A conformation-dependent epitope of human platelet glycoprotein IIIa.

This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion. Platelets express 40,000-50,000 GP IIb-IIIa molecules in their surface membranes. However, resting platelets only bound approximately 5,000 D3GP3 molecules/platelet. D3GP3 binding to platelets could be increased 2-3-fold by dissociation of the GP IIb-IIIa complex with 5 mM EDTA or by occupying the fibrinogen receptor with either RGDS peptides or fibrinogen. Platelet stimulation with ADP in the absence of fibrinogen did not cause increased D3GP3 binding above control levels. These data suggest that 1) GP IIb-IIIa can exist in multiple conformations in the platelet membrane, 2) D3GP3 binding to GP IIIa can expose the fibrinogen receptor, 3) the binding of either RGDS peptides or fibrinogen causes exposure of the D3GP3 epitope, and 4) platelet activation in the absence of ligand does not induce the same conformational changes in GP IIb-IIIa as does receptor occupancy by RGDS peptides or fibrinogen.     

Identification and characterization of fragments of major glycoproteins from platelet membrane after chymotrypsin treatment

Human platelets were surface-labeled by the periodate/NaB3H4 method or by lactoperoxidase-catalysed iodination with 125I. The labeled platelets were treated with chymotrypsin under conditions known to give platelets which aggregate with fibrinogen without stimulation with ADP. Platelets and supernatant were then analysed by various gel electrophoretic techniques including isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions and two-dimensional non-reduced/reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography or indirect autoradiography. Chymotrypsin-treatment of surface-labeled platelets degraded the major glycoproteins Ib, IIb and IIIa but also GP120(4.9-5.4), GPIc and GPV. The membrane-bound fragments of GPIb, IIb and IIIa could be identified and also the supernatant fragments of GPIb and GPV. GPIIIa was also cleaved within a loop structure formed by disulfide bond(s). The fact that remnants of both GPIIb and IIIa are left on chymotrypsin-treated platelets which aggregate spontaneously with fibrinogen may indicate that a complex formed by these remnants constitutes the fibrinogen-binding site on platelets.     

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.     

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.     

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.     

Abnormal Whole Blood Thrombi in Humans with Inherited Platelet Receptor Defects

To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann’s Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.     

Involvement of platelet glycoprotein Ib in platelet microparticle mediated neutrophil activation

Summary Platelet microparticles (MPs) are membrane vesicles shed by platelets after activation, and carry antigens characteristic of intact platelets, such as glycoprotein (GP) IIb/IIIa, GPIb and P-selectin. Elevated platelet MPs have been observed in many disorders in which platelet activation is documented. Recently, platelet GPIb has been implicated in the mediation of platelet–leukocyte interaction via binding to its ligand Mac-1 on leukocyte. The role of GPIb for mediating adhesion-activation interactions between platelet MPs and leukocytes has not been clarified. In this study we investigate the role of GPIb in the interplay between platelet MPs and neutrophils. Platelet MPs were obtained from collagen-stimulated platelet-rich plasma (PRP). In a study model of neutrophil aggregation, platelet MPs can serve a bridge to support neutrophil aggregation under venous level shear stress, suggesting that platelet MPs may enhance leukocyte aggregation, which would bear clinical relevance in diseases where the platelet MPs are elevated. The level of aggregation can be reduced by GPIb blocking antibodies, AP1 and SZ2, but not by anti-CD18 mAb. The GPIb blocking antibodies also decreased platelet MP-mediated neutrophil activation, including β2 integrin expression, adherence-dependent superoxide release and platelet MP-mediated neutrophil adherence to immobilized fibrinogen. Our data provide the evidence for the involvement of GPIb–Mac-1 interaction in the cross-talk between platelet MPs and neutrophils.     

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.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

Homologous recombination among three intragene Alu sequences causes an inversion-deletion resulting in the hereditary bleeding disorder Glanzmann thrombasthenia.

The crucial role of the human platelet fibrinogen receptor in maintaining normal hemostasis is best exemplified by the autosomal recessive bleeding disorder Glanzmann thrombasthenia (GT). The platelet fibrinogen receptor is a heterodimer composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Platelets from patients with GT have a quantitative or qualitative abnormality in GPIIb and GPIIIa and can neither bind fibrinogen nor aggregate. Very few genetic defects have been identified that cause this disorder. We describe a kindred with GT in which the affected individuals have a unique inversion-deletion mutation in the gene for GPIIIa. Patient platelets lacked both GPIIIa protein and mRNA. Southern blots of patient genomic DNA probed with an internal 1.0-kb GPIIIa cDNA suggested a large rearrangement of this gene but were normal when probed with small GPIIIa cDNA fragments that were outside the mutation. Cytogenetics and pulsed-field gel analysis of the GPIIIa gene were normal, making a translocation or a very large rearrangement unlikely. Additional Southern analyses suggested that the abnormality was not a small insertion. We constructed a patient genomic DNA library and isolated fragments containing the 5' and 3' breakpoints of the mutation. The nucleotide sequence from these genomic clones was determined and revealed that, relative to the normal gene, the mutant allele contained a 1-kb deletion immediately preceding a 15-kb inversion. The DNA breaks occurred in two inverted and one forward Alu sequence within the gene for GPIIIa and in the left, right, and left arms, respectively, of these sequences. There was a 5-bp repeat at the 3' terminus of the inversion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depression is associated with an increase in the expression of the platelet adhesion receptor glycoprotein Ib

There is a significant association between cardiovascular disease and depression. Previous studies have documented changes in platelets in depression. It is unknown if depression causes functional changes in platelet surface receptors. Therefore, we analyzed (1) the surface expression of glycoprotein (GP)Ib and the integrin receptor _IIbβ_IIIa, receptors involved in platelet adhesion and aggregation, (2) CD62 (P-selectin) and CD63, integral granule proteins translocated during platelet activation, (3) platelet aggregation in response to ADP and (4) plasma levels of glycocalicin and von Willebrand factor (vWF), in depressed patients compared to healthy volunteers. Fifteen depressed patients with a Hamilton depression score of at least 22 and fifteen control subjects were studied. Platelets were assessed for surface expression levels of GPIb, _IIbβ_IIIa, CD62 and CD63 by flow cytometry. Genomic DNA was isolated to investigate a recently described polymorphism in the 5’ untranslated region of the GPIb gene. The number of GPIb receptors was significantly increased on the surface of platelets from patients with depression compared to control subjects. Surface expression of CD62 was also significantly increased in the depressed patients versus control subjects. There was no significant difference between depressed patients and healthy volunteers in the surface expression of _IIbβ_IIIa or CD63, or in glycocalicin or vWF plasma concentration, or ADP-induced aggregation. There was no difference in allele frequency of the Kozak region polymorphism of the GPIb gene, which can affect GPIb expression. The results of this study demonstrate that the number of GPIb receptors on platelets are increased in depression and suggest a novel risk factor for thrombosis in patients with depression.     

Variations of glycoprotein IIb–IIIa (αIIb/β3-integrin) content in healthy donors. The influence on platelet aggregation activity and efficacy of aspirin action

The effects of content of a fibrinogen receptor, glycoprotein (GP) IIb–IIIa (αIIb/β3-integrin), GP IIIa genetic polymorphism (substitution Leu33Pro), and fibrinogen concentration in blood plasma on platelet aggregation activity have been investigated in a group of healthy volunteers. In 35 examined donors the GP IIb–IIIa content on platelet surface varied from 40 to 71 × 10³ per platelet. Repeated measurements revealed that the GP IIb–IIIa content coefficient of variation was 9.5%, and deviations from mean levels did not exceed 20%. The level and the rate of platelet aggregation induced by ADP (1.25–20 μM) correlated with GP IIb–IIIa number (r from 0.315 to 0.591) and were higher in the group of donors with high in comparison with low GP IIb–IIIa content (>60 and (40–50) × 10^?3 per platelet, respectively). Aspirin, the inhibitor of thromboxane A₂ synthesis, partially suppressed ADP-induced platelet aggregation. The level of residual aggregation in the presence of aspirin also correlated with GP IIb–IIIa content and increased in subjects with high receptor content. Parameters of ADP-induced aggregation did not differ in donors with genotypes GP IIIa Pro33(?) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotype. GP IIb–IIIa content was also not affected by GP IIIa polymorphism. No significant correlations were found between the level and rate of platelet aggregation and fibrinogen concentration in blood plasma. The data obtained indicate that the effects of variations of GP IIb–IIIa content on platelet aggregation are higher than GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration. High GP IIb–IIIa content is associated with increased platelet aggregation activity and decreased efficacy of aggregation inhibition by aspirin.     

Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding 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/microns 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 alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained 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 alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-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 alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies

Platelet membrane glycoprotein IIb-IIIa exists as a calcium-dependent complex of two large peptides (designated IIb and IIIa) in Triton X-100 solutions, but it remains unknown if these peptides are subunits of one glycoprotein or are actually two individual glycoproteins in the intact platelet membrane. We used crossed immunoelectrophoresis to define the epitopes of two monoclonal antibodies to IIb-IIIa, then used these antibodies to study the structural and functional organization of IIb and IIIa in the platelet membrane. Human platelets solubilized in Triton X-100 were electrophoresed through an intermediate gel containing 125I-monoclonal IgG, then into an upper gel containing rabbit anti-human platelet antibodies. Our previously characterized antibody. Tab, and a new monoclonal antibody, T10, both bound to the immunoprecipitate corresponding to the IIb-IIIa complex. When platelets were electrophoresed after solubilization in 5 mM EDTA, 125I-Tab bound to the dissociated IIb polypeptide, but not to IIIa. In contrast, 125-I-T10 did not react with either IIb or IIIa. Thus, Tab recognizes a determinant on IIb, while T10 recognizes a determinant created only after the association of IIb and IIIa. Gel-filtered platelets from six normal donors bound 50,600 +/- 5,600 125I-T10 molecules/platelet and 47,800 +/- 11,200 125I-Tab molecules/platelet, consistent with IIb-IIIa being a heterodimer. 125I-T10 binding was identical in unactivated platelets and platelets stimulated with 10 microM ADP. However, platelets did not aggregate or bind 125I-fibrinogen until ADP was added. T10, but not Tab or nonimmune mouse antibody, inhibited ADP-induced platelet aggregation and 125I-fibrinogen binding. Our findings suggest that IIb and IIIa exist as subunits of a single membrane glycoprotein in unstimulated platelets. Fibrinogen binding appears to require not only the interaction of IIb and IIIa, but also some additional change occurring after platelet activation.     

Shear stress-induced binding of von willebrand factor to platelets

Shear stress-induced platelet aggregation requires von Willebrand factor (vWF), platelet glycoprotein (GP) Ib, GPIIb-IIIa, Ca²⁺, and adenosine diphosphate (ADP). Recent reports using vWF labeled with either ¹²⁵I or fluorescein isothiocyanate (FITC) have demonstrated that in shear-fields, vWF binds to both GPIb and GPHb-IIIa. The sequence of the vWF binding to the two platelet receptors has not been precisely determined in these reports. In this study, a flow cytometry technique using a primary anti-vWF antibody and a secondary FITC IgG antibody was used to measure shear stress-induced vWF binding to platelets. Washed normal platelets suspended at 50,000/μl with purified large VWF multimers were exposed to laminar shear stresses of 15 to 120 dynes/cm² for 30 sec. At this low platelet count, little or no aggregation occurred in the shear fields. A significant increase in post-shear vWF-positive platelets was consistently observed. Experiments with platelets from normal and severe von Willebrand's disease (vWD) (which lack plasma and platelet α-granule vWF) demonstrated that exogenous vWF predominately contributed to the platelet-vWF binding. Blockade of platelet GPIb with the monoclonal anti-GPIb antibody, 6D1, completely inhibited shear stress-induced platelet-vWF attachment. In contrast, blockade of GPIIb-IIIa with monoclonal anti-GPIIb-IIIa antibodies, 10E5 or c7E3, or with the GPIIb-IIIa-blocking tetrapeptide, RGDS, had little or no inhibitory effect on platelet-vWF binding. These data demonstrate that the binding of vWF to GPIb is likely to be the initial shear-induced platelet-ligand binding event. © 1997 Elsevier Science Ltd     

Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib

Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that alpha-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second alpha-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative alpha-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, gamma407, with a deletion of the gamma-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to gamma-thrombin, an autoproteolytic product of alpha-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-alpha-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient.     

Demonstration of feline and canine platelet glycoproteins by immuno- and lectin histochemistry

Canine and feline platelet cytocentrifuge preparations (CCPs), cryostat and paraffin-embedded bone marrow sections were used in this study. We evaluated whether platelets, megakaryocytes and megakaryocyte precursor cells could be labelled by monoclonal antibodies (Y2/51, CLB-thromb/1, HPL1) against human platelet membrane glycoprotein GP IIIa and the GP IIb/IIIa complex or by the following 10 biotinylated lectins: concanavalin A (Con A), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PsA), wheat germ agglutinin (WGA), peanut agglutinin (PNA), Phaseolus vulgaris lectin (PHA-L), Ricinus communis agglutinin 120 (RCA₁₂₀), Ulex europaeus agglutinin — I(UEA-1), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). Monoclonal antibodies Y2/51 and HPL1 cross reacted with platelets and megakaryocytic cells from both species, whereas CLB-thromb/1 was unreactive with canine preparations. Only Y2/51 labelled megakaryocytic cells in paraffin-embedded samples. LCA, PSA, WGA and PHA-L labelled feline and canine platelets and different numbers of morphologically identifiable megakaryocytes and numerous other, mostly myeloid, cells. Immunoblots of dog and cat platelet lysates using Y2/51 visualized a single protein of 95 kDa (unreduced), a mol·wt value within the range of those reported for GP IIIa. Some of the platelet (but not necessarily megakaryocyte) glycoproteins reacting with LCA, PSA and WGA could be identified in lectin blots following one- or two (nonreduced/reduced)-dimensional sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). Thus in dogs and cats, the immunohistochemical detection of GP IIIa (and eventually GP IIb/IIIa) rather than lectin binding patterns could be important for the diagnosis of megakaryoblastic leukaemias.     

Ca2+ transport across the platelet plasma membrane. A role for membrane glycoproteins IIB and IIIA

Human platelets maintain a low cytosolic free Ca2+ concentration in part by controlling plasma membrane Ca2+ transport. The present studies examine the role in this process of two well-characterized membrane proteins: glycoproteins IIb and IIIa. These glycoproteins form a Ca2+-dependent complex which serves as both the platelet fibrinogen receptor and the principle site for high affinity Ca2+ binding on the platelet surface. The kinetics of plasma membrane Ca2+ exchange were compared in normal platelets and in thrombasthenic platelets, which lack the IIb X IIIa complex. Under steady-state conditions, the maximum rate of plasma membrane Ca2+ exchange in the thrombasthenic platelets was half the rate observed in normal platelets. The size of the cytosolic exchangeable Ca2+ pool and the cytosolic free Ca2+ concentration, however, were normal. A quantitatively similar decrease in plasma membrane Ca2+ exchange was seen in normal platelets after incubation with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 37 degrees C, conditions that dissociate the IIb X IIIa complex. This decrease in the Ca2+ exchange rate in normal platelets could be prevented by preincubating platelets with a complex-specific anti-IIb X IIIa monoclonal antibody, but not by preincubating platelets with an anti-IIIa monoclonal antibody. In order to determine whether loss of the IIb X IIIa complex primarily affects Ca2+ influx or Ca2+ efflux, both processes were also examined under nonsteady-state conditions. An immediate decrease in the 45Ca2+ influx rate was seen when Ca2+ was added back to platelets preincubated with EGTA at 37 degrees C. The 45Ca2+ efflux rate, on the other hand, was not immediately affected. These data suggest, therefore, that an intact IIb X IIIa complex is necessary for normal Ca2+ homeostasis in platelets.     

The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells

Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterized by a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin alpha(IIb)beta(3)) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin alpha(IIb)beta(3). vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1, 2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.     

Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex

Triflavin, an antiplatelet peptide containing Arg-Gly-Asp, purified from Trimeresurus flavoviridis venom, inhibits aggregation of human platelets stimulated by a variety of agonists. It blocks aggregation through interference with fibrinogen binding to its specific receptor on the platelet surface membrane in a competitive manner, but it has no apparent effect on intracellular events, such as thromboxane B2 formation, phosphoinositides breakdown and intracellular Ca2+ mobilization of thrombin-activated platelets. In this study, we determined the complete sequence of triflavin, which is composed of a single polypeptide chain of 70 amino acids. Its sequence is rich in cysteine and contains Arg-Gly-Asp at residues 49-51 in the carboxy-terminal domain. Triflavin shows about 68% identity of amino acid sequence with trigramin, which is a specific antagonist of the fibrinogen receptor associated with glycoprotein IIb/IIIa complex. [125I]Triflavin binds to unstimulated and ADP-stimulated platelets in a saturable manner and its Kd values are estimated to be 76 and 74 nM, respectively; the corresponding numbers of binding sites are 31,029 and 34,863 per platelet, respectively. [125I]Triflavin binding is blocked by Gly-Arg-Gly-Asp-Ser in a competitive manner. EDTA, the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, trigramin and rhodostomin), and monoclonal antibody, 7E3, raised against GP IIb/IIIa complex, inhibit [125I]triflavin binding to unstimulated and ADP-stimulated human platelets. In conclusion, triflavin specifically binds to fibrinogen receptor associated with GP IIb/IIIa complex and its binding site is located at or near GP IIb/IIIa complex, overlapping with those of 7E3 and another Arg-Gly-Asp-containing polypeptide, rhodostomin.(ABSTRACT TRUNCATED AT 250 WORDS)