THROMBOPHILIA-HYPOFIBRINOLYSIS, REDUCED NITRIC OXIDE PRODUCTION, AND OSTEONECROSIS
Charles J Glueck MD, * Richard A Freiberg MD, † and Ping Wang PhD*
From the *Cholesterol Center, Jewish Hospital, Cincinnati, Ohio, and the †Cincinnati Veterans Administration Hospital, Cincinnati, Ohio
Gene mutations leading to thrombophilia, hypofibrinolysis, and reduced nitric oxide production are risk factors for osteonecrosis. The importance of the diagnosis of thrombophilia-hypofibrinolysis in idiopathic osteonecrosis lies in the potential to stop the progression of osteonecrosis when Enoxaparin is started at Ficat stages I or II before irreversible segmental collapse of the head of the femur.
RISK FACTORS FOR OSTEONECROSIS: THROMBOPHILIA, HYPOFIBRINOLYSIS, AND REDUCED NITRIC OXIDE PRODUCTION
Gene mutations leading to thrombophilia and hypofibrinolysis [1-10] and reduced nitric oxide production [11-13] associated with eNOS gene polymorphisms are risk factors for osteonecrosis (ON), . Polymorphisms are changes in the genetic code (like mutations) that occur commonly enough in the population such that they are considered a variation on normal. These may be harmful, helpful or neither to the persons who have a polymorphism. The importance of the diagnosis for thrombophilia-hypofibrinolysis in idiopathic ON lies in the potential to stop the progression of ON provided that Enoxaparin is started at Ficat stages I or II  before irreversible segmental collapse of the head of the femur . Moreover, the diagnosis of thrombophilia-hypofibrinolysis in ON is important because of associations with other venous thrombosis , for screening first degree relatives of affected probands for heritable thrombophilia-hypofibrinolysis [5, 15], and in identifying patients at high risk for deep venous thrombosis and pulmonary emboli after hip-knee replacement, for whom longer term (at least 3 months) post-operative thromboprophylaxis is warranted [16-19].
By current convention, ON of the hip is either described as “secondary” (high dose corticosteroids, alcoholism, lupus erythematosus, hip dislocation or fracture, chemotherapy, HIV-AIDS, dysbaria, estrogen replacement therapy, pregnancy, [ 1]), or “idiopathic” when there is no known etiology [2, 20-22]. In 1993, we reported on two brothers with idiopathic bilateral hip ON who were homozygous for the hypofibrinolytic 4G/4G polymorphism of the plasminogen activator inhibitor-1 gene (PAI-1 gene) and had very high levels of the PAI-1 gene product, plasminogen activator inhibitor (PAI-Fx) . This kindred  and 4 similar cases reported by Van Veldhuisen in 1993  led us to the hypothesis that ON of the hip and jaw is caused by familial or acquired hypofibrinolysis-thrombophilia [6-10, 24-35].
We  initially, and subsequently others [2, 23, 34-53] hypothesized that venous thrombosis in the femoral head leads to increased intraosseous venous pressure, and, thence, to impaired arterial flow, osseous hypoxia, and, bone death ( 1). Beyond hip ON, [2, 5-9, 23, 31-33, 36, 38-40, 42, 46-51] thrombophilia-hypofibrinolysis also appears to be pathoetiologic for Legg-Calve-Perthes disease [24, 26, 28, 29, 34, 35, 52, 53] and for ON of the jaw [25, 27, 54].
Preservation of the femoral head is the ultimate goal of treatment of ON of the hip, but, as recently summarized by Lieberman  “…development of successful strategies to treat this disease has been difficult to do because ON is associated with numerous different diseases and neither the etiology nor the natural history have been delineated clearly.” Further, as noted by Assouline-Dayan et al  “…management of osteonecrosis is primarily palliative and does not necessarily halt or retard the progression of the disease. Treatment options focus on repairing the secondary changes that develop in the femoral head and not on reversing the idiopathic pathology.”
We believe that understanding the associations of thrombophilia-hypofibrinolysis with ON opens avenues for improved treatment, with the ultimate goal of preserving the femoral head. We have shown that anticoagulation [10, 32, 33, 42] with enoxaparin [56-61] for the same duration as used in deep vein thrombosis in the leg  stops the progression of idiopathic hip ON  in cases with thrombophilia-hypofibrinolysis, decreasing the frequency of total hip replacement .
THROMBOPHILIA-HYPOFIBRINOLYSIS AND OSTEONECROSIS
The PCR and serologic measurements which are currently thought to be important in investigating the etiology of ON are summarized in Table 1. All of the PCR and serologic measurements displayed in Table 1 should be available through major national laboratory networks, and in many large general hospital laboratories, excepting the plasminogen activator inhibitor-1 4G5G mutation and the eNOS T786C polymorphism which are available through Molecular Diagnostic Laboratories of Cincinnati, Ohio (firstname.lastname@example.org). These laboratory results should be easily interpreted by Hematologists, and, after minimal experience, by orthopedists.
Plasminogen activator inhibitor activity and plasminogen activator inhibitor-1 gene 4G4G mutation
G1691A Factor V Leiden and G20210A Prothrombin gene mutations
The G1691A Factor V Leiden and G20210A prothrombin gene mutations, the most common mutations associated with thrombophilia, are associated both with osteonecrosis of the hip and knee  and with Legg-Calve’-Perthes disease [28, 35], . Bjorkman et al  reported a higher prevalence of the V Leiden mutation in idiopathic (but not secondary) ON cases than in the Swedish population. In patients given long term (>6 years) anticoagulation with Coumadin, who presented with Ficat I-II idiopathic ON, the Factor V Leiden mutation, and other venous thrombotic events, our unpublished clinical experience is that the progression of ON may be stopped.
Elevated homocysteine and MTHFR C677T-A1298C mutations
The methylenetetrahydrofolate reductase (MTHFR) gene mutation and high levels of the gene product, homocysteine, are associated with ON () as we  and others [3, 69] have previously described. Homozygosity for the MTHFR C677T mutation or compound C677T/A1298C mutations is usually associated with abnormalities in homocysteine metabolism, even if serum homocysteine is normal, and, like high homocysteine, should be treated with folic acid 5 mg, Vitamin B6 100 mg, and Vitamin B12 2000 mcg/day . Our unpublished clinical experience in patients with idiopathic ON, the MTHFR gene mutations, and high homocysteine is that long term (>6 years) normalization of homocysteine appears to stop progression of Ficat stages I-II osteonecrosis. Kutlar et al  have reported that the MTHFR C677T mutation is associated with ON which develops in patients with sickle cell disease.
High Factor VIII (s 1,3) is inherited as a dominant trait closely associated with thrombosis [71-75]. In our recent studies , high levels of Factor VIII were more common in men with idiopathic ON than in controls (23% vs 2%, p = .0041), and Factor VIII was also higher in men with secondary ON than in controls (29% vs 2%, p = .0014). Our finding that high Factor VIII was associated with idiopathic and secondary ON is congruent with reported associations of familial thrombophilias (Factor V Leiden, Prothrombin gene, Factor XI, low protein C, low protein S [31, 51]) with idiopathic ON in adults , and with associations of familial thrombophilia (Factor V Leiden , low protein S ) with Legg-Calve-Perthes disease in children.
Familial high Lipoprotein (a) [Lp(a)] ( 3) has previously been reported as a risk factor for idiopathic ON . Lp(a) has been associated with familial clustering of bone marrow edema of the hip in 3 female siblings . Bone marrow edema of the hip may be a forme fruste of ON .
High levels of anticardiolipin antibodies IgG and IgM ( 3) have been associated with ON [3, 38, 39] particularly in osteonecrosis secondary to lupus erythematosus. The antiphospholipid antibody syndrome is also associated with development of ON . Nagasawa et al  have reported preliminary but promising effects of anticoagulation with warfarin in newly diagnosed patients with systemic lupus erythematosus and, 2) the lupus anticoagulant in reducing development of ON and decreasing the development of symptomatic ON.
Exogenous estrogens and pregnancy
Previous studies have demonstrated an association of ON with exogenous estrogens or pregnancy [7, 9, 27, 31]. This suggests that an estrogen-mediated increase in the tendency to thrombosis may promote development of osteonecrosis particularly in the presence of underlying thrombophilia. When estrogen-induced thrombophilia  is superimposed on familial thrombophilia-hypofibrinolysis, we believe that intraosseous thrombosis is promoted, facilitating development of ON [9, 27, 30, 31, 33].
eNOS T-786C polymorphism, 4a intron 4 polymorphism, nitric oxide production, and osteonecrosis
Beyond thrombophilia-hypofibrinolysis, another genetic mechanism for development of idiopathic ON appears to involve mutations in the endothelial nitric oxide synthase (eNOS) gene that controls nitric oxide production, . Koo et al  have recently reported that the frequency of the 27-bp repeat polymorphism in intron 4, a non coding part of the eNOS gene, was higher in Koreans with idiopathic ON of the hip than in controls (9% vs 2.4%, p =.03). Since this 4a polymorphism is associated with reduced synthesis of eNOS, Koo et al  concluded, “…carrier state of 4a allele in intron 4 might be a genetic risk factor of FHON (femoral head osteonecrosis), … and could provide insight into the protective role of nitric oxide in the pathogenesis of FHON.” In the study by Koo et al , the frequency of the intron 4 polymorphism did not differ between cases with ON of the hip secondary to steroids or alcohol and controls . Working for 11 months before the report by Koo et al  on a different eNOS polymorphism (T-786C) and its relationship to ON and to Buerger’s syndrome , we have recently  reported a strong association between the T-786C eNOS polymorphism and idiopathic ON of the hip. Impairment of nitric oxide production by the T-786C eNOS polymorphism [83, 84] promotes vasoconstriction and platelet activation-recruitment-aggregation  and diminishes angiogenesis [86, 87] and bone formation .
ENOXAPARIN THERAPY IN PATIENTS WITH THROMBOPHILIA-HYPOFIBRINOLYSIS AND FICAT STAGES I-II OSTEONECROSIS
As previously described, in our prospective enoxaparin treatment study , patients were selected by ≥1 thrombophilic-hypofibrinolytic disorder and Ficat  Stages I-II ON of ≥ 1 hip(s). Enoxaparin (60 mg/day) was given for the first 12 weeks of the study. Sixteen patients (25 hips) with ≥ 1 thrombophilia-hypofibrinolysis and Ficat Stages I-II ON of ≥1 hip were initially followed for ≥ 2 years . Our follow-up of yearly AP and frog-leg lateral x-rays is now 5-7 years (average 6 years). In our current study, as in the original report , maintenance of Ficat Stages I-II versus progression to Stages III-IV or total hip replacement was the significant outcome, 4. Based on intent to treat, 17 of 25 hips (68%) have remained Ficat Stages I-II at average 6-year follow-up. Compared with untreated historical controls (approximately 20% 2 year hip survival) [89-91], survival of 17/25 hips (68%), indicates that the original 12 week enoxaparin thromboprophylaxis produced lasting benefit in idiopathic ON in patients with heritable thrombophilia and hypofibrinolysis . Enoxaparin seemingly prevents progression of idiopathic hip ON, causing a decrease in the incidence of total hip replacement .
ON of the femoral head has been shown to be caused by familial or acquired thrombophilia-hypofibrinolysis [2, 5-10, 23, 24, 31-33, 36, 40, 42, 46-51, 53]. Thrombophilia-hypofibrinolysis has also been associated with Legg-Calve-Perthes disease in many [7, 24, 26, 28, 29, 34, 35, 52, 53] but not all  reports. To evaluate the pathophysiology of osteonecrosis and to open avenues to medical treatment, we suggest PCR studies of the G1691A Factor V Leiden, G20210A Prothrombin, C677T-A1298C methylenetetrahydrofolate reductase genes associated with thrombophilia and the plasminogen activator inhibitor-1 4G4G mutation associated with hypofibrinolysis. We also suggest PCR studies of the T786C polymorphism and the 4a polymorphism of the eNOS gene in idiopathic ON, . It is also important to carry out serologic studies of thrombophilia (Factor VIII, Factor XI, proteins C, S (total and free), antithrombin III, homocysteine, anticardiolipin antibody IgG and IgM, lupus anticoagulant), and serologic studies of hypofibrinolysis (Lp[a], PAI-Fx), Table 1.
Experimental models of ON [43, 45, 93-96] and of Legg-Calve-Perthes disease  implicate venous occlusion as a precipitating event, with subsequent increased intraosseous pressure, reduced arterial inflow, ischemia, and infarction, 1. We believe that thrombophilia-hypofibrinolysis, by promoting deep osseous venous thrombosis, initiates this cascade. [10, 32, 33, 35-37, 42-44, 96]. In patients with thrombophilia-hypofibrinolysis and Ficat Stages I-II ON, Enoxaparin seemingly prevents progression of idiopathic hip ON, causing a decrease in the incidence of total hip replacement .
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