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Platelet von Willebrand factor: Comparison with plasma von Willebrand factor
THROMBOSIS RESEARCH 38; 623-633, 1985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (cl 1985 Pergamon Press Ltd. All rights reserved.
PLATELET VON WILLEBRAND FACTOR: COMPARISON WITH PLASMA VON WILLEBRAND FACTOR
Harvey R. Gralnick, Hematology
Sybil B. Williams, Laurie P. McKeown, Dennis H. Krizek, Brenda C. Shafer and Margaret E. Rick Service, Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland
U.S.A. (Received 15.1.1985; Accepted in revised form 22.2.1985 by Editor J.G. White) ABSTRACT Platelet von Willebrand factor (vWf) was compared to its plasma The platelet vWf was different from plasma vWf in that counterpart. .l) the multimeric organization was different, 2) larger multimers were present, and 3) the ratio of vWf activity to antigen was higher. Platelet and plasma vWf were similar in their antigenic reactivity in the electroimmunoassay and by liquid phase radioimmunoassay. The amount of vWf activity in large platelets was significantly higher than in normal platelets while the antigen content, although somewhat higher, was not significant. These studies show differences between normal platelet and plasma vWf, and also suggest that platelet size must be considered when platelet vWf is measured in disease states.
INTRODUCTION The plasma von Willebrand factor (vWf) has been characterized as a macromolecular glycoprotein. This protein has a multimeric organization with the molecular weight varying from the smallest plasma multimer of approximately 800,000 daltons to the largest molecular weight multimers ranging between 5-20 million daltons (l-3). vWf also has been localized in platelets and the platelet vWf is synthesized in the megakaryocyte, while the plasma vWf is thought to originate in the endothelial cell (4-13). Differences between normal platelet and plasma vWf have been reported with the major difference observed being the presence of larger niolecular weight multimers in the platelets as compared to plasma (1,2,14). We have undertaken a study to further characterize the difference(s) between the normal platelet and plasma VWf. Key words: Platelet von Willebrand factor , plasma von Willebrand factor,
platelet size
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MATERIALS AND METHODS Blood was obtained from 15 normal blood donors by a two-syringe technique using a 19-gauge butterfly needle and polypropylene syringes. Three of the 15 normal blood donors were known to have an increased mean platelet size. Donors were fully informed regarding the potential risk of venipuncture. The first 5 ml of blood were drawn for blood counts, and the subsequent samples were utilized for the studies described below. Blood for plasma separation was collected into polypropylene tubes containing sodium citrate (0.32% final concentration, 10.9 mM) or the same citrate anticoagulant containing 5 mM EDTA, 1 mM leupeptin and 6 mM n-ethylmaleimide (Aldrich Chemical Co., Milwaukee, WI). The blood was centrifuged at 2000 g for 15 minutes at 4"C, and the cell-free plasma was removed and used fresh or frozen at -7O*C for subsequent testing. Blood for the platelet studies was collected in an anticoagulant of 109 mM sodium citrate and 1 mM EDTA (final concentration 10.9 mM sodium citrate and 1 mM EDTA) and in an anticoagulant of sodium citrate-EDTA, leupeptin, and n-ethylmaleimide (final concentration 10.9 mM sodium cirate, 5 mM EDTA, 1 mM leupeptin and 6 mM n-ethylmaleimide). Platelet-rich plasma (PRP) was prepared by centrifugation at 750 g for 3 minutes at room temperature. After removal of the PRP, the red cells were washed once with Tris-NaCl-EDTA-bovine serum albumin, pH 6.8 (Tris 0.1 M, 0.15 M NaCl, 0.003 H EDTA, 0.5% bovine serum albumin; Pentex Bovine Serum Albumin, Fraction V, Miles Laboratories, Naperville, IL) and the wash was added to the PRP. Over 85% of the total platelet population was recovered. The PRP and wash were separated from plasma proteins on a discontinuous arabinogalactan gradient (3 ml of 20% and 5 ml of 10% arabinogalactan). The gradient was centrifuged at 2500 RCF for 30 minutes at 24“C. The platelets were removed and resuspended in the TrisNaCl-EDTA-bovine serum albumin buffer and washed once with the same buffer (2000 RCT for 20 minutes at 24°C. After the wash, the platelets were resuspended in a Tris-NaCl-bovine serum albumin buffer, pH 7.4 (Tris 0.1 H, The platelets NaCl 0.15 M, bovine serum albumin 3%), at 37°C for 15 minutes. were then counted and diluted in the same buffer to a final concentration of 106/Pl. The platelets were lysed by the addition of l/40 volume of 20% Triton The frozen X 100, incubated for 1 hour at 37'C, then frozen at -70°C. samples, they were centrifuged for 30 minutes at 10,000 RCF at 4°C and the supernatant removed and analyzed. The f.VIII-related antigen (VIII R:Ag) assays were performed by the electroimmunoassay employing a goat anti-f.VIII/vWf antibody (15) and by radioimmunoassay utilizing a rabbit anti-f.VIII/vWf antibody (16). vWf activity was assayed using the formalin-fixed platelet assay as previously described (15). A pool of normal plasma (50 donors) was used in establishing the standard curves for the antigen and vWf activities. In determining the level of VIII R:Ag and vWf, only those points which fell on the parallel At least 2 diluportion of the curve were used for estimation of potency. tions of plasma and platelet lysate were assayed for VIII R:Ag and at least 3 dilutions of plasma or platelet lysate (in duplicate) were analyzed in the vWf assay. Normal plasma and platelet vWf were prepared and analyzed on 3 separate occasions on two normals. Glyoxyl agarose electrophoresis was performed using a modification of the technique described by Hoyer and Shainoff (17). The modification consisted of using 0.5 mm thick gels and a plying only 10 pl of sample in the wells. The gels were incubated with an 155 I-labelled immunopurified rabbit anti-human
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factor VIIIlvon Willebrand factor (f.VIII/vWf) antibody, and the radioautograph developed on XAR-2 film (Eastman Kodak, Rochester, NY). In experiments to determine if Triton X-100 influenced the measurement of vWf antigen or activity, we added l/40 volume of 20% Triton X-100 to plasma and the vWf antigen and activity were measured. Controls were plasma to which l/40 volume of 0.14 NaCl was added. Platelets (log/ml) or the Triton-X-100 lysate of platelets from a patient with severe vWd (bleeding time >30 minutes, factor VIII C <5%, factor VIII:RAg <5% and vWf activity <5%) were incubated with either normal plasma (1:l ratio) or labelled or unlabelled purified plasma vWf (2-4 ng) diluted in the plasma of a patient with severe vWd or buffer. We incubated the mixture at 37°C (same temperature as we incubate our platelets) and analyzed the plasma vWf multimeric structure by agarose gel electrophoreeis over time. Similar studies were performed with 1251 plasma vWf and normal platelet lysate. Platelet size was measured on a Cellozone Cell Counter (Particle Data, Elmhurst, IL) as previously described (18,19). Platelet associated IgG and platelet aggregation studies were performed as previously described (15). Platelet
protein
was measured
by the method of Lowry et al. (20). RESULTS
The mean plasma VIII R:Ag was 0.96 U/ml (range 0.55-1.68, n=12) when measured by the Laurel1 electroimmunoassay and 1.11 U/ml (range 0.71-1.47) by the radioimmunoassay. The mean plasma vWf activity was 1.07 U/ml (range 0.771.33, n-12). The mean antigen content by electroimmunoassay was 0.44 U/log platelets (range 0.26-0.58, n=12). The vWf activity was 0.91 U/log platelets (range 0.67-1.27, n=12, Table 1). The mean ratio of plasma vWf to plasma VIII R:Ag was 1.11 when the antigen was measured by the Laurel1 technique; in
TABLE 1 von Willebrand
Plasma (U/ml)
Platelet (U/109 platelets)
Factor Antigen
and Activity
in Normal Plasma and Platelets
VWf
VIIIR:Ag*
1.07 (0.77-1.33)
0.96 (0.55-1.68)
0.91 (0.67-1.27)
0.44t (0.26-0.58)
*Antigen assays by electroimmunoassay. ilOg platelets/ml. n = 12 normal donors with normal platelet
size.
Ratio vWf/Ag
1.11 (0.63-1.54)
2.07 (1.57-2.89)
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platelets, this ratio was 2.07. Similar results were seen in studies when the antigen data obtained by radioimmunoassay were analyzed. The ratio of plasma vWf to VIII R:Ag was 0.96 and in platelets the ratio was 1.52. The differences noted are primarily due to the relative reduction in VIII R:Ag in platelets. No differences in antigen levels or vWf activity were seen when plasma or platelets were collected in the presence or absence of the proteolytic inhibitors. Triton X-100 added to normal plasma did not change the vWf antigen level or the vWf activity. The mean platelet size of 3 of the normal individuals was high, i.e., 9.86, 9.49, 10.07~~. respectively (normal range derived from mean f.2 SD; 6.56 ? 1.64 u3). The mean platelet size for the other donors was normal, i.e., greater than 4.92 u3 and less than 8.20 P3. Platelet associated IgG and platelet aggregation studies have been repeatedly normal in these 3 individuals, and they have no clinical bleeding history despite major surgical challenges. The protein content of the latelets from these 3 individual was within our normal range (mean 1.92 mg/lO6 platelets, range 1.68-2.18). The values for these three individuals were 1.76, 2.07, and 1.87 mg/lOg platelets, respectively. The plasma vWf, VIII R:Ag and mean ratio of vWf to VIII R:Ag of the 3 donors with large platelets were similar to the results of the other normals. In contrast, the platelet values were higher than normal (Table 2). In particular, the platelet vWf activity of all 3 normals with lar e platelets was greater than the normal range, i.e., 1.30, 1.45, and 1.98 U/10B platelets vs. 0.67-1.27 U/log platelets normal range, and the platelet VIII R:Ag was near or outside the upper limit of the normal range (0.44, 0.52 and 0.83 U/ml; normal range 0.26-O-58 U/ml).
TABLE 2 von Willebrand Factor Antigen and Activity in Patients with Increased Platelet Volume
Platelet Size P3
Plasma (U/ml) VWf VIIIR:Ag
Platelet (U/log platelets)? VWf VIIIR:Ag
LP I
8.86
0.71
0.79
1.98
0.83
LP II
9.49
0.88
0.77
1.30
0.44
LP III
10.07
1.12
1.18
1.45
0.52
Nl
4.92-8.20
1.07 (0.77-1.33)
0.96 (0.55-1.68)
LP = normal donor with large platelets. Nl - normal, n=12. tlOg platelets/ml.
0.91 (0.67-1.27)
0.44 (0.26-0.58)
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627
B
41 FIG. 1 Glyoxyl agarose electrophoresis of platelet and plasma vWf. Platelet (lane A) and plasma (lane B) vWf were analyzed by glyoxyl agarose electrophoresis. Identical amounts of antigen were placed in the two wells. The gels were run at a constant voltage of 2.5 volts per cm. Twenty ul samples were applied to each well. Each sample contained 4% 0.5% bromophenyl blue. The gels were run until the bromophenyl blue had migrated approximately 35-45 ems usually within 3 l/2-4 hours. After electrophoresis the gels were fixed, incubated with an immunopurified 12? rabbit anti-human f.VIII/vWf antibody, and the radioautographs were developed. The arrows and numbers on the right represent the different bands of multimers. Note that multimers 1 and 2 are more lightly stained in the platelet (lane A) than in plasma (lane band 1 migrates faster than the B). It is difficult to see that platelet plasma band as does band 2. Band 3 of plasma and platelets have approximately the same electrophoretic mobility; however, bands 4, 5, and 6 of the platelet migrate slower than their respective plasma counterparts. The anode is at the bottom.
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FIG. 2 Glyoxyl agarose electrophoresis analysis of platelet vWf prepared in the presence of lane A, and absence of lane B proteolytic inhibitors. The technique is as described for Figure 1. In lane a, the blood for preparation of platelet vWf was collected into a citrate-EDTA-leupeptin-N-ethylmaleimide anticoagulant (see Methods). For comparison blood was collected into a citrate-EDTA buffer (see Methods) and the platelet vWf prepared. The inhibitors were present in the wash buffer. No discernible difference can be seen between lane A and lane B. The anode is at the bottom.
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FIG. 3 lz51 plasma vWf was added in equal Glyoxyl agarose electrophoresis. volume to the platelet lysate (109/m1) of a patient with severe vWd. The Samples were taken at intervals mixture was incubated at 37'C for 4 hours. Lane 1 is the purified vWf prior to for multimer analysis and vWf activity. Lane 2 is the analysis of the plasma vWf addition to the platelet lysate. Lane 3 is after 1 hour after being left with the lysate for 30 seconds. incubation; Lane 4 is after 2 hours incubation; Lane 5 is after 4 hours No changes are noted in the multimeric structure or vWf activity. incubation. The plasma vWf baseline activity was 87% prior to addition; at 30 seconds after addition it was 88%. at 1 hour 84%. at 2 hours 82% and at 4 hours 93%. Identical results were seen when unlabelled vWf was incubated with the platelet lysate of a patient with severe vWd or when radiolabelled vWf was When normal plasma was incubated with the added to normal platelet lysate. platelet lysate of the patient with severe vWd, no changes in multimeric structure or vWf activity were seen.
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Reproducibility of the platelet vWf and VIII R:Ag was good. The two normals who had their 4 different preparations and determinations of their platelet activities show a mean VIII R:A of 0.40 U/lo9 platelets (range 0.34-0.48 U/lo9 platelets) and 0.31 U/l0 B platelets (range 0.28-0.38 U/lo9 platelets). The platelet vWf activity was 0.95 U/lo9 platelets, mean (range 0.88-1.06 U/lo9 platelets) and 1.04 U/lo9 platelets mean (range 0.81-1.35). respectively. Glyoxyl agarose electrophoresis of plasma and platelet vWf showed distinct differences in three areas (Figure 1): 1) the platelet vWf contained slower migrating multimers than did the plasma, 2) the multimeric organization of platelet vWf appeared to be different from plasma in that the smaller multimers (Figure 1, bands 1 and 2) of the platelet vWf migrated more rapidly than did the plasma vWf, and 3) the larger multimer (bands 4-6) of the platelet vWf migrated slower (less) than the plasma vWf. These findings were consistently seen in all the normals studied. No differences in the migration of the platelet vWf multimers were seen whether the platelets were collected in the presence (B) or absence (A) of proteolytic inhibitors (Fig. 2). The multimeric structure of the plasma vWf incubated with either the intact platelets or the platelet lysate from the patient with severe vWd was not modified, and the above noted differences between the normal plasma and the normal platelet vWf were observed. This experiment was carried out at the same temperature (37°C) that we use to isolate normal platelet vWf. In this experiment we incubated the purified plasma vWf with the platelets for 4 hours (usual incubation of normal platelet lysate is 60 minutes). This did not modify the results of the added plasma vWf activity or the multimeric structure (Figure 3). Identical results were observed when the purified vWf in severe vWd plasma or buffer, or normal plasma were incubated with these platelets. The multimer patterns of the platelet vWf of the 3 individuals platelets were indistinguishable from the other normals.
with large
DISCUSSION Howard and coworkers were the first to describe platelet-related vWf (i.e, VIII R:Ag) (4). Since that time, numerous groups have studied vWf by immunofluorescence, electrophoretic techniques and by measuring the antigenic content in intact and lysed platelets (4-11). Assays of the VIII R:Ag in lysed platelets have ranged from 0.11-0.61 U/lo9 platelets (2,4,5,8,9,13). Previously, only three groups detected vWf activity, i.e., ristocetin cofactor activity, of the platelets (9-11). In one study, it was detected without attempts at quantitation (11). and in another study the ristocetin cofactor activity was found to be 0.12 U/mg of platelet protein (9). In the same study, the VIII R:Ag was 0.15 U/mg protein. These values correspond to approximately 0.24 U and 0.30 U/lo9 platelets for ristocetin cofactor and VIII R:Ag, respectively (9). In the third study, the vWf activity was 0.56 U/lo9 platelets (10).
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We have found that the ratio of platelet vWf-to-antigen is l-1/2 to 2 times greater than that seen in normal plasma. Although we found approximately the same amount of vWf activity in 109 platelets as in 1 ml of plasma, the platelet vWf antigen was only 50% of the seen in plasma. We initially considered that this may have been due to the fact that the larger multimers actually reduced the migration of the platelet antigen in the electroimmunoHowever, when the antigen was analyzed by radioimmunoassay, a assay. technique not dependent on migration in gels, we found only slightly higher antigenic levels (mean 0.60 for radioimmunoassay vs. 0.44 for the electroimmunoassay). The ratio of vWf activity to antigen was still higher in platelet vWf than in plasma vWf. The increased vWf activity and VIII R:Ag in the platelets of the 3 normals with increased platelet volume may represent a shift in the platelet population to younger platelets, although recent work has raised questions concerning the relationship of platelet age, size and density (18,19,21-23). Nevertheless, in measuring platelet vWf and VIII R:Ag in disease states, one must take platelet size into consideration in the interpretation of the results. Studies of the structure of the platelet vWf and plasma vWf have revealed both similarities and differences. Nachman and Jaffe (11) first reported the platelet and plasma vWf had a subunit of similar, if not identical, size when analyzed after disulfide reduction on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Subsequently, three reports employing agarose gel electrophoresis with autoradiography have indicated that the platelet vWf contains slower migrating multimers, i.e, larger multimers than No other differences have been noted in the does the plasma vWf (1,2,24). migration between plasma and platelet multimers. This study confirms the presence of larger multimers within platelets, i.e., slower migrating forms, than that seen in plasma. However, we also observed a different multimeric organization of the platelet vWf as compared to plasma. The fastest platelet band (Fig. 1, band 1) consistently showed a slightly faster migration than the lowest band of the plasma vWf. The two fastest migrating bands of platelet vWf migrate further than those of plasma vWf while band 3 of both plasma and platelets appears to have an identical migration. The hierarchy of the larger multimers in platelets migrates more slowly when compared to the comparable plasma multimers (Fig. 1, bands 4,5,6). This may represent structural differences in the subunit or may represent varying degrees of polymerization which may occur at different intervals than with plasma vWf. These possibilities may account for the findings of Nachman and Jaffe (14) who demonstrated that plasma vWf revealed 7 radioactive peptide spots which were not present in the fingerprint of platelet vWf. The possibility that our results of the multimeric organization of the platelet vWf 1) are due to proteolytic modification of the platelet vWf seems unlikely since the platelet vWf antigen collected in the presence of protease inhibitors has the same multimeric organization, biologic activity and antigenie reactivity as those collected in the presence of citrate-EDTA only and 2) the multimeric organization of plasma vWf did not change after being placed in an environment of whole platelets or the Triton X-100 lysate of platelets from a patient with severe vWd. These studies do not rule out the possibility that the multimeric differences seen between platelet and plasma vWf are due The plasma vWf we studied has to intracellular processing prior to release. already undergone intracellular processing prior to release from endothelial cells and while the platelet vWf is recovered from lysed platelets.
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The possibility exists that the platelet vWf may be a gene product different from the plasma vWf or more likely it may be an identical gene product. If it is an identical gene product then it may undergo a posttranslational modification in the megakaryocyte or platelet to which it has not been exposed to by our technique of isolation from platelets. Either of these two possibilities would result in the observed differences from plasma VWf. ACKNOWLEDGMENTS The authors wish to thank Mrs. Lynda Ray for secretarial
assistance.
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RUGGERI, Z.M. and ZIMMERMAN, T.S. Variant von Willebrand's disease. Characterization of two subtypes by analysis of multimeric composition factor VIII/van Willebrand factor in plasma and platelets. J. Clin. Invest. 65, 1318, 1980.
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WEISS, H.J., PIETLJ, G., RABINOWITZ, R., GIRMA, J.-P., ROGERS, J. and MEYER, D. Heterogeneous abnormalities in the multimeric structure, antigenic properties and plasma-platelet content of factor VIII/van Willebrand factor in subtypes of classic (type I) and variant (type IIA) von Willebrand's disease. J. Lab. Clin. Med. E, 411, 1983.
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OHMORI, K., FRETTO, L.J., HARRISON, R.L., SWITZER, M.E.P., ERICKSON, H.P. and MC KEE, P.A. Electron microscopy of human factor VIIIlvon Willebrand glycoprotein: Effect of reducing reagents on structure and function. J. Cell. Biol. 95, 632, 1982.
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HOWARD, M.A., MONTGOMERY, D.C. and HARDISTY, R.M. antigen in platelets. Thromb. Res. 5, 617, 1974.
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BOUMA, B.N., HORDIJK-HOS, J.M., DE GRAAF, S. and SIXMA, J.J. Presence of factor VIII-related antigen in blood platelets of patients with von Willebrand's disease. Nature 257, 510, 1975.
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SHEARN, S.A.M., GIDDINGS, J.C., PEAKE, I.R. and Bloom, A.L. A comparison of five different rabbit antisera to factor VIII and the demonstration of a factor VIII related antigen in normal and von Willebrand's disease platelets. Thromb. Res. 1, 585, 1974.
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Studies on the-factor COLLER, B.S., HIRSCHMAN, R.J. and GRALNICK, H.R. VIII/van Willebrand factor antigen in human platelets. Thromb. Res. 6, 469, 1975.
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SULTAN, Y., BOUMA, B.N., DE GRAAF, S., SIMBON, J., CAEN, J.P. and SIXMA, J.J. Factor VIII related antigen in platelets of patients with von Willebrand's disease. Thromb. Res. 11, 23, 1977.
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RUGGERI, Z.M., MANNUCCI, P.M., BADER, R. and BARBUI, T. Factor VIIIrelated properties in platelets from patients with von Willebrand's disease. J. Lab. Clin. Med. 91, 132, 1978.
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SULTAN, Y., MAISSONEUVE, P. and ANGLES-CANO, E. Release of VIII R:Ag and VIII R:WF during thrombin and collagen induced aggregation. Thromb. Res. l-5, 415, 1979.
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NACHMAN, R.L. and JAFFE, E.A. Subcellular platelet factor VIII antigen and von Willebrand factor. J. Exper. Med. 141, 1101, 1975.
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ZUCKER, M.B., BROEKMAN, M.J. and KAPLAN, K.L. Factor VIII-related antigen in human blood platelets. J. Lab. Clin. Med. 94, 675, 1979.
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NACHMAN, R., LEVINE, R. and JAFFE, E.A. Synthesis of factor VIII antigen by cultured guinea pig megakaryocytes. J. Clin. Invest. 60, 914, 1977.
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NACHMAN, R.L., JAFFE, E.A. and FERRIS, B. Peptide map analysis of normal plasma and platelet factor VIII antigen. Biochem. Biophys. Res. Commun. 92, 1208, 1980.
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GRALNICK, H.R., COLLER, B.S. and SULTAN, Y. Studies of the human factor VIII/van Willebrand factor protein. III. Qualitative defects in von Willebrand's disease. J. Clin. Invest. 6, 814, 1975.
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HOYER, L.W. Immunologic studies of antihemophilic factor (AHF, factor VIII). IV. Radioimmunoassay of AHF antigen. J. Lab. Clin. Med. 80, 822, 1972.
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HOYER, L.W. and SHAINOFF, J.R. Factor VIII-related protein circulates in normal human plasma as high molecular weight multimers. -Blood 55, 1056, 1980.
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CORASH, L., TAN, H. and GRALNICK, H.R. Heterogeneity of human whole blood platelet subpopulations. I. Relationship between buoyant density, cell volume and ultrastructure. -Blood 49, 71, 1977.
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CORASH, L., SHAFER, B. and PERLOW, H. Heterogeneity of human whole blood platelet subpopulations. II. Use of a subhuman primate model to analyze the relationship between density and platelet age. -Blood 52, 726, 1978.
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LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265, 1951.
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PLATELET VON WILLEBRAND FACTOR: COMPARISON WITH PLASMA VON WILLEBRAND FACTOR
Harvey R. Gralnick, Hematology
Sybil B. Williams, Laurie P. McKeown, Dennis H. Krizek, Brenda C. Shafer and Margaret E. Rick Service, Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland
U.S.A. (Received 15.1.1985; Accepted in revised form 22.2.1985 by Editor J.G. White) ABSTRACT Platelet von Willebrand factor (vWf) was compared to its plasma The platelet vWf was different from plasma vWf in that counterpart. .l) the multimeric organization was different, 2) larger multimers were present, and 3) the ratio of vWf activity to antigen was higher. Platelet and plasma vWf were similar in their antigenic reactivity in the electroimmunoassay and by liquid phase radioimmunoassay. The amount of vWf activity in large platelets was significantly higher than in normal platelets while the antigen content, although somewhat higher, was not significant. These studies show differences between normal platelet and plasma vWf, and also suggest that platelet size must be considered when platelet vWf is measured in disease states.
INTRODUCTION The plasma von Willebrand factor (vWf) has been characterized as a macromolecular glycoprotein. This protein has a multimeric organization with the molecular weight varying from the smallest plasma multimer of approximately 800,000 daltons to the largest molecular weight multimers ranging between 5-20 million daltons (l-3). vWf also has been localized in platelets and the platelet vWf is synthesized in the megakaryocyte, while the plasma vWf is thought to originate in the endothelial cell (4-13). Differences between normal platelet and plasma vWf have been reported with the major difference observed being the presence of larger niolecular weight multimers in the platelets as compared to plasma (1,2,14). We have undertaken a study to further characterize the difference(s) between the normal platelet and plasma VWf. Key words: Platelet von Willebrand factor , plasma von Willebrand factor,
platelet size
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MATERIALS AND METHODS Blood was obtained from 15 normal blood donors by a two-syringe technique using a 19-gauge butterfly needle and polypropylene syringes. Three of the 15 normal blood donors were known to have an increased mean platelet size. Donors were fully informed regarding the potential risk of venipuncture. The first 5 ml of blood were drawn for blood counts, and the subsequent samples were utilized for the studies described below. Blood for plasma separation was collected into polypropylene tubes containing sodium citrate (0.32% final concentration, 10.9 mM) or the same citrate anticoagulant containing 5 mM EDTA, 1 mM leupeptin and 6 mM n-ethylmaleimide (Aldrich Chemical Co., Milwaukee, WI). The blood was centrifuged at 2000 g for 15 minutes at 4"C, and the cell-free plasma was removed and used fresh or frozen at -7O*C for subsequent testing. Blood for the platelet studies was collected in an anticoagulant of 109 mM sodium citrate and 1 mM EDTA (final concentration 10.9 mM sodium citrate and 1 mM EDTA) and in an anticoagulant of sodium citrate-EDTA, leupeptin, and n-ethylmaleimide (final concentration 10.9 mM sodium cirate, 5 mM EDTA, 1 mM leupeptin and 6 mM n-ethylmaleimide). Platelet-rich plasma (PRP) was prepared by centrifugation at 750 g for 3 minutes at room temperature. After removal of the PRP, the red cells were washed once with Tris-NaCl-EDTA-bovine serum albumin, pH 6.8 (Tris 0.1 M, 0.15 M NaCl, 0.003 H EDTA, 0.5% bovine serum albumin; Pentex Bovine Serum Albumin, Fraction V, Miles Laboratories, Naperville, IL) and the wash was added to the PRP. Over 85% of the total platelet population was recovered. The PRP and wash were separated from plasma proteins on a discontinuous arabinogalactan gradient (3 ml of 20% and 5 ml of 10% arabinogalactan). The gradient was centrifuged at 2500 RCF for 30 minutes at 24“C. The platelets were removed and resuspended in the TrisNaCl-EDTA-bovine serum albumin buffer and washed once with the same buffer (2000 RCT for 20 minutes at 24°C. After the wash, the platelets were resuspended in a Tris-NaCl-bovine serum albumin buffer, pH 7.4 (Tris 0.1 H, The platelets NaCl 0.15 M, bovine serum albumin 3%), at 37°C for 15 minutes. were then counted and diluted in the same buffer to a final concentration of 106/Pl. The platelets were lysed by the addition of l/40 volume of 20% Triton The frozen X 100, incubated for 1 hour at 37'C, then frozen at -70°C. samples, they were centrifuged for 30 minutes at 10,000 RCF at 4°C and the supernatant removed and analyzed. The f.VIII-related antigen (VIII R:Ag) assays were performed by the electroimmunoassay employing a goat anti-f.VIII/vWf antibody (15) and by radioimmunoassay utilizing a rabbit anti-f.VIII/vWf antibody (16). vWf activity was assayed using the formalin-fixed platelet assay as previously described (15). A pool of normal plasma (50 donors) was used in establishing the standard curves for the antigen and vWf activities. In determining the level of VIII R:Ag and vWf, only those points which fell on the parallel At least 2 diluportion of the curve were used for estimation of potency. tions of plasma and platelet lysate were assayed for VIII R:Ag and at least 3 dilutions of plasma or platelet lysate (in duplicate) were analyzed in the vWf assay. Normal plasma and platelet vWf were prepared and analyzed on 3 separate occasions on two normals. Glyoxyl agarose electrophoresis was performed using a modification of the technique described by Hoyer and Shainoff (17). The modification consisted of using 0.5 mm thick gels and a plying only 10 pl of sample in the wells. The gels were incubated with an 155 I-labelled immunopurified rabbit anti-human
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factor VIIIlvon Willebrand factor (f.VIII/vWf) antibody, and the radioautograph developed on XAR-2 film (Eastman Kodak, Rochester, NY). In experiments to determine if Triton X-100 influenced the measurement of vWf antigen or activity, we added l/40 volume of 20% Triton X-100 to plasma and the vWf antigen and activity were measured. Controls were plasma to which l/40 volume of 0.14 NaCl was added. Platelets (log/ml) or the Triton-X-100 lysate of platelets from a patient with severe vWd (bleeding time >30 minutes, factor VIII C <5%, factor VIII:RAg <5% and vWf activity <5%) were incubated with either normal plasma (1:l ratio) or labelled or unlabelled purified plasma vWf (2-4 ng) diluted in the plasma of a patient with severe vWd or buffer. We incubated the mixture at 37°C (same temperature as we incubate our platelets) and analyzed the plasma vWf multimeric structure by agarose gel electrophoreeis over time. Similar studies were performed with 1251 plasma vWf and normal platelet lysate. Platelet size was measured on a Cellozone Cell Counter (Particle Data, Elmhurst, IL) as previously described (18,19). Platelet associated IgG and platelet aggregation studies were performed as previously described (15). Platelet
protein
was measured
by the method of Lowry et al. (20). RESULTS
The mean plasma VIII R:Ag was 0.96 U/ml (range 0.55-1.68, n=12) when measured by the Laurel1 electroimmunoassay and 1.11 U/ml (range 0.71-1.47) by the radioimmunoassay. The mean plasma vWf activity was 1.07 U/ml (range 0.771.33, n-12). The mean antigen content by electroimmunoassay was 0.44 U/log platelets (range 0.26-0.58, n=12). The vWf activity was 0.91 U/log platelets (range 0.67-1.27, n=12, Table 1). The mean ratio of plasma vWf to plasma VIII R:Ag was 1.11 when the antigen was measured by the Laurel1 technique; in
TABLE 1 von Willebrand
Plasma (U/ml)
Platelet (U/109 platelets)
Factor Antigen
and Activity
in Normal Plasma and Platelets
VWf
VIIIR:Ag*
1.07 (0.77-1.33)
0.96 (0.55-1.68)
0.91 (0.67-1.27)
0.44t (0.26-0.58)
*Antigen assays by electroimmunoassay. ilOg platelets/ml. n = 12 normal donors with normal platelet
size.
Ratio vWf/Ag
1.11 (0.63-1.54)
2.07 (1.57-2.89)
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platelets, this ratio was 2.07. Similar results were seen in studies when the antigen data obtained by radioimmunoassay were analyzed. The ratio of plasma vWf to VIII R:Ag was 0.96 and in platelets the ratio was 1.52. The differences noted are primarily due to the relative reduction in VIII R:Ag in platelets. No differences in antigen levels or vWf activity were seen when plasma or platelets were collected in the presence or absence of the proteolytic inhibitors. Triton X-100 added to normal plasma did not change the vWf antigen level or the vWf activity. The mean platelet size of 3 of the normal individuals was high, i.e., 9.86, 9.49, 10.07~~. respectively (normal range derived from mean f.2 SD; 6.56 ? 1.64 u3). The mean platelet size for the other donors was normal, i.e., greater than 4.92 u3 and less than 8.20 P3. Platelet associated IgG and platelet aggregation studies have been repeatedly normal in these 3 individuals, and they have no clinical bleeding history despite major surgical challenges. The protein content of the latelets from these 3 individual was within our normal range (mean 1.92 mg/lO6 platelets, range 1.68-2.18). The values for these three individuals were 1.76, 2.07, and 1.87 mg/lOg platelets, respectively. The plasma vWf, VIII R:Ag and mean ratio of vWf to VIII R:Ag of the 3 donors with large platelets were similar to the results of the other normals. In contrast, the platelet values were higher than normal (Table 2). In particular, the platelet vWf activity of all 3 normals with lar e platelets was greater than the normal range, i.e., 1.30, 1.45, and 1.98 U/10B platelets vs. 0.67-1.27 U/log platelets normal range, and the platelet VIII R:Ag was near or outside the upper limit of the normal range (0.44, 0.52 and 0.83 U/ml; normal range 0.26-O-58 U/ml).
TABLE 2 von Willebrand Factor Antigen and Activity in Patients with Increased Platelet Volume
Platelet Size P3
Plasma (U/ml) VWf VIIIR:Ag
Platelet (U/log platelets)? VWf VIIIR:Ag
LP I
8.86
0.71
0.79
1.98
0.83
LP II
9.49
0.88
0.77
1.30
0.44
LP III
10.07
1.12
1.18
1.45
0.52
Nl
4.92-8.20
1.07 (0.77-1.33)
0.96 (0.55-1.68)
LP = normal donor with large platelets. Nl - normal, n=12. tlOg platelets/ml.
0.91 (0.67-1.27)
0.44 (0.26-0.58)
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627
B
41 FIG. 1 Glyoxyl agarose electrophoresis of platelet and plasma vWf. Platelet (lane A) and plasma (lane B) vWf were analyzed by glyoxyl agarose electrophoresis. Identical amounts of antigen were placed in the two wells. The gels were run at a constant voltage of 2.5 volts per cm. Twenty ul samples were applied to each well. Each sample contained 4% 0.5% bromophenyl blue. The gels were run until the bromophenyl blue had migrated approximately 35-45 ems usually within 3 l/2-4 hours. After electrophoresis the gels were fixed, incubated with an immunopurified 12? rabbit anti-human f.VIII/vWf antibody, and the radioautographs were developed. The arrows and numbers on the right represent the different bands of multimers. Note that multimers 1 and 2 are more lightly stained in the platelet (lane A) than in plasma (lane band 1 migrates faster than the B). It is difficult to see that platelet plasma band as does band 2. Band 3 of plasma and platelets have approximately the same electrophoretic mobility; however, bands 4, 5, and 6 of the platelet migrate slower than their respective plasma counterparts. The anode is at the bottom.
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PLATELET vWF & PLASMA vWF
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B
FIG. 2 Glyoxyl agarose electrophoresis analysis of platelet vWf prepared in the presence of lane A, and absence of lane B proteolytic inhibitors. The technique is as described for Figure 1. In lane a, the blood for preparation of platelet vWf was collected into a citrate-EDTA-leupeptin-N-ethylmaleimide anticoagulant (see Methods). For comparison blood was collected into a citrate-EDTA buffer (see Methods) and the platelet vWf prepared. The inhibitors were present in the wash buffer. No discernible difference can be seen between lane A and lane B. The anode is at the bottom.
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FIG. 3 lz51 plasma vWf was added in equal Glyoxyl agarose electrophoresis. volume to the platelet lysate (109/m1) of a patient with severe vWd. The Samples were taken at intervals mixture was incubated at 37'C for 4 hours. Lane 1 is the purified vWf prior to for multimer analysis and vWf activity. Lane 2 is the analysis of the plasma vWf addition to the platelet lysate. Lane 3 is after 1 hour after being left with the lysate for 30 seconds. incubation; Lane 4 is after 2 hours incubation; Lane 5 is after 4 hours No changes are noted in the multimeric structure or vWf activity. incubation. The plasma vWf baseline activity was 87% prior to addition; at 30 seconds after addition it was 88%. at 1 hour 84%. at 2 hours 82% and at 4 hours 93%. Identical results were seen when unlabelled vWf was incubated with the platelet lysate of a patient with severe vWd or when radiolabelled vWf was When normal plasma was incubated with the added to normal platelet lysate. platelet lysate of the patient with severe vWd, no changes in multimeric structure or vWf activity were seen.
PLATELET vWF & PLASMA vWF
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Reproducibility of the platelet vWf and VIII R:Ag was good. The two normals who had their 4 different preparations and determinations of their platelet activities show a mean VIII R:A of 0.40 U/lo9 platelets (range 0.34-0.48 U/lo9 platelets) and 0.31 U/l0 B platelets (range 0.28-0.38 U/lo9 platelets). The platelet vWf activity was 0.95 U/lo9 platelets, mean (range 0.88-1.06 U/lo9 platelets) and 1.04 U/lo9 platelets mean (range 0.81-1.35). respectively. Glyoxyl agarose electrophoresis of plasma and platelet vWf showed distinct differences in three areas (Figure 1): 1) the platelet vWf contained slower migrating multimers than did the plasma, 2) the multimeric organization of platelet vWf appeared to be different from plasma in that the smaller multimers (Figure 1, bands 1 and 2) of the platelet vWf migrated more rapidly than did the plasma vWf, and 3) the larger multimer (bands 4-6) of the platelet vWf migrated slower (less) than the plasma vWf. These findings were consistently seen in all the normals studied. No differences in the migration of the platelet vWf multimers were seen whether the platelets were collected in the presence (B) or absence (A) of proteolytic inhibitors (Fig. 2). The multimeric structure of the plasma vWf incubated with either the intact platelets or the platelet lysate from the patient with severe vWd was not modified, and the above noted differences between the normal plasma and the normal platelet vWf were observed. This experiment was carried out at the same temperature (37°C) that we use to isolate normal platelet vWf. In this experiment we incubated the purified plasma vWf with the platelets for 4 hours (usual incubation of normal platelet lysate is 60 minutes). This did not modify the results of the added plasma vWf activity or the multimeric structure (Figure 3). Identical results were observed when the purified vWf in severe vWd plasma or buffer, or normal plasma were incubated with these platelets. The multimer patterns of the platelet vWf of the 3 individuals platelets were indistinguishable from the other normals.
with large
DISCUSSION Howard and coworkers were the first to describe platelet-related vWf (i.e, VIII R:Ag) (4). Since that time, numerous groups have studied vWf by immunofluorescence, electrophoretic techniques and by measuring the antigenic content in intact and lysed platelets (4-11). Assays of the VIII R:Ag in lysed platelets have ranged from 0.11-0.61 U/lo9 platelets (2,4,5,8,9,13). Previously, only three groups detected vWf activity, i.e., ristocetin cofactor activity, of the platelets (9-11). In one study, it was detected without attempts at quantitation (11). and in another study the ristocetin cofactor activity was found to be 0.12 U/mg of platelet protein (9). In the same study, the VIII R:Ag was 0.15 U/mg protein. These values correspond to approximately 0.24 U and 0.30 U/lo9 platelets for ristocetin cofactor and VIII R:Ag, respectively (9). In the third study, the vWf activity was 0.56 U/lo9 platelets (10).
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We have found that the ratio of platelet vWf-to-antigen is l-1/2 to 2 times greater than that seen in normal plasma. Although we found approximately the same amount of vWf activity in 109 platelets as in 1 ml of plasma, the platelet vWf antigen was only 50% of the seen in plasma. We initially considered that this may have been due to the fact that the larger multimers actually reduced the migration of the platelet antigen in the electroimmunoHowever, when the antigen was analyzed by radioimmunoassay, a assay. technique not dependent on migration in gels, we found only slightly higher antigenic levels (mean 0.60 for radioimmunoassay vs. 0.44 for the electroimmunoassay). The ratio of vWf activity to antigen was still higher in platelet vWf than in plasma vWf. The increased vWf activity and VIII R:Ag in the platelets of the 3 normals with increased platelet volume may represent a shift in the platelet population to younger platelets, although recent work has raised questions concerning the relationship of platelet age, size and density (18,19,21-23). Nevertheless, in measuring platelet vWf and VIII R:Ag in disease states, one must take platelet size into consideration in the interpretation of the results. Studies of the structure of the platelet vWf and plasma vWf have revealed both similarities and differences. Nachman and Jaffe (11) first reported the platelet and plasma vWf had a subunit of similar, if not identical, size when analyzed after disulfide reduction on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Subsequently, three reports employing agarose gel electrophoresis with autoradiography have indicated that the platelet vWf contains slower migrating multimers, i.e, larger multimers than No other differences have been noted in the does the plasma vWf (1,2,24). migration between plasma and platelet multimers. This study confirms the presence of larger multimers within platelets, i.e., slower migrating forms, than that seen in plasma. However, we also observed a different multimeric organization of the platelet vWf as compared to plasma. The fastest platelet band (Fig. 1, band 1) consistently showed a slightly faster migration than the lowest band of the plasma vWf. The two fastest migrating bands of platelet vWf migrate further than those of plasma vWf while band 3 of both plasma and platelets appears to have an identical migration. The hierarchy of the larger multimers in platelets migrates more slowly when compared to the comparable plasma multimers (Fig. 1, bands 4,5,6). This may represent structural differences in the subunit or may represent varying degrees of polymerization which may occur at different intervals than with plasma vWf. These possibilities may account for the findings of Nachman and Jaffe (14) who demonstrated that plasma vWf revealed 7 radioactive peptide spots which were not present in the fingerprint of platelet vWf. The possibility that our results of the multimeric organization of the platelet vWf 1) are due to proteolytic modification of the platelet vWf seems unlikely since the platelet vWf antigen collected in the presence of protease inhibitors has the same multimeric organization, biologic activity and antigenie reactivity as those collected in the presence of citrate-EDTA only and 2) the multimeric organization of plasma vWf did not change after being placed in an environment of whole platelets or the Triton X-100 lysate of platelets from a patient with severe vWd. These studies do not rule out the possibility that the multimeric differences seen between platelet and plasma vWf are due The plasma vWf we studied has to intracellular processing prior to release. already undergone intracellular processing prior to release from endothelial cells and while the platelet vWf is recovered from lysed platelets.
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The possibility exists that the platelet vWf may be a gene product different from the plasma vWf or more likely it may be an identical gene product. If it is an identical gene product then it may undergo a posttranslational modification in the megakaryocyte or platelet to which it has not been exposed to by our technique of isolation from platelets. Either of these two possibilities would result in the observed differences from plasma VWf. ACKNOWLEDGMENTS The authors wish to thank Mrs. Lynda Ray for secretarial
assistance.
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