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Activated protein C resistance and anticardiolipin antibodies in patients with venous leg ulcers
Activated protein C resistance and anticardiolipin antibodies in patients with venous leg ulcers Douglas Grossman, MD, PhD,a Peter W. Heald, MD,a Chao Wang, MS,b and Henry M. Rinder, MDb New Haven, Connecticut Background: Hypercoagulable states, which include the presence of anticardiolipin antibodies (ACAs), have been associated with skin ulceration. Resistance to activated protein C (APe), resulting from the factor V Leiden mutation, is a common risk factor for venous thrombosis. Its prevalence among patients with venous leg ulcers is not known. Objective: Our purpose was to determine the prevalence of factor V Leiden and ACA in patients with venous leg ulceration. Methods: Twenty-nine consecutive patients with venous leg ulcers were studied. Resistance to APC was first determined by functional assay based on the partial thromboplastin time. Patients with an abnormally low APC ratio were then subjected to molecular analysis for confirmation of factor V Leiden. Measurements of ACA were performed by enzymelinked immunosorbent assay. Results: APC resistance was detected in 11 of 26 patients. However, only 2 of these 1I patients (7.7% overall) were found to be heterozygous for the factor V Leiden mutation. ACA was present in neither patient with the Leiden mutation but was found in 6 of 21 patients tested (29% overall). Conclusion: The factor V Leiden mutation, unlike ACA, may not be more prevalent in patients with venous leg ulcers than in the general population. Our results emphasize the importance of molecular analysis for factor V Leiden in patients with APC resistance. (J Am Acad Dermatol 1997;37:409-13.)
Resistance to activated protein C (APC) occurs in 2% to 5% of the U.S. population and represents the most common heritable cause of hypercoagulability.I In essentially all patients, APC resistance is caused by a single point mutation at nucleotide position 1691 (Leiden mutation) in the gene encoding coagulation factor V, resulting in substitution of glutamine for arginine at position 506 in the APC-cleavage site on factor V.2,3 Heterozygosity for factor V Leiden is associated with a fivefold to tenfold increased risk of venous thrombosis," Most leg ulcers occur in patients with venous insufficiency.f However, most patients with venous insufficiency do not develop leg ulcers, which suggests that additional factors may be involved in their pathogenesis. In many patients From the Departments ofDermatology' and Laboratory Medicine," Yale University School ofMedicine. Accepted for publication May 12, 1997. Reprints not available from the authors. Copyright © 1997 by the American Academy of Dermatology, Inc. 0190-9622/97/$5.00 + 0 16/1/83205
there is evidence of prior venous thrombosis.l Hypercoagulable states such as protein C and S deficiency," antithrombin III deficiency," tissue plasminogen activator imbalance.f-? and antiphospholipid antibody syndrornel" have been associated with both venous thrombosis and skin ulceration. In fact, previous studies found anticardiolipin antibodies (ACAs) in almost 40% of patients with venous leg ulcers.II Recent reports suggest that APC resistance may be an important risk factor for cutaneous necrosiS l2 and leg ulceration.':' Recently, Munkvad and Jorgensen!" reported thatAPC resistance, based on the functional assay alone, occurred in 26% of leg ulcer patients. We have subsequently examined patients with venous leg ulcers for the presence of APC resistance, determined by both functional and molecular assays, and for the presence of ACA. METHODS Patients From October 1995 to August 1996, 29 consecutive patients with venous leg ulcers were studied (Table I).
409
Journal ofthe American Academy of Dermatology September 1997
410 Grossman et al. Table I. Patients included in this study Patient No.
Age (yr)/Race/Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
48/W/M 6l/W/M 5l/W/M 801BIM 70/W/F 33/W1F 78/W1F 68/W1F 37/W/M 3l/W/F 87/W1F 63/W/F 54/W/M 78/W1F 55/W1F 67/W1F 72/W/F 34/B/M 69/W/F 92/W/M 38/W1F 57/W1F 46/W/M 541B1F 63/W1F 77/W1F 47/W1F 72/W1F 58/W1F
Additional clinical information
Rheumatoid arthritis, h/o phlebitis h/o phlebitis
Low PIT
Decreased protein C (51% normal)
Scleroderma
Leldenf
IgM#, IgG## Negative
1.49 1.78 2.08 2.09 2.12 2.13 2.16 2.17 2.19 2.22 2.29 2.36 2.37 2.40 2.42 2.46 2.53 2.53 2.54 2.57 2.61 2.77 2.78 2.85 2.86 2.87
Absent Heterozyg Heterozyg Absent Absent Absent Absent Absent Absent Absent Absent
IgG### Negative
HIV infection
h/o phlebitis
APC ratio"
Negative Negative IgM# Negative IgM## Negative Negative Negative IgM# Negative
Rheumatoid arthritis Rheumatoid arthritis, low PIT
Ulcerative colitis
ACA*
Negative Negative Negative Negative Negative IgG# Negative
Absent
*Anticardiolipin antibodies, isotype detected, reported as negative, low positive," moderate.f" or high positive### as described in the "Methods" section. t APC ratios, values ~ 2.30 considered normal. *Leiden mutation, reported absent or present (heterozyg), based on molecular analysis.
Patients with other causes for leg ulceration such as pyoderma gangrenosum, cryoglobulinemia, thalassemia, polycythemia vera, necrobiosis lipoidica, sarcoid, morphea, and factitial dermatitis were excluded. None of the study patients had an elevated partial thromboplastin time at baseline, and none was receiving heparin or warfarin. All patients improved with standard compression dressings (Unna boots). Informed consent was obtained from all patients, and this study was approved by the Yale University Human Investigation Committee.
Determination of APC resistance Blood was collected into sodium citrate-containing glass tubes, and platelet-free plasma was isolated by centrifugation. The APC ratio was derived from partial thromboplastin time values obtained with and without addition of a standard amount of APC, as described
previously. 15 On the basis of control studies of healthy subjects, APC ratios more than 2.30 are normal and ratios less than 2.30 are consistent with functional APC resistance. Studies in our laboratory have determined that although this functional assay is highly sensitive, it is not specific; all patients with the factor V Leiden mutation have an APC ratio of less than 2.30, but we found that the Leiden mutation was present in only 32% of patients with an APC ratio of less than 2.30.
Determination of ACA Blood was collected into glass tubes without anticoagulant and allowed to clot. Serum was immediately isolated and analyzed for the presence of ACA by enzyme-linked immunoabsorbent assay as described. 16 ACA titers of IgM and IgG were reported as negative (<10 MPLlGPL units), low positive (10 to 20 units), moderate (20 to 80 units), or high positive (>80 units).
Journal of the American Academy of Dermatology Volume 37, Number 3, Part I
Determination of the factor V Leiden mutation Blood was collected into potassium EDTA-eontaining glass tubes and mononuclear leukocytes (I x 106) were isolated by Ficoll-Hypaque density gradient centrifugation. Genomic DNA was prepared by standard procedures. The factor V gene exon 10 was amplified by polymerase chain reaction as described,17·18 using sense and antisense oligonucleotide primers corresponding to nucleotides 1581-1602 and nucleotides 127-146 in intron 10, respectively. The 276 bp amplified fragment was then digested with the restriction endonuclease Mnl I under standard conditions and electrophoresed through a 3% agarose gel. The amplified fragment from wild-type individuals contains two Mnl 1 restriction sites yielding three fragments of 163 bp, 76 bp, and 37 bp. In the mutant V Leiden, a G to A transition at nucleotide 1691 disrupts one of the Mnl I sites, resulting in 200 bp and 76 bp fragments after Mnl I digestion.
Grossman et al.
411
ABC D ../276 -200 '163 Fig. 1. Molecular analysis for the factor V Leiden mutation. PCR products from a negative control (A), patient 2 in this study (B), and a positive control for homozygous factor V Leiden (C) were subjected to digestion with Mnl 1 as described in the "Methods" section. Undigested PCR product from the negative control is shown in lane D. Complete endonuclease digestion is confirmed by the presence of only the 163 bp fragment in lane A. The presence of the 163 bp and 200 bp fragments in lane B confirm the heterozygosity for factor V Leiden in patient 2.
RESULTS
The APC ratios in this group of patients ranged from 1.49 to 2.87 (Table I). Fifteen patients (58% of 26 tested) were normal and 11 (42%) had an APC ratio of less than 2.30, suggesting functional APC resistance. Molecular analysis revealed that only 2 of these 11 patients (7.7% overall) had the factor V Leiden mutation, and both patients were heterozygous (Fig. 1). Both patients also had a history of thrombophlebitis. Of interest, the patient with the lowest APC ratio (patient 1) did not have the Leiden mutation. ACAs were detected in 6 of 21 patients tested (29% overall) (Table I). Three patients had a low positive titer and one patient had a moderate titer of IgM ACA. With respect to IgG titers, one patient each had low-positive, moderate, and high-positive titers, respectively. The patient with moderate IgG titer and low positive IgM titer (patient 1) also had the lowest APC ratio and was negative for the Leiden mutation, as already noted. There was no clear predominance of IgM compared with IgG isotypes of ACA. Both patients with the Leiden mutation were negative for ACA. We observed no differences in presentation or response to treatment between patients with and without APC resistance or the factor V Leiden mutation. In addition, the presence of ACA did not appear to have any prognostic significance in this group of patients.
DISCUSSION
We found that APC resistance caused by the Leiden mutation of factor V occurred in 7.7% of patients with venous leg ulcers. These results are based on a small number of patients seen at a tertiary care and referral center; larger studies will be required to determine whether factor V Leiden is more prevalent in this group of patients than the 2% to 5% reported in the general population. I This study found a similar prevalence of APC resistance in patients with venous ulcers as in other studies 14; however, previous investigators relied on the functional assay alone. In our study, patients demonstrating APC resistance by functional assay were further subjected to molecular analysis for confirmation of factor V Leiden. Several investigators have reported that this functional assay for APC resistance has a relatively poor positive predictive value for factor V Leiden,'? and we found that 9 of 11 patients with APC resistance did not have factor V Leiden. Thus molecular analysis for factor V Leiden is required for confirmation of APC resistance. We have no explanation for the low APC ratios, particularly the extremely low ratio found in one patient (patient 1), in the absence of the Leiden mutation. Although a confounding role for antiphospholipid antibodies in APC resistance has been suggested.I? these antibodies have not been
412 Grossman et al.
shown to interfere with the functional assay for APC resistance. Moreover, half our patients with ACA had a normal APC ratio. It is possible that the low APC ratios reflect an undefined inhibitor of APC function in plasma or, more likely, relatively increased sensitivity of the functional assay to fluctuations in factor levels necessary for the prothrombinase complex (factors V and VIII). In fact, several reports indicate that functional APC resistance assays performed using dilutions of patient plasma with factor V-deficient plasma have a much higher positive predictive value for the factor V Leiden mutation" Finally, there may be unknown factor V mutations that result in APC resistance. We have sequenced the region of factor V containing the major protein C cleavage site in patient #1 and found no mutations (M. Rose, D. Grossman, unpublished observation). We anticipated that APC resistance caused by factor V Leiden would prove to be more common in patients with venous leg ulcers, given its strong association with venous thrombosis. Indeed, both leg ulcer patients with factor V Leiden mutation had a history of thrombophlebitis, suggesting that the Leiden mutation is likely to occur only when both venous thrombosis and leg ulceration are present. However, the frequency of factor V Leiden I and venous insufficiency' found in normal persons suggests the importance of additional factors in the development of leg ulcers. The fact that the Leiden mutation was not associated with more severe disease or altered response to therapy in these two patients suggests that factor V Leiden may not be a significant risk factor for the development or chronicity of venous leg ulcers. We detected ACA in almost one third of our patients. There was no correlation between the presence of ACA and APC resistance because patients with ACA had a spectrum of APC ratios. The relatively high prevalence of ACA in our patients is comparable to that reported by Barbaud et al.!' In that study, only the IgG isotype of ACA was examined; we found both IgM and IgG isotypes in this study, although neither appeared to be predominant. In contrast, Alarcon-Segovia et a1. 22 found that in patients with systemic lupus erythematosus, the presence of leg ulcers was most strongly associated with IgM ACA rather than the IgG or IgA isotypes. Thus the presence of ACA, rather than APC resistance, appears to be the more prevalent
Journal of the American Academy of Dermatology September 1997
hypercoagulable factor in patients with venous leg ulcers. Although venous leg ulcers may result from multiple causes, an evaluation of the patient with leg ulcers should include screening tests for ACA and, if there is history of thrombophlebitis, APC resistance as well. If the latter is identified, the patient should be further examined by molecular analysis for the factor V Leiden mutation. REFERENCES 1. Nichols WL, Heit JA. Activated protein C resistance and thrombosis. Mayo Clin Proc 1996;71:897-8. 2. Greengard JS, Sun X, Xu X, et al. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 1994;343: 1362-3. 3. Zoller B, Svensson PJ, He X, et al. Identification of the same factor V gene mutation in 47 out of 50 thrombosisprone families with inherited resistance to activated protein C. J Clin Invest 1994;94:2521-4. 4. Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994;330:517-21. 5. Ouahes N, Phillips TJ. Leg ulcers. Curr Probl Dermatol 1995;7:109-42. 6. Falanga V, Bontempo FA, Eaglstein WHo Protein C and protein S plasma levels in patients with lipodermatosclerosis and venous ulceration. Arch Dermatol 1990;126:1995-7. 7. Bazex J, Freour E, Adoue D, et al. Leg ulcers: vascular and thrombotic factors. Ann Dermatol Venereol 1979;106:565-7. 8. Pizzo SV, Fuchs HE, Doman KA, et aI. Release of tissue plasminogen activator and its fast-acting inhibitor in defective fibrinolysis. Arch Intern Med 1986;146:18891. 9. Margolis DJ, Kruithof EKO, Barnard M, Howe K, Lazarus GS. Fibrinolytic abnormalities in two different cutaneous manifestations of venous disease. J Am Acad Dermatol 1996;34:204-8. 10. Asherson RA, Cervera R. Antiphospholipid syndrome. J Invest Dermatol 1993;I00(Suppl):21 S-7S. II. Barbaud AM, Gobert B, Reichert S, et al. Anticardiolipin antibodies and ulcerations of the leg. J Am Acad Dermatol 1994;31:670-1. 12. Perkins W, Downie I, Keefe M, et al. Cutaneous necrosis in pregnancy secondary to activated protein C resistance in hereditary angioedema. J R Soc Med 1995;88:229-30. 13. Peus D, Schmiedeberg S, Pier A, et al. Coagulation factor V gene mutation associated with activated protein C resistance leading to recurrent thrombosis, leg ulcers, and lymphedema: successful treatment with intermittent compression. J Am Acad Dermatol 1996;35:306-9. 14. Munkvad S, Jorgensen M. Resistance to activated protein C: a common anticoagulant deficiency in patients with venous leg ulceration. Br J Dermatol 1996;134:2968. 15. Griffin JH, Evatt B, Wideman C, et aI. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood 1993;82: 1989-93. 16. Harris EN, Gharavi AE, Patel SP, et al. Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin Exp Immunol 1987;68:215-22.
Journal of the American Academy of Dermatology Volume 37, Number 3, Part I
Grossman et al. 413
17. Jenny RJ, Pittman DD, Toole JJ, et al, Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987;84:4846-50. 18. Bertina RM, Koeleman BPC, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64-7. 19. Dahlback B. Resistance to activated protein C, the Arg 506 to GIn mutation in the factor V gene, and venous thrombosis: functional tests and DNA-based assays, pros and cons. Thromb Haernost 1995;73:739-42. 20. Hampton KK, Preston FE, Greaves M. Resistance to activated protein C [letter]. N Engl J Med 1994;331: 130.
21. Alhenc-Gelas M, Aillaud MF, Bonvarlet MN, et a1. Specificity of an assay based on factor V-deplered plasma in patients carrying the Arg506G1n mutation. Thromb Haemost 1996;75:976-7. 22. Alarcon-Segovia D, Deleze M, Oria CV, et al. Antiphospholipid antibodies and the anti phospholipid syndrome in systemic lupus erythematosus: a prospective analysis of 500 consecutive patients. Medicine (Baltimore) 1989;68:353-65.
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Resistance to activated protein C (APC) occurs in 2% to 5% of the U.S. population and represents the most common heritable cause of hypercoagulability.I In essentially all patients, APC resistance is caused by a single point mutation at nucleotide position 1691 (Leiden mutation) in the gene encoding coagulation factor V, resulting in substitution of glutamine for arginine at position 506 in the APC-cleavage site on factor V.2,3 Heterozygosity for factor V Leiden is associated with a fivefold to tenfold increased risk of venous thrombosis," Most leg ulcers occur in patients with venous insufficiency.f However, most patients with venous insufficiency do not develop leg ulcers, which suggests that additional factors may be involved in their pathogenesis. In many patients From the Departments ofDermatology' and Laboratory Medicine," Yale University School ofMedicine. Accepted for publication May 12, 1997. Reprints not available from the authors. Copyright © 1997 by the American Academy of Dermatology, Inc. 0190-9622/97/$5.00 + 0 16/1/83205
there is evidence of prior venous thrombosis.l Hypercoagulable states such as protein C and S deficiency," antithrombin III deficiency," tissue plasminogen activator imbalance.f-? and antiphospholipid antibody syndrornel" have been associated with both venous thrombosis and skin ulceration. In fact, previous studies found anticardiolipin antibodies (ACAs) in almost 40% of patients with venous leg ulcers.II Recent reports suggest that APC resistance may be an important risk factor for cutaneous necrosiS l2 and leg ulceration.':' Recently, Munkvad and Jorgensen!" reported thatAPC resistance, based on the functional assay alone, occurred in 26% of leg ulcer patients. We have subsequently examined patients with venous leg ulcers for the presence of APC resistance, determined by both functional and molecular assays, and for the presence of ACA. METHODS Patients From October 1995 to August 1996, 29 consecutive patients with venous leg ulcers were studied (Table I).
409
Journal ofthe American Academy of Dermatology September 1997
410 Grossman et al. Table I. Patients included in this study Patient No.
Age (yr)/Race/Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
48/W/M 6l/W/M 5l/W/M 801BIM 70/W/F 33/W1F 78/W1F 68/W1F 37/W/M 3l/W/F 87/W1F 63/W/F 54/W/M 78/W1F 55/W1F 67/W1F 72/W/F 34/B/M 69/W/F 92/W/M 38/W1F 57/W1F 46/W/M 541B1F 63/W1F 77/W1F 47/W1F 72/W1F 58/W1F
Additional clinical information
Rheumatoid arthritis, h/o phlebitis h/o phlebitis
Low PIT
Decreased protein C (51% normal)
Scleroderma
Leldenf
IgM#, IgG## Negative
1.49 1.78 2.08 2.09 2.12 2.13 2.16 2.17 2.19 2.22 2.29 2.36 2.37 2.40 2.42 2.46 2.53 2.53 2.54 2.57 2.61 2.77 2.78 2.85 2.86 2.87
Absent Heterozyg Heterozyg Absent Absent Absent Absent Absent Absent Absent Absent
IgG### Negative
HIV infection
h/o phlebitis
APC ratio"
Negative Negative IgM# Negative IgM## Negative Negative Negative IgM# Negative
Rheumatoid arthritis Rheumatoid arthritis, low PIT
Ulcerative colitis
ACA*
Negative Negative Negative Negative Negative IgG# Negative
Absent
*Anticardiolipin antibodies, isotype detected, reported as negative, low positive," moderate.f" or high positive### as described in the "Methods" section. t APC ratios, values ~ 2.30 considered normal. *Leiden mutation, reported absent or present (heterozyg), based on molecular analysis.
Patients with other causes for leg ulceration such as pyoderma gangrenosum, cryoglobulinemia, thalassemia, polycythemia vera, necrobiosis lipoidica, sarcoid, morphea, and factitial dermatitis were excluded. None of the study patients had an elevated partial thromboplastin time at baseline, and none was receiving heparin or warfarin. All patients improved with standard compression dressings (Unna boots). Informed consent was obtained from all patients, and this study was approved by the Yale University Human Investigation Committee.
Determination of APC resistance Blood was collected into sodium citrate-containing glass tubes, and platelet-free plasma was isolated by centrifugation. The APC ratio was derived from partial thromboplastin time values obtained with and without addition of a standard amount of APC, as described
previously. 15 On the basis of control studies of healthy subjects, APC ratios more than 2.30 are normal and ratios less than 2.30 are consistent with functional APC resistance. Studies in our laboratory have determined that although this functional assay is highly sensitive, it is not specific; all patients with the factor V Leiden mutation have an APC ratio of less than 2.30, but we found that the Leiden mutation was present in only 32% of patients with an APC ratio of less than 2.30.
Determination of ACA Blood was collected into glass tubes without anticoagulant and allowed to clot. Serum was immediately isolated and analyzed for the presence of ACA by enzyme-linked immunoabsorbent assay as described. 16 ACA titers of IgM and IgG were reported as negative (<10 MPLlGPL units), low positive (10 to 20 units), moderate (20 to 80 units), or high positive (>80 units).
Journal of the American Academy of Dermatology Volume 37, Number 3, Part I
Determination of the factor V Leiden mutation Blood was collected into potassium EDTA-eontaining glass tubes and mononuclear leukocytes (I x 106) were isolated by Ficoll-Hypaque density gradient centrifugation. Genomic DNA was prepared by standard procedures. The factor V gene exon 10 was amplified by polymerase chain reaction as described,17·18 using sense and antisense oligonucleotide primers corresponding to nucleotides 1581-1602 and nucleotides 127-146 in intron 10, respectively. The 276 bp amplified fragment was then digested with the restriction endonuclease Mnl I under standard conditions and electrophoresed through a 3% agarose gel. The amplified fragment from wild-type individuals contains two Mnl 1 restriction sites yielding three fragments of 163 bp, 76 bp, and 37 bp. In the mutant V Leiden, a G to A transition at nucleotide 1691 disrupts one of the Mnl I sites, resulting in 200 bp and 76 bp fragments after Mnl I digestion.
Grossman et al.
411
ABC D ../276 -200 '163 Fig. 1. Molecular analysis for the factor V Leiden mutation. PCR products from a negative control (A), patient 2 in this study (B), and a positive control for homozygous factor V Leiden (C) were subjected to digestion with Mnl 1 as described in the "Methods" section. Undigested PCR product from the negative control is shown in lane D. Complete endonuclease digestion is confirmed by the presence of only the 163 bp fragment in lane A. The presence of the 163 bp and 200 bp fragments in lane B confirm the heterozygosity for factor V Leiden in patient 2.
RESULTS
The APC ratios in this group of patients ranged from 1.49 to 2.87 (Table I). Fifteen patients (58% of 26 tested) were normal and 11 (42%) had an APC ratio of less than 2.30, suggesting functional APC resistance. Molecular analysis revealed that only 2 of these 11 patients (7.7% overall) had the factor V Leiden mutation, and both patients were heterozygous (Fig. 1). Both patients also had a history of thrombophlebitis. Of interest, the patient with the lowest APC ratio (patient 1) did not have the Leiden mutation. ACAs were detected in 6 of 21 patients tested (29% overall) (Table I). Three patients had a low positive titer and one patient had a moderate titer of IgM ACA. With respect to IgG titers, one patient each had low-positive, moderate, and high-positive titers, respectively. The patient with moderate IgG titer and low positive IgM titer (patient 1) also had the lowest APC ratio and was negative for the Leiden mutation, as already noted. There was no clear predominance of IgM compared with IgG isotypes of ACA. Both patients with the Leiden mutation were negative for ACA. We observed no differences in presentation or response to treatment between patients with and without APC resistance or the factor V Leiden mutation. In addition, the presence of ACA did not appear to have any prognostic significance in this group of patients.
DISCUSSION
We found that APC resistance caused by the Leiden mutation of factor V occurred in 7.7% of patients with venous leg ulcers. These results are based on a small number of patients seen at a tertiary care and referral center; larger studies will be required to determine whether factor V Leiden is more prevalent in this group of patients than the 2% to 5% reported in the general population. I This study found a similar prevalence of APC resistance in patients with venous ulcers as in other studies 14; however, previous investigators relied on the functional assay alone. In our study, patients demonstrating APC resistance by functional assay were further subjected to molecular analysis for confirmation of factor V Leiden. Several investigators have reported that this functional assay for APC resistance has a relatively poor positive predictive value for factor V Leiden,'? and we found that 9 of 11 patients with APC resistance did not have factor V Leiden. Thus molecular analysis for factor V Leiden is required for confirmation of APC resistance. We have no explanation for the low APC ratios, particularly the extremely low ratio found in one patient (patient 1), in the absence of the Leiden mutation. Although a confounding role for antiphospholipid antibodies in APC resistance has been suggested.I? these antibodies have not been
412 Grossman et al.
shown to interfere with the functional assay for APC resistance. Moreover, half our patients with ACA had a normal APC ratio. It is possible that the low APC ratios reflect an undefined inhibitor of APC function in plasma or, more likely, relatively increased sensitivity of the functional assay to fluctuations in factor levels necessary for the prothrombinase complex (factors V and VIII). In fact, several reports indicate that functional APC resistance assays performed using dilutions of patient plasma with factor V-deficient plasma have a much higher positive predictive value for the factor V Leiden mutation" Finally, there may be unknown factor V mutations that result in APC resistance. We have sequenced the region of factor V containing the major protein C cleavage site in patient #1 and found no mutations (M. Rose, D. Grossman, unpublished observation). We anticipated that APC resistance caused by factor V Leiden would prove to be more common in patients with venous leg ulcers, given its strong association with venous thrombosis. Indeed, both leg ulcer patients with factor V Leiden mutation had a history of thrombophlebitis, suggesting that the Leiden mutation is likely to occur only when both venous thrombosis and leg ulceration are present. However, the frequency of factor V Leiden I and venous insufficiency' found in normal persons suggests the importance of additional factors in the development of leg ulcers. The fact that the Leiden mutation was not associated with more severe disease or altered response to therapy in these two patients suggests that factor V Leiden may not be a significant risk factor for the development or chronicity of venous leg ulcers. We detected ACA in almost one third of our patients. There was no correlation between the presence of ACA and APC resistance because patients with ACA had a spectrum of APC ratios. The relatively high prevalence of ACA in our patients is comparable to that reported by Barbaud et al.!' In that study, only the IgG isotype of ACA was examined; we found both IgM and IgG isotypes in this study, although neither appeared to be predominant. In contrast, Alarcon-Segovia et a1. 22 found that in patients with systemic lupus erythematosus, the presence of leg ulcers was most strongly associated with IgM ACA rather than the IgG or IgA isotypes. Thus the presence of ACA, rather than APC resistance, appears to be the more prevalent
Journal of the American Academy of Dermatology September 1997
hypercoagulable factor in patients with venous leg ulcers. Although venous leg ulcers may result from multiple causes, an evaluation of the patient with leg ulcers should include screening tests for ACA and, if there is history of thrombophlebitis, APC resistance as well. If the latter is identified, the patient should be further examined by molecular analysis for the factor V Leiden mutation. REFERENCES 1. Nichols WL, Heit JA. Activated protein C resistance and thrombosis. Mayo Clin Proc 1996;71:897-8. 2. Greengard JS, Sun X, Xu X, et al. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 1994;343: 1362-3. 3. Zoller B, Svensson PJ, He X, et al. Identification of the same factor V gene mutation in 47 out of 50 thrombosisprone families with inherited resistance to activated protein C. J Clin Invest 1994;94:2521-4. 4. Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994;330:517-21. 5. Ouahes N, Phillips TJ. Leg ulcers. Curr Probl Dermatol 1995;7:109-42. 6. Falanga V, Bontempo FA, Eaglstein WHo Protein C and protein S plasma levels in patients with lipodermatosclerosis and venous ulceration. Arch Dermatol 1990;126:1995-7. 7. Bazex J, Freour E, Adoue D, et al. Leg ulcers: vascular and thrombotic factors. Ann Dermatol Venereol 1979;106:565-7. 8. Pizzo SV, Fuchs HE, Doman KA, et aI. Release of tissue plasminogen activator and its fast-acting inhibitor in defective fibrinolysis. Arch Intern Med 1986;146:18891. 9. Margolis DJ, Kruithof EKO, Barnard M, Howe K, Lazarus GS. Fibrinolytic abnormalities in two different cutaneous manifestations of venous disease. J Am Acad Dermatol 1996;34:204-8. 10. Asherson RA, Cervera R. Antiphospholipid syndrome. J Invest Dermatol 1993;I00(Suppl):21 S-7S. II. Barbaud AM, Gobert B, Reichert S, et al. Anticardiolipin antibodies and ulcerations of the leg. J Am Acad Dermatol 1994;31:670-1. 12. Perkins W, Downie I, Keefe M, et al. Cutaneous necrosis in pregnancy secondary to activated protein C resistance in hereditary angioedema. J R Soc Med 1995;88:229-30. 13. Peus D, Schmiedeberg S, Pier A, et al. Coagulation factor V gene mutation associated with activated protein C resistance leading to recurrent thrombosis, leg ulcers, and lymphedema: successful treatment with intermittent compression. J Am Acad Dermatol 1996;35:306-9. 14. Munkvad S, Jorgensen M. Resistance to activated protein C: a common anticoagulant deficiency in patients with venous leg ulceration. Br J Dermatol 1996;134:2968. 15. Griffin JH, Evatt B, Wideman C, et aI. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood 1993;82: 1989-93. 16. Harris EN, Gharavi AE, Patel SP, et al. Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin Exp Immunol 1987;68:215-22.
Journal of the American Academy of Dermatology Volume 37, Number 3, Part I
Grossman et al. 413
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