Induction of GPIb/IX-vWF Receptor-Ligand Translocation on Surface-Activated Platelets
Abstract Multimers of von Willebrand factor (vWF) readily bind to glycoprotein (GP) Ib/IX receptors on spread human platelets and cover the cell from edge to edge. Addition of anti-vWF antibody to spread platelets covered with vWF caused the multimers to move from peripheral margins into caps over platelet centers. Despite almost complete centralization of receptor-ligand complexes, a significant number of GPIb/IX receptors capable of binding multimers remained available on the peripheral zone. Fixation followed by a second incubation with vWF, anti-vWF, and staph protein A coupled to 5-nm gold particles (PAG5) revealed multimers extending from the centrally concentrated cap of vWF to cell margins. If spread platelets with central caps of vWF were exposed a second time to multimers and anti-vWF antibody before fixation and stained with PAG5 after, the residual GPIb/IX receptors and second wave of vWF formed a ring around the cap, leaving a clear margin. If after fixation and staining with PAG5 the grids with caps and rings of vWF were washed, exposed a third time to vWF, refixed, and then incubated with anti-vWF and PAG10, the clear margin was covered with multimers of vWF forming a second ring around the first circle of receptor-ligand complexes. Thin sections of spread platelets with central caps of GPIb/IX-vWF complexes revealed only rare examples of uptake by the open canalicular system. The interaction of GPIb/IX with vWF multimers observed in the present study suggests a mechanism by which platelets under high shear forces may adhere and attach firmly to a denuded vascular surface.
- Received July 27, 1994.
- Accepted February 16, 1995.
Investigations using isotopic techniques,1 flow cytometry,2 3 and ultrastructural immunogold cytochemistry4 5 6 7 8 have suggested that 60% to 80% of glycoprotein (GP) Ib/IX receptors are downregulated or cleared from the surfaces of thrombin-activated platelets to channels of the open canalicular system (OCS).9 Studies in our laboratory, however, have been unable to confirm the reported phenomenon.10 11 12 13 Use of a polyclonal rather than a monoclonal antibody to study downregulation of GPIb/IX following thrombin activation of platelets in suspension revealed no decrease in fluorescence intensity.11 Immunogold techniques on frozen thin sections of resting and thrombin-activated platelets in suspension, together with thrombin stimulation of platelets spread on surfaces, were unable to demonstrate any decrease in GPIb/IX receptors. More recently we have shown that the combination of thrombin activation in suspension, spreading on formvar or glass surfaces, and receptor occupancy by von Willebrand factor (vWF) for intervals up to 1 hour does not result in downregulation or clearance of GPIb/IX receptor-ligand complexes from activated platelet surfaces to the OCS.12 13
We have, however, found one way to induce the translocation of GPIb/IX receptors on platelet surfaces,13 confirming that the receptor-ligand complexes are mobile.5 When previously spread platelets coated with vWF multimers are exposed to an anti-vWF antibody, the multimers move from peripheral margins into caps over cell centers. The present study has investigated the antibody-induced translocation of GPIb/IX-vWF complexes on spread platelets.
Preparation of Platelets
Blood for the present study was obtained after informed consent from donors used regularly in our studies and known to be free of all medications. Blood obtained by venipuncture was mixed immediately with citrate–citric acid dextrose (CCD; 93 mmol/L sodium citrate, 7.0 mmol/L citric acid, and 140 mmol/L dextrose), pH 6.5, in a ratio of 9 parts blood to 1 part anticoagulant.14 Platelet-rich plasma (PRP) was obtained by centrifugation of the whole blood for 20 minutes at 100g. Platelets from PRP were washed twice with an equal volume of CCD with 70 mmol/L adenosine and 3 mmol/L theophylline added.15 The final pellet was resuspended in Hanks’ balanced salt solution (HBSS) without calcium and adjusted to 50 000 platelets per microliter.
Preparation of Plasmas
Human platelet-poor plasma (PPP) was prepared from PRP by centrifugation at 1000g for 10 minutes. Human PPP was incubated with ristocetin (1.2 mg/mL) antibiotic before use. Bovine blood was obtained from healthy adult cattle housed under supervision of the School of Veterinary Sciences at the University of Minnesota. Samples aspirated from the external jugular vein were mixed immediately with the CCD anticoagulant described above in a ratio of 9:1.16 Bovine PPP was prepared in the same manner as human PPP for vWF. Immunoblots of the human and bovine plasmas were prepared to be sure their content of vWF was comparable (data not shown). Multimers of human and bovine vWF are virtually identical.
Small drops, 10 μL in volume, of washed platelet suspension were placed on carbon-stabilized, formvar-coated grids and allowed to interact with this surface for 20 minutes at 37°C.17 After this period unattached platelets were washed away by passing the grid through successive drops of HBSS. For evaluation of vWF multimer frequency and distribution, grids of spread platelets were fixed in 0.01% glutaraldehyde in HBSS for 30 minutes at 37°C.
Interaction of Bovine and Ristocetin-Activated Human Plasma vWF With Spread Human Platelets
Grids of platelets fixed after spreading were incubated with bovine plasma or ristocetin-activated human plasma for 15 minutes, washed in drops of HBSS, and then exposed to a polyclonal anti-vWF (Dako A082, Dako) antibody in various dilutions, usually 1:50.10 11 After 15 minutes the antibody was removed, and the grid was washed in several changes of HBSS. The grids were then incubated with staph protein A coupled to either 5- or 10-nm gold particles (PAG5 and PAG10, respectively) (Amersham) for 15 minutes. After 15 minutes the grids were rinsed several times, fixed in 2% glutaraldehyde in cacodylate buffer for 30 minutes, and rinsed in buffer. Excess fluid was removed from the grid edge with filter paper, and the grid was air dried.
Organization of vWF Multimers on Unfixed, Spread Platelets
Formvar grids incubated with platelets for 20 minutes were rinsed in HBSS and combined with either human or bovine plasma for 15, 30, 45, or 60 minutes before being rinsed in HBSS and fixed in 0.01% glutaraldehyde.12 13 After fixation the grids were washed, exposed to anti-vWF antibody for 15 minutes, rinsed, and combined with 5 or 10 nm protein A gold. The grids were rinsed and fixed again in 2% glutaraldehyde in cacodylate buffer as described above.
Influence of Anti-vWF Antibody on Organization of Multimers
Platelets spread for 20 minutes were incubated with bovine plasma for 15 minutes and then exposed to anti-vWF antibody for 15 minutes before fixation. The fixed samples were stained with PAG10 and fixed again in 2% glutaraldehyde.
Second Exposure to Bovine Plasma vWF After Fixation
Spread platelets combined with bovine plasma for 15 minutes followed by anti-vWF antibody for 15 minutes were fixed and incubated with PAG10. The grids were washed and incubated with bovine plasma a second time, washed, fixed again, and stained with anti-vWF antibody and PAG5. The grids were fixed once more in 2.0% glutaraldehyde in 0.1 mol/L cacodylate buffer, rinsed, and air dried.
Second Exposure to vWF and Anti-vWF Antibody Before Fixation
Spread platelets with central caps of GPIb/IX-vWF complexes induced by anti-vWF antibody were washed and covered a second time with vWF for 15 minutes, washed, and exposed to anti-vWF antibody for 15 minutes, washed, fixed in 0.01% glutaraldehyde, and stained with PAG5 before a final fix in 2% glutaraldehyde.
Third Exposure to vWF
Spread platelets with caps and rings of GPIb/IX-vWF caused by exposure to anti-vWF antibody and stained respectively with PAG10 and PAG5 before fixation were washed, fixed in 0.01% glutaraldehyde, incubated with vWF for 15 minutes, washed, refixed in 0.01% glutaraldehyde, and stained with anti-vWF antibody and PAG10 before final fixation in 2% glutaraldehyde.
Effects of Antibodies on Second Deposition of vWF
The possibility that GPIIb/IIIa or a nonspecific receptor was binding the second wave of vWF after the initial GPIb/IX-vWF complexes had been cleared to the spread cell centers by the influence of anti-vWF antibody required consideration.10 Grids of spread platelets incubated with bovine vWF for 15 minutes and anti-vWF for 15 minutes were fixed in 0.01% glutaraldehyde, rinsed and exposed to either AP1, AP2, or anti-glycocalicin antibody at dilutions of 1:100 for 15 minutes, rinsed and incubated with bovine vWF a second time, fixed again, and stained with anti-vWF antibody followed by PAG before the final fixation in 2% glutaraldehyde. AP1, a monclonal antibody to GPIb/IX, and AP2, a monoclonal antibody specific for GPIIb/IIIa, were kindly provided by Dr Tom Kunicki, Scripps Institute, La Jolla, Calif. Anti-glycocalicin antibody was provided by Dr Kenneth Clemetson, Theodor-Kocher Institute, Bern, Switzerland.
Preparation of Cross Sections of Spread Platelets
Lab-Tek 4 chamber slides (Nunc, Inc) were used to prepare cross sections of surface-activated platelets. The glass surface of each chamber was coated initially with 0.1% polylysine for 5 minutes and washed several times before being air dried. Volumes (0.5 mL) of washed platelet suspension prepared as described above were added to each well and incubated at 37°C for 20 minutes. The chambers of spread platelets were rinsed well and then exposed to bovine or ristocetin-activated human plasmas, anti-vWF antibody, PAG5, and PAG10 in the same manner as described for studies on formvar grids (see above). After completion of incubation, fixation, and immunocytochemical procedures, the chambers were fixed again in 3% glutaraldehyde in White’s saline16 17 18 for 15 minutes and then with 1% osmic acid combined with 1.5% potassium ferrocyanide in distilled water for 30 minutes at room temperature.18 After fixation the samples were washed and dehydrated in a series of alcohols and exposed to increasing concentrations of epon in alcohol. After the final rinse in 100% epon, the chambers were drained, and 0.3 mL of 100% epon was added to each chamber. Some of the epon was saved in a syringe for final embedding. The chambers were placed in a 45°C oven overnight and in a 60°C oven for 1 to 2 days. The slab of embedded platelets was cut into rectangular wedges, mounted in holders, and sectioned on an ultramicrotome. Thin sections were stained with lead citrate and uranyl acetate to enhance contact.
Each experiment was performed in duplicate and repeated on at least three different occasions. After final fixation and air drying the grids were evaluated in a Philips 301 electron microscope.
Interaction of Platelets With Formvar Grids
Washed, discoid platelets interact quickly with formvar grids and other foreign surfaces; about 90% of platelets are fully spread on the formvar surface19 after a 20-minute exposure at 37°C. The remaining 10% reveal various stages in conversion from discoid cells to dendritic forms and fully spread forms. Spread platelets are typically thin films, round in shape, with a central density representing the site of the granulomere. However, some fully spread cells may be irregular in form. The basis for variations in spread platelet shape are not known.
Organization of vWF on Human Platelets Fixed After Spreading
Spread human platelets have the ability to bind vWF from bovine plasma, ristocetin-treated human plasma, and purified vWF (kindly provided by David Fass, Mayo Clinic, Rochester, Minn).10 Multimers of vWF selectively stained by anti-vWF antibody and PAG10 covered surface-activated fixed platelets from edge to edge (Fig 1⇓). The multimers presented a linear, serpentine appearance, resembling a mosaic of rope-like proteins. Cross sections of spread platelets covered with vWF revealed the multimers stained by anti-vWF antibody as discrete patches. The appearance indicates that the rope-like multimers lie in the plane of the surface and are not perpendicular to it20 (Fig 2⇓).
Organization of vWF on Unfixed, Spread Human Platelets
The density and distribution of vWF multimers on platelets spread on formvar grids without fixation and incubated with vWF from bovine or ristocetin-treated human plasma for 15 to 60 minutes were indistinguishable from the organization of vWF multimers on fixed, spread platelets.12 13 Multimers covered the spread platelet surfaces from edge to edge. No evidence of multimer movement away from peripheral margins and/or into channels of the OCS could be identified (Fig 3⇓).
Effects of Anti-vWF Antibody on Organization of vWF
Grids of spread platelets exposed to vWF from ristocetin-activated human plasma or bovine plasma for 15 minutes and then combined with anti-vWF antibody for 15 minutes before fixation and staining revealed a dramatic reorganization of multimers (Fig 4⇓). Linear, serpentine multimers of vWF were cleared from spread cell margins and peripheral zones to cell centers. Multimers in cell centers often appeared to coalesce (Fig 5⇓). The extent of clearance to central zones varied from cell to cell, but every platelet was affected. In contrast to the response of GPIIb/IIIa bound to fibrinogen-coated gold particles,21 resting the grids of spread platelets combined with vWF and the antibody to vWF on drops of HBSS had no apparent effect on the extent of vWF multimer concentration in platelet centers. Cross sections of spread platelets exposed to vWF for 15 minutes and anti-vWF antibody and PAG5 for 15 minutes before fixation revealed central caps of GPIb/IX-vWF multimers. However, despite centralization of vWF on virtually every spread platelet, only two platelets in over 300 cross sections revealed any uptake of receptor-ligand complexes into the OCS (Figs 6⇓ and 7⇓).
Second Exposure of Spread Platelets With Centrally Concentrated Multimers to vWF After Fixation
Spread platelets combined with bovine or ristocetin-activated human plasma and anti-vWF antibody to concentrate multimers in cell centers, then fixed and incubated with either plasma a second time and stained with PAG5 revealed the persistence of GPIb/IX receptors over peripheral zones of the spread cells (Fig 8⇓). Centrally concentrated vWF multimers marked by PAG5 and PAG10 were surrounded by multimers stained by PAG5 extending to the edge of the cells. Cross sections of spread platelets prepared in an identical manner in glass chambers revealed a similar appearance (Fig 9⇓). The first wave of vWF bound to GPIb/IX and stimulated to move by anti-vWF was concentrated into caps stained by PAG10 over platelet centers. The second wave of vWF was deposited as linear polymers stained by PAG5 extending from central caps of vWF to platelet edges.
Second Exposure of Spread Platelets to vWF and Anti-vWF Antibody Before Fixation
Spread platelets with central caps of GPIb/IX-vWF complexes moved by treatment with anti-vWF and exposed to a second wave of vWF, anti-vWF, and PAG5 revealed rings around the PAG10-stained central mass (Figs 10⇓ and 11⇓). A space between the ring and platelet margins was clear of receptor-ligand complexes. Thus, anti-vWF was able to trigger translocation of GPIb/IX-vWF from the peripheral zone a second time.
Third Exposure to vWF
Spread platelets with caps and rings of vWF caused by double exposure to multimers and anti-vWF antibody, fixed, incubated with vWF, fixed again, and re-treated with anti-vWF and PAG10 revealed a second ring. The peripheral zone from the cell edge to the ring of multimers stained mainly by PAG5 surrounding the central cap marked by PAG10 contained multimers of vWF stained by anti-vWF and PAG10 (Fig 12⇓). The last exposure to PAG10 also stained the first ring. However, the PAG5 marking ring 1 was not obscured by PAG10.
Influence of Monoclonal Antibodies on the Second Exposure to vWF
The influence of specific antibodies on the second exposure to vWF was tested on spread platelets covered with multimers centralized into caps by anti-vWF antibody and fixed. Grids of platelets prepared in this manner and incubated with either AP1 or AP2, monoclonal antibodies against GPIb/IX and GPIIb/IIIa, respectively, or with anti-glycocalicin, the polyclonal antibody against GPIb/IX, before the second exposure to bovine vWF gave sharply contrasting results. Incubation with AP2 had no effect on the subsequent deposition of vWF multimers on the peripheral zone (Fig 13A⇓). Prior exposure to AP1 (Fig 13B⇓) or anti-glycocalicin antibody (Fig 13C⇓) prevented binding of vWF multimers to the cleared peripheral zone. PAG5 did bind to the anti-glycocalicin coupled to GPIb/IX, adding further support to the presence of GPIb/IX receptors remaining on the peripheral zone (Fig 13C⇓).
The present study evaluated the anti-vWF antibody–induced translocation of GPIb/IX complexes on the plasma membranes of surface-activated, spread platelets. Fifteen minutes after exposure to anti-vWF antibody the multimers of bovine or ristocetin-activated human vWF covering the surface of spread platelets from edge to edge moved from peripheral margins into caps over the central zone. Peripheral and intermediate zones appeared devoid of multimers stained by the anti-vWF antibody and made visible by PAG10 and PAG5. However, a second exposure to human or bovine vWF after fixation followed by restaining with the anti-vWF antibody and PAG demonstrated that GPIb/IX or some other surface receptor remained available to bind multimers during the second incubation with vWF. The vWF multimers stained by anti-vWF antibody and PAG extended from the central caps formed by the first wave to the peripheral margins of the spread cells.
The residual GPIb/IX receptors on the peripheral zone not only bound vWF but were also able to translocate the multimers. Spread platelets with central caps of GPIb/IX-vWF complexes covered a second time with vWF and anti-vWF antibody formed rings of multimers stained by 5-nm PAG particles around the PAG10-marked central caps, leaving clear areas extending to cell margins.
Yet the cleared areas after two exposures to vWF and anti-vWF antibody were not devoid of GPIb/IX receptors. Incubation of spread platelets with central caps of vWF, surrounded by rings of multimers, to vWF a third time after fixation revealed another ring of vWF attached to GPIb/IX extending from the edge of the first ring around the central cap to the cell margin.
The persistence of receptor binding sites for vWF on areas cleared of GPIb/IX-vWF complexes after two exposures to vWF and anti-vWF antibody is reminiscent of the tenacity of GPIIb/IIIa receptors.22 Loftus and Albrecht23 and later Albrecht and coworkers24 demonstrated that exposure of spread platelets to fibrinogen-coated gold particles (Fgn/Au) resulted in translocation of GPIIb/IIIa-Fgn/Au complexes toward cell centers. Work in our laboratory noted that the receptor-ligand complexes could be moved into caps over spread platelet central zones by incubating the grids on drops of HBSS for 10 to 15 minutes.17 21 The cleared peripheral and intermediate zones, however, were not devoid of receptors for Fgn/Au. Fixation and exposure to a second wave of Fgn/Au resulted in deposit of the electron-dense probes for GPIIb/IIIa from the central cap of receptor-ligand complexes to the cell margins.17 21 22
Exposure of spread platelets with caps of GPIIb/IIIa-Fgn/Au complexes to alternating waves of small latex particles and Fgn/Au did not exhaust the binding capacity for Fgn/Au.22 As many as two rings of Fgn/Au bound to GPIIb/IIIa separated by circles of latex surrounding the central cap of GPIIb/IIIa-Fgn/Au complexes could be deposited on single spread platelets. The receptors binding the rings of Fgn/Au outside the central cap of receptor-ligand complexes were shown to be GPIIb/IIIa.22
The present study has demonstrated that the receptors remaining over cleared areas on platelets with central caps of GPIb/IX-vWF complexes are GPIb/IX. AP2 had no effect on the frequency or distribution of multimers deposited on the peripheral and intermediate zone of spread platelets with central zones occupied by GPIb/IX-vWF complexes. Exposure of spread platelets with centrally concentrated masses of receptor-ligand complexes to AP1 completely inhibited deposition of multimers during the second exposure to human or bovine vWF. The polyclonal antibody glycocalicin also blocked binding of multimers to the peripheral zone and demonstrated persistence of GPIb/IX receptors remaining in cleared areas.
The results indicate, as they did for GPIIb/IIIa,21 22 that complete clearance of GPIb/IX from the platelet surface is virtually impossible. Despite the translocation of significant numbers of GPIb/IX receptors to cell centers with vWF multimers, many remained available in the cleared zones to bind additional vWF. As in the case of the GPIIb/IIIa receptors replacing those cleared from peripheral and intermediate zones and concentrated in caps over centers of spread platelets,17 21 the origin of the GPIb/IX receptors binding the second and third waves of vWF is uncertain.
However, two mechanisms might explain the persistence of GPIb/IX receptors in the twice-cleared zones. Lu and colleagues7 have shown that downregulation of GPIb/IX caused by thrombin in suspension is almost completely reversible. Their findings have been confirmed by Michelson and associates.25 To evaluate that possibility, samples exposed to vWF and anti-vWF antibody were stained with PAG10, fixed, and exposed to vWF, anti-vWF, and PAG5 for intervals of 5, 10, 15, 20, and 30 minutes. The cleared area became more extensive with each successive exposure up to 15 minutes, but staining the fixed cell with a second wave of vWF after fixation revealed the same extent of residual GPIb/IX receptors covering the peripheral zone at each stage in formation of the central caps of vWF multimers. Thus, clearance followed by a time-dependent reversal does not readily explain the findings observed in the present study.
Another mechanism that might explain restoration of GPIb/IX to cleared areas on spread platelets previously exposed to vWF and anti-vWF antibody is receptor cycling. Wencel-Drake and colleagues26 27 have proposed that GPIIb/IIIa receptors on discoid platelets are in a constant state of flux. A significant number appear to be taken up into the platelet each hour, and a similar number are returned to the outside surface over the same interval. Behnke28 as well as Morgenstern et al29 report that plasma-borne particulates and antibodies binding to platelets are taken up into clathrin-coated vesicles and transferred to alpha granules. Thus, uptake of receptors by platelets stimulated by thrombin and a vesicular mechanism that could facilitate transfer from channels of the OCS through the platelet cytoplasm under the surface membrane and back to the spread exterior surface appear to exist.
However, nearly all of the OCS channels have been evaginated to contribute to increased surface areas of fully spread platelets.19 Cross sections of over 300 platelets revealed only two examples in which GPIb/IX-vWF complexes marked by PAG5 were in the process of uptake into OCS channels and transfer to vesicles or alpha granules. No evidence of coated or uncoated vesicle formation from OCS channels or development on the undersides of spread cell membranes was observed in our studies. Thus, receptor recycling does not appear to provide a plausible explanation for the origin of residual GPIb/IX receptors present on peripheral zones of spread platelets cleared by prior exposure to vWF and anti-vWF antibody.
If neither recycling nor reversal adequately explain the persistence of GPIb/IX on peripheral zones of spread platelets, the possibility that the continuing availability of receptors is due to the fact that they never left must be considered. The size of the serpentine multimers may result in coverage of the platelet surface without contacting all of the available GPIb/IX receptors.30 Even a second exposure to vWF may fail to bind all the remaining receptors, leaving some available to interact with the third wave of multimers.
The present results show that GPIb/IX receptors coupled to vWF, their natural ligand, can move on spread platelet surfaces, even though they do not appear to do so as a result of thrombin activation in suspension, surface activation, receptor occupancy, or a combination of all three stimuli.13 Demonstration that GPIb/IX attached to its ligand vWF can move in the plane of the exposed membrane on surface-activated, spread platelets is important to understanding the role of the receptor in hemostatic physiology. The importance, however, does not reside in the ability of the receptor-ligand complexes to move for long distances on the free surface. Rather, just as in the case of GPIIb/IIIa,31 the fundamental value of the observation relates to the downside of platelet-vessel wall interaction.
Platelets driven to an area of denuded vascular surface under high shear force have only microseconds during which to interact. Mobile but resilient GPIb/IX receptors can soften the impact by moving on attachment to vWF. Giving on contact would slow the cell sufficiently to permit it to bind more multimers and enhance adhesion. If the receptors were rigid, interaction time would be reduced, and the platelet would be more likely to bounce off the damaged surface. On the other hand, if the GPIb/IX receptors were as mobile as other studies have suggested,2 3 4 5 6 7 8 9 they might not hold the platelet firmly enough or long enough to facilitate adhesion. The loosely attached cell might tether under high shear force and break loose, leaving a vesicular remnant on the damaged surface.
The ability of GPIb/IX-vWF complexes to move in the lipid bilayer of the exposed surface of spread platelets has an even more important relation to events following attachment than to the process of adhesion itself.32 Following the binding of discoid platelets to the damaged vascular surface, the cell must spread in order to establish a hemostatic plug. Attachment of the mobile receptor, GPIb/IX, to vWF multimers covering the damaged site makes spreading possible.
Multimers of vWF on the injured surface are unlikely to move.32 As a result, the GPIb/IX receptors form immobile associations with vWF. The receptor-ligand complexes cannot move in the plane of the downside platelet surface to channels of the OCS. However, the downside surface and membranes lining the channels of the OCS can move through the immobilized GPIb/IX-vWF complexes, thus permitting the platelet to spread during assembly of new actin filaments.31 If the GPIb/IX receptors were not mobile, then the platelet surface membrane could not move through them, and spreading would be limited or impossible. Further experiments employing fixed and chemically stabilized or softened platelets under high shear conditions in the Baumgartner chamber are in progress to test this hypothesis.
This study was supported by grants from the NIH (HL11880 and HL49556) and the March of Dimes Birth Defects Foundation (FY94-0900).
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