Inhibition of fibrinogen binding to human platelets by blockage of Na+/H+ exchange

Binding of ADP to platelets enhances the binding of fibrinogen to Gp IIb-IIIa, the specific platelet receptor for adhesive proteins. The linkage between ADP and fibrinogen binding is indirect since ADP does not bind to the same receptor as fibrinogen. We have recently proposed that a third component, once affected by ADP binding, induces a conformational transition of the fibrinogen receptor from the low to the high affinity state, which is responsible for platelet aggregation [De Cristofaro, R., Landolfi, R., Castagnola, M., De Candia, E., Di Cera, E., & Wyman, J. (1988) Proc. Natl. Acad. Sci. USA 85, 8473-8476]. In the present study we provide evidence that this component should be identified with the platelet Na+/H+ antiport. Inhibition of the antiport by pharmacological agents such as amiloride, or else by decreasing extracellular Na+, results in a marked decrease of fibrinogen binding to platelets.     

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.     

Lysine binding to activated human platelets and its similarity to fibrinogen binding

Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of ¹²⁵I-fibrinogen and [³H]lysine with platelets activated by the Ca²⁺ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. (a) Marked enhancement by platelet activation; (b) deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; (c) saturability (fibrinogen binding approaches saturation at more than 12 μM, within 10 min; lysine binding at more than 100 mM within 1 min); (d) Ca²⁺-dependence (at 1 mM Ca²⁺ lysine binding is minute and fibrinogen binding is half-saturated); (e) reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; (f) inhibition by the oligoamines His-Lys and Lys₄; (g) inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; (h) specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated; Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa.     

Lysine binding to activated human platelets and its similarity to fibrinogen binding

Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of 125I-fibrinogen and [3H]lysine with platelets activated by the Ca2+ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. Marked enhancement by platelet activation; deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; saturability (fibrinogen binding approaches saturation at more than 12 microM, within 10 min; lysine binding at more than 100 mM within 1 min); Ca2+-dependence (at 1 mM Ca2+ lysine binding is minute and fibrinogen binding is half-saturated); reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; inhibition by the oligoamines His-Lys and Lys4; inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated. Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa.     

The role of ligand-ligand interactions in competition by fibrinogen and fibrin degradation products for fibrinogen binding to human platelets

Binding to human platelets of radioiodinated human fibrinogen and fragments X, Y, D, D₁ dimer and E was studied to determine the domain of the fibrinogen molecule responsible for binding to the platelet receptor. Although the fragments did not bind, some wer able to complete for the binding of fibrinogen to platelets. It was postulated that the fragments bound to fibrinogen and subsequently interfered with its binding to the receptor. Two approaches were developed to test this hypothesis. In the first technique, molecular exclusion on Sephacryl S-200 superfine was utilized to examine the interaction of radiolabeled fragments with fibrinogen. In the second seties of studies, fibrinogen-Sepharose was prepared and the binding of degradation products directly determined. A spin dialysis apparatus was employed in each case to achieve rapid separation of bound and free radioligand. These studies demonstrated that fragments D and E bind to fibrinogen. Therefore, the mechanism by which degradation products interfere with fibrinogen binding to the platelet receptor is ligand-ligand interaction rather than binding of the fragments to the receptor. Since none of the radiolabeled degradation products bound to platelets, it appears that receptor recognition requires the intact molecule.     

Kinetics of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine-induced fibrinogen binding to human platelets

Platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether) triggers exposure of fibrinogen binding sites on platelets via binding to specific receptors. Comparison of [3H]PAF-acether binding with 125I-fibrinogen binding shows that the rate with which PAF-acether binds to a number of receptors and not the degree of receptor occupancy determines how much fibrinogen binds. At low concentrations of PAF-acether (0.1-1.0 nM) binding site exposure is incomplete and parallels the rate of formation of the PAF-acether-receptor complex. Fibrinogen binding then primarily depends on the concentration of PAF-acether. At a high concentration of PAF-acether (500 nM) binding site exposure is complete within 2-5 min. Fibrinogen binding then depends on the concentration of fibrinogen. Exposure of binding sites in the absence of fibrinogen leads to disappearance of accessible binding sites. At 500 nM PAF-acether, this disappearance is exponential in nature and shows the same characteristics after 5-15 min incubation with fibrinogen as after 60 min. Exposure of binding sites is then complete within 5 min and their disappearance is not disturbed by other processes. At 0.5 nM PAF-acether, the same characteristics are found after 60 min incubation with fibrinogen, but shorter incubation times reveal an ongoing binding site exposure that interferes with the disappearance process. These results demonstrate close coupling between the PAF-acether receptors and fibrinogen binding sites and indicate that the rate of formation of the PAF-acether-receptor complex is a major factor in the regulation of binding site exposure.     

Lactoferrin binding to human platelets

• 1.1. Platelets bind specifically lactoferrin.
• 2.2. The lactoferrin binding to the platelets depends on the concentration of labelled lactoferrin, the number of platelets, the time of incubation and pH.
• 3.3. The binding was characterized by two types of binding site: one with high affinity and low capacity, and another with low affinity and high capacity (respectively k_{aff 1} = 13.6 × 10⁹1/mol and about 40 binding sites, and K_{aff 2} = 1.23 × 10⁹1/mol and about 135 binding sites per platelet).
• 4.4. Both human transferrin and bovine lactoferrin compete with human lactoferrin for the receptors.
• 5.5. The presence of lactoferrin receptors on the platelet membrane surface is connected most probably with the effect(s) on the cell function(s) of these cells.

     

Cytochalasin B binding by human platelets

Intact human platelets bind cytochalasin B (CB) with a capacity of 100– 120 p mols CB/mg protein or approximately 7 × 10⁴ molecules/cell and dissociation constants (K_D) ranging from 2 × 10^?8 to 10^?6 M. Up to 85% of this saturable binding is displaced by 10^?5 M cytochalasin E (CE). This CE-sensitive binding also appears heterogeneous with K_D similar to those of the overall binding. The CE-insensitive binding, however, appears as a single component with K_D ≌ 4 × 10^?7 M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with K_D ranging from 2.4 × 10^?8 to 1.5 × 10^?6 M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60–80%) of this CB binding is displaceable by 500 mM D-glucose and has a K_D of 1.5 × 10^?6M, while only 10–15% is CE-sensitive with a K_D of 2.4 ± 10^?8 M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80–85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane-associated binding. If the CE-sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.     

Differential structural requirements for fibrinogen binding to platelets and to endothelial cells

The cytoadhesins represent a group of RGD receptors that belongs to the integrin superfamily of adhesion molecules. Members of this cytoadhesin family include the platelet GPIIb-IIIa and the vitronectin receptors. These glycoproteins share the same beta-subunit, which is associated with different alpha subunits to form an alpha/beta heterodimer. In the present study, we have analyzed the fine recognition specificy of the cytoadhesins from platelets and endothelial cells for the adhesive protein, fibrinogen. Two sets of synthetic peptides, RGDX peptides and peptides corresponding to the COOH terminus of the fibrinogen gamma chain, were compared for their structure-function relationships in the two cellular systems. The results indicate that: (a) both RGDX and gamma-chain peptides inhibit the binding of fibrinogen to platelets and endothelial cells; (b) a marked influence of the residue at the COOH- and NH2-terminal positions of each peptide set can be demonstrated on the two types; and (c) RGDX and gamma peptides have differential effects on platelets and endothelial cells with respect to fine structural requirements. These results clearly indicate that while the platelet and endothelial cytoadhesins may interact with similar peptidic sequences, they express a different fine structural recognition.     

Inhibition of fibrinogen binding to GP IIb-IIIa by a GP IIIa peptide.

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) mediates platelet aggregation and is a member of the cytoadhesin family of receptors that bind adhesive proteins such as fibrinogen, fibronectin, and von Willebrand factor. Despite the wide range of cell-substrate interactions mediated by these receptors, ligand binding domains have not yet been identified on any of the integrins. The present study was designed to determine potential fibrinogen binding domain(s) on the GP IIb-IIIa complex. Synthetic peptides derived from residues 1-288 of the amino-terminal portion of GP IIIa were tested for their abilities to block the binding of fibrinogen to purified GP IIb-IIIa in a solid-phase microtiter assay. Two overlapping peptides encompassing residues 204-229 of GP IIIa were identified which blocked fibrinogen binding in this assay. Polyclonal antibodies to these peptides blocked fibrinogen binding to purified GP IIb-IIIa as well as platelet aggregation. The overlapping residues of these two peptides GP IIIa (211-222), SVSRNRDAPEGG-NH2, blocked the binding of fibronectin, von Willebrand factor, and vitronectin to purified GP IIb-IIIa. Finally, direct binding of GP IIIa (204-229) to fibrinogen and fibronectin was demonstrated by enzyme-linked immunosorbent assay. We conclude from these studies that the amino acid sequence 211-222 of GP IIIa is critically involved in adhesive protein binding, and may represent an important portion of the GP IIb-IIIa ligand binding domain.     

Preferred conformations of RGDX tetrapeptides to inhibit the binding of fibrinogen to platelets

The conformational study on Arg-Gly-Asp (RGD)-containing tetrapeptides in the unhydrated and hydrated states has been carried out using the force field ECEPP/3 and the hydration shell model. The tetrapeptides studied here are H-RGDX-OH (X = Trp, Tyr, Phe, Leu, Val, Cys, Gln, and Ser), which show the inhibitory activity for binding of fibrinogen to platelets in the order of RGDW approximately equal to RGDY approximately equal to RGDF approximately equal to RGDL > RGDV > or = RGDC > or = RGDQ > or = RGDS. The backbone conformations with two C(7) backbone-to-backbone hydrogen bonds between Asp and Arg residues and between Xaa and Gly residues are in common most probable for the RGD sequence of RGDX tetrapeptides in the hydrated state. The dominant beta-turns for RGDX are found to be the types V' and IV at Gly-Asp and Asp-Xaa sequences, respectively, which are quite similar to the types II' and I (or II), respectively. However, it cannot be ruled out that the extended conformations are also remarkably feasible for RGDX tetrapeptides in water by peering the distributions of backbone conformations. These calculated results are consistent with the experimental results on RGD-containing proteins and conformationally constrained RGD-containing peptides. The reason why the RGDX becomes more potent as the side chain of the X residue is more hydrophobic may be ascribed to that the more hydrophobic is the residue X, the more populated are beta-turn structures for the Gly-Asp sequence. The hydrophobic side chain of X residue exposed to water is likely to interact with the hydrophobic region of receptor easily.     

Immunogold-surface replica study of ADP-induced ligand binding and fibrinogen receptor clustering in human platelets

Platelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP-activated human platelets. We found that the gold prove was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa binding domains of fibrinogen--namely, the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.     

High affinity binding of thrombin to platelets. Inhibition by tetranitromethane and heparin

[¹²⁵I] iodo-α-thrombin has been modified at the macromolecular substrate binding site in order to study the importance of this region in the platelet-thrombin interaction. Modification was effected by the nitration of tyrosine residues with tetranitromethane. This chemical modification abolished the ability of the enzyme to bind with a high affinity to the platelet surface but did not significantly alter low affinity binding. The presence of heparin was also found to inhibit high affinity binding. These results indicate that the high affinity binding site interacts with the fibrinogen binding region of the thrombin molecule and suggests that there are two distinct classes of binding sites for thrombin on the platelet membrane.     

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.     

Induction of the fibrinogen receptor on human platelets by intracellular mediators

We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes the exposure of surface receptors for fibrinogen. Receptor exposure was detected using 125I-fibrinogen and 125I-PAC1, a monoclonal antibody specific for the activated form of the fibrinogen receptor. The potential mediators that were studied included guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and guanosine 5'O-(thiotriphosphate) (GTP gamma S), which cause G protein-dependent phospholipase C activation in platelets; inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release from the platelet dense tubular system; and diacylglycerol and phorbol ester, which activate protein kinase C. Each of these molecules caused fibrinogen and PAC1 binding. The effect of IP3 was mimicked by raising the cytosolic free Ca2+ concentration in the permeabilized platelets. However, IP3 and Ca2+-induced PAC1 binding were abolished by indomethacin or aspirin, which had no effect on PAC1 binding caused by Gpp(NH)p, phorbol ester, or diacylglycerol. This suggests that the response to IP3 and Ca2+ is due to the formation of metabolites of arachidonic acid. One such metabolite, TxA2, is believed to activate platelets by stimulating G protein-dependent phosphoinositide hydrolysis. Indeed, we found that the G protein inhibitor guanyl-5'-yl thiophosphate (GDP beta S) inhibited PAC1 binding caused by a thromboxane A2 analog (U46619), IP3, and Ca2+, but had no effect on diacylglycerol or phorbol ester-induced PAC1 binding. Thrombin-induced PAC1 binding and phosphoinositide hydrolysis were also inhibited by GDP beta S and by pertussis toxin. Increasing the thrombin concentration overcame the inhibition of PAC1 binding caused by GDP beta S but did not overcome the inhibition of phosphoinositide hydrolysis. These observations demonstrate that fibrinogen receptor exposure occurs by at least two routes. One of these, in response to agonists such as thrombin and U46619, is initiated by G protein-dependent phosphoinositide hydrolysis and involves the formation of IP3 and diacylglycerol. IP3 appears to act by stimulating Ca2+-dependent arachidonic acid metabolism which, in turn, triggers further phosphoinositide hydrolysis. Diacylglycerol acts by stimulating protein kinase C. A second route is activated by high concentrations of thrombin and is independent of phosphoinositide hydrolysis.     

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.     

Evidence that a collagen derived octapeptide inhibits fibrinogen binding to platelets stimulated by collagen and not by ADP

A synthetic octapeptide derived from type III collagen which specifically inhibits the activation and aggregation of platelets by collagen without affecting their adhesion was assayed on the collagen and ADP dependent fibrinogen binding to platelets. With 20 micrograms/ml collagen, the octapeptide (6 mM) inhibited by 68% the fibrinogen binding: this inhibition was correlated (p less than 0.01) to a decrease in the velocity of aggregation, suggesting that the fibrinogen binding might influence this parameter. The octapeptide did not affect the ADP-induced platelet aggregation and fibrinogen binding. This indicates that the octapeptide does not inhibit the binding of fibrinogen to its receptor directly, but interferes with some step(s) preceding the collagen-induced expression of the fibrinogen receptor.     

Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms.

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [( 3H]arachidonate mobilization and thromboxane synthesis) and secretion [( 14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.