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.     

Activation of platelets by alpha-thrombin is a receptor-mediated event. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha-tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor

Competition binding studies have been carried out to evaluate the antagonism of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated thrombin) with alpha-thrombin using computer-assisted analysis of the binding isotherms (LIGAND). alpha-Thrombin bound to high, moderate, and low affinity sites as previously described (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). PPACK-thrombin bound to all three sites accessible to alpha-thrombin (K1, 7 nM; R1, 20 sites/platelet; K2, 3 nM; R2, 1800 sites/platelet; K3, 510 nM; R3, 84,000 sites/platelet) as well as to a separate fourth site (Kx, 0.4 nM; Rx, 20 sites/platelet) for PPACK-thrombin that was not accessible to alpha-thrombin. In contrast, TLCK-thrombin did not bind to the high affinity site for alpha-thrombin but bound to the moderate and low affinity sites for alpha-thrombin with similar affinity (K2, 2 nM; R2, 890 sites/platelet; K3, 900 nM; R3, 100,000 sites/platelet) and to another site (Ky, 0.03 nM; Ry, 10 sites/platelet) which was not accessible to alpha-thrombin. As predicted from these binding studies, TLCK-thrombin did not compete with alpha-thrombin for platelet activation at concentrations as high as 1000 nM (500-fold excess). In contrast a 300-fold excess of PPACK-thrombin (670 nM) totally inhibited platelet activation by 2 nM thrombin. These results demonstrate that the high affinity binding site for thrombin on human platelets is a classical receptor, occupancy of which is necessary for platelet activation by low concentrations of thrombin; that TLCK-thrombin does not occupy this high affinity site and hence cannot inhibit platelet activation by alpha-thrombin; and that PPACK-thrombin does compete with alpha-thrombin at the high affinity site and is an antagonist of alpha-thrombin induced activation.     

Platelet activation by thrombin in the absence of the high-affinity thrombin receptor

The receptor status of the moderate-affinity platelet binding site for alpha-thrombin has been established by treating platelets with Serratia marcescens protease under conditions causing cleavage of 95-97% glycoprotein Ib (2.5 micrograms for 30 min). High-affinity binding was lost under these conditions, but the platelets continued to show moderate-affinity binding (Kd1 = 16 +/- 5 nM; 930 +/- 300 sites/platelet) and low-affinity binding (Kd2 = 4.6 +/- 3 microM; 170,000 +/- 90,000 sites/platelet), in good agreement with the values previously obtained for moderate- and low-affinity binding in intact platelets [Harmon, J.T., & Jamieson, G.A. (1986) J. Biol. Chem. 261, 15928-15933]. Platelets treated with Serratia protease under these conditions were about 4-fold less sensitive to activation by alpha-thrombin, as measured by serotonin secretion. Crossover studies with analogues showed that binding of alpha-thrombin was compatible by both D-phenyl-alanyl-L-prolyl-L-arginine chloromethyl ketone treated thrombin and N alpha-p-tosyl-L-lysine chloromethyl ketone treated thrombin, and both analogues were capable of inhibiting activation of Serratia-proteolyzed platelets by alpha-thrombin. These studies establish that the moderate-affinity platelet binding site for alpha-thrombin is a receptor, occupancy of which is required for platelet activation in the absence of the high-affinity receptor.     

Thrombin interaction with platelets. Influence of a platelet protease nexin

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.     

Thrombin-platelet interactions: an assessment of the roles of saturable and nonsaturable binding in platelet activation

Thrombin binds to platelets and induces platelet activation, but the relationship of binding to activation is not clear. To better define this relationship, we have analyzed parameters of binding and activation by alpha-thrombin and by three analogous proteases that activate platelets somewhat differently. The proteases were nitro-alpha-thrombin, a derivative with nitrated tyrosine, gamma-thrombin, a product of partial proteolysis of alpha-thrombin, and trypsin, a homologous protease. Nitro-alpha-thrombin and native alpha-thrombin activated platelets similarly, whereas gamma-thrombin and trypsin activated to a slightly lesser extent than alpha-thrombin and only after a distinctive delay. alpha-Thrombin and nitro-alpha-thrombin bound to platelets to about the same extent, but only alpha-thrombin showed evidence of saturable binding. Hirudin, a thrombin inhibitor, blocked both platelet activation and saturable binding by alpha-thrombin. With nitro-alpha-thrombin, hirudin blocked platelet activation, but it had no effect on binding. gamma-Thrombin and trypsin bound less than alpha-thrombin and with no evidence of saturable binding. There were identical relationships between the total amount bound and the extent of platelet activation for the four proteases (some show no saturable binding) but distinct differences in the relationships of total amount bound and the rate of activation; similar rates of activation required the binding of three to five times more gamma-thrombin or trypsin than alpha-thrombin. That is, without saturable binding, activation was slower. These data thus show a correlation between total amount bound and extent of activation but no correlation between amount saturably bound and the extent of platelet activation. Conversely, the rate of activation is more closely correlated with saturable binding than with total binding. We conclude that high-affinity saturable binding is not essential for thrombin-induced platelet activation but that it may accelerate the reaction.     

Function of glycoprotein Ib alpha in platelet activation induced by alpha-thrombin.

We have obtained evidence that selective inhibition of high affinity thrombin-binding sites located in the amino-terminal domain of the membrane glycoprotein (GP) Ib alpha results in impaired platelet activation, as shown by abrogation or reduction of the following responses induced in normal platelets by exposure to less than 1 nM alpha-thrombin: (i) increase in intracellular ionized calcium concentration ([Ca2+]i), (ii) release of dense granule content, (iii) binding of fibrinogen, (iv) aggregation. An anti-GP Ib monoclonal antibody, LJ-Ib 10, which does not inhibit von Willebrand factor binding to platelets, obliterated the high affinity alpha-thrombin-binding sites on normal platelets. Isotherms of alpha-thrombin binding to normal platelets treated with saturating amounts of the antibody were virtually identical to those obtained with platelets from a patient with classical Bernard-Soulier syndrome. In parallel with decreased binding of the agonist, this antibody caused 50% inhibition of the maximal extent of platelet aggregation and 90% inhibition of ATP release induced by 0.3 nM alpha-thrombin. By inhibiting alpha-thrombin binding to GP Ib, the antibody prevented the activation of platelets exposed to low concentrations of the agonist, as demonstrated by abrogation of the increase in intraplatelet ionized calcium concentration induced in control platelets by 0.18 nM alpha-thrombin; under these conditions, fibrinogen binding was inhibited by 84%. Therefore, there is a correlation between occupancy of the high affinity sites for alpha-thrombin on GP Ib alpha and platelet activation, secretion, and aggregation, suggesting that GP Ib alpha is part of an alpha-thrombin receptor relevant for platelet function.     

Modulation of the platelet aggregation capacity by modified forms of thrombin

It was found that human platelets possess a high sensitivity towards alpha-thrombin (Km = 2 nM). Modified thrombin forms (beta/gamma-thrombin) with an impaired recognition site of high molecular weight substrates and DIP-alpha-thrombin and trypsin are incapable of inducing platelet aggregation when taken at concentrations corresponding to effective concentrations of alpha-thrombin. Beta/gamma-Thrombin and trypsin, unlike DIP-alpha-thrombin, cause platelet aggregation at concentrations of 100-200 nM. Studies on the modulating effects of modified thrombin forms, alpha-thrombin and trypsin, on platelet aggregation induced by alpha-thrombin revealed that beta/gamma-thrombin, alpha-thrombin and trypsin at concentrations causing no cell aggregation potentiate the platelet response after 2 min incubation and inhibit platelet aggregation upon prolonged (15 min) incubation. However, DIP-alpha-thrombin, irrespective of the incubation time (up to 30 min) increased the sensitivity of platelets to alpha-thrombin-induced aggregation. The activating effect of DIP-alpha-thrombin is characterized by an equilibrium constant (KA) of 17 nM. The experimental data confirm the hypothesis that the necessary prerequisite for an adequate physiological response of platelets to alpha-thrombin is the maintenance in the thrombin molecule of an intact active center and a recognition site for high molecular weight substrates. The specificity of thrombin as a potent platelet aggregation inducer is determined by the recognition site for high molecular weight substrates.     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

Thrombin binds to a high-affinity approximately 900 000-dalton site on human platelets

The functional sizes of the binding sites for thrombin on human platelets and isolated membranes have been determined by the technique of radiation inactivation: similar results were obtained. Independent studies using different radiation doses (0, 3, and 48 Mrad) and different thrombin concentrations (10(-10), 10(-8), and 10(-6) M) confirmed the presence of three binding sites with functional sizes of 900 000, 30 000, and 4000 daltons. The binding site of lowest apparent size (4000 daltons) probably corresponds to what has been termed nonspecific binding since its dissociation constant (2900 nM) is well outside the physiological range. The site of intermediate size (30 000 daltons) is also probably not involved in platelet activation since its dissociation constant (11 nM) is also beyond the concentration range required for activation, although it may be involved in other aspects of platelet-thrombin interaction. The sites with the largest functional size are probably important in platelet function since their dissociation constant (0.3 nM) is in the range required for platelet activation. The functional size of these sites (900 000 daltons) suggests that the high-affinity site for thrombin binding to platelets may involve a multimolecular complex of membrane components.     

The reaction of thrombin with platelet-derived nexin requires a secondary recognition site in addition to the catalytic site

A protease nexin released by activated platelets forms stable complexes with alpha-thrombin. Active-site-blocked thrombin does not form the stable complex, but it inhibits formation of the stable complex by active alpha-thrombin. gamma-Thrombin, which has a damaged substrate recognition site (the anion-binding exosite), did not form the complex and did not inhibit formation of the stable complex by alpha-thrombin. Complex formation was inhibited by the C-terminal dodecapeptide of hirudin, which has been shown to bind to the anion-binding exosite. A monoclonal antibody that blocks reactions of thrombin that involve the anion-binding exosite also inhibited formation of a stable complex of alpha-thrombin and the platelet-derived protease nexin. It is concluded that the anion-binding exosite of thrombin, a site that confers a high degree of specificity for substrates with a complementary site, binds to the platelet nexin prior to reaction of the catalytic site with the serpin.     

Covalent binding of human thrombin to a human endothelial cell-associated protein

Binding of 125I-thrombin to endothelial cells derived from human umbilical vein was studied in tissue culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed covalent binding of thrombin in a 72-kDa complex. This binding is specific and requires the catalytically active site of the enzyme. Formation of the complex could be detected as early as 3 min after addition of thrombin or with a thrombin concentration as low as 0.5 nM. This irreversible binding exhibits thrombin dose-dependence and reaches maximum levels at a concentration of 50 nM (10 fmol/10(5) cells). Some characteristics of the 72-kDa complex were compared to those of the complexes formed between thrombin and protease nexin originating from fibroblasts or platelets: (i) its electrophoretic mobility on SDS-PAGE is identical to that of the thrombin-platelet protease nexin complex, (ii) heparin prevents the appearance of the complex on the cell surface, (iii) plasmin in a 100-fold molar excess prevents the covalent linkage of thrombin, suggesting that the protease specificity of the endothelial component involved in the complex might not be restricted to thrombin. Yet no release, nor any secretion of the endothelial protein, could be detected. These results indicate that active thrombin binds covalently to a specific endothelial protein that is in several respects similar to fibroblast or platelet protease nexin and provides a thrombin binding site distinct from thrombomodulin and glycosaminoglycans.     

Structural and functional characterization of platelet receptor-mediated factor VIII binding

Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.     

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by alpha-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibalpha also has a high affinity binding site for alpha-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbalpha may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by alpha-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k(cat)/K(m) value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 +/- 0.1 x 10(7) m(-1) sec(-1). The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbalpha on platelet membranes by mocarhagin treatment reduced the k(cat)/K(m) value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k(cat)/K(m) value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbalpha, reduced the apparent k(cat)/K(m) value by about 5-fold. 4) Displacement of alpha-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparent k(cat)/K(m) value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparent k(cat)/K(m) value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a "cofactor" for PAR-1 activation by thrombin.     

The thrombin high-affinity binding site on platelets is a negative regulator of thrombin-induced platelet activation. Structure-function studies using two mutant thrombins, Quick I and Quick II.

To elucidate the thrombin domains required for high-affinity binding and platelet activation, the platelet binding properties of thrombin and two mutant thrombins, thrombin Quick I and Quick II, were compared to their agonist effects in elevating intraplatelet [Ca2+]. In Quick I, a mutation within the fibrinogen binding groove results in decreased clotting and platelet aggregating activities, whereas in Quick II, a mutation in the primary substrate binding pocket abolishes both activities. Dysthrombin binding was decreased compared to thrombin. The fibrinogen binding groove appeared more important than the primary substrate pocket for high-affinity binding since Quick I showed drastically reduced, and Quick II only slightly reduced, binding affinity (Kd approximately 200 and approximately 10 nM, respectively). The deduced interaction of thrombin with its high-affinity binding site indicated that the thrombin catalytic site is directed toward the platelet surface and therefore, when bound, is proteolytically inactive. Quick I (0.5-5 nM) elicited intraplatelet [Ca2+] fluxes at concentrations where high-affinity binding was undetectable. Saturation of high-affinity binding sites with active-site-modified thrombin did not affect thrombin-induced (0.5 nM) or Quick I-induced (5 nM) responses. In contrast, addition of D-Phe-Pro-Arg chloromethyl ketone (FPRCK) subsequent to thrombin or Quick I stimulation of platelets abolished agonist-induced responses. Since Quick I was only 10-17% as effective as thrombin in increasing intraplatelet [Ca2+], our data support a model in which thrombin acts enzymatically on a platelet membrane "substrate", through an interaction mediated in part by the fibrinogen binding groove of thrombin. This conclusion is consistent with the inhibition observed with high concentrations (greater than 100 nM) of Quick II and FPRCK-modified thrombin (FPR-thrombin) in platelets stimulated with low concentrations of thrombin (less than 0.5 nM) or Quick I (less than 2 nM), consistent with inhibition by substrate depletion. In contrast, concentrations of FPR-thrombin or Quick II (less than 100 nM), which saturated predominantly the high-affinity binding sites, enhanced the platelet responses induced by thrombin (less than 0.5 nM). Thus, occupation of the high-affinity sites with inactive thrombin increased the concentration of active thrombin available for substrate interaction. Quick I-induced responses were not enhanced, consistent with its inability to interact with the high-affinity site. Since thrombin bound to the high-affinity site is proteolytically inactive, we hypothesize that the thrombin high-affinity binding site on platelets functions to alter thrombin activity and platelet activation.     

Identification of a binding site for glycoprotein Ibalpha in the Apple 3 domain of factor XI

Factor XI (FXI) is a homodimeric plasma zymogen that is cleaved at two internal Arg(369)-Ile(370) bonds by thrombin, factor XIIa, or factor XIa. FXI circulates as a complex with the glycoprotein high molecular weight kininogen (HK). FXI binds to specific sites (K(d) = approximately 10 nM, B(max) = approximately 1,500/platelet) on the surface of stimulated platelets, where it is efficiently activated by thrombin. The FXI Apple 3 (A3) domain mediates binding to platelets in the presence of HK and zinc ions (Zn(2+)) or prothrombin and calcium ions. The platelet glycoprotein (GP) Ib-IX-V complex is the receptor for FXI. Using surface plasmon resonance, we determined that FXI binds specifically to glycocalicin, the extracellular domain of GPIbalpha, in a Zn(2+)-dependent fashion (K(d) = approximately 52 nM). We now show that recombinant FXI A3 domain inhibits FXI inbinding to glycocalicin in the presence of Zn(2+), whereas the recombinant FXI A1, A2, or A4 domains have no effect. Experiments with full-length recombinant FXI mutants show that, in the presence of Zn(2+), glycocalicin binds FXI at a heparin-binding site in A3 (Lys(252) and Lys(253)) and not by amino acids previously shown to be required for platelet binding (Ser(248), Arg(250), Lys(255), Phe(260), and Gln(263)). However, binding in the presence of HK and Zn(2+) requires Ser(248), Arg(250), Lys(255), Phe(260), and GLn(263) and not Lys(252) and Lys(253). Thus, binding of FXI to GPIbalpha is mediated by amino acids in the A3 domain in the presence or absence of HK. This interaction is important for the initiation of the consolidation phase of blood coagulation and the generation of thrombin at sites of platelet thrombus formation.     

Structure-function relationships in the interaction of alpha-thrombin with blood platelets.

Highly purified alpha-thrombin has been chemically modified in an attempt to determine which features of the molecule are important for normal platelet-thrombin interactions. Modifying agents included diisopropylphosphorofluoridate and 1-chloro-3-tosylamido-7-amino-L-2-heptanone, which modify serine and histidine, respectively, at the catalytic site, as well as N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, which modify a single tryptophan at or near the fibrinogen-binding site. Active site-directed modification did not appreciably affect the binding characteristics, but prevented platelet activation. In contrast, modification of tryptophan at the macromolecular substrate-binding site resulted in the loss of high affinity binding of thrombin to platelets, while low affinity binding was apparently unaffected. This modification altered but did not abolish the ability of thrombin to effect platelet aggregation and release of [14C]serotonin. These results suggest that residues at the catalytic site are not involved in binding and that the macromolecular substrate-binding site of alpha-thrombin participates in high affinity binding to platelets. These data are also consistent with the existence of at least two types of binding sites for thrombin on the platelet surface as well as more than one platelet-binding region on the thrombin molecule.     

Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets

Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.     

Exposure of binding sites for vitronectin on platelets following stimulation

Vitronectin is a glycoprotein that mediates cell adhesion and spreading in a number of cell culture systems. Liposomes containing platelet glycoproteins IIb-IIIa complex have been shown to bind vitronectin-coated surfaces through an Arg-Gly-Asp cell attachment mechanism. We examined the expression of the binding sites for vitronectin on the surface of intact, resting platelets and following stimulation. 125I-Labeled vitronectin bound specifically in a saturable manner to platelets treated with physiological concentrations of thrombin. The binding reached saturation at 100 nM concentration, and, at saturation, approximately 5000 specific binding sites were detected per platelet. The binding was divalent cation-dependent and only partially reversible after complete saturation. A synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited vitronectin binding to platelets. A monoclonal antibody against platelet glycoprotein IIb-IIIa complex also inhibited the binding of vitronectin to stimulated platelets. These data suggest that platelets possess an inducible divalent cation-dependent receptor for vitronectin and that the glycoprotein IIb-IIIa complex is involved in the expression of the vitronectin receptor.     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.