å¡ CASE REPORT å¡ Inherited Heterozygous Protein C Deficiency and Dysfunctional Protein S with Recurrent Venous Thrombotic Diseases: A Study of Three Generations of a Japanese Family Toshikazu Hashizume, Nobuko Tsushima, Hiroshi Matsuo, Akira Pro, Keizaburo Oozono and Toshiyuki Sakata* We describe a rare occurence of a family affected with venous thrombosis, exhibiting a protein C (PC) deficiency and dysfunctional protein S (PS). The propositus and his father developed recurrent venous thrombosis. Their PC deficiency was characterized by low levels of both antigen and activity, and their dysfunctional PS was suggested by low PS activities despite the presence of normal free PS antigen. Over three generations, six family members had a PC deficiency, and three had both a PC deficiency and a dysfunctional PS. The mode of inheritance of PC appears (Internal Medicine 31:deficiency 1197- 1200, 1992)to be autosomal dominant. Key words: anticoagulant, blood coagulation system
Introduction 2. Measurement of the level of PC activity and antigen We measured the levels of amidolytic activity of PC Protein (PS) is a vitamin K-dependent plasma using aSPC activator ("Protac", Pentapharm, Basel, protein (1), that serves as a co factor of activated protein Switzerland), isolated from the venom of Agkistrodon CContortrix, (APC) forand the aanti-coagulant. APC is a serine protease chromogenic substrate (S-2366, Kabi, which enzymatically Va was and Villa (2), Stockholm, Sweden)inactivates (3). The PCfactor antigen measured and thus functions as an anticoagulant. using a one step sandwich enzyme immunoassay (EIA) A(Protein hereditary deficiency of either PC oremploying PS is nowtwo reC test Teijin, Teijin, Japan), cognized as a cause of recurrent venous thrombotic dismonoclonal antibodies recognize Gla (gamma ease, but there have beenwhich no reports of occurence of carboxyglutamic acid) domain and a peptide by abnormalities of both PC and PS in the same released individual. We activation report here (4). The a family results with were recurring expressed pulmonary as percent thromof boembolism (PTE) and deep vein thrombosis (DVT) the normal control. 3. Measurement thedeficiency levels of PS and antigen associated with aofPC andactivity a dysfunctional PS. PS is present free and in a complex with C4b-binding Methods protein. We measured the total PS and free PS antigen. The total PS antigen was measured by a sandwich type enzyme-linked immunosorbent assay (ELISA), 1. Plasmas Venous (Asserachroom blood was PS,collected Diagnostica in one-tenth Stago, France). volumeFree of PS 3.8% sodium citrate. Platelet-poor plasma was obtained antigen was measured using a one-step sandwich EIA by centrifugation of citrated at 3 ,000 g. All plasmas (Protein S test Teijin, Teijin,blood Japan). were stored in aliquots at -80°C until use. Standard4. Measurement(Behringwerke) of anticoagulantwas activity Human-Plasma used as a standard We measured the anticoagulant activity of PS by using plasma in the following assays. STACLOT PROTAC (Diagnostrica Stago, France)
From the Division of Cardiology, Department of Internal Medicine and *the Department of Clinical Laboratory, National Cardiovascular Center, Suita Received for publication September 24, 1991; Accepted for publication July 20, 1992 Reprint requests should be addressed to Dr. Toshikazu Hashizume, the Division of Cardiology, Department of Internal Medicine, Nationa Cardiovascular Center, Suita, Osaka 565, Japan Internal Medicine Vol. 31, No. 10 (October 1992) 1197
Hashizume et al (5). The results were expressed as percent of the normal control Case Reports
Patient 1 A 25-year-old male suddenly developed right-sided chest pain and dyspnea in May 1981. He was admitted to a neighboring hospital and diagnosed as having PTE and He previous was referred DVT, because to this hospital of swelling for further of the examination. right leg. Digital subtraction angiography and radioisotope (RI) venography of the right leg revealed partial obstruction of the right femoral vein (Fig. 1), and a pulmonary perfusion scintigram showed perfusion defects in the lateral sides of the right middle lung field. He was treated with urokinase (600,000 units/day) for one week, and then received warfarin (3 mg/day) and ticlopidine HC1 (200mg/day) for several years. Only ticlopidine was administered after 1988, since no thrombotic symptoms were evident. In 1989, when he was 34 years old, his levels ofthese PC and activity inand the PS absence of warfarin Despite lowPS levels of PC activity, he has were low, 47 and 65% of normal levels, respectively. remained free of thrombotic symptoms without warfarin. Patient 2 A 57-year-old male, the father of patient 1, suddenly developed swelling and pain of the right leg in 1988. These symptoms disappeared gradually, but two months
later left localized chest pain, swelling of the right leg and hemosputum occurred without a proximate cause. He was admitted to a neighboring hospital, and was diagnosed as having DVT and PTE. Chest pain, hemos putum and leg swelling recurred several times. In June 1990, his levels of PC and PS activity without warfarin were low, 50 and 53% of normal, respectively. He had no abnormality of liver function and no signs of dis seminated intravascular coagulation. He was admitted to this hospital for further examination and therapy in June 1990. A radioisotope venograph did not reveal any significant stenosis or obstruction of either leg, but a pulmonary perfusion scintigram disclosed a perfusion defect, Study suggesting the left lingular segment (Fig. 2). Family Studies on plasma samples from the propositus and his family members are summarized in Table 1 and Fig. 3. In this family, there was a consanguineous marriage (Fig. 3). The distinctive features common to those affected with PTE and DVT were low levels of PC activity and antigen, and a low level of PS activity. A heterozygous PC deficiency was suspected because levels of PC antigen and activity were 2 S.D. below the mean of a normal control group and the concentrations of the other vitamin K-dependent clotting factors were within the normal range. Six family members over three generations ex hibited low levels of PC activity and antigen, and three of them (propositus, his father and nephew) exhibited
Fig. 1. Digital subtraction angiography of right leg (patient 1 at 25 years old), showing partial obstruction of the right femoral vein 1198
Internal
Medicine
Vol. 31, No. 10 (October
1992)
PC Deficiency and Dysfunctional
PS
might have been associated with these abnormalities of regulatory factors of the blood coagulation system. However PC deficiency coupled with dysfunctional PS has not been reported previously. We described here a rare case of a PC deficiency associated with dysfunctional PS, exhibiting venous thrombotic disease. In this family, dysfunctional PS was suggested by low levels of PS activity despite normal levels of free PS antigen. Comp reported in detail, dysfunctional protein S with the dissociation between the levels of PS activity and PS antigen (10). He classified dysfunctional protein S into three subtypes as shown in Table 2. According to this classification, the present patients had a type lib ab normality; which has not been previously reported to venous thrombotic disease in this family was Fig. 2. a) Radioisotope venography (patient 1 at 25 years old), The our knowledge. showing partial obstruction of the right femoral vein, b) Pulmonary not as severe as expected from the low levels of PC and PS. Only two members suffered from manifest venous perfusion scintigram of patient 1 taken at age 25, showing low perfusion area at the lateral side of the right middle lung field, c) pulmonary thrombotic disease and no thrombotic symptoms were perfusion scintigram of patient 2, showing a perfusion defect suggesting evident in other members with low levels of PC and/or the left lingular segment. PS. Since PS is a co factor ofAPC, a concomitant decrease of PS activity equivalent to the decrease in PC may not low levels of PS activity despite normal PS antigen cause results, a furtherhowever, decreaseindicated of the anticoagulant activity. levels, suggesting the presence of dysfunctional PS. These the difficulty of evalu The inheritance of the PC deficiency was consistent with ating the relationship between abnormalities among the an autosomal dominant trait. Among the members regulatory factors of the blood coagulation system and with PC deficiency and/or dysfunctional PS, only two the severity of venous thrombotic disease. Miletich et al (propositus and his father) were affected with venous reported that in 5422 healthy adults without a history of thrombotic disease. venous thrombotic disease, 79 of them had low levels (under 65%) of PC activity, and the levels in 10 were Discussion from 33 to 51% of normal (ll). They concluded that a heterozygous deficiency of PC is not always associated Cases of PC, PS, antithrombin III deficiency with In theapresent regardless of not only the with risk of patients, venous thrombosis. venous thrombotic disease have been reported previously heterozygous PC deficiency but also the deficient PS (6-9). These deficiencies were consistent with an inherited activity, the complications of venous thrombotic disease deficiency and unknown venous thrombotic disease
Table 1.
rpP_ oe sl d,..igtlO.r en e *
TI-3 I I -4 I I -5 T I -7 I I I -3 T I I -4 * * I T I -6 IV -1 N o rm al
Concentration
of Protein
A . tog e /._S e x
C and Protein S in Plasmas of the Propositus
A . Pn rt[roig .Ec teIxe AniAn ]l(/ %oC / ),
5 5 /F 6 0 /M 6 2 /M 5 2 /F 3 7 /F 3 4 /M 3 5 /M 1 4 /M ra n g e (n = 2 7 )
1 13 58 45 33 40 38 33 6 2 - 12 5
and His Family (Oct.
P r o te in C
P r o te i n S
Aa m i daA o lic yt ivti * cit Jy v( % AA') Pn T T
T^ o t. aA li nPrtｻcSig6 e n Frv( r% e ef) Pr ｻcS
1 14 47 87 53 33 47 41 31 6 6 - 1 79
116 47 84 49 30 50 38 29 62 - 1 2 2
[E L I S A ]
[E I A ]
100 103 10 4 62 99 10 0 85 67 65 - 118
99
1990)
A . pc tiv r .o it te. jyin. v( %S_ /)
10 0 53
87 84 81 79 79 63 - 123
62 10 0 55 45 70 6 5 - 13 1
* Pedigree positions correspond to those in Fig. 3. ** Propositus. ElA: enzyme immunoassay, ELISA: enzyme-linked immunosorbent assay, APTT: activated prothrombin time, PS: protein S. Internal
Medicine
Vol. 31, No. 10 (October
1992)
1199
Hashizume et al
Ill
IV
normal protein C deficiency dysfunctional protein S not tested deceased clinical
The arrow indicates Fig.
manifestations
of thrombotic
disease
the propositus 3.
Family
pedigree.
Table 2. Classification of Dysfunctional Protein S Based on theReferences Levels of Free and Total Protein S* 1) Di Seipio RG, Hermodson MA, Yates SG, et al. A comparison F ree pro tein S P ro tein S T o tal pro tein S of human prothrombin, factor IX (Christmas factor), factor X antigen activity an tigen (Stuart factor), and protein S. Biochemistry 16: 698, 1977. 2) Esmon CT, Sten flo J, Suttie JW, et al. A new vitamin K dependent T yp e I L ow L ow L ow protein. A phospholipid-binding zymogen of a serine esterase. T yp e H a L ow L ow N o rm al J Bio Chem 251: 3052, 1976. T yp e lib N o rm al L ow N o rm al 3) Martinoli JL, Stocker K. Fast functional protein C assay using Protac, a novel protein C activator. Thromb Res 43: 253, 1986. *A fter C om p (10). 4) Espana F, Estelles A, Aznar J, Gilabert J. Assay of protein C in human plasma: Comparison of amidolytic, coagulation and immunochemical assays. Thromb Res 44: 771, 1986. 5) Suzuki K, Nishioka J. Plasma protein S activity measured using were not too severe. These findings point to the difficulty Protac. a snake venon derived activator of protein C. Thromb in the explanation of the association between the ab Res 49: 241, 1988. 6) Griffin JG, Evatt B, Zimmerman TS, et al. Deficiency of protein normalities of PC, PS and the frequency and severity of C in congenital thrombotic disease. J Clin Invest 88: 1370, 1981. venous thrombotic disease. However, it is suspected 7) Broekmans AW, Veltkamp JJ, Bertina RM. Congenital protein that the abnormalities in the genetic constitution of C deficiency and venous thromboembolism. N Engl J Med 309: 340, 1983. both PC and PS are somehow different, and that other 8) Schwarz HP, Fischer M, Hepmeier P, et al. Plasma protein S abnormalities of the blood coagulation system might deficiency in familial thrombotic disease. Blood 64: 1297, 1984. enhance the risk of venous thrombotic disease, including 9) Egeberg O. Inherited antithrombin deficiency causing throm bophilia. Thromb Diath Haemorrh 13: 516, 1965. those of thrombomodulin and heparin co factor II, for 10) Comp PC. Laboratory evaluation of protein S status. Semin example. Further studies will be required to determine Thromb Hemost 16: 177, 1990. the relationship between the cause of venous thrombotic ll) Miletich J, Sherman L, Broze G Jr. Absence of thrombosis in disease and deficiencies of regulatory factors of the subjects with heterozygous protein C deficiency. N Engl J Med 317: 991, 1987. blood coagulation system.
1200
Internal
Medicine
Vol. 31, No. 10 (October
1992)
Introduction 2. Measurement of the level of PC activity and antigen We measured the levels of amidolytic activity of PC Protein (PS) is a vitamin K-dependent plasma using aSPC activator ("Protac", Pentapharm, Basel, protein (1), that serves as a co factor of activated protein Switzerland), isolated from the venom of Agkistrodon CContortrix, (APC) forand the aanti-coagulant. APC is a serine protease chromogenic substrate (S-2366, Kabi, which enzymatically Va was and Villa (2), Stockholm, Sweden)inactivates (3). The PCfactor antigen measured and thus functions as an anticoagulant. using a one step sandwich enzyme immunoassay (EIA) A(Protein hereditary deficiency of either PC oremploying PS is nowtwo reC test Teijin, Teijin, Japan), cognized as a cause of recurrent venous thrombotic dismonoclonal antibodies recognize Gla (gamma ease, but there have beenwhich no reports of occurence of carboxyglutamic acid) domain and a peptide by abnormalities of both PC and PS in the same released individual. We activation report here (4). The a family results with were recurring expressed pulmonary as percent thromof boembolism (PTE) and deep vein thrombosis (DVT) the normal control. 3. Measurement thedeficiency levels of PS and antigen associated with aofPC andactivity a dysfunctional PS. PS is present free and in a complex with C4b-binding Methods protein. We measured the total PS and free PS antigen. The total PS antigen was measured by a sandwich type enzyme-linked immunosorbent assay (ELISA), 1. Plasmas Venous (Asserachroom blood was PS,collected Diagnostica in one-tenth Stago, France). volumeFree of PS 3.8% sodium citrate. Platelet-poor plasma was obtained antigen was measured using a one-step sandwich EIA by centrifugation of citrated at 3 ,000 g. All plasmas (Protein S test Teijin, Teijin,blood Japan). were stored in aliquots at -80°C until use. Standard4. Measurement(Behringwerke) of anticoagulantwas activity Human-Plasma used as a standard We measured the anticoagulant activity of PS by using plasma in the following assays. STACLOT PROTAC (Diagnostrica Stago, France)
From the Division of Cardiology, Department of Internal Medicine and *the Department of Clinical Laboratory, National Cardiovascular Center, Suita Received for publication September 24, 1991; Accepted for publication July 20, 1992 Reprint requests should be addressed to Dr. Toshikazu Hashizume, the Division of Cardiology, Department of Internal Medicine, Nationa Cardiovascular Center, Suita, Osaka 565, Japan Internal Medicine Vol. 31, No. 10 (October 1992) 1197
Hashizume et al (5). The results were expressed as percent of the normal control Case Reports
Patient 1 A 25-year-old male suddenly developed right-sided chest pain and dyspnea in May 1981. He was admitted to a neighboring hospital and diagnosed as having PTE and He previous was referred DVT, because to this hospital of swelling for further of the examination. right leg. Digital subtraction angiography and radioisotope (RI) venography of the right leg revealed partial obstruction of the right femoral vein (Fig. 1), and a pulmonary perfusion scintigram showed perfusion defects in the lateral sides of the right middle lung field. He was treated with urokinase (600,000 units/day) for one week, and then received warfarin (3 mg/day) and ticlopidine HC1 (200mg/day) for several years. Only ticlopidine was administered after 1988, since no thrombotic symptoms were evident. In 1989, when he was 34 years old, his levels ofthese PC and activity inand the PS absence of warfarin Despite lowPS levels of PC activity, he has were low, 47 and 65% of normal levels, respectively. remained free of thrombotic symptoms without warfarin. Patient 2 A 57-year-old male, the father of patient 1, suddenly developed swelling and pain of the right leg in 1988. These symptoms disappeared gradually, but two months
later left localized chest pain, swelling of the right leg and hemosputum occurred without a proximate cause. He was admitted to a neighboring hospital, and was diagnosed as having DVT and PTE. Chest pain, hemos putum and leg swelling recurred several times. In June 1990, his levels of PC and PS activity without warfarin were low, 50 and 53% of normal, respectively. He had no abnormality of liver function and no signs of dis seminated intravascular coagulation. He was admitted to this hospital for further examination and therapy in June 1990. A radioisotope venograph did not reveal any significant stenosis or obstruction of either leg, but a pulmonary perfusion scintigram disclosed a perfusion defect, Study suggesting the left lingular segment (Fig. 2). Family Studies on plasma samples from the propositus and his family members are summarized in Table 1 and Fig. 3. In this family, there was a consanguineous marriage (Fig. 3). The distinctive features common to those affected with PTE and DVT were low levels of PC activity and antigen, and a low level of PS activity. A heterozygous PC deficiency was suspected because levels of PC antigen and activity were 2 S.D. below the mean of a normal control group and the concentrations of the other vitamin K-dependent clotting factors were within the normal range. Six family members over three generations ex hibited low levels of PC activity and antigen, and three of them (propositus, his father and nephew) exhibited
Fig. 1. Digital subtraction angiography of right leg (patient 1 at 25 years old), showing partial obstruction of the right femoral vein 1198
Internal
Medicine
Vol. 31, No. 10 (October
1992)
PC Deficiency and Dysfunctional
PS
might have been associated with these abnormalities of regulatory factors of the blood coagulation system. However PC deficiency coupled with dysfunctional PS has not been reported previously. We described here a rare case of a PC deficiency associated with dysfunctional PS, exhibiting venous thrombotic disease. In this family, dysfunctional PS was suggested by low levels of PS activity despite normal levels of free PS antigen. Comp reported in detail, dysfunctional protein S with the dissociation between the levels of PS activity and PS antigen (10). He classified dysfunctional protein S into three subtypes as shown in Table 2. According to this classification, the present patients had a type lib ab normality; which has not been previously reported to venous thrombotic disease in this family was Fig. 2. a) Radioisotope venography (patient 1 at 25 years old), The our knowledge. showing partial obstruction of the right femoral vein, b) Pulmonary not as severe as expected from the low levels of PC and PS. Only two members suffered from manifest venous perfusion scintigram of patient 1 taken at age 25, showing low perfusion area at the lateral side of the right middle lung field, c) pulmonary thrombotic disease and no thrombotic symptoms were perfusion scintigram of patient 2, showing a perfusion defect suggesting evident in other members with low levels of PC and/or the left lingular segment. PS. Since PS is a co factor ofAPC, a concomitant decrease of PS activity equivalent to the decrease in PC may not low levels of PS activity despite normal PS antigen cause results, a furtherhowever, decreaseindicated of the anticoagulant activity. levels, suggesting the presence of dysfunctional PS. These the difficulty of evalu The inheritance of the PC deficiency was consistent with ating the relationship between abnormalities among the an autosomal dominant trait. Among the members regulatory factors of the blood coagulation system and with PC deficiency and/or dysfunctional PS, only two the severity of venous thrombotic disease. Miletich et al (propositus and his father) were affected with venous reported that in 5422 healthy adults without a history of thrombotic disease. venous thrombotic disease, 79 of them had low levels (under 65%) of PC activity, and the levels in 10 were Discussion from 33 to 51% of normal (ll). They concluded that a heterozygous deficiency of PC is not always associated Cases of PC, PS, antithrombin III deficiency with In theapresent regardless of not only the with risk of patients, venous thrombosis. venous thrombotic disease have been reported previously heterozygous PC deficiency but also the deficient PS (6-9). These deficiencies were consistent with an inherited activity, the complications of venous thrombotic disease deficiency and unknown venous thrombotic disease
Table 1.
rpP_ oe sl d,..igtlO.r en e *
TI-3 I I -4 I I -5 T I -7 I I I -3 T I I -4 * * I T I -6 IV -1 N o rm al
Concentration
of Protein
A . tog e /._S e x
C and Protein S in Plasmas of the Propositus
A . Pn rt[roig .Ec teIxe AniAn ]l(/ %oC / ),
5 5 /F 6 0 /M 6 2 /M 5 2 /F 3 7 /F 3 4 /M 3 5 /M 1 4 /M ra n g e (n = 2 7 )
1 13 58 45 33 40 38 33 6 2 - 12 5
and His Family (Oct.
P r o te in C
P r o te i n S
Aa m i daA o lic yt ivti * cit Jy v( % AA') Pn T T
T^ o t. aA li nPrtｻcSig6 e n Frv( r% e ef) Pr ｻcS
1 14 47 87 53 33 47 41 31 6 6 - 1 79
116 47 84 49 30 50 38 29 62 - 1 2 2
[E L I S A ]
[E I A ]
100 103 10 4 62 99 10 0 85 67 65 - 118
99
1990)
A . pc tiv r .o it te. jyin. v( %S_ /)
10 0 53
87 84 81 79 79 63 - 123
62 10 0 55 45 70 6 5 - 13 1
* Pedigree positions correspond to those in Fig. 3. ** Propositus. ElA: enzyme immunoassay, ELISA: enzyme-linked immunosorbent assay, APTT: activated prothrombin time, PS: protein S. Internal
Medicine
Vol. 31, No. 10 (October
1992)
1199
Hashizume et al
Ill
IV
normal protein C deficiency dysfunctional protein S not tested deceased clinical
The arrow indicates Fig.
manifestations
of thrombotic
disease
the propositus 3.
Family
pedigree.
Table 2. Classification of Dysfunctional Protein S Based on theReferences Levels of Free and Total Protein S* 1) Di Seipio RG, Hermodson MA, Yates SG, et al. A comparison F ree pro tein S P ro tein S T o tal pro tein S of human prothrombin, factor IX (Christmas factor), factor X antigen activity an tigen (Stuart factor), and protein S. Biochemistry 16: 698, 1977. 2) Esmon CT, Sten flo J, Suttie JW, et al. A new vitamin K dependent T yp e I L ow L ow L ow protein. A phospholipid-binding zymogen of a serine esterase. T yp e H a L ow L ow N o rm al J Bio Chem 251: 3052, 1976. T yp e lib N o rm al L ow N o rm al 3) Martinoli JL, Stocker K. Fast functional protein C assay using Protac, a novel protein C activator. Thromb Res 43: 253, 1986. *A fter C om p (10). 4) Espana F, Estelles A, Aznar J, Gilabert J. Assay of protein C in human plasma: Comparison of amidolytic, coagulation and immunochemical assays. Thromb Res 44: 771, 1986. 5) Suzuki K, Nishioka J. Plasma protein S activity measured using were not too severe. These findings point to the difficulty Protac. a snake venon derived activator of protein C. Thromb in the explanation of the association between the ab Res 49: 241, 1988. 6) Griffin JG, Evatt B, Zimmerman TS, et al. Deficiency of protein normalities of PC, PS and the frequency and severity of C in congenital thrombotic disease. J Clin Invest 88: 1370, 1981. venous thrombotic disease. However, it is suspected 7) Broekmans AW, Veltkamp JJ, Bertina RM. Congenital protein that the abnormalities in the genetic constitution of C deficiency and venous thromboembolism. N Engl J Med 309: 340, 1983. both PC and PS are somehow different, and that other 8) Schwarz HP, Fischer M, Hepmeier P, et al. Plasma protein S abnormalities of the blood coagulation system might deficiency in familial thrombotic disease. Blood 64: 1297, 1984. enhance the risk of venous thrombotic disease, including 9) Egeberg O. Inherited antithrombin deficiency causing throm bophilia. Thromb Diath Haemorrh 13: 516, 1965. those of thrombomodulin and heparin co factor II, for 10) Comp PC. Laboratory evaluation of protein S status. Semin example. Further studies will be required to determine Thromb Hemost 16: 177, 1990. the relationship between the cause of venous thrombotic ll) Miletich J, Sherman L, Broze G Jr. Absence of thrombosis in disease and deficiencies of regulatory factors of the subjects with heterozygous protein C deficiency. N Engl J Med 317: 991, 1987. blood coagulation system.
1200
Internal
Medicine
Vol. 31, No. 10 (October
1992)
