THROMBOSIS RESEARCH 68; 393-398, 1992 00493848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
INTRAPLATELET
VON
WILLEBRAND
FACTOR
AND
ABO
BLOOD
GROUP
Joseph D. Sweeney and Lynne A. Hoernig Hemophilia Center of Western New York and Department of Laboratory Medicine, Roswell Park Cancer Institute, Buff'alo, NY 14263 USA (Received 31.8.1992; accepted in revised form 5.10.1992 by Editor D.K. Galarrakis) ABElTRACT
Levels of plasma Von Willebrand Factor (vWF) are known to be influenced by ABO Blood Group but such an influence on platelet vWF is not known. Forty-three healthy donors had blood drawn for measurement of plasma and platelet vWF, both antigenic (vWF:Ag) and functional (RCo) . Twenty-six were Group 0 and seventeen were Group A. Groups did not differ in age, platelet count, hemoglobin, white cell counts, platelet rich plasma counts nor length of in vitro storage of samples prior to assay. Plasma levels of vWF:Ag and RCo was lower in Group 0 as expected. Platelet RCo was lower in Group 0 and such a trend was present for vWF:Ag. This influence of ABO Groups on platelet vWF was modest compared to that on plasma vWF.
Von Willebrand Factor (vWF) is a glycoprotein which is known to be synthesized by megakaryocytes and endothelial cells, and in blood is present in both platelets and plasma. vWF exists in multimeric forms with molecular weight from 500-20,000 Kd,, and is an important molecule in the adhesion of platelets to surfaces under certain conditions of flow.(l) Von Willebrand Factor can be measured in platelet poor plasma antigenically, commonly by electroimmunodiffusion or enzyme immunoassay (EIA) or, functionally in a ristocetin cofactor assay and function is also assessed in whole blood or platelet rich plasma by the agglutination of platelets by ristocetin (RIPA). The pla:sma level of vWF is influenced by ABO blood group, Group 0 individuals having lower levels than either A, B or AB.(2,3) Despite this, RIPA in Group 0 subjects as measured in an The impedance aggregometer appears superior to Group A.(3) explanation of this is unclear, but an influence of intraplatelet vWF is a possibility. This study was conducted to examine for an effect of ABO group on intraplatelet vWF. Key words:
Platelets,
Von Willebrand
393
Factor, Blood Group
394
PLATELETS, vWF AND BLOOD GROUP
Vol. 68, Nos. 415
METHODS Blood was collected from plateletpheresis donors, all either Group 0 or Group A. All donors are carefully questioned for a history of a bleeding disorder. A first sample was collected in a vacutainer tube containing 3.8% buffered trisodium citrate and a second sample into a vacutainer tube containing 3.2% citrate, (Diatube H, American theophylline, adenosine and dipyridamole. Bioproducts, Parsipanny, NJ) and suitable for assay of platelet The first tube was spun at 3,000g for 15 cc-granule content. minutes and the platelet poor plasma (PPP) removed and stored in two aliquots at -7OOC until assayed. The latter tubes were spun at 110 g for 10 minutes, and the platelet rich plasma (PRP) removed. A platelet count was performed in the PRP using an The PRP was then acidified using 1M citric acid Ortho ELT-8d/s. (0.09 ,,l/ml) and subsequently washed twice in a buffer containing sodium citrate, trizma base (O.OlM), chymostatin (5 vg/ml), aprotinin (5 pg/ml), leupeptin (5 uM), pepstatin (5 u M), PMSF (1mM) and EDTA (1mM). After the final resuspension, the platelets were spun, the supernatant discarded and the pellet frozen at -7OOC. Frozen platelet pellets were thawed, then sonicated three times using a sonicator model W220 (Heat System Untrasonics, Inc., Farmingdale, NY) for 15 seconds at setting #2. The disrupted platelets were then ultracentrifuged at 100,000 g for 1 hour, and the supernatant referred to as platlelet supernatant (PS) removed for assay. PPP and PS were assayed for vWF:Ag by enzyme immunoassay using a commercially available kit (American Bioproducts), and for RCo activity using formaldehyde fixed O-platelets in an aggregometer.(5) In this enzyme immunoassay five dilutions of the plasma standard and two dilutions of the test plasma are made. A blank (diluent only) is included. Absorbance is correlated linearly with vWF concentration. In the plasma RCo Assay, 3 dilutions of the standard are made and 2 dilutions of test PPP plasma. For PS RCo Assay, only undiluted and one dilution PS were used in the assay, since levels measured were so low. Statistical evaluation was done using a t-test. The same standard was used in all assays and was calibrated against the WHO standard (87/718). Assay r sults for PPP were expressed as U/dL and for PS as U/dL per 10 8 platelets. RESULTS 43 healthy subjects were studied, 17 Group A and 26 Group 0. The mean age, hemoglobin, white cell count or platelet count did not differ between the Group A and Group 0 subjects.
Vol. 68, Nos. 415
395
PLATELETS, vWF AND BLOOD GROUP
Results of the vWF levels between Group A and Group 0 are given in table 1. TABLE I
Group A Group 0 P
No.
Plasma(U/dL) vWF:Aq RCo
17 26
128+29 99-116 co.01
Platelets(U/dL per log platelets) vWF:Aq RCo
119*37 71?r24 co.05
13.2+3 12.2+3 0.06
Differences in vWF levels in plasma and platelets and Group 0.
8.355 7.0?4 co.05 between Group A
Highly significant differences are present in PPP as previously reported. Platelet vWF levels also show the same trend but to a lesser extent; this is statistically significant with RCo. Plasma vWF:Ag Correlations were sought between these measures. correlated with plasma RCo (r = 0.74,p
INTRAPLATELET
VON
WILLEBRAND
FACTOR
AND
ABO
BLOOD
GROUP
Joseph D. Sweeney and Lynne A. Hoernig Hemophilia Center of Western New York and Department of Laboratory Medicine, Roswell Park Cancer Institute, Buff'alo, NY 14263 USA (Received 31.8.1992; accepted in revised form 5.10.1992 by Editor D.K. Galarrakis) ABElTRACT
Levels of plasma Von Willebrand Factor (vWF) are known to be influenced by ABO Blood Group but such an influence on platelet vWF is not known. Forty-three healthy donors had blood drawn for measurement of plasma and platelet vWF, both antigenic (vWF:Ag) and functional (RCo) . Twenty-six were Group 0 and seventeen were Group A. Groups did not differ in age, platelet count, hemoglobin, white cell counts, platelet rich plasma counts nor length of in vitro storage of samples prior to assay. Plasma levels of vWF:Ag and RCo was lower in Group 0 as expected. Platelet RCo was lower in Group 0 and such a trend was present for vWF:Ag. This influence of ABO Groups on platelet vWF was modest compared to that on plasma vWF.
Von Willebrand Factor (vWF) is a glycoprotein which is known to be synthesized by megakaryocytes and endothelial cells, and in blood is present in both platelets and plasma. vWF exists in multimeric forms with molecular weight from 500-20,000 Kd,, and is an important molecule in the adhesion of platelets to surfaces under certain conditions of flow.(l) Von Willebrand Factor can be measured in platelet poor plasma antigenically, commonly by electroimmunodiffusion or enzyme immunoassay (EIA) or, functionally in a ristocetin cofactor assay and function is also assessed in whole blood or platelet rich plasma by the agglutination of platelets by ristocetin (RIPA). The pla:sma level of vWF is influenced by ABO blood group, Group 0 individuals having lower levels than either A, B or AB.(2,3) Despite this, RIPA in Group 0 subjects as measured in an The impedance aggregometer appears superior to Group A.(3) explanation of this is unclear, but an influence of intraplatelet vWF is a possibility. This study was conducted to examine for an effect of ABO group on intraplatelet vWF. Key words:
Platelets,
Von Willebrand
393
Factor, Blood Group
394
PLATELETS, vWF AND BLOOD GROUP
Vol. 68, Nos. 415
METHODS Blood was collected from plateletpheresis donors, all either Group 0 or Group A. All donors are carefully questioned for a history of a bleeding disorder. A first sample was collected in a vacutainer tube containing 3.8% buffered trisodium citrate and a second sample into a vacutainer tube containing 3.2% citrate, (Diatube H, American theophylline, adenosine and dipyridamole. Bioproducts, Parsipanny, NJ) and suitable for assay of platelet The first tube was spun at 3,000g for 15 cc-granule content. minutes and the platelet poor plasma (PPP) removed and stored in two aliquots at -7OOC until assayed. The latter tubes were spun at 110 g for 10 minutes, and the platelet rich plasma (PRP) removed. A platelet count was performed in the PRP using an The PRP was then acidified using 1M citric acid Ortho ELT-8d/s. (0.09 ,,l/ml) and subsequently washed twice in a buffer containing sodium citrate, trizma base (O.OlM), chymostatin (5 vg/ml), aprotinin (5 pg/ml), leupeptin (5 uM), pepstatin (5 u M), PMSF (1mM) and EDTA (1mM). After the final resuspension, the platelets were spun, the supernatant discarded and the pellet frozen at -7OOC. Frozen platelet pellets were thawed, then sonicated three times using a sonicator model W220 (Heat System Untrasonics, Inc., Farmingdale, NY) for 15 seconds at setting #2. The disrupted platelets were then ultracentrifuged at 100,000 g for 1 hour, and the supernatant referred to as platlelet supernatant (PS) removed for assay. PPP and PS were assayed for vWF:Ag by enzyme immunoassay using a commercially available kit (American Bioproducts), and for RCo activity using formaldehyde fixed O-platelets in an aggregometer.(5) In this enzyme immunoassay five dilutions of the plasma standard and two dilutions of the test plasma are made. A blank (diluent only) is included. Absorbance is correlated linearly with vWF concentration. In the plasma RCo Assay, 3 dilutions of the standard are made and 2 dilutions of test PPP plasma. For PS RCo Assay, only undiluted and one dilution PS were used in the assay, since levels measured were so low. Statistical evaluation was done using a t-test. The same standard was used in all assays and was calibrated against the WHO standard (87/718). Assay r sults for PPP were expressed as U/dL and for PS as U/dL per 10 8 platelets. RESULTS 43 healthy subjects were studied, 17 Group A and 26 Group 0. The mean age, hemoglobin, white cell count or platelet count did not differ between the Group A and Group 0 subjects.
Vol. 68, Nos. 415
395
PLATELETS, vWF AND BLOOD GROUP
Results of the vWF levels between Group A and Group 0 are given in table 1. TABLE I
Group A Group 0 P
No.
Plasma(U/dL) vWF:Aq RCo
17 26
128+29 99-116 co.01
Platelets(U/dL per log platelets) vWF:Aq RCo
119*37 71?r24 co.05
13.2+3 12.2+3 0.06
Differences in vWF levels in plasma and platelets and Group 0.
8.355 7.0?4 co.05 between Group A
Highly significant differences are present in PPP as previously reported. Platelet vWF levels also show the same trend but to a lesser extent; this is statistically significant with RCo. Plasma vWF:Ag Correlations were sought between these measures. correlated with plasma RCo (r = 0.74,p
