Mediates Platelet Aggregation Research Articles

Isoxazoline GPIIb/IIIa antagonists bearing a phosphoramidate

Isoxazolinylacetamides bearing a phosphoramidate group α- to the carboxylate moiety ( 3) were prepared and evaluated for in vitro antiplatelet efficacy. They were found to bind GPIIb/IIIa with high affinity and were potent antagonists of ADP mediated platelet aggregation.

Evidence for Two Distinct G-protein-coupled ADP Receptors Mediating Platelet Activation

The identity of the receptors mediating platelet activation by ADP remains elusive. To distinguish between platelet ADP receptor subtypes, the effects of antagonists on platelet responses and the cloned P2Y1 receptor, a putative platelet ADP receptor, have been investigated. 2-methylthio-AMP (2MeSAMP), an inhibitor of ADP-dependent platelet aggregation, antagonized ADP-mediated inhibition of adenylyl cyclase, competed with binding of [3H]2-methylthio-ADP and inhibited the stimulation of [35S]GTPgammaS binding. 2MeSAMP did not inhibit platelet shape change and was only a weak antagonist of intracellular calcium mobilization in platelets or in cells expressing the cloned human P2YI receptor. By contrast, the P2Y1 receptor antagonist adenosine 3',5'-diphosphate (A3P5P) inhibited ADP-induced platelet aggregation, completely abolished shape change, but did not antagonize ADP effects on cyclic AMP generation or [3H]2-methylthio-ADP binding. However, A3P5P antagonized intracellular calcium mobilization in platelets and cells expressing the cloned P2Y1 receptor. Furthermore, using a specific monoclonal antibody and flow cytometry, P2Y1 receptor protein was detected on human platelets. These results support the existence of two G protein-coupled ADP receptors mediating platelet aggregation, one of which is coupled to Gi proteins and blocked by 2MeSAMP, whereas the second receptor is similar or identical to P2Y1 and coupled to Gq.

Signal Transduction Pathways for Mouse Platelet Membrane Adhesion Receptors

IntroductionThe study of genetic bleeding disorders provided the first link between platelet functions and specific membrane glycoproteins. Two examples are well known and have been the subject of numerous reviews. First, Glanzmann’s thrombasthenia is a bleeding disorder caused by a defect of platelet aggregation in which the glycoprotein αIIbβ3 (GP IIb-IIIa) is either lacking or is expressed but is defective.1 We now know that αIIbβ3 exists on the surface of unstimulated platelets in an inactive form but, through a process known as “inside-out” signaling, responds to platelet stimulation to become a receptor for soluble fibrinogen and von Willebrand factor (vWF) to mediate platelet aggregation. αIIbβ3 is also known to bind immobilized fibrinogen and, through a process known as “outside-in” signaling, to induce platelet stimulation.2 A second example is Bernard-Soulier syndrome, a bleeding disorder caused by the failure of platelets to bind to subendothelial matrices due to the lack of or defective GP Ib-IX-V.3 It is now known that GP Ib-IX-V binds to vWF to mediate the adhesion of unstimulated platelets to injured blood vessel walls.4,5 GP Ib-IX-V interactions also induce platelet stimulation, a process mediated by signaling through GP Ib-IX-V.6 The mechanisms responsible for the binding of adhesive proteins to αIIbβ3 and GP Ib-IX-V are beginning to be understood and, as such, targets for therapeutic intervention have been identified. Three parenteral αIIbβ3 antagonists have demonstrated a therapeutic benefit in large-scale clinical trials of acute coronary syndromes, including unstable angina, non Q-wave myocardial infarction, and percutaneous intervention, and are now commercially available.7 Many orally available αIIbβ3 antagonists are presently in clinical trials. Although GP Ib antagonists have not been pursued as aggressively, animal studies have shown that they do have a proven antithrombotic benefit.8 Despite these advances in the understanding of glycoprotein ligand binding and development of therapeutic antagonists of adhesive protein receptors, the mechanisms responsible for transducing signals through these receptors have remained elusive.It is now established that signal transduction reactions through αIIbβ3 and GP Ib-IX-V are not only involved in platelet aggregation to cause vessel occlusions, but also that glycoprotein signaling affects thrombus growth and stability, as well as the biology and perhaps the pathology of the vessels in which aggregates occur. In one example, platelet-derived growth factor (PDGF), secreted in response to αIIbβ3 signaling from the α-granules of aggregated platelets, is a primary smooth muscle cell mitogen and is believed to be involved not only in the response to vascular injury but also in atherosclerotic lesion progression.9,10 In another example, CD 154 (previously termed CD40 ligand) redistributes from α-granule membranes to the surface of aggregated platelets in response to αIIbβ3 signaling.11 CD 154 is an important inflammatory mediator that induces the release of cytokines from endothelial and smooth muscle cells, initiates vascular inflammation, and participates in atherosclerotic lesion progression.12 A third example involves the assembly of prothrombinase and factor Xase on the surface of aggregated platelets, enabling platelet thrombi to be procoagulant and accounting for the apparent anticoagulant activity of αIIbβ3 antagonists.13,14 In addition, platelet aggregates also display fibrinogen and vWF bound to platelet membrane glycoproteins that function to recruit additional platelets and, therefore, enhance thrombus growth.15 More recent data also indicate that platelet aggregation induces de novo protein synthesis.16,17 These and other events are secondary to the initial adhesion and aggregation reactions of platelets and are consequences of signaling reactions induced by the adhesion and aggregation receptors. Thus, characterization of the membrane glycoprotein signal transduction pathways has become essential, not only to understand platelet function, but also to determine whether there are additional ways by which platelet-mediated pathologies can be regulated.Platelet membrane glycoprotein signaling reactions either do not occur in nucleated cells normally used for transfection studies or are insufficiently characterized. Accordingly, the use of genetics to study mechanisms of platelet adhesive protein receptor signaling has been limited. The advent of technologies that facilitate genetic manipulations in the mouse genome has produced new ways to define protein function and determine the structure-function relationships of individual proteins and is proving of value in unraveling signal transduction pathways in platelets. Although one should always be cautious in extrapolating data from mouse to human platelets (as demonstrated by the PAR receptors, see below), it is impressive that much of what has been learned about platelets appears to apply to both mouse and human. Indeed, this review summarizes the status of genetic manipulations of the mouse genome that have contributed to our understanding of platelet membrane adhesion receptor signaling in platelets.

The fibrinogen RIBS-I epitope (γ373–385) appears proximate to the γ408–411 adhesive domain but is not involved in interaction between receptor-bound or surface-adsorbed fibrinogen and platelet GPIIbIIIa

The carboxyl terminus of the fibrinogen (Fg) γ chain (γ400–411) is necessary and sufficient to support platelet aggregation and adhesion. However, a monoclonal antibody (mAb) to the Fg RIBS-I epitope (γ373–385), the anti-Fg-RIBS-I, which binds only to platelet-bound or surface-adsorbed Fg but not soluble Fg, inhibits platelet aggregation. In this study, we showed that this same antibody also inhibits the adhesion of platelets to Fg-coated polystyrene beads. We then investigated the mechanisms by which the anti-Fg-RIBS-I antibody inhibits platelet aggregation and adhesion. The Fg RIBS-I epitope does not interact with platelet GPIIbIIIa, since recombinant Fg missing the last four amino acids, the Ala-Gly-Asp-Val, on the carboxyl terminus of its γ chains supports neither platelet aggregation nor adhesion to surfaces, nor GPIIbIIIa binding, while it binds anti-Fg-RIBS-I normally. Purified, soluble GPIIbIIIa (265 kDa) inhibits the binding of both the anti-Fg-RIBS-I and 4A5 (a mAb specific to γ408–411 of Fg); however, peptide G13 (1.5 kDa), corresponding to the Fg γ chain binding domain on GPIIba (GPIIb300–312), only inhibits the binding of 4A5, and does not affect the binding of the anti-Fg-RIBS-I to Fg. The anti-Fg-RIBS-I reduces the on-rate of the 4A5 binding to Fg with no measurable changes in the dissociation of the Fg-bound 4A5. These data indicate that the inhibition of platelet aggregation and adhesion by the anti-Fg-RIBS-I antibody is due to the steric hindrance of the Fg γ400–411 to platelet GPIIbIIIa. Thus the Fg RIBS-I epitope (γ373–385) does not appear to be involved in direct interaction with platelet GPIIbIIIa, leaving the γ408–411 of Fg as the sole domain mediating platelet aggregation and adhesion.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin αIIb Subunit in a Patient With a Glanzmann's Thrombasthenia-Like Syndrome

AbstractThe integrin αIIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of αIIb and β3. We now report the molecular analysis of αIIbβ3 from a patient with a Glanzmann's thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of αIIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of αIIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the αIIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated αIIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type αIIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the αIIb cytoplasmic domain.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin IIb Subunit in a Patient With a Glanzmann’s Thrombasthenia-Like Syndrome

The integrin IIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and β3. We now report the molecular analysis of IIbβ3 from a patient with a Glanzmann’s thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

Priming of platelet alphaIIbbeta3 by oxidants is associated with tyrosine phosphorylation of beta3.

Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H2O2, a major cellular oxidant. Exposure to H2O2 triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced99032416 required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO4) and was dependent on the participation of integrin alphaIIbbeta3 (glycoprotein IIb-IIIa). A specific inhibitor of alphaIIbbeta3 blocked platelet aggregation induced by H2O2 and NaVO4, thus confirming that aggregation requires this receptor. In the presence of H2O2 and NaVO4, multiple platelet substrates were phosphorylated on tyrosine. Such tyrosine kinase response was necessary but not sufficient to activate alphaIIbbeta3, as detected by binding of soluble fibrinogen to platelets. Stirring of the platelets exposed to H2O2 and NaVO4 was also needed to allow for binding of fibrinogen to alphaIIbbeta3. The tyrosine kinase inhibitor genistein was able to block platelet aggregation induced by H2O2 and NaVO4, thus confirming that tyrosine kinase activity was needed to trigger alphaIIbbeta3 activation on stirring. N-Acetyl-L-cysteine, a cell-permeant antioxidant, blocked the tyrosine phosphorylation of platelet substrates and also the platelet aggregation induced by H2O2 and NaVO4. We found that beta3 was phosphorylated on tyrosine in platelets exposed to H2O2 and NaVO4, even in the absence of aggregation. Hence, tyrosine phosphorylation of beta3 might contribute to the "priming" of alphaIIbbeta3 induced by H2O2 and NaVO4, whereby the receptor can become activated on stirring of the platelets.

Pharmacokinetics and pharmacodynamics of sibrafiban (Ro 48-3657), an orally active IIb/IIIa antagonist, administered alone or in combination with heparin, aspirin, and recombinant tissue-type plasminogen activator in beagles.

This study characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of sibrafiban (Ro 48-3657) in the presence of aspirin, heparin, and recombinant tissue-type plasminogen activator (rt-PA) in beagles. Sibrafiban is a double prodrug that undergoes bioconversion to the inactive prodrug Ro 48-3656 and to the active IIb/IIIa antagonist, Ro 44-3888, after oral administration. After oral sibrafiban, peak Ro 48-3656 plasma concentrations were observed earlier than Ro 44-3888 and were five- to sixfold higher than Ro 44-3888 peak concentrations. Administration of sibrafiban with heparin and aspirin or heparin and rt-PA did not alter sibrafiban PK. Ro 48-3656 and Ro 44-3888 PK and inhibition of platelet-aggregation profiles in groups treated with sibrafiban and heparin/aspirin or sibrafiban and heparin/rt-PA were similar to those of the group receiving sibrafiban alone. Sibrafiban resulted in >80% inhibition of adenosine diphosphate (ADP)-mediated platelet aggregation and an approximate sixfold increase in bleeding time (BT) compared with baseline measurements. The BT increase was greater in the sibrafiban, heparin, and rt-PA-treated group, during rt-PA administration, compared with the group treated with sibrafiban alone. The recovery of platelet aggregation may be slower after administration of sibrafiban with heparin and rt-PA. Sibrafiban had no effect on rt-PA PK or heparin PD.

Adenosine 5′-diphosphate as a factor in platelet aggregation induced by human plasma remnant lipoproteins

The action of lipoprotein lipase on chylomicrons (CM) and very low density lipoproteins (VLDL) produces remnant lipoproteins (RLP) which are rich in triglycerides, cholesterol and apolipoprotein E (apo E). Apo E serves as a ligand for uptake of RLP by macrophages, platelets, endothelial cells and other cells expressing the LDL-receptor or the remnant receptor, thus having a major role in the clearance of plasma cholesterol and triglycerides, but at the same time, uptake of apo E-bearing RLP can profoundly alter the physiology of these cells and promote atherosclerosis. Like RLP, blood platelets also have roles in atherosclerosis and thrombosis, hence it is likely that RLP influence platelet activity as well. RLP derived from normal human plasma VLDL and CM were prepared using two monoclonal antibodies, antiapo B-100 (JI-H) and anti-apo A-I (H-12) coupled to Sepharose 4B gel to form an immunoaffinity column. Lipoproteins containing apo B-100 including VLDL and LDL adsorb to (JI-H)-gel, while CM and HDL with apo A-I adsorb to (H-12)-gel. The particles in the unbound fraction (RLP) are rich in apo B-48, apo E and apo B-100 containing particles with multiple molecules of apo E. The RLP fraction with a total triglyceride of 14 ± 3.2 mg/ml; cholesterol, 0.39 ± 0.1 mg/ml and protein, 0.78 ± 0.24 mg/ml (n = 19) was added to aliquots of blood of man, rabbits, guinea pigs, mice, and rats at protein equivalents of 0.98 to 78 μg/ml blood and agitated gently at 37 °C for 40 sec. Platelet aggregation was measured as a fall in single platelet count. RLP induced aggregation of platelets in man (p < 0.005) rabbit (p < 0.0005), guinea pig (p < 0.002) and mouse (p < 0.0001), but no RLP induced platelet aggregation was observed in the rat blood. Scanning electron microscopy revealed that in the presence of RLP, platelets had adhered to and formed aggregates on red cells. The platelet response to RLP was inhibited by apyrase known to scavenge ADP, by 5μM 2-chloroadenosine, a platelet ADP receptor antagonist and by 3.4μM cilostazol, a phosphodiesterase type III inhibitor known to raise cyclic AMP level in platelets. It is thought that RLP cause leakage of ADP from red cells which then mediates platelet aggregation.

Pl a1/a2 polymorphism of glycoprotein IIIa and risk of coronary artery disease and restenosis following coronary angioplasty

Glycoprotein IIIa (GpIIIa), a platelet protein that mediates platelet aggregation and thrombus formation, has attained recent widespread interest following a report that a common genetic variant of GpIIIa, known as Pl a2, is an inherited risk factor for the development of premature coronary artery disease (CAD). We determined the frequency of the Pl a2 allele in 589 prospectively recruited subjects aged <50 years presenting with symptomatic CAD with or without myocardial infarction and documented by coronary angiography (group I), 207 subjects investigated prospectively for restenosis 6 months after coronary balloon angioplasty (group II), and 570 control subjects without a history of angina or myocardial infarction, randomly selected from the community. Detection of the Pl a2 allele was based on MspI digestion of polymerase chain reaction (PCR) amplified deoxyribonucleic acid (DNA) spanning the Pl a1/a2 locus. The accuracy of genotyping was verified with a second independent method based on BstXI digestion of DNA amplified with mutagenic PCR primers. The frequency of the Pl a2 allele was similar (p >0.1) in group I (170 of 1,178, 14%), group II (49 of 414, 12%), and control subjects (160 of 1,140, 14%). Among group I subjects, there was no relation (p >0.1) between the Pl a2 allele frequency and the number of coronary vessels with >50% diameter obstruction, and current or previous myocardial infarction; among group II subjects, there was no difference between those with and without restenosis after angioplasty (26 of 242 and 23 of 172, respectively, p >0.1). We conclude that the Pl a2 allele is not associated with a significantly elevated risk of premature CAD, myocardial infarction, or restenosis after coronary angioplasty.

Complementary roles for receptor clustering and conformational change in the adhesive and signaling functions of integrin alphaIIb beta3.

Integrin alphaIIb beta3 mediates platelet aggregation and "outside-in" signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to alphaIIb beta3 function, alphaIIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified alphaIIb subunits were expressed with beta3 in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25-50% of that observed when alphaIIb beta3 affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of alphaIIb beta3 also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72(Syk) and fibrinogen-dependent phosphorylation of pp125(FAK), even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in alphaIIb beta3 function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of alphaIIb beta3 in platelets.

Role of the γ chain Ala–Gly–Asp–Val and A α chain Arg–Gly–Asp–Ser sites of fibrinogen in coaggregation of platelets and fibrinogen-coated beads

Fibrinogen (Fg) mediates platelet aggregation and adhesion to artificial surfaces. The carboxyl terminus of the γ chain of Fg (residues AGDV at γ408–411) is known to play an exclusive role in platelet aggregation, while there is no known role for the consensus RGD sites in the A α chain. In this study, we used flow cytometry to measure the coaggregation (CA) of platelets with Fg-coated beads, and investigated which domains in surface-immobilized Fg support platelet adhesion. CA of platelets with Fg-beads was nearly abolished in the presence of 4A5, a monoclonal antibody (mAb) whose epitope includes AGDV, while Z69/8, a mAb that also binds to the γ chain carboxyl terminus but does not cover AGDV, had little effect. When beads were coated with recombinant Fg (rFg) lacking AGDV, CA was similarly abolished. In contrast, beads coated with Fg that lacked the RGDS site, supported platelet CA as did intact Fg. These results were confirmed in experiments that measured the binding of activated soluble glycoprotein IIb and IIIa (GPIIbIIIa), the platelet membrane glycoprotein complex known to be the Fg receptor, to immobilized Fg. This binding was inhibited by mAb 4A5, but not by mAb Z69/8. Binding was totally retained when beads were coated with Fg lacking RGDS, but was completely lost when beads were coated with Fg lacking AGDV. These results demonstrated that the AGDV sequence on the carboxyl terminus of the γ chain of Fg plays an exclusive role in platelet adhesion to surface-immobilized Fg, while the carboxyl terminus of the A α chain, including a consensus RGD site, is not required.

Platelet Cytosolic Calcium Responses to Serotonin in Depressed Patients and Controls: Relationship to Symptomatology and Medication

Background: Serotonin produces an exaggerated rise in platelet cytosolic calcium (Δ[Ca ++] i) in patients with mood disorders. Studies on patients with bipolar disorder consistently demonstrate calcium abnormalities. By comparison, data on patients with major depression are more variable. Methods: To determine causes of variability, we utilized Fura-2 loaded platelets to compare changes in platelet intracellular calcium levels (Δ[Ca ++] i) following serotonin stimulation in 24 patients with major depression and in 20 controls. We also sought relationships between the Δ[Ca ++] i responses and scores on clinical depression and anxiety scales. Results: We found positive correlations between Δ[Ca ++] i responses and the clinical scales across all subjects. Furthermore, depressed patients with high anxiety had significantly increased Δ[Ca ++] i responses compared to depressed patients with low anxiety. In addition, patients receiving selective-serotonin reuptake inhibitors (SSRIs) demonstrated reduced Δ[Ca ++] i responses compared to patients not on SSRIs. Conclusions: Since elevations in [Ca ++] i mediate platelet aggregation and secretion cascades, the enhanced responsivity observed in depressed, and in particular anxious, depressed patients may contribute to their increased risk for vascular disease.

Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions.

Platelet aggregation contributes to arresting bleeding at wound sites, but may cause occlusion of atherosclerotic vessels, thus curtailing blood flow to vital organs. According to current dogma, the integrin alphaIIbbeta3 plays an exclusive role in linking platelets to one another through interactions with fibrinogen or vWf. We demonstrate here that, depending on shearing flow conditions, this process may require vWf binding to glycoprotein Ibalpha, even when alphaIIbbeta3 is competent to bind adhesive ligands. Platelet activation induced solely by high shear stress is initiated by glycoprotein Ibalpha interaction with vWf, but results in aggregation only if the latter can bind concurrently to alphaIIbbeta3. In contrast, platelets exposed to high shear rate after activation by exogenous agonists such as ADP and epinephrine can aggregate when fibrinogen is the alphaIIbbeta3 adhesive ligand, yet only if vWf binding to glycoprotein Ibalpha can also occur. Thus, the latter interaction appears to provide a bond with biomechanical properties necessary to overcome the effects of high shear rate and initiate interplatelet cohesion. These findings highlight the distinct function of two adhesive receptors mediating platelet aggregation under varying fluid dynamic conditions, and modify the current interpretation of a crucial event in hemostasis and thrombosis.

Purification of the Porcine Platelet GP IIb-IIIa Complex and the Propolypeptide of von Willebrand Factor

Platelet membrane glycoproteins (GP) are involved in platelet adhesion and aggregation. The glycoprotein IIb-IIa complex (GP IIb-IIIa) is a Ca2+-dependent heterodimer that binds fibrinogen and other adhesive proteins, thereby mediating platelet aggregation and adhesion. We have purified two major glycoproteins from pig platelets by Concanavalin A-Sepharose, Heparin-Sepharose and Sephacryl S-300 HR chromatography (Fitzgerald et al. Anal Biochem, 1985): i) the GP IIb-IIIa complex, GP IIb Mr = 140,000 and GP IIIa a single chain of Mr = 95,000-100,000; and ii) a predominant glycoprotein of high molecular weight, the propolypeptide of von Willebrand factor (Mr = 80,000-100,000). Western-blot analysis of the purified GP IIb-IIIa showed that only certain monoclonal antibodies against the human receptor specifically recognize the porcine complex. Differences between the porcine and human GP IIb-IIIa glycoproteins could partially explain the decreased inhibitory effects of GP IIb/IIIa-antagonists (against the human receptor) in porcine platelets.

Identification of a P 2X1Purinoceptor Expressed on Human Platelets

It has been proposed that platelets possess a P 2X1-purinoceptor-like ligand-gated cation channel, through which Ca 2+enters platelets from the extracellular medium upon ADP or ATP stimulation. In this paper we describe the cloning of human P 2X1-specific cDNA from human platelets, K562 and human erythroleukaemic cell lines. Sequence analyses of these cDNAs show 100% nucleotide sequence identity with that of human P 2X1cloned from urinary bladder. Western blotting of platelet lysates separated by SDS-PAGE and probed with anti-P 2X1IgG shows the expected protein with a molecular mass of 60kDa and a second protein of 45kDa. These data confirm that platelets possess at least two distinct purinoceptors: a P 2Tpurinoceptor which mediates platelet aggregation, inhibition of adenylate cyclase, and release of intracellular Ca 2+stores and a platelet P 2X1purinoceptor which upon ATP and ADP stimulation mediates the rapid entry of extracellular Ca 2+into platelets.

Ristocetin- and Thrombin-induced Platelet Aggregation at Physiological Shear Rates: Differential Roles for GPIb and GPIIb-IIIa Receptor

We recently reported that washed platelets (WP) activated with ADP and expressing surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette device at physiological shear rates of G = 300 s(-1)-1000 s(-1) in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa totally required for the aggregation. We have now extended these studies to aggregation of platelets "activated" with ristocetin or thrombin. Washed platelet suspensions with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin (0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at uniform shear rate, G = 1000 s(-1). The collision capture efficiency (alphaG) with which small aggregates form (= experimental/calculated initial rates of aggregation) was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb interaction was exclusively able to support ristocetin-mediated shear aggregation of metabolically active platelets, with very few vWF monomer equivalents bound per platelet (representing < or = 10 molecules of 10 million Da) required to yield high capture efficiencies (alphaG = 0.38+/-.02; n = 11), suggesting rapid and stable bond formations between vWF and GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to washed platelets, was found to mediate platelet aggregation with alphaG = 0.08+/-.01 (n = 6), surprisingly comparable to that previously reported for WP and ADP activation. Blocking the GPIIb-Illa receptor decreased alphaG by 95+/-3% (n =3), while a monoclonal antibody to the vWF site on GPIb caused a 49+/-7% (n = 8) decrease in alphaG. The partial role for GPIb thus appears to reflect a facilitative function for increasing contact time between flowing platelets, and allowing engagement of the GPIIb-IIa receptor to yield stable attachment.

The AGDV residues on the gamma chain carboxyl terminus of platelet-bound fibrinogen are needed for platelet aggregation

It has been clear that only the carboxyl terminus of fibrinogen (Fg) γ chain is required for the initial binding of Fg from solution to its GPIIbIIIa (glycoprotein IIb and IIIa) receptor on activated platelets, whereas the two RGD sites on the A α chain do not play any role. In this study, we examined the role of these three putative adhesive domains on Fg already bound to its receptors in mediating platelet aggregation. Activated platelets were first incubated with Fg to let the Fg bind, then with monoclonal antibodies (mAb) to block the putative adhesive domains, and the platelet suspension was then sheared or stirred to induce aggregation. The mAb 4A5, which recognizes the last four amino acid residues (AGDV) in a dodecapeptide (H12) on the carboxyl terminus of the Fg γ chain, markedly inhibited platelet aggregation. Z69/8, a mAb whose epitope is also on the dodecapeptide but does not recognize the AGDV residues, did not have any inhibitory effect on aggregation. The anti-RGDS and anti-RGDF mAbs did not affect both macro- and micro-aggregation at all, whether tested singly or together. These results demonstrate that, similar to the situation for the initial binding of soluble Fg, only the γ chain carboxyl terminus with the AGDV residues are needed for platelet-bound Fg to support aggregation, while the RGD sites on the A α chain do not seem to be required.

Integrin-mediated Activation of Focal Adhesion Kinase Is Independent of Focal Adhesion Formation or Integrin Activation: STUDIES WITH ACTIVATED AND INHIBITORY β3CYTOPLASMIC DOMAIN MUTANTS

Integrin alphaIIbbeta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either "inside-out" or "outside-in" signaling through alphaIIbbeta3 is the phosphorylation of focal adhesion kinase (pp125(FAK)) and the association of pp125(FAK) with cytoskeletal components. To examine the role of pp125(FAK) in these integrin-mediated events, pp125(FAK) phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of alphaIIbbeta3: alphaIIbbeta3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125(FAK) is altered, and alphaIIbbeta3(F727A/K729E/F730A), in which the putative binding site for alpha-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing alphaIIbbeta3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing alphaIIbbeta3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125(FAK) is recruited to focal adhesions in adherent cells expressing alphaIIbbeta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing alphaIIbbeta3(F727A/K729E/F730A), pp125(FAK) was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125(FAK) can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.

Metal-ion catalyzed oxidation affects fibrinogen activity on platelet aggregation and adhesion

Exposure of fibrinogen to the Fe 3+/ascorbate oxidative system resulted in structural modifications and altered functionality of the glycoprotein. The overnight treatment of fibrinogen by oxidants caused a 20-fold increase of carbonyl content with respect to the native protein. Formation of dityrosines as well as loss of tryptophan following fibrinogen oxidation were observed. The occurrence of conformational changes of the fibrinogen molecule as a consequence of the oxidative treatment was also established. Oxidized fibrinogen showed a distinct capability from the native molecule to mediate platelet aggregation and adhesion. The percentage of ADP-induced platelet aggregation decreased as a function of fibrinogen oxidative damage. Further, both unstimulated platelets and ADP-activated platelets showed a reduced ability to adhere to oxidized fibrinogen than to the native protein. These results suggest that oxidative treatment alters fibrinogen domains involved in the recognition and the binding of this molecule by the platelet receptor GP IIb/IIIa.