Gaston Vilaire

Exposure of platelet fibrinogen receptors by ADP and epinephrine.

The role of fibrinogen as a cofactor for platelet aggregation was examined by measuring the binding of 125I-labeled human fibrinogen to gel-filtered human platelets both before and after platelet stimulation by ADP and epinephrine. Platelet stimulation by ADP resulted in the rapid, reversible binding of fibrinogen to receptors on the platelet surface. Fibrinogen binding increased as the concentration of ADP was increased from 0.1 to 2 microM, reaching a plateau at higher ADP concentrations. Binding occurred only after platelet stimulation and in the presence of divalent cations. However, fibrinogen binding did not occur to ADP-stimulated platelets from three patients with Glanzmanns thrombasthenia. Analysis of fibrinogen binding as a function of increasing fibrinogen concentration demonstrated that maximal platelet stimulation exposed approximately or equal to 45,000 binding sites per platelet with a dissociation constant of 80--170 nM. These fibrinogen binding parameters were essentially the same whether ADP or epinephrine was the platelet-stimulating agent. Thus, these studies demonstrate that platelet stimulation by ADP and epinephrine exposes a limited number of fibrinogen receptors on the platelet surface. Furthermore, these data suggest that the fibrinogen molecules bound to the platelet as a consequence of platelet stimulation are directly involved in the platelet aggregation response.

Platelet-derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells.

OBJECTIVE Peripheral blood platelet-derived microparticles (PMPs) circulate in blood and may interact directly with target cells affecting their various biological functions. METHODS To investigate the effect of human PMPs on hematopoiesis, we first phenotyped them for expression of various surface molecules and subsequently studied various biological responses of normal stem/progenitor (CD34(+)) and more differentiated precursor cells as well as several leukemic cell lines to PMPs. RESULTS We found that, in addition to platelet-endothelium attachment receptors (CD41, CD61 and CD62), PMPs express G-protein-coupled seven transmembrane-span receptors such as CXCR4 and PAR-1; cytokine receptors including TNF-RI, TNF-RII, and CD95; and ligands such as CD40L and PF-4. Moreover, we found that several of these receptors could be transferred by PMPs to the membranes of normal as well as malignant cells and observed that PMPs: 1) chemoattract these cells, 2) increase their adhesion, proliferation, and survival, and 3) activate in these cells various intracellular signaling cascades including MAPK p42/44, PI-3K-AKT, and STAT proteins. The biological effects of PMPs were only partly reduced by heat inactivation or trypsin digest, indicating that, in addition to the protein components of PMPs, lipid components are also responsible for their biological activity. CONCLUSIONS We conclude that PMPs modulate biological functions of hematopoietic cells and postulate that they play an important but as yet not fully understood role in intercellular cross-talk in hematopoiesis. Further studies, however, are needed to identify the PMP components that exert specific biological effects.

Computational Design of Peptides That Target Transmembrane Helices

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 (αIIbβ3 and αvβ3) 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.

Agonist-activated αvβ3 on Platelets and Lymphocytes Binds to the Matrix Protein Osteopontin

The phosphorylated acidic glycoprotein osteopontin is present in the extracellular matrix of atherosclerotic plaques and the wall of injured but not normal arteries. To determine if osteopontin could serve as a substrate for platelet adhesion, we measured the adherence of resting and agonist-stimulated human platelets to immobilized recombinant human osteopontin. Agonist-stimulated but not resting platelets bound to osteopontin by a process that was mediated primarily by αvβ3. αvβ3-mediated adherence occurred at physiologic concentrations of calcium and was inhibited by an αvβ3-selective cyclic peptide. Assays using phorbol myristate acetate-stimulated transfected B lymphocytes expressing both αvβ3 and αIIbβ3 confirmed that activated αvβ3 not activated αIIbβ3 was responsible for the cellular adherence we measured. These studies indicate that αvβ3 can reside on the cell surface in an inactive state and can be converted to a ligand binding conformation by cellular agonists. Moreover, they suggest that platelet adherence to osteopontin mediated by activated αvβ3 could play a role in anchoring platelets to disrupted atherosclerotic plaques and the walls of injured arteries. By inhibiting αvβ3 function, it may be possible to inhibit platelet-mediated vascular occlusion with a minimal effect on primary hemostasis.

The Platelet Cytoskeleton Regulates the Affinity of the Integrin αIIbβ3 for Fibrinogen

Agonist-generated inside-out signals enable the platelet integrin αIIbβ3 to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to αIIbβ3. By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating αIIbβ3 function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains αIIbβ3 in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on αIIbβ3, allowing it to assume the high affinity conformation required for soluble ligand binding.

Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.

The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.

A Role for Prostaglandins and Thromboxanes in the Exposure of Platelet Fibrinogen Receptors

Exposure of fibrinogen receptors by a variety of agonists is a prerequisite for platelet aggregation. Because the synthesis of prostaglandins and thromboxane A2 also occurs during platelet aggregation we wondered whether these agents participate in the exposure of platelet fibrinogen receptors. Therefore, we measured the binding of human 125I-fibrinogen to gel-filtered normal human platelets after prostaglandin and thromboxane synthesis had been inhibited by aspirin or indomethacin. The fibrinogen binding assay was performed at 37 degrees C but without stirring to prevent the formation of platelet aggregates. Platelet secretion, measured with [14C]serotonin, did not occur during the procedure. Aspirin or indomethacin inhibited fibrinogen binding stimulated by 10 microM epinephrine by 53%, and inhibited fibrinogen binding stimulated by 1-2 microM ADP by 37.1%. However, ADP at concentrations greater than 2 microM returned fibrinogen binding toward control values. Scatchard analysis demonstrated that aspirin decreased the number but not the affinity of the exposed fibrinogen receptors. To determine whether prostaglandins are capable of directly exposing fibrinogen receptors, prostaglandin H2 was used to stimulate platelets in the fibrinogen binding assay. Prostaglandin H2 exposed approximately 54,000 fibrinogen receptors/platelet and corrected the deficit in receptor exposure induced by aspirin. These studies demonstrate that platelet prostaglandins or thromboxane A2 can play a direct role in the exposure of platelet fibrinogen receptors. In addition, they suggest that the synthesis of prostaglandins and thromboxane A2 by stimulated platelets may be all that is required for optimal secondary platelet aggregation.

RGD-containing Peptides Inhibit Fibrinogen Binding to Platelet αIIbβ3by Inducing an Allosteric Change in the Amino-terminal Portion of αIIb

To determine the molecular basis for the insensitivity of rat αIIbβ3 to inhibition by RGD-containing peptides, hybrids of human and rat αIIbβ3 and chimeras of αIIbβ3 in which αIIb was composed of portions of human and rat αIIb were expressed in Chinese hamster ovary cells and B lymphocytes, and the ability of the tetrapeptide RGDS to inhibit fibrinogen binding to the various forms of αIIbβ3 was measured. These measurements indicated that sequences regulating the sensitivity of αIIbβ3 to RGDS are located in the seven amino-terminal repeats of αIIb. Moreover, replacing the first three or four (but not the first two) repeats of rat αIIb with the corresponding human sequences enhanced sensitivity to RGDS, whereas replacing the first two or three repeats of human αIIb with the corresponding rat sequences had little or no effect. Nevertheless, RGDS bound to Chinese hamster ovary cells expressing αIIbβ3 regardless whether the αIIb in the heterodimers was human, rat, or a rat-human chimera. These results indicate that the sequences determining the sensitivity of αIIbβ3 to RGD-containing peptides are located in the third and fourth amino-terminal repeats of αIIb. Because RGDS binds to both human and rat αIIbβ3, the results suggest that differences in RGDS sensitivity result from differences in the allosteric changes induced in these repeats following RGDS binding.

Activation of Platelet αIIbβ3 by an Exogenous Peptide Corresponding to the Transmembrane Domain of αIIb

A transmembrane domain heterodimer, acting in concert with a membrane-proximal cytoplasmic domain clasp, is thought to maintain integrins in a low affinity state. To test whether helix-helix interactions between the αIIb and β3 transmembrane domains regulate the activity of integrin αIIbβ3, we synthesized a soluble peptide corresponding to the αIIb transmembrane domain, designated αIIb-TM, and we studied its ability to affect αIIbβ3 activity in human platelets. αIIb-TM was α-helical in detergent micelles and phospholipid vesicles, readily inserted into membrane bilayers, bound to intact purified αIIbβ3, and specifically associated with the transmembrane domain of αIIb, rather than the transmembrane domains of β3, α2, and β1, other integrin subunits present in platelets. When added to suspensions of gel-filtered platelets, αIIb-TM rapidly induced platelet aggregation that was not inhibited by preincubating platelets with the prostaglandin E1 or the ADP scavenger apyrase but was prevented by the divalent cation chelator EDTA. Furthermore, αIIb-TM induced fibrinogen binding to platelets but not the binding of osteopontin, a specific ligand for platelet αvβ3. The peptide also induced fibrinogen binding to recombinant αIIbβ3 expressed by Chinese hamster ovary cells, confirming that its effect was independent of platelet signal transduction. Finally, transmission electron microscopy of purified αIIbβ3 revealed that αIIb-TM shifted the integrin from a closed configuration with its stalks touching to an open configuration with separated stalks. These observations demonstrate that transmembrane domain interactions regulate integrin function in situ and that it is possible to target intra-membranous protein-protein interactions in a way that can have functional consequences.

Effect of Cytoplasmic Domain Mutations on the Agonist-stimulated Ligand Binding Activity of the Platelet Integrin αIIbβ3

Function of the platelet integrin αIIbβ3 is regulated by agonist-generated signals interacting with its cytoplasmic tails. When αIIbβ3 is expressed in Epstein-Barr virus-transformed B lymphocytes, stimulation of the cells with phorbol 12-myristate 13-acetate results in αIIbβ3-mediated lymphocyte adherence to immobilized fibrinogen, as well as soluble fibrinogen binding to αIIbβ3, indicating that agonists increase the affinity of αIIbβ3 for fibrinogen in these cells. To address the contribution of the αIIb and β3 cytoplasmic tails to this process, we mutated each tail and expressed the mutants in B lymphocytes. Truncation of the αIIb tail did not impair unstimulated or stimulated lymphocyte adherence to fibrinogen, regardless whether the truncation was proximal or distal to the conserved GFFKR sequence. However, deleting GFFKR or replacing it with alanines markedly reduced αIIbβ3 expression due to impaired intracellular assembly of αIIbβ3 heterodimers, probably due to a mutation-induced change in the conformation of αIIb. Introducing β3 mutations known to impair αIIbβ3 function in platelets into the cytoplasmic tail of β3 in lymphocytes also impaired αIIbβ3 function in these cells. These studies demonstrate that the cytoplasmic tail of αIIb is not required for αIIbβ3 function in lymphocytes, although the presence of GFFKR in the αIIb tail is required for αIIb to interact with β3. Additionally, they indicate that signals interacting with the β3 cytoplasmic tail are responsible for the ability of agonists to stimulate αIIbβ3 function.