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)     

The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit

The extreme carboxyl-terminal amino acid sequence of the gamma chain of fibrinogen is involved in the binding of this adhesive protein to the platelet integrin glycoprotein (GP) IIb-IIIa, and synthetic peptides corresponding to this region inhibit fibrinogen as well as fibronectin and von Willebrand factor binding to platelets. A chemical cross-linking approach was used to characterize the interaction of a 16-amino acid fibrinogen gamma chain peptide with platelets and to localize the site of its binding to GPIIb-IIIa. This peptide became specifically cross-linked to GPIIb, and platelet stimulation selectively enhanced its cross-linking to this alpha subunit. The cross-linking reaction was specifically inhibited by fibrinogen and an Arg-Gly-Asp peptide but not by an unrelated protein or a substituted peptide. Utilizing a combination of immunochemical mapping, enzymatic and chemical digestions, and amino acid sequencing, the cross-linking site of the gamma chain peptide in GPIIb was localized to a stretch of 21 amino acids. The identified region, GPIIb 294-314, contains the second putative calcium binding domain within GPIIb. The primary structure of this region is highly conserved among alpha subunits of other integrin adhesion receptors. These results identify a discrete region of GPIIb that resides in close proximity to a ligand binding site within GPIIb-IIIa. The homologous region may be involved in the functions of other integrin receptors.     

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.     

Inhibition of fibrinogen binding to human platelets by S-nitroso-N-acetylcysteine

Because of the central role of fibrinogen binding in platelet aggregation and recent evidence implicating S-nitrosothiol compounds in the platelet inhibitory effects of endogenous and exogenous organic nitrate compounds, we examined the effect of the S-nitrosothiol S-nitroso-N-acetylcysteine (SNOAC) on fibrinogen binding to gel-filtered human platelets. We found that SNOAC markedly inhibited the binding of fibrinogen to normal human platelets in a dose-dependent fashion and that this inhibitory effect was the result of both an increase in the apparent Kd of the platelet receptor for the fibrinogen molecule (from 6.8 x 10(-7) to 1.8 x 10(-6) M, a 2.7-fold increase) and a decrease in the total number of fibrinogen molecules bound to the platelet (from 76,200 to 38,250, a 50% decrease). In addition, we noted a rapid, dose-dependent rise in platelet cyclic GMP levels following exposure of platelets to SNOAC which was significantly inversely correlated with fibrinogen binding and was accompanied by inhibition of intracellular calcium flux in response to a variety of platelet agonists. Similar dose-dependent inhibition of fibrinogen binding was found in the presence of cyclic GMP analogues and was significantly enhanced by inhibition of platelet cyclic GMP phosphodiesterase. These results describe the inhibition of platelet fibrinogen binding by an S-nitrosothiol compound, help define the biochemical mechanism by which S-nitrosothiols inhibit platelet aggregation, and lend support to the view that cyclic GMP is an important inhibitory intracellular mediator in human platelets.     

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.     

Characteristics of collagen-induced fibrinogen binding to human platelets

Polymerized type I calf skin collagen induced a time-dependent specific binding of 125I-fibrinogen to washed human platelets. Binding occurred more rapidly in a shaken rather than in an unstirred system. It was linear in the range 0.05-0.3 microM added fibrinogen and was saturated at higher fibrinogen concentrations (more than 0.8 microM). Scatchard analysis showed a single population of binding sites (16530 +/- 5410 per platelet) with a Kd = 0.53 +/- 0.23 microM. Collagen-induced 125I-fibrinogen binding to platelets was completely inhibited by ADP antagonists such as creatine phosphate/creatine phosphokinase and AMP, and partially inhibited by pretreatment of the platelets with aspirin. With both normal and aspirin-treated platelets a close correlation was observed between the amount of 125I-fibrinogen bound and the extent of dense granule secretion. Our results confirm that fibrinogen becomes bound to platelet surface receptors during collagen-induced platelet aggregation and suggest that secreted ADP is an essential cofactor in this process.     

Mechanism of action of the antiplatelet peptide, arietin, from Bitis arietans venom

Arietin, an Arg-Gly-Asp containing peptide from venom of Bitis arietans, inhibited aggregation of platelets stimulated by a variety of agonists with a similar IC50, 1.3-2.7.10(-7) M. It blocked aggregation through the interference of fibrinogen binding to fibrinogen receptors on platelet surface. In this paper, we further demonstrated that arietin had no significant effect on the intracellular mobilization of Ca2+ in Quin2-AM-loaded platelets stimulated by thrombin. It inhibited 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50, 1.1.10(-7) M). 125I-arietin bound to unstimulated, ADP-stimulated and elastase-treated platelets in a saturable manner and its Kd values were estimated to be 3.4.10(-7), 3.4.10(-8) and 6.5.10(-8) M, respectively, while the corresponding binding sites were 46,904, 48,958 and 34,817 per platelet, respectively. Arg-Gly-Asp-Ser (RGDS) inhibited 125I-arietin binding to ADP-stimulated platelets in a competitive manner. RGD-containing peptides, including trigramin and rhodostomin, EDTA and monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex, inhibited 125I-arietin binding to ADP-stimulated platelets, indicating that the binding sites of arietin appear to be located at or near glycoprotein IIb-IIIa complex. In conclusion, arietin and other RGD-containing trigramin-like peptides preferentially bind to the fibrinogen receptors associated with glycoprotein IIb-IIIa complex of the activated platelets, thus leading to the blockade of fibrinogen binding to its receptors and subsequent aggregation. The presence of RGD of arietin is essential for the expression of its biological activity. Its binding sites are overlapped with those of trigramin, rhodostomin and the monoclonal antibody, 7E3.     

Incorporation of thrombospondin into fibrin clots

Thrombospondin is a major platelet glycoprotein which is released from platelets during blood coagulation. We examined the interaction of thrombospondin with polymerizing fibrin. Thrombospondin, purified from human platelets and labeled with 125I, became incorporated into clots formed from both plasma and purified fibrinogen. Plasma clots contained somewhat less thrombospondin than clots formed from equivalent concentrations of fibrinogen. In plasma clots and fibrin clots formed in the presence of factor XIII, thrombospondin was cross-linked in the clot; thrombospondin in the supernatant remained largely monomeric. Cross-linking of thrombospondin by factor XIII, however, only slightly increased the amount of thrombospondin which was incorporated into the clot. In contrast, incorporation of 125I-fibronectin into clots was dependent upon cross-linking. Most of the incorporation of 125I-thrombospondin occurred during fibrin polymerization as judged by parallel studies of the incorporation of 125I-fibrinogen. The amount of thrombospondin incorporated into a clot was directly related to thrombospondin concentration and was only weakly dependent on fibrinogen concentration. Incorporation was not saturated at thrombospondin:fibrin (mol/mol) ratios as high as 2/1. Thrombospondin, however, modified the final structure of fibrin clots in a concentration-dependent manner as monitored by opacity. When tryptic digests of 125I-thrombospondin were studied, the 270-kilodalton core became incorporated into fibrin whereas the 30-kilodalton heparin binding fragment was excluded. These results indicate that thrombospondin specifically co-polymerizes with fibrin during blood coagulation and may be an important modulator of clot structure.     

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.     

Binding of fibrinogen molecules to pig platelets and their membranes

Following addition of ADP, 125I-labelled fibrinogen binds specifically to pig platelets. This binding is completely inhibited by the unlabelled fibrinogen. Quantitative analysis indicates the presence of 12,400-25,000 molecules of fibrinogen which can be bound with an association constant of 5 . 10(8) M-1 to platelets. Fibrinogen receptors were found to be active in the isolated platelet membranes as well. Quantitative analysis of the saturable binding of fibrinogen to the platelet membranes showed that these receptors react with the same affinity with fibrinogen molecules. In contrast to the intact platelets, the platelet membranes can specifically bind fibrinogen in the absence of ADP. We conclude that a specific receptor for fibrinogen is exposed on the surface as a result of cell damage which is the first step of the platelet membrane isolation.     

Cross-linking of alpha and gamma-thrombin to distinct binding sites on human platelets

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane-impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross-linking of membrane glycoproteins. The proteins covalently linked to 125I-labelled alpha and gamma-thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I-alpha-thrombin was detected in high-molecular-mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr approximately equal to 400,000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78-kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active alpha-thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine-protease complex. The 240-kDa complex was formed with 0.8-100 nM alpha-thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl-inactivated alpha-thrombin. After analysis of Triton-X-100-soluble extracts of cross-linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I-alpha-thrombin. These were a precipitate containing GPIIb-IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240-kDa complex. Significantly, 125I-gamma-thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240-kDa and 78-kDa molecular mass range were not observed. We conclude that several binding sites for alpha-thrombin are present at the platelet surface, and that GPIb is one of them. The studies with gamma-thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.     

Identification of the fibrinogen receptor on human platelets by photoaffinity labeling

Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. In order to identify the platelet fibrinogen receptor, we modified fibrinogen with the photoreactive, heterobifunctional cross-linking reagent methyl 4-azidobenzoimidate (MABI). MABI-fibrinogen was fully clottable and able to support platelet aggregation. To photoaffinity label the fibrinogen receptor, gel-filtered human platelets were incubated at 37 degrees C in the dark with 200 micrograms/ml of MABI-fibrinogen, 10 microM ADP, and 0.5 mM calcium. Irradiation of these platelets with ultraviolet light resulted in the incorporation of MABI-fibrinogen into the platelet surface. Incorporation could be prevented by excess native fibrinogen suggesting that MABI-fibrinogen had interacted with the fibrinogen receptor before photolysis. Examination of the irradiated platelets by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the photoactivated MABI-fibrinogen had been incorporated into a 105,000 molecular weight membrane polypeptide that also contained the PlA1 antigen. Thus, this polypeptide has the characteristics of the membrane glycoprotein IIIa. Previous studies have shown that thrombasthenic platelets lack this glycoprotein and fail to bind fibrinogen after stimulation by ADP. Consequently, our data suggest that glycoprotein IIIa constitutes at least one component of the platelet fibrinogen receptor.     

Specific binding of blood coagulation factor XIIIa to thrombin-stimulated platelets

We studied the binding of 125I-platelet and plasma Factor XIII (125I-Factor XIII) to human platelets. When 125I-Factor XIII was incubated with gel-filtered platelets, calcium chloride (5 mM) and thrombin (1 unit/ml) at 37 degrees C, saturable binding was observed. Half-maximal binding occurred at 1 min. Binding was inhibited 93% by a 100-fold molar excess of unlabeled ligand but not by other purified proteins. Greater than 87% of platelet-bound radioactivity migrated as thrombin-cleaved a-chains (a'-chains) in sodium dodecyl sulfate-polyacrylamide gels indicating that Factor XIIIa but not Factor XIII binds to platelets. 125I-Factor XIIIa does not bind to unstimulated platelets. When platelet secretion was blocked, binding was markedly inhibited. 125I-Factor XIIIa bound minimally to platelets stimulated with agonists other than thrombin. Thus, binding is dependent on platelet activation, as well as modification of platelets by thrombin. 125I-Factor XIIIa bound to gamma-thrombin-stimulated platelets, at concentrations which did not clot fibrinogen. Therefore, Factor XIIIa is not bound to fibrin associated with platelets. Binding was only partially reversible. Approximately 12,000 molecules of Factor XIIIa were bound per platelet. 125I-Factor XIIIa bound normally to platelets from patients with severe Glanzmann's thrombasthenia indicating that 125I-Factor XIIIa does not bind to platelet glycoproteins IIb or IIIa, or platelet-bound fibrinogen. Chymotrypsin treatment of platelets inhibited 125I-Factor XIIIa binding by 78% without inhibiting secretion. Methylamine and putrescine, Factor XIIIa substrates, and N-ethylmaleimide, an active site inhibitor, did not inhibit binding. Factor XIIIa bound to platelets was enzymatically active and catalyzed [3H]putrescine incorporation into platelet proteins. The specific binding of Factor XIIIa to platelets suggests it may play a role in physiologic reactions involving platelets.     

Induction of the fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP

The capacity of epinephrine alone and the combination of low dose epinephrine and ADP to support the binding of fibrinogen to washed human platelets has been examined, 125I-Fibrinogen was bound to epinephrine-stimulated platelets, but 90 min were required to achieve maximal binding at 22 degrees C in contrast to 20 to 30 min with ADP. The overall rate of interaction appeared to reflect the slow binding of fibrinogen to epinephrine-stimulated platelets as opposed to the rate of stimulation of the cell. Divalent ions were required for binding of fibrinogen to epinephrine-stimulated platelets, and both calcium and magnesium supported binding with a prolonged time course. Fibrinogen binding was maximally supported by 20 to 30 microM epinephrine. The combination of low dose epinephrine (5 microM) and low dose ADP (0.5 microM), which acted synergistically to induce platelet aggregation, supported the rapid (10 min) binding of fibrinogen to platelets. With 4 microM epinephrine, more fibrinogen bound per platelet at all ADP doses than with ADP alone. With all the stimuli, saturable binding of fibrinogen to the platelet was observed, and Scatchard plots were linear, yielding very similar apparent association constants. The number of molecules bound per cell was stimulus-dependent, with 30 microM epinephrine inducing the binding of fewer fibrinogen molecules per cell (mean = 20,400) than 10 microM ADP (mean = 35,900) or the combination of 5 microM epinephrine + 0.5 microM ADP (mean = 43,600). The participation of endogenous ADP in fibrinogen binding to epinephrine-stimulated platelets was suggested since enzymes which remove ADP, apyrase, and creatine phosphate/creatine phosphokinase, and the ADP analogue, 2-chloroadenosine, completely inhibited the binding of fibrinogen to the platelet.     

Fibronectin-binding properties of the purified platelet glycoprotein IIb-IIIa complex

Fibronectin binds to specific receptors on the surface of washed, thrombin-activated platelets. Evidence suggests that these receptors are closely associated with the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). To determine whether GP IIb-IIIa itself can form a platelet receptor for fibronectin, we used a filtration assay to examine the interaction of purified fibronectin with purified GP IIb-IIIa incorporated into phospholipid vesicles. 125I-Fibronectin binding to the phospholipid vesicles required the presence of incorporated GP IIb-IIIa and was specific, time-dependent, reversible, saturable, and divalent cation-dependent (Mg2+ greater than Ca2+). The dissociation constant for 125I-fibronectin binding to the GP IIb-IIIa-containing vesicles in the presence of 2 mM MgCl2 was 87 nM. Proteins or peptides that inhibit 125I-fibronectin binding to whole platelets also inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. Thus, specific 125I-fibronectin binding was inhibited by excess unlabeled fibrinogen or fibronectin, the anti-GP IIb-IIIa monoclonal antibody 10E5, the decapeptide from the carboxyl terminus of the fibrinogen gamma-chain, and the tetrapeptide Arg-Gly-Asp-Ser from the cell-binding domain of fibronectin. In contrast to results obtained using whole platelets, unlabeled fibronectin inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. These results show that 125I-fibronectin binds directly to purified GP IIb-IIIa with most of the previously reported properties of 125I-fibronectin binding to washed, thrombin-stimulated platelets. Thus, GP IIb-IIIa has the potential to function as a platelet receptor for fibronectin as well as for fibrinogen.     

Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein IIb-IIIa complex

Several lines of evidence indicate that the platelet membrane glycoprotein IIb-IIIa complex (GP IIb-IIIa) is necessary for the expression of platelet fibrinogen receptors. The purpose of the present study was to determine whether purified GP IIb-IIIa retains the properties of the fibrinogen receptor on platelets. Glycoprotein IIb-IIIa was incorporated by detergent dialysis into phospholipid vesicles composed of 30% phosphatidylcholine and 70% phosphatidylserine. 125I-Fibrinogen binding to the GP IIb-IIIa vesicles, as measured by filtration, had many of the characteristics of 125I-fibrinogen binding to whole platelets or isolated platelet plasma membranes: binding was specific, saturable, reversible, time dependent, and Ca2+ dependent. The apparent dissociation constant for 125I-fibrinogen binding to GP IIb-IIIa vesicles was 15 nM, and the maximal binding capacity was 0.1 mol of 125I-fibrinogen/mol of GP IIb-IIIa. 125I-Fibrinogen binding was inhibited by amino sugars, the GP IIb and/or IIIa monoclonal antibody 10E5, and the decapeptide from the carboxyl terminus of the fibrinogen gamma chain. Furthermore, little or no 125I-fibrinogen bound to phospholipid vesicles lacking protein or containing proteins other than GP IIb-IIIa (i.e. bacteriorhodopsin, apolipoprotein A-I, or glycophorin). Also, other 125I-labeled plasma proteins (transferrin, orosomucoid) did not bind to the GP IIb-IIIa vesicles. These results demonstrate that GP IIb-IIIa contains the platelet fibrinogen receptor.     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.     

Transglutaminase-mediated cross-linking of fibrinogen by human umbilical vein endothelial cells

The interaction of endothelial cells with soluble or substrate-immobilized 125I-labeled fibrinogen (125I-FGN) was analyzed. Binding experiments involved incubation of 125I-FGN with cell suspensions at 4 degrees C. Bound ligand was quantitated by centrifugation of cells through silicone oil followed by scintillation analysis of the cell pellet. Calcium-dependent binding of 125I-FGN reached a maximum after 3 h and represented about 60% of the total. Half-maximal saturation occurred at 60 nM, and about 9 x 10(4) molecules were bound/cell at saturation (approximately 100 nM). Calcium-dependent binding was completely inhibited by unlabeled fibrinogen, partially inhibited by a monoclonal antibody (7E3) against glycoprotein IIb-IIIa, but not inhibited by fibrinogen fragments D or E, an anti-glycoprotein IIIa polyclonal antibody, or the Arg-Gly-Asp-Ser tetrapeptide. In contrast, the Arg-Gly-Asp-Ser tetrapeptide as well as the monoclonal antibody 7E3 markedly inhibited attachment of endothelial cells to substrate-immobilized fibrinogen, whereas fragment D or E did not. Both in suspension and monolayer, the 125I-FGN underwent cross-linking involving principally the A alpha chain. The transglutaminase inhibitors putrescine, histamine, and cystamine interfered with 125I-FGN binding and cross-linking by suspended cells. Since cross-linking in suspension was limited to bound 125I-FGN and since transglutaminase activity was not detectable in the binding buffer, cross-linking may have been mediated by a cell-associated transglutaminase.     

Activation of human platelets by a stimulatory monoclonal antibody

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.     

High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.