A B S T R A C T The role of fibrinogen as a cofactor for platelet aggregation was examiined by measuring the binding of 1251-labeled hulnan fibrinogen to gelfiltered human platelets both before and after platelet stimulation by ADP and

Transmembrane helices of integrin alpha and beta subunits have been implicated in the regulation of integrin activity. Two mutations, glycine-708 to asparagine-708 (G708N)and methionine-701 to asparagine-701, in the transmembrane helix of the beta3 subunit enabled integrin alphaIIbbeta3 to constitutively bind soluble fibrinogen. Further characterization of the G708N mutant revealed that it induced alphaIIbbeta3 clustering and constitutive phosphorylation of focal adhesion kinase. This mutation also enhanced the tendency of the transmembrane helix to form homotrimers. These results suggest that homomeric associations involving transmembrane domains provide a driving force for integrin activation. They also suggest a structural basis for the coincidence of integrin activation and clustering.

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins; however, companion methods for targeting transmembrane (TM) regions are not available. Here, we describe a method for the computational design of peptides that target TM helices in a sequence-specific manner. To illustrate the method, peptides were designed that specifically recognize the TM helices of two closely related integrins (alphaIIbbeta3 and alphavbeta3) in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target-host complex.

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The platelet integrin α IIb β 3 is required for platelet aggregation. Like other integrins, α IIb β 3 resides on cell surfaces in an equilibrium between inactive and active conformations. Recent experiments suggest that the shift between these conformations involves a global reorganization of the α IIb β 3 molecule and disruption of constraints imposed by the heteromeric association of the α IIb and β 3 transmembrane and cytoplasmic domains. The biochemical, biophysical, and ultrastructural results that support this conclusion are discussed in this Review.

Integrin activation states determine the ability of these receptors to mediate cell-matrix and cell-cell interactions. The prototypic example of this phenomenon is the platelet integrin, ␣IIb␤3. In unstimulated platelets, ␣IIb␤3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables ␣IIb␤3 to bind ligands such as fibrinogen and von Willebrand factor. To study the regulation of integrin activation states at the level of single molecules, we developed a model system based on laser tweezers, enabling us to determine the rupture forces required to separate single ligand-receptor pairs by using either purified proteins or intact living cells. Here, we show that rupture forces of individual fibrinogen molecules and either purified ␣IIb␤3 or ␣IIb␤3 on the surface of living platelets were 60 to 150 pN with a peak yield strength of 80 -100 pN. Platelet stimulation using either ADP or the thrombin receptor-activating peptide enhanced the accessibility but not the adhesion strength of single ␣IIb␤3 molecules, indicating that there are only two states of ␣IIb␤3 activation. Thus, we found it possible to use laser tweezers to measure the regulation of forces between individual ligand-receptor pairs on living cells. This methodology can be applied to the study of other regulated cell membrane receptors using the ligand-receptor yield strength as a direct measure of receptor activation͞inactivation state.T he detailed biochemistry of receptor-ligand interactions can be determined from solution and͞or surface studies, but these results do not take into account the response of receptormediated cell functions to externally applied forces encountered in vivo. This consideration is particularly relevant for integrins because of the cellular capacity to regulate the state of integrin activation. The extent to which cells regulate integrin function is highly variable. In some cellular environments, the ability of a specific integrin to support cellular adhesion may be constitutive, whereas in others, the integrin may be inactive or only partially active (1). Thus, it has been concluded that integrins exist in a variety of activation states, although the basis for this conclusion has generally been indirect, based on whole-cell adhesion assays and the interaction of integrins with specific monoclonal antibodies. Accordingly, direct studies of the activation states of individual receptors are important. In addition, such studies can reveal the relative contribution to integrin regulation of changes in receptor conformation (affinity modulation) vs. receptor clustering (avidity; ref. 2).The platelet integrin ␣IIb␤3 (GPIIb͞IIIa), which is inactive on resting platelets but is activated by agonists such as ADP and thrombin, is the prototypic example of adhesion receptor modulation. This tight regulation of ␣IIb␤3 activity is imperative to prevent the spontaneous formation of thrombi. In this paper, we demonstrate a model employing laser tweezers to determine the force between single ligand-receptor bonds eithe...

A B S T R A C T Platelets from individuals with familial hypercholesterolemia show increased sensitivity to the aggregating agents, epinephrine and ADP. Since the mechanism of this abnormal sensitivity is unknown, we examined, in vitro, the influence of the plasma lipid environment on the function of platelets. The composition of plasma lipids was altered by the addition of sonicated cholesterol-dipalmitoyl lecithin liposomes which were "cholesterol normal" (cholesterol-phospholipid mole ratio [C/P] = 1.0), "cholesterol rich" (C/P = 2.2), or "cholesterol poor" (C/P = 0). Cholesterolnormal liposomes had no influence on platelet lipids or platelet function. In contrast, after incubation for 5 h at 370C with cholesterol-rich liposomes, normal platelets acquired 39.2% excess cholesterol with no change in phospholipids or protein. The percent increase in platelet membrane cholesterol was threefold that of the granule fraction. The acquisition of cholesterol by platelets was associated with a 35-fold increase in sensitivity to epinephrine-induced aggregation (P < 0.001) and 15-fold increase to ADP aggregation (P <0.01), as determined both by aggregometry and by [14C]serotonin release. Response to thrombin or collagen was unchanged. Platelets incubated with cholesterol-poor liposomes underwent a selective loss of 21.4% cholesterol and this was associated with an 18-fold reduction in their sensitivity to epinephrine. (5,6). These studies have demonstrated that platelets from individuals with type II hyperlipoproteinemia have an increase in both platelet factor 3 availability and phospholipid content (5) and an increased sensitivity to aggregating agents, particularly epinephrine (6). However, it is not clear whether these findings are due to an intrinsic platelet defect or are related to the platelet's lipid-rich plasma environment. Furthermore, the relevance of these observations to the in vivo situation is unknown.The abnormal lipid composition of lipoproteins in certain individuals with alcoholic cirrhosis profoundly influences erythrocyte membrane lipid composition and membrane function leading to the disorder, spur cell anemia (7,8 METHODSPlatelet preparation. All blood donors were fasting for 12 h, had abstained from medications for at least 2 wk before blood donation, and had normal serum lipoproteins according to standard criteria (10), except for one who conformed to type Ha. Venous blood was collected through siliconized needles into plastic syringes and anticoagulated by mixing 9 vol of blood with 1 vol citrate-phosphate-dextrose (trisodium citrate 0.0894 M, citric acid 0.0156 M, monobasic sodium phosphate 0.0161 M, dextrose 0.1418 M). All blood processing was carried out in plastic-ware at room temperature. Platelet-rich plasma was obtained by centrifugation of samples for 10 min at 100 g. The remaining blood was centrifuged for 15 min at 1,800 g to obtain platelet-poor plasma which contained less than 20,000 platelets per A1. Platelets were counted (11) and their volume distributions measured using a Coul...

Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.Epidemiological studies have indicated that increased levels of circulating fibrinogen is an independent predictor of coronary heart disease and in some cases of premature death from cardiovascular disease, although a causative relationship between high levels of fibrinogen and cardiovascular disease has not been firmly established (1-4). Fibrinogen is a multifunctional protein essential for hemostasis. It is a 340-kDa glycoprotein, consisting of three non-identical peptide chains A␣, B␤, and ␥, which are linked together by 29 disulfide bonds (5). During coagulation, the soluble fibrinogen is converted to insoluble fibrin polymers. The process is initiated by thrombin, a serine protease, which catalyzes the cleavage first of two fibrinopeptides from the amino termini of the A␣ chains and then two fibrinopeptides from the amino termini B␤ chains. Upon release of the fibrinopeptides, the remaining fibrin monomers aggregate spontaneously to form ordered fibrin polymers (5). The clot is stabilized by the formation of covalent bonds introduced by the action of a transglutaminase, factor XIII (6). Under physiological conditions, fibrinolysis is dependent on the binding of circulating plasminogen and tissue-type plasminogen activator (tPA) 1 to fibrin clots. Urokinase and tPA convert plasminogen to the active protease plasmin, which then cleaves fibrin polymers to soluble fragments completing the coagulation and clot resolution cycle.A major cause...