Immunocytochemical evidence for the translocation of α-granule membrane glycoprotein IIb/IIIa (integrin αIIbβ3) of human platelets to the surface membrane during the release reaction

Summary The localization of glycoprotein (GP) IIb/IIIa (integrin _IIb3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the pre-embedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0±2.7 gold particles/m of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of -granules, and the density of gold particles was 19.7±3.6 particles/m. At 5 min, the -granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0±3.7 particles/m; p<0.01). In immunostained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the -granule membranes of resting platelets. At 30 s after thrombin stimulation the -granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that -granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS. Also the translocation of -granule membrane GPIIb/IIIa gives rise to an actual increase in GPIIb/IIIa on the surface membrane during the release reaction induced by thrombin.     

Topographic distribution of a granule membrane protein (GMP-140) that is expressed on the platelet surface after activation: an immunogold-surface replica study

Monoclonal and polyclonal antibodies have been developed that recognize a 140 kD glycoprotein on the plasma membrane of activated, but not unstimulated, platelets. This glycoprotein is found in resting platelets as an alpha-granule membrane protein and has therefore been named GMP-140. After thrombin stimulation, alpha-granules fuse with the surface-connected canalicular system and GMP-140 is redistributed to the plasma membrane. In the present study, we immunolabeled unstimulated and activated human platelets and analyzed the distribution of GMP-140 over broad expanses of the plasma membrane using surface replication techniques. Fixed platelets were allowed to settle onto poly-L-lysine-coated coverslips and immunolabeled with polyclonal anti-GMP-140, followed by protein A gold. After critical-point drying, rotary-shadowed surface replicas were made. GMP-140 was not present on the surfaces of unstimulated platelets, but thrombin stimulation resulted in the massive expression of GMP-140 on the cell surface, with the immunogold label monodispersed. In contrast, we recently found that GPIIb-IIIa, the fibrinogen receptor, is monodispersed on unstimulated platelets and clustered on activated platelets. Although GMP-140's hemostatic function is unknown, its monodispersed surface pattern implies significant differences form GPIIb-IIIa with respect to ligand binding and/or cytoskeletal interaction.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20-40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10-70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Characterization of the proteins of isolated human platelet alpha-granules. Evidence for a separate alpha-granule-pool of the glycoproteins IIb and IIIa

The protein composition of a well-defined alpha-granule preparation isolated from human platelets has been studied. Crossed immunoelectrophoresis against polyspecific platelet antibodies revealed more than 20 immunoprecipitates. The glycoprotein IIb-IIIa complex represented a major antigen in the Triton X-100-solubilized alpha-granule preparation and cross-reacted with the corresponding platelet membrane antigen. Furthermore, after lactoperoxidase-catalyzed 125I-iodination of whole platelets it was not labelled, in contrast to its membrane-located counterpart. This indicates an intracellular location of glycoproteins IIb and IIIa, probably as constituents of the alpha-granules. Fibrinogen, platelet factor 4, albumin, factor VIII-related antigen and the main granule glycoprotein (thrombinsensitive protein, thrombospondin) were identified in the alpha-granule preparation by the crossed immunoelectrophoresis technique. Crossed affinity immunoelectrophoresis using lectins revealed the presence of at least seven glycoproteins, and six sialoglycoproteins were identified by their altered electrophoretic mobility after neuraminidase treatment. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of reduced samples of the alpha-granules revealed at least 15 Coomassie Brilliant Blue-staining polypeptide bands, one of which comigrated with myosin heavy chain. No prominent band was observed in the actin region. Five glycopolypeptide bands were observed after periodic acid-Schiff staining. The dominant three represented the main granule glycoprotein, glycoprotein IIb and glycoprotein IIIa, respectively. More glycoproteins seem to be present in the alpha-granules than was previously recognized.     

The platelet open canalicular system: a final common pathway.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

Summary We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20–40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10–70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Differences between platelet and microparticle glycoprotein IIb/IIIa.

Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene-diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC-conjugated anti-GPIb antibody (NNKY5-5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EDTA at 37 degrees C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles).     

Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.     

Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.     

Role of calcium and calpain in complement-induced vesiculation of the platelet plasma membrane and in the exposure of the platelet factor Va receptor

The role of calcium and intracellular calpains in the expression of platelet prothrombinase activity was investigated. Incubation of gel-filtered platelets with complement proteins C5b-9 resulted in alpha-granule and dense granule secretion and exposure of membrane binding sites for coagulation factors Va and Xa. This was accompanied by the release of microparticles from the cell surface that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa and the alpha-granule membrane protein GMP-140. Generation of these membrane microparticles was dependent on the presence of extracellular calcium and was accompanied by proteolytic degradation of the cytoskeletal proteins, actin binding protein (ABP), talin, and myosin heavy chain. Microparticle formation was also detected when unstirred platelets were activated by thrombin plus collagen, although proteolysis of ABP, talin, or myosin was not observed. Preincorporation of the calpain inhibitor leupeptin into the platelet cytosol completely blocked C5b-9-induced proteolysis of ABP, talin, and myosin. However, inhibition of this calpain-mediated proteolysis had no effect on platelet secretion, the generation of microparticles, the exposure of membrane sites for factors Va and Xa, or the expression of prothrombinase activity. Furthermore, the microparticles that formed in the presence of leupeptin contained intact ABP, talin, and myosin heavy chain. Prior depletion of ATP with metabolic inhibitors eliminated all platelet responses to thrombin plus collagen, but did not affect C5b-9-induced microparticle formation or exposure of binding sites for factor Va on the microparticles. These data indicate that the formation of microparticles and the expression of platelet prothrombinase activity in response to C5b-9 are dependent upon an influx of calcium into the platelet cytosol, but do not require metabolic energy or calpain-mediated proteolysis of cytoskeletal proteins.     

Platelet membrane glycoprotein IIb-IIIa complex incorporated into phospholipid vesicles. Preparation and morphology

Platelet membrane glycoproteins (GP) IIb and IIIa have been identified as platelet aggregation sites. These glycoproteins form a heterodimer complex (GP IIb-IIIa) in the presence of Ca2+. To study the morphology of this glycoprotein complex in membranes, we incorporated GP IIb-IIIa into artificial phospholipid vesicles using a detergent (octyl glucoside) dialysis procedure. Phosphatidylserine-enriched vesicles (70% phosphatidylserine, 30% phosphatidylcholine) incorporated approximately 90% of the GP IIb-IIIa as determined by sucrose flotation. Glycoprotein IIb-IIIa incorporation into the vesicles was unaffected by ionic strength, suggesting a hydrophobic interaction between the glycoprotein and the phospholipid. In both intact platelets or phospholipid vesicles, GP IIb was susceptible to neuraminidase hydrolysis, indicating that most of the glycoprotein complexes were oriented toward the outside of the platelets or vesicles. The morphology of GP IIb-IIIa in the phospholipid vesicles was observed by negative staining electron microscopy. Individual GP IIb-IIIa complexes appeared as spikes protruding as much as 20 nm from the vesicle surface. Each spike consisted of a GP IIb "head," which was distal to the vesicle and was supported by the GP IIIa "tails." The GP IIb-IIIa complex appeared to be attached to the vesicle membrane by the tips of the GP IIIa tails. Treatment of vesicles with EGTA dissociated the GP IIb-IIIa complex. The dissociated glycoproteins remained attached to the phospholipid vesicles, indicating that both GP IIb and GP IIIa contain membrane-attachment sites. These data suggest a possible structural arrangement of the GP IIb-IIIa complex in whole platelets.     

Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity

We have investigated the composition and function of membrane microparticles released from platelets exposed to the C5b-9 proteins of the complement system. Gel-filtered human platelets were incubated with sub-lytic amounts of the purified C5b-9 proteins and the distribution of surface antigens was analyzed using monoclonal antibodies and flow cytometry. C5b-9 assembly caused secretory fusion of the alpha-granule membrane with the plasma membrane and the release of membrane vesicles (approximately 0.1-micron diameter) that contained the plasma membrane glycoproteins (GP) GP Ib and GP IIb-IIIa as well as the alpha-granule membrane protein GMP-140. These microparticles were highly enriched in the C9 neoantigen of the C5b-9 complex. The apparent surface density of C5b-9 on the microparticles was approximately 10(3)-fold higher than on the platelet itself, suggesting that the vesicles were selectively shed from the plasma membrane at the site of C5b-9 insertion. C5b-9 induced the expression of an activation-dependent epitope (recognized by monoclonal antibody, PAC1) in GP IIb-IIIa on the platelet surface but not in GP IIb-IIIa on the microparticles. The surface of the microparticles was also highly enriched in alpha-granule-derived coagulation factor V (or Va), accounting for nearly half of all the membrane-bound factor V detected. The number of potential membrane binding sites for factor Va was probed by adding saturating concentrations of factor Va light chain. Under these conditions, the density of factor Va binding sites on the microparticle surface exceeded that on the C5b-9-treated platelet by three to four orders of magnitude. Moreover, the microparticles provided most of the membrane surface for conversion of prothrombin to thrombin by VaXa. These studies demonstrate that the microparticles shed by C5b-9-treated platelets (and not the platelets themselves) provide the principal binding sites for coagulation factor Va and the principal catalytic surface for the prothrombinase complex. Platelet-derived microparticles formed during complement activation in vivo could provide a membrane surface that facilitates the assembly and dissemination of procoagulant enzyme complexes.     

Activation of platelet concentrate during preparation and storage.

This review will discuss how stored platelets become activated and will examine their ability to function and survive in vivo, posttransfusion. Experimental methods which have been shown to alter platelets during storage will be detailed. Using beta-thromboglobulin (beta-TG) and surface adhesion receptors as markers, investigators have examined the activation changes in platelet concentrates during preparation and storage. Resuspension of the platelet pellet after isolation of platelet-rich plasma appears to play a major role in producing platelet activation and beta-TG release during preparation. However, there is a significant amount of interdonor variability in platelet activation even at this early stage of storage. Over 5 days of storage, platelets release approximately 50% of their beta-TG contents. Furthermore, between 40% and 60% of the platelets express the alpha-granule membrane protein, P-selectin (GMP-140), during storage, which is also indicative of platelet activation. These activation changes correlate to some degree with platelet recovery posttransfusion but clearly do not explain the full lesion of platelet storage. The surface density of two platelet membrane receptors, glycoproteins (GP) Ib and IIb/IIIa, also change with activation, although in opposite directions. Platelet surface GPIb decreases initially with storage and then recovers, perhaps due to its relocation to the platelet surface from an intracellular pool. In contrast to GPIb, mean platelet surface GPIIb/IIIa increases slightly during storage, probably as a consequence of platelet activation and release of alpha-granule GPIIb/IIIa to the surface. Some hypotheses are offered regarding how these activated platelets can continue to circulate after transfusion. Further exploration of the platelet storage lesion will hopefully provide needed answers and thus permit better treatment of hemostatic disorders in the future.     

Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa

Human umbilical vein endothelial (HUVE) and bovine aortic endothelial (BAE) cells in culture were examined to determine whether membrane proteins similar to human platelet glycoproteins (GP) IIb and IIIa were present. The HUVE and BAE cells were either 125I-surface labeled or metabolically labeled. Triton X-100 lysates of labeled cells were immunoprecipitated with polyclonal antibodies prepared against purified human platelet GP IIb-IIIa complex. Two membrane proteins were detected on both HUVE (Mr = 130,000 and 110,000) and BAE (Mr = 135,000 and 105,000) cells, which were similar to human platelet GP IIb (Mr = 125,000) and GP IIIa (Mr = 108,000). The two membrane proteins from HUVE cells and the two from BAE cells cosedimented in sucrose gradients, indicating that they exist as a complex. Unlike the human platelet GP IIb-IIIa complex, the HUVE and BAE membrane protein complexes were not dissociated by chelation of Ca2+. Platelet GP IIb and GP IIIa and the related membrane proteins on both HUVE and BAE cells showed similar changes in electrophoretic mobility upon disulfide reduction. These data demonstrate that human and bovine endothelial cells synthesize membrane proteins that have properties similar to the platelet membrane GP IIb-IIIa complex.     

Annexin V relocates to the periphery of activated platelets following thrombin activation: an ultrastructural immunohistochemical approach

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1% +/- 0.78 of annexin V is present at the plasma membrane and 15.0% +/- 0.82 in the region corresponding to the membrane cytoskeleton (10-80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7% +/- 0.22 and 40.4% +/- 0.53, respectively.     

Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex

Platelet activation converts the membrane GP IIb-IIIa complex into a functional receptor for fibrinogen, but the mechanism is poorly understood. We asked whether induction of receptor competency coincides with a conformational change affecting the spatial arrangement of exoplasmic domains of the IIb and IIIa subunits. Epitopes on these subunits were labeled with monoclonal antibodies conjugated to either a donor fluorescein (FITC) or an acceptor tetramethylrhodamine (TR) chromophore. Then, fluorescence resonance energy transfer (RET) between platelet-bound FITC and TR was measured by flow cytometry. In unstimulated platelets, 6-8% RET efficiency was detected between antibody B1B5, bound to GP IIb, and antibody SSA6, bound to GP IIIa, regardless of which antibody served as RET donor. RET was also observed between these antibodies and A2A9, an antibody specific for the GP IIb-IIIa complex. Cell stimulation by thrombin, ADP plus epinephrine or phorbol-ester caused up to a 2-fold increase in RET between chromophore-labeled, platelet-bound B1B5, SSA6, and A2A9 (p less than or equal to 0.05), suggesting a change in the separation or orientation of these epitopes within the GP IIb-IIIa complex. The activation-related conformational change detected by the increase in RET between antibody B1B5 and SSA6 was independent of receptor occupancy since it was unaffected by the addition of fibrinogen or by the inhibition of fibrinogen binding by the antibody, A2A9, or the peptide, RGDS. In contrast to these results with antibodies bound to different epitopes within GP IIb-IIIa, no RET was observed between FITC-A2A9 and TR-A2A9 bound to different GP IIb-IIIa complexes or between a TR-labeled GP Ib antibody and FITC-labeled GP IIb-IIIa antibodies. These studies demonstrate that platelet activation causes a change in the spatial separation or orientation of exoplasmic domains within GP IIb and IIIa, which may serve to convert this integrin into a functional adhesion receptor.     

Identification of PlAl alloantigen domain on a 66 kDa protein derived from glycoprotein IIIa of human platelets

Incubation of platelets with chymotryptin leads to the exposure of fibrinogen receptors and to the appearance of a 66 kDa membrane component on the surface of platelets. Both glycoprotein IIIa (GP IIIa) and a 66 kDa component were precipitated from detergent extracts of solubilized, surface radiolabeled chymotrypsin-treated platelets by human anti-PlAl antisera. Moreover, the presence of the P1A1 antigen was identified on GP IIIa (but not on GP IIb) and on a 66 kDa protein by means of immunoblot procedures using platelet Triton X-114 extracts and these purified proteins. Anti-PlAl antiserum did not recognize GP IIIa on the surface of intact (untreated) platelets nor the 66 kDa protein on the surface of chymotrypsin-treated platelets of PlAl-negative individuals. The present data demonstrate directly that the 66 kDa protein is derived from GP IIIa and contains the PlAl alloantigen.     

Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.     

Comparative study of the major glycoproteins of the plasma membrane and secretory granule membranes of porcine platelets

The major glycoproteins of porcine platelet plasma membranes and alpha-granule membranes were compared. Significant cross-contamination of the two preparations was ruled out by surface labeling and proteolysis experiments. At least four of the major glycoproteins of the alpha-granule membrane had counterparts in the plasma membranes with identical molecular weight and lectin-binding properties. Two of these (mol. wts 110,000 and 125,000) were further analysed by one-dimensional peptide mapping. The results confirmed that there are two distinct pools of identical glycoproteins: one on the surface membrane and the other on the alpha-granular membrane. The 110,000 and 125,000 mol. wt glycoproteins are probably equivalent to glycoproteins IIb and IIIa of the human platelet and may therefore be involved in fibrinogen binding.     

Thrombin-stimulated glutamate uptake in human platelets is predominantly mediated by the glial glutamate transporter EAAT2

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.