- Email: [email protected]
Degradation products of fibrin and of fibrinogen in synovial fluid and in plasma of patients with rheumatoid arthritis
Piprinofysis (1989) 3, 183-186 0 1989 Longman Group UK Ltd
0268~9499/89,WO3-0183
S10.00
Degradation Products of Fibrin and of Fibrinogen in Synovial Fluid and in Plasma of Patients with Rheumatoid Arthritis
J. Jespersen, E. J. P. Brommer, F. Haverkate, W. Nieuwenhuizen SUMMAR Y. Degradation products of fibrin and of fibrinogen, together (FDP) and separately, were measured in plasma and synovial fluid of patients with rheumatoid arthritis using specific enzyme immunoassays. Considerable amounts of fibrin degradation products (fibrin-DP) were found in the synovial fluid; only 1 case yielded some fibrinogen-derived products (fibrinogen-DP). In plasma relatively small amounts of degradation products, both fibrin-DP and fibrinogen-DP, were present. From the discrepancy between the amount and composition of degradation products in synovial fluid and in plasma we conclude that there are extra-articular sites of fibrin(ogen) breakdown in rheumatoid arthritis. The new specific methods may contribute to the understanding of the role of fibrinolysis in rheumatoid disease and in inflammation in general. KEYWORDS.
Synovial Fluids. RA. Fibrin and Fibrinogen degradation products.
PATIENTS
Fibrin deposition is a conspicious feature of joint inflammation in rheumatoid arthritis (RA), and degradation products of fibrin or fibrinogen can be detected in the synovial fluid of the inflamed joints.ls2 Several investigators have demonstrated the presence of fibrinolytic enzymes in the fluid, and this could give rise to formation of the degradation products in the joint cavity.3-5 The recent introduction of quantitative immunoassays6-* based on monoclonal antibodies specific for these degradation products has now allowed a distinction between split products of fibrin (fibrin-DP) and of fibrinogen (fibrinogen-DP) in the synovial fluid from patients with chronic rheumatoid arthritis. We attempt to answer the question of whether it is fibrin or fibrinogen which is being broken down in the joint cavity. Because the split products might dissipate into the circulating blood, we have also determined the degradation products in samples of plasma from some of the patients. In all but 1 patient only fibrin degradation products were detectable in the synovial fluid. In the circulating blood, however, both fibrinogen-DPs and Fibrin-DPs were detectable. The different composition of the split products in the two compartments suggests differences in mode or site of degradation.
AND METHODS
We studied 12 patients with long-standing RA as defined according to ARA criteria, including the presence of circulating rheuma-factors. Relevant clinical and laboratory data are given in Tables 1 and 2. Haemoglobin, total white cell counts and platelet counts were determined on Coulter S plus IV (Coulter). Fibrinogen was determined by a thrombin time assay.g The duration of the symptoms ranged from 1 to almost 30 years (median 4.5 years). At the time of investigation 4 patients (D, E, F and H) had extra-articular manifestations of the rheumatic disease. Synovial fluid obtained by routine knee puncture and collected in tubes (9 volumes) containing 1 volume of trisodium citrate (0.13 mol/l) and aprotinin lo6 KIU/l was centrifuged to remove cells and other corpuscular elements. The supernatant was kept frozen (< -20°C) until analysis. For demonstration of clottable fibrinogen, thrombin (10 NIH U/ml tinal concentration) was added to the undiluted synovial fluid. Clotting was monitored manually by means of a wire hook. Plasma was prepared from titrated blood (9 volumes collected in ice-cold tubes containing 1 volume of trisodium citrate, 0.13 mol/l, and aprotinin lo6 KIU/l) by centrifugation at 2000g for 30min at 4°C and kept frozen (< - 20°C) until analysis. Diisopropylfluorophosphate (DFP; 0.1 mol/l) 0.1 volume was added to all samples before freezing. In addition to the assessment of the total amount of
J. Jespersen, Dept. of Clinical Chemistry and Internal Medicine, Ribe County Hospital Esbjerg, Denmark, EL J. P. Brommer, F. Have&ate, W. Nieuwenbuizeo, Gaubius Institute TNO, PO Box
612,230O AP Leiden, The Netherlands. 183
184
Degradation
Products
of Fibrin and of Fibrinogen
Table 1 Clinical data of patients
with theumatoid
in Synovial
Fluid
arthritis
Patient
Sex
Born
Age at debut (y)
A
M
260440
38
Prednisolone 5 mg x 3 Imurel50 mg x 5 Dextropropoxifen 65 mg x 4
B
F
050218
59
Dimethylcystein 125 mg Brufen 400 mg x 3 Naprosyn 500 mg Doloxene 100 mg x 3
C
M
100818
60
Dextropropoxifen
D
F
210713
66
Brufen 400 mg x 3
Noduli at the hand area
E
F
021008
44
Confortid 100 mg Dextroproxifen 65 mg x 6
Myositis interstit.
F
F
150832
47
Dextropropoxifen 65 mg x 34 Brufen 400 mg x 3
Noduli, and Baker cyst
G
M
261015
50
Prednisolone 5 mg Ibuprofen 400 mg x 4 Dextropropoxifen 65 mg x 4
H
M
110733
30
Dimethylcystein Dextropropoxifen
I
F
290409
61
Paracetamol 1 g p.n. Panodril 1 g x 3 Dextropropoxifen 65 mg x 4
3
F
080421
63
Atamir 450 mg Ibuprofen 400 mg x 2-3
K
F
130535
45
Atamir 300 mg Felden 20 mg
L
M
241122
57
Imurel50 mg Ibuprofen 400 mg x 3 Dextropropoxifen 65 mg (max. x 4) Prednisolone 10 mg
Table 2 Analysis of blood and plasma in patients
RA-related
with rheumatoid
Notes
treatment
65 mg x 4
Noduli at the areas of the knee and elbow
125 mg 65 mg x 4
arthritis
Patient
Year of study
Haemoglobin (mmol/l)
Leucocytes (109/1)
Platelets (109P)
Fibrinogen Olmol/l)
ESR (arb, units)
8.2 7.1 7.9 7.0 6.4 7.7 6.5 9.1 6.1 7.3 7.1 7.8
7.5 9.2 12.6 8.8 5.5 10.2 9.3
312 266 343 392 320 463 262
K L
1981 1981 1981 1981 1981 1982 1982 1985 1985 1985 1985 1985
9.3 5.4 4.7 9.1
432 417 295 421
35.0 13.5 17.7 14.7 10.7 14.4 12.2 13.3 17.3 16.3 11.2 18.8
73 48 66 74 43 53 73 24 86 58 25 51
- = not determined.
degradation products from fibrin and fibrinogen (FDP), specific determinations of fibrinogen-DP and fibrin-DP were made in some of the samples. All three assays are sandwich EM’s (Organon Teknika, Turnhout, Belgium) based on a monoclonal, catching antibody (FDP-14) against a neoantigenic determinant in the E-fragment. FDP-14 reacts with degradation products of fibrin and of fibrinogen, but not with the intact parent molecules.* For the specific, separate
assay of fibrinogen degradation products a horseradish peroxidase-conjugated monoclonal antibody (‘Y 18’)8 reactive with covalently bound fibrinopeptide A’ is used as tagging antibody. For the specific fibrinDP assay of a horseradish peroxidase (HRP) conjugates of a monoclonal antibody elicited against the Ddimer (‘DD13’),” is used as the tagging antibody.” For the FDP assay, i.e. the total of fibrin-DP and fibrinogen-DP, either a polyclonal rabbit antifibrino-
Fibrinolysis
185
Table 3 Fibrin and fibrinogen degradation products in synovial fluid and plasma from patients with rheumatoid arthritis
Patient
Synovial fluid TDP FbDP mg/l mg/l
FgDP mg/l
A B C D E F G H I J K L
340 470 346 513 146 227 115 157 923 414 151 156
_* _* _* _* _* _* 101 61.1 522 148 118 93
Plasma TDP mg/i
FbDP mg/l
6.4 4.6 1.1 6.2 0.5 8.2
_
27.9 5.4 2.7 1.8 4.9
1.2 <0.5 1.2 <0.5 1.0
_ _
FgDP mg/l 1.1 10.5 0.8 <0.5
-=not determined. TDP = total of fibrinogen-DP and fibrin-DP; FbDP = fibrin-DP; FgDP = fibrinogen-DP. * =synovial fluid samples of these patients were not available for testing with the fibrin-DP test.
gen antibody, conjugated with HRP, or a mixture of monoclonals ‘Y18’8 and ‘DD13”O (both HRP conjugated) was used as the tagging antibody.6 These tests are not influenced by the rheumatoid factor; normal values for FDP, fibrinogen-DP and Fibrin-DP in plasma are below 0.5 mg/l. l1
RESULTS Synovial fluid was obtained from 12 patients by puncture of inflamed knee joints. The high viscosity of the fluid did not hamper the assays after dilution, as usual for plasma. Clottable fibrinogen was not detected in any of the synovial fluid samples. The other data are given in Table 3. High levels of degradation products (FDP) were present in all samples, varying from 115 to 923 mg/l. Assays of fibrin-DP and fibrinogen-DP separately in 6 patients revealed that the bulk of the degradation products consisted of fibrin degradation products; fibrinogen-DP were virtually absent from synovial fluid in all but 1 patient. The FDP assay using polyclonal antibodies as tagging antibodies instead of the mixture of two ,monoclonals confirmed the same high concentrations of FDP in the synovial fluid. Plasma samples showed lower concentrations of fibrin-DP and FDP (Table 3) but higher levels of fibrinogen-DP in comparison with the synovial fluids.
DISCUSSION Diagnostic tools, based on specific monoclonal antibodies, provided us with means to measure fibrin-DP and fibrinogen-DP separately in samples of synovial fluid from patients with rheumatoid arthritis. For
obvious reasons, no normal reference values are available. The enzyme immunoassays have been devised for the use in plasma, but they can be applied to other body fluids as well. It is not clear why the amount of FDP exceeds that of the sum of fibrin-DP and fibrinogen-DP. In synovial fluid, the presence of proteolytic enzymes, different from plasmin may play a role. Elastase is a possible candidate. However, the monoclonal antibodies used in the enzyme immunoassays hardly react with degradation products produced by elastase, at least in a purified system.6 Whichever enzymes are involved, the results show that the degradation products in the joint fluid proved to lack specific fibrinogen epitopes and react like plasmin-induced fibrin-DP. This indicates that thrombin-like activity had been high enough to convert all fibrinogen, or (if the latter is digested by other proteases before thrombin came into action) its early degradation products, to fibrin or fibrin-like products. This is in agreement with the absence of clottable fibrinogen in the samples of synovial fluid. In the plasma samples only relatively low levels of split products were found, and their composition did not reflect that of the synovial fluid. Since the samples were obtained in the presence of aprotinin, and kept frozen in the presence of DFP, a potent protease inhibitor, it is highly unlikely that they are artefacts induced in vitro. FDPs were present in plasma in most patients. In contrast to synovial fluid, most plasma samples also contained small but measurable amounts of fibrinogenDP. Since fibrinogen-DPs were virtually absent from the synovial fluid, the fibrinogen-DP in the samples of venous blood is probably derived from other sources, such as the inflamed tissue around the inflamed joint or extra-articular sites of rheumatic inflammation. Patient F, who had the highest level of fibrinogen-DP in plasma showed extra-articular manifestations of the
186
Degradation
Products
of Fibrin and of Fibrinogen
in Synovial
disease. He also had the highest platelet count, another indication of disease activity. Interestingly, he was the only patient with fibrinogen-DP detectable in the knee joint. Fibrinolytic enzymes may play several roles in RA. One of the proteins degraded by plasmin in the joint cavity is fibrin. Plasmin is also involved in the activation of procollagenase to collagenase in rheumatoid synovial cells. I2 This presumably promotes the invasion of the pannus into the cartilage causing a destruction of the articular surface of the joint. To what extent these two effects of plasmin might represent a balance, and whether a shift in this may contribute favourably to the disease is still unknown. Belch et alI3 have measured fibrinolytic factors in blood and synovial fluid of patients with RA. They found low levels of plasminogen activator and high plasminogen concentrations in the blood of their patients, especially in the most severely affected cases. Arguing that the low blood fibrinolytic activity might be a cause of the fibrin deposition within the joints they treated their patients with stanozolol, an anabolic steroid able to raise the fibrinolytic activity by suppressing inhibitor levels in the blood.14 Apart from normalising the blood fibrinolytic activity, the treatment raised the plasminogen levels both in plasma and in the synovial fluid. Although the disease activity decreased during the treatment period of 6 months, it is not known if this was due to a rise in the rate of disappearance of fibrin-related material from the synovial cavity. However, the observations might suggest that formation of plasmin could be beneficial and reduce disease activity. We conclude from this study that the enzyme immunoassays for FDP, fibrinogen-DP and fibrinDP, based on specific monoclonal antibodies, allow for the study of intra-articular breakdown of fibrinogen and fibrin. Our preliminary experience suggests that it might be possible to estimate extra-articular disease activity separate from joint inflammation. Future studies should include non-rheumatic arthritis and should pay attention to correlations between clinical manifestations and the level and composition of degradation products of fibrin and of fibrinogen, especially in plasma of treated and non-treated patients with RA.
Received: 16 January 1989 Accepted after revision: 4 April 1989 Offprint orders to: Dr. J. Jespersen, Section of Coagulation Fibrinolysis, Department of Clinical Chemistry, Ribe County pital in Esbjerg, DK-6700 Esbjerg, Denmark.
and Hos-
Fluid
ACKNOWLEDGEMENT The authors wish to thank E. Hoegee-de Nobel for technical ance and C. and M. Horsting for typing the manuscript.
assist-
REFERENCES 1. Barnhart M I, Riddle J M, Bluhm G B, Quintana C 1967 Fibrin promotion and lysis in arthritic joints. Ann Rheum Dis 26:206218 2. Clemmensen I, Andersen R B 1978 Properties of fibrinogenantigenic material on the rheumatoid synovial membrane and in the rheumatoid synovial fluid. J Lab Clin Med 92:678689 3. Van de Putte L B A, Kluft C, Noordhoek Hegt V, et al 1977 Activators and inhibitors of fibrinolysis in rheumatoid joint inflammation. In: Willoughby D A et al (eds), Perspectives in Inflammation. MTP Press, Lancaster. vv 499-503 4 Mochan E, Uhl J 1984 Elevation in syndvial fluid plasminogen activator in patients with rheumatoid arthritis. J Rheumatol 11:123-128 5 Inman R D, Harpcl PC 1985 cc,-Plasmin inhibitor-plasmin complexes in synovial fluid. J Rheumatol 13:535-537 6 Koopman J, Haverkate F, Koppert P, et al 1987 New immunoassay of fibrin(ogen) degradation products in plasma using a monoclonal antibody. J Lab Clin Med 109:75-84 I Koppert P, Kuipcrs W. Hoegee-de Nobel B, et al 1987 A quantitative enzyme immunoassay for primary librinogenolysis products in plasma. Thromb Haemost 57:25-28 W 1985 8 Koppert P W, Huijsmans C M G, Nieuwenhuizen A monoclonal antibody specific for human fibrinogen, fibrinopeptide A-containing fragments and not reacting with free fibrinopeptide A. Blood 66: 503-507 9. Jespersen J, Sidelmann J 1982 A study of the conditions and accuracy of the thrombin time assay of plasma fibrinogen. Acta Haematol67:2-7 W 1988 10. Koppcrt P W, Hoegee-de Nobel B, Nieuwenhuizen A new quantitative enzyme-immuno-assay for fibrin degradation products (Fibrin-DP) in plasma, based on monoclonal antibodies. Thromb Haemost 58:31@315 W 1987 A double blind cooperative study of six 11. Nieuwenhuizen monoclonal antibody-based plasma assays for fibrinogen derivatives. In: Lowe G D 0 et al (eds) Fibrinogen 2. Biochemistry, physiology and clinical relevance. Elsevier Science Publishers, Amsterdam, pp 181-186 12. Werb Z, Mainardi C L, Vater C A, Harris E D 1977 Endogenous activation of latent collagenase by rheumatoid synovial cells. Evidence for a role of plasminogen activator. N Engl J Med 2%: 1017-1023 K, Capcll 13, Belch J J F, McArdle B, Madhok R, McLaughlin H A, Forbes C D, Sturrock R D 1984 Decreased plasma fibrinolysis in patients with rheumatoid arthritis. Ann Rheuma Dis 43: 774777 14. Verheijen J H, Rijken D C, Chang G T G, Preston F E, Kluft C 1985 Decreased level of rapid tissue-type plasminogen activator inhibitor by oral administration of stanozolol. In: Davidson J F et al (eds) Progress in Fibrinolysis VII. Churchill Livingstone, Edinburgh, pp 102-104
0268~9499/89,WO3-0183
S10.00
Degradation Products of Fibrin and of Fibrinogen in Synovial Fluid and in Plasma of Patients with Rheumatoid Arthritis
J. Jespersen, E. J. P. Brommer, F. Haverkate, W. Nieuwenhuizen SUMMAR Y. Degradation products of fibrin and of fibrinogen, together (FDP) and separately, were measured in plasma and synovial fluid of patients with rheumatoid arthritis using specific enzyme immunoassays. Considerable amounts of fibrin degradation products (fibrin-DP) were found in the synovial fluid; only 1 case yielded some fibrinogen-derived products (fibrinogen-DP). In plasma relatively small amounts of degradation products, both fibrin-DP and fibrinogen-DP, were present. From the discrepancy between the amount and composition of degradation products in synovial fluid and in plasma we conclude that there are extra-articular sites of fibrin(ogen) breakdown in rheumatoid arthritis. The new specific methods may contribute to the understanding of the role of fibrinolysis in rheumatoid disease and in inflammation in general. KEYWORDS.
Synovial Fluids. RA. Fibrin and Fibrinogen degradation products.
PATIENTS
Fibrin deposition is a conspicious feature of joint inflammation in rheumatoid arthritis (RA), and degradation products of fibrin or fibrinogen can be detected in the synovial fluid of the inflamed joints.ls2 Several investigators have demonstrated the presence of fibrinolytic enzymes in the fluid, and this could give rise to formation of the degradation products in the joint cavity.3-5 The recent introduction of quantitative immunoassays6-* based on monoclonal antibodies specific for these degradation products has now allowed a distinction between split products of fibrin (fibrin-DP) and of fibrinogen (fibrinogen-DP) in the synovial fluid from patients with chronic rheumatoid arthritis. We attempt to answer the question of whether it is fibrin or fibrinogen which is being broken down in the joint cavity. Because the split products might dissipate into the circulating blood, we have also determined the degradation products in samples of plasma from some of the patients. In all but 1 patient only fibrin degradation products were detectable in the synovial fluid. In the circulating blood, however, both fibrinogen-DPs and Fibrin-DPs were detectable. The different composition of the split products in the two compartments suggests differences in mode or site of degradation.
AND METHODS
We studied 12 patients with long-standing RA as defined according to ARA criteria, including the presence of circulating rheuma-factors. Relevant clinical and laboratory data are given in Tables 1 and 2. Haemoglobin, total white cell counts and platelet counts were determined on Coulter S plus IV (Coulter). Fibrinogen was determined by a thrombin time assay.g The duration of the symptoms ranged from 1 to almost 30 years (median 4.5 years). At the time of investigation 4 patients (D, E, F and H) had extra-articular manifestations of the rheumatic disease. Synovial fluid obtained by routine knee puncture and collected in tubes (9 volumes) containing 1 volume of trisodium citrate (0.13 mol/l) and aprotinin lo6 KIU/l was centrifuged to remove cells and other corpuscular elements. The supernatant was kept frozen (< -20°C) until analysis. For demonstration of clottable fibrinogen, thrombin (10 NIH U/ml tinal concentration) was added to the undiluted synovial fluid. Clotting was monitored manually by means of a wire hook. Plasma was prepared from titrated blood (9 volumes collected in ice-cold tubes containing 1 volume of trisodium citrate, 0.13 mol/l, and aprotinin lo6 KIU/l) by centrifugation at 2000g for 30min at 4°C and kept frozen (< - 20°C) until analysis. Diisopropylfluorophosphate (DFP; 0.1 mol/l) 0.1 volume was added to all samples before freezing. In addition to the assessment of the total amount of
J. Jespersen, Dept. of Clinical Chemistry and Internal Medicine, Ribe County Hospital Esbjerg, Denmark, EL J. P. Brommer, F. Have&ate, W. Nieuwenbuizeo, Gaubius Institute TNO, PO Box
612,230O AP Leiden, The Netherlands. 183
184
Degradation
Products
of Fibrin and of Fibrinogen
Table 1 Clinical data of patients
with theumatoid
in Synovial
Fluid
arthritis
Patient
Sex
Born
Age at debut (y)
A
M
260440
38
Prednisolone 5 mg x 3 Imurel50 mg x 5 Dextropropoxifen 65 mg x 4
B
F
050218
59
Dimethylcystein 125 mg Brufen 400 mg x 3 Naprosyn 500 mg Doloxene 100 mg x 3
C
M
100818
60
Dextropropoxifen
D
F
210713
66
Brufen 400 mg x 3
Noduli at the hand area
E
F
021008
44
Confortid 100 mg Dextroproxifen 65 mg x 6
Myositis interstit.
F
F
150832
47
Dextropropoxifen 65 mg x 34 Brufen 400 mg x 3
Noduli, and Baker cyst
G
M
261015
50
Prednisolone 5 mg Ibuprofen 400 mg x 4 Dextropropoxifen 65 mg x 4
H
M
110733
30
Dimethylcystein Dextropropoxifen
I
F
290409
61
Paracetamol 1 g p.n. Panodril 1 g x 3 Dextropropoxifen 65 mg x 4
3
F
080421
63
Atamir 450 mg Ibuprofen 400 mg x 2-3
K
F
130535
45
Atamir 300 mg Felden 20 mg
L
M
241122
57
Imurel50 mg Ibuprofen 400 mg x 3 Dextropropoxifen 65 mg (max. x 4) Prednisolone 10 mg
Table 2 Analysis of blood and plasma in patients
RA-related
with rheumatoid
Notes
treatment
65 mg x 4
Noduli at the areas of the knee and elbow
125 mg 65 mg x 4
arthritis
Patient
Year of study
Haemoglobin (mmol/l)
Leucocytes (109/1)
Platelets (109P)
Fibrinogen Olmol/l)
ESR (arb, units)
8.2 7.1 7.9 7.0 6.4 7.7 6.5 9.1 6.1 7.3 7.1 7.8
7.5 9.2 12.6 8.8 5.5 10.2 9.3
312 266 343 392 320 463 262
K L
1981 1981 1981 1981 1981 1982 1982 1985 1985 1985 1985 1985
9.3 5.4 4.7 9.1
432 417 295 421
35.0 13.5 17.7 14.7 10.7 14.4 12.2 13.3 17.3 16.3 11.2 18.8
73 48 66 74 43 53 73 24 86 58 25 51
- = not determined.
degradation products from fibrin and fibrinogen (FDP), specific determinations of fibrinogen-DP and fibrin-DP were made in some of the samples. All three assays are sandwich EM’s (Organon Teknika, Turnhout, Belgium) based on a monoclonal, catching antibody (FDP-14) against a neoantigenic determinant in the E-fragment. FDP-14 reacts with degradation products of fibrin and of fibrinogen, but not with the intact parent molecules.* For the specific, separate
assay of fibrinogen degradation products a horseradish peroxidase-conjugated monoclonal antibody (‘Y 18’)8 reactive with covalently bound fibrinopeptide A’ is used as tagging antibody. For the specific fibrinDP assay of a horseradish peroxidase (HRP) conjugates of a monoclonal antibody elicited against the Ddimer (‘DD13’),” is used as the tagging antibody.” For the FDP assay, i.e. the total of fibrin-DP and fibrinogen-DP, either a polyclonal rabbit antifibrino-
Fibrinolysis
185
Table 3 Fibrin and fibrinogen degradation products in synovial fluid and plasma from patients with rheumatoid arthritis
Patient
Synovial fluid TDP FbDP mg/l mg/l
FgDP mg/l
A B C D E F G H I J K L
340 470 346 513 146 227 115 157 923 414 151 156
_* _* _* _* _* _* 101 61.1 522 148 118 93
Plasma TDP mg/i
FbDP mg/l
6.4 4.6 1.1 6.2 0.5 8.2
_
27.9 5.4 2.7 1.8 4.9
1.2 <0.5 1.2 <0.5 1.0
_ _
FgDP mg/l 1.1 10.5 0.8 <0.5
-=not determined. TDP = total of fibrinogen-DP and fibrin-DP; FbDP = fibrin-DP; FgDP = fibrinogen-DP. * =synovial fluid samples of these patients were not available for testing with the fibrin-DP test.
gen antibody, conjugated with HRP, or a mixture of monoclonals ‘Y18’8 and ‘DD13”O (both HRP conjugated) was used as the tagging antibody.6 These tests are not influenced by the rheumatoid factor; normal values for FDP, fibrinogen-DP and Fibrin-DP in plasma are below 0.5 mg/l. l1
RESULTS Synovial fluid was obtained from 12 patients by puncture of inflamed knee joints. The high viscosity of the fluid did not hamper the assays after dilution, as usual for plasma. Clottable fibrinogen was not detected in any of the synovial fluid samples. The other data are given in Table 3. High levels of degradation products (FDP) were present in all samples, varying from 115 to 923 mg/l. Assays of fibrin-DP and fibrinogen-DP separately in 6 patients revealed that the bulk of the degradation products consisted of fibrin degradation products; fibrinogen-DP were virtually absent from synovial fluid in all but 1 patient. The FDP assay using polyclonal antibodies as tagging antibodies instead of the mixture of two ,monoclonals confirmed the same high concentrations of FDP in the synovial fluid. Plasma samples showed lower concentrations of fibrin-DP and FDP (Table 3) but higher levels of fibrinogen-DP in comparison with the synovial fluids.
DISCUSSION Diagnostic tools, based on specific monoclonal antibodies, provided us with means to measure fibrin-DP and fibrinogen-DP separately in samples of synovial fluid from patients with rheumatoid arthritis. For
obvious reasons, no normal reference values are available. The enzyme immunoassays have been devised for the use in plasma, but they can be applied to other body fluids as well. It is not clear why the amount of FDP exceeds that of the sum of fibrin-DP and fibrinogen-DP. In synovial fluid, the presence of proteolytic enzymes, different from plasmin may play a role. Elastase is a possible candidate. However, the monoclonal antibodies used in the enzyme immunoassays hardly react with degradation products produced by elastase, at least in a purified system.6 Whichever enzymes are involved, the results show that the degradation products in the joint fluid proved to lack specific fibrinogen epitopes and react like plasmin-induced fibrin-DP. This indicates that thrombin-like activity had been high enough to convert all fibrinogen, or (if the latter is digested by other proteases before thrombin came into action) its early degradation products, to fibrin or fibrin-like products. This is in agreement with the absence of clottable fibrinogen in the samples of synovial fluid. In the plasma samples only relatively low levels of split products were found, and their composition did not reflect that of the synovial fluid. Since the samples were obtained in the presence of aprotinin, and kept frozen in the presence of DFP, a potent protease inhibitor, it is highly unlikely that they are artefacts induced in vitro. FDPs were present in plasma in most patients. In contrast to synovial fluid, most plasma samples also contained small but measurable amounts of fibrinogenDP. Since fibrinogen-DPs were virtually absent from the synovial fluid, the fibrinogen-DP in the samples of venous blood is probably derived from other sources, such as the inflamed tissue around the inflamed joint or extra-articular sites of rheumatic inflammation. Patient F, who had the highest level of fibrinogen-DP in plasma showed extra-articular manifestations of the
186
Degradation
Products
of Fibrin and of Fibrinogen
in Synovial
disease. He also had the highest platelet count, another indication of disease activity. Interestingly, he was the only patient with fibrinogen-DP detectable in the knee joint. Fibrinolytic enzymes may play several roles in RA. One of the proteins degraded by plasmin in the joint cavity is fibrin. Plasmin is also involved in the activation of procollagenase to collagenase in rheumatoid synovial cells. I2 This presumably promotes the invasion of the pannus into the cartilage causing a destruction of the articular surface of the joint. To what extent these two effects of plasmin might represent a balance, and whether a shift in this may contribute favourably to the disease is still unknown. Belch et alI3 have measured fibrinolytic factors in blood and synovial fluid of patients with RA. They found low levels of plasminogen activator and high plasminogen concentrations in the blood of their patients, especially in the most severely affected cases. Arguing that the low blood fibrinolytic activity might be a cause of the fibrin deposition within the joints they treated their patients with stanozolol, an anabolic steroid able to raise the fibrinolytic activity by suppressing inhibitor levels in the blood.14 Apart from normalising the blood fibrinolytic activity, the treatment raised the plasminogen levels both in plasma and in the synovial fluid. Although the disease activity decreased during the treatment period of 6 months, it is not known if this was due to a rise in the rate of disappearance of fibrin-related material from the synovial cavity. However, the observations might suggest that formation of plasmin could be beneficial and reduce disease activity. We conclude from this study that the enzyme immunoassays for FDP, fibrinogen-DP and fibrinDP, based on specific monoclonal antibodies, allow for the study of intra-articular breakdown of fibrinogen and fibrin. Our preliminary experience suggests that it might be possible to estimate extra-articular disease activity separate from joint inflammation. Future studies should include non-rheumatic arthritis and should pay attention to correlations between clinical manifestations and the level and composition of degradation products of fibrin and of fibrinogen, especially in plasma of treated and non-treated patients with RA.
Received: 16 January 1989 Accepted after revision: 4 April 1989 Offprint orders to: Dr. J. Jespersen, Section of Coagulation Fibrinolysis, Department of Clinical Chemistry, Ribe County pital in Esbjerg, DK-6700 Esbjerg, Denmark.
and Hos-
Fluid
ACKNOWLEDGEMENT The authors wish to thank E. Hoegee-de Nobel for technical ance and C. and M. Horsting for typing the manuscript.
assist-
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