Glycoprotein IIb-IIIa Complex Research Articles

Procoagulant activity and active calpain in platelet-derived microparticles

Microparticles are released during in vitro platelet activation and have been detected in vivo in several pathologies. Their characterization is of interest as they may play a potential role in hemostasis. Here, we report the formation of microparticles as the result of increases in intracellular Ca 2+ brought about by inhibition of Ca 2+-ATPases. They were isolated following centrifugation of the activated platelet suspension over a sucrose layer. Flow cytometric studies using annexin V-FITC as a probe for aminophospholipids, prothrombinase activity measurements and annexin V inhibition experiments enabled us to evaluate the procoagulant activity of microparticles prepared in this way. The efficiency of the annexin V inhibition (IC 50=10–20 nM) of this activity confirmed significant anticoagulant properties for this protein. Microparticles also contained the glycoprotein IIb-IIIa complex, detected in flow cytometry at a density higher than on the remnant platelets. The activation of calpain, a Ca 2+-dependent protease, in platelets was shown to be more efficient under conditions of a sudden Ca 2+ influx. The microparticles contained only the active form of calpain detected by Western blotting using a monoclonal antibody able to recognize both the unactivated and the activated catalytic subunit of the enzyme. However, flow cytometry failed to find significant amounts of active calpain on the microparticle or on the platelet surface. Our results, while confirming the procoagulant activity of microparticles, also document for the first time the exclusive presence of the activated form of calpain, inferring a possible role for this protease in microparticle-mediated functions.

Visualization of activation-dependent epitopes on glycoprotein IIb-IIIa complexes of platelets stimulated by thrombin: immunogold staining of ultrathin sections.

An immunoglobulin M monoclonal antibody (IgM MAb; AP-6) recognizing the sequence 211-221 of glycoprotein (GP) IIIa enabled a study of the distribution of this epitope on unstimulated or thrombin-activated platelets. Flow cytometry was used to evaluate the expression of this epitope on platelets and immunogold staining on ultrathin sections to analyze its distribution within the cell. There was little or no binding of AP-6 to unstimulated platelets, but immunogold staining showed labeling associated with the membranes of alpha-granules. The binding of AP-6 to thrombin-stimulated platelets was compared with that of anti-RIBS MAbs and antifibrinogen polyclonal antibodies. An increased expression of the AP6 epitope was observed on membranes of the surface-connected canalicular system and at the periphery of the cell as early as 10 to 15 seconds after platelet activation by thrombin. At the same time, binding of anti-RIBS MAbs confirmed that at least part of the endogenous fibrinogen had left the alpha-granules and was bound to platelet membranes. Staining with polyclonal antifibrinogen antibody also revealed fibrinogen in vesicles resulting from granule fusion. Rapidly, the pool of internal membranes with bound fibrinogen became exposed to the outside of the platelet, a process involving the unfolding of membranes and pseudopod formation. Our results provide evidence that activation of GP IIb-IIIa and binding of fibrinogen to platelet membranes can occur before their expression at the platelet surface. They also suggest the presence of an alpha-granule pool of GP IIb-IIIa linked to fibrinogen in unstimulated platelets.

Abnormal Inside-Out Signal Transduction-Dependent Activation of Glycoprotein IIb-IIIa in a Patient With Impaired Pleckstrin Phosphorylation

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year-old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction-independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.

A close spatial relationship between GP IIb—IIIa complexes and CD9 antigen as demonstrated by the MAIPA technique

CD9 is a well-defined component of the platelet plasma membrane and has a copy number almost equivalent to that of glycoprotein (GP) IIb-IIIa complexes, the aggregation receptor on platelets. It has an apparent molecular mass of 24 kD and is otherwise known as p24. Stimulation of p24 by monoclonal antibodies (MAb) induces platelet aggregation and granule release, involves FcγRII, and is mainly mediated through the stimulation of phospholipase C. In accordance with a signalling function, p24 has been reported to associate with small GTP-binding proteins and to GP IIb-IIIa complexes upon activation. We now report further evidence of a strong relationship between p24 and GP IIb-IIIa in platelets. Using the MAIPA (monoclonal antibody immobilization of platelet antigens) assay in the screening of human antibodies to platelet glycoproteins, we found that GP IIb-IIIa-antibody complexes were almost invariably associated with p24 in the harvested detergent-soluble fraction of platelet lysates. Thus, associated human antibodies were detected following the targeting of either GP IIb-IIIa or p24 by monospecific murine monoclonal antibodies (MAbs). This is a point to bear in mind when assessing for antibodies to p24 or GP IIb-IIIa in immune thrombocytopenias.

Porcine von Willebrand Factor Binding to Human Platelet GPIb Induces Transmembrane Calcium Influx

Porcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) Ib and, upon stirring (1500 rpm/min) at 37 degrees C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP Ib, obtained with anti GP Ib monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP Ib monoclonal antibody which does not inhibit the vWF binding to GP Ib. An anti GP IIb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP Ib occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP Ib, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxygenase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

A monoclonal antibody (SZ21) specific for platelet GPIIIa distinguishes P1A1 from P1A2.

Neonatal alloimmune thrombocytopenia (NATP) and post-transfusion purpura (PTP) are acquired bleeding disorders caused by alloimmune thrombocytopenia. In most cases, the thrombocytopenia is due to an alloantibody directed against the platelet glycoprotein IIb-IIIa (GPIIb-IIIa) complex. During the course of routine studies on the role of GPIIb-IIIa in inherited and acquired bleeding and thrombotic disorders, we unexpectedly identified an individual whose platelets reacted by non-reduced Western blot analysis with anti-GPIIIa polyclonal antisera, but did not react with a commercially available monoclonal antibody (SZ21) specific for GPIIIa. We screened all 14 GPIIIa exons for possible nucleotide changes which might alter amino acids and found variations in only exons 3 and 10. Nucleotide sequencing revealed that only the exon 3 alteration changed the predicted amino acid sequence. This variation was caused by homozygosity for the uncommon P1A2 allele of the GPIIIa gene. Platelets from two additional unrelated normal individuals known to be homozygous for P1A2 also lacked reactivity with SZ21 by Western blot. Using flow cytometry with intact platelets, we observed a markedly reduced binding of SZ21 to platelets with the P1A2 genotype. Scatchard analyses indicated that SZ21 bound to P1A1/A1 platelets with a Kd of approximately 8.26 x 10(-10) M, and to P1A2/A2 platelets with a Kd of approximately 5.58 x 10(-9) M. Thus, we have characterized a readily available monoclonal antibody able to distinguish between the two P1A alleles of the GPIIIa gene. Because incompatibility for this platelet polymorphism is the most common cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura, and because platelet immunophenotyping reagents lack specificity and are not easily available, this monoclonal antibody could facilitate the management of patients with these disorders.

Crotavirin, a potent platelet aggregation inhibitor purified from the venom of the snake Crotalus viridis

A potent platelet aggregation inhibitor in the venom of Crotalus viridis snake was purified to homogeneity by gel filtration chromatography and reverse phase high-performance liquid chromatography. This purified principle, named crotavirin, is a single-chain polypeptide with a mol. wt of 9200 as estimated by SDS-polyacrylamide gel electrophoresis. It inhibited the aggregation of human washed platelets induced by collagen, thrombin and thomboxane analogue (U46619) with a similar IC 50 (~ 1.0 μg/ml, 0.11 μM). The binding of fluorescein isothiocyanate-conjugated crotavirin to platelets was abolished in the presence of divalent cation chelator, EDTA, indicating that divalent cation is essential for crotavirin's binding. A monoclonal antibody, 7E3, raised against platelet glycoprotein IIb-IIIa complex blocked the binding of fluorescein isothiocyanate-conjugated crotavirin to platelets, whereas the other monoclonal antibody against glycoprotein IIb-IIIa, 10E5, had no inhibitory effect. In addition, crotavirin inhibited in a concentration-dependent manner the binding of fluorescein isothiocyanate-conjugated rhodostomin, a member of the disintegrin family, to platelets. Its binding to platelets was blocked by disintegrins e.g. trigramin and rhodostomin. It is concluded that crotavirin is a potent platelet aggregation inhibitor, which acts specifically on an epitope of glycoprotein IIb-IIIa, leading to the blockade of fibrinogen binding to glycoprotein IIb-IIIa and eventually the blockade of platelet aggregation.

50-kD Integrin-Associated Protein Does Not Detectably Influence Several Functions of Glycoprotein IIb-IIIa Complex in Human Platelets

A 50-kD integrin-associated protein (IAP) has been reported to be associated with beta 3 integrins and to modulate their function, especially vitronectin receptor in human erythroleukemia (HEL) cells and leukocyte response integrin in neutrophils. We studied the involvement of IAP in the function of platelet beta 3 integrin, glycoprotein (GP) IIb-IIIa complex. IAP was a widely distributed protein and was also expressed in the cells that do not have beta 3 integrin. Platelets from a patient with thrombasthenia, which lack GPIIb and IIIa, expressed IAP as well as normal platelets. Neither platelet aggregation nor intracellular Ca2+ elevation after stimulation was influenced by the anti-IAP antibody, B6H12, which was reported to be inhibitory for other beta 3 integrins. The expression level of GPIIb-IIIa complex was not influenced by coexpression of human IAP in the transfected Chinese hamster ovary (CHO) cells. IAP did not facilitate the binding of soluble fibrinogen to the CHO cells expressing GPIIb-IIIa complex. Furthermore, cell adhesion onto the immobilized fibrinogen via GPIIb-IIIa complex was not inhibited by B6H12 in HEL cells and was not altered by coexpression of human IAP in CHO cells. We concluded that expression of IAP is regulated independently with that of GPIIb-IIIa complex and that IAP does not influence the function of GPIIb-IIIa complex.

Maxa, A New Low-Frequency Platelet-Specific Antigen Localized on Glycoprotein IIb, Is Associated With Neonatal Alloimmune Thrombocytopenia

We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti-Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse-transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA-3 allele.

Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia

We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti- Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse- transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA- 3 allele.

Gly-Pro-Arg-Pro (GPRP) enhances free thrombin

Fibrinogen-related peptides, which inhibit the interaction of fibrinogen with the platelet membrane glycoprotein IIb IIIa complex ( GPIIb IIIa ) are under preclinical investigation now (1). Whereas peptides containing the Arg-Gly-Asp (RGD) sequence inhibit fibrinogen-dependent platelet aggregation by direct binding to GPIIb IIIa (2), GPRP inhibits fibrinogen polymerisation by direct binding to the fibrinogen polymerisation sites and modifying the glutamine residues in the α- and γ-chains of fibrinogen (3, 4). Since GPRP has been shown to inhibit ADP-induced platelet aggregation it has been suggested as antithrombotic agent (5). It has been demonstrated that in defibrinated plasma the amount of free thrombin generated after clotting activation is significantly higher than in normal plasma (6). The explanation for this observation is that in normal plasma free thrombin is partially adsorbed on generated fibrin (7). Since GPRP inhibits fibrinogen polymerisation, we investigated the generation of thrombin in platelet-rich plasma (PRP) containing GPRP.

An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.

Much discussion has concerned the central role of ADP in platelet aggregation. We now describe a patient (M.L.) with an inherited bleeding disorder whose specific feature is that ADP induces a limited and rapidly reversible platelet aggregation even at high doses. Platelet shape change and other hemostatic parameters were unmodified. A receptor defect was indicated, for, while epinephrine normally lowered cAMP levels of PGE1-treated (M.L.) platelets, ADP was without effect. The binding of [3H]2-methylthio-ADP decreased from 836 +/- 126 molecules/platelet for normals to 30 +/- 17 molecules/platelet for the patient. Flow cytometry confirmed that ADP induced a much lower fibrinogen binding to (M.L.) platelets. Nonetheless, the binding in whole blood of activation-dependent monoclonal antibodies showed that some activation of GP IIb-IIIa complexes by ADP was occurring. Platelets of a patient with type I Glanzmann's thrombasthenia bound [3H]2-methylthio-ADP and responded normally to ADP in the presence of PGE1. Electron microscopy showed that ADP-induced aggregates of (M. L.) platelets were composed of loosely bound shape-changed platelets with few contact points. Thus this receptor defect has a direct influence on the capacity of platelets to bind to each other in response to ADP.

A comparison of the kinetics of thrombin- and adenosine diphosphate-induced aggregation of human platelets in poiseuille flow

The molecular basis of platelet-fibrin binding has been elucidated by studying interactions between platelets and protofibrils, soluble two-stranded polymers of fibrin which are intermediates on the fibrin assembly pathway. The fibrinogen degradation product, fragment D, has been used to block fibrin assembly, thus enabling the preparation of stable solutions of short protofibrils, composed of fewer than twenty fibrin monomer molecules per polymer. Fibrin protofibrils bound to ADP-activated platelets in a time- and concentration-dependent process which was effectively blocked by excess unlabelled fibrinogen, i.e., the binding was specific and appeared to involve a common receptor. ADP-stimulated cells bound approx. 3 μg of fibrin protofibrils/108 platelets, compared to 4 μg of fibrinogen/108 cells, following a 30-min incubation period at room temperature. Binding of both ligands was inhibited by high concentrations of fragment D, further indicating a similar mechanism. The kinetic data obtained were well described by an apparent first-order mechanism in which the rate constant for fibrin protofibril binding was found to be 5-fold slower than that measured for fibrinogen. Two monoclonal antibodies, each directed against the platelet glycoprotein IIb-IIIa complex, inhibited the binding of fibrin protofibrils and fibrinogen in a similar, concentration-dependent manner, providing strong evidence for a common receptor. Binding of GPRP-fibrin (soluble fibrin oligomers formed in the presence of 1 mM Gly-Pro-Arg-Pro) to ADP-stimulated platelets was also inhibited by a monoclonal antibody directed against the GPIIb-IIIa complex. Neither fibrin protofibrils nor fibrinogen bound to Glanzmann's thrombasthenic platelets, which lack normal quantities of functional glycoprotein IIb-IIIa complex, further supporting the hypothesis that fibrinogen and fibrin bind to a common platelet receptor present on the glycoprotein IIb-IIIa complex.

Mechanisms Involved in Platelet Vessel Wall Interaction

The molecular mechanisms underlying formation of platelet thrombi constitute one of the central problems in vascular biology and medicine. Fibrinogen binding to its platelet integrin receptor alpha IIb beta 3 (glycoproteins IIb-IIIa complex) plays a pivotal role in formation of platelet thrombi by providing molecular bridges spanning platelets and by contributing to its receptor-mediated outside-in signaling. Conversely, fibrinogen binding to integrin alpha IIb beta 3 requires inside-out signaling mediated by intraplatelet signal transducers activated in response to external signals generated at the site of vascular injury. This tightly regulated fibrinogen-integrin alpha IIb beta 3 interaction constitutes a fundamental platelet mechanism of response to vascular injuries such as accidental and surgical wounds, rupture of atherosclerotic plaques in coronary and cerebral atherosclerosis and microvascular endothelial desquamation in septic shock. After mapping the fibrinogen site on the gamma chain responsible for recognition of platelet integrin alpha IIb beta 3 and the development of synthetic peptide inhibitors, we solved the 3D structure of the carboxy-terminal segment of the human fibrinogen gamma chain using our new technology of carrier protein-driven crystallization. Fundamental knowledge concerning a molecular 3D model of fibrinogen-integrin alpha IIb beta 3 interaction, coupled to the mechanism of its inside-out and outside-in regulation, will lead to development of a new generation of platelet-selective antithrombotic drugs.

Regulation of platelet anchorage by integrins

Interactions between human blood platelets and fibrin have been visualized by light microscopy, and quantitative details of the extent, rate and specificity of fibrin-platelet binding obtained by microfluorimetry. Adhesion of fluorescein-labelled fibrin to activated platelets yielded brightly fluorescent fibrin-platelet aggregates, which emitted light at an intensity 4–7-fold greater than that due to nonspecific association of fluorescein-fibrin with unstimulated fixed cells. The intensity of fluorescent light emitted from fibrin-platelet aggregates increased as a function of time, reaching a plateau after about 1 h under physiological buffer and temperature conditions. Two monoclonal antibodies directed against the glycoprotein IIb-IIIa complex, which have been shown to inhibit the binding of fibrinogen and soluble fibrin oligomers to ADP-stimulated platelets, were employed to further probe the specificity of this adhesive interaction. In contrast to the results with the soluble ligands, one of these antibodies (HP1-1D) was capable of fully inhibiting the attachment of fluorescent fibrin to ADP-activated cells while the other (AP-2) was much less effective.

Detection of biotinylated proteins in crossed immunoelectrophoresis gels: studies on platelet membrane receptors and microparticles.

Biotinylation can be used as an alternative for surface labeling of cell membrane proteins. The use of the water soluble N-hydroxysulfosuccinimide (NHSS)-biotin or the more lipophilic N-hydroxysuccinimide (NHS)-biotin reagent has been investigated in the present study labeling two central receptor complexes on the platelet surface, i.e. the glycoprotein (GP) Ib-IX and the GP IIb-IIIa complexes involved in platelet adhesion and aggregation. Lack of labeling of the intracellularly located albumin was used as a negative control. The labeling has been studied using crossed immunoelectrophoresis in the PhastSystem format after extraction of the labeled cells in Triton X-100, and it is shown that, using enzyme-conjugated avidin and chromogenic substrates, the biotinylated proteins can be visualized directly in the dried electrophoresis gel without the need for a transfer to a blotting membrane as is used after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Suitable conditions for biotinylation and for visualization in the crossed immunoelectrophoresis gels are described. Further, surface-biotinylation of platelets was used to observe shedding of microparticles as a consequence of formation of the complement membrane attack complex. For this purpose the formation and composition of the biotinylated microparticles were observed by flow cytometry and crossed immunoelectrophoresis.

Inhibition of platelet aggregation by monoclonal antibodies against glycoprotein IIb–IIIa complex

Monoclonal antibodies CRC64 are obtained against Ca2+-dependent glycoprotein IIb–IIIa complex of the platelet membrane which possess the ability to inhibit completely fibrinogen-dependent platelet aggregation. CRC64 is directed against the epitope formed by the glycoprotein IIb–IIIa complex and does not interact with proteins isolated after platelets are treated with ethylenediamine tetraacetate. Complete, reproducible blockade of platelet aggregation caused by 5 μM adenosine diphosphate is noted in an MCA concentration of 3 μg/ml, while in the case of a stronger inductor, namely 1 U/ml thrombin, platelet aggregation is inhibited in a concentration of 5 μg/ml. F(ab′)2 fragments are also able to inhibit platelet aggregation completely and are usually effective in concentrations lower than native monoclonal antibodies.

Phosphorylation of Focal Adhesion Vasodilator‐Stimulated Phosphoprotein at Ser157 in Intact Human Platelets Correlates with Fibrinogen Receptor Inhibition

Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin alpha IIb beta 3) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, cAMP-elevating agents [e.g. prostaglandin E1 and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of cAMP- and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet cAMP- or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.

Human platelet alloantigen typing: PCR analysis is not a substitute for serological methods.

Currently, five platelet alloantigen (alloAg) systems have been established (HPA-1, -2, -3, -4, -5). Three of these are expressed on the glycoprotein (GP) IIb-IIIa complex, HPA-1, HPA-3 and HPA-4, inherited in an autosomal codominant mode. Recent investigations of the molecular basis of these platelet alloantigen systems have shown that only one nucleic acid base substitution in the genes encoding for GP IIb and GP IIIa is responsible for the polymorphism. This substitution is reflected in a difference in restriction enzyme recognition allowing platelet alloantigen typing by restriction fragment length polymorphism (RFLP) analysis of DNA amplified by the polymerase chain reaction (PCR). To validate the PCR technology for platelet typing, we have compared PCR-RFLP with monoclonal-antibody-specific immobilization of platelet antigens (MAIPA). For this purpose, we have studied different Glanzmann thrombasthenic families and particularly heterozygous individuals, who are not lacking GP IIb-IIIa, as a model to detect the occurrence of discrepancies between these two technologies. In two families, we have found differences between molecular biology and serological methods with the lack of expression of one antigen on the platelet membrane surface. In the first family, the abnormality is related to the HPA-1 alloantigen system with three informative members; in the second, the HPA-3 alloantigen system is concerned with two informative members. Considering these results, there may not always be a perfect correlation between molecular biology and serological methods, as an unknown molecular defect could interfere with the PCR results and lead to false platelet typing.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of glycoprotein IIb as the lipoprotein(a)-binding protein on platelets. Lipoprotein(a) binding is independent of an arginyl-glycyl-aspartate tripeptide located in apolipoprotein(a).

Lipoprotein(a) [Lp(a)] plays an important role in atherosclerosis. The amino acid sequence of apolipoprotein(a) [apo(a)] reveals an arginyl-glycyl-aspartate (RGD) tripeptide that is the consensus sequence for binding of adhesive plasma proteins of the fibrinolytic system, such as fibrinogen and von Willebrand factor, to the platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex. Therefore, we undertook the present study to further investigate the role of Lp(a) in hemostasis. Binding of 125I-Lp(a) to a single platelet membrane-associated protein (137 +/- 6 kD) comigrating with platelet GPIIb (140 kD) was found to be specific, saturable, and Ca2+ independent. Binding of 125I-Lp(a) to resting human blood platelets was saturable, insensitive to temperature, and independent of the apo(a) isoform (B, S1 through S3). Scatchard analysis revealed a Kd of 7.2 +/- 1.8 x 10(-9) mol/L, with 729 +/- 313 Lp(a) molecules bound per platelet. Monoclonal anti-GPIIb IgG diminished Lp(a) binding by approximately 80%, monoclonal anti-GPIIb-IIIa IgG by 60%, and anti-GPIIIa IgG by just 15%. 125I-Lp(a) binding was competitively inhibited to the same extent by either unlabeled Lp(a) or fibrinogen. Low- and high-density lipoproteins were much weaker competitors. A polyclonal antibody raised against the RGDGQSYRGT sequence of apo(a) was used to verify the presence of an RGD sequence in the different Lp(a) preparations investigated. However, two lines of evidence indicated that the RGD sequence is not the binding domain mediating Lp(a) binding to platelets. First, incubation of platelets with isolated RGD tripeptide did not influence Lp(a) binding.(ABSTRACT TRUNCATED AT 250 WORDS)