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Acute bullous purpura associated with hyperhomocysteinemia and antiphospholipid antibodies
Acute bullous purpura associated with hyperhomocysteinemia and antiphospholipid antibodies Ludovic Martin, MD, Pierre Armingaud, MD, Voichita Georgescu, MD, Fre´de´ric Maıˆtre, MD, Eric Legac, MD, and Eric Este`ve, MD Orle´ans, France We describe a female patient with an acute purpuric and bullous eruption mainly affecting the lower aspect of the legs. Skin biopsy specimens demonstrated microvascular occlusions with fibrin thrombi but no dermal inflammation. Intermediate hyperhomocysteinemia and transient antiphospholipid antibodies were evidenced as factors of thrombophilia. The responsibility of the latter in the onset of the cutaneous lesions is discussed. (J Am Acad Dermatol 2003;49:S161-3.)
H
omocysteine (Hcy) is a sulfur-containing amino acid derived from the demethylation of dietary methionin. Increased plasma levels of Hcy (hyperhomocysteinemia) are independently associated with an increased risk of atherothrombotic events (coronary heart disease and stroke)1,2 and, to a lesser extent, deep venous thrombosis.2-4 Cutaneous changes of microvascular origin have rarely been reported with hyperhomocysteinemia.5,6 We describe a female patient with hyperhomocysteinemia who had acute bullous purpura. The cutaneous lesions seemed to be precipitated by the transient presence of antiphospholipid antibodies. To our knowledge, such an observation has never previously been reported.
CASE REPORT A 73-year-old Caucasian woman was referred for evaluation of a recent extensive, purpuric, and hemorrhagic skin eruption. Examination demonstrated bullous purpura on the lower aspect of the limbs (Fig 1), and petechiae on the dorsal aspect of both hands and the nose (Fig 2). Bullae were the consequence of epidermal detachment. There was no mucosal involvement. The patient was overall in good condition and had no fever. A biopsy was performed of a leg lesion. The epidermis was microscopically unremarkable, but examination of the papillary dermal microvasculature demonstrated segmental intravascular hyalinized material evocative of cryoglobulinemia or fibrin thrombi (Fig 3). The hypodermal vasculature was not involved. Vasculitis and leukocytoclasia were consistently absent despite serial examination of sections. Interstitial edema and infiltrate were moderate and direct immunofluorescence produced negative findings. Routine laboratory tests, including platelet count, prothrombin time, and activated partial thromboplastin time, were all within normal limits. There was no cryoglobulinemia. This supplement is made possible through an unrestricted educational grant from Stiefel Laboratories to the American Academy of Dermatology. From the Departments of Dermatology, Pathology, and Hematology, CHR d’Orle´ans. Reprint requests: Ludovic Martin, MD, Service de Dermatologie, Hoˆpital Porte-Madeleine, BP 2439, CHR d’Orle´ans, 45032 Orle´ans Cedex, France. E-mail: [email protected]. Copyright © 2003 by the American Academy of Dermatology, Inc. 0190-9622/2003/$30.00 ⫹ 0 doi:10.1067/mjd.2003.340
Fig 1. Extensive bullous purpura on leg.
Fig 2. Purpuric patches on nose.
No infectious or drug causes were identified. The patient, a son, and 2 sisters had experienced several episodes of deep venous thrombosis and pulmonary embolism, prompting us to look for inherited or acquired thrombophilia in view of the cutaneous microvascular thromboses in the patient. Informed consent was obtained and DNA was extracted according to usual procedures. DNA analysis for factor V Leiden and factor II (prothrombin) G20210A mutation, and N5,N10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism were performed according to the reverse hybridization procedure (prothrombin, factor V, and MTHFR gene mutation assay, ViennaLab, Vienna, Aus-
S161
S162 Martin et al
Fig 3. Photomicrograph of skin biopsy specimen (leg). Dermal microvascular occlusion with fibrin thrombi without inflammation. (Hematoxylin-eosin stain; original magnification ⫻200).
tria). Levels of protein C, free protein S, and antithrombin were normal. Factor VIII was high (292%; N ⬍ 150%). Resistance to activated protein C, factor V Leiden, and factor II G20210A mutations were absent. Fibrinolysis tests (tissue plasminogen activator, plasminogen activator inhibitor, plasminogen) were unremarkable. Intermediate fasting hyperhomocysteinemia (40 mol/L; N ⬍ 15) was demonstrated twice. MTHFR C677T was found in a homozygous state. Antinuclear antibodies, anti-DNA antibodies, and antineutrophil cytoplasmic autoantibodies were absent. Serum complement was normal. Antiphospholipid antibodies were positive with anticardiolipin IgG ⫽ 50 U/mL (N ⬍ 10) (Cardiolisa, Biomedical Diagnostics, Marne-la-Valle´ e, France). Their origin was not demonstrated. Antibodies to 2 glycoprotein-1 (2-lisa, Biomedical Diagnostics) were negative. Thrombophilia was also investigated in the son and he was found to have moderate hyperhomocysteinemia. The skin condition improved in 2 weeks with prednisone therapy (1/2 mg/ kg/d), as antiphospholipid antibodies disappeared in the serum. Hcy-lowering treatment was started with folinic acid (5 mg/d), yielding normal Hcy concentrations in a few weeks. There was no relapse after 1.5 years of follow-up. Reticulate scars persisted but there was no livedo reticularis or atrophie blanche and no leg ulcers. An additional skin biopsy specimen was obtained 3 months after healing, revealing that the dermal microvasculature was normal.
DISCUSSION The 2 clinical diagnoses first suspected in this patient were leukocytoclastic vasculitis and cryoglobulinemia type I. Leukocytoclastic vasculitis was definitely ruled out by pathology findings. Occlusive material in the dermal vessels was pathologically consistent with cryoglobulinemia but this paraprotein was absent in the serum and finally it became clear that the cutaneous
J AM ACAD DERMATOL AUGUST 2003
changes were related to acute segmental occlusion of the dermal microvasculature with fibrin thrombi. Intermediate hyperhomocysteinemia and antiphospholipid antibodies were demonstrated when we addressed the possibility of thrombophilia in this patient. We assume that in this case both were involved in the pathogenesis of vascular thrombi and the resulting purpura. Hcy is present in various forms in the plasma and the term “hyperhomocysteinemia” refers to any raised plasma level of all these forms above the 90th or 95th percentile values in the general population.1,2 The plasma level of Hcy is demonstrated by fasting measurement, and normal values commonly range from 5 to 15 mol/L.7 Higher fasting values are usually referred to as moderate (16-30 mol/L), intermediate (31-100 mol/L), and severe (⬎100 mol/L). Hyperhomocysteinemia occurs in various conditions such as chronic renal failure, folate or vitamin B12 (and to a lesser extent vitamin B6) deficiencies, malignancy, and drug intake.1,2 Genetic variants may also influence Hcy metabolism and induce hyperhomocysteinemia. Homozygous deficiency of cystathionine -synthase (with considerably increased fasting Hcy) is responsible for the majority of cases of the rare condition referred to as homocystinuria.8 However, the most common enzyme defect associated with moderately or intermediately raised Hcy is a point polymorphism (C677T) in the MTHFR gene.9 The resulting enzyme, so-called thermolabile variant, is present in a homozygous state in 5% to 15% of white patients and is associated with half normal activity. Our patient had intermediate hyperhomocysteinemia probably because she was a homozygous carrier of the MTHFR C677T variant. Vitamin B deficiencies and other conditions were not demonstrated as cofactors. The mechanisms by which Hcy (even moderate levels) leads to vascular events are numerous, including both vascular injury and promotion of thrombosis. Indeed, it has been shown that Hcy is directly toxic to the vasculature, alters endothelium-dependent vasodilatation and thromboresistance, promotes platelet aggregation and adhesion to the subendothelium, enhances proliferation of vascular smooth muscle cells, activates coagulation, and eventually inhibits the fibrinolytic system.10-14 One major restriction is that most key abnormalities have not been specifically evidenced in the skin microvasculature and cannot be extrapolated to the present situation. Most results have been demonstrated in large arterial vessels or with in vitro experiments. Cutaneous changes have rarely been related to hyperhomocysteinemia. In a small series of female patients, Gibson et al5 reported that an association might exist between plasma Hcy levels and livedoid vasculitis, a cutaneous noninflammatory thrombotic vasculopathy involving dermal arterioles. The authors showed that fasting Hcy was higher in patients with livedoid vasculitis (8.7 ⫾ 3.1 mol/L; N ⫽ 19) than in nonagematched control patients (7 ⫾ 2.9; N ⫽ 46) (P ⫽ .03). Pathology findings in livedoid vasculitis are identical to those observed in our patient but features of cutaneous microvascular thrombotic occlusion may be seen in various prothrombotic conditions with skin manifestations. Didier et al6 described a female patient with plantar nodules and forefoot skin necrosis. The pathology studies revealed subcutaneous thrombophlebitis in a nodule, and postmethionine-loading hyperhomocysteinemia was demonstrated. Hcy might, therefore, also be deleterious for the cutaneous microvasculature. However, hyperhomocysteinemia was probably long-standing in our patient and its single and direct responsibility in the pathogenesis of microvascular occlusion is questionable. In addition, purpura is absent in most patients with intermediate hyperhomocysteinemia and even in homo-
Martin et al S163
J AM ACAD DERMATOL VOLUME 49, NUMBER 2
cystinuria. This is in keeping with the doubtful single responsibility of Hcy. It seemed in the episode of severe purpura of our patient that antiphospholipid antibodies triggered thrombogenesis and precipitated the lesions. The transient presence of antiphospholipid antibodies is usually related to infection. In this situation antiphospholipid antibodies are not usually thrombogenic. However, the resulting additive thrombogenic effect of several simultaneous causes of thrombophilia is well known for large venous or arterial vessels15,16 and may be discussed in terms of the skin vasculature. To our knowledge, no previous study has addressed the possible contribution of the combination of 2 or more types of thrombophilia to thrombotic occlusion of dermal microvessels and skin changes. REFERENCES 1. Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998;338:1042-50. 2. Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407-13. 3. Den Heijer M, Blom HJ, Gerrits WB, Rosendaal FR, Haak HL, Wijermans PW, et al. Is hyperhomocysteinaemia a risk factor for recurrent venous thrombosis? Lancet 1995;345:882-5. 4. Arruda VR, Von Zuben PM, Chiaparini LC, Annichino-Bizzacchi JM, Costa FF. The mutation Ala677 Val in the methylene tetrahydrofolate reductase gene: a risk factor for arterial disease and venous thrombosis. Thromb Haemost 1997;77:818-21. 5. Gibson GE, Li H, Pittelkow MR. Homocysteinemia and livedoid vasculitis. J Am Acad Dermatol 1999;40:279-81. 6. Didier AF, Touraud JP, Collet E, Dalac S, Becker F, Lambert D. Ne´crose
7.
8. 9.
10. 11. 12.
13.
14.
15. 16.
cutane´e distale, une e´tiologie inhabituelle: l’hyperhomocyste´ine´mie [in French]. Ann Dermatol Venereol 1999;126:822-5. Selhub J, Jacques PF, Rosenberg IH, Rogers G, Bowman BA, Gunter EW, et al. Serum total homocysteine concentrations in the third national health and nutrition examination survey (1991-1994): population reference ranges and contribution of vitamin status to high serum concentrations. Ann Intern Med 1999;131:331-9. Online Mendelian Inheritance in Man. Available at: http://www.ncbi.nlm.nih.gov/Omim. Accessed June 2, 2001. Kang SS, Zhou J, Wong PWK, Kowasilyn J, Strokosch G. Intermediate homocysteinemia: a thermolabile variant of methylene tetrahydrofolate reductase. Am J Hum Genet 1988;43:414-21. Bellamy MF, McDowell IF. Putative mechanisms for vascular damage by homocysteine. J Inherit Metab Dis 1997;20:307-15. Tyagi SC. Homocysteine redox receptor and regulation of extracellular matrix components in vascular cells. Am J Physiol 1998;274:C396-405. De Jong SC, van den Berg M, Rauwerda JA, Stehouwer CD. Hyperhomocysteinemia and atherothrombotic disease. Semin Thromb Hemost 1998;24:381-5. Khajuria A, Houston DS. Induction of monocyte tissue factor expression by homocysteine: a possible mechanism for thrombosis. Blood 2000; 96:966-72. Coppola A, Davi G, De Stefano V, Mancini FP, Cerbone AM, Di Minno G. Homocysteine, coagulation, platelet function, and thrombosis. Semin Thromb Hemost 2000;26:243-54. Seligsohn U, Zivelin A. Thrombophilia as a multigenic disorder. Thromb Haemost 1997;78:297-301. Bertina RM. Molecular risk factors for thrombosis. Thromb Haemost 1999;82:601-9.
H
omocysteine (Hcy) is a sulfur-containing amino acid derived from the demethylation of dietary methionin. Increased plasma levels of Hcy (hyperhomocysteinemia) are independently associated with an increased risk of atherothrombotic events (coronary heart disease and stroke)1,2 and, to a lesser extent, deep venous thrombosis.2-4 Cutaneous changes of microvascular origin have rarely been reported with hyperhomocysteinemia.5,6 We describe a female patient with hyperhomocysteinemia who had acute bullous purpura. The cutaneous lesions seemed to be precipitated by the transient presence of antiphospholipid antibodies. To our knowledge, such an observation has never previously been reported.
CASE REPORT A 73-year-old Caucasian woman was referred for evaluation of a recent extensive, purpuric, and hemorrhagic skin eruption. Examination demonstrated bullous purpura on the lower aspect of the limbs (Fig 1), and petechiae on the dorsal aspect of both hands and the nose (Fig 2). Bullae were the consequence of epidermal detachment. There was no mucosal involvement. The patient was overall in good condition and had no fever. A biopsy was performed of a leg lesion. The epidermis was microscopically unremarkable, but examination of the papillary dermal microvasculature demonstrated segmental intravascular hyalinized material evocative of cryoglobulinemia or fibrin thrombi (Fig 3). The hypodermal vasculature was not involved. Vasculitis and leukocytoclasia were consistently absent despite serial examination of sections. Interstitial edema and infiltrate were moderate and direct immunofluorescence produced negative findings. Routine laboratory tests, including platelet count, prothrombin time, and activated partial thromboplastin time, were all within normal limits. There was no cryoglobulinemia. This supplement is made possible through an unrestricted educational grant from Stiefel Laboratories to the American Academy of Dermatology. From the Departments of Dermatology, Pathology, and Hematology, CHR d’Orle´ans. Reprint requests: Ludovic Martin, MD, Service de Dermatologie, Hoˆpital Porte-Madeleine, BP 2439, CHR d’Orle´ans, 45032 Orle´ans Cedex, France. E-mail: [email protected]. Copyright © 2003 by the American Academy of Dermatology, Inc. 0190-9622/2003/$30.00 ⫹ 0 doi:10.1067/mjd.2003.340
Fig 1. Extensive bullous purpura on leg.
Fig 2. Purpuric patches on nose.
No infectious or drug causes were identified. The patient, a son, and 2 sisters had experienced several episodes of deep venous thrombosis and pulmonary embolism, prompting us to look for inherited or acquired thrombophilia in view of the cutaneous microvascular thromboses in the patient. Informed consent was obtained and DNA was extracted according to usual procedures. DNA analysis for factor V Leiden and factor II (prothrombin) G20210A mutation, and N5,N10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism were performed according to the reverse hybridization procedure (prothrombin, factor V, and MTHFR gene mutation assay, ViennaLab, Vienna, Aus-
S161
S162 Martin et al
Fig 3. Photomicrograph of skin biopsy specimen (leg). Dermal microvascular occlusion with fibrin thrombi without inflammation. (Hematoxylin-eosin stain; original magnification ⫻200).
tria). Levels of protein C, free protein S, and antithrombin were normal. Factor VIII was high (292%; N ⬍ 150%). Resistance to activated protein C, factor V Leiden, and factor II G20210A mutations were absent. Fibrinolysis tests (tissue plasminogen activator, plasminogen activator inhibitor, plasminogen) were unremarkable. Intermediate fasting hyperhomocysteinemia (40 mol/L; N ⬍ 15) was demonstrated twice. MTHFR C677T was found in a homozygous state. Antinuclear antibodies, anti-DNA antibodies, and antineutrophil cytoplasmic autoantibodies were absent. Serum complement was normal. Antiphospholipid antibodies were positive with anticardiolipin IgG ⫽ 50 U/mL (N ⬍ 10) (Cardiolisa, Biomedical Diagnostics, Marne-la-Valle´ e, France). Their origin was not demonstrated. Antibodies to 2 glycoprotein-1 (2-lisa, Biomedical Diagnostics) were negative. Thrombophilia was also investigated in the son and he was found to have moderate hyperhomocysteinemia. The skin condition improved in 2 weeks with prednisone therapy (1/2 mg/ kg/d), as antiphospholipid antibodies disappeared in the serum. Hcy-lowering treatment was started with folinic acid (5 mg/d), yielding normal Hcy concentrations in a few weeks. There was no relapse after 1.5 years of follow-up. Reticulate scars persisted but there was no livedo reticularis or atrophie blanche and no leg ulcers. An additional skin biopsy specimen was obtained 3 months after healing, revealing that the dermal microvasculature was normal.
DISCUSSION The 2 clinical diagnoses first suspected in this patient were leukocytoclastic vasculitis and cryoglobulinemia type I. Leukocytoclastic vasculitis was definitely ruled out by pathology findings. Occlusive material in the dermal vessels was pathologically consistent with cryoglobulinemia but this paraprotein was absent in the serum and finally it became clear that the cutaneous
J AM ACAD DERMATOL AUGUST 2003
changes were related to acute segmental occlusion of the dermal microvasculature with fibrin thrombi. Intermediate hyperhomocysteinemia and antiphospholipid antibodies were demonstrated when we addressed the possibility of thrombophilia in this patient. We assume that in this case both were involved in the pathogenesis of vascular thrombi and the resulting purpura. Hcy is present in various forms in the plasma and the term “hyperhomocysteinemia” refers to any raised plasma level of all these forms above the 90th or 95th percentile values in the general population.1,2 The plasma level of Hcy is demonstrated by fasting measurement, and normal values commonly range from 5 to 15 mol/L.7 Higher fasting values are usually referred to as moderate (16-30 mol/L), intermediate (31-100 mol/L), and severe (⬎100 mol/L). Hyperhomocysteinemia occurs in various conditions such as chronic renal failure, folate or vitamin B12 (and to a lesser extent vitamin B6) deficiencies, malignancy, and drug intake.1,2 Genetic variants may also influence Hcy metabolism and induce hyperhomocysteinemia. Homozygous deficiency of cystathionine -synthase (with considerably increased fasting Hcy) is responsible for the majority of cases of the rare condition referred to as homocystinuria.8 However, the most common enzyme defect associated with moderately or intermediately raised Hcy is a point polymorphism (C677T) in the MTHFR gene.9 The resulting enzyme, so-called thermolabile variant, is present in a homozygous state in 5% to 15% of white patients and is associated with half normal activity. Our patient had intermediate hyperhomocysteinemia probably because she was a homozygous carrier of the MTHFR C677T variant. Vitamin B deficiencies and other conditions were not demonstrated as cofactors. The mechanisms by which Hcy (even moderate levels) leads to vascular events are numerous, including both vascular injury and promotion of thrombosis. Indeed, it has been shown that Hcy is directly toxic to the vasculature, alters endothelium-dependent vasodilatation and thromboresistance, promotes platelet aggregation and adhesion to the subendothelium, enhances proliferation of vascular smooth muscle cells, activates coagulation, and eventually inhibits the fibrinolytic system.10-14 One major restriction is that most key abnormalities have not been specifically evidenced in the skin microvasculature and cannot be extrapolated to the present situation. Most results have been demonstrated in large arterial vessels or with in vitro experiments. Cutaneous changes have rarely been related to hyperhomocysteinemia. In a small series of female patients, Gibson et al5 reported that an association might exist between plasma Hcy levels and livedoid vasculitis, a cutaneous noninflammatory thrombotic vasculopathy involving dermal arterioles. The authors showed that fasting Hcy was higher in patients with livedoid vasculitis (8.7 ⫾ 3.1 mol/L; N ⫽ 19) than in nonagematched control patients (7 ⫾ 2.9; N ⫽ 46) (P ⫽ .03). Pathology findings in livedoid vasculitis are identical to those observed in our patient but features of cutaneous microvascular thrombotic occlusion may be seen in various prothrombotic conditions with skin manifestations. Didier et al6 described a female patient with plantar nodules and forefoot skin necrosis. The pathology studies revealed subcutaneous thrombophlebitis in a nodule, and postmethionine-loading hyperhomocysteinemia was demonstrated. Hcy might, therefore, also be deleterious for the cutaneous microvasculature. However, hyperhomocysteinemia was probably long-standing in our patient and its single and direct responsibility in the pathogenesis of microvascular occlusion is questionable. In addition, purpura is absent in most patients with intermediate hyperhomocysteinemia and even in homo-
Martin et al S163
J AM ACAD DERMATOL VOLUME 49, NUMBER 2
cystinuria. This is in keeping with the doubtful single responsibility of Hcy. It seemed in the episode of severe purpura of our patient that antiphospholipid antibodies triggered thrombogenesis and precipitated the lesions. The transient presence of antiphospholipid antibodies is usually related to infection. In this situation antiphospholipid antibodies are not usually thrombogenic. However, the resulting additive thrombogenic effect of several simultaneous causes of thrombophilia is well known for large venous or arterial vessels15,16 and may be discussed in terms of the skin vasculature. To our knowledge, no previous study has addressed the possible contribution of the combination of 2 or more types of thrombophilia to thrombotic occlusion of dermal microvessels and skin changes. REFERENCES 1. Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998;338:1042-50. 2. Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407-13. 3. Den Heijer M, Blom HJ, Gerrits WB, Rosendaal FR, Haak HL, Wijermans PW, et al. Is hyperhomocysteinaemia a risk factor for recurrent venous thrombosis? Lancet 1995;345:882-5. 4. Arruda VR, Von Zuben PM, Chiaparini LC, Annichino-Bizzacchi JM, Costa FF. The mutation Ala677 Val in the methylene tetrahydrofolate reductase gene: a risk factor for arterial disease and venous thrombosis. Thromb Haemost 1997;77:818-21. 5. Gibson GE, Li H, Pittelkow MR. Homocysteinemia and livedoid vasculitis. J Am Acad Dermatol 1999;40:279-81. 6. Didier AF, Touraud JP, Collet E, Dalac S, Becker F, Lambert D. Ne´crose
7.
8. 9.
10. 11. 12.
13.
14.
15. 16.
cutane´e distale, une e´tiologie inhabituelle: l’hyperhomocyste´ine´mie [in French]. Ann Dermatol Venereol 1999;126:822-5. Selhub J, Jacques PF, Rosenberg IH, Rogers G, Bowman BA, Gunter EW, et al. Serum total homocysteine concentrations in the third national health and nutrition examination survey (1991-1994): population reference ranges and contribution of vitamin status to high serum concentrations. Ann Intern Med 1999;131:331-9. Online Mendelian Inheritance in Man. Available at: http://www.ncbi.nlm.nih.gov/Omim. Accessed June 2, 2001. Kang SS, Zhou J, Wong PWK, Kowasilyn J, Strokosch G. Intermediate homocysteinemia: a thermolabile variant of methylene tetrahydrofolate reductase. Am J Hum Genet 1988;43:414-21. Bellamy MF, McDowell IF. Putative mechanisms for vascular damage by homocysteine. J Inherit Metab Dis 1997;20:307-15. Tyagi SC. Homocysteine redox receptor and regulation of extracellular matrix components in vascular cells. Am J Physiol 1998;274:C396-405. De Jong SC, van den Berg M, Rauwerda JA, Stehouwer CD. Hyperhomocysteinemia and atherothrombotic disease. Semin Thromb Hemost 1998;24:381-5. Khajuria A, Houston DS. Induction of monocyte tissue factor expression by homocysteine: a possible mechanism for thrombosis. Blood 2000; 96:966-72. Coppola A, Davi G, De Stefano V, Mancini FP, Cerbone AM, Di Minno G. Homocysteine, coagulation, platelet function, and thrombosis. Semin Thromb Hemost 2000;26:243-54. Seligsohn U, Zivelin A. Thrombophilia as a multigenic disorder. Thromb Haemost 1997;78:297-301. Bertina RM. Molecular risk factors for thrombosis. Thromb Haemost 1999;82:601-9.