© 1996 American Heart Association, Inc.
Articles |
Uptake of vWF–Anti-vWF Complexes by Platelets in Suspension
the Departments of Laboratory Medicine and Pathology, Pediatrics, University of Minnesota Medical School, Minneapolis (J.G.W., M.D.K., D.J.C.-J.), and Servicio de Hemoterapia y Hemostasia, Hospital Clinico y Provincial, Barcelona, Spain (G.E.).
Correspondence to James G. White, MD, Departments of Laboratory Medicine and Pathology, Pediatrics, University of Minnesota Medical School, 420 Delaware St SE, UMHC Box 490, Minneapolis, MN 55455.
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Abstract |
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Efforts to identify the translocation of glycoprotein (GP) Ib/IX receptors, either bound to von Willebrand factor (vWF) or not, from exposed surfaces to interior membranes of thrombin-activated platelets in suspension have been unsuccessful. To observe vWF uptake by platelets, we added an anti-vWF antibody and staphylococcal protein A–gold (to act as a marker for the antibody) to an incubation medium containing washed platelets and bovine plasma vWF or ristocetin-activated human vWF. Thin sections of platelets incubated for 10, 20, or 30 minutes with vWF but without antibody revealed no internalization and minimal changes in the original discoid form. Over the same 30-minute period with anti-vWF, however, GPIb/IX-vWF–anti-vWF complexes were cleared from cell exteriors to channels of the open canalicular system. Engorgement of the open canalicular system with vWF multimers resulted in changes in shape, internal transformation, and degranulation. Physical changes associated with anti-vWF–induced uptake of vWF are not seen in platelets that are involved in hemostatic plug formation or clot retraction.
Key Words: vWF–anti-vWF complexes • platelets • GPIb/IX • staphylococcal protein A–gold • open canalicular system
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Introduction |
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GPIb/IX complexes are critical for the interaction between blood platelets and damaged vascular surfaces in vivo.1 2 3 Therefore, it is important to know whether or not GPIb/IX receptors are available for platelet hemostatic reactions.4 5 6 Recent studies have suggested that GPIb/IX receptors, either alone or coupled to their natural ligand, vWF, are rapidly cleared from the external surfaces of blood platelets to the channels of the surface-connected OCS after exposure to thrombin and other platelet-activating agents.7 8 9 10 11 12 13 14 Proponents of this hypothesis have suggested that downregulation and clearance of GPIb/IX on platelets during hemostasis favor upregulation of GPIIb/IIIa, the fibrinogen receptor; aggregation; and thrombus formation.9 13 14 15
Although this concept is attractive, several investigations have suggested that GPIb/IX, though possibly downregulated on activated platelets in suspension under certain conditions,8 is not cleared from external surfaces to the channels of the OCS.16 17 18 Platelets that were (1) exposed to thrombin before or after attachment to Formvar grids, (2) allowed to spread fully, and (3) then covered with bovine- or ristocetin-activated human plasma were covered from edge to edge with vWF multimers.16 This event could not have occurred if the GPIb/IX receptors had been cleared from cell surfaces by suspension activation, surface activation, or a combination of the two stimuli.17 18 The only way to cause GPIb/IX movement was to bind vWF to spread cells and then add anti-vWF antibody before fixation.18 19 The vWF antibody caused GPIb/IX-vWF complexes to move from the cell margins into the "caps" overlying the central zones of spread platelets. Despite clearance of the receptor-ligand-antibody complexes from the peripheral two thirds of cell surface membranes, GPIb/IX receptors capable of binding a second and even a third wave of vWF multimers remained.19
The present investigation used a similar approach to study vWF uptake by platelets in suspension. Platelets that were incubated for as long as 30 minutes with various sources of vWF did not take up vWF multimers. However, addition of an anti-vWF antibody to the platelet-vWF suspension resulted in rapid accumulation of GPIb/IX–vWF–anti-vWF complexes stained with PAG particles that were found to be localized in the channels of the OCS.
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Methods |
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Preparation of Platelets
After verbal informed consent was obtained, blood for the present study was drawn from donors who were regular participants in our studies and who were known to be free of all medications. Venous blood was immediately mixed with CCD (93 mmol/L sodium citrate, 7.0 mmol/L citric acid, and 140 mmol/L dextrose), pH 6.5, at a 9:1 ratio of blood to anticoagulant.20 PRP was obtained by centrifugation of whole blood for 20 minutes at 100g. Platelets from PRP were washed twice with an equal volume of CCD; 70 mmol/L adenosine and 3 mmol/L theophylline were also added.21 The final pellet was resuspended in HBSS without calcium, adjusted to a density of 5x104/µL, and maintained at 37°C for at least 30 minutes before use.
Preparation of Plasmas
Human PPP was prepared from PRP by centrifugation at 1000g for 10 minutes and then incubated with ristocetin antibiotic (1.2 mg/mL) before use.17 Bovine blood was obtained from healthy, adult cattle that were housed under the supervision of the School of Veterinary Sciences at the University of Minnesota.22 Samples aspirated from the external jugular vein were mixed immediately with CCD at a 9:1 ratio of blood to anticoagulant. Bovine PPP was prepared in the same manner as human PPP for use as a source of vWF. Humate-P (Beringwerke AG), a fibrinogen-free partially purified source of human vWF that is often used for intravenous therapy,23 was also used. Immunoblots of Humate-P and of human and bovine plasma revealed that the vWF multimers were essentially identical in all three sources (data not shown).
Antibodies
A rabbit polyclonal antibody against the glycocalicin portion of GPIb
was kindly provided by Dr Ken Clemetson of the Theodor Kocher Institut, Berne, Switzerland. This antibody blocks ristocetin-induced human plasma or purified vWF-induced platelet agglutination and prevents deposition of vWF multimers on spread cells.16 17 The anti-glycocalicin antibody has been shown to be an excellent, highly specific probe for GPIb/IX on human platelets.24 25 AP1 and 6D1, monoclonal antibodies against GPIb that were used in earlier studies,16 17 18 19 were kindly provided by Dr Tom Kunicki, Scripps Clinic, La Jolla, Calif, and Dr Barry Coller, Mt Sinai Medical Center, New York, NY, respectively. The polyclonal rabbit anti-human vWF antibody was obtained from Dako, and its specificity for human, bovine, and porcine vWF has been demonstrated in several previous studies.16 17 18 19
Incubation of Platelets With vWF
Samples of washed platelets in HBSS were combined with GPRP (Calbiochem; final concentration, 2.5 mmol/L) to inhibit fibrin formation and platelet aggregation.26 Bovine plasma (0.1 mL), ristocetin (1.2 mg/mL)-activated human plasma, or a 1/100 dilution of Humate-P activated by 1.2 mg/mL ristocetin was added to 1 mL of platelet suspension. Replicate samples were maintained at 37°C for 10, 20, and 30 minutes. After incubation the samples were fixed in an equal volume of 0.1% glutaraldehyde in cacodylate buffer for 30 minutes at room temperature. The platelets were centrifuged to a pellet, and the supernatant fixative was discarded and replaced by 3% glutaraldehyde in White's saline (a 10% solution of a 1:1 mixture of [a] 2.4 mmol/L NaCl, 0.1 mmol/L KCl, 46 mmol/L MgSO4, and 64 mmol/L Ca(NO3)2 · 4H2O and [b] 0.13 mmol/L NaHCO3, 8.4 mmol/L NaH2PO4, and 0.1 g/L phenol red, pH 7.4).27 After 30 minutes at 4°C the pellet was rinsed, and the supernatant was discarded and replaced with either 1% osmic acid in Zetterquist's buffer or 1% osmic acid in distilled water containing 1.5% K3Fe(CN)6 for 1 hour at 4°C. Samples were dehydrated in a graded series of alcohols and embedded in epoxy resin (Epon 812). Thin sections were cut from the plastic blocks on an ultramicrotome and examined either unstained or after staining with uranyl acetate and lead citrate to enhance the contrast. Examination was performed in a Philips 301 electron microscope.
Other samples of washed platelets that were incubated with one of the three sources of vWF for 10, 20, or 30 minutes were fixed in an equal volume of 1% paraformaldehyde and 0.05% glutaraldehyde in cacodylate buffer for 15 minutes; centrifuged to pellets; washed and resuspended in HBSS containing 0.1% BSA three times for 5 minutes each; centrifuged again; and resuspended in 0.5 mL buffer containing a 1/50 dilution of a polyclonal rabbit anti-vWF antibody (Dako A082).28 After 15 minutes the samples were sedimented to pellets, washed, resuspended in HBSS with 0.1% BSA, and resuspended in a 0.5-mL volume of buffer containing a 1/25 dilution of stock PAG10 (Auroprobe, Amersham). After 15 minutes the sample was pelleted, washed and resuspended three times in HBSS, and centrifuged to a pellet again. Supernatant was discarded and replaced with 3% glutaraldehyde in White's saline; fixation with osmic acid, dehydration, and embedding were performed as described above.
Incubation With vWF, Anti-vWF, and PAG
Additional washed platelet samples in HBSS with GPRP were combined with one of the sources of vWF, 0.1 mL of the 1/50 dilution of the anti-vWF antibody, and 0.1 mL PAG10. The samples were mixed once and incubated at 37°C for 10, 20, or 30 minutes. After incubation the samples were fixed in glutaraldehyde/osmic acid, dehydrated, embedded, and sectioned for electron microscopy study as described above. In other experiments either AP1 or the anti-glycocalicin antibody was added to the platelet suspension before the vWF source, the anti-vWF antibody, and PAG10 were added. These samples were incubated for 30 minutes and prepared for electron microscopy as described above.
Influence of Cytochalasins on Uptake of GPIb/IX-vWF–anti-vWF–PAG Complexes
Cytochalasins at low concentrations inhibit the assembly of actin filaments from molecular species and can disassemble the newly formed actin filaments.29 30 At higher concentrations cytochalasins will inhibit the function of previously formed actin filaments that constitute the platelet membrane "motor."31 Cytochalasin E is 100 to 1000 times more potent than cytochalasin B,32 and various concentrations of each agent were combined and incubated with washed platelets for 15 minutes before exposure to one of the sources of vWF, anti-vWF, and PAG10. Incubation continued at 37°C for 15 or 30 minutes and samples were prepared for thin section electron microscopy as above.
Cryosection Preparation and Immunogold Labeling
Samples of washed platelets (1 mL) were combined with 0.1 mL bovine- or ristocetin-activated human plasma or Humate-P, 0.1 mL anti-vWF antibody (1/50), 0.1 mL PAG10, and 40 µL GPRP and incubated at 37°C for 30 minutes. The incubated samples were combined with an equal volume of 1% paraformaldehyde and 0.05% glutaraldehyde in 0.1 mol/L cacodylate buffer for 15 minutes and then concentrated to pellets by centrifugation. The supernatant was removed, fresh fixative was added, and pellet fixation was continued for 24 hours at 4°C. After this period small portions of the pellets were washed and infiltrated for 2 hours in a mixture of polyvinylpyrrolidone and sucrose as recommended by Tokuyasu.33 Pyramidal blocks of the infiltrated pellets were cut, mounted on metal stubs, and frozen in liquid N2.16 Frozen sections were cut from these blocks by ultracryomicrotome techniques described elsewhere. In brief, the sections were cut at -90°C with an MT 6000-XL ultracryotome with a CR2000 cryounit (RMC, Inc) and stored on Formvar-coated copper grids.
Indirect immunocytochemistry was used to localize GPIb/IX. Sections were first incubated in 1:100 dilution of the anti-GPIb (anti-glycocalicin) antibody for 45 minutes.16 After repeated washes with PBS, the location of the primary antibody was visualized by incubation with protein A–colloidal gold (for polyclonal antibodies) for 60 minutes (Amersham International plc). Excess gold was removed by washing the sections in PBS. Finally, labeled grids were stained and embedded in a mixture of 2% polyvinyl alcohol and 0.3% uranyl acetate34 35 before examination in the electron microscope. All experiments in this study were repeated at least once.
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Results |
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Incubation With vWF
Incubation of washed platelet suspensions with human plasma, Humate-P activated by ristocetin, or bovine plasma without ristocetin in the absence of stirring for 10, 20, or 30 minutes caused only modest changes in morphology11 ; ie, most platelets retained their discoid shape. Some platelets were irregularly shaped and small agglutinates were occasionally observed when Humate-P or bovine plasma was the source for vWF. Differences from washed control platelets incubated for similar intervals were not significant.
Identification of vWF Multimers on Platelets Incubated With vWF
After incubation of platelets for 10, 20, or 30 minutes, vWF multimers from ristocetin-activated human plasma and Humate-P or bovine vWF were identified on exposed platelet surfaces. Thin section study revealed that the vWF multimers occurred in discrete masses, as evidenced by anti-vWF and PAG staining (Fig 1
). The distribution of these multimers was relatively uniform on discoid platelets regardless of the duration of incubation. There was no suggestion that GPIb/IX-vWF multimers were being cleared from platelet surfaces or accumulating over channels of the OCS.
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Platelets Incubated With vWF–Anti-vWF and PAG10
Addition of the anti-vWF antibody to platelet suspensions containing vWF from ristocetin-activated human plasma and Humate-P or bovine plasma resulted in rapid uptake of receptor-ligand complexes. The initial association of the vWF–anti-vWF complexes appeared to be with the external surfaces of discoid platelets (Figs 2 through 4). Complexes of vWF–anti-vWF that were marked by PAG10 covered the external surfaces of the platelets. Although discoid platelets were more common in the 10-minute samples, they were also found in the 20- and 30-minute samples.
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; magnification x42 500).
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Uptake of PAG10-marked vWF–anti-vWF complexes into the platelet interior was apparent even in the 10-minute samples. PAG-marked GPIb/IX-vWF–anti-vWF complexes appeared to have been translocated to the platelet interior through the channels of the OCS (Figs 2 through 6
). Uptake by coated or uncoated vesicles was not observed.36
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Changes in platelet shape that were associated with uptake of the vWF–anti-vWF complexes were evident in 10-minute samples (Figs 3, 5, and 6) but were more prominent after incubation for 20 (Figs 7 through 12) and 30 (Figs 13 through 16) minutes. Increases in the number of OCS channels filled with GPIb/IX-vWF–anti-vWF complexes and in channel distention appeared to be associated with changes in shape (Figs 7 through 12). The physical alterations resembled those caused by activation with potent agents, such as thrombin,27 or uptake of foreign particles.37
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Platelet activation due to uptake of complexes caused degranulation (Figs 7 through 12). However, -granule products destined for secretion appeared to be trapped by complex-filled channels of the OCS (Figs 13 through 16). As a result, PAG-marked vWF–anti-vWF complexes that filled OCS channels also appeared in distended -granules and in some profiles, may have labeled the granule membrane (Figs 8 through 12).
The change in shape, internal transformation, and degranulation that followed uptake of complexes into the OCS were also associated with clearance of the exposed platelet surfaces (Figs 7 through 12). Though more prominent at 30 minutes (Figs 13 through 16), clearance of GPIb/IX-vWF–anti-vWF complexes from cell surfaces to the interior membranes was evident in samples incubated for 10 or 20 minutes.
After a 30-minute incubation with vWF–anti-vWF, most platelets were irregular or relatively spherical (Figs 13 through 16) and exposed surfaces had been almost completely cleared of GPIb/IX-vWF–anti-vWF complexes. The OCS channels were distended with multimers of vWF and anti-vWF marked by PAG particles. Mitochondria (Fig 14) were intact in the altered platelet cytoplasm, but the -granules were absent. Their membranes and contents had most likely fused to swollen OCS channels (Figs 15 and 16).
Platelet suspensions combined with AP1 or anti-glycocalicin antibody before vWF, anti-vWF, and PAG10 were added that were then incubated for 30 minutes did not take up the GPIb/IX-vWF–anti-vWF complexes.
Localization of GPIb/IX After Uptake of vWF–Anti-vWF Complexes
Cryosections of platelets that were incubated with either source of vWF, together with anti-vWF and PAG10, for 30 minutes before fixation and freezing and then stained with anti-glycocalicin antibody and PAG3 or a "cocktail" of AP1 or 6D1 followed by a goat anti-mouse IgG coupled to gold particles appeared to have residual GPIb/IX receptors on exposed cell surfaces and lining the channels of the OCS (Fig 17). Thus, translocation did not result in complete clearance of GPIb/IX receptors to the platelet interior.
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Influence of Cytochalasins on Uptake of GPIb/IX-vWF–Anti-vWF–PAG Complexes
Exposure of platelets to cytochalasin B (10-6 or 10-5 mol/L) or cytochalasin E (10-7 mol/L) for 15 minutes before incubation with bovine vWF, anti-vWF, and PAG10 for 30 minutes had no effect on the clearance of receptor-ligand complexes. Cytochalasin B at 10-4 mol/L appeared to inhibit but did not prevent uptake. Cytochalasin E at 10-6 mol/L inhibited and at 10-5 mol/L blocked clearance of GPIb/IX-vWF–anti-vWF–PAG complexes from the exterior to interior membranes (data not shown).
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Discussion |
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Results of the present investigation have shown that human platelets in suspension can take up vWF multimers. Incubation with only bovine plasma vWF, ristocetin-activated human plasma vWF, or ristocetin-activated Humate-P,23 a partially purified preparation of vWF, resulted in labeling of platelet surfaces but did not cause translocation of vWF multimers to the platelet interior. Platelet uptake of vWF during incubation required addition of an anti-vWF antibody to the medium containing platelets and vWF. The surfaces of discoid platelets were more densely labeled by vWF–anti-vWF complexes than were lentiform cells that had been exposed to one of the sources of vWF but without the anti-vWF antibody. Conversion of vWF on exposed platelet surfaces to a vWF–anti-vWF immune complex resulted in rapid clearance of GPIb/IX-vWF–anti-vWF complexes marked by PAG10. These complexes appeared to enter the discoid platelets via channels of the surface-connected OCS. No evidence of entry by clathrin-coated or uncoated vesicles that formed at the cell surface was noted.36
Transfer of large quantities of GPIb/IX-vWF–anti-vWF complexes from the cell exterior to the OCS was associated with apparent distention of the OCS channels. Channel distention of granule and OCS elements was associated with changes in shape, internal transformation, and degranulation.27 37 During degranulation distended channels became connected to -granules, and gold markers often appeared within distended granules.
Internalization of GPIb/IX-vWF–anti-vWF complexes was associated with their clearance from exposed surface membranes. Involvement of GPIb/IX in the binding and clearance of complexes was demonstrated by exposing the platelets to either AP1 or anti-glycocalicin (antibodies to GPIb/IX) before adding vWF and anti-vWF. Both AP1 and anti-glycocalicin blocked uptake of vWF–anti-vWF complexes during a 30-minute incubation. However, cryosections of 30-minute samples that were stained for GPIb/IX revealed that their receptors remained on exposed surfaces. As a result, we concluded that translocation of GPIb/IX-vWF–anti-vWF complexes from exposed surfaces to interior membranes was not associated with complete clearance of GPIb/IX receptors.
Ultrastructural gold immunocytochemistry that had been used in earlier studies failed to show that any significant change had occurred in the frequency or organization of GPIb/IX and GPIIb/IIIa receptors on thrombin-activated platelets in suspension or on platelets treated with thrombin after they had been spread on Formvar grids.16 17 Additional investigations with human and bovine vWF have shown that GPIb/IX receptor occupancy on spread platelets does not cause any decrease in frequency, movement into the OCS, or reorganization of GPIb/IX-vWF receptor complexes.18 Furthermore, binding of vWF to discoid platelets (as a marker for GPIb/IX) and fibrinogen-coated gold particles (to identify GPIIb/IIIa) that are subsequently allowed to spread revealed that fibrinogen-gold moved into the caps that overlie spread cell centers and OCS channels while the vWF attached to GPIb/IX spread edge to edge with the surface membrane as the cell expanded.38 These findings support the concept that GPIb/IX, compared with GPIIb/IIIa, is a relatively tenacious receptor and does not move easily on platelet surfaces before or after stimulation.39 40 41 42
The mechanism involved in the translocation and uptake of GPIb/IX-vWF–anti-vWF–PAG may utilize the membrane motor. Earlier studies31 32 demonstrated that a membrane motor, relatively resistant to the action of cytochalasins B and E, was responsible for clearing GPIIb/IIIa-fibrinogen-gold from exterior surfaces to the OCS channels. Transport of GPIb/IX receptor-ligand complexes was similarly resistant to low concentrations of cytochalasins B and E but was inhibited or blocked by larger amounts.
Only the addition of anti-vWF18 19 or, in more recent studies, anti-glycocalicin antibody,43 caused movement of GPIb/IX on spread platelets. Success in mobilizing vWF-bound GPIb/IX by exposure to anti-vWF on spread platelets suggested this antibody might be useful for studying GPIb/IX movement on platelets in suspension. Movement of GPIb/IX-vWF complexes that was induced by anti-vWF on spread platelets did not cause complete loss of GPIb/IX from the cleared areas.18 19 Residual GPIb/IX could be identified on cleared margins after one and even two translocations induced by anti-vWF.19 The present study, which involved use of cryosections and immunogold techniques, has shown that movement of GPIb/IX-vWF complexes, after exposure to anti-vWF, from exposed surfaces to interior membranes also leaves behind GPIb/IX receptors, just as in previous investigations.18 19
Thus, we have confirmed that GPIb/IX receptors on surfaces of platelets in suspension can move and be cleared to the OCS channels. Hourdille and colleagues have reported that GPIb/IX receptors, either alone or occupied by vWF, are almost completely cleared from exterior surfaces to the OCS channels after exposure of the platelets to thrombin in suspension.10 11 12 13 We have tried to corroborate their findings in a number of investigations.16 18 28 Frozen thin sections of resting platelets and cells that were activated by thrombin for as long as 30 minutes and later stained with an anti-glycocalicin antibody and PAG revealed no loss of GPIb/IX from activated platelet surfaces or an increase in density of receptors lining the channels of the OCS.28 Thin sections of thrombin-activated resting cells and platelets that had been deposited on plastic chambers for as long as 30 minutes; fixed; and sequentially exposed to vWF, anti-vWF, and PAG revealed discrete masses and linear fragments of vWF (marking the sites of GPIb/IX) over the entire platelet surface. No differences in vWF-GPIb/IX binding between control and thrombin-activated platelets were observed.
In more recent studies resting platelets have been incubated with anti-glycocalicin for 30 minutes before exposure to thrombin for 5 or 10 minutes. After fixation at these times the cells were stained with PAG.44 Other platelets without prior exposure to anti-glycocalicin were exposed to thrombin for 5 or 10 minutes, fixed, and stained with anti-glycocalicin and PAG to locate GPIb/IX. There was no difference in the frequency of GPIb/IX receptors between platelets that had been exposed to anti-glycocalicin before exposure to thrombin and those that were exposed to the antibody after activation by serine protease.
Thus, our attempts to confirm earlier observations, which suggest that GPIb/IX receptors, coupled to vWF or not, are cleared from exposed platelet surfaces to the OCS channels after activation by thrombin in suspension, have failed.10 11 12 13 14 As a result, we decided to use an anti-vWF antibody. Although converting vWF to an immune complex by exposure to a polyclonal antibody may be unphysiological, the results indicate that GPIb/IX-vWF complexes can be cleared from platelet surfaces to the OCS. In addition, our findings indicate what the OCS channels and platelet interiors would look like if GPIb/IX-vWF multimers were cleared from platelet surfaces to internal membranes after thrombin activation. The high-molecular-weight multimers distend the OCS channels, thereby producing changes in their physical appearance and organization that are not seen in thrombi formed at sites of vascular injury.3 45
In summary, the present investigation has followed the uptake of GPIb/IX-vWF complexes by platelets in suspension. Incubation for 30 minutes with bovine plasma, ristocetin-activated human plasma, or ristocetin-activated Humate-P did not cause internalization of GPIb/IX-bound vWF on platelet surfaces. However, addition of anti-vWF to the vWF source–+atelet system resulted in translocation of GPIb/IX-vWF–anti-vWF complexes (marked by PAG) from exterior surfaces to the channels of the OCS. Incorporation of large vWF multimers caused distention of OCS channels and consequent changes in shape, internal transformation, and degranulation. OCS channels that are filled with vWF multimers (as observed in the present study) are not found in thrombus-forming platelets on denuded subendothelium or in isometric clot retraction.3 45 46 Thus, while it is possible for platelets to internalize GPIb/IX-vWF complexes, it is unlikely that such uptake occurs under other conditions in vivo or in vitro.
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL11880 and HL49556 and grant FY940900 from the March of Dimes Birth Defects Foundation.
Received July 13, 1995; accepted February 27, 1996.
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References |
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