstetrics & Gynecology
Discussion
Figure 1. Normal values of activated protein C ratios (APC-ratios) in plasma samples of healthy, nonpregnant women. A subpopulation of values (90%) were normally distributed, varying between 2.35 and 2.95. A subpopulation of samples (5%) had a manifest activated protein C resistance (activated protein C ratio less than 2.0). Another subpopulation (5%) had subnormal activated protein C ratios between 2.0 and 2.3.
ples (5%) had activated protein C resistance values above the 2.0 cutoff value that were subnormal values in comparison with those of the normally distributed population (values about 2.1; Figure 1). On the basis of the activated protein C resistance measurements in the normal population, activated protein C ratios were determined in 21 women who previously had developed HELLP syndrome. Of the 21 women, seven (33%) had a detectable activated protein C resistance (activated protein C ratio less than 2). Another eight women (38%) had subnormal activated protein C ratios between 2.0 and 2.3. Only six women (29%) had activated protein C ratios above 2.3 that matched the normally distributed values of the control population. Taken together, women who previously had developed HELLP syndrome had significantly reduced activated protein C ratios compared with the randomly collected control population (P , .001). One woman with an activated protein C resistance had a protein S deficiency, and one woman without an activated protein C resistance received a diagnosis of protein C deficiency. None of the patients had an antithrombin deficiency. An apparent relationship between activated protein C resistance and laboratory findings that led to the diagnosis of HELLP syndrome could not be detected. Genomic analysis for the presence of the Factor V Leiden mutation identified four (19%) of 21 women who previously had developed HELLP syndrome as heterozygous carriers of the Factor V Leiden mutation. All of the heterozygous carriers of the Factor V Leiden mutation also had activated protein C ratios of less than 2 (means of two independent measurements 1.67, 1.74, 1.45, and 1.46).
VOL. 92, NO. 3, SEPTEMBER 1998
Thrombin converts fibrinogen to fibrin and activates coagulation Factors V and VIII. At the same time, excessive coagulation is balanced by a number of anticoagulative mechanisms. These include antithrombin and protein C, which act as key regulators of the coagulation system.11 Protein C is activated on the binding of thrombin to the endothelial cell surface protein thrombomodulin. Activated protein C inhibits the coagulation cascade by cleaving Factor Va and Factor VIIIa, which leads to the inactivation of these factors. The anticoagulative activity of activated protein C is enhanced by protein S, which acts as a cofactor. A deficiency of protein C, protein S, or antithrombin results in a significantly increased risk for thrombosis.8,14 The incidence of a heterozygous deficiency of protein C in patients with thromboembolic disease is estimated to range between 2% and 5%.15 A similar incidence has been reported for the presence of a protein S deficiency.15 The incidence of a heterozygous protein C deficiency in the total population varies between 0.1% and 0.3%.15 Levels of protein C and antithrombin do not vary during normal pregnancy. In contrast, levels of protein S are reduced in the first trimester of pregnancy.9 Women with preeclampsia have been shown to have reduced levels of antithrombin and protein C.8 Postpartum analysis of coagulation parameters (at least 10 weeks after parturition) in women who had developed preeclampsia during pregnancy identified a protein S deficiency in 24.7% of the analyzed patients.8 Only 1.2% of patients had a protein C deficiency or an antithrombin deficiency, respectively.8 These findings indicate that women with preeclampsia have a higher risk of developing thrombosis. Analysis of coagulation parameters in women with HELLP syndrome confirmed these observations: of 21 analyzed patients, one (5%) was identified with a protein S deficiency and one woman (5%) received a diagnosis of protein C deficiency. It is estimated that the incidence of an activated protein C resistance in the central European population is approximately 4%.13 Recently, a rate as high as 60% was reported in women who developed thrombosis during pregnancy.16 It is estimated that the Factor V Leiden mutation accounts for up to 90% of all cases of activated protein C resistance.11 Other yet unidentified genetic defects have been hypothesized to account for the remaining cases of activated protein C resistance, those not associated with the Factor V Leiden mutation.11 Dizon-Townson et al17 reported an incidence of 8.9% of Factor V Leiden mutations in women with severe preeclampsia, compared with an incidence of 4.2% in
Krauss et al
Factor V Leiden in HELLP Syndrome
459
the healthy control population. Activated protein C ratios were not determined in this study. However, an increased rate of activated protein C resistance (16%) in women with preeclampsia was reported by others.8 An association between the Factor V Leiden mutation leading to activated protein C resistance and HELLP syndrome recently has been reported in two women.18 In the present study, four (19%) of 21 patients who previously had developed HELLP syndrome were identified as carriers of the Factor V Leiden mutation, a higher frequency of this mutation than in the normal central European population.13 Furthermore, we identified an activated protein C resistance in 33% of women who previously had developed HELLP syndrome (7 of 21). Another 38% of patients with HELLP syndrome had subnormal values (between 2.0 and 2.3). Activated protein C resistance measurements are known to be influenced significantly by a number of technical factors, including the applied preanalytical conditions (centrifugation, storage, fresh or frozen samples) as well as the choice of the reagents (PTT reagent, activated protein C, CaCl2 concentration).19 Nevertheless, even when the limitations in interpretation of activated protein C ratios are considered, the high percentages of HELLP syndrome patients with activated protein C resistance as determined in the present study suggest an association between an increased risk for developing thrombosis and HELLP syndrome. Most interestingly, however, the Factor V Leiden mutation was present only in four (57%) of the seven HELLP syndrome patients with an activated protein C resistance, and none of the women with subnormal ratios between 2.0 and 2.3 were carriers of the Factor V Leiden mutation. This apparent discrepancy indicates that the Factor V Leiden mutation is only partially responsible for the thrombophilia observed in women who previously had developed HELLP syndrome.
6. Wilke G, Rath W, Schu¨tz E, Armstrong VW, Kuhn W. Haptoglobin as a sensitive marker of hemolysis in HELLP syndrome. Int J Gynaecol Obstet 1992;39:29 –34. 7. de Boer K, Bu¨ller HR, Ten Cate JW, Treffers PE. Coagulation studies in the syndrome of hemolysis, elevated liver enzymes and low platelets. Br J Obstet Gynaecol 1991;98:42–7. 8. Dekker GA, de Vries JI, Doelitzsch PM, Huijgens PC, von Blomberg BM, Jakobs C, et al. Underlying disorders associated with severe early-onset preeclampsia. Am J Obstet Gynecol 1995; 173:1042– 8. 9. Faught W, Garner P, Jones G, Ivey B. Changes in protein C and protein S levels in normal pregnancy. Am J Obstet Gynecol 1995;172:147–50. 10. Dahlba¨ck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C. Proc Natl Acad Sci U S A 1993;90:1004 – 8. 11. Dahlba¨ck B. Factor V gene mutation causing inherited resistance to activated protein C as a basis for venous thromboembolism. J Intern Med 1995;237:221–7. 12. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, et al. Mutation in blood coagulation Factor V associated with resistance to activated protein C. Nature 1994;369:64 –7. 13. Rees DC, Cox M, Clegg JB. World distribution of Factor V Leiden. Lancet 1995;346:1133– 4. 14. Pabinger I, Kyrle PA, Heistinger M, Eichinger S, Wittmann E, Lechner K. The risk of thromboembolism in asymptomatic patients with protein C and protein S deficiency: A prospective cohort study. Thromb Haemost 1994;71:441–5. 15. Miletich J, Sherman L, Broze G. Absence of thrombosis in subjects with heterozygous protein C deficiency. N Engl J Med 1987;317: 991– 6. 16. Hellgren M, Svensson PJ, Dahlba¨ck B. Resistance to activated protein C as a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Am J Obstet Gynecol 1995;173: 210 –3. 17. Dizon-Townson DS, Nelson LM, Easton K, Ward K. The Factor V Leiden mutation may predispose women to severe preeclampsia. Am J Obstet Gynecol 1996;175:902–5. 18. Brenner B, Lanir N, Thaler I. HELLP syndrome associated with Factor V R506Q mutation. Br J Haematol 1996;92:999 –1001. 19. Bertina RM. Laboratory findings of resistance to activated protein C (APC-resistance). Thromb Haemost 1997;78:478 – 82.
References
Address reprint requests to:
1. Sibai BM, Ramadan MK, Usta I, Salama M, Mercer BM, Friedman SA. Maternal morbidity and mortality in 442 pregnancies with hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome). Am J Obstet Gynecol 1993;169:1000 – 6. 2. Meden H, Armstrong VW, Arendt H, Cunze T, Kuhn W. HELLP syndrome: Prognosis and parameters of hemolysis with special regard to haptoglobin. Singapore J Obstet Gynaecol 1996;27:42– 8. 3. Weinstein L. Syndrome of hemolysis, elevated liver enzymes, and low platelet count: A severe consequence of hypertension in pregnancy. Am J Obstet Gynecol 1982;142:159 – 67. 4. Rath W, Loos W, Kuhn W, Graeff H. The importance of early laboratory screening methods for maternal and fetal outcome in cases of HELLP syndrome. Eur J Obstet Gynecol Reprod Biol 1990;36:43–51. 5. Sibai BM. The HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets): Much ado about nothing? Am J Obstet Gynecol 1990;162:311– 6.
460 Krauss et al
Factor V Leiden in HELLP Syndrome
Thomas Krauss, MD Department of Gynecology and Obstetrics University of Go¨ttingen Robert-Koch-Str. 40 Go¨ttingen 37075 Germany E-mail: [email protected]
Received January 6, 1998. Received in revised form April 1, 1998. Accepted April 10, 1998. Copyright © 1998 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
Obstetrics & Gynecology
Objective: Hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is characterized by a distinct activation of the coagulation system. A mutation of the gene coding for coagulation Factor V (Factor V Leiden) has been identified as the most frequent risk factor for thrombosis. To identify risk factors for HELLP syndrome, we determined coagulation parameters and the Factor V Leiden mutation in women who previously had developed HELLP syndrome. Methods: Coagulation parameters (activated protein C resistance, antithrombin, protein C, protein S) were determined in 21 women 6 months to 9 years after they had developed HELLP syndrome in the third trimester. In addition, these women were analyzed for the presence of the Factor V Leiden mutation. Results: Of these analyzed women, 33% (seven of 21) had an activated protein C resistance (activated protein C ratio less than 2.0). Another 38% of the women had subnormal activated protein C ratios (2.0 –2.3). Only 57% of the women with an activated protein C resistance were identified as heterozygous carriers of the Factor V Leiden mutation (four of seven). Conclusion: Women with HELLP syndrome have a higher incidence of Factor V Leiden mutations. This increased incidence does not, however, account fully for the increased frequency of activated protein C resistance in these patients. (Obstet Gynecol 1998;92:457– 60. © 1998 by The American College of Obstetricians and Gynecologists.)
Hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is a severe, life-threatening disorder of pregnancy with a perinatal maternal mortality of 0 –24%1,2 and a perinatal infant mortality of 7.7– 60%.1 It From the Departments of Gynecology and Obstetrics and Hematology and Oncology, University of Go¨ttingen, Go¨ttingen, Germany. Supported by a grant from the Deutsche Forschungsgemeinschaft Kr1571/1-2.
VOL. 92, NO. 3, SEPTEMBER 1998
is a severe manifestation of preeclampsia with a typical constellation of laboratory parameters (hemolysis, elevated liver enzymes, low platelet count).3– 6 Both etiology and pathogenesis of HELLP syndrome remain unknown. The clinical manifestation of HELLP syndrome involves an activation of the coagulation system that may lead to microthrombi and consequently to a compensated disseminated intravascular coagulopathy.7 A number of genetic defects have been identified in patients with hereditary thrombophilia. These include a deficiency of protein S, protein C, and antithrombin. Changes of these parameters also have been reported to be involved in preeclampsia.8,9 In 1993, Dahlba¨ck et al10 first described the phenomenon known as activated protein C resistance. Activated protein C resistance is defined by an insufficient prolongation of partial thromboplastin time (PTT) after addition of activated protein C compared with basal PTT. Activated protein C resistance or a deficiency of protein C, protein S, or antithrombin accounts for 60 –70% of all cases of hereditary thrombophilia.11 Genetic analysis of a family with activated protein C resistance identified a point mutation within the Factor V gene.12 This mutation is characterized by an exchange of bp1691 from guanine to adenine, resulting in an arginine 3 glutamine replacement of aa506. As a consequence of this mutation, Factor Va becomes resistant to activated protein C. On the basis of the characteristic disturbance of the coagulation system in women with HELLP syndrome involving intravascular fibrin deposition7 and the identification of the Factor V Leiden mutation as the most frequent risk factor for thrombosis, we hypothesized
0029-7844/98/$19.00 PII S0029-7844(98)00208-7
457
Table 1. Clinical and Laboratory Findings of 21 Patients 6 Months to 9 Years After Development of Hemolysis, Elevated Liver Enzymes, Low Platelets Syndrome
Mean Range
Syst. blood pressure (mmHg)
Diast. blood pressure (mmHg)
Thrombocyte count* (cells/mL)
ASAT† (U/L)
ALAT† (U/L)
LDH‡ (U/L)
Haptoglobin§ (g/L)
147 110 –260
96 80 –130
83,500 18,000 –140,000
89 28 –192
98 24 –236
455 293–999
0.27 0.00 –1.09
Syst. 5 systolic; Diast. 5 diastolic; ASAT 5 asparate aminotransferase; ALAT 5 alanine aminotransferase; LDH 5 lactate dehydrogenase. Normal reference values *150 – 400 3 103 cells/mL, † , 17 U/L, ‡ 90 –200 U/L, § 0.45–2.05 g/L.
that women who are heterozygous carriers of the Factor V Leiden mutation may have an increased risk of developing HELLP syndrome during pregnancy.
Materials and Methods A total of 80 patients with HELLP syndrome were diagnosed and treated at the University of Go¨ttingen Medical School between 1987 and 1996. Of these, 21 women could be identified 6 months to 9 years after parturition who still lived in the Go¨ttingen area, were willing to participate in the study, and were not users of oral contraceptives at the time of the study. Because none of these selection criteria had a direct relationship to any of the experimental parameters of the study, this selection was considered random. The age range of the 21 women who participated in this study was 26 – 45 years (median 31) at the time of sample collection. Two independent plasma samples from each woman were collected and analyzed, and the minimum time between sampling was 2 weeks. The age range of the women at parturition was 18 –36 years (median 28). Median gestational age of these women at the time of developing clinical symptoms of HELLP syndrome was 33 weeks (range 26 – 41). Of the 21 women, 18 were primigravidas. The laboratory parameters of the patients that had led to the diagnosis of HELLP syndrome are summarized in Table 1. All patients with HELLP syndrome had significant proteinuria (more than 0.3 g/L). Given the diagnosis HELLP syndrome, all newborns were delivered by cesarean delivery. To determine normal values, we determined activated protein C ratios in 70 female healthy, nonpregnant blood donors and 70 male healthy blood donors. The age range of the blood donors was 20 – 60 years (median 32). Protein C, protein S, and antithrombin were determined in samples of citrate plasma with the use of commercially available test systems (activated protein C resistance: Chromogenics, Mo¨lndal, Sweden; protein C and protein S: Immuno AG, Vienna, Austria; assays performed using a BCS analyzer: Behring, Marburg, Germany). All measurements were performed using
458 Krauss et al
Factor V Leiden in HELLP Syndrome
Factor V– deficient plasma. Prothrombin time, coagulation Factors II and V, and fibrinogen also were determined, to exclude hepatic changes and as control parameters for the assessment of sample quality (Behring). The presence of an activated protein C resistance was defined as an activated protein C ratio of less than 2 in repeated measurements of two independent samples. Likewise, a deficiency of protein C or protein S was defined as a measurement of less than 70% in repeated measurements or 60% in a single measurement (duplicate determinations). The normal reference levels in our laboratory for protein C and protein S are 110 6 36% and 97 6 16%, respectively. Normal values for antithrombin vary between 80% and 130%. For the detection of the Factor V Leiden mutation, a 287-bp fragment of the Factor V gene flanking the mutation site at nucleotide 1691 was amplified by polymerase chain reaction (PCR) essentially as described previously.11 The PCR product was digested with the restriction enzyme Mnl I, and the resulting DNA fragments were resolved on a 3% agarose gel. When the Factor V gene is mutated, one Mnl I site is lost, resulting in fragments of 157 bp and 130 bp. Differences in the activated protein C ratios of normal control women and women who previously had developed HELLP syndrome were analyzed by Wilcoxon rank-sum test.
Results Activated protein C resistance determinations now are employed widely. Nevertheless, results are known to vary significantly and the analytic systems are not standardized. We consequently determined the frequency distribution of activated protein C ratios in randomly selected plasma sample donors in the Go¨ttingen (central Germany) area. As shown in Figure 1, a subgroup of 90% of these values were normally distributed, varying around a mean of 2.65. There was no difference between male and female donors. When a cutoff value of 2.0 was applied, approximately 5% of plasma donors had an activated protein C resistance, corresponding to the reported values in the central European population.13 Another subpopulation of sam-
Obstetrics & Gynecology
Discussion
Figure 1. Normal values of activated protein C ratios (APC-ratios) in plasma samples of healthy, nonpregnant women. A subpopulation of values (90%) were normally distributed, varying between 2.35 and 2.95. A subpopulation of samples (5%) had a manifest activated protein C resistance (activated protein C ratio less than 2.0). Another subpopulation (5%) had subnormal activated protein C ratios between 2.0 and 2.3.
ples (5%) had activated protein C resistance values above the 2.0 cutoff value that were subnormal values in comparison with those of the normally distributed population (values about 2.1; Figure 1). On the basis of the activated protein C resistance measurements in the normal population, activated protein C ratios were determined in 21 women who previously had developed HELLP syndrome. Of the 21 women, seven (33%) had a detectable activated protein C resistance (activated protein C ratio less than 2). Another eight women (38%) had subnormal activated protein C ratios between 2.0 and 2.3. Only six women (29%) had activated protein C ratios above 2.3 that matched the normally distributed values of the control population. Taken together, women who previously had developed HELLP syndrome had significantly reduced activated protein C ratios compared with the randomly collected control population (P , .001). One woman with an activated protein C resistance had a protein S deficiency, and one woman without an activated protein C resistance received a diagnosis of protein C deficiency. None of the patients had an antithrombin deficiency. An apparent relationship between activated protein C resistance and laboratory findings that led to the diagnosis of HELLP syndrome could not be detected. Genomic analysis for the presence of the Factor V Leiden mutation identified four (19%) of 21 women who previously had developed HELLP syndrome as heterozygous carriers of the Factor V Leiden mutation. All of the heterozygous carriers of the Factor V Leiden mutation also had activated protein C ratios of less than 2 (means of two independent measurements 1.67, 1.74, 1.45, and 1.46).
VOL. 92, NO. 3, SEPTEMBER 1998
Thrombin converts fibrinogen to fibrin and activates coagulation Factors V and VIII. At the same time, excessive coagulation is balanced by a number of anticoagulative mechanisms. These include antithrombin and protein C, which act as key regulators of the coagulation system.11 Protein C is activated on the binding of thrombin to the endothelial cell surface protein thrombomodulin. Activated protein C inhibits the coagulation cascade by cleaving Factor Va and Factor VIIIa, which leads to the inactivation of these factors. The anticoagulative activity of activated protein C is enhanced by protein S, which acts as a cofactor. A deficiency of protein C, protein S, or antithrombin results in a significantly increased risk for thrombosis.8,14 The incidence of a heterozygous deficiency of protein C in patients with thromboembolic disease is estimated to range between 2% and 5%.15 A similar incidence has been reported for the presence of a protein S deficiency.15 The incidence of a heterozygous protein C deficiency in the total population varies between 0.1% and 0.3%.15 Levels of protein C and antithrombin do not vary during normal pregnancy. In contrast, levels of protein S are reduced in the first trimester of pregnancy.9 Women with preeclampsia have been shown to have reduced levels of antithrombin and protein C.8 Postpartum analysis of coagulation parameters (at least 10 weeks after parturition) in women who had developed preeclampsia during pregnancy identified a protein S deficiency in 24.7% of the analyzed patients.8 Only 1.2% of patients had a protein C deficiency or an antithrombin deficiency, respectively.8 These findings indicate that women with preeclampsia have a higher risk of developing thrombosis. Analysis of coagulation parameters in women with HELLP syndrome confirmed these observations: of 21 analyzed patients, one (5%) was identified with a protein S deficiency and one woman (5%) received a diagnosis of protein C deficiency. It is estimated that the incidence of an activated protein C resistance in the central European population is approximately 4%.13 Recently, a rate as high as 60% was reported in women who developed thrombosis during pregnancy.16 It is estimated that the Factor V Leiden mutation accounts for up to 90% of all cases of activated protein C resistance.11 Other yet unidentified genetic defects have been hypothesized to account for the remaining cases of activated protein C resistance, those not associated with the Factor V Leiden mutation.11 Dizon-Townson et al17 reported an incidence of 8.9% of Factor V Leiden mutations in women with severe preeclampsia, compared with an incidence of 4.2% in
Krauss et al
Factor V Leiden in HELLP Syndrome
459
the healthy control population. Activated protein C ratios were not determined in this study. However, an increased rate of activated protein C resistance (16%) in women with preeclampsia was reported by others.8 An association between the Factor V Leiden mutation leading to activated protein C resistance and HELLP syndrome recently has been reported in two women.18 In the present study, four (19%) of 21 patients who previously had developed HELLP syndrome were identified as carriers of the Factor V Leiden mutation, a higher frequency of this mutation than in the normal central European population.13 Furthermore, we identified an activated protein C resistance in 33% of women who previously had developed HELLP syndrome (7 of 21). Another 38% of patients with HELLP syndrome had subnormal values (between 2.0 and 2.3). Activated protein C resistance measurements are known to be influenced significantly by a number of technical factors, including the applied preanalytical conditions (centrifugation, storage, fresh or frozen samples) as well as the choice of the reagents (PTT reagent, activated protein C, CaCl2 concentration).19 Nevertheless, even when the limitations in interpretation of activated protein C ratios are considered, the high percentages of HELLP syndrome patients with activated protein C resistance as determined in the present study suggest an association between an increased risk for developing thrombosis and HELLP syndrome. Most interestingly, however, the Factor V Leiden mutation was present only in four (57%) of the seven HELLP syndrome patients with an activated protein C resistance, and none of the women with subnormal ratios between 2.0 and 2.3 were carriers of the Factor V Leiden mutation. This apparent discrepancy indicates that the Factor V Leiden mutation is only partially responsible for the thrombophilia observed in women who previously had developed HELLP syndrome.
6. Wilke G, Rath W, Schu¨tz E, Armstrong VW, Kuhn W. Haptoglobin as a sensitive marker of hemolysis in HELLP syndrome. Int J Gynaecol Obstet 1992;39:29 –34. 7. de Boer K, Bu¨ller HR, Ten Cate JW, Treffers PE. Coagulation studies in the syndrome of hemolysis, elevated liver enzymes and low platelets. Br J Obstet Gynaecol 1991;98:42–7. 8. Dekker GA, de Vries JI, Doelitzsch PM, Huijgens PC, von Blomberg BM, Jakobs C, et al. Underlying disorders associated with severe early-onset preeclampsia. Am J Obstet Gynecol 1995; 173:1042– 8. 9. Faught W, Garner P, Jones G, Ivey B. Changes in protein C and protein S levels in normal pregnancy. Am J Obstet Gynecol 1995;172:147–50. 10. Dahlba¨ck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C. Proc Natl Acad Sci U S A 1993;90:1004 – 8. 11. Dahlba¨ck B. Factor V gene mutation causing inherited resistance to activated protein C as a basis for venous thromboembolism. J Intern Med 1995;237:221–7. 12. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, et al. Mutation in blood coagulation Factor V associated with resistance to activated protein C. Nature 1994;369:64 –7. 13. Rees DC, Cox M, Clegg JB. World distribution of Factor V Leiden. Lancet 1995;346:1133– 4. 14. Pabinger I, Kyrle PA, Heistinger M, Eichinger S, Wittmann E, Lechner K. The risk of thromboembolism in asymptomatic patients with protein C and protein S deficiency: A prospective cohort study. Thromb Haemost 1994;71:441–5. 15. Miletich J, Sherman L, Broze G. Absence of thrombosis in subjects with heterozygous protein C deficiency. N Engl J Med 1987;317: 991– 6. 16. Hellgren M, Svensson PJ, Dahlba¨ck B. Resistance to activated protein C as a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Am J Obstet Gynecol 1995;173: 210 –3. 17. Dizon-Townson DS, Nelson LM, Easton K, Ward K. The Factor V Leiden mutation may predispose women to severe preeclampsia. Am J Obstet Gynecol 1996;175:902–5. 18. Brenner B, Lanir N, Thaler I. HELLP syndrome associated with Factor V R506Q mutation. Br J Haematol 1996;92:999 –1001. 19. Bertina RM. Laboratory findings of resistance to activated protein C (APC-resistance). Thromb Haemost 1997;78:478 – 82.
References
Address reprint requests to:
1. Sibai BM, Ramadan MK, Usta I, Salama M, Mercer BM, Friedman SA. Maternal morbidity and mortality in 442 pregnancies with hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome). Am J Obstet Gynecol 1993;169:1000 – 6. 2. Meden H, Armstrong VW, Arendt H, Cunze T, Kuhn W. HELLP syndrome: Prognosis and parameters of hemolysis with special regard to haptoglobin. Singapore J Obstet Gynaecol 1996;27:42– 8. 3. Weinstein L. Syndrome of hemolysis, elevated liver enzymes, and low platelet count: A severe consequence of hypertension in pregnancy. Am J Obstet Gynecol 1982;142:159 – 67. 4. Rath W, Loos W, Kuhn W, Graeff H. The importance of early laboratory screening methods for maternal and fetal outcome in cases of HELLP syndrome. Eur J Obstet Gynecol Reprod Biol 1990;36:43–51. 5. Sibai BM. The HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets): Much ado about nothing? Am J Obstet Gynecol 1990;162:311– 6.
460 Krauss et al
Factor V Leiden in HELLP Syndrome
Thomas Krauss, MD Department of Gynecology and Obstetrics University of Go¨ttingen Robert-Koch-Str. 40 Go¨ttingen 37075 Germany E-mail: [email protected]
Received January 6, 1998. Received in revised form April 1, 1998. Accepted April 10, 1998. Copyright © 1998 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
Obstetrics & Gynecology