AlphaIIbbeta3 priming and clustering by orally active and intravenous integrin antagonists.

BACKGROUND: Drugs that block platelet-platelet and platelet-fibrin interactions via the alpha(IIb)beta(3) (glycoprotein IIb/IIIa) receptor are used daily in patients undergoing percutaneous coronary interventions. Along with expected increases in spontaneous bleeding, clinical trials have revealed a surprising increase in thrombosis when these drugs are used without other anticoagulants. A better understanding of their mechanisms can minimize these risks. OBJECTIVES: This study tested the hypothesis that interventions designed to block fibrinogen binding inevitably leave the alpha(IIb)beta(3) receptor in an activated state. It compared the effects on platelet function and alpha(IIb)beta(3) conformation of the orally active compounds orbofiban and roxifiban, the i.v. agents eptifibatide and tirofiban, and echistatin, an arginine-glycine-aspartate (RGD) disintegrin. METHODS: The integrin antagonist concentrations required to saturate platelets and to block platelet-platelet and platelet-fibrin interactions were determined by flow cytometry, aggregometry, and clot-based adhesion assays, respectively. Analytical ultracentrifugation measured each antagonist's effects on the solution structure of alpha(IIb)beta(3). Fluorescence anisotropy provided equilibrium and kinetic data for integrin:antagonist interactions. RESULTS: Both orally active drugs bound more tightly and inhibited platelet aggregation and adhesion to fibrin more effectively than echistatin. Analytical ultracentrifugation yielded this order for perturbing alpha(IIb)beta(3) conformation (priming) and promoting oligomerization (clustering): echistatin > eptifibatide > orbofiban > tirofiban > roxifiban. Roxifiban was also most effective at disrupting the rapidly forming/slowly dissociating alpha(IIb)beta(3):echistatin complex. CONCLUSIONS: Our results suggest that the same molecular mechanisms that enable glycoprotein IIb/IIIa inhibitors to bind tightly to the alpha(IIb)beta(3) receptor and block fibrinogen binding contribute to their ability to perturb the resting integrin's conformation, thus limiting the safety and efficacy of both oral and i.v. integrin antagonists.

A comparison of the mechanical and structural properties of fibrin fibers with other protein fibers.

In the past few years a great deal of progress has been made in studying the mechanical and structural properties of biological protein fibers. Here, we compare and review the stiffness (Young's modulus, E) and breaking strain (also called rupture strain or extensibility, epsilon(max)) of numerous biological protein fibers in light of the recently reported mechanical properties of fibrin fibers. Emphasis is also placed on the structural features and molecular mechanisms that endow biological protein fibers with their respective mechanical properties. Generally, stiff biological protein fibers have a Young's modulus on the order of a few Gigapascal and are not very extensible (epsilon(max) < 20%). They also display a very regular arrangement of their monomeric units. Soft biological protein fibers have a Young's modulus on the order of a few Megapascal and are very extensible (epsilon(max) > 100%). These soft, extensible fibers employ a variety of molecular mechanisms, such as extending amorphous regions or unfolding protein domains, to accommodate large strains. We conclude our review by proposing a novel model of how fibrin fibers might achieve their extremely large extensibility, despite the regular arrangement of the monomeric fibrin units within a fiber. We propose that fibrin fibers accommodate large strains by two major mechanisms: (1) an alpha-helix to beta-strand conversion of the coiled coils; (2) a partial unfolding of the globular C-terminal domain of the gamma-chain.

A study of the kinetics and mechanism of ADP-triggered platelet aggregation.

The time-course of ADP-triggered aggregation of human blood platelets has been followed by sensitive right-angle light scattering intensity measurements as a function of the platelet and fibrinogen concentrations. Rayleigh-Gans light scattering theory has been combined with the Smoluchowski aggregation model to predict the dependence of the right-angle scattering intensity on particle size and concentration as well as the time-dependent changes during aggregation. The validity of the calculations was confirmed by measuring the scattering intensity with suspensions of polystyrene microspheres of known radius, as well as the time-dependent changes in the 90 degrees scattering intensity during aggregation of these particles. However, in contrast to the predictions of the model, the time-course of the scattering intensity changes during platelet aggregation was characterized by single exponential decay with a rate constant which reached a limiting value of 0.017 s-1 at high platelet concentrations. The value of kagg was also independent of the fibrinogen concentration over a 30-fold range. Covalently cross-linked fibrinogen dimers and Fragment D-inhibited fibrin protofibrils yielded aggregation rates that agreed with those measured with fibrinogen. The results indicate that the rate of platelet aggregation is not limited by either the rate of fibrinogen binding or the frequency of platelet-platelet collisions under these conditions.

Fibrin protofibril and fibrinogen binding to ADP-stimulated platelets: evidence for a common mechanism.

The molecular basis of platelet-fibrin binding has been elucidated by studying interactions between platelets and protofibrils, soluble two-stranded polymers of fibrin which are intermediates on the fibrin assembly pathway. The fibrinogen degradation product, fragment D, has been used to block fibrin assembly, thus enabling the preparation of stable solutions of short protofibrils, composed of fewer than twenty fibrin monomer molecules per polymer. Fibrin protofibrils bound to ADP-activated platelets in a time- and concentration-dependent process which was effectively blocked by excess unlabelled fibrinogen, i.e., the binding was specific and appeared to involve a common receptor. ADP-stimulated cells bound approx. 3 micrograms of fibrin protofibrils/10(8) platelets, compared to 4 micrograms of fibrinogen/10(8) cells, following a 30-min incubation period at room temperature. Binding of both ligands was inhibited by high concentrations of fragment D, further indicating a similar mechanism. The kinetic data obtained were well described by an apparent first-order mechanism in which the rate constant for fibrin protofibril binding was found to be 5-fold slower than that measured for fibrinogen. Two monoclonal antibodies, each directed against the platelet glycoprotein IIb-IIIa complex, inhibited the binding of fibrin protofibrils and fibrinogen in a similar, concentration-dependent manner, providing strong evidence for a common receptor. Binding of GPRP-fibrin (soluble fibrin oligomers formed in the presence of 1 mM Gly-Pro-Arg-Pro) to ADP-stimulated platelets was also inhibited by a monoclonal antibody directed against the GPIIb-IIIa complex. Neither fibrin protofibrils nor fibrinogen bound to Glanzmann's thrombasthenic platelets, which lack normal quantities of functional glycoprotein IIb-IIIa complex, further supporting the hypothesis that fibrinogen and fibrin bind to a common platelet receptor present on the glycoprotein IIb-IIIa complex.

Localization of the domains of fibrin involved in binding to platelets.

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of fibrin protofibril and fibrinogen binding to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides of sequence RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen alpha- and gamma-chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 10 and 40 microM, respectively. Synthetic GPRP and GHRP, corresponding to the N-terminal tripeptide sequence of the fibrin alpha-chains and the tetrapeptide sequence of the beta-chains, respectively, were minimally effective in blocking soluble fibrin polymer binding to ADP-stimulated platelets. Platelet functions which are unique to the three-dimensional fibrin network were examined by measurements of the extent of adhesion of fluorophore-labelled fibrin to platelets with a microfluorimetric technique and by light scattering measurements of the time course of clot retraction. Inhibition of fibrin-platelet adhesion by RGDS, HHLGGAKQAGDV and GHRP exhibited a similar, linear dependence reaching 1/2 maximum at about 200 microM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin-platelet adhesion. Only RGDS effected clot retraction, causing a 4-6-fold decrease in rate at 230 microM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates on the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the alpha- and gamma-chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three-dimensional fibrin network and stimulated platelets.

An investigation of fibrin-platelet adhesive interactions by microfluorimetry.

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

Integrin clustering mechanisms explored with a soluble alphaIIbbeta3 ectodomain construct.

The purpose of this study was to test the hypothesis that residues critical for ligand- and temperature-induced clustering of integrin alphaIIbbeta3 are present on its extracellular domain. Sucrose density gradient sedimentation was used to examine the effects of ligand-mimetic peptides and physiological temperature on the oligomeric state of a soluble recombinant ectodomain variant of the alphaIIbbeta3 integrin, alphaIIbDelta962beta3Delta692, and its full-length counterpart. Both the ectodomain construct, isolated from High Five insect cell culture supernatants, and alphaIIbbeta3, isolated from human blood platelets, exhibited similar weight-average sedimentation coefficients at 23 degrees C, in the absence and presence of the ligand-mimetic peptide eptifibatide. These observations indicate that alphaIIbbeta3's ectodomain exhibits a similar extended conformation in both its free and ligand-bound states. Oligomerization was examined by incubation of both alphaIIbDelta962beta3Delta692 and full-length receptors at 37 degrees C, in the presence or absence of ligand-mimetic. Minimal oligomerization was observed with alphaIIbDelta962beta3Delta692. In contrast, full-length alphaIIbbeta3 exhibited substantial temperature-induced increases in its distribution of sedimenting species, indicative of thermal aggregation. These observations suggest that optimum oligomerization requires the participation of the integrin's transmembrane and cytoplasmic regions. In vivo, clustering of ligand-bound integrins may enhance signaling by increasing the local concentration of intracellular integrin-associated proteins.

AlphaIIb's cytoplasmic domain is not required for ligand-induced clustering of integrin alphaIIbbeta3.

The platelet integrin alphaIIbbeta3 exhibits bidirectional signaling, in that intracellular messengers enable adhesive macromolecules to bind to its ectodomain, while ligation promotes the association of cytoskeletal proteins with its cytoplasmic domains. In order to understand the linkage between these distant regions, we investigated the effects of receptor occupancy on the solution structure of both full-length recombinant alphaIIbbeta3 and alphaIIbDelta991beta3, an integrin truncation mutant which lacks one cytoplasmic domain. Lysates of (35)S-labeled human A549 cells expressing either full-length alphaIIbbeta3 or alphaIIbDelta991beta3 were examined by sucrose density gradient sedimentation followed by immunoprecipitation to determine the distributions of integrin protomers and oligomers. Recombinant alphaIIbbeta3 exhibited a weight-average sedimentation coefficient, S(w)=11.3+/-1.4 S with 73% sedimenting as protomers/dimers (9.1+/-1.0 S) and 27% as oligomers (15.4+/-0.4 S). Truncation mutant alphaIIbDelta991beta3 exhibited a similar pattern with 65% sedimenting as protomers/dimers. Upon ligation with eptifibatide, both full-length alphaIIbbeta3 and alphaIIbDelta991beta3 sedimented mainly at >14 S, indicating 2-3-fold increased oligomerization. Thus we have demonstrated that alphaIIb's cytoplasmic region is not required for integrin clustering, a key event in outside-in signaling.

Modeling the alpha IIb beta 3 integrin solution conformation.

The alpha IIb beta 3 platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of alpha and beta subunits. Electron microscopic (EM) images of the alpha IIb beta 3 complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross-linking studies rather suggest a somewhat more compact conformation for the alpha IIb beta 3 complex. We have studied the shape of detergent-solubilized alpha IIb beta 3 by employing a low-resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three-dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and -18% from the experimental values, determined, respectively, in octyl glucoside and Triton X-100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by -7% (octyl glucoside) to -6% (Triton X-100). Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent-solubilized alpha IIb beta 3 complex, where the relative spatial location of specific domains the size of 5-10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.

Binding of a fibrinogen mimetic stabilizes integrin alphaIIbbeta3's open conformation.

The platelet integrin alphaIIbbeta3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of alphaIIbbeta3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for alphaIIbbeta3. Eptifibatide binding promptly and reversibly perturbed the conformation of the alphaIIbbeta3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted alphaIIbbeta3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected alphaIIbbeta3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to alphaIIbbeta3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects alphaIIbbeta3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.

Platelets and endothelial cells act in concert to delay thrombolysis--evidence from an in vitro model of the human occlusive thrombus.

The molecular and cellular mechanisms that over a period of hours render a human thrombus progressively resistant to fibrinolysis have been probed with a novel in vitro model. The kinetics of clot formation and fibrinolysis were monitored by laser light scattering with platelet-rich model thrombi contained in cylindrical flow chambers. In selected experiments, human umbilical vein endothelial cells were also cultured to confluence on the inner walls of these "glass blood vessels". Following an "aging" period (0.5, 2 or 4 h), each thrombus was gently perfused with a bolus of plasminogen/recombinant tissue plasminogen activator to induce fibrinolysis. Platelets delayed lysis of 2 h-aged thrombi by approximately 70% and (non-stimulated) endothelial cells by approximately 30%, compared to cell-free control clots. However, even greater lytic delays (approximately 260%) resulted when both vascular cells were present in the same 2 h-aged thrombus. In contrast, rapid lysis was consistently achieved with R298E,R299E t-PA, a genetically engineered plasminogen activator that is insensitive to inhibition by plasminogen activator inhibitor type 1. These observations suggest platelets and endothelial cells act in concert to enrich the fibrin scaffold of an aging human thrombus in plasminogen activator inhibitor. We propose that the presence of both platelets and endothelial cells may contribute to progressive thrombolytic resistance.

Glycoprotein IIb/IIIa blockade inhibits platelet-mediated force development and reduces gel elastic modulus.

The effects of GPIIb/IIIa blockade on clot retraction were studied utilizing an instrument which directly measures force produced by platelets. GPIIb/IIIa disruption by calcium chelation, and GPIIb/IIIa blockade by peptides and anti-GPIIb/IIIa antibodies were investigated. One mM EDTA suppressed ADP-induced platelet aggregation by 72% and reduced force developed at 1200 s by 33%. At 234 microM, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) suppressed platelet aggregation by 74%, reduced force at 1200 s by 45% and reduced gel elastic modulus by 19%. At 10 microM, the peptide D-Arg-Gly-L-Asp-L-Try (D-RGDW) completely suppressed platelet aggregation, reduced force development by 38% and reduced gel elastic modulus by 29%. At 0.133 microM, monoclonal anti-GPIIIa antibody (AP-3) reduced force development by 74% and reduced gel modulus by 60%. Murine antiGPIIb/IIIa antibodies 10E5 and 7E3 markedly suppressed force development. At 0.133 microM, 10E5 reduced force by 89% and reduced gel modulus by 67%. At 0.053 microM, 7E3 completely stopped force development and reduced gel modulus by 46%. Platelet aggregation was blocked by 0.027 microM 7E3. Selective GPIIb blockade by antibodies did not affect force development. None of the agents studied altered fibrin structure as monitored by effects of fibrin mass/length ratios. Suppression of platelet aggregation occurred at inhibitor concentrations substantially lower than those required to suppress force development. Complete suppression of platelet aggregation did not assure inhibition of clot retraction probably due to profound platelet activation by thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of platelet adhesion to fibrin(ogen) in flowing whole blood by Arg-Gly-Asp and fibrinogen gamma-chain carboxy terminal peptides.

We have employed synthetic peptides with sequences corresponding to the integrin receptor-recognition regions of fibrinogen as inhibitors of platelet aggregation and adhesion to fibrinogen- and fibrin-coated surfaces in flowing whole blood, using a rectangular perfusion chamber at wall shear rates of 300 s-1 and 1,300 s-1. D-RGDW caused substantial inhibition of platelet aggregation and adhesion to fibrinogen and fibrin at both shear rates, although it was least effective at blocking platelet adhesion to fibrin at 300 s-1. RGDS was a weaker inhibitor, and produced a biphasic dose-response curve; SDRG was inactive. HHLGGAKQAGDV partially inhibited platelet aggregation and adhesion to fibrin(ogen) at both shear rates. These results support the identification of an RGD-specific receptor, most likely the platelet integrin glycoprotein IIb:IIIa, as the primary receptor responsible for platelet:fibrin(ogen) adhesive interactions under flow conditions, and indicate that platelet adhesion to surface bound fibrin(ogen) is stabilized by multivalent receptor-ligand contacts.

Fibrinogen Longmont. A heterozygous abnormal fibrinogen with B beta Arg-166 to Cys substitution associated with defective fibrin polymerization.

B beta Arg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo-Cys residues were found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Thrombin and batroxobin induced fibrin polymerization were impaired, despite normal release of fibrinopeptides A and B. Moreover, the polymerization defect was not corrected by removing the dimeric species or adding calcium. Fibrinogen Longmont had normal polymerization site a, as evidenced by normal GPRP-peptide binding. Thus, the sites A and a can interact to form protofibrils, as evidenced by dynamic light scattering measurements. These protofibrils, however, do not associate laterally in a normal manner, leading to an abnormal clot formation.

Inhibition of platelet-mediated clot retraction by integrin antagonists.

The effects of the platelet alphaIIb beta3 integrin (GPIIb/IIIa) antagonists XV459 (non-peptide), c7E3 (Fab monoclonal antibody) and DMP728 (cyclic peptide) as well as the alpha(v)beta3 integrin antagonists, LM609 (monoclonal antibody) and XT199 (non-peptide) on clotting and platelet-mediated clot retraction were examined. While 30 nM of XV459 had no significant effect on the kinetics of coagulation, platelet-mediated clot retraction was nearly fully inhibited at this concentration (Relative Retraction Rate = 0.09). XV459 resulted in a concentration related-response curve. Other experiments demonstrated that platelet aggregation was maximally inhibited at XV459 concentrations ranging from 30-50 nM. Similarly, c7E3 demonstrated comparable inhibitory efficacy in inhibiting either clot retraction or platelet aggregation. In contrast, DMP728, an equally potent anti-aggregatory agent with an IC50 of 20-50 nM in inhibiting platelet aggregation induced by various agonists, was found to be a less potent inhibitor of platelet-mediated clot retraction with a half-maximal inhibition of clot retraction at approximately 0.7 microM, and maximum effects at concentrations of 10 microM. The alpha(v)beta3 integrin antagonists, LM609 or XT199 were without any significant effects on either platelet-mediated clot retraction or platelet aggregation. In conclusion, these data suggest a differential efficacy among different GPIIb/IIIa antagonists in inhibiting platelet-mediated clot retraction in spite of the equivalent anti-aggregatory potency. Additionally, the alpha(v)beta3 integrin antagonists do not affect platelet-mediated clot retraction or aggregation. Further studies with the previously described alphaIIb beta3 integrin antagonists as well as others revealed a distinct correlation between the Kd to resting and activated platelets and the efficacy in inhibiting platelet-mediated clot retraction.

Inhibition of fibrin(ogen) binding to stimulated platelets by a monoclonal antibody specific for a conformational determinant of GPIIIa.

We have purified the integrin GPIIb:IIIa from the membrane fraction of human blood platelets by lentil lectin affinity chromatography followed by gel filtration chromatography. With purified GPIIb:IIIa as an antigen, we have produced monoclonal antibody CS-1, which immunoblotting demonstrates to be specific for native GPIIIa; disulfide bond reduction of GPIIIa resulted in loss of immunoreactivity. Radiolabelled ligand binding studies revealed that CS-1 recognized approximately 55,000 sites per platelet and bound with a Kd in the nanomolar range, independent of the state of platelet activation. Binding of CS-1 or its Fab fragment to ADP- and thrombin-stimulated gel filtered platelets caused a 2-3 fold inhibition of binding the soluble ligands fibrinogen and fibrin protofibrils. CS-1 also inhibited aggregation of ADP- and thrombin-stimulated platelets by 2- and 4-fold, respectively. Since CS-1 inhibits fibrin(ogen) interactions with GPIIb:IIIa, we propose that the conformationally dependent epitope on GPIIIa recognized by CS-1 constitutes a region of the receptor which is involved in fibrin(ogen) binding.

Differential effects of c7E3 Fab on thrombus formation and rt-PA-Mediated thrombolysis under flow conditions.

Although the Fab fragment of the mouse-human chimeric anti-alphaIIbbeta3 (GP IIb/IIIa) monoclonal antibody (MoAb) c7E3 facilitates recombinant tissue-type plasminogen activator (rt-PA)-mediated thrombolysis, it is not clear whether this is due to inhibition of new clot formation and/or a direct effect on the lysis rate. We employed an in vitro flow (re)circulation model to investigate how c7E3 Fab affected (a) platelet adhesion to clotted fibrin substrates under laminar flow at wall shear rates of 100 or 500 s(-1) and (b) rt-PA-induced lysis of preformed mural platelet-fibrin substrates at 500 s(-1). c7E3 Fab dose-dependently (0.5-5 microg/ml) inhibited platelet adhesion from flowing whole blood onto fibrin substrates ( approximately 14 microm thick) at each wall shear rate. When at 5 min after the onset of flow, c7E3 Fab (0.1-10 microg/ml) and rt-PA (1 microg/ml) were coinjected in flowing blood, it was found that modest fibrinolysis caused major platelet release from fibrin substrates and there was no difference in the lysis rate in the presence of rt-PA + c7E3 Fab compared to rt-PA alone. Platelet pretreatment with c7E3 Fab (10 microg/ml) had no effect on the lysis rate of thin ( approximately 40 microm), and slightly delayed the lysis rate of thick (< 250 microm), platelet-fibrin substrates containing evenly dispersed platelets (10(9)/ml). When the platelets within thick platelet-fibrin substrates were organized in platelet-rich regions ("residual thrombi"), these substrates followed a nonuniform lysis pattern, where fibrin between the thrombi lysed first and the residual thrombi lysed at a slower rate. Platelet pretreatment with c7E3 Fab (10 microg/ml) abolished the formation of the lytic-resistant residual thrombi and the associated platelet-protected fibrin zones. Hence, treatment with c7E3 Fab has no direct effect on the rate of rt-PA-mediated lysis, but is expected to block platelet-fibrin interactions that lead to clot retraction, thus maintaining a fibrin architecture that is more susceptible to lysis.

Fibrinogen New Britain: characterization of an A alpha Arg 16-->His variant human fibrinogen.

Routine laboratory screening identified an abnormal fibrinogen in an asymptomatic patient from New Britain, Connecticut. This paper reports the characterization of the abnormal fibrinogen. Clinical clotting studies showed marked prolongation of both the thrombin and Reptilase times. A discrepancy was noted between the fibrinogen concentration as determined by a rate-dependent clotting assay (0.73 milligrams) vs immunological determination (2.27 milligrams). Polymerase chain reaction (PCR) based DNA sequencing was used to identify the underlying molecular defect. Fibrinogen New Britain was found to have a point mutation in exon 2 of the A alpha chain. This mutation results in the amino acid substitution Arg-->His at position 16 of the A alpha chain, the site of thrombin cleavage. The substitution results in delayed polymerization of fibrin clot due to impaired release of fibrinopeptide A by thrombin. Studies of fibrin fibre dimensions and structure showed New Britain fibrin fibres to have a larger mean diameter and an increased mass/length ratio when compared with normal fibrin fibres formed under the same conditions.

Platelet adhesion to fibrin(ogen).

We have found that glycoprotein IIb:IIIa (GPIIb:IIIa) expressed on nonstimulated platelets is the primary receptor for platelet adhesion to immobilized fibrinogen or fibrin. At low shear rates of the blood the interaction between GPIIb:IIIa and fibrin(ogen) is strong enough to resist the shear forces exerted on the platelet as was shown with experiments with antibodies against platelet membrane glycoproteins and perfusion studies with blood from patients lacking platelet membrane receptors. Impaired platelet adhesion to fibrin(ogen) was found with blood from a patient with Glanzmann's thrombasthenia (lacking GPIIb:IIIa), blood from patients with the Bernard-Soulier syndrome (lacking GPIb) and blood from patients with severe von Willebrand's disease. This indicates that at higher shear rates additional interactions via GPIb on the platelet and von Willebrand factor originating from plasma or platelets are necessary to increase the affinity of the platelet for fibrin(ogen).