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.     

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.     

Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes

Using an immunogold staining technique and electron microscopy, we investigated the localization of the alpha-granule pool of glycoprotein (GP) IIb-IIIa in normal platelets and maturing megakaryocytes (MK), in pathologic platelets from a patient with type I Glanzmann's thrombasthenia (GT), and from three patients with the gray platelet syndrome (GPS). In normal resting platelets, GPIIb-IIIa was observed on the plasmatic side of the plasma membrane, the open canicular system (OCS) membranes, and along the internal face of the alpha-granule membrane. This location was found with three monospecific polyclonal antibodies: one anti-GPIIb-IIIa antibody, the second specific for GPIIb, and the third specific for GPIIIa. After thrombin stimulation, the alpha-granule labeling disappeared whereas membrane labeling increased. Platelets from GT did not display labeling on plasma membranes, OCS membranes, or alpha-granule membranes. Platelets from the three patients with GPS displayed intense labeling of the plasma membrane and the OCS membrane, as well as the abnormal small alpha-granules and along the inside of large vacuoles (which contain the granule membrane protein [GMP]-140). In cultured immature MK from normal progenitors, both peptide components of GPIIb-IIIa appeared in the Golgi saccules and vesicles, and in the small precursors of alpha-granules, labeling both their membranes and their matrix. It was then observed only on the membrane of the mature MK alpha-granules, although labeling was less consistent than on the platelet granules. The MK plasma membrane and demarcation membrane system also displayed GPIIb-IIIa labeling. In conclusion, this study demonstrates that GPIIb-IIIa is present on the internal face of the alpha-granule membranes of platelets (where it appears early during MK maturation) as well as in the abnormal alpha-granules of gray platelets; it is absent from GT type I platelets.     

Ultrastructural demonstration of CD36 in the alpha-granule membrane of human platelets and megakaryocytes

CD36 (glycoprotein [GP] IV) is a membrane GP of 88 kD found on monocytes, endothelial cells, and platelets. It may serve as a receptor for collagen and is also able to bind thrombospondin (TSP), because a monoclonal antibody to CD36 inhibits TSP binding to thrombin-stimulated platelets. In the following study, we investigated the subcellular distribution of CD36 within normal resting platelets, thrombin- stimulated platelets, and in cultured megakaryocytes (MK) by an immunogold staining technique and electron microscopy. We used an affinity-purified monospecific polyclonal antibody showing a single major band of precipitation at 88 kD via immunoblot analysis. In normal platelets, ultrastructural observation detected immunolabeling for CD36, homogeneously distributed along the platelet plasma membrane and in the luminal side of the open canalicular system (OCS). Moreover, some labeling was found around the alpha-granules along the inner face of their limiting membrane. An average of 70% of granules were labeled. The granule-associated pool of CD36 was estimated at approximately 25% of the total cell content. To exclude the possibility of a cross- reaction with GPIIb-IIIa, platelets from a patient with type I Glanzmann's thrombasthenia (which completely lack GPIIb-IIIa) were studied and showed a similar subcellular distribution of CD36, including alpha-granule membrane labeling. In activated platelets, CD36 was shown to be redistributed to the OCS and pseudopods of the plasma membrane. Platelets from a patient with the Gray platelet syndrome expressed CD36 on their plasma membrane, and some immunolabeling was also found within small abnormal alpha-granules. In cultured MK, CD36 immunolabeling was detected in the Golgi saccules, associated vesicles, immature alpha-granules, and demarcation membranes. In conclusion, this study shows the existence of a significant intragranular pool of CD36 in platelets that may play a critical role in the surface expression of alpha-granule TSP during platelet 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.     

Immunolocalization of beta 1 integrins in platelets and platelet- derived microvesicles

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.     

Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate.

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.     

Cilostazol inhibits platelet-leukocyte interaction by suppression of platelet activation

The influence of three anti-platelet drugs, cilostazol, aspirin, and tirofiban, was investigated on platelet-leukocyte interaction by flow cytometry. When platelets and leukocytes were pre-incubated with anti-platelet drugs and stimulated by thrombin or collagen, cilostazol was found to inhibit platelet adhesion to monocytes and polymorphonuclear cells (PMNs). Similar effects were observed with anti-CD62P antibody, while aspirin and tirofiban did not appear to interfere with interaction between platelets and leukocytes. In the platelets pre-incubated with anti-platelet drugs, cilostazol significantly reduced CD62P expression and GPIIb/IIIa activation on platelet surface stimulated by thrombin or collagen. Aspirin inhibited CD62P expression and GPIIb/IIIa activation induced by collagen, but not thrombin. Tirofiban significantly blocked GPIIb/IIIa activation induced with both, and weakly inhibited CD62P expression induced by collagen. When added after stimulation of platelets, cilostazol again significantly inhibited CD62P expression and GPIIb/IIIa activation, although to a lesser extent than in the pre-incubation study. Aspirin hardly inhibited CD62P expression or GPIIb/IIIa activation, while tirofiban strongly blocked GPIIb/IIIa activation induced by thrombin or collagen, but had little effects on CD62P expression. In conclusion, our results suggest that cilostazol inhibits platelet-leukocyte interaction by reducing CD62P expression on the platelet surface.     

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.     

Absence of tubular structures and immunolabeling for von Willebrand factor in the platelet alpha-granules from porcine von Willebrand disease [published erratum appears in Blood 1987 Feb;69(2):707]

The electron microscopic localization of von Willebrand factor (vWF) was studied in platelets from normal and von Willebrand disease (vWD) pigs. In normal pig platelets, immunolabeling for vWF was far more intense and extensive than in human platelets and was either localized at one pole of the alpha-granule or all along its periphery or long axis. As in human platelets, this immunolabeling coincided with the presence of tubules about 200 nm in diameter. These structures were more numerous than in human platelets, with up to 30 tubules per alpha- granule. They were easily identified either in transverse sections, usually grouped in a less electron-dense part of the matrix at one pole of the alpha-granule, or in longitudinal sections parallel to the long axis of the elongated granules, or coiled around the alpha-granule core. They closely resemble those structures found in Weibel-Palade bodies. In platelets from pigs with severe vWD, these structures were absent, as was the immunolabeling for vWF; however, cytoplasmic microtubules were normally present in these platelets. Thus, the granule-associated tubules can be distinguished from the microtubules, which are larger in diameter (250 nm), are present in both normal and vWD platelets, and do not stain for vWF. These results strongly suggest that the tubular structures present in the alpha-granules of normal porcine platelets correspond to the vWF molecule itself.     

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.     

Differential inhibition of adenosine diphosphate- versus thrombin receptor-activating peptide-stimulated platelet fibrinogen binding by abciximab due to different glycoprotein IIb/IIIa activation kinetics

The exposure of internal glycoprotein (GP) IIb/IIIa receptors has been proposed to explain the incomplete inhibition of aggregation of thrombin receptor-activating peptide (TRAP)-stimulated platelets by abciximab. However, a marked and rapid externalization of GPIIb/IIIa was also observed upon stimulation with 30 microM adenosine diphosphate (ADP). ADP-induced fibrinogen binding was completely inhibited by 10 microg/mL abciximab, 30 nM tirofiban, or 3 microg/mL eptifibatide, while fibrinogen binding induced by 100 microM TRAP was inhibited only by 50%. Interestingly, striking differences in fibrinogen binding kinetics in ADP- versus TRAP-stimulated platelets were observed. ADP-induced fibrinogen binding was much slower than that of abciximab. These differences in the fibrinogen binding rate were due to differential GPIIb/IIIa activation kinetics because the actual fibrinogen binding rate (measured by adding fibrinogen after platelet activation) was similar in ADP- and TRAP-stimulated platelets. Thus, the TRAP-induced GPIIb/IIIa activation rate would allow significant amounts of fibrinogen to occupy externalized GPIIb/IIIa receptors even in the presence of the inhibitor.     

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.     

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.     

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.     

Platelet dense granule membranes contain both granulophysin and P-selectin (GMP-140).

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.     

Aspirin inhibits surface glycoprotein IIb/IIIa, P-selectin, CD63, and CD107a receptor expression on human platelets.

Platelet inhibition after aspirin therapy reduces the risk for the development of acute coronary syndromes. However, the mechanism by which aspirin affect platelets other than by prostaglandin blockade is unclear. We sought to determine the in vitro effects of aspirin on the surface expression of nine platelet receptors using whole blood flow cytometry. Blood from 24 healthy volunteers was incubated for 30 min with 1.8 and 7.2 mg/l phosphate-buffered saline-diluted acetylsalicylic acid in the presence or absence of apyrase. Platelet serotonin release, and the surface expression of platelet receptors with or without apyrase were determined using the following monoclonal antibodies: anit-CD41 [glycoprotein (GP)IIb/IIIa], CD42b (GPIb), CD62p (P-selectin), CD51/CD61 (vitronectin receptor), CD31 [platelet/endothelial cellular adhesion molecule-1 (PECAM-1)], CD107a [lysosomal associated membrane protein (LAMP)-1], CD107b (LAMP-2), CD63 (LIMP or LAMP-3), and CD151 (PETA-3). Samples were then immediately fixed with 2% paraformaldehyde, and run on the flow cytometer within 48 h. Aspirin does not affect serotonin release from human platelets. Dose-dependent inhibition of GPIIb/IIIa, P-selectin, CD63, and CD107a receptor expression was observed in the aspirin-treated whole-blood samples. Apyrase potentiates the effects of aspirin, and independently inhibits PECAM-1. In addition to the known effect of irreversibly inhibiting platelet cyclooxygenase-1, thereby blocking thromboxane A(2) synthesis, it appears that aspirin exhibits direct effects on selective major platelet receptors.     

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.     

Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets

Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha- thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400-411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha- thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.     

Advances in Antiplatelet Therapy in Coronary Artery Disease: Importance of the Platelet GPIIb/IIIa Receptor

It is now widely agreed that platelets are intimately involved in and contribute to the pathogenesis of acute coronary thrombosis. Aspirin, a relatively weak inhibitor of platelet activation, saves lives when administered early after acute myocardial infarction and should be routinely used as lifelong therapy in patients with coronary atherosclerosis. Ticlopidine has a mechanism of action distinct from and additive to that of aspirin; it inhibits activation of platelets mediated by the agonist, adenosine diphosphate (ADP). The reduction in subacute coronary thrombosis attained by the use of combination therapy with aspirin and ticlopidine (for 2–4 weeks) after intracoronary stenting is further evidence of the role of platelets in mediating acute arterial thrombosis. Potent platelet agonists (like thrombin) can override the effect of aspirin and ticlopidine; therefore these agents are of limited efficacy. In contrast, inhibitors of the platelet glycoprotein (GP) IIb/IIIa receptor are potentially more potent inhibitors of adhesive platelet interaction and may therefore be effective in blocking adhesive platelet interactions irrespective of the activating agonist. The GPIIb/IIIa receptor mediates the bridging of platelets (platelet aggregation) via fibrinogen, thus allowing platelet to bind other platelets at the injured vessel wall. Antagonists of this receptor are thus capable of blocking the “effector function” by acting at a step that is downstream to platelet activation. By abrogating the final common pathway of platelet aggregation, antagonists of GPIIb/IIIa also affect the most proximal step in thrombin generation (that most efficiently occurs on the membrane surface provided by platelets). Accordingly, these agents can profoundly inhibit arterial thrombosis. The clinical use of the antibody fragment directed against the GPIIb/IIIa receptor (c7E3 Fab) has truly revolutionized the practice of interventional cardiology and has the potential to effectively treat heparin-resistant intracoronary thrombosis. Synthetic antagonists of fibrinogen binding to the GPIIb/IIIa receptor (the “fibans”) are currently under initial clinical testing.