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© 1996 The Journal of Bone and Joint Surgery, Inc. Association of Antithrombotic Factor Deficiencies and Hypofibrinolysis with Legg-Perthes Disease*   , CINCINNATI, OHIOInvestigation performed at the Cholesterol Center and the Department of Orthopedics, Jewish Hospital; the Department of Orthopedics, Children's Hospital; and the Departments of Pathology and Laboratory of Medicine, University of Cincinnati College of Medicine, Cincinnati
Thirty-three (75 per cent) of forty-four unselected children who had Legg-Perthes disease were found to have coagulation abnormalities. Twenty-three children had thrombophilia (a deficiency in antithrombotic factor C or S, with an increased tendency toward thrombosis); nineteen of the twenty-three children had protein-C deficiency and four had protein-S deficiency. Seven children had a high level (0.25 gram per liter or more) of lipoprotein(a), a thrombogenic, atherogenic lipoprotein associated with osteonecrosis in adults. Three children had hypofibrinolysis (a reduced ability to lyse clots). The mean age of the children when the Legg-Perthes disease was first diagnosed was 5.8 ± 2.7 years, and the mean age at the time of the present study was 10.1 ± 4.4 years. At least one of the first-degree relatives of eleven of the nineteen probands who had a low protein-C level had a low protein-C level as well; all of these low levels represented previously undiagnosed familial protein-C deficiency. The eleven probands who had familial protein-C deficiency were more likely to have early onset of Legg-Perthes disease (at or before the age of five years) than the eleven children who had normal levels of protein C, protein S, and lipoprotein(a) as well as normal fibrinolytic activity (chi-square = 6.6; p = 0.01). At least one first-degree relative of one of the four probands who had a low protein-S level had a low protein-S level and previously undiagnosed familial protein-S deficiency. At least one first-degree relative of six of the seven probands who had a high level of lipoprotein(a) had a familial high level of lipoprotein(a). Six of the seven children who had a high level of lipoprotein(a) also had a low level of stimulated tissue-plasminogen activator activity, the major initiator of fibrinolysis. At least one first-degree relative of one of the three probands who had normal levels of protein C, protein S, and lipoprotein(a) but low stimulated tissue-plasminogen activator activity also had low stimulated tissue-plasminogen activator activity (familial hypofibrinolysis). Legg-Perthes disease, thrombophlebitis, premature myocardial infarction, and stroke, which are ramifications of the familial thrombophilic-hypofibrinolytic disorders, were common in the first and second-degree relatives of the thirty-three children with Legg-Perthes disease who also had thrombophilic-hypofibrinolytic disorders. CLINICAL RELEVANCE: Protein-C or S deficiency, hypofibrinolysis, or a high level of lipoprotein(a) may result in thrombotic venous occlusion of the femur, which leads to the venous hypertension and osteonecrosis of the femoral head characteristic of Legg-Perthes disease. When Legg-Perthes disease develops in a child, the levels of proteins C and S, lipoprotein(a), and stimulated fibrinolysis should be measured. Early diagnosis of protein-C or S deficiency, hypofibrinolysis, or a high level of lipoprotein(a) in such children may open avenues for pharmacological preventive therapy to reduce thrombophilia, stimulate fibrinolysis, or lower the level of lipoprotein(a), potentially ameliorating the Legg-Perthes disease process.
Legg-Perthes disease is a pediatric disorder similar to idiopathic osteonecrosis of the femoral head in adults, with considerable morbidity, and it often necessitates operative intervention3,7,11-13,19,26,27. One study showed that when venous drainage of the neck of the femur was obstructed in dogs, osteonecrosis resembling that seen in Legg-Perthes disease developed22. It has been postulated that Legg-Perthes disease may be caused by intravascular thrombosis due to reduced fibrinolysis14. We previously reported that five of eight patients with Legg-Perthes disease had a coagulation abnormality11. Four patients had deficiency of the antithrombotic protein C or S (two had a low level of protein C, one had a familial low level of protein C, and one had a familial low level of protein S), and one patient had hypofibrinolysis11. Our earlier observations of hypofibrinolysis or deficiency of antithrombotic protein C or S in five of eight patients who had Legg-Perthes disease11 motivated us to perform a systematic study of the roles of hypofibrinolysis and thrombophilia in a larger number of patients who had Legg-Perthes disease. Forty-four children were included in the present study. Conditions associated with an increased tendency for thrombosis (intravascular thrombi) are considered as thrombophilic or hypercoagulable states, while impairment of intravascular lysis of clots is called hypofibrinolysis5. Normally, there is a balance between thrombosis and fibrinolysis1,4-12,15,18,20,28-32. When there are heritable or acquired excesses of plasminogen activator inhibitor-1, the major intrinsic inhibitor of fibrinolysis, then tissue-plasminogen activator activity, the major stimulator of fibrinolysis, is reduced, leading to reduced lysis of clots (hypofibrinolysis)5,7,8,10-12,29,31. Thrombosis may be caused by heritable or acquired deficiencies of the vitamin-K-dependent antithrombotic factors5, such as proteins C and S. Protein C, synthesized in the liver, is present in the plasma in the inactive state as a proenzyme5. Once activated by thrombin, protein C forms a complex with free protein S, its cofactor. The activated protein C then neutralizes two prothrombotic factors (Va and VIIIa), thus preventing additional formation of thrombin5. When levels of protein C are low (usually less than 65 per cent of normal), factors Va and VIIIa are relatively uninhibited, increasing the likelihood of thrombosis5. Protein-S deficiency produces an increased tendency for thrombosis similar to that of protein-C deficiency5. Familial protein-C and S deficiencies (autosomal dominant traits28,30) are rare, with estimates of heterozygote prevalence ranging widely from less than one in 200,000 (as reported by McKee23), to one in 16,000 (as reported by Broekmans et al.6), to one in 200 to 300 (as reported by Miletich et al.25). Even in patients who have thrombophlebitis, familial protein-C and S deficiencies are rare, accounting for less than 5 per cent of cases4,15,18,28-30. Reported estimates of the prevalence of Legg-Perthes disease have ranged from one in 1200 (as reported by Molloy and MacMahon26), to one in 1400 (as reported by Gray et al.13), to one in 4750 (as reported by Harper et al.19), to one in 12,500 (as reported by Barker et al.3). By chance alone, the estimated likelihood of a child having Legg-Perthes disease and concurrent heterozygous protein-C deficiency is one in 325,000 (as we reported previously11). Since hypofibrinolysis, low levels of proteins C and S, and a high level of lipoprotein(a) play major pathophysiological roles in adult idiopathic and secondary osteonecrosis7,10,12,31, our goal was to assess these roles in forty-four children who had Legg-Perthes disease.
Children Who Had Legg-Perthes Disease After the patient had fasted overnight, we measured fibrinolytic activity7,8,12; the levels of protein C, protein S, C4b-binding protein, antithrombin III11,17,21, lipids8, and lipoprotein(a)2; and the prothrombin time. A lipoprotein(a) level of 0.25 gram per liter or more was considered high. We recorded the age at which the Legg-Perthes disease was first diagnosed as well as any diseases or drugs that could produce secondary osteonecrosis7,10-12,24. We systematically sought evidence of current infection (which might have raised the level of C4b-binding protein with a decrease in the level of free protein S), malabsorption, obstructive jaundice, other liver disease, or antibiotic therapy that may have produced vitamin-K deficiency. A detailed family history was obtained. Thirty-three of the children who had Legg-Perthes disease had a low level of protein C or S, a high level of lipoprotein(a), or a low level of stimulated fibrinolysis, and vigorous efforts were made to obtain samples from all of their living first-degree relatives and, when possible, second and third-degree relatives, to determine whether these disorders were familial.
Controls
Measurement of Fibrinolytic Activity and Levels of Protein C, Protein S, and Antithrombin III The concentrations of all factors in healthy individuals and the lower and upper limits of the concentrations (reference range) have been determined statistically from a normal, or so-called gaussian, distribution with use of the middle 80, 90, or 95 per cent of values. Tissue-plasminogen activator activity, the major stimulator of fibrinolysis, was measured by chromogenic assay (Biopool Spectrolyse/Fibrin; Biopool, Umea, Sweden)8. The within-day coefficient of variation was 7.7 per cent and the between-day coefficient of variation was 15.2 per cent. The bottom-to-top decile reference range for stimulated tissue-plasminogen activator activity that included 80 per cent of the thirty controls was 2.33 to 9.57 international units per milliliter, which is comparable with that in adults (2.28 to 11.30 international units per milliliter)11. The reference range for stimulated tissue-plasminogen activator activity that included 90 per cent of the thirty controls was 2.19 to 17.03 international units per milliliter. Plasminogen activator-inhibitor activity, the major inhibitor of fibrinolysis, was measured by chromogenic assay (Biopool Spectrolyse pL; Biopool)8. The within-day coefficient of variation was 5.6 per cent, and the between-day coefficient of variation was 7.7 per cent. The bottom-to-top decile reference range for basal plasminogen activator-inhibitor activity that included 80 per cent of the thirty controls was 5.5 to 19.6 units per milliliter, similar to that in adults (5.2 to 19.9 units per milliliter)12. The reference range for plasminogen activator-inhibitor activity that included 90 per cent of the thirty controls was 4.3 to 24.0 units per milliliter. For the analysis of the levels of antithrombin III, protein C, and protein S, plasma samples from thirty-three healthy, normal adults were pooled and were used for standard dilution curves by making serial dilutions of the pooled normal plasma. The optical density reading for the undiluted pooled normal plasma is defined as 100 per cent of normal. The optical density readings of plasma levels of antithrombin III, protein C, and protein S in the thirty control children and the forty-four patients who had Legg-Perthes disease were read against these dilution curves to determine their concentrations, with the unit of measurement being the percentage of normal. The protein-C level was measured by enzyme-linked immunosorbent assay (Asserachrom Protein C; Diagnostica Stago, distributed by American Bioproducts, Parsippany, New Jersey). The within-day coefficients of variation for low (53 per cent of normal) and normal (92 per cent of normal) protein-C plasma pools were 6.7 and 3.6 per cent, respectively, and the between-day coefficients of variation were 10.7 and 7.6 per cent, respectively. The bottom-to-top decile reference range for adults is 70 to 140 per cent of normal. The bottom-to-top decile reference range that included 80 per cent of the thirty controls was 70 to 106 per cent of normal, the reference range that included 90 per cent of the controls was 65 to 114 per cent of normal, and the reference range that included 95 per cent of the controls was 54 to 114 per cent of normal. Depending on the reference interval used, protein-C deficiency was identified when the protein-C level in a child who had Legg-Perthes disease was less than 70, 65, and 54 per cent of normal, respectively. The protein-S level was measured by enzyme-linked immunosorbent assay (Asserchrom Protein S; American Bioproducts). The within-day coefficients of variation for low (42 per cent of normal) and normal (87 per cent of normal) protein-S plasma pools were 8.2 and 7.2 per cent, respectively, and the between-day coefficients of variation were 8.9 and 10.7 per cent, respectively. The bottom-to-top decile reference range for adults is 70 to 140 per cent of normal. The bottom-to-top decile reference range that included 80 per cent of the thirty controls was 76 to 134 per cent of normal, the reference range that included 90 per cent of the controls was 76 to 150 per cent of normal, and the reference range that included 95 per cent of the controls was 43 to 151 per cent of normal. Depending on the reference interval used, protein-S deficiency was identified when the protein-S level in a child who had Legg-Perthes disease was less than 76, 76, and 43 per cent of normal, respectively. The level of antithrombin III was measured with use of chromogenic assay (Dade antithrombin III; Baxter Healthcare, Miami, Florida). The bottom-to-top decile reference range for adults is 80 to 120 per cent of normal. Antithrombin-III deficiency is recognized when the measurement is in the bottom decile for normal (less than 80 per cent of normal). The level of C4b-binding protein was quantitated by latex immunoassay (Liatest C4b-BP; American Bioproducts). Free protein-S and protein-E activity assays were not performed.
We chose not to display post-occlusion values for fibrinogen, plasminogen, and
Statistical Methods
Children Who Had Legg-Perthes Disease
With regard to the age at the onset of the disease, sex, race, or the percentage who had unilateral or bilateral disease, these forty-four children did not differ significantly (p Only eleven (25 per cent) of the forty-four children who had Legg-Perthes disease were entirely normal in that they had normal levels of lipoprotein(a), protein C, and protein S; normal stimulated tissue-plasminogen activator activity; and normal plasminogen activator-inhibitor activity. In the thirty-three families in which the proband had a low level of protein C or S, hypofibrinolysis, or a high level of lipoprotein(a) (thought to increase the risk for Legg-Perthes disease11), sixty-one children (including the thirty-three probands) were tested. Fifty-two (85 per cent) of them had a low level of protein C or S, hypofibrinolysis, or a high level of lipoprotein(a); thirty-three (63 per cent) of these fifty-two children had Legg-Perthes disease.
Family Studies
Deficiency in Antithrombotic Factors C and S Although the mean age (and standard deviation) at the time of the diagnosis of Legg-Perthes disease was 5.8 ± 2.7 years (range, two to fourteen years), the patients were first seen by us at a mean age of 10.1 ± 4.4 years (range, two to twenty-four years), which is comparable with the mean age of the controls (10.8 ± 4.9 years). The protein-C level and the lower limit of the normal range increase with age in childhood1, being lowest at the ages of one to five years and more closely approximating adult values by the ages of eleven to sixteen years1. Of the nineteen children with Legg-Perthes disease whom we identified as having a low level of protein C, seven were more than eleven years old when they were studied and ten were six to ten years old (Table I). Although the protein-S level also increases with age in childhood, it does not differ significantly from that in adulthood1. Two of the four children who had a low level of protein S were sixteen years old at the time of the study, one was six years old, and one was five years old (Table II).
Of the forty-four children who had Legg-Perthes disease, twenty-three had thrombophilia (nineteen had a low level of protein C and four had a low level of protein S), seven had a high level of lipoprotein(a), and three had hypofibrinolysis (low stimulated tissue-plasminogen activator activity and a normal level of lipoprotein[a]) (Tables I, II, III through IV). At least one first-degree relative of eleven (C1 through C11) of the nineteen probands who had a low level of protein C also had a low level of protein C (Table I and Fig. 1). Consequently, previously undiagnosed familial protein-C deficiency was identified in these individuals. Conversely, both parents and at least one brother of five (C12 and C14 through C17) of the remaining eight probands had a normal level of protein C (Table I). None of these five probands had a sister.
Of the eight probands (C12 through C19) who had a low level of protein C and in whose families protein-C deficiency could not be demonstrated (Table I), four (C13, C14, C17, and C18) are of particular interest as they were very young (three years old or less) when the Legg-Perthes disease was first diagnosed. Four probands had a low level of protein S (Table II). At least one relative of one (S1) of these four probands also had a low level of protein S (Table II and Fig. 1). Of the three probands (S2, S3, and S4) who had a low level of protein S and in whose families familial protein-S deficiency could not be demonstrated, two (S2 and S4) were very young (four years old or less) when the Legg-Perthes disease was first diagnosed.
High Levels of Lipoprotein(a) or Hypofibrinolysis, or Both
Three probands had a normal level of lipoprotein(a) and low stimulated tissue-plasminogen activator activity (Table III). One of these probands (H1), along with her father, also had high plasminogen activator-inhibitor activity, indicating hereditary hypofibrinolysis (Fig. 5).
Race, Sex, Age at Onset, and Nature of Legg-Perthes Disease
Familial Aggregation of Legg-Perthes Disease, Thrombophlebitis, Premature Myocardial Infarction, and Stroke At least one first-degree relative of two of the four probands who had protein-S deficiency had thrombophlebitis, and in two families at least one member had had a myocardial infarction or stroke before the age of sixty-five years. A maternal uncle in one family (the kindred of L3) of the six probands who had a familial high level of lipoprotein(a) had Legg-Perthes disease (Fig. 3). Thrombophlebitis was diagnosed in at least one member in two of the families, and at least one member in five of the families had had a myocardial infarction or stroke before the age of sixty-five years. One relative of one of the three probands who had hypofibrinolysis had Legg-Perthes disease and three other relatives had adult idiopathic osteonecrosis (two paternal aunts, one paternal uncle, and one maternal aunt were affected). At least one member in two of the families had had a myocardial infarction or stroke before the age of sixty-five years. In one of these two families, all eight brothers and sisters of the maternal grandfather of the proband had had a myocardial infarction before the age of sixty-five years. Over-all, four members of the twenty-four families just mentioned had abnormalities of coagulation, which increase the risk of osteonecrosis; four families had a history of Legg-Perthes disease; and three members of one family had adult idiopathic osteonecrosis. In aggregate, 58 per cent (sixty-seven) of the 116 adult members in families with familial hypofibrinolysis, thrombophilia, or a high level of lipoprotein(a) had had a myocardial infarction or stroke before the age of sixty-five years. This high rate was comparable with that (50 per cent9) in adult first-degree relatives of 432 hyperlipidemic patients at high risk for myocardial infarction and stroke.
We7,10,12 and others31 have recently shown that heritable, high plasminogen activator-inhibitor activity with consequent hypofibrinolysis in adults is associated with idiopathic osteonecrosis. We studied thirty adults who had osteonecrosis of the hip (twelve of whom had idiopathic osteonecrosis and eighteen of whom had secondary osteonecrosis)12. Nine of the twelve patients who had idiopathic osteonecrosis had very high plasminogen activator-inhibitor activity, with consequent hypofibrinolysis. The remaining three patients had normal plasminogen activator-inhibitor activity and normal fibrinolysis. These three patients and fourteen of the eighteen patients who had secondary osteonecrosis had a high level of lipoprotein(a). We speculated that hypofibrinolysis mediated by high plasminogen activator-inhibitor activity or a high level of lipoprotein(a) could cause inadequate lysis of venous thrombi in the head of the femur, obstruction of osseous venous drainage, and venous hypertension of bone leading to osteonecrosis7,10,12. In our recent study of eight children who had Legg-Perthes disease11, we found that three had protein-C deficiency (it was familial in one), one had familial protein-S deficiency, and one had hypofibrinolysis. In the present study, we analyzed the data regarding proteins C and S with two different values to identify the lower limit of normal (less than the fifth or tenth percentile) from normal age-matched controls, and with either approach many of the children who had Legg-Perthes disease had lower-than-normal values for protein C or S. Conventionally4,6-8,10-12,25,28-31, the fifth or tenth percentile, not the 2.5th percentile1, has been used as the lower normal limit to identify low levels of proteins C and S. If, however, we had used the 2.5th percentile, as Andrew et al.1 did, for a lower limit, five of eleven children whom we identified as having familial protein-C deficiency would still have been identified as having a low level, while six whose values might be considered borderline would have been recategorized1 as having a low-normal level. However, with the use of any lower normal limit for proteins C and S, many affected children and families would still have been identified. With this caveat1,32 in mind, of the forty-four children who had Legg-Perthes disease, eleven (25 per cent) were shown by extensive family studies to have familial protein-C deficiency and one (2 per cent) had familial protein-S deficiency. Our finding11 that 25 to 27 per cent of children who have Legg-Perthes disease have familial protein-C or S deficiency is much higher than would be suggested by chance alone (one in 325,000)6,11,13,23,25,26 and strongly implies causality, particularly since the children in our study were chosen solely on the basis of their having Legg-Perthes disease. Familial occurrence of Legg-Perthes disease has been described previously, although the etiology of this heritability was not known16. We speculate that familial protein-C or S deficiency, hypofibrinolysis, or a high level of lipoprotein(a), as in the current study, account in part for the familial occurrence of Legg-Perthes disease. Four of the eight children in whom the low level of protein C could not be demonstrated as being familial, and two of the three in whom the low level of protein S could not be demonstrated as being familial, were three years old or less when the Legg-Perthes disease was first diagnosed. We speculate that these children may have had age-related1 low protein-C or S levels superimposed on the intrinsic low protein-C or S levels, with the aggregate very low levels resulting in Legg-Perthes disease. A low level of protein C or S, hypofibrinolysis, or a high level of lipoprotein(a) may facilitate thrombotic venous occlusion of the femur, leading to venous hypertension in the femoral head and consequently to Legg-Perthes disease in children11 and osteonecrosis in adults7,10,12,31. Except for the osteonecrosis, other clinical manifestations of thrombi (such as thrombophlebitis and pulmonary emboli) were not revealed in the thirty-three children with Legg-Perthes disease who had a low level of protein C or S, a high level of lipoprotein(a), or hypofibrinolysis. It is unclear why some family members who have protein-C or S deficiency or others who have a high level of lipoprotein(a) and hypofibrinolysis display different manifestations of thrombosis, such as Legg-Perthes disease, thrombophlebitis, premature myocardial infarction, and stroke. In four families, we found a total of four other members who had Legg-Perthes disease, and in one family, we found three adults who had idiopathic osteonecrosis of the femoral head. In our study of twelve adults who had idiopathic osteonecrosis, the apparent etiological disorder in three was a high level of lipoprotein(a)12. By virtue of its structural similarity to plasminogen, lipoprotein(a) in high levels should inhibit fibrinolysis; nevertheless, in vivo studies in humans have failed to demonstrate this effect8. In the closed space of the femur, hypofibrinolytic activity mediated by a high level of lipoprotein(a) might contribute to venous obstruction, as in adult idiopathic osteonecrosis7,12. Interestingly, in the six families with familial high levels of lipoprotein(a), five of the six probands had hypofibrinolysis and three of the five had familial hypofibrinolysis. On the basis of the observations presented here, when Legg-Perthes disease develops in a child, the levels of protein C, protein S, and lipoprotein(a); plasminogen activator-inhibitor activity; and stimulated tissue-plasminogen activator activity should be measured. Early diagnosis of thrombophilia, hypofibrinolysis, or a high level of lipoprotein(a) in children who have Legg-Perthes disease may open avenues for pharmacological preventive therapy12 (stimulation of fibrinolysis or reduction of thrombophilia) that has the potential to ameliorate the process of the Legg-Perthes disease.
*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were Jewish Hospital Medical Research Council Grants 733 and 2733.
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Abstract
Introduction
2-antiplasmin since they are primarily affected by hemoconcentration and are not released from the endothelium in response to increased vascular transmural pressure
0.09) from the sixty-eight children with Legg-Perthes disease who did not respond to the invitation to participate in the study. Thus, there was no self-selection bias on the part of the forty-four participants. 
ski, M.; Stolecka, D.; Kandzierski, G.; and |and |Szponar, M.: Ischemia of the femoral head in Perthes' disease: is the cause intra- or extravascular?. J. Pediat. Orthop., 9: 160-162, 1989.
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