THROMBOSIS RESEARCH 61; 271-278,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
CIRCULATING
ACTIVATED
PLATELETS DISORDERS
IN MYELOPROLIFERATIVE
A. Wehmeier*, D. Tschijpe **, J. Esser**, C. Menzel*, H.K. Nieuwenhuis*** and W. Schneiddc Department of Haematology, Oncology and Clinical Immunology (*), Heinrich-Heine University, Moorenstr. 5, D-4000 Diisseldorf, FRG, Diabetes Research Institute (**), Heinrich-Heine University, Diisseldorf, FRG, Department of Haematology ( ***), University Hospital Utrecht, The Netherlands (Received
24.9.1990;
accepted
in revised form 14.11 .1990 by Editor H. Pirkle)
ABSTRACT Platelet activation in patients with myeloproliferative disorders is often suggested by increased platelet a-granule secretion and an acquired storage pool defect of dense granules. To determine whether activated platelets circulate in patients with chronic myeloproliierative disorders, we evaluated the binding of monoclonal antibodies against activation-dependent epitopes on resting platelets (P 12, CD 63 , and CD 62 ) in 12 patients with prominent megakazyocytic proliferation (8 patients with essential thrombocythemia, 2 with chronic myeloid leukemia, and 2 patients with polycythemia rubra Vera). In addition, platelet aggregation in response to collagen, adenosine diphosphate, platelet activating factor, and agglutination with ristocetin was investigated. In 3 patients there was an increased percentage of platelets binding at least 1 activation marker. In 2 other patients, a trend towards increased antibody binding was observed. Binding of the antibody to thrombospondin (P 12) was related to expression of the GMP 140 protein (CD 62, r= 0.76, p= 0.004). There was no correlation of platelet aggregation defects in vitro to increased expression of platelet activation markers or to thmmbohaemorrhagic complications. However, circulating activated platelets were detected in three out of five patients with a history of bleeding or thrombotic complications. The results of this preliminary study suggest that some but not all patients with myeloproliferative disorders showed increased amounts of circulating activated platelets. The relation of bleeding and thrombotic complications to the expression of activation-dependent epitopes on platelets in myeloproliferative disorders requires further investigation.
Key words:
Platelets, myeloproliferative disorders, platelet activation, GMP 140 protein, thrombospondin 271
272
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
UVTRODUCUQN
Bleeding and thrombosis in myeloprohferative disorders (MPD) have been attributed to platelet abnormalities as a consequence of clonal megakaryocyte proliferation (review: 1). It has been hypothesized that platelet activation, followed by platelet secretion and recirculation of degranulated platelets are a major cause of thrombohaemorrhagic complications (2). This view is supported by the observation that most patients with MPD have elevated plasma levels of platelet a-granule proteins and a dense granule storage pool defect (3,4). However, the acquired storage pool defect of MPD platelets may be due to an intrinsic abnormality of storage organelles (5) rather than to platelet activation in the circulation. Flow cytometric determination of activation-dependent platelet antibody binding has been shown to detect platelet activation in vivo, e.g. in patients with extracorporeal circulation and diabetes mellitus (6-9). To investigate whether circulating activated platelets are detectable in MPD, and whether they may be related to thrombohaemorrhagic complications, we determined the percentage of thrombospondin, CD 63 and CD 62 positive platelets in relation to platelet function and clinical patient characteristics.
Patientsand controlsubjects Twelve patients with MPD were investigated, the majority classified as essential thrombocythemia (ET, n=8) according to established clinical criteria (10) and the results of a bone marrow biopsy. Two patients suffered from chronic myelogenous leukemia (CML, 1 Philadelphia chromosome positive, 1 Philadelphia status unknown) and 2 from polycythemia rubra Vera (PV), All patients had a prominent megakaryocytic proliferation on bone marrow aspirates. Three patients had a history of thrombosis (ET 1, ET 2, PV 2), and three patients (ET 5, ET 7, PV 2) had experienced bleeding complications. Cytoreductive therapy was discontinued at least two weeks before the investigation. One patient (ET 6) was taking low dose aspirin (100 mg/day) and two patients (ET 2, ET 8) were on oral anticoagulants when studied (Table 1). Fifteen healthy subjects, mostly from the hospital staff, served as a control group for the aggregation parameters. Reference values for the activation markers were obtained from another group of 30 healthy volunteers. Control subjects had not taken platelet-inhibitory drugs at least 14 days before the investigation. Plateletaggregation Platelet aggregation was performed according to the method of Born (11) using a Chrono-Log aggregometer (Model 530, Chrono-Log, Havertown, PA., USA). Platelet rich plasma was prepared from titrated blood (0.38%) by centrifugation at 120 g for 10 min. The platelet count was adjusted to 25O,OOO/plwith autologous platelet poor plasma. Results were judged to be normal or abnormal on the basis of maximal deflection of the aggregation tracing in comparison to control subjects.
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
273
Platelet antibodies Antibody 2.28 (CD 63) binds to a 53 kD protein which has been localised in platelet lysosomes (7). Antibody 2.17 (CD 62) binds to a 140 kD protein (GMP 140) which is present in the a-granule membrane of platelets (13). Both subcellular antigenic epitopes have been shown to be detectable on the outer platelet membrane only after platelet activation (7,12-14). Monoclonal antibody against thrombospondin (P12) was obtained from Immunotech (Marseille, France) (16).
Clinical data of patients with myeloproliferative disorders Sex
Patients [n--121
Age bears]
CMLl cML2 ET 1 ET2
73 36 42 32
Y m
ET3
65
f
ET4 ET 5 ET6 ET7 ET8 PV 1 PV 2
2 33 35 69 73 45
f f f f f m
m
Follow-up [months]
17 20 43 7
Bleeding/Thrombosis
none none Thrombosis venae portae Angina pwtoris
Therapy
BUdfan
a-IFN ncne a-EN, Melphalan, AnticoagulanIs
1 286 4 1 1 108 ~24
none
&Y-
none Postoperative bleeding none ThrombosYFf the aorta, gastrointestinal bleeding
Mer& none Aspirin Anticz&nts Venesection Venesection
Abbreviations: a-IPN = a-Interferon, CML = chronic myelocytic leukemia, ET = essential thrombocythemia, PV = polycythemia vet-a.
Flowcytometric assay The flowcytometric determination of platelet antibody binding was modified according to Tschiipe et al. (16). Fixed platelets were washed twice with sodium-citrate (3.8%) -phosphate buffered saline (PBS) and sodium-citrate (3.8 %)-PBS containing 10% (volume)of rabbit serum (Behringwerke, Marburg, FRG). 200 ~1 of washed platelets were diluted to SO.OOO/~land simultaneously stained by 50 ~1 of 2.28 and 2.17 monoclonal antibody (5 cLg/ml) for 60 min at room temperature. Isotype-matched mouse IgG (1 pg/ml, Coulter Electronics, Hialeah, FL.,USA) was used for unspecific control staining. For fluorescence labelling 100 ~1 sheep antimouse F(ab)2-FITC fragments (62.5 pg/ml, Sigma Chemicals, St. Louis, MO., USA) were added for a further 30-min incubation. Finally, platelets were washed twice in sodium-citrate (3.8%)-PBS buffer and resuspended in Isoton-II-solution (Coulter Electronics). Immediately after staining fluorescence of 10,000 platelets was measured with a FACS-Star instrument (Becton-Dickinson, Mountain View, CA., USA) at an analysing rate of 1000/s and at 488 nm excitation wave length (200 mW laser power). All data were digitally stored and processed as list mode files using Consort 30 and Paint-A-Gate software (Becton Dickinson). The percentage of specific fluorescence-positive platelets was obtained after subtraction of unspecific MsIgG
CIRCULATING ACTIVATED PLATELETS
274
Vol. 61, No. 3
binding. Fluorescence signal discrimination and intensity were calibrated daily using fluorescent microbead standards conjugated with definite numbers of FITC molecules (Plowcytometry Standards Corporation, NC., USA) at logarithmic photomultiplier settings. statistical analysis
Linear regression analysis was performed using a statistical software package (StatView, Abacus Concepts, Berkeley, CA., USA) on a microcomputer. As the percentage of platelets expressing activation-dependent epitopes was not normally distributed in control subjects, the geometric mean of these parameters is reported. Accordingly, the upper limit of normal was defined as geometric mean x (standarddeviation facto@.
ULTS Plateletjknctionin vitro
Platelet function defects were mainly observed with low concentrations of collagen (1 @/ml) and upon agglutination of platelets with ristocetin (1.2 mg/ml). The most severe aggregation abnormalities were found in patients CML 1, ET 2, ET 6, PV 1 and PV 2 (Table 2).
Blood count and platelet aggregation parameters in myeloproliferative disorders Patient
CMLl
cML2 ET1 ET2 ET3 ET4 ET5 ET 6* ET7 ET8 PV 1 PV2
Blood count WBC
Hct
109/l
%
61.3 571.0
53.4
14.0
39.9 48.1 50.5 34.4 57.2 39.6 34.1 43.9 56.0 58.1
;-:. 2415 16.1 12.0 21-I 27:9 11.5
Platelet aggregation
Platelets 109/l
445 191 476 826 z 1149 939 3100 943 459 414
Collagen 1 pg/ml
NA ii NA NA N z
_ _ N NA
PM 1 pg/ml
RistoCdl
1.2 mg/ml
N
NA N
N N N N
:;
z
N _ _
fl
N A NAY _ Ni N
Abbreviations: WBG white blood cell count, Hcb haematocd, N= Normal aggregation response, A= Abnormaf aggregation response. NA= No aggregation response, *= patient was on aspirin (100 mg/day).
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
275
10 iz 8
8 6
10
5 aA
20
15
25
30
GMxSDF2
Thrombospondln
FIGURE Correlation between binding of thrombospondin (P 12) and CD 62 antibodies (% of platelets binding the antibody) in 12 patients with myeloproliferative disorders. Patients with bleeding or thrombotic complications are marked by a solid square. The geometric mean (GM) and upper limit of normal (GM x SD@) are indicated for both parameters. 30
0
k cu v) 25 II
?? ? ?? ?? ?? ??
....~.....
i 20. 8 8
1
15.
10.
0 fi
5
0 GM
??
10
0
a
15
Thrombospondin
20
25
30
GMxSDF2
FIGURE Correlation between binding of thrombospondin (P 12) and CD 63 antibodies (% of platelets binding the antibody) in 12 patients with myeloprolifemtive disorders. Patients with bleeding or thrombotic complications are marked by a solid square. The geometric mean (GM) and upper limit of normal (GM x SD@) are indicated for both parameters.
276
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
Platelet antibody binding There was only one patient (PV 1) showing increased percentage of platelet binding with two antibodies (CD 62 and P 12). Another two patients (CML 2, ET 5) had increased platelet binding of CD 63. There was also a trend towards increased antibody binding (not exceeding the upper limit of normal) in patients ET 1 (P 12), PV 2 (all three antibodies) and of P 12 in patient ET 5. Binding of the antibody P 12 was correlated with binding of the CD 62 antibody (r=O.76, p=O.O04, Figure 1) but not with binding of the CD 63 antibody (Figure 2). There was a correlation (although not statistically significant for antibodies P 12 and CD 62) between haematocrit levels and platelet antibody binding (P 12: r=O.46, p=O.16; CD 62: t=O.56, p=O.O8; CD 63: r=O.6, p=O.O5). One patient with increased platelet binding of CD 63 (CML 2) had a low haematocrit but a very high white blood cell count. Platelet antibody binding was not related to platelet aggregation defects in vitro.
In this group of 12 patients with MPD, there was evidence of circulating activated platelets determined by the binding of antibodies to activation-dependent platelet epitopes in at least three patients. Two other patients had elevated antibody binding below the upper limit of the control values. Three of these patients had an increased red cell mass with a haematocrit of >55%. Two patients had polycythemia Vera, and one was classified as essential thrombocythemia because of his very prominent megakaryocytopoiesis, but also had a less prominent proliferation of the white cell and red cell lines. Platelet activation because of rheologic abnormalities has been well documented in patients with high haematocrit, and platelet dysfunction and thrombotic complications are more often encountered in polycythemia rubra vera than in other entities of MPD (4). One patient with untreated CML and very high WBC had a high binding of the antibody CD 63 but normal binding of the other two markers. Binding of the P 12 antibody was related to expression of the GMP 140 protein (located on the a-granule membrane) but not to binding of the CD 63 antibody. In this series, 5 patients had a history of bleeding or thrombotic complications, and 3 of these patients were found to have circulating activated platelets. However, the expression of activationdependent epitopes on platelets from patients with a history of bleeding or thrombotic complications was slightly but not significantly higher than from patients without complications. Most patients were given a diagnosis of ET, and this group may have a relatively low risk of thrombohaemorrhagic complications especially at young age (17). Many patients had some platelet aggregation abnormalities, and most had a high platelet count at the time of investigation. However, there was no correlation between thrombohaemorrhagic complications and platelet function abnormalities in vitro. Platelet function defects in vitro, increased plasma levels of platelet a-granule proteins, and the acquired platelet storage pool defect may be different expressions of the underlying clonal megakaryocyte abnormality. It is as yet unknown whether platelet activation as evidenced by expression of activation-dependent membrane epitopes is related to these intrinsic abnormalities. It also remains to be established in a larger patient population whether antibody binding to activation-dependent platelet epitopes is predictive of thrombohaemorrhagic complications, and which combination of antibodies may be useful to determine platelet activation in this heterogeneous group of stem cell disorders.
Vol. 61, No. 3
CIRCULATING ACTIVATED PLATELETS
277
1.
SCHAFER, A. I. Bleeding and thrombosis in the myeloproliferative 1-12, 1984
disorders. Blood 64,
2.
BOUGHTON, B. J., CORBETI’, W. E. N., GINSBURG, A. D. Myeloproliferative disorders: a paradox of in-viva and in-vitro platelet function. J. Clin. Path. 30, 228-234, 1977
3.
PARETI, F. I., GUGLIOTTA, L., MANNUCCI, L., GUARINI, A., MANNUCCI, P. M. Biochemical and metabolic aspects of platelet dysfunction in chronic myeloproliferative disorders. Thromb. Haemostas. 47, 84-89, 1982
4.
WEHMEIER, A., SCHARF, R. E., FRICKE, S., SCHNEIDER, W. Bleeding and thrombosis in chronic myeloproliferative disorders: Relation of platelet disorders to clinical aspects of the disease. Haemostasis 19,251-259, 1989
5.
CARANOBE, C., SIE, P., FERNANDEZ, F., PRIS, J., MOAT-II, S., BONEU, B. Abnormal platelet serotonin uptake and binding sites in myeloproliferative disorders. Thromb. Haemostas. 51,349-353,1984
6.
BERMAN, C. L., YEO, E. L., WENCEL-DRAKE, J .D., FURIE, B. C., GINSBURG, M. H., FURIE, B. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein. J. Inva 78,130-137,1986
7.
NIEUWENHUIS, H. K., VAN OOSTERHOUT, J. J. G., ROZEMULLER, E., VAN IWAARDEN, F., SIXMA, J. J. Studies with a monoclonal antibody against activated platelets: Evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. &$ 70,838-845, 1987
8.
LEGRAND, C., DUBERNARD, V., KIEFFER, N., NURDEN, A. T. Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation. Eur. J. Biochem. 171,393-399, 1988
9.
TSCHOEPE, D., ESSER, J., ROESEN, P., SCHWIPPERT, B., GRIES, F.A . Evidence for enhanced “in viva” platelet activation in diabetes by direct flow-cytometric analysis of the functional platelet status. Diabetolozia 7, 1989
10.
MURPHY, S., ILAND, H., ROSENTHAL, D., LASZLO, J. Essential thrombocythemia: An interim report from the polycythemia Vera study group. Semin. Hematol. 23, 177- 182, 1986
11.
BORN, G.V.R. Aggregation of blood platelets by adenosine diphosphate and its reversal, Nature 194,927-929,1962
278
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
12.
METZELAAR, M.J., SIKMA, J.J., NIEUWENHUIS, H.K. Activation dependent mAb recognizing a 140 kD platelet alpha-granule membrane protein, expressed after activation. In: Knapp W, D&ken B, Rieber P, Schmidt RE, Stein H, von dem Borne AEGK (Eds.) Leukocyte typing IV. White cell differentiation antigens. Oxford University Press, Oxford, New York, Tokyo 1989, p. 1039-1040
13.
STENBERG, P.E., McEVER, P.R., SHUMAN, M.A., JACQUES, Y.V., BAINTON, D.F. A platelet a-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J. Cell I&&101,880-886,198s
14.
McEVER, R.P. Properties of GMP-140, an inducible granule membrane protein of platelets and endothelium. Blood CeuS 16,73-83,199O
15.
CLEZARDIN, PH., McGREGOR, J.L., LYON, M., CLEMETSON, K.J., HUPPERT, J. Characterisation of two murine monoclonals (P 10, P 12) directed against different determinants on human blood platelet thrombospondin. m. r 154, 95102, 1986
16.
TSCHOEPE, D., SPANGENBERG, P., ESSER, J., SCHWIPPERT, B., KEHREL, B., ROESEN, P., GRIES, F.A. Flow-cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets. cvtometrv 11, 652-656, 1990
17.
HOAGLAND, H.C., SILVERSTEIN, M. N. Primary thrombocythemia patient. Mavo Clin. Proc. 53,578-580,1978
in the young
CIRCULATING
ACTIVATED
PLATELETS DISORDERS
IN MYELOPROLIFERATIVE
A. Wehmeier*, D. Tschijpe **, J. Esser**, C. Menzel*, H.K. Nieuwenhuis*** and W. Schneiddc Department of Haematology, Oncology and Clinical Immunology (*), Heinrich-Heine University, Moorenstr. 5, D-4000 Diisseldorf, FRG, Diabetes Research Institute (**), Heinrich-Heine University, Diisseldorf, FRG, Department of Haematology ( ***), University Hospital Utrecht, The Netherlands (Received
24.9.1990;
accepted
in revised form 14.11 .1990 by Editor H. Pirkle)
ABSTRACT Platelet activation in patients with myeloproliferative disorders is often suggested by increased platelet a-granule secretion and an acquired storage pool defect of dense granules. To determine whether activated platelets circulate in patients with chronic myeloproliierative disorders, we evaluated the binding of monoclonal antibodies against activation-dependent epitopes on resting platelets (P 12, CD 63 , and CD 62 ) in 12 patients with prominent megakazyocytic proliferation (8 patients with essential thrombocythemia, 2 with chronic myeloid leukemia, and 2 patients with polycythemia rubra Vera). In addition, platelet aggregation in response to collagen, adenosine diphosphate, platelet activating factor, and agglutination with ristocetin was investigated. In 3 patients there was an increased percentage of platelets binding at least 1 activation marker. In 2 other patients, a trend towards increased antibody binding was observed. Binding of the antibody to thrombospondin (P 12) was related to expression of the GMP 140 protein (CD 62, r= 0.76, p= 0.004). There was no correlation of platelet aggregation defects in vitro to increased expression of platelet activation markers or to thmmbohaemorrhagic complications. However, circulating activated platelets were detected in three out of five patients with a history of bleeding or thrombotic complications. The results of this preliminary study suggest that some but not all patients with myeloproliferative disorders showed increased amounts of circulating activated platelets. The relation of bleeding and thrombotic complications to the expression of activation-dependent epitopes on platelets in myeloproliferative disorders requires further investigation.
Key words:
Platelets, myeloproliferative disorders, platelet activation, GMP 140 protein, thrombospondin 271
272
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
UVTRODUCUQN
Bleeding and thrombosis in myeloprohferative disorders (MPD) have been attributed to platelet abnormalities as a consequence of clonal megakaryocyte proliferation (review: 1). It has been hypothesized that platelet activation, followed by platelet secretion and recirculation of degranulated platelets are a major cause of thrombohaemorrhagic complications (2). This view is supported by the observation that most patients with MPD have elevated plasma levels of platelet a-granule proteins and a dense granule storage pool defect (3,4). However, the acquired storage pool defect of MPD platelets may be due to an intrinsic abnormality of storage organelles (5) rather than to platelet activation in the circulation. Flow cytometric determination of activation-dependent platelet antibody binding has been shown to detect platelet activation in vivo, e.g. in patients with extracorporeal circulation and diabetes mellitus (6-9). To investigate whether circulating activated platelets are detectable in MPD, and whether they may be related to thrombohaemorrhagic complications, we determined the percentage of thrombospondin, CD 63 and CD 62 positive platelets in relation to platelet function and clinical patient characteristics.
Patientsand controlsubjects Twelve patients with MPD were investigated, the majority classified as essential thrombocythemia (ET, n=8) according to established clinical criteria (10) and the results of a bone marrow biopsy. Two patients suffered from chronic myelogenous leukemia (CML, 1 Philadelphia chromosome positive, 1 Philadelphia status unknown) and 2 from polycythemia rubra Vera (PV), All patients had a prominent megakaryocytic proliferation on bone marrow aspirates. Three patients had a history of thrombosis (ET 1, ET 2, PV 2), and three patients (ET 5, ET 7, PV 2) had experienced bleeding complications. Cytoreductive therapy was discontinued at least two weeks before the investigation. One patient (ET 6) was taking low dose aspirin (100 mg/day) and two patients (ET 2, ET 8) were on oral anticoagulants when studied (Table 1). Fifteen healthy subjects, mostly from the hospital staff, served as a control group for the aggregation parameters. Reference values for the activation markers were obtained from another group of 30 healthy volunteers. Control subjects had not taken platelet-inhibitory drugs at least 14 days before the investigation. Plateletaggregation Platelet aggregation was performed according to the method of Born (11) using a Chrono-Log aggregometer (Model 530, Chrono-Log, Havertown, PA., USA). Platelet rich plasma was prepared from titrated blood (0.38%) by centrifugation at 120 g for 10 min. The platelet count was adjusted to 25O,OOO/plwith autologous platelet poor plasma. Results were judged to be normal or abnormal on the basis of maximal deflection of the aggregation tracing in comparison to control subjects.
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
273
Platelet antibodies Antibody 2.28 (CD 63) binds to a 53 kD protein which has been localised in platelet lysosomes (7). Antibody 2.17 (CD 62) binds to a 140 kD protein (GMP 140) which is present in the a-granule membrane of platelets (13). Both subcellular antigenic epitopes have been shown to be detectable on the outer platelet membrane only after platelet activation (7,12-14). Monoclonal antibody against thrombospondin (P12) was obtained from Immunotech (Marseille, France) (16).
Clinical data of patients with myeloproliferative disorders Sex
Patients [n--121
Age bears]
CMLl cML2 ET 1 ET2
73 36 42 32
Y m
ET3
65
f
ET4 ET 5 ET6 ET7 ET8 PV 1 PV 2
2 33 35 69 73 45
f f f f f m
m
Follow-up [months]
17 20 43 7
Bleeding/Thrombosis
none none Thrombosis venae portae Angina pwtoris
Therapy
BUdfan
a-IFN ncne a-EN, Melphalan, AnticoagulanIs
1 286 4 1 1 108 ~24
none
&Y-
none Postoperative bleeding none ThrombosYFf the aorta, gastrointestinal bleeding
Mer& none Aspirin Anticz&nts Venesection Venesection
Abbreviations: a-IPN = a-Interferon, CML = chronic myelocytic leukemia, ET = essential thrombocythemia, PV = polycythemia vet-a.
Flowcytometric assay The flowcytometric determination of platelet antibody binding was modified according to Tschiipe et al. (16). Fixed platelets were washed twice with sodium-citrate (3.8%) -phosphate buffered saline (PBS) and sodium-citrate (3.8 %)-PBS containing 10% (volume)of rabbit serum (Behringwerke, Marburg, FRG). 200 ~1 of washed platelets were diluted to SO.OOO/~land simultaneously stained by 50 ~1 of 2.28 and 2.17 monoclonal antibody (5 cLg/ml) for 60 min at room temperature. Isotype-matched mouse IgG (1 pg/ml, Coulter Electronics, Hialeah, FL.,USA) was used for unspecific control staining. For fluorescence labelling 100 ~1 sheep antimouse F(ab)2-FITC fragments (62.5 pg/ml, Sigma Chemicals, St. Louis, MO., USA) were added for a further 30-min incubation. Finally, platelets were washed twice in sodium-citrate (3.8%)-PBS buffer and resuspended in Isoton-II-solution (Coulter Electronics). Immediately after staining fluorescence of 10,000 platelets was measured with a FACS-Star instrument (Becton-Dickinson, Mountain View, CA., USA) at an analysing rate of 1000/s and at 488 nm excitation wave length (200 mW laser power). All data were digitally stored and processed as list mode files using Consort 30 and Paint-A-Gate software (Becton Dickinson). The percentage of specific fluorescence-positive platelets was obtained after subtraction of unspecific MsIgG
CIRCULATING ACTIVATED PLATELETS
274
Vol. 61, No. 3
binding. Fluorescence signal discrimination and intensity were calibrated daily using fluorescent microbead standards conjugated with definite numbers of FITC molecules (Plowcytometry Standards Corporation, NC., USA) at logarithmic photomultiplier settings. statistical analysis
Linear regression analysis was performed using a statistical software package (StatView, Abacus Concepts, Berkeley, CA., USA) on a microcomputer. As the percentage of platelets expressing activation-dependent epitopes was not normally distributed in control subjects, the geometric mean of these parameters is reported. Accordingly, the upper limit of normal was defined as geometric mean x (standarddeviation facto@.
ULTS Plateletjknctionin vitro
Platelet function defects were mainly observed with low concentrations of collagen (1 @/ml) and upon agglutination of platelets with ristocetin (1.2 mg/ml). The most severe aggregation abnormalities were found in patients CML 1, ET 2, ET 6, PV 1 and PV 2 (Table 2).
Blood count and platelet aggregation parameters in myeloproliferative disorders Patient
CMLl
cML2 ET1 ET2 ET3 ET4 ET5 ET 6* ET7 ET8 PV 1 PV2
Blood count WBC
Hct
109/l
%
61.3 571.0
53.4
14.0
39.9 48.1 50.5 34.4 57.2 39.6 34.1 43.9 56.0 58.1
;-:. 2415 16.1 12.0 21-I 27:9 11.5
Platelet aggregation
Platelets 109/l
445 191 476 826 z 1149 939 3100 943 459 414
Collagen 1 pg/ml
NA ii NA NA N z
_ _ N NA
PM 1 pg/ml
RistoCdl
1.2 mg/ml
N
NA N
N N N N
:;
z
N _ _
fl
N A NAY _ Ni N
Abbreviations: WBG white blood cell count, Hcb haematocd, N= Normal aggregation response, A= Abnormaf aggregation response. NA= No aggregation response, *= patient was on aspirin (100 mg/day).
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
275
10 iz 8
8 6
10
5 aA
20
15
25
30
GMxSDF2
Thrombospondln
FIGURE Correlation between binding of thrombospondin (P 12) and CD 62 antibodies (% of platelets binding the antibody) in 12 patients with myeloproliferative disorders. Patients with bleeding or thrombotic complications are marked by a solid square. The geometric mean (GM) and upper limit of normal (GM x SD@) are indicated for both parameters. 30
0
k cu v) 25 II
?? ? ?? ?? ?? ??
....~.....
i 20. 8 8
1
15.
10.
0 fi
5
0 GM
??
10
0
a
15
Thrombospondin
20
25
30
GMxSDF2
FIGURE Correlation between binding of thrombospondin (P 12) and CD 63 antibodies (% of platelets binding the antibody) in 12 patients with myeloprolifemtive disorders. Patients with bleeding or thrombotic complications are marked by a solid square. The geometric mean (GM) and upper limit of normal (GM x SD@) are indicated for both parameters.
276
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
Platelet antibody binding There was only one patient (PV 1) showing increased percentage of platelet binding with two antibodies (CD 62 and P 12). Another two patients (CML 2, ET 5) had increased platelet binding of CD 63. There was also a trend towards increased antibody binding (not exceeding the upper limit of normal) in patients ET 1 (P 12), PV 2 (all three antibodies) and of P 12 in patient ET 5. Binding of the antibody P 12 was correlated with binding of the CD 62 antibody (r=O.76, p=O.O04, Figure 1) but not with binding of the CD 63 antibody (Figure 2). There was a correlation (although not statistically significant for antibodies P 12 and CD 62) between haematocrit levels and platelet antibody binding (P 12: r=O.46, p=O.16; CD 62: t=O.56, p=O.O8; CD 63: r=O.6, p=O.O5). One patient with increased platelet binding of CD 63 (CML 2) had a low haematocrit but a very high white blood cell count. Platelet antibody binding was not related to platelet aggregation defects in vitro.
In this group of 12 patients with MPD, there was evidence of circulating activated platelets determined by the binding of antibodies to activation-dependent platelet epitopes in at least three patients. Two other patients had elevated antibody binding below the upper limit of the control values. Three of these patients had an increased red cell mass with a haematocrit of >55%. Two patients had polycythemia Vera, and one was classified as essential thrombocythemia because of his very prominent megakaryocytopoiesis, but also had a less prominent proliferation of the white cell and red cell lines. Platelet activation because of rheologic abnormalities has been well documented in patients with high haematocrit, and platelet dysfunction and thrombotic complications are more often encountered in polycythemia rubra vera than in other entities of MPD (4). One patient with untreated CML and very high WBC had a high binding of the antibody CD 63 but normal binding of the other two markers. Binding of the P 12 antibody was related to expression of the GMP 140 protein (located on the a-granule membrane) but not to binding of the CD 63 antibody. In this series, 5 patients had a history of bleeding or thrombotic complications, and 3 of these patients were found to have circulating activated platelets. However, the expression of activationdependent epitopes on platelets from patients with a history of bleeding or thrombotic complications was slightly but not significantly higher than from patients without complications. Most patients were given a diagnosis of ET, and this group may have a relatively low risk of thrombohaemorrhagic complications especially at young age (17). Many patients had some platelet aggregation abnormalities, and most had a high platelet count at the time of investigation. However, there was no correlation between thrombohaemorrhagic complications and platelet function abnormalities in vitro. Platelet function defects in vitro, increased plasma levels of platelet a-granule proteins, and the acquired platelet storage pool defect may be different expressions of the underlying clonal megakaryocyte abnormality. It is as yet unknown whether platelet activation as evidenced by expression of activation-dependent membrane epitopes is related to these intrinsic abnormalities. It also remains to be established in a larger patient population whether antibody binding to activation-dependent platelet epitopes is predictive of thrombohaemorrhagic complications, and which combination of antibodies may be useful to determine platelet activation in this heterogeneous group of stem cell disorders.
Vol. 61, No. 3
CIRCULATING ACTIVATED PLATELETS
277
1.
SCHAFER, A. I. Bleeding and thrombosis in the myeloproliferative 1-12, 1984
disorders. Blood 64,
2.
BOUGHTON, B. J., CORBETI’, W. E. N., GINSBURG, A. D. Myeloproliferative disorders: a paradox of in-viva and in-vitro platelet function. J. Clin. Path. 30, 228-234, 1977
3.
PARETI, F. I., GUGLIOTTA, L., MANNUCCI, L., GUARINI, A., MANNUCCI, P. M. Biochemical and metabolic aspects of platelet dysfunction in chronic myeloproliferative disorders. Thromb. Haemostas. 47, 84-89, 1982
4.
WEHMEIER, A., SCHARF, R. E., FRICKE, S., SCHNEIDER, W. Bleeding and thrombosis in chronic myeloproliferative disorders: Relation of platelet disorders to clinical aspects of the disease. Haemostasis 19,251-259, 1989
5.
CARANOBE, C., SIE, P., FERNANDEZ, F., PRIS, J., MOAT-II, S., BONEU, B. Abnormal platelet serotonin uptake and binding sites in myeloproliferative disorders. Thromb. Haemostas. 51,349-353,1984
6.
BERMAN, C. L., YEO, E. L., WENCEL-DRAKE, J .D., FURIE, B. C., GINSBURG, M. H., FURIE, B. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein. J. Inva 78,130-137,1986
7.
NIEUWENHUIS, H. K., VAN OOSTERHOUT, J. J. G., ROZEMULLER, E., VAN IWAARDEN, F., SIXMA, J. J. Studies with a monoclonal antibody against activated platelets: Evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. &$ 70,838-845, 1987
8.
LEGRAND, C., DUBERNARD, V., KIEFFER, N., NURDEN, A. T. Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation. Eur. J. Biochem. 171,393-399, 1988
9.
TSCHOEPE, D., ESSER, J., ROESEN, P., SCHWIPPERT, B., GRIES, F.A . Evidence for enhanced “in viva” platelet activation in diabetes by direct flow-cytometric analysis of the functional platelet status. Diabetolozia 7, 1989
10.
MURPHY, S., ILAND, H., ROSENTHAL, D., LASZLO, J. Essential thrombocythemia: An interim report from the polycythemia Vera study group. Semin. Hematol. 23, 177- 182, 1986
11.
BORN, G.V.R. Aggregation of blood platelets by adenosine diphosphate and its reversal, Nature 194,927-929,1962
278
CIRCULATING ACTIVATED PLATELETS
Vol. 61, No. 3
12.
METZELAAR, M.J., SIKMA, J.J., NIEUWENHUIS, H.K. Activation dependent mAb recognizing a 140 kD platelet alpha-granule membrane protein, expressed after activation. In: Knapp W, D&ken B, Rieber P, Schmidt RE, Stein H, von dem Borne AEGK (Eds.) Leukocyte typing IV. White cell differentiation antigens. Oxford University Press, Oxford, New York, Tokyo 1989, p. 1039-1040
13.
STENBERG, P.E., McEVER, P.R., SHUMAN, M.A., JACQUES, Y.V., BAINTON, D.F. A platelet a-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J. Cell I&&101,880-886,198s
14.
McEVER, R.P. Properties of GMP-140, an inducible granule membrane protein of platelets and endothelium. Blood CeuS 16,73-83,199O
15.
CLEZARDIN, PH., McGREGOR, J.L., LYON, M., CLEMETSON, K.J., HUPPERT, J. Characterisation of two murine monoclonals (P 10, P 12) directed against different determinants on human blood platelet thrombospondin. m. r 154, 95102, 1986
16.
TSCHOEPE, D., SPANGENBERG, P., ESSER, J., SCHWIPPERT, B., KEHREL, B., ROESEN, P., GRIES, F.A. Flow-cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets. cvtometrv 11, 652-656, 1990
17.
HOAGLAND, H.C., SILVERSTEIN, M. N. Primary thrombocythemia patient. Mavo Clin. Proc. 53,578-580,1978
in the young
