BritishJournalof Haematology, 1979,qz.585-591.
Correlation between Serum IgG, Platelet Membrane IgG, and Platelet Function in Hypergammaglobulinaemic States K. M. MCGRATH,* J. J. STUART A N D F. RICHARDS,I1 Department of Medicine, Section of HernatologylOncology, Bowman G r a y School ofMedicine, Winston-Salem, North Carolina (Received30 September 1978; acceptedforpublication 16 October 1978)
SUMMARY. Platelet membrane IgG levels were measured by the complement lysis inhibition test, and were correlated with serum IgG levels in 29 patients with elevated immunoglobulins due to multiple myeloma, benign monoclonal gammopathy, systemic lupus erythematosis, rheumatoid arthritis, benign hypergammaglobulinaemic purpura and systemic infection. Platelet membrane IgG was increased in proportion to the elevation of serum IgG with a highly significant correlation coefficient (0.5615 , P I O.OI), irrespective of the underlying disease state. Further washing of the platelets did not significantly alter the amount of membrane bound IgG. Qualitative platelet defects were present in nine of the 10myeloma patients in whom platelet aggregation, platelet factor 3 availability, platelet adhesion to glass beads and bleeding times were studied. The most severe abnormalities occurred in those patients having the highest levels of platelet membrane IgG. Platelet function was normal despite markedly elevated platelet bound IgG in one patient with benign hypergammaglobulinaemic purpura. These findings indicate that elevation of serum IgG must be excluded before inferring the presence of platelet autoantibodies in patients with elevated platelet bound IgG. They also support the concept that the qualitative platelet abnormalities seen in multiple myeloma result from platelet coating by abnormal immunoglobulins or are secondary to myeloma effect on bone marrow. The recent description by Dixon et a1 (1975) of a complement lysis inhibition assay for measurement of surface bound platelet IgG has provided a sensitive method for the detection of antiplatelet antibodies in idiopathic autoimmune thrombocytopenia, systemic lupus erythematosis (SLE) and lymphoma (Dixon et al, 1975;Hegde et al, 1977). The thrombocytopenia seen in patients with SLE and lymphoproliferative disorders clinically mimics the idiopathic variety and is considered to result from platelet autoantibody production (Dixon et ul, 1975b;
* Present address: Department of Haematology, Royal Infirmary, Sheffield, U.K. Correspondence:Dr Frederick Richards, 11, Department of Medicine, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, North Carolina 27103,U.S.A. 0007-1048/79/0800-oj85$02.00 01979Blackwell Scientific Publications 585
586
Platelet Membrane IgC
Hegde et al, 1977; Dixon & Rosse, 1975). However, elevated levelsof platelet bound IgG have been reported in SLE in association with a normal platelet count (Dixon et al, 1975;Karpatkin et al, 1972). This has been attributed to compensated thrombolysis in the presence of antiplatelet antibody (Dixon & Rosse, I975), but the effect ofincreased serum IgG on this assay has not been determined. In normal or thrombocytopenic patients, platelet surface bound IgG decreased with up to four washes, and thereafter remained constant (Dixon et al, 1975),and it would be reasonable to assume that nonspecifically bound IgG in patients with increased serum levels would be removed by this step. However, following the finding of increased platelet bound IgG in a patient having thrombocytopenia and an IgG myeloma, and the return of both serum and platelet IgG to normal with treatment, we investigated the relationship between increased serum IgG and platelet bound IgG. Platelet function abnormalities have been reported in association with the bleeding diasthesis known to occur in multiple myeloma, and have been attributed to possible coating of the platelet by abnormal immunoglobulins (Perkins et a l , 1970; Cohen et al, 1970). Therefore a correlation of platelet function with platelet bound IgG was also investigated. These relationships were further studied in one case of benign hypergammaglobulinaemic purpura, a disease in which a polyclonal increase in serum IgG is associated with the development of dependent purpura, in the presence of a normal platelet count.
MATERIALS AND METHODS Patients. Blood was obtained from 29 patients with increased serum immunoglobulins: 19 patients with IgG myeloma, five patients with IgA or light chain myeloma; one patient with each of the following disorders: SLE, rheumatoid arthritis, benign IgG monoclonal gammopathy, benign hypergammaglobulinaemic purpura, and erysipelas. All patients with myeloma were receiving therapy with alkeran, prednisone, BCNU, adriamycin, cytoxan, bleomycin or vincristine, alone or in combination. Platelet function studies were performed in 10patients with IgG myeloma, and one patient with benign hypergammaglobulinaemic purpura. Patients who had taken drugs known to affect platelet function were excluded from this part of the study. Two of the myeloma patients studied gave a history of bleeding: epistaxis (A.C.) and easy bruising (C.H.). Methods. Platelet counts on whole blood were performed on a Technicon AutoAnalyzer TM. Platelet surface bound IgG was measured by the method ofDixon etaf (1975),as modified by Hegde et a1 (1977). A normal control was run in parallel. In a group of 25 healthy controls platelet bound IgG was always less than 50 ng/107 platelets. Serum immunoglobulins were quantitated by an immunonephelometric assay (Lizana & Hellsing, 1974) using a Hyland laser nephelometer. Serum immunoelectrophoresis was carried out in I % agarose containing barbital buffer pH 8.6, 0.1 M (Osserman & Lawlor, 1961). Platelet function studies were performed as follows: Modified Ivy bleeding time (Mielke et al, 1969), platelet adhesion to glass beads by the method of Hume (1967). platelet factor 3 availability by the method of Hardisty & Hutton (1965)~ and platelet aggregation according to the method of Born (1962) using a Chronolog aggregometer, with ADP (2 PM, 3 PM), epinephrine ( 5 PM), and collagen (bovine achilles tendon, Sigma) as aggregating agents.
K. M. McGrath,].].
587
Stuart and F. Richards, II
Aggregation results were expressed as a percentage of a normal control plasma having the same platelet concentration as the patient. RESULTS The correlation between serum and platelet bound IgG is shown in Table I and Fig I . 15 of the 19 patients with IgG myeloma had elevated platelet bound IgG. The remaining four patients (A.S., A.P., G.M., S.J.) had elevated serum IgG but normal platelet IgG. All patients with light chain or IgA myeloma had normal serum and platelet bound IgG. All three patients with polyclonal increases in IgG due to infection or collagen-vascular diseases, and patient M.L. with benign monoclonal IgG gammopathy had increased platelet bound IgG. Patient F.H. had 28.98 g/l serum IgG and 188 ng/107 platelets when first diagnosed as having multiple TABLE I. Relationship between serum IgG, platelet bound IgG, and platelet count
Disease
Patient
IgG myeloma
C.P. G.C. J.S. W.W. R.C. L.M. R.B. A.C. C.H. F.H.* F.H.t Y.M. AS. W.K. E.Y. S.J. L.W. A.P. E.S. G.M. SLE D.R. Rheumatoid arthritis T.F. Erysipelas J.W. Benign monoclonal gammopathy M.L. Hypergammaglobulinaemic purpura L.M.W. Non-IgG myeloma R.L. A.M. H.T. D.P.
F.T. Normal ranges
Serum IgG Platelet-bound IgC Platelet count (gll) (ngllo7$3) ( x 10~11) 86.00 74.80 64.00 61.00 43.80 37.69 3 7.60 37.00 37.00 29.90 6.50
218
65
5 20
35
280
50
166 450 266 60 219 99
230
230 200 130
65 250
I88
40
I0
I20 250
20.I 0
184 33 62 81
20.00
28
25.80 25.00
24.40 19.80 15.60 11.00
10.40 16.80 12.50
52
38 I45 47 72
205 225
187 495 23 5 225
35 0 I60 lo00
200
22.00
I SO I00
29.20 20.40
158 600
5.20
5.90 4.60
45 26 38
200
4.40
20
I20
6.25
48
I 80
4.5-9.8
50
I 50-400
Before treatment; t after treatment.
250 200
295
46 3 75
Platelet Membrane IgG
588
myeloma. Both levels returned to normal after 4 months treatment with alkeran and prednisone. The increase in platelet bound IgG in all cases was proportional to the increase in serum levels with a highly significant correlation coefficient 0.5615 ( P I 0.01). Platelets from four patients with IgG myeloma and elevated platelet bound IgG were separated into two aliquots and washed five and nine times respectively in complement fixation test buffer. The levels of platelet bound IgG after five and nine washes were as follows: (W.W.) 166, 93; (J.S.) 280, 290; (C.P.) 2 1 8 , 190; and (R.C.) 450, 260 ng IgG/107 platelets. Platelet bound IgG did not return to normal with further washing.
0000 -
moo 6000&
a
6 5000-
/
0
2ool
0
1000
1
1
1
1
1
1
1
1
I
1
400 450 500 550 600 PLATELET-BOUND I ~ G1ng/107 pits)
FIG I . Correlation between serum and platelet bound IgG in patients with IgG myeloma
(0). light
chain or IgA myeloma (0) and other diseases (0)as specified in the methods.
Platelet function was investigated in 10 patients with IgG myeloma (Table 11). Four patients had > 200 ng IgG/107 platelets. Three of these patients were abnormal in most of the platelet function tests. The fourth patient had the highest level of platelet bound IgG, but had normal platelet function tests. In six patients in whom the level of platelet bound IgG was normal or only slightly elevated, occasional abnormalities of platelet function were observed, particularly in collagen-induced aggregation. There was no correlation between the platelet count and levels of platelet bound IgG. Patient L.M.W. had been diagnosed in 1975 as having benign hypergammaglobulinaemic purpura on the basis of a diffuse polyclonal increase in IgA, IgM and IgG, prominent brownish discoloration of the lower limbs following recurrent purpuric rashes in the same area, mild anaemia, elevated ESR, positive rheumatoid factor, normal platelet count and normal bone marrow biopsy. These findings have remained constant without therapy. Currently, her serum IgG level is 20.40 g/l, platelet bound IgG is >600 ng/ro7 platelets, after five or nine washes, and no abnormality of platelet function could be demonstrated.
K . M . McGrath,].j. Stuart and F. Richards, 11
589
TABLE 11. Relationships between platelet bound IgG and platelet function in IgG myeloma
Patient
Platelet bound IgG (nglio’ plts)
ADP (2 pi)
A.C.
219
9,
J.S. C.P. R.C. C.H. E.Y. R.B. L.W. S.J.
280
o*
AS.
Normal range (mean? I SD)
SO* 32*
75 *
218
I37
450 99 81 60
160
52
80
28 33
ND
< 50
Bleeding time Platelet Platelet (3 p ~ ) (5 p ~ ) Collagen (min) factor 3 adhesion
ADP
125
68* 85 132
110
136 108
69* 115
Epinephrine
0,
0,
IO*
roo*t
I7* 8
N
0,
1’
72
29*
12.
N N N N N
51*
109 I33
63 75 I00
100
83 100 83 71-111% 92-116% 61-105%
150
34* 71, 29* 3 I* 67* 7s 82-114%
2 2
I4* 8
ND 5 9 IIZ* 2-9
~
1’
N N
History of bleeding
Epistaxis
13*
46 O*
Easy bruising
40 38 46 I9* 3@80% ~
N D =not done; N =normal; 1=decreased. * Abnormal; t prolonged lag phase.
DISCUSSION This study demonstrates that in patients with increased serum IgG the level of platelet bound IgG is proportional to the amount of free IgG in the corresponding plasma. This relationship applies irrespective of the underlying disease state. Thus patients with IgG myeloma, benign monoclonal IgG gammopathies, infections and collagen-vascular diseases may all have elevated levels of platelet bound IgG as a consequence of their disease. The amount of IgG on the platelet surface showed a highly significant correlation with the increase in serum IgG, and in one patient returned to normal when the serum IgG level became normal with treatment. The nature of the bond between the IgG and the platelet membrane in these diseases remains uncertain. In patients with autoimmune thrombocytopenia the level of platelet bound IgG correlates with the platelet count as well as the response to treatment (Dixon et af, 1975; Hegde et al, 1977; Luiken et al, 1977). There was no correlation between the platelet count and the membrane bound IgG in our patients. Further, the majority of our patients were not thrombocytopenic, and had no history of abnormal bleeding, even though the levels of platelet bound IgG were in the same range as those seen in autoimmune thrombocytopenia. Thus an antigen-antibody relationship is unlikely. Nonspecific coating of the platelets has been previously suggested to occur in association with high concentrations of paraproteins (Perkins et al, 1970); however, such proteins would normally be expected to be removed by washing. We were unable to remove the excess membrane bound IgG by increasing the number of washes from five to nine, in four patients. Patients with SLE have previously been found to have high levels of platelet bound IgG in the absence of thrombocytopenia (Dixon & Rosse, 1975). Decreased platelet survival and
590
Platelet Membrane IgC
serum antiplatelet antibodies, detected by platelet factor 3 release, have been reported in these patients (Weiss et a l , 1963).In SLE the IgG appears to be in the form of an immune complex (Dixon & Rosse, 1975) which attaches to the platelet membrane and results in increased thrombolysis. A similar mechanism could also be responsible for the increased platelet membrane IgG in rheumatoid arthritis, benign hypergammaglobulinaemic purpura and systemic infections. In paraproteinaemias, however, there is no evidence to suggest immune complex formation. It may be that the attachment of abnormal IgG to the platelet membrane results in increased platelet destruction; however, confirmation of this requires further investigation. The association of high concentrations of paraproteins and abnormal bleeding is well recognized. The bleeding time (Perkins er all 1970),platelet adhesion (Perkins et al, rg~o), platelet factor 3 availability (Pachter et a l , 1959),clot retraction (Cohen et al, 1970),and platelet aggregation in a cone rotational viscometer (Rosenberg & Dintenfass, 1965)have all been reported to be abnormal in patients with multiple myeloma or Waldenstrom’s macroglobulinaemia. Nine of 10 patients in this study demonstrated varying abnormalities of platelet function. Usually, the most severe defects were seen in those patients having the highest levels of platelet bound IgG. Aggregation in response to collagen was the only parameter which was consistently abnormal in all nine patients and was absent in the one patient (A.C.) presenting with frank haemorrhage. The bleeding time, platelet adhesion, platelet aggregation to ADP and epinephrine, and platelet factor 3 availability were each abnormal in 20-30% of the patients, and did not appear to correlate with the bleeding tendency. These findings are consistent with the concept that abnormal platelet function seen in multiple myeloma is a consequence of coating of the platelet by the abnormal protein, but structural and/or metabolic abnormalities in the platelet induced by the malignancy in the bone marrow may also play a role. One patient with benign hypergammaglobulinaemia purpura was investigated to determine whether abnormalities of platelet function are involved in the development of the purpuric lesions seen in this disease. The platelet bound IgG was markedly elevated in this patient (>600 ng IgG/107 platelets) in association with a moderate increase in serum IgG. However, there was no evidence of abnormal platelet function. Decreased platelet factor 3 has been reported in previous studies and attributed to coating of the platelets by the immunoglobulin (Weiss et al, 1963), but was normal in our patient. In most studies the purpura has been attributed to a localized vascular lesion (Baughan et al, 1971;Kyle et all 1971).Thus the contribution of increased platelet IgG to the pathogenesis of the purpura in this disease remains uncertain. These studies demonstrate that increased platelet bound IgG, in the absence of thrombocytopenia, can occur in many conditions associated with increased serum IgG, and therefore the latter should be considered before the presence of an antiplatelet antibody can be confirmed.
ACKNOWLEDGMENTS
Supported by Biomedical Research Support Grant RR-5404 and Forsyth Cancer Service Grant. We are grateful to Mrs Karen Spaugh for her excellent secretarial work.
I(. M.
McCrath,J. J . Stuart and F. Richards, I I
591
REFERENCES BAUGHAN, M.A., DANIELS, J.C., LEVIN,W.C. & RrrzMAN, S.E. (1971) Hyperglobulinemic purpura (Waldenstrom’s). A report of 4 cases and review of the literature. Texas Reports on Biolofy and Medicine, 29,14~161. BORN,G.V.R. (1962) Aggregation of blood platelets by adenosine disphosphate and its reversal. Nature, 1941Y27-929. COHEN,I., AMIR,J., BEN-SHAUL, Y., PICK,A. & D E VRIES,A. (1970) Plasma cell myeloma associated with an unusual myeloma protein causing impairment of fibrin aggregation and platelet function in a patient with multiple malignancy. Americanjournal of Medicine, 48, 766-776. DIXON,R.H. & ROSSE,W.F. (1975) Platelet antibody in autoimmune thrombocytopenia. (Annotation). British journal offfaematology, 31, 129-134. DIXON,R.H., ROSSE,W.F. & EBBERT, L. (1975) Quantitative determination of antibody in idiopathic thrombocytopenic purpura. Correlation of serum and platelet-bound antibody with clinical response. N e w England Journal of Medicine, 292, 230-236. HARDISTY, R.M. & HUITON, R.A. (1965) The kaolin clotting time of platelet-rich plasma: a test of platelet factor-3 availability. British Journal of Haematology, 11, 258-268. HEGDE,U.M., GORDON-SMRH, E.C. & WORLLEDGE, S. (1977) Platelet antibodies in thrombocytopenic patients. British Journal offfaematology, 35, I 13-122. HUME,M . (1967) Platelet entrapment in glass spheres: clinical and experimental application. Anna15 ofSur,pry, 166, 5 ~ 5 4 . KARPATKIN, S., STRICK,N., KARPATKIN, M.B. & SrsKIND, G.W. (1972) Cumulative experience in the detection of antiplatelet antibody in 234 patients with idiopathic thrombocytopenic purpura, systemic lupus erythematosus and other clinical disorders. AmericanJournal of Medicine, 5 2 , 776-785.
KYLE,R.A., GLEICH,G.J., BAYRD,E.D. & VAUGHAN, J.H. (1971) Benign hypergammaglobulinemic purpura of Waldenstrom. Medicine, 50, I 13-123. LIZANA, J. & HELLSING, K. (1974)Manual immunonephelometric assay of proteins, with use of polymer enhancement. Clinical Chemistry, 20, I 181-1 186. LUIKEN, G.A., MCMILLAN, R., LIGHTSEY, A.L., GORDON,P., ZEVELY, S., SCHULMAN, I., GRIBBLE, T.J. & LONGMIRE, R.L. (1977) Platelet-associated IgC in immune thrombocytopenic purpura. Blood, 50, 3 17-325. MIELKE,C.H., JR, KANESHIRO, M.M., MAHER,I.A., WEINER, J.M. & RAPAPOKT, S.I. (196y)The standardized normal Ivy bleeding time and its prolongation by aspirin. Blood, 34, 204-215. OSSERMAN, E.F. & LAWLOR, D. (1961) Immunoelectrophoretic characterization of the serum and urinary proteins in plasma cell myeloma and Waldenstrom’s macroglobulinemia. Annals ofthe N e w York Academy of Sciences, 94, 93-109. PACHTER,M.R., JOHNSON, S.A., NEBLEIT, T.R. 81 TRUANT, J.P. (1959) Bleeding platelets and macroglobulinemia. AmericanJournal ofclinical Pathology, 31,467-482. PERKINS,H.A., MACKENZIE, M.R. & FUDENBERG, H.H. (1970) Hemostatic defectsin dysproteinemias. Blood, 35.695-707ROSENBERG, M.C. & DINTENFASS, L. (1965) Platelet aggregation in Waldenstrom’s macroglobulinaemia, Thrombosis et Diathesis Haemorrhagica, 14. 202-208.
WEISS,H.J., DEMIS,D.J., ELGART. M.L., BROWN, C.S. & CROSBY, W.H. (1963) Treatment of two cases of hyperglobulinemic purpura with thioguanine. N e w England Journal ofMedicine, 268, 753-756.
Correlation between Serum IgG, Platelet Membrane IgG, and Platelet Function in Hypergammaglobulinaemic States K. M. MCGRATH,* J. J. STUART A N D F. RICHARDS,I1 Department of Medicine, Section of HernatologylOncology, Bowman G r a y School ofMedicine, Winston-Salem, North Carolina (Received30 September 1978; acceptedforpublication 16 October 1978)
SUMMARY. Platelet membrane IgG levels were measured by the complement lysis inhibition test, and were correlated with serum IgG levels in 29 patients with elevated immunoglobulins due to multiple myeloma, benign monoclonal gammopathy, systemic lupus erythematosis, rheumatoid arthritis, benign hypergammaglobulinaemic purpura and systemic infection. Platelet membrane IgG was increased in proportion to the elevation of serum IgG with a highly significant correlation coefficient (0.5615 , P I O.OI), irrespective of the underlying disease state. Further washing of the platelets did not significantly alter the amount of membrane bound IgG. Qualitative platelet defects were present in nine of the 10myeloma patients in whom platelet aggregation, platelet factor 3 availability, platelet adhesion to glass beads and bleeding times were studied. The most severe abnormalities occurred in those patients having the highest levels of platelet membrane IgG. Platelet function was normal despite markedly elevated platelet bound IgG in one patient with benign hypergammaglobulinaemic purpura. These findings indicate that elevation of serum IgG must be excluded before inferring the presence of platelet autoantibodies in patients with elevated platelet bound IgG. They also support the concept that the qualitative platelet abnormalities seen in multiple myeloma result from platelet coating by abnormal immunoglobulins or are secondary to myeloma effect on bone marrow. The recent description by Dixon et a1 (1975) of a complement lysis inhibition assay for measurement of surface bound platelet IgG has provided a sensitive method for the detection of antiplatelet antibodies in idiopathic autoimmune thrombocytopenia, systemic lupus erythematosis (SLE) and lymphoma (Dixon et al, 1975;Hegde et al, 1977). The thrombocytopenia seen in patients with SLE and lymphoproliferative disorders clinically mimics the idiopathic variety and is considered to result from platelet autoantibody production (Dixon et ul, 1975b;
* Present address: Department of Haematology, Royal Infirmary, Sheffield, U.K. Correspondence:Dr Frederick Richards, 11, Department of Medicine, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, North Carolina 27103,U.S.A. 0007-1048/79/0800-oj85$02.00 01979Blackwell Scientific Publications 585
586
Platelet Membrane IgC
Hegde et al, 1977; Dixon & Rosse, 1975). However, elevated levelsof platelet bound IgG have been reported in SLE in association with a normal platelet count (Dixon et al, 1975;Karpatkin et al, 1972). This has been attributed to compensated thrombolysis in the presence of antiplatelet antibody (Dixon & Rosse, I975), but the effect ofincreased serum IgG on this assay has not been determined. In normal or thrombocytopenic patients, platelet surface bound IgG decreased with up to four washes, and thereafter remained constant (Dixon et al, 1975),and it would be reasonable to assume that nonspecifically bound IgG in patients with increased serum levels would be removed by this step. However, following the finding of increased platelet bound IgG in a patient having thrombocytopenia and an IgG myeloma, and the return of both serum and platelet IgG to normal with treatment, we investigated the relationship between increased serum IgG and platelet bound IgG. Platelet function abnormalities have been reported in association with the bleeding diasthesis known to occur in multiple myeloma, and have been attributed to possible coating of the platelet by abnormal immunoglobulins (Perkins et a l , 1970; Cohen et al, 1970). Therefore a correlation of platelet function with platelet bound IgG was also investigated. These relationships were further studied in one case of benign hypergammaglobulinaemic purpura, a disease in which a polyclonal increase in serum IgG is associated with the development of dependent purpura, in the presence of a normal platelet count.
MATERIALS AND METHODS Patients. Blood was obtained from 29 patients with increased serum immunoglobulins: 19 patients with IgG myeloma, five patients with IgA or light chain myeloma; one patient with each of the following disorders: SLE, rheumatoid arthritis, benign IgG monoclonal gammopathy, benign hypergammaglobulinaemic purpura, and erysipelas. All patients with myeloma were receiving therapy with alkeran, prednisone, BCNU, adriamycin, cytoxan, bleomycin or vincristine, alone or in combination. Platelet function studies were performed in 10patients with IgG myeloma, and one patient with benign hypergammaglobulinaemic purpura. Patients who had taken drugs known to affect platelet function were excluded from this part of the study. Two of the myeloma patients studied gave a history of bleeding: epistaxis (A.C.) and easy bruising (C.H.). Methods. Platelet counts on whole blood were performed on a Technicon AutoAnalyzer TM. Platelet surface bound IgG was measured by the method ofDixon etaf (1975),as modified by Hegde et a1 (1977). A normal control was run in parallel. In a group of 25 healthy controls platelet bound IgG was always less than 50 ng/107 platelets. Serum immunoglobulins were quantitated by an immunonephelometric assay (Lizana & Hellsing, 1974) using a Hyland laser nephelometer. Serum immunoelectrophoresis was carried out in I % agarose containing barbital buffer pH 8.6, 0.1 M (Osserman & Lawlor, 1961). Platelet function studies were performed as follows: Modified Ivy bleeding time (Mielke et al, 1969), platelet adhesion to glass beads by the method of Hume (1967). platelet factor 3 availability by the method of Hardisty & Hutton (1965)~ and platelet aggregation according to the method of Born (1962) using a Chronolog aggregometer, with ADP (2 PM, 3 PM), epinephrine ( 5 PM), and collagen (bovine achilles tendon, Sigma) as aggregating agents.
K. M. McGrath,].].
587
Stuart and F. Richards, II
Aggregation results were expressed as a percentage of a normal control plasma having the same platelet concentration as the patient. RESULTS The correlation between serum and platelet bound IgG is shown in Table I and Fig I . 15 of the 19 patients with IgG myeloma had elevated platelet bound IgG. The remaining four patients (A.S., A.P., G.M., S.J.) had elevated serum IgG but normal platelet IgG. All patients with light chain or IgA myeloma had normal serum and platelet bound IgG. All three patients with polyclonal increases in IgG due to infection or collagen-vascular diseases, and patient M.L. with benign monoclonal IgG gammopathy had increased platelet bound IgG. Patient F.H. had 28.98 g/l serum IgG and 188 ng/107 platelets when first diagnosed as having multiple TABLE I. Relationship between serum IgG, platelet bound IgG, and platelet count
Disease
Patient
IgG myeloma
C.P. G.C. J.S. W.W. R.C. L.M. R.B. A.C. C.H. F.H.* F.H.t Y.M. AS. W.K. E.Y. S.J. L.W. A.P. E.S. G.M. SLE D.R. Rheumatoid arthritis T.F. Erysipelas J.W. Benign monoclonal gammopathy M.L. Hypergammaglobulinaemic purpura L.M.W. Non-IgG myeloma R.L. A.M. H.T. D.P.
F.T. Normal ranges
Serum IgG Platelet-bound IgC Platelet count (gll) (ngllo7$3) ( x 10~11) 86.00 74.80 64.00 61.00 43.80 37.69 3 7.60 37.00 37.00 29.90 6.50
218
65
5 20
35
280
50
166 450 266 60 219 99
230
230 200 130
65 250
I88
40
I0
I20 250
20.I 0
184 33 62 81
20.00
28
25.80 25.00
24.40 19.80 15.60 11.00
10.40 16.80 12.50
52
38 I45 47 72
205 225
187 495 23 5 225
35 0 I60 lo00
200
22.00
I SO I00
29.20 20.40
158 600
5.20
5.90 4.60
45 26 38
200
4.40
20
I20
6.25
48
I 80
4.5-9.8
50
I 50-400
Before treatment; t after treatment.
250 200
295
46 3 75
Platelet Membrane IgG
588
myeloma. Both levels returned to normal after 4 months treatment with alkeran and prednisone. The increase in platelet bound IgG in all cases was proportional to the increase in serum levels with a highly significant correlation coefficient 0.5615 ( P I 0.01). Platelets from four patients with IgG myeloma and elevated platelet bound IgG were separated into two aliquots and washed five and nine times respectively in complement fixation test buffer. The levels of platelet bound IgG after five and nine washes were as follows: (W.W.) 166, 93; (J.S.) 280, 290; (C.P.) 2 1 8 , 190; and (R.C.) 450, 260 ng IgG/107 platelets. Platelet bound IgG did not return to normal with further washing.
0000 -
moo 6000&
a
6 5000-
/
0
2ool
0
1000
1
1
1
1
1
1
1
1
I
1
400 450 500 550 600 PLATELET-BOUND I ~ G1ng/107 pits)
FIG I . Correlation between serum and platelet bound IgG in patients with IgG myeloma
(0). light
chain or IgA myeloma (0) and other diseases (0)as specified in the methods.
Platelet function was investigated in 10 patients with IgG myeloma (Table 11). Four patients had > 200 ng IgG/107 platelets. Three of these patients were abnormal in most of the platelet function tests. The fourth patient had the highest level of platelet bound IgG, but had normal platelet function tests. In six patients in whom the level of platelet bound IgG was normal or only slightly elevated, occasional abnormalities of platelet function were observed, particularly in collagen-induced aggregation. There was no correlation between the platelet count and levels of platelet bound IgG. Patient L.M.W. had been diagnosed in 1975 as having benign hypergammaglobulinaemic purpura on the basis of a diffuse polyclonal increase in IgA, IgM and IgG, prominent brownish discoloration of the lower limbs following recurrent purpuric rashes in the same area, mild anaemia, elevated ESR, positive rheumatoid factor, normal platelet count and normal bone marrow biopsy. These findings have remained constant without therapy. Currently, her serum IgG level is 20.40 g/l, platelet bound IgG is >600 ng/ro7 platelets, after five or nine washes, and no abnormality of platelet function could be demonstrated.
K . M . McGrath,].j. Stuart and F. Richards, 11
589
TABLE 11. Relationships between platelet bound IgG and platelet function in IgG myeloma
Patient
Platelet bound IgG (nglio’ plts)
ADP (2 pi)
A.C.
219
9,
J.S. C.P. R.C. C.H. E.Y. R.B. L.W. S.J.
280
o*
AS.
Normal range (mean? I SD)
SO* 32*
75 *
218
I37
450 99 81 60
160
52
80
28 33
ND
< 50
Bleeding time Platelet Platelet (3 p ~ ) (5 p ~ ) Collagen (min) factor 3 adhesion
ADP
125
68* 85 132
110
136 108
69* 115
Epinephrine
0,
0,
IO*
roo*t
I7* 8
N
0,
1’
72
29*
12.
N N N N N
51*
109 I33
63 75 I00
100
83 100 83 71-111% 92-116% 61-105%
150
34* 71, 29* 3 I* 67* 7s 82-114%
2 2
I4* 8
ND 5 9 IIZ* 2-9
~
1’
N N
History of bleeding
Epistaxis
13*
46 O*
Easy bruising
40 38 46 I9* 3@80% ~
N D =not done; N =normal; 1=decreased. * Abnormal; t prolonged lag phase.
DISCUSSION This study demonstrates that in patients with increased serum IgG the level of platelet bound IgG is proportional to the amount of free IgG in the corresponding plasma. This relationship applies irrespective of the underlying disease state. Thus patients with IgG myeloma, benign monoclonal IgG gammopathies, infections and collagen-vascular diseases may all have elevated levels of platelet bound IgG as a consequence of their disease. The amount of IgG on the platelet surface showed a highly significant correlation with the increase in serum IgG, and in one patient returned to normal when the serum IgG level became normal with treatment. The nature of the bond between the IgG and the platelet membrane in these diseases remains uncertain. In patients with autoimmune thrombocytopenia the level of platelet bound IgG correlates with the platelet count as well as the response to treatment (Dixon et af, 1975; Hegde et al, 1977; Luiken et al, 1977). There was no correlation between the platelet count and the membrane bound IgG in our patients. Further, the majority of our patients were not thrombocytopenic, and had no history of abnormal bleeding, even though the levels of platelet bound IgG were in the same range as those seen in autoimmune thrombocytopenia. Thus an antigen-antibody relationship is unlikely. Nonspecific coating of the platelets has been previously suggested to occur in association with high concentrations of paraproteins (Perkins et al, 1970); however, such proteins would normally be expected to be removed by washing. We were unable to remove the excess membrane bound IgG by increasing the number of washes from five to nine, in four patients. Patients with SLE have previously been found to have high levels of platelet bound IgG in the absence of thrombocytopenia (Dixon & Rosse, 1975). Decreased platelet survival and
590
Platelet Membrane IgC
serum antiplatelet antibodies, detected by platelet factor 3 release, have been reported in these patients (Weiss et a l , 1963).In SLE the IgG appears to be in the form of an immune complex (Dixon & Rosse, 1975) which attaches to the platelet membrane and results in increased thrombolysis. A similar mechanism could also be responsible for the increased platelet membrane IgG in rheumatoid arthritis, benign hypergammaglobulinaemic purpura and systemic infections. In paraproteinaemias, however, there is no evidence to suggest immune complex formation. It may be that the attachment of abnormal IgG to the platelet membrane results in increased platelet destruction; however, confirmation of this requires further investigation. The association of high concentrations of paraproteins and abnormal bleeding is well recognized. The bleeding time (Perkins er all 1970),platelet adhesion (Perkins et al, rg~o), platelet factor 3 availability (Pachter et a l , 1959),clot retraction (Cohen et al, 1970),and platelet aggregation in a cone rotational viscometer (Rosenberg & Dintenfass, 1965)have all been reported to be abnormal in patients with multiple myeloma or Waldenstrom’s macroglobulinaemia. Nine of 10 patients in this study demonstrated varying abnormalities of platelet function. Usually, the most severe defects were seen in those patients having the highest levels of platelet bound IgG. Aggregation in response to collagen was the only parameter which was consistently abnormal in all nine patients and was absent in the one patient (A.C.) presenting with frank haemorrhage. The bleeding time, platelet adhesion, platelet aggregation to ADP and epinephrine, and platelet factor 3 availability were each abnormal in 20-30% of the patients, and did not appear to correlate with the bleeding tendency. These findings are consistent with the concept that abnormal platelet function seen in multiple myeloma is a consequence of coating of the platelet by the abnormal protein, but structural and/or metabolic abnormalities in the platelet induced by the malignancy in the bone marrow may also play a role. One patient with benign hypergammaglobulinaemia purpura was investigated to determine whether abnormalities of platelet function are involved in the development of the purpuric lesions seen in this disease. The platelet bound IgG was markedly elevated in this patient (>600 ng IgG/107 platelets) in association with a moderate increase in serum IgG. However, there was no evidence of abnormal platelet function. Decreased platelet factor 3 has been reported in previous studies and attributed to coating of the platelets by the immunoglobulin (Weiss et al, 1963), but was normal in our patient. In most studies the purpura has been attributed to a localized vascular lesion (Baughan et al, 1971;Kyle et all 1971).Thus the contribution of increased platelet IgG to the pathogenesis of the purpura in this disease remains uncertain. These studies demonstrate that increased platelet bound IgG, in the absence of thrombocytopenia, can occur in many conditions associated with increased serum IgG, and therefore the latter should be considered before the presence of an antiplatelet antibody can be confirmed.
ACKNOWLEDGMENTS
Supported by Biomedical Research Support Grant RR-5404 and Forsyth Cancer Service Grant. We are grateful to Mrs Karen Spaugh for her excellent secretarial work.
I(. M.
McCrath,J. J . Stuart and F. Richards, I I
591
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