Thrombin-induced inhibition of platelet agglutination by von Willebrand factor (vWF): reversal by ionized calcium

The present study has used washed human platelets combined with ethylene diamine tetracetic acid (EDTA) to determine the influence of calcium ions on thrombin-induced down-regulation of glycoprotein (GP) Ib/IX, the platelet surface receptor for von Willebrand factor (vWF). Bovine plasma vWF (BvWF) does not require the antibiotic, ristocetin, or calcium ions to cause agglutination of platelets. Thus, EDTA platelets agglutinate as well with BvWF as platelets in the absence of the chelating agent. Thrombin treatment of EDTA platelets prevented subsequent agglutination by BvWF. However, the addition of calcium ions to the sample restores sensitivity of the six PIb/IX receptors, and irreversible agglutination occurs when BvWF is added. Monoclonal antibodies to GPIb/IX and GPIIb/IIIa demonstrated that restoration of refractory platelet sensitivity to BvWF was related to GPIb/IX, not to GPIIb/IIIa. Experimental results suggest that GPIb/IX receptors on thrombin-treated EDTA platelets can be down-regulated by thrombin, but are not cleared from the surface to internal membranes.     

Localization of GPIb/IX and GPIIb/IIIa on Discoid Platelets

The organization and reorganization of mobile receptors, GPIIb/IIIa and GPIb/IX, on surface- and suspension-activated platelets have been studied in detail, but their distribution on resting, discoid platelets is uncertain. The present study has treated platelets in suspension with cytochalasin E before mounting on formvar grids or glass slide fragments in order to preserve their discoid appearance, then probed the organization of GPIIb/IIIa with fibrinogen coupled to gold particles (Fgn/Au) and GPIb/IX with bovine or ristocetin-activated human plasma detected by combined anti-vWF antibody and protein A coupled to gold particles. Multimers of vWF had the same tortuous, linear distribution from edge to edge observed previously on surface-activated platelets. However, the gold particles marking the complex of vWF-anti-vWF bound to GPIb/M were closer together on the discoid cells. Fgn/Au particles bound to GPIIb/IIIa receptors were uniformly distributed from edge to edge on many discoid platelets. On others they tended to clump or cluster in strips or patches. The latter organization of Fgn/Au-GPIIb/IIIa receptors may be due to the rugose nature of the discoid platelet surface or an influence of cytochalasin E. Definition of mobile receptor organization on discoid cells provides a useful baseline for determining their fate following surface or suspension activation.     

Spontaneous platelet aggregation during pregnancy in a patient with von Willebrand disease type IIB can be blocked by monoclonal antibodies to both platelet glycoproteins Ib and IIb/IIIa

Spontaneous platelet aggregation appeared in a patient with von Willebrand disease type IIB during the 37th week of pregnancy. This phenomenon was not associated with symptoms of thrombosis and the patient delivered by caesarean section with no complications. Her platelet-poor plasma (PPP) aggregated normal platelet-rich plasma (PRP) and washed platelets. Aggregation was inhibited by monoclonal antibodies with known specificity for the platelet receptors of von Willebrand factor (vWF), i.e. the glycoprotein Ib (GPIb) and the GPIIb/IIIa complex. A monoclonal antibody, which selectively inhibits the binding of vWF to the GPIIb/IIIa complex, did not block aggregation, suggesting that spontaneous aggregation is not dependent on the binding to GPIIb/IIIa of vWF from patient plasma. Aggregation induced by patient plasma could also be blocked either by two monoclonal antibodies raised against vWF or by a fragment derived from trypsin digestion of normal vWF which blocks the ristocetin-induced binding of normal vWF to platelets. These findings indicate that the spontaneous platelet aggregation in this patient results from the binding of her vWF to GPIb but is independent from the binding of her vWF to GPIIb/IIIa.     

Redistribution of GPIb/IX and GPIIb/IIIa during spreading of discoid platelets

Summary. The purpose of the present study was to prelabel mobile receptors on discoid platelets with specific ligands identifiable in the electron microscope and follow their redistribution during spreading. Platelets were incubated in suspension with cytochalasin E (CE) to preserve discoid form, chilled and mounted on cold formvar grids or glass slide fragments to inhibit receptor movement, covered with cold bovine or ristocetin-activated human plasmas as sources of vWF to bind GPIb/IX, fibrinogen-coated gold particles (Fgn/ Au) to couple GPIIb/IIIa, or both probes simultaneously, washed to remove CE and rewarmed to 37°C for intervals up to 30min to stimulate spreading. After brief fixation grids and glass fragments were incubated with anti-vWF antibody and, subsequently, staphylococcal protein A coupled to 5 nm and 10 nm gold particles to detect vWF multimers. Virtually all of the CE-treated chilled platelets retained their discoid shape. Half of the discs (53-3%) bound Fgn/Au, and all bound vWF. Receptors for both ligands were randomly dispersed on discoid cells from edge to edge. During re warming discoid platelets expanded into spread forms. Fgn/Au-GPIIb/IIIa complexes moved into caps over cell centres and into residual channels of the open canalicular system (OCS). vWF bound to GPIb/IX moved with the cell membrane as the surface expanded during spreading. Discoid platelets prelabelled with both ligands demonstrated similar findings. During rewarming Fgn/Au-GPIIb/IIIa complexes moved to cell centres and into OCS channels. vWF multimers bound to GPIb/IX moved apart from each other toward peripheral margins of the spread cells. Thus, surface activation resulting in conversion of discoid platelets to spread forms does not cause clearance of GPIb/IX receptors to cell centres and channels of the OCS in the manner that GPIIb/IIIa receptors coupled to Fgn/Au are simultaneously translocated and concentrated in OCS channels.     

Influence of combined thrombin stimulation, surface activation, and receptor occupancy on organization of GPIb/IX receptors on human platelets

Summary. Down-regulation and clearance of as many as 60–80% of GPIb/IX receptors from exposed surfaces on thrombin-activated platelets to channels of the open canalicular system (OCS) is considered to be a fundamental mechanism regulating platelet adhesivity in vitro and in vivo . The present study has combined thrombin stimulation in suspension, surface activation on formvar grids, receptor occupancy by von Willebrand factor (vWF) and exposure to anti-vWF antibody in an effort to demonstrate the removal of GPIb/IX receptors from activated cells. Individually the stimuli failed to cause any change in the frequency of GPIb/ IX receptors. Combined, the stimuli were no more effective than when each was used alone. The only way to cause GPIb/IX to move was to add anti-vWF to thrombin-activated platelets allowed to spread on formvar grids and covered with multimers of ristocetin-activated human or bovine vWF. Translocation of GPIb/IX-vWF-anti-vWF complexes from peripheral margins into caps over cell centres, however, did not clear the peripheral zone of vWF binding capacity. Exposure of capped platelets after fixation to a second incubation with vWF demonstrated as many multimers extending from the central cap to the peripheral margins as were seen on platelets exposed a single time to vWF. Antibodies to GPIb, but not to GPIIb/IIIA, prevented the second labelling by vWF. Down-regulation or clearance of GPIb/IX, in light of this study, does not appear to be a fundamental mechanism modulating platelet adhesivity.     

The Membrane Glycoprotein IIb/IIIa Complex Mediates Deposition of Thrombin-stimulated Blood Platelets on Polystyrene Plastic Under Static Conditions

A major challenge in the use of artificial materials for implant devices, artificial organs, and extra-corporeal circulation systems, is the adhesion of platelets and the subsequent formation of platelet aggregates on the non-biological surface. The mechanism of platelet attachment to artificial surfaces is not completely understood. Using an enzyme immunoassay, we examined platelet deposition to the polystyrene plastic of microtiter plate wells under static conditions. Following thrombin stimulation, platelets adhered to the wells. This adhesion process was suppressible by the use of different substances known to interfere with the function of the platelet surface glycoprotein IIb/IIIa complex (GPIIb/IIIa). The substances we used were ethylenediaminetetraacetic acid (EDTA), tetrapeptide RGDS (Arg-Gly-Asp-Ser), and a monoclonal antibody directed against the IIIa moiety of the GPIIb/IIIa complex. Our results indicate that the GPIIb/IIIa complex is the platelet receptor which mediates platelet adhesion to polystyrene plastic under such static conditions. The GPIIb/IIIa complex should consequently be regarded as a multifunctional platelet regulator which, depending on the circumstances, may support platelet adhesion as well as platelet aggregation. By contrast, a monoclonal antibody directed against the platelet surface glycoprotein complex Ib/IX (GPIb/IX) did not under the same static conditions inhibit platelet deposition to the polystyrene plastic. In the microtiter wells, platelet alpha-granular proteins were detected either on the surface of adherent platelets or, when platelet deposition was inhibited by EDTA directly on the polystyrene plastic. In the latter case, fibrinogen and thrombospondin were definitely the dominating proteins. The presence of platelet-derived proteins in the microtiter wells significantly enhanced the adhesion of thrombin-stimulated platelets but not of non-stimulated platelets.     

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

Interaction of porcine von Willebrand factor with the platelet glycoproteins Ib and IIb/IIIa complex

Porcine von Willebrand factor (PvWF) induces platelet aggregation which is thought to be responsible for the thrombocytopenia that occurs in haemophilic patients treated with commercial preparations of porcine factor VIII. This study demonstrates that such aggregation can be completely inhibited by a monoclonal antibody against human platelet glycoprotein GPIb and partially inhibited by an antibody directed against platelet GPIIb/IIIa. The interaction of PvWF with GPIb is also demonstrated by the inhibitory effect of purified glycocalycin on aggregation. The binding site of PvWF to GPIb is very close to that of human vWF, since a recombinant peptide blocks the binding of both molecules to GPIb. When platelets are incubated with PvWF, the GPIIb/IIIa receptor is activated and binds fibrinogen. PvWF also binds to GPIIb/IIIa when platelets are stimulated with thrombin, suggesting that the molecule has the same RGD sequence as other adhesive proteins (human vWF, fibrinogen, fibronectin and vitronectin). These findings identify the dual mechanisms responsible for in vivo platelet aggregation induced by PvWF, i.e. binding to GPIb and activation of the GPIIb/IIIa receptor.     

A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.     

Absence of Effect of DDAVP Infusion on Platelet Glycoprotein Ib/IX and IIb/IIIa Complexes, and their Interaction with Newly Released von Willebrand Factor

Several possibilities have been raised to explain the beneficial effect of l-deamino-8-D-arginine vasopressin (DDAVP) in several hemostatic disorders but, so far, its exact mechanism(s) of action is still unknown. Aiming to throw new light on the problem, we have investigated: (a) whether DDAVP induces platelet activation or quantitative/qualitative modifications of GPs Ib/IX and IIb/IIIa, and (b) the binding to these glycoprotein receptors of von Willebrand factor (vWF) purified from blood obtained before and after administration of DDAVP. Analysis of the expression of GMP 140 and thrombospondin demonstrated no platelet activation following administration of DDAVP. Binding assays and flow cytometry with antibodies against GPs Ib/IX and IIb/IIIa, the study of the ristocetin-dependent vWF binding, and immunoblotting with an anti-GPIb/IX antibody, demonstrated no quantitative or functional changes of these complexes after the infusion of DDAVP. Finally, native vWF purified from cryoprecipitate, and vWF purified from plasma of one DDAVP-infused volunteer, showed similar binding properties to GPIb/IX and GPIIb/IIIa. These results suggest that DDAVP is quite inert on platelet glycoproteins, and the drug-induced appearance of 'supranormal' high molecular weight vWF multimers seems not to modify the interaction of vWF with its main platelet receptors.     

Differential redistribution of platelet glycoproteins Ib and IIb-IIIa after plasmin stimulation.

The subcellular localization of the platelet membrane receptors glycoproteins (GP) Ib and IIb/IIIa [corrected] has been studied within resting platelets by a combination of biochemical and cytochemical techniques. While both GPIb and GPIIb/IIIa are localized within the plasma membrane and surface-connected canalicular system (SCCS) membranes, only GPIIb/IIIa is present within the internal face of alpha-granular membranes. Previous studies demonstrated that plasmin can induce platelet stimulation and also decrease ristocetin-induced platelet aggregation; it was suggested that this was because of GPIb degradation by plasmin. In this study, the respective localizations of both GPIb and GPIIb/IIIa were visualized during in vitro plasmin stimulation of platelets. Generally, plasmin induced shape change, pseudopod formation, organelle centralization either with or without alpha-granule release depending on the conditions of stimulation. Plasmin treatment of platelets at 37 degrees C resulted in the disappearance of GPIb from the cell surface and its subsequent redistribution into the channels and vesicles of the SCCS with no significant modification of GPIIb/IIIa remaining on the plasma membrane. Within degranulated platelets, GPIIb/IIIa was expressed on the plasma membrane and within membranes of large vacuoles containing the alpha-granule proteins. GPIb was virtually absent from these structures and mainly restricted to the SCCS. Addition of cytochalasin D inhibited the migration of GPIb to the SCCS. Biochemical measurements confirmed that no important hydrolysis of GPIb had occurred because only very little amounts of glycocalicin were generated during the reaction. In conclusion, in plasmin-treated platelets GPIIb/IIIa is externalized to the plasma membrane while GPIb is internalized into the SCCS. Although previous studies have suggested that plasmin degrades GPIb, the reduction in ristocetin-induced aggregation may be explained by its apparent redistribution within the membranes of the SCCS.     

Mapping of distinct von Willebrand factor domains interacting with platelet GPIb and GPIIb/IIIa and with collagen using monoclonal antibodies

We have used monoclonal antibodies (M Abs) and proteolytic fragmentation to localize structurally the functional sites of human von Willebrand factor (vWF) responsible for interaction with membrane glycoproteins GPIb, GPIIb/IIIa, and with collagen. SpII (215 kd) and SpIII (320 kd), the S aureus V-8 protease homodimeric fragments representing the carboxy-terminal and amino-terminal segments of the vWF subunit, competitively inhibited the binding of multimeric vWF to thrombin-stimulated or ristocetin-stimulated platelets, respectively. Specific saturable binding of each fragment was observed to stimulate platelets appropriately and was inhibited only by selected M Abs that both bound to the specific fragment and inhibited the corresponding function. M Ab 9, which blocks thrombin-induced binding of vWF to platelets, inhibited binding of SpII to platelets and bound to SpII as well as to a dimeric, 86-kd thermolysin fragment composed of 42-kd and 23-kd subunits, each possessing the epitope. Binding of SpII was also inhibited by a M Ab to GPIIb/IIIa. Thus, it appears that a portion of the carboxy-terminal end of vWF contains the ligand site for the GPIIb/IIIa receptor. In contrast, M Ab H9, which blocks ristocetin- induced binding of vWF to platelets, inhibited binding of SpIII to platelets and bound to SpIII as well as to monomeric 33-kd and 28-kd subtilisin fragments. Binding of SpIII to platelets was also inhibited by a M Ab to GPIb. Thus, it appears that a small segment of the amino- terminal part of vWF contains the ligand for the platelet GPIb receptor. The collagen binding site of vWF was localized with M Ab B203, which inhibits vWF interaction with collagen. This M Ab also bound to SpIII as well as to monomeric 26-kd and 23-kd subtilisin fragments. Thus, the third functional site responsible for collagen binding appears to be localized on the amino-terminal portion of vWF, in a linear sequence different from those responsible for interaction with either of the platelet receptors. These assignments of functional sites should facilitate the localization of structural defects of vWF in the various forms of vWD and support the role of vWF as an adhesive protein with multiple interactive sites.     

Receptor patching and capping of platelet membranes induced by monoclonal antibodies

Redistribution of glycoproteins (GP) Ib, glycocalicin, IIb, and IIIa on the surface of human platelets in response to stimulation with corresponding monoclonal antibodies (MoAb) and a polyclonal antiglycocalicin antibody was studied by immunofluorescence, immunoelectron microscopy, and a quantitative radioimmune assay. Immobilization of the antigens by prefixation with formaldehyde showed a uniform distribution over the surface of the platelet. Incubation of unfixed platelets with specific MoAb against various epitopes on GPIIb and/or IIIa resulted in a time-dependent patching, subsequent capping, and after prolonged exposure to the antibody/label complex, internalization of the complex, possibly by endocytosis. In contrast, GPIb could not be shown to cap. From these results we conclude that platelet GPIIb and/or IIIa undergo spatial rearrangement in a manner analogous to that observed in lymphocytes, whereas GPIb does not. Since both GPIb and GPIIb and/or IIIa seem to be transmembraneous GP associated with the cytoskeleton, a special, though unidentified, role of GPIIb/IIIa in the induction of lateral membrane mobility is postulated.     

Activation of plasminogen by tissue plasminogen activator on normal and thrombasthenic platelets: effects on surface proteins and platelet aggregation

Tissue plasminogen activator (TPA) converts plasminogen to plasmin within the fibrin clot, thus localizing activation of fibrinolysis. To determine the extent to which platelets promote activation of plasminogen by TPA, we studied the interaction of TPA and plasminogen with unstimulated platelets. Normal washed platelets incubated in the presence of physiologic concentrations of plasminogen (180 micrograms/mL) and TPA (20 ng/mL) failed to generate plasmin activity. In contrast, incubation of platelets with TPA concentrations achieved during thrombolytic therapy (40 to 800 ng/mL) produced a tenfold to 50- fold increase in plasmin activity. After exposure to plasminogen and 200 ng/mL of TPA for one hour, platelets failed to agglutinate in the presence of ristocetin. Incubation of platelets suspended in autologous plasma with 400 ng/mL of TPA for one hour also inhibited ristocetin- induced agglutination. Exposure of platelets to plasminogen and increasing concentrations of TPA correlated with a decrease in glycoprotein Ib (GPIb) and an increase in glycocalicin, as shown by immunoblotting. The glycoprotein IIb/IIIa (GPIIb/IIIa) complex and a 250,000-dalton protein also disappeared from washed platelets after incubation with plasminogen and 200 ng/mL of TPA for one hour. These platelets failed to aggregate in the presence of adenosine diphosphate (ADP) or gamma thrombin, although aggregation in response to calcium ionophore A23187 and arachidonic acid remained intact. However, aggregation in response to all four agonists was normal when platelets were incubated with TPA in the presence of autologous plasma. Platelets from a patient with Glanzmann's thrombasthenia also generated plasmin in the presence of TPA. Hydrolysis of GPIb and inhibition of ristocetin- induced agglutination occurred to a lesser extent with these platelets than with control platelets. We conclude that platelets provide a surface for activation of plasminogen by pharmacologic amounts of TPA. Plasmin generation leads to degradation of GPIb and decreased ristocetin-induced agglutination in normal and thrombasthenic platelets, as well as degradation of GPIIb/IIIa in normal washed platelets and inhibition of ADP and gamma thrombin-induced aggregation. These findings suggest that pharmacologic concentrations of TPA may cause platelet dysfunction due to plasmin generation on the platelet surface.     

Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.

Von Willebrand factor (vWF) was purified from the plasma of a patient with type IIB von Willebrand disease (vWF from such a patient, IIB vWF) who had a normal platelet count and showed no evidence of spontaneous platelet aggregation. Large multimers of IIB vWF were absent from purified preparations and from plasma. Ristocetin-induced platelet aggregation was enhanced by purified IIB vWF. The aggregation of washed normal platelets mixed with IIB vWF (0.4 microgram/ml) required lower amounts of ristocetin than the aggregation of normal platelets mixed with the same concentrations of normal vWF. Moreover, purified IIB vWF alone induced aggregation of platelet-rich plasma at concentrations as low as 10 micrograms of IIB vWF/ml in the absence of any other agonist. Aggregation was blocked by a monoclonal antibody against the platelet membrane glycoprotein, GPIb, as well as by an anti-GPIIb/IIIa antibody. Washed platelet suspensions were promptly aggregated by IIB vWF only when fibrinogen and CaCl2 were added to the mixture. Purified IIB vWF induces the binding of fibrinogen to platelets. Such binding was blocked by the anti-GPIb monoclonal antibody as well as by the anti-GPIIb/IIIa monoclonal antibody that inhibited aggregation. A second anti-GPIIb/IIIa antibody, which has the property of blocking vWF but not fibrinogen binding to platelets, blocked neither aggregation nor fibrinogen binding induced by IIB vWF. These studies demonstrate that platelet aggregation is triggered by the initial interaction of IIB vWF with GPIb which is followed by exposure of fibrinogen binding sites on GPIIb/IIIa. Fibrinogen binds to these sites and acts as a necessary cofactor for the aggregation response.     

Quinine- and quinidine platelet antibodies can react with GPIIb/IIIa

Quinine- and quinidine-dependent antiplatelet antibodies are generally believed to bind to the membrane glycoprotein complex, GPIb/IX. However, we and others have found that some drug-dependent antibodies bind to platelets from patients with the Bernard-Soulier syndrome which lack these glycoproteins. We therefore studied the reactions of a group of these antibodies with normal and Bernard-Soulier platelets and their membrane proteins. As indicated by rosette formation of the sensitized platelets around protein A-Sepharose beads, two quinine- and two quinidine-dependent antibodies reacted with both normal and Bernard-Soulier syndrome platelets at a high (300 microM) concentration of drug. At a pharmacologic drug concentration (10 microM), all four antibodies reacted with normal platelets but only the two quinine-induced antibodies reacted with Bernard-Soulier platelets. Immunoprecipitation studies with solubilized, tritium-labelled normal platelets, at both high and low drug concentrations, showed that each of the four antibodies precipitated proteins corresponding to GPIb and GPIX. Fainter bands corresponding to glycoproteins IIb and IIIa, which do not label well with tritium, were also detected. With radioiodinated normal platelets, it was found that each of the four antibodies was capable of precipitating GPIIb/IIIa, but only in the presence of drug. The four antibodies also promoted drug-dependent precipitation of GPIIb and GPIIIa from lysates of radioiodinated Bernard-Soulier platelets. The two quinine-dependent antibodies precipitated these glycoproteins at both high and low drug concentrations, while the quinidine-dependent antibodies reacted much more strongly at the higher drug concentration. Precipitation of GPIb/IX was not observed with BSS platelets. Absorption of a quinine-induced antibody with Bernard-Soulier platelets in the presence of drug eliminated its ability to precipitate GPIIb and GPIIIa. However, the absorbed antibody retained the ability to precipitate GPIb from solubilized normal platelets. Thus, at least two drug-dependent antibodies were present, one specific for GPIb/IX and the other for GPIIb/IIIa. These findings indicate that glycoproteins IIb and/or IIIa, in addition to the GPIb/IX complex, can serve as targets for drug-dependent antibodies in both intact and detergent-solubilized platelet preparations.     

Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets

Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.     

Platelet membrane abnormalities in myeloproliferative disorders: decrease in glycoproteins Ib and IIb/IIIa complex is associated with deficient receptor function

The number and functional activity of membrane glycoproteins (GP) Ib and IIb/IIIa were investigated in platelets from 11 patients with myeloproliferative disorders (MPD). Three patients had essential thrombocythaemia, two had chronic myeloid leukaemia and six had polycythaemia vera. The numbers of GPIb and GPIIb/IIIa molecules were detected on the platelet surface using different 125I-labelled monoclonal antibodies. The functional properties of GPIb and GPIIb/IIIa were evaluated using purified 125I-labelled asialo von Willebrand factor (vWF) and purified 125I-labelled fibrinogen, respectively, in a binding assay. Binding of the anti-GPIIb/IIIa antibody was decreased by 40% in almost all patients studied and, when measured, it was accompanied by decreased fibrinogen binding to activated platelets. Binding of anti-GPIb antibodies to platelets was also slightly decreased or virtually the same in eight out of 11 patients. The decrease correlated with decreased binding of asialo vWF. The increased plasma glycocalicin levels, measured in four patients, depended on the high platelet count. Scatchard analysis revealed normal receptor binding affinity for all ligands tested in all but one patient. In this report we demonstrate that abnormalities in the concentrations of GPIIb/IIIa membrane proteins are commonly present in patients with MPD, while a decrease in GPIb concentration is also seen, although in fewer patients. These abnormalities are accompanied by a concurrent decrease in the respective receptor functions. These findings may explain part of the haemorrhagic tendency often encountered in MPD.     

Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.     

von Willebrand factor competes with fibrin for occupancy of GPIIb:IIIa on thrombin-stimulated platelets

We have investigated two major questions related to the molecular basis of interactions between the three-dimensional fibrin network and thrombin-stimulated human platelets. First, what are the roles played by glycoproteins (GP) Ib and IIb:IIIa in linking the fibrin clot tightly to the platelet surface? Second, does von Willebrand factor (vWF) modulate the extent of platelet-fibrin interactions? Quantitative fluorescence microscopy (microfluorimetry) has been used to determine the quantity of fluorescein-labeled fibrin bound to the surface of thrombin-stimulated, gel-filtered platelets (the supernatants of which contained small quantities of vWF) in the presence/absence of receptor-specific and vWF-specific monoclonal antibodies (MoAbs), as well as exogenous vWF. A MoAb specific for the GPIIb:IIIa complex exhibited a concentration-dependent inhibition of fibrin binding, whereas a MoAb specific for GPIb was ineffective in this regard. Similarly, a MoAb that recognizes the N-terminal region of vWF involved in GPIb binding did not influence fibrin binding. In contrast, a MoAb that binds to a C-terminal region of vWF involved in GPIIb:IIIa recognition caused a specific, concentration-dependent increase in the quantity of platelet-bound fibrin. We also found that exogenous vWF caused a concentration-dependent decrease in fibrin binding. These results support the hypothesis that vWF and fibrin, both of which are multimeric adhesive ligands, compete for occupancy of the GPIIb:IIIa complex on thrombin-stimulated platelets.