Trigramin. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex

Trigramin, a highly specific inhibitor of fibrinogen binding to platelet receptors, was purified to homogeneity from Trimeresurus gramineus snake venom. Trigramin is a single chain (approximately 9 kDa) cysteine-rich peptide with the Glu-Ala-Gly-Glu-Asp-Cys-Asp-Cys-Gly-Ser-Pro-Ala NH2-terminal sequence. Chymotryptic fragmentation showed the Arg-Gly-Asp sequence in trigramin. Trigramin inhibited fibrinogen-induced aggregation of platelets stimulated by ADP (IC50 = 1.3 X 10(-7)M) and aggregation of chymotrypsin-treated platelets. It did not affect the platelet secretion. Trigramin was a competitive inhibitor of the 125I-fibrinogen binding to ADP-stimulated platelets (Ki = 2 X 10(-8) M). 125I-Trigramin bound to resting platelets (Kd = 1.7 X 10(-7) M; n = 16,500), to ADP-stimulated platelets (Kd = 2.1 X 10(-8) M; n = 17,600), and to chymotrypsin-treated platelets (Kd = 8.8 X 10(-8) M; n = 13,800) in a saturable manner. The number of 125I-trigramin binding sites on thrombasthenic platelets amounted to 2.7-5.4% of control values obtained for normal platelets and correlated with the reduced number of GPIIb-GPIIIa molecules on the platelet surface. EDTA, monoclonal antibodies directed against the GPIIb-GPIIIa complex, and synthetic peptides (Arg-Gly-Asp-Ser and Tyr-Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val) blocked both 125I-fibrinogen binding and 125I-trigramin binding to platelets. Fibrinogen binding was more readily inhibited by these compounds than was trigramin binding. Monoclonal antibodies directed either against GPIIb or GPIIIa molecules did not block the interaction of either ligand with platelets. Reduced, S-pyridylethyl, trigramin did not inhibit platelet aggregation and fibrinogen binding to platelets and it did not bind to platelets, suggesting that the secondary structure of this molecule is critical for expression of its biological activity.     

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.     

Association of fibrin with the platelet cytoskeleton

We have previously postulated that surface membrane proteins become specifically associated with the internal platelet cytoskeleton upon platelet activation (Tuszynski, G.P., Walsh, P.N., Piperno, J., and Koshy, A. (1982) J. Biol. Chem. 257, 4557-4563). Four lines of evidence are in support of this general hypothesis since we now show that platelet surface receptors for fibrin become specifically associated with the platelet Triton-insoluble cytoskeleton. 1) Fibrin was detected immunologically in the washed Triton-insoluble cytoskeletons of thrombin-activated platelets under conditions where fibrin polymerization and resultant precipitation was blocked with Gly-Pro-Arg-Pro, a synthetic peptide that inhibits polymerization of fibrin monomer. 2) Radiolabeled fibrin bound to thrombin-activated platelets and became associated with the cytoskeleton. 3) The amount of radiolabeled fibrin bound to thrombin-activated thrombasthenic platelets and their cytoskeletons amounted to about 20% of the fibrin bound to thrombin-activated control platelets and their cytoskeletons. 4) The association of fibrin with cytoskeletons and with the platelet surface was nearly quantitatively blocked by an antibody prepared against cytoskeletons (anti-C), an antibody against isolated membranes of Pronase-treated platelets (anti-M1), and a monoclonal antibody to the platelet surface glycoprotein complex, GPIIb-GPIII (anti-GPIII). These antibodies blocked ADP and thrombin-induced platelet aggregation as well as thrombin-induced clot retraction. Analysis of the immunoprecipitates obtained with anti-C, anti-M1, and anti-GPIII from detergent extracts of 125I-surface labeled platelets revealed that these antibodies recognized GPIIb-GPIII. These data suggest that thrombin activation of platelets results in the specific association of fibrin with the platelet cytoskeleton, that this association may be mediated by the GPIIb-GPIII complex, and that these mechanisms may play an important role in platelet aggregation and clot retraction induced by thrombin.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Activation of the fibrinogen receptor on human platelets exposed to alpha chymotrypsin. Relationship with a major proteolytic cleavage at the carboxyterminus of the membrane glycoprotein IIb heavy chain

The serine proteinase alpha chymotrypsin from bovine pancreas (CT) is known to expose fibrinogen binding sites on the surface of human platelets in the absence of cell activation and granular secretion. This is accompanied by the appearance of membrane-bound chymotryptic fragments of both glycoprotein (GP) IIb and GPIIIa, the two subunits of the platelet fibrinogen receptor, the GPIIb-IIIa complex. However, no clear relationship between discrete proteolytic event(s) within GPIIb-IIIa and fibrinogen-binding-site expression has yet been established. We have now evaluated the proteolysis of GPIIb-IIIa by CT by Western blot analyses using a panel of polyclonal and monoclonal antibodies against GPIIb or GPIIIa. The different proteolytic events were then correlated with the kinetics of the expression of active fibrinogen binding sites on platelets, as measured through the binding of 125I-labelled purified fibrinogen and to the capacity of CT-treated platelets to aggregate. Treatment of platelets with CT at 22 degrees C resulted in the expression of fibrinogen binding sites prior to cleavage of GPIIIa (Mr approximately 90,000) into a previously described, major membrane-bound fragment with Mr 60,000. In contrast, fibrinogen receptor expression closely paralleled a proteolytic cleavage at the carboxy terminus of the GPIIb heavy chain (Mr approximately 120,000), which was converted into a faster migrating species with Mr approximately 115,000). This proteolysis resulted in the release of a soluble peptide with an expected molecular mass of less than 3.7 kDa. Quantitation of this peptide using a competitive immunoenzymatic assay, confirmed that its release from the platelet surface correlated with the expression of fibrinogen binding sites and aggregability. When platelets were exposed to CT at 37 degrees C, a prompt increase in fibrinogen binding sites and platelet aggregability was observed, whereas the GPIIb heavy chain was rapidly converted into the carboxy-terminal-cleaved form. However, incubation at 37 degrees C for longer than 10 min resulted in extensive and simultaneous degradation of both the GPIIb heavy and light chains and of GPIIIa, with the latter being converted into the 60-kDa fragment. These later events were associated with a sharp decline of platelet aggregability and a reduction in the number of fibrinogen binding sites. These data allow us to propose that an early and limited proteolytic processing of the GPIIb component of the platelet fibrinogen receptor is associated with a shift of this receptor complex into a state which expresses specific binding sites for fibrinogen. Further cleavage of GPIIIa to generate the 60-kDa fragment results in loss of receptor activity.     

Evidence that monoclonal antibodies against CD9 antigen induce specific association between CD9 and the platelet glycoprotein IIb-IIIa complex

Monoclonal antibodies to the CD9 antigen are powerful platelet agonists. We report here the novel finding that the anti-CD9 monoclonal antibodies 50H.19 and ALB6 promote physical association between CD9 antigen and the glycoprotein IIb-IIIa complex (GPIIb-IIIa) component of the platelet fibrinogen receptor. The monoclonal antibodies do not consistently immunoprecipitate proteins other than CD9 from 125I-labeled human platelets even if the platelets are first treated with the homobifunctional cross-linking reagent dithiobis(succinimidyl propionate), indicating that CD9 antigen is not physically associated with other membrane proteins in the resting state. However, the addition of agonistic concentrations of either monoclonal antibody before cross-linking results in the coprecipitation of proteins corresponding in mobility and peptide composition to GPIIb, and GPIIIa. The association of CD9 with the GPIIb-IIIa complex is unaffected by a combination of aspirin and ADP scavengers sufficient to abrogate anti-CD9 monoclonal antibody-induced platelet aggregation, and is therefore not dependent upon thromboxane- and ADP-mediated pathways of intracellular signalling. The specificity of the association is demonstrated by the lack of other coprecipitating major proteins, by the requirement for induction by anti-CD9 monoclonal antibodies, and by the failure to promote reciprocal association with either of the anti-GPIIb-IIIa complex monoclonal antibodies P2 or HuP1-m1a.     

Platelet autoantigens: identification and characterization using immunoblotting

Antiplatelet autoantibodies are important in the etiology of idiopathic (or immune) thrombocytopenic purpura (ITP). Studies using immunoblotting techniques have been helpful in identifying the antigenic target proteins for the antibodies. Antibodies against the glycoprotein (GP) IIIa portion of the GPIIb/IIIa complex were the first to be demonstrated by this approach. Similar GPIIIa autoantigens have also been found to be the most frequent targets of ITP antibodies. Not all anti-GPIIIa antibodies are directed against the same epitope on GPIIIa. A subset of anti-GPIIIa antibodies found in patients with an acquired qualitative platelet dysfunction actually interfere with fibrinogen binding to normal platelets. Antibodies directed against targets on GPV have been found in patients with acute ITP of childhood. In patients with ITP associated with lupus erythematosus, antibodies which bind to intracellular proteins of apparent molecular weights of 66 and 108 kDa have been detected. Thus, ITP antibodies can have a variety of target antigens. Study of larger series of patients will determine whether identification of platelet autoantigens correlates with clinical course of ITP.     

Reversal of platelet aggregation by aortic microsomes

The microsomal fraction of dog aortas inhibited human platelet aggregation induced by arachidonic acid, ADP, or thrombin. When aortic microsomes were added to a preparation of irreversibly aggregated platelets, the aggregates dispersed after 4–6 minutes. The fact that aortic microsomes inhibit platelet aggregation induced by ADP suggests that its effect is probably on the cellular function of platelets and not in direct competition against thromboxane A₂.     

Distinction between glycoprotein IIIa and the 100-kDa membrane protein (aggregin) mediating ADP-induced platelet activation

Previous studies from our laboratories showed that 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) inhibits ADP-induced platelet shape change, aggregation, and exposure of fibrinogen sites while covalently binding to 100-kDa platelet membrane protein (aggregin) on the intact platelet. Chymotrypsin digests aggregin to a fragment of 70 kDa, abolishing the inhibition, and also cleaves platelet glycoprotein IIIa (GPIIIa) (100 kDa) to a 70-kDa fragment containing the P1A1 epitope. We questioned whether these platelet membrane proteins were distinct. Both 5'-p-[3H]sulfonylbenzoyl adenosine (SBA)-labeled aggregin and 125I-GPIIIa were precipitated by polyclonal antibodies to a 100-kDa fraction of platelet membranes, but aggregin was not precipitated by a monospecific antibody to P1A1 which precipitates GPIIIa. Further a monospecific polyclonal antibody to immunopurified GPIIIa coupled to protein A-Sepharose adsorbed GPIIIa but not aggregin. Similarly, both aggregin and GPIIIa were precipitated by a polyclonal antibody to an isolated 70-kDa component of platelet membrane but only GPIIIa was precipitated by the monoclonal antibody to GPIIIa, (SSA6). Two patients with Glanzman's thrombasthenia whose platelet membranes contained less than 5% GPIIIa as assayed by monoclonal antibody binding (A2A6), incorporated [3H]SBA to the same extent as normal individuals. Furthermore, FSBA inhibited ADP-induced shape change with a similar concentration dependence for both thrombasthenic and normal platelets. Finally, mobility of GPIIIa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was decreased following reduction with dithiothreitol whereas that of [3H]SBA-labeled MP 100 was not altered. We conclude that GPIIIa and aggregin are distinct platelet membrane proteins.     

Characterization of a monoclonal antibody which is an activator of rabbit platelets

A monoclonal antibody (MAb) raised against rabbit platelet membranes was shown to be a strong agonist to induce platelet aggregation and secretion. This MAb, designated 19CB-1, was identified as an IgM and purified to near homogeneity by ammonium sulfate precipitation and Q-sepharose column chromatography. Aggregation induced by 19CB-1 was only slightly affected in the presence of creatine phosphate/creatine phosphokinase and aspirin, indicating that it was not mediated through the cyclooxygenase pathway and the release of ADP. 19CB-1 Fab fragments did not induce platelet aggregation. However, 19CB-1-induced aggregation was inhibited by these Fab fragments. 19CB-1 also elicited a rise in cytoplasmic calcium concentration in fura-2 loaded platelets. In the absence of external calcium, a substantial calcium signal remained to be observed, suggesting the release of calcium from intracellular stores in response to 19CB-1. This MAb reacted primarily with a polypeptide of Mr = 57,000, as revealed by immunoblotting. These results suggest that the 57 kDa antigen is one of the platelet surface proteins directly involved in the activation of rabbit platelets.     

Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin

We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49-66. Surprisingly, binding avidity for GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99-102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution.     

Synthesis and use of a new spin-labeled analogue of ADP with platelet-aggregating activity.

A new spin-labeled derivative of ADP, 2-(4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl)thioadenosine-5'-diphosphate, has been synthesized. The compound causes both the reversible and irreversible phases of aggregation of human blood platelets at concentrations similar to those required for similar phases of aggregation by ADP itself. The spin-labeled ADP also rivals ADP as a substrate for pyruvate kinase. The interaction of intact human blood platelets and of isolated platelet membranes with the platelet-aggregating spin-labeled derivatives of ADP has been studied. The dramatic decrease in the ESR signal of the spin label is primarily due to chemical reduction of the nitroxide, rather than immobilization of the label. When platelets and spin-labeled ADP are mixed, a rapid burst of nitroxide reduction occurs, followed by a much slower reduction similar in time course to that seen for other spin labels. The rapid burst of reduction, but not the slow reduction, is inhibited by adenosine, an inhibitor of ADP-induced platelet aggregation, and by sulfhydryl-blocking agents. Experiments conducted with Ellman's reagent and platelet membranes or washed platelets revealed a 10 to 30% increase in the number of reactive membrane sulfhydryl groups when ADP was present. These results indicate that there is an increase in the number of reactive sulfhydryl groups on the platelet surface when platelets or membranes are stimulated by ADP.     

Dependence of the thrombocyte aggregation reaction on plasma fibronectin and the tripeptide arginyl-glycyl-asparagilin

If was shown that the addition of fibronectin antibodies exerted the inhibition of platelet aggregation. The tripeptide RGD inhibited the platelet aggregation induced by the same agents (ADP, epinephrine, thrombin, collagen) both in blood plasma and in suspension of washed platelets.     

Purinergic Receptors in Human Blood Platelets: Chemical Modification and Cloning Investigations

Platelet aggregation is important for maintaining normal hemostasis. However, aberrant platelet aggegation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is one of the earliest and most important platelet agonists. ADP induces platelet aggregation, shape change, secretion, influx and intracellular mobilization of Ca²⁺, and inhibition of the adenylyl cyclase stimulated by prostaglandins. Binding of ADP to purinergic receptor(s) is required for elicitation of the ADP-induced platelet responses. But the platelet ADP receptor(s) has not been purified, largely due to the unavailability of the reagents that can be used to selectively label the platelet ADP receptor. The ADP receptor responsible for the ADP-induced platelet aggregation and inhibition of stimulated adenylyl cyclase activity has not been cloned due to difficulties in screening responsive clones generated from a cDNA library. Since the purified ADP-receptor protein is not available, antibodies that can be used as alternative tools to purify the ADP receptor or screen the clones expressing the receptor could not be made. In addition, the problem may be compounded by the low copy number and the susceptibility of the receptor to proteolysis. Therefore, signal transduction mechanisms underlying biochemical transformations in ADP-induced platelet responses remain less well defined and/less well understood. In the past decade efforts have been made to identify a platelet ADP receptor(s) by photoaffinity as well as affinity labeling by the ADP-affinity analogs. More recently efforts have been directed to clone the platelet ADP receptors. These investigations, however, have not produced definite results. The purpose of this review is to examine the results obtained by the photoaffinity- and affinity-labeling investigations and cloning experiments to identify a platelet ADP receptor(s).     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

Interrelation of platelet aggregation, release reaction and thromboxane A2 production

Aggregation of platelets, stimulated by different agonists, was inhibited by omitting sample stirring or by preincubation of platelets with a monoclonal antibody against glycoproteins IIb-IIIa or with a pentapeptide containing the sequence Arg-Gly-Asp-Ser. In platelets stimulated by collagen, ADP and epinephrine, the inhibition of aggregation paralleled a reduction of both release reaction and thromboxane A2 formation. When thrombin was the stimulus, ATP release and thromboxane A2 production were unaffected (or only slightly modified) by the inhibition of platelet aggregation. These data add further evidence to the hypothesis that aggregation supports the activation of platelets stimulated by weak agonists.     

Platelet ADP receptor and alpha 2-adrenoreceptor interaction. Evidence for an ADP requirement for epinephrine-induced platelet activation and an influence of epinephrine on ADP binding

The nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) is a potent irreversible inhibitor of ADP-mediated platelet activation. Utilizing this compound, the role of ADP in epinephrine-mediated platelet activation was evaluated. Pretreatment of platelets with FSBA under conditions producing covalent incorporation was able to completely block epinephrine-stimulated aggregation of human platelets. In addition, the exposure of latent fibrinogen-binding sites by epinephrine was also inhibited in platelets modified by FSBA. The inhibition of epinephrine-mediated activation of the cells was time dependent, reflecting the need for covalent modification of the ADP receptor by FSBA. The inhibitory effect of FSBA was not due to effects on the affinity of binding methyl [3H]yohimbine or the number of platelet alpha 2-adrenergic receptors. Studies of the effect of epinephrine on the ability of ADP to protect against FSBA incorporation demonstrated that epinephrine can increase the affinity of ADP for its receptor 10-fold without affecting the total amount of FSBA covalently bound. This effect of epinephrine is mediated through the alpha 2-adrenoreceptor since the effect can be reversed by the competitive antagonist, methyl yohimbine. These results suggest that promotion of platelet aggregation and the exposure of fibrinogen receptors by epinephrine is dependent on ADP. The mechanism by which epinephrine renders low concentrations of ADP effective appears to be mediated by an increased avidity of the ADP receptor for the nucleotide.     

Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets

A chemical cross-linking approach has been used to characterize the interaction of platelets with small peptides of 7 and 14 residues containing the arginyl-glycyl-aspartic acid (RGD) sequence recognized by a variety of cellular adhesion receptors. The radioiodinated peptides were bound to platelets, and chemical cross-linking was attained by subsequent addition of bifunctional reagents. Three different cross-linking reagents coupled the RGD-containing peptides to platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa), and both subunits of this platelet membrane glycoprotein became radiolabeled with the RGD peptides. Platelet stimulation with agonists including thrombin, phorbol myristrate acetate, and ADP increased the extent of cross-linking by predominantly enhancing the coupling of the RGD peptides to the GPIIIa subunit. Cross-linking of the labeled RGD peptides to GPIIb and GPIIIa on stimulated and nonstimulated platelets exhibited structural specificity and was inhibited by excess nonlabeled RGD peptides. The interactions were inhibited by nonlabeled RGD peptides and a peptide with an amino acid sequence corresponding to the carboxyl terminus of the gamma chain of fibrinogen but less effectively by an arginyl-glycyl-glutamic acid peptide. Cross-linking of the RGD peptides to GPIIb-IIIa was divalent ion-dependent and, on stimulated platelets, was inhibited by the adhesive proteins fibrinogen and fibronectin, but not by albumin. These results indicate that the RGD-binding sites on platelets reside in close proximity to both subunits of GPIIb-IIIa and that platelet stimulation alters the topography of these sites such that the peptides become more efficiently cross-linked to GPIIIa.     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C_8 reverse phase HPLC. The purified activity, named longieornin, is a protein of moleeular weight 16 000 on SDS-PAGE under both reduced and nonredueed conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC_(50) was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effeetors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longieonin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intraeellar Ca~(2 ) level of platelets in response to collagen were com     

Purification and identification of the lipoprotein-binding proteins from human blood platelet membrane

As reported previously, homologous plasma lipoproteins specifically bind to the plasma membrane of human blood platelets. The two major lipoprotein-binding membrane glycoproteins were purified to apparent homogeneity and identified by their mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both in the nonreduced and reduced state, by specific antibodies against glycoproteins IIb (GPIIb) and IIIa (GPIIIa), respectively, including the alloantibody anti-PlA1 and monoclonal antibodies. Furthermore, lipoprotein binding to intact platelets is also inhibited in a dose-dependent fashion by preincubation of the platelets with antibodies against these glycoproteins. From these experiments it can be concluded that lipoproteins bind to both components of the glycoprotein IIb-IIIa complex in isolated membranes and intact platelets. High density lipoprotein and low density lipoprotein bind to GPIIIa blotted to nitrocellulose in a way that binding of one species interferes with the binding of the other. Addition of fibrinogen significantly inhibits this binding. The specific binding of fibrinogen to GPIIIa is strongly inhibited in the presence of either of the two lipoproteins. LDL and HDL are specifically bound by isolated GPIIb, too. In our blotting experiments fibrinogen shows no binding to this membrane glycoprotein. On the other hand, fibrinogen significantly interferes with the interaction between GPIIb and the lipoproteins.