Prevalence of Activated Protein C Resistance in Acute Myocardial Infarction in Japan

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1 Clinical Studies Prevalence of Activated Protein C Resistance in Acute Myocardial Infarction in Japan Kazunori HAYASHI, MD, Takahito SONE,1 MD, Junichiro KONDOH,1 MD, Hideyuki TSUBOI,1 MD, Hiromi SASSA,1 MD, Yasushi NUMAGUCHI, MD, Yukio TOKI, MD, Kenji OKUMURA, MD, Takayuki ITO, MD and Tetsuo HAYAKAWA, MD SUMMARY To investigate the possibility that activated protein C (APC) resistance due to the factor V could be an important predisposing factor in acute myocardial infarction (AMI), we have retrospectively examined the prevalence of APC resistance with protein C, protein S and antithrombin III deficiency and antiphospholipid antibody syndrome in AMI patients ( 50 years) admitted to our hospital over the past 7 years. Forty-seven patients were enrolled in the study. We divided the patients into two groups, warfarin group (group A) and a non-warfarin group (group B). APC resistance is defined as when the APC ratio is below or equal to the cut-off value 2. APC resistance was not detected in either group. The prevalence of an APC ratio below or equal to 2.5 was 16.7% (1 case) in group A and 24.4% (10 cases) in group B. The prevalence of protein C deficiency was 5.0% (2 cases) in group B. Two cases (5.0%) in group B had protein S deficiency. Antithrombin III deficiency was not detected in either group. The prevalence of antiphospholipid antibody syndrome measured by APTT was 40.4% (19 cases). We compared the AMI patients with 97 healthy volunteers ( 50 years old) without any thromboembolic events or bleeding tendency in their past history. No significant difference were found between these groups and the volunteers. APC resistance is a major cause of venous thromboembolism in Europe and the United States, while in Japan it is believed to be a minor cause of arterial thromboembolism. (Jpn Heart J 1997; 38: ) Key words: APC resistance, Antiphospholipid antibody syndrome, AMI A LTHOUGH predicting markers for thrombophilia have been extensively investigated, well-explained biochemical defects, impairing the natural From Department of Internal Medicine II, Nagoya University School of Medicine, Nagoya, and 1Department of Cardiology, Ogaki Municipal Hospital, Ogaki, Japan. Address for correspondence: Kazunori Hayashi, MD, Department of Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan. Received for publication June 5, Accepted July 30,

2 770 HAYASHI ET AL Jpn Heart J November 1997 antithrombotic mechanism capable of physiologically limiting thrombin formation, have been found only in a relatively small percentage of patients with familial thrombosis. Thrombophilia is associated with acquired disorders, including cancer, DIC and lupus anticoagulant or inherited deficiencies of protein C, protein S and antithrombin III (AT III).1-3) Taken together, these inherited deficiencies are estimated to account for about 15% of the prevalence of thromboembolic disease4) and a laboratory search for other inherited causes of thrombophilia including defects in heparin cofactor II, abnormalities in fibrinogen or in the fibrinolytic system offers only a slight increase in the diagnostic Yield.5) In 1993 Dahlback et al. reported the case of a middle-aged man with a history of recurrent venous thrombosis.6) In normal plasma, the addition of activated protein C (APC) induced a prolongation of APTT because of cleavage and inhibition of factors Va and VIIIa; in the proband the prolongation of APTT in the presence of APC was much shorter than in control plasma. Most of the proband's relatives manifested a poor anticoagulant response to APC, so a genetic basis for the alteration could be hypothesized. Bertina et al. demonstrated that resistance to activated protein C (APC resistance) was due to a single base point mutation in the factor V gene resulting in the mutation of amino acid 506 Arg Gln, thereby rendering factor Va resistant to inactivation by APC.7) It seems that this disorder is the most frequent cause of hereditary thrombophilia (19-37%) and is also found in 5% of normal subjects in Europe and the United States.8-10) Apart from the venous thromboembolism, there are several reports of arterial thromboembolism associated with APC resistance.11-13) However, reports of no association with APC resistance also have been made.14-16) Therefore, to elucidate the relationship between APC resistance and the onset of myocardial infarction, we retrospectively investigated 47 survivors of myocardial infarction whose age of onset were below or equal to 50 years and 97 healthy controls without any thromboembolic events or bleeding tendency in their history for the prevalence of APC resistance. Furthermore, we also examined the incidences of protein C deficiency, protein S deficiency, AT III deficiency and anti-phospholipid antibody syndrome in the myocardial infarction group. METHODS Patients: Forty-seven survivors among the 98 patients 50 years old or younger admitted to Ogaki Municipal Hospital for acute myocardial infarction (AMI) from January 1989 to August 1995 were enrolled in the study. Patients were divided into two groups depending on whether they were taking warfarin or not, a warfarin group (group A) and a non-warfarin group (group B). Group A con-

3 Vol 38 APC RESISTANCE IN AMI IN JAPAN 771 No 6 sisted of 6 men with a mean age of 44. The infarct-related artery was the left anterior descending coronary artery in 4, the left circumflex coronary artery in 1 and the right coronary artery in 1. A patient who experienced thrombotic myocardial infarction twice was included in group A. Group B consisted of 41 patients (39 men and 2 women) with a mean age of 44. One group B patient was a 22 year-old woman. The infarct-related artery was the left anterior descending coronary artery in 17, the left circumflex coronary artery in 5 and the right coronary artery in 19. A patient who experienced 3 thrombotic myocardial infarction was included in group B. Controls: The control population consisted of 97 healthy volunteers (44 women not taking oral contraceptives or postmenopausal estrogen replacement and 53 men) without any thromboembolic events or bleeding tendency in their history. The ages ranged from 20 to 47 years (mean 33 years). APC resistance: Blood was drawn from an antecubital vein to measure the anticoagulant response in plasma to activated protein C. Nine volumes of blood was mixed with 0.1mol/l sodium citrate (1 volume) and centrifuged at 2000 ~g for 20 minutes. The plasma was separated from the cells within 30 minutes. The patients were at rest for 20 minutes before sampling. The sample was frozen rapidly at -70 Ž until used for the assays. Frozen plasma samples were rapidly thawed at 37 Ž in a standardized way ensuring negligible loss of activity of the labile coagulation factors. After prewarming a sufficient volume of CaCl2 and APC/CaCl2 at 37 }0.5 Ž, one volume of plasma was added to a test tube or cuvette and then an equal volume of APTT reagent added. After incubating at 37 Ž for 5 minutes, one volume of CaCl2 was added and the timing of clot formation simultaneously begun. The time for clot formation (T1) was recorded. A second analysis of the plasma, exchanging CaCl2 with APC/CaCl2, was performed and the time for clot formation (T2) recorded. The APC ratio (T2/T1) was calculated. APC resistance is indicated when the APC ratio is 2 or less. All procedures were performed using the Coatest APC resistance kit (Chromogenix, Molndal, Sweden). Table I. Criteria for the Antiphospholipid Antibody Syndrome* *Patients with at least one clinical plus one serologic feature are classified as having the antiphospholipid antibody syndrome. #Test must be positive on at least two occasions more than 3 months apart.

4 772 HAYASHI ET AL Jpn Heart J November 1997 Protein C, protein S, AT III assay: Protein C and protein S (total and free) levels were measured by ELISA. Protein C activity and AT III activity were measured using the choromogenic substrate method. Protein C deficiency was defined when protein C activities or levels of protein C antigens against the normal subjects was lower than 70%. Protein S deficiency was diagnosed when the levels of either total or free protein S were lower than the normal subjects. Every assay in group A was performed under warfarin administration. Antiphospholipid antibody syndrome: Lupus anticoagulant activity was determined by the APTT method after confirmation of the absence of APTT prolongation in all patients. Anticardiolipin antibodies were determined as lgg and IgM isotypes by ELISA, (anticardiolipin kit, Chromogenix, Molndal, Sweden). The criteria for antiphospholipid antibody syndrome were selected according to Harris' proposal shown in Table I.17) Statistics: The values of the APC ratio were compared between the AMI patients and control groups and between the AMI patients with and without antiphospholipid antibody using the unpaired Student's t-test. Statistical significance was accepted at p<0.05. RESULTS As shown in Figure 1, no patients in the control group, group A or group B belonged to the category of APC resistance when the APC resistance was defined as 2 or less. One case (16.7%) in group A and 10 cases (24.4%) in group B had Figure 1. Dot plot of APC ratio in patients in group A and group B (left side). Open circles represent group A and closed circles represent group B. Dot plot of APC ratio in control group (right side). No significant differences in the APC ratios were detected between the groups.

5 Vol 38 APC RESISTANCE IN AMI IN JAPAN 773 No 6 Figure 2. Dot plot of APC ratio in AMI patients with and without antiphospholipid antibody syndrome. Open squares represent the patients on warfarin who were lupus anticoagulant positive and open triangles are non-warfarin patients who were with lupus anticoagulant positive. Open circles are anticardiolipin antibody positive patients, while closed circles are antiphospholipid antibody negative patients. Table II. Prevalence of Protein C, Protein S and AT III Deficiency in AMI Patients ( 50) APC ratios below 2.5. The patient with an APC ratio of 2.02 was included in group B. The mean value of the APC ratio in group A and group B was 2.93, while it was 3.03 in the control group. The value was higher in the control group, however, no significant differences were found between the groups. Figure 2 demonstrates the APC ratio in patients with antiphospholipid antibody syndrome. Patients with anticardiolipin antibody positive did not receive warfarinization. The incidence of antiphospholipid antibodies found was 25.5% (12 cases) for lupus anticoagulant and 7 cases (14.9%) for anticardiolipin antibody, which represents an overall percentage of 40.4% for antiphospholipid antibody syndrome in patients with AMI. There were no patients who were positive for both anticardiolipin antibody and lupus anticoagulant. No significant difference in the value of the APC ratio was found between AMI patients with and without antiphospholipid antibody. The incidences of protein C, protein S and AT III deficiency are shown in Table II. Protein C deficiency was detected in 3 cases (50%) in group A and 2 cases (5.0%) in group B. Protein S deficiency was detected in 4 cases (66.7%) in group A and 2 cases (5.0%) in group B. No patient with both protein C and

6 774 HAYASHI ET AL Jpn Heart J November 1997 protein S deficiency was observed. AT III deficiency was not detected in either group. DISCUSSION Figure 3 is a schematic diagram of the blood coagulation system, in particular the protein C pathway. When bound to thrombomodulin, a thrombin receptor located in the surface of the endothelial membrane, protein C is converted to an active serine protease, APC, by thrombin. APC then inhibits coagulation by specifically inactivating factors Va and VIIIa, bringing the coagulation pathway to an end. The anticoagulant effect of APC requires the presence of the cofactor protein S in its unbound form. A deficiency in protein C or protein S induces thromboembolic events because a lack of either of these protein cannot put the brakes on the coagulation pathway.1,2) A deficiency in AT III also brings about recurrent thrombosis.3) AT III inactivates thrombin and factor Xa and, to a lesser extent, other serine proteases (VIIa, IXa, XIa, XIIa). However, these deficiencies accounted for only about 15% of the prevalence of thromboembolic disease.4) In 1993 Dahlback demonstrated the existence of a new defect associated with an elevated frequency of deep vein thrombosis (DVT). A poor anticoagulant response to APC (APC resistance), which does not prolong the APTT by inactivating factor Va and VIIIa, was the causes of this phenomenon.6) Genetic mutation involving the locus for the factor V gene was subsequently demonstrated to be associated in heterozygosity or homozygosity with the phenotype of APC resistance in patients with thromboembolism.7,9) The association of APC resis- Figure 3. Schematic diagram of the blood coagulation system, in particular the protein C pathway.

7 Vol 38 No 6 APC RESISTANCE IN AMI IN JAPAN 775 tance with venous thromboembolism is well-defined but the prevalence of venous thrombosis or pulmpnary thromboembolism based on APC resistance in Japan is reported to be low in comparison with that in Europe.18) The association of APC resistance with arterial thromboembolism is still in a controversy even in Europe. Concerning the relationship between APC resistance and the onset of myocardial infarction, we retrospectively investigated 47 survivors among young AMI patients ( 50) in Japan. We selected AMI patients whose ages of onset were below or equal to 50 because an inherited disorder could cause thromboembolism at younger ages. The prevalence of APC resistance is shown in Figure 1. No patient was classified as APC resistant when the APC ratio was defined as below or equal to 2. The cut-off value 2 was calculated in Europe from the analysis of plasmas from 52 healthy individuals (23 men, 29 women, age years). Therefore, we examined the APC ratio among 97 healthy Japanese volunteers without any thrombotic events or bleeding tendency in their past history. The minimum value of the APC ratio in this group was 2.19 and the mean Our statistical analysis indicated an APC ratio below or equal to 2.2 was positive (mean }2SD). According to this criteria, 2 cases in group A and 3 cases in group B may be APC resistant. Six patients in group A had been receiving warfarin anticoagulant therapy for the prevention of recurrent myocardial infarction. The APC resistance assay in patients on warfarin is influenced by reductions in the concentration of factors II, X, IX and VIII that lead to an increased APC ratio. Therefore, modified assays based on the dilution of test plasma in factor V deficient plasma have been reported to be highly reliable in such patients.19,20) One patient in group A had an APC ratio of This patient may show a lower APC ratio based on the factor V mutation when measured by the modified assay, however, the details are unknown because molecular analysis of factor V gene using polymerase chain amplification from genomic DNA and modified assay were not performed in our study. Holm et al. reported two cases of myocardial infarction in young women aged 34 and 33 years-old who were homozygous.12) Marz et al. demonstrated that 9% of the patients had a heterozygous factor V mutation among 224 angiographically diagnosed coronary artery disease patients, a significantly higher prevalence compared with control subjects.21) Both of these studies reported an association between myocardial infarction and APC resistance, however, Cushman et al. found no difference in the prevalence of APC resistance in a cohort of patients with arterial thrombosis and in healthy controls.14) Samani et al. and Emmerich et al. reported a similar prevalence of factor V mutation in patients with myocardial infarction and in healthy age-matched controls.15,16) The incidence of factor V mutation in patients with myocardial infarction reported by Marz, Samani and Emmerich et al. were almost the same. The deviations in the results and the conclusions to be drawn from them are due mainly to the different

8 776 HAYASHI ET AL Jpn Heart J November 1997 mutation frequencies in the control groups. These data may reflect racial diversity in Europe, but technical procedures including improper hybridization or polymerase chain reaction, may account for the differences. The prevalence of homozygosity in factor V mutation is much lower compared with that of heterozygosity. The presence of homozygosity in APC resistance does not always induce thromboembolic events, but the past clinical reports, including Holm et al. and Lindblad et al.,13) support the idea of the presence of homozygosity being a risk factor of arterial thromboembolism. Judging from previous studies, APC resistance does not seem to be a risk factor for myocardial infarction, at least in heterozygous individuals. Hereditary and acquired coagulation abnormalities are not often investigated simultaneously in thrombophilic patients. Therefore, we also examined protein C, protein S and AT III as a hereditary factor and antiphospholipid antibody as an acquired factor as well as the prevalence of APC resistance. Table II presents the prevalences of protein C, protein S and AT III deficiency. It is well-known that the serum concentrations or activities of protein C and protein S decrease in patients on warfarin because protein C and protein S are the vitamin K-dependent plasma proteins, so the incidences of protein C and protein S in group A were high. Clinical symptoms and features of protein C, protein S and AT III deficiency are reportedly similar, while arterial thromboembolism is reported to be rare in patients with AT III deficiency in comparison with protein C and protein S deficiency,22) which supports our clinical data. Although antiphospholipid binds to phospholipid complexes and causes a prolongation of coagulation time, it is clinically associated with thrombosis. Direct binding of phospholipid-bound protein C and/or protein S, inhibition of protein C activation by thrombomodulin and inhibition of APC-mediated degradation of factor Va have been described.23,24) The interference of antiphospholipid antibody with the APC resistance test should be taken into consideration if this mechanism is true. In fact, the APC ratio in patients with antiphospholipid antibody syndrome is reported to decrease in comparison with the actual APC ratio. Bokarewa et al. reported that APC resistance was induced only by IgG fractions with both anticardiolipin and lupus anticoagulant activity.25) The underlying mechanism was postulated to be a negative interaction between IgG and the APC-binding sites on factor Va molecules. We had 3 cases who were IgM-anticardiolipin antibody positive and they did not show APC resistance. The remaining antiphospholipid antibody positive patients were not APC resistant because our APC ratio already greater than 2. As shown in Figure 2, no correlation was observed between the APC ratio and the presence and absence of antiphospholipid antibody in AMI patients. We did not examine the prevalence of antiphospholipid antibody syndrome in healthy volunteers, so we

9 Vol 38 No 6 APC RESISTANCE IN AMI IN JAPAN 777 cannot comment on whether the prevalence of antiphospholipid antibody syndrome is high or not. However, the data indicate the prevalence of APC resistance in Japan is low even in patients who are anticardiolipin antibody positive. Anticardiolipin antibody was reported to be an independent risk factor of myocardial infarction in a prospective cohort study,26) however, further prospective evaluation of the correlation between antiphospholipid antibody and the fibrinolytic and coagulation system, as well as the correlation between thrombotic risk and APC resistance will be needed to establish the thrombotic risk of antiphospholipid and APC resistance. In conclusion, it is unlikely that APC resistance contributes to the occurrence of AMI in Japan. REFERENCES 1. Comp PC, Nixon RR, Cooper MR, Emson CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984; 74: Allaart CF, Poort SR, Rosendaal FR, Reitsma PH, Bertina RM, Briet E. Increased risk of venous thrombosis in carriers of hereditary protein C deficiency defect. Lancet 1993; 341: Thaler E, Lechner K. Antithrombin III deficiency and thromboembolism. Clin Haematol 1981; 10: Bauer KA. Hypercoagulability: a new cofactor in the protein C anticoagulant pathway. N Engl J Med 1994; 330: Mannucci PM, Tripodi A. Laboratory screening of inherited thrombotic syndromes. Thromb Haemostas 1987; 57: Dahlback 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 USA 1993; 90: Bertina RM, Koelemann BPC, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: Cadroy Y, Sic P, Boneu B. Frequency of a defective response to activated protein C in patients with a history of venous thrombosis. Blood 1994; 83: Koster T, Rosendaal RS, de Tonde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet 1993; 342: Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: Halbmayer WM, Haushofer A, Schon R, Fischer M. The prevalence of poor anticoagulant response to activated protein C (APC resistance) among patients suffering from stroke or venous thrombosis and among healthy subjects. Blood Coag Fibrinol 1994; 5: Holm J, Zoller B, Svensson PJ, Berntorp E, Erhardt L, Dahlback B. Myocardial infarction associated with homozygous resistance to activated protein C. Lancet 1994; 344: Lindblad B, Svensson PJ, Dahlback B. Arterial and venous thromboembolism with fatal outcome and resistance to activated protein C. Lancet 1994; 343: Cushman M, Bhushan F, Bovill E, Tracy R. Plasma resistance to activated protein C in venous and arterial thrombosis. Thromb Haemostas 1994; 72: Samani NJ, Lodwick D, Martin D, Kimber P. Resistance to activated protein C and risk of premature myocardial infarction. Lancet 1994; 344: Emmerich J, Poirier O, Evans A, et al. Myocardial infarction, Arg 506 to Gln factor V mutation, and activated protein C resistance. Lancet 1995; 345: 321.

10 778 HAYASHI ET AL Jpn Heart J November Harris EN. Antiphospholipid antibodies. Br J Haematol 1990; 74: Zama T, Murata M, Ono F, Watanabe K, et al. Low prevalence of activated protein C resistance and coagulation factor V Arg 506 to Gln mutation among Japanese patients with various forms of thrombosis and normal individuals. Int J Hematol 1996; 65: Trossaert M, Conard J, Horellou MH, et al. Modified APC resistance assay for patients on oral anticoagulants. Lancet 1994; 344: Denson KWE, Reed SV, Haddon ME. The modified APC resistance test. Thromb Haematostas 1995; 74: Marz W, Seydewitz H, Winkelmann B, Chen M, Nauck M, Witt I. Mutation in coagulation factor V associated with resistance to activated protein C in patients with coronary artery disease. Lancet 1995; 345: Hirsh J, Piovella S, Pini M. Congenital antithrombin III deficiency. Am J Med 1989; 87 (suppl 3B): Borrell M, Sal N, de Carsellarnau C, Lopez S, Fontcuberta J. Immunoglobulin fractions isolated from patients with antiphospholipid antibodies prevent the inactivation of factor Va by activated protein C on human endothelial cells. Thromb Haemost 1992; 68: Oosting JD, Derksen RHWM, Bobbing WG, Hackeng TM, Bouma BM, de Groot PhG. Antiphospholipid antibodies directed against a combination of phospholipids with prothrombin, protein C or protein S: an explanation for their pathogenic mechanism? Blood 1993; 81: Bokarewa MI, Blomback M, Egberg N, Rosen S. A new variant of interaction between phospholipid antibodies and the protein C system. Blood Coag Fibrinol 1994; 5: Vaarala O, Manttari M, Manninen V, et al. Anticardiolipin antibodies and risk of myocardial infarction in a prospective cohort of middle-aged men. Circulation 1995; 91: 23-7.