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.     

pH and magnesium alter 45calcium binding to platelets at sites other than glycoproteins I or IIb/IIIa

Calcium is a cofactor of human platelet aggregation. Moreover a direct correlation between the ability of platelets to bind this divalent cation and to aggregate has been demonstrated. Since magnesium can substitute for calcium in supporting aggregation, especially in the presence of low calcium concentrations, and platelet aggregation is inhibited at low pH, the present study was designed to examine the effects of magnesium and low pH on 45calcium binding to human platelets, and to determine whether such effects might be associated with calcium binding to glycoproteins I (GPI) or IIb/IIIa (GPIIb/IIIa), the putative fibrinogen receptor. 45Calcium binding to aspirin-treated platelets that had been depleted of surface-associated calcium by brief exposure to EDTA was evaluated. Magnesium (5-10 mM) or a change in hydrogen ion concentration to decrease the pH from 7.5 to 6.0 was found to inhibit the binding of 45calcium to platelets from healthy donors by 34 +/- 6 and 32 +/- 8% (mean +/- SD, n = 13), respectively. Similar results were obtained with platelets incubated with chymotrypsin to selectively remove GPI or platelets from a patient with the Bernard Soulier Syndrome, congenitally deficient in GPI. In contrast, calcium binding to platelets from two patients with thrombasthenia, lacking GPIIb/IIIa, was reduced 49 +/- 6% and 42 +/- 8% (n = 4) by magnesium and hydrogen ions, respectively. This apparently increased inhibition was attributed to the combined effects of an overall decrease (approximately 50%) in calcium binding to thrombasthenic platelets compared with that in control platelets, and a similar absolute reduction in calcium binding in the presence of magnesium and/or hydrogen ions. No additional inhibition of 45calcium binding was noted in the presence of magnesium and at low pH, indicating that magnesium and hydrogen ions may affect the same platelet membrane binding sites. The data suggest that although modulation of platelet aggregation by magnesium and pH is accompanied by changes in platelet-associated calcium, calcium binding to the three major platelet membrane glycoproteins, GPI, IIb, and IIIa is unaffected.     

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.     

Inhibition of human tumor cell induced platelet aggregation by antibodies to platelet glycoproteins Ib and IIb/IIIa

Tumor cell induced platelet aggregation was shown to be inhibited in a dose dependent manner by preincubation of human platelets with antibodies to platelet glycoprotein Ib and the IIb/IIIa complex. Combination of antibody to Ib and antibody to the IIb/IIIa complex at concentrations which produced half maximal inhibition of platelet aggregation alone caused complete inhibition of tumor cell induced platelet aggregation. Antibodies to platelet glycoproteins Ib and the IIb/IIIa complex also inhibited platelet synthesis of thromboxane A2, but not synthesis of 12-hydroxyeicosatrienoic acid. Inhibition of tumor cell induced platelet aggregation with antibodies against platelet glycoproteins suggests a role for these glycoproteins in tumor cell-platelet interactions and possibly platelet facilitated tumor cell metastasis.     

Design, synthesis, and structure-activity relationships of potent GPIIb/IIIa antagonists: discovery of FK419

The discovery of the non-peptide antiplatelet injectable agent FK419 is reported. Based on the beta-turn structure of RGD peptide sequences in the alpha chain of fibrinogen, which binds the glycoprotein IIb/IIIa (GPIIb/IIIa) on the surface of platelets to induce platelet aggregation, the prototype 2 was designed. After further substituent effects were investigated at the alpha-position of the carboxylic acid in 2, we enhanced platelet aggregation inhibition, and discovered the useful feature of reduced prolongation of bleeding time. Finally, the potent platelet aggregation inhibitor FK419 (3) could be discovered. FK419 shows a safe feature of reduced prolongation of bleeding time, as well as potent inhibition of platelet aggregation.     

Thrombosis of microvascular anastomoses in traumatized vessels: fibrin versus platelets

Thrombosis is the end result of two closely interrelated processes: the coagulation cascade and the platelet aggregation process. To determine their relative contribution, we used pharmacologic agents that selectively block each process. The specific effect of each pharmacologic agent on either fibrin deposition or platelet activity was confirmed morphologically by scanning electron microscopy and was substantiated with ADP-induced platelet aggregation and blood clotting time determinations. Forty-two rats had both femoral arteries subjected to a standardized crush-avulsion injury. A total of 84 femoral microvascular anastomoses were subsequently performed. None of the 24 control anastomoses treated with saline remained patent, whereas 6 of 24 of the anastomoses treated with dazmagrel (a selective thromboxane synthetase and platelet aggregation inhibitor), 2.5 mg/kg IV, remained patent and 18 of 24 of those treated with a single dose of heparin, 200 U/kg IV, remained patent. All 12 anastomoses treated with both drugs remained patent but developed a 33 percent hematoma rate. We conclude that in this microvascular model, fibrin mesh deposition is a more significant factor than platelet aggregation in the pathogenesis of occlusional thrombosis within traumatized arteries. Its temporary inhibition with a single dose of heparin yielded a 75 percent improvement in patency rate.     

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.     

Reactive thrombocytosis alone does not affect the patency of microvascular anastomosis in the splenectomy rat

Vascular thrombosis is a harbinger of failure in microsurgery. However, there is still controversy regarding the correlation of the complications of thrombocytosis and thrombosis. Some evidence indicates that patients with elevated platelet counts tend to have a higher flap failure rate, and surgeons usually hesitate to operate on patients with thrombocytosis. Nevertheless, the authors have experienced successful free tissue transfer in seven patients with thrombocytosis resulting from traumatic splenectomy or multiple trauma. On the basis of clinical observation, the authors investigated whether reactive thrombocytosis contributes to the patency of a microvascular anastomosis. In a rodent splenectomy-induced thrombocytosis model (n = 40), stable reactive thrombocytosis occurred after postoperative days 5 to 10, with the peak on postoperative day 7. Femoral artery division and reanastomosis was performed in rats with or without splenectomy-induced thrombocytosis, and vascular patency was assessed. Platelet counts and platelet activation were studied in correlation to microvascular patency. Platelet activation as demonstrated by CD62P expression on platelets was not significantly different between rats with and without thrombocytosis (6.41 +/- 0.95 percent versus 4.51 +/- 0.55 percent, respectively; p = 0.089). As immature platelets were not increased (2.86 +/- 0.33 percent versus 1.99 +/- 0.32 percent, p = 0.074), it seems that the splenectomy-induced thrombocytosis is the result of redistribution of platelets instead of an increase in bone marrow production. There were no significant differences in the patency rates or perfusion units of femoral artery after arterial anastomosis between rats with and without thrombocytosis (90 percent and 95 percent, respectively; p = 0.561). In conclusion, this study demonstrates that microvascular anastomosis can be performed safely in patients with reactive thrombocytosis without platelet activation.     

Human cytomegalovirus infection of endothelial cells triggers platelet adhesion and aggregation

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of vascular diseases. HCMV infection of endothelial cells may lead to vascular damage in vitro, and acute-phase HCMV infection has been associated with thrombosis. We hypothesized that viral infection of endothelial cells activates coagulation cascades and contributes to thrombus formation and acute vascular catastrophes in patients with atherosclerotic disease. To assess the effects of HCMV on thrombogenesis, we examined the adhesion and aggregation of blood platelets to uninfected and HCMV-infected endothelial cells. At 7 days after infection, platelet adherence and aggregation were greater in infected than in uninfected cultures (2,000 platelets/100 cells and 225 +/- 15 [mean +/- standard error of the mean] aggregates/five microscopic fields versus 100 platelets/100 cells and no aggregates). von Willebrand factor (vWF), ICAM-1, and VCAM-1 but not collagen IV, E-selectin, P-selectin, CD13, and CD31 were expressed at higher levels on infected cells than on uninfected cells. Platelet aggregation was inhibited by blocking of platelet GPIb (with blocking antibodies) or GPIIb/IIIa (with ReoPro) or by blocking of vWF (with polyclonal antibodies to vWF). Furthermore, blocking of vWF, platelet GPIb, and ICAM-1 but not of the endothelial cell marker CD13, alpha(5)beta(3)-integrin, or HCMV glycoprotein B reduced platelet adherence to infected cells by 75% +/- 5%, 74% +/- 5%, or 18% +/- 5%, respectively. The increased thrombogenicity was dependent on active virus replication and could be inhibited by foscarnet and ganciclovir; these results suggest that a late viral gene may be mediating this phenomenon, which may contribute to vascular catastrophes in patients with atherosclerotic disease.     

Ring constrained analogues of beta-alanine-containing GPIIb/IIIa receptor antagonists

A series of ring constrained analogues of the GPIIb/IIIa receptor antagonist XR299 (1) was investigated as potential inhibitors of glycoprotein IIb/IIIa, a platelet receptor that plays a key role in platelet aggregation and platelet adhesion. Ring size was found to have a large effect on in vitro potency. Selected compounds showed good in vitro activity, a preference for binding to activated platelets, and modest duration of action when dosed i.v. as a racemate in a canine model.     

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.     

Aminopeptidase resistant Arg-Gly-Asp analogs are stable in plasma and inhibit platelet aggregation.

Tetrapeptides containing the sequence Arg-Gly-Asp (RGD) antagonize fibrinogen binding to its platelet receptor (gp IIb/IIIa, integrin alpha IIb beta 3) and inhibit platelet aggregation in vitro. The peptides RGDS and RGDY(Me)-NH2 were rapidly degraded when incubated in human, rat, and dog plasma. HPLC analysis indicated that amino acids were sequentially removed from the peptide N-terminus, and this degradation was prevented by the aminopeptidase inhibitor bestatin. Analogs of RGDY(Me)-NH2 with an acetylated or deleted alpha-amino group were prepared. Both analogs were stable when incubated in plasma, blocked 125I-fibrinogen binding to activated platelets (IC50 = 10-30 microM) and inhibited ADP induced platelet aggregation (IC50 = 10-30 microM). This study concludes that aminopeptidase rapidly degrades RGD peptides in plasma, an important issue for in vivo testing of RGD peptides and analogs. RGD analogs intrinsically stabilized against aminopeptidase are stable in plasma and are important tools for antithrombotic studies involving antagonism of gp IIb/IIIa.     

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)     

Characterization of platelet abnormalities of Tester Moriyama (TM) rats with storage pool deficiency

Platelet abnormalities of Tester Moriyama (TM) rats, which have prolonged bleeding time with normal platelet count, were characterized by comparison with those of fawn-hooded (FH) rats with platelet storage pool deficiency (SPD). Morphologically, the dense granules were virtually lacking in platelets from TM and FH rats. Platelets from TM and FH rats aggregated in response to adenosine diphosphate (ADP), but failed to have secondary aggregation. In contrast, platelet aggregation was completely absent in response to 1 to 20 micrograms of collagen/ml, although partial aggregation was observed at the higher dosage of 50 micrograms/ml. Normal amounts of platelet membrane glycoproteins IIb/IIIa were expressed in TM and FH rats, but platelet adenosine triphosphate (ATP) and ADP contents were lower than those in platelets from control Wistar rats. Platelet ATP-to-ADP ratio of TM and FH rats was significantly higher than that of Wistar rats. Serotonin content in platelets from TM and FH rats was 20 to 25% that of Wistar rat platelets. These results suggested that platelet abnormalities of TM rats are a typical characteristic of platelet SPD and are similar to those of FH rats, which are genetically different from TM rats. Therefore, TM rats may serve as a useful animal model for the study of platelet SPD.     

An ethanol extract of Ramulus mori improves blood circulation by inhibiting platelet aggregation

Inappropriate platelet aggregation can cause blood coagulation and thrombosis. In this study, the effect of an ethanol extract of Ramulus mori (ERM) on blood circulation was investigated. The antithrombotic activity of ERM on rat carotid arterial thrombosis was evaluated in vivo, and the effect of ERM on platelet aggregation and blood coagulation time was evaluated ex vivo. To evaluate the safety of ERM, its cytotoxicity to platelets and its effect on tail bleeding time were assessed; ERM was not toxic to rat platelets and did not prolong bleeding time. Moreover, administering ERM to rats had a significant preventive effect on carotid arterial thrombosis in vivo, and significantly inhibited adenosine diphosphate- and collagen-induced platelet aggregation ex vivo, whereas it did not prolong coagulation periods, such as prothrombin time and activated partial thromboplastin time. The results suggest that ERM is effective in improving blood circulation via antiplatelet activity rather than anticoagulation activity.     

Platelet responses in health and disease

Summary This article summarizes recent ultrastructure findings from our laboratory and documents some of the information accumulated primarily since 1975 from many laboratories. Special attention is given to documentation by scanning electron microscopy which affords insight into platelet activation (adhesion, aggregation, release/secretion) and especially platelet-vessel wall interactions. Structural physiology of platelets is considered in some detail as a basis for understanding platelet disorders contributing to clinical problems of thrombosis and hemorrhage. The impaired ability of vonWillebrand platelets to adhere to injured vessel wall is reported using the human umbilical vein perfusion model. Relationships between platelets and blood coagulation factors focus on the exquisite sensitivity of platelets to minute amounts of thrombin. Unmasking of platelet factor 3 sites is identified on activated platelets, after glutaraldehyde fixation, by their reaction to latex bearing anti-platelet factor 3 markers. The basis for platelet-collagen interactions is reviewed. Conditions for and possible mechanisms behind platelet interaction with vessel wall are discussed. Ex vivo flowing blood-vessel wall models offer opportunities for improved understanding of the platelets role(s) in vascular diseases.     

The expression of glycoproteins on single blood platelets from healthy individuals and from patients with congenital bleeding disorders

Glycoproteins present on the surface of blood platelets are fundamental to normal blood platelet behaviour. We have used monoclonal antibodies and flow cytofluorimetry to study the expression of glycoproteins on single platelets from normal subjects, and from patients with Glanzmann's thrombasthenia and the Bernard-Soulier syndrome. We show that normal platelets are heterogeneous in that individual cells display markedly different numbers of glycoprotein IIb/IIIa complex and glycoprotein Ib molecules. We also show that the two congenital bleeding disorders are associated with markedly reduced numbers of glycoprotein IIb/IIIa complex or glycoprotein Ib molecules on all the platelets rather than the difference residing in a sub-population.     

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)     

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.     

In vitro binding of an IgE protein to human platelets

Bronchoconstriction in extrinsic asthma is initiated by mediators released from IgE-sensitized leukocytes after contact with polyvalent antigen. Because platelets also contain soluble mediators that can cause bronchoconstriction, platelet activation and release of the contents of platelet granules may play a role in IgE-mediated responses under some circumstances. We therefore sought to determine if platelets are capable of binding IgE and if cross-linking this cell-bound IgE initiates secretion of platelet granule contents. Platelets from 10 normal donors were studied by using automated fluorescence analysis and fluorescence microscopy. We detected binding of a purified myeloma IgE protein to 24.1 +/- 9.6% (mean +/- 2 SD) of the gel-filtered platelets from these normal individuals. Although we could detect the binding of IgE and anti-IgE to a minority of cells, every normal individual had a population of platelets that bound IgE. The amount of IgE that bound to normal platelets appeared to be distributed heterogeneously among the IgE-positive platelet population. Platelets from two individuals with type II Glanzmann's thrombasthenia bound normal amounts of heat-aggregated IgG, but less than 3% of the platelets bound detectable IgE. Moreover, a combination of monoclonal antibodies to glycoproteins IIb and IIIa inhibited the binding of the IgE protein to normal platelets but did not affect the binding of aggregated IgG. Thus, the binding of IgE to human platelets appeared to require the presence of the glycoprotein IIb-IIIa complex. Binding of monomeric IgE to platelets, by itself, did not initiate either platelet aggregation or release of 14C-serotonin. However, both aggregation and secretion of serotonin followed the addition of anti-IgE to IgE-sensitized platelets. These studies indicate that human platelets can bind an IgE myeloma protein in vitro and that cross-linking of surface-bound IgE with anti-IgE initiates aggregation and secretion. If platelets have a similar capacity to bind normal IgE in vivo, it is possible that platelets may participate directly in several atopic or inflammatory disorders in man mediated by this class of antibody.