Analysis of the process of localization of fertilin to the sperm posterior head plasma membrane domain during sperm maturation in the epididymis

Fertilin is a heterodimeric (subunits alpha and beta) sperm plasma membrane protein. Both subunits belong to the ADAM protein family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in sperm-egg fusion by binding the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta. On testicular sperm of guinea pig, fertilin is distributed on the plasma membrane over the entire sperm head, but is found only on the posterior head once sperm have passed through the epididymis. This redistribution of fertilin to the posterior head can be partially mimicked in vitro if testicular sperm are briefly treated with trypsin. In this study we used immunofluorescence and digital image analysis to analyze how fertilin becomes restricted to the posterior head. We found that fertilin became restricted to the posterior head by migration of anterior head fertilin molecules into the posterior head domain. Comparison of immunofluorescence patterns and immunoblots of fertilin from seven regions of the epididymis showed a temporal correlation between the beginning of fertilin's migration to the posterior head and the proteolytic processing of the full-length fertilin beta precursor (the 85-kDa pro-beta form) to a 75-kDa intermediate, pro-beta*. Completion of the migration coincided with the further cleavage of pro-beta* to the 25- to 28-kDa mature form. Our data suggest that the cleavage of fertilin pro-beta to pro-beta* may initiate fertilin's migration into the posterior head domain and, after localization to that membrane domain, pro-beta* is cleaved to mature beta. We also report evidence that a common mechanism may be used to change the localization pattern of other sperm surface molecules. Other surface proteins were shown to become localized to either the posterior or the anterior head membrane domains on sperm at the same time fertilin became localized to the posterior head. These restrictions of surface protein localizations were also shown to immediately precede the development of the sperm's ability to swim and undergo the acrosome reaction, and thus redistribution of surface proteins may be necessary before sperm become functional.     

Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg

The sperm surface has an active role in the events of fertilization. The definition of the sperm surface in both its composition and domain organization begins during spermatogenesis and continues until the moment of sperm-egg fusion. Alterations of the surface proceed as a result of internal programming and environmental cues from both the male and female reproductive tracts, including interactions with the egg itself. We have investigated the sperm surface to understand its domain organization and the ongoing changes in this organization as well as the role of specific surface proteins in fertilization. Much of our research has concentrated on two surface proteins: PH-20 and fertilin. PH-20 is a single-chain protein, anchored in the membrane via a glycosyl phosphatidylinositol (GPI) anchor. The N-terminal domain of the molecule has a hyaluronidase activity. The hyaluronidase activity of PH-20 on the sperm plasma membrane enables sperm to penetrate the layer of cumulus cells surrounding the oocyte. PH-20 has a second function, unrelated to its hyaluronidase activity, in the binding of acrosome-reacted sperm to the zona pellucida (secondary sperm-zona binding). The fertilin molecule is an alpha,beta heterodimer whose two subunits are closely related transmembrane proteins. Fertilin beta has a disintegrin domain that has high sequence homology with the snake disintegrins, a known class of soluble integrin ligands. The binding site of the beta disintegrin domain functions to bind sperm to the egg plasma membrane via a mechanism that leads to sperm-egg fusion. The precursor of fertilin alpha, made in the testis, has an active metalloprotease site that could function in spermatogenesis. This metalloprotease domain is removed by proteolytic processing in the testis. Mature fertilin alpha on sperm also has a hydrophobic, putative "fusion peptide" that may promote the process of lipid bilayer fusion between sperm and egg plasma membranes. Fertilin alpha and beta are the first identified members of a new gene family of transmembrane proteins, the ADAM family, so called because they contain A Disintegrin And Metalloprotease domain. Many distinct ADAMs have now been found in diverse tissues and species (Drosophila to human) and are proposed to have a variety of functions in development and the adult. In addition to fertilin, other ADAMs are also present on the sperm plasma membrane and may participate with fertilin in sperm-egg fusion.     

Characterization of the binding of recombinant mouse sperm fertilin alpha subunit to mouse eggs: evidence for function as a cell adhesion molecule in sperm-egg binding

Fertilin (previously known as PH-30) is a sperm protein that is a candidate molecule for mediating the binding and fusion of the sperm and egg plasma membranes. Fertilin is a heterodimer, with a beta subunit that has a region of homology to the disintegrin family of integrin ligands and an alpha subunit that has a region of homology to viral fusion peptides. It has been hypothesized that fertilin beta and alpha subunits mediate the interactions between sperm and egg plasma membranes, namely, binding and fusion, respectively. To address this hypothesis and to examine specifically the role of fertilin alpha in fertilization, we have expressed the predicted extracellular domain of mouse fertilin alpha as a bacterial fusion protein with maltose-binding protein. This fusion protein (hereafter referred to as recombinant fertilin alpha-EC) binds to the microvillar region of zona pellucida (ZP)-free eggs, the region of the membrane to which sperm bind. This binding is reduced in the absence of divalent cations and is supported by Ca2+, Mg2+, or Mn2+. Eggs that have been treated with chymotrypsin bind less recombinant fertilin alpha-EC than do untreated eggs, suggesting that a chymotrypsin-sensitive binding site for recombinant fertilin alpha-EC is present on egg surfaces. Binding to eggs is also affected by the method used to remove the ZP. Finally, recombinant fertilin alpha-EC inhibits the binding of sperm to eggs during in vitro fertilization of ZP-free eggs. These data are the first evidence to suggest that fertilin alpha can function as a cell adhesion molecule during fertilization, mediating the binding of sperm and egg plasma membranes.     

Structural properties of the putative fusion peptide of fertilin, a protein active in sperm-egg fusion, upon interaction with the lipid bilayer

We recently demonstrated that a peptide representing the putative fusion domain of fertilin, a surface membrane protein of sperm involved in sperm-egg fusion, induces fusion of large unilamellar vesicles containing negatively charged lipids [Martin, I., and Ruysschaert, J. M. (1997) FEBS Lett. 405, 351-355]. In the present work, we demonstrate that increasing the concentration in negatively charged lipids strongly enhances the binding of the fertilin fusion peptide to the membrane, suggesting that electrostatic attractions play a crucial role in the binding process. While no significant change of the secondary structure content is observed by increasing the amounts of negatively charged lipids in the bilayer, the orientation of the alpha-helix changes from a parallel to an oblique orientation in the membrane. This topological change is confirmed by amide II hydrogen/deuterium exchange measurements that monitor the accessibility of the peptide to the water medium. Differential scanning calorimetry data also suggest that the fertilin fusion peptide lowers the bilayer to hexagonal phase transition temperature of model membranes composed of mixtures of dipalmitoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylserine and therefore promotes negative curvature in lipid vesicles. A comparison of the biophysical properties and the membrane-perturbing activities of fertilin and of viral fusion peptides is discussed in terms of sperm-egg fusion and virus cell fusion.     

Evidence for a polyspermy block at the level of sperm-egg plasma membrane fusion in Urechis caupo

The results of sperm binding experiments reveal no change in the sperm binding properties of the egg surface coat at fertilization of Urechis caupo eggs. When fertilized eggs are reinseminated, sperm continue to attach to the egg surface coat. The acrosomal tubules of supernumerary sperm are observed in the perivitelline space closely apposed to the egg membrane. Thus, the polyspermy block in Urechis eggs involves neither alteration of sperm binding sites nor inhibition of the acrosome reaction. Our results suggest that the block is at the level of sperm-egg membrane fusion.     

Perioperative outcomes of major hepatic resections under low central venous pressure anesthesia: blood loss, blood transfusion, and the risk of postoperative renal dysfunction

Fish sperm head plasma membranes have been demonstrated to contain syndecan (transmembrane heparan sulfate proteoglycan) immunofluorohistochemically and using immunoblot analysis and transglutaminase (TGase) by histochemistry. In order to examine the involvement of syndecan in fertilization, mature eggs were inseminated by direct mixing with untreated sperm or with sperm pretreated with an antiheparan sulfate (HS) antibody monoclonal (mAb), bovine serum albumin, human transferrin, or a TGase inhibitor (monodansylcadaverine, cystamine, and iodoacetamide). The fertilization rates of eggs inseminated with untreated and albumin-pretreated sperm were approximately 99.3% and 94.7%, respectively. Those of eggs pretreated with the anti-HS antibody and transferrin were 0% and 5.4%, respectively, whereas use of sperm pretreated with TGase inhibitors resulted in fertilization rates of approximately 13.2-17.8%. These results indicate that sperm head syndecan play an important role in fish sperm-egg contact and/or binding and that TGase inhibitors may reduce the fertilization rate by inhibiting sperm motility.     

Surface retention of an inactivating lutropin receptor mutant in exoloop 3

The heptahelical lutropin receptor (LHR) signals primarily via the Gs-adenylyl cyclase pathway and undergoes ligand-mediated receptor desensitization and internalization. A loss-of-function rat LHR mutant was recently described in which a single amino acid residue replacement in exoloop 3, K583E, had no effect on human choriogonadotropin (hCG) binding but essentially abolished signaling. This LHR mutant is a prime candidate for which to study hCG-mediated receptor internalization since it is highly unlikely that an amino acid residue in exoloop 3 , i.e. an extracellular portion of LHR connecting transmembrane helices 6 and 7, could have any direct interaction with Galpha(s), which is located on the cytoplasmic face of the plasma membrane. A method to study endocytosis was adapted that involves concanavalin A binding to the glycoproteins on the cell surface, thus facilitating separation of the plasma membrane fraction from other cellular membrane fractions by sucrose gradient centrifugation. Conditions were used such that a single round of endocytosis could be determined with [125I]hCG. Endocytic rate constants of 0.03 and O min(-1) were obtained for LHR and the mutant, respectively, in transfected human embryonic kidney 293 cells; moreover, internalization of the mutant could not be restored by the addition of 8-Br-cAMP. Thus, the presence of the second messenger cAMP is not sufficient for internalization of ligand-occupied LHR. Rather, it appears that ligand-mediated activation and subsequent internalization of LHR results from an altered conformational state or a conformation-dependent post-ligand binding modification such as phosphorylation.     

Differential membrane binding of the human immunodeficiency virus type 1 matrix protein

The human immunodeficiency virus type 1 matrix protein (p17MA) plays a central role at both the early and late stages of the virus life cycle. During viral assembly, the p17MA domain of Pr55gag promotes membrane association, which is essential for the formation of viral particles. When viral infection occurs, the mature p17MA dissociates from the plasma membrane and participates in the nuclear targeting process. Thus, p17MA contains a reversible membrane binding signal to govern its differential subcellular localization and biological functions. We previously identified a membrane binding signal within the amino-terminal 31 amino acids of the matrix domain of human immunodeficiency virus type 1 Gag, consisting of myristate and a highly basic region (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). Here we show that exposure of this membrane binding signal is regulated in different Gag protein contexts. Within full-length Pr55gag, the membrane targeting signal is exposed and can direct Pr55gag as well as heterologous proteins to the plasma membrane. However, in the context of p17MA alone, this signal is hidden and unable to confer plasma membrane binding. To investigate the molecular mechanism for regulation of membrane binding, a series of deletions within p17MA was generated by sequentially removing alpha-helical regions defined by the nuclear magnetic resonance structure. Removal of the last alpha helix (amino acids 97 to 109) of p17MA was associated with enhancement of binding to biological membranes in vitro and in vivo. Liposome binding experiments indicated that the C-terminal region of p17MA exerts a negative effect on the N-terminal MA membrane targeting domain by sequestering the myristate signal. We propose that mature p17MA adopts a conformation different from that of the p17MA domain within Pr55gag and present evidence to support this hypothesis. It is likely that such a conformational change results in an N-terminal myristyl switch which governs differential membrane binding.     

Palmitoyl-CoA stimulates cellular uptake and plasma membrane binding of carnitine

Carnitine cellular uptake and plasma membrane binding was investigated in S49 lymphoma cells. Palmitoyl-CoA was found to increase membrane binding of carnitine from 506 +/- 48 to 8,690 +/- 235 pmol/mg membrane protein. Palmitate and CoA acted synergistically and increased carnitine binding to plasma membranes but could not replace palmitoyl-CoA. The effect of palmitoyl-CoA on membrane binding of carnitine was maximal at 10 microM and required the presence of ATP. Palmitoyl-CoA increased the cellular uptake rate of carnitine from 181 +/- 5 to 884 +/- 25 amol/cell and h-1. We conclude that palmitoyl-CoA is a major regulator of cellular uptake of carnitine and, based on quantitative estimations, that the carnitine carrier binds more than one carnitine molecule.     

Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions

The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.     

Molecular mechanisms of sperm-egg interactions and egg activation

The binding of acrosome reacted mammalian sperm to the egg plasma membrane initiates a series of signaling events in the egg, termed "egg activation", which lead to the completion of meiosis II and the initiation of a mitotic cell cycle. Many of these signaling events have characteristics of classical signal transduction events in somatic cells. Currently, there are two hypotheses for how sperm-induced egg activation is initiated. In the "receptor" hypothesis, the fertilizing sperm interacts with a specific egg surface receptor, and this interaction leads to signal transduction and effector activation. In the "fusion" hypothesis it is postulated that following fusion of the sperm and egg plasma membranes a soluble sperm-derived factor enters the egg's cytoplasm and activates pathways leading to egg activation. This chapter will provide an overview of the processes of cell-cell interaction and signal transduction leading to mammalian egg activation. It will concentrate on specific molecules proposed to be involved in sperm-egg interaction, signal transduction and effector mechanisms involved in egg activation, and a discussion of sperm-associated factors that have been implicated in egg activation.     

Familial brain wave patterns: study of a 12-sib family

To identify peptide-specific antibodies which define sperm surface antigens, hybridomas were derived from the splenocytes of mice immunized with swollen human spermatozoa which had been subjected to limited proteolytic cleavage under reducing conditions prior to immunization. A total of 13.7% of the hybrid clones secreted antibodies which reacted with deglycosylated human seminal plasma glycoproteins when screened by an ELISA. A monoclonal antibody, designated mAb 4A8 sp., specifying a peptide epitope of human epididymal and a sperm surface glycoprotein was selected which inhibited human sperm-egg binding in a dose-dependent manner, and totally blocked sperm penetration in vitro. This inhibition did not result from an effect of the antibody on the motility of spermatozoa, nor was it due to premature induction of the acrosome reaction. Exclusion of oligosaccharide chains by chemical hydrolysis with trifluoromethane sulphonic acid (TFMS), enzymatic degradation and binding of lectins, did not abrogate the reactivity of mAb 4A8 to the cognate epitope whereas antibody binding was precluded upon digestion with proteolytic enzymes. In Western immunoblots of human seminal plasma glycoproteins, the antigen presented as a set of immunoreactive polypeptides, a major glycoprotein of M(r) 78 kDa and less prominent bands of M(r) 56 and 44 kDa. Immunocytochemical staining of a number of human reproductive and somatic tissues revealed strong immunostaining of the luminal epithelium of the epididymis as well as of spermatozoa in the lumen. Immunolocalization to the plasma membrane of ejaculated human spermatozoa was demonstrated by immunofluorescence, although on undigested spermatozoa the antigen epitope was less accessible. Upon capacitation the antigen persisted on the sperm surface and was present on the head of capacitated acrosome-intact spermatozoa. The pronounced peripheral immunostaining of the sperm head was accentuated after DTT/trypsin treatment, implicating the dynamic accessibility of the epitope on the plasma membrane of capacitated spermatozoa. It is suggested that the protein in question appears on the sperm membrane as a consequence of its modification in the epididymis (insertion and processing), and may be involved in the processes leading to sperm attachment and interaction with the human zona pellucida.     

Effect of angiotensin converting enzyme (ACE) and angiotensins on human sperm functions

The presence of components of the renin angiotensin system (RAS) and specific receptors of angiotensin II in the female and male reproductive tract supports the hypothesis that reproductive functions may be controlled by RAS. Therefore, the present study investigated the influence of ACE and angiotensins on sperm functions and the sperm-egg interaction. The experiments did not indicate direct effects of ACE on the capacitation process or acrosome reaction. Release of ACE from human spermatozoa during capacitation was not related to their ability to undergo acrosome reaction after stimulation with ionophore. Therefore, ACE release does not seem to be a useful clinical marker for human sperm capacitation. However, decreased binding of human spermatozoa to the oolemma of zonafree hamster oocytes after inhibition of ACE by captopril indicates that kininase II is involved in sperm-egg interactions. In contrast to other studies, incubation with captopril had no influence on sperm binding to the zona pellucida. Because effects of ACE on sperm-egg interactions but not on capacitation or acrosome reaction were observed, several experiments were performed to study the influence of substrates and products on the acrosome reaction. Angiotensin II induced the acrosome reaction dose-dependently, whereas angiotensin I had no effect on the acrosome reaction. The effect of angiotensin II on acrosome reaction seems to be calcium-dependent and mediated by protein kinases. Since a specific type 2 angiotensin II receptor inhibits the acrosome reaction induced by angiotensin II, this subtype of receptors may be present at the surface of sperm heads. Another clue for the presence of type 2 receptors on human spermatozoa is the finding that pertussis toxin did not inhibit the angiotensin II induced acrosome reaction. In contrast to type 1 angiotensin II receptors, type 2 receptors are known to be G-protein independent.     

The fertilization potential provides a fast block to polyspermy in lamprey eggs

At fertilization, the membrane potential of the egg of the lamprey, Lampetra japonica, shifted rapidly from its resting value of -12 to +36 mV and gradually returned to about the same resting level (fertilization potential). The amplitude of depolarization was influenced by the external Cl- concentration and by an anion channel blocker, DIDS, indicating that the positive shift of membrane potential resulted from Cl- efflux. A similar change in membrane potential (activation potential) was observed when the unfertilized egg was pricked with a fine needle or treated with A23187 to induce parthenogenetic activation. Pricking at the animal pole region (predetermined site for sperm entry) resulted in the occurrence of an immediate activation potential and the initiation of cortical granule exocytosis. A time lag between the pricking and the occurrence of the activation potential was observed when the egg was pricked at a distance from the animal pole. In this instance, the activation potential was produced immediately before the propagating cortical granule exocytosis initiated at the pricked site reached the animal pole region. Sperm-egg fusion was blocked in eggs voltage-clamped at +20 to +40 mV and inseminated, whereas it took place in eggs clamped at -60 to 0 mV. However, most eggs clamped at +20 to +40 mV did activate, indicating that the voltage dependence of egg activation differs from that of sperm-egg fusion. Although eggs voltage-clamped at negative membrane potentials permitted multiple sperm to fuse with the egg plasma membrane, the nucleus of the fused sperm did not necessarily enter the ooplasm. We conclude that: (1) A fast electrical block against polyspermy operates in this species and is effective for about 160 sec of the onset of the positive shift; (2) the opening of Cl- channels is responsible for the potential change; (3) the channels are largely localized in the animal pole region; (4) during voltage clamp at positive potentials, eggs can be activated without sperm-egg fusion; and (5) during voltage clamp at negative potentials, sperm-egg fusion occurs, but sperm entry into the egg cytoplasm does not always proceed.     

The effect of substitutions at position three on the binding and bioactivity of proctolin in locust hindgut and oviduct

A number of proctolin analogs modified at position three were analyzed for their relative binding affinities and biological activity on locust hindgut and oviduct. A decrease in chain length at this position (from Leu, Ile to Val) or an increase in hydrophobicity alone (Glu) or combined with a decrease in chain length (Val, Ser, Thr and Asp) decreased bioactivity but not necessarily binding. (Ser3)-proctolin had a higher affinity than proctolin for both hindgut and oviduct membranes but was less biologically active than proctolin in both tissues. Several other analogs bound with a similar affinity to proctolin but were significantly less biologically active, particularly on locust oviduct. These results suggest that the position three leucine of proctolin is more important for bioactivity than for binding in both oviduct and hindgut. The data also suggest the presence of two proctolin receptor subtypes on oviduct but not on hindgut membranes. Position three proctolin analogs may be useful in more precisely distinguishing these subtypes.     

Immunolocalization of CD44 and the ERM family in bone cells of mouse tibiae

We studied the immunohistochemical localization of CD44, hyaluronate receptor, and the ezrin-radixin-moesin (ERM) family, actin binding proteins, in bone cells using confocal laser scanning microscopy and transmission electron microscopy to clarify the mechanism of the organization of their cytoskeletons. In osteoclasts, intense immunoreactivity to CD44 could be detected on their basolateral plasma membranes. There was less reactivity observed in the area of the plasma membrane in direct contact with the bone surface. The immunogold electron-microscopical method revealed that CD44 was mainly localized on the microvilli of the basolateral plasma membrane. The plasma membrane of the clear zone and the ruffled border were not immunolabeled with CD44. As for the ERM family, the basolateral plasma membrane of osteoclasts was stained with antimoesin monoclonal antibody, but not with ezrin or radixin. In osteoblasts attached to the bone surface, immunoreactivity to CD44 was restricted to their cytoplasmic processes. They showed immunoreactivities to radixin and moesin on the cytoplasmic side of their plasma membrane when in contact with each other. However, although osteocytes in the bone matrix demonstrate an intense immunolabeling with CD44 on their plasma membrane, they scarcely show immunoreactivity to the ERM family. These findings suggest that: (1) the CD44-moesin-actin filament system is involved in the organization of cytoskeletons in the basolateral plasma membrane of osteoclasts; and (2) other mechanisms, rather than the CD44 and the ERM family, may be involved in the cells of osteoblast lineage.     

In vitro binding of synaptic vesicles to the synaptic plasma membrane: lack of effect of beta-bungarotoxin

To help characterize the mechanisms of neurotransmitter release, and the role of the specific neurotoxin beta-bungarotoxin in inhibiting release, the interaction of synaptic vesicles with the synaptic plasma membrane was investigated using two in vitro systems. Binding of radiolabeled synaptic vesicles to immobilized synaptic plasma membrane was specific, protein-dependent, and modulated by phosphorylation of membrane proteins. Stimulation of phosphorylation by phorbol ester increased binding, and reduction of phosphorylation by alkaline phosphatase or staurosporine reduced binding. beta-Bungarotoxin did not alter basal binding of synaptic vesicles to synaptic plasma membrane, nor did it affect the increase in binding induced by phorbol esters. Under conditions which stimulate acetylcholine release from synaptosomes, both phorbol ester and 4-aminopyridine caused an increase in attachment of the synaptic vesicle marker protein synaptophysin to the synaptic plasma membrane. beta-Bungarotoxin did not alter the change in localization of synaptophysin induced by either drug, under conditions in which it inhibits ACh release induced by 4-aminopyridine. It is concluded that beta-bungarotoxin inhibition probably does not occur at the level of the interaction of the synaptic vesicle and the synaptic plasma membrane, but occurs at an earlier stage in the neurotransmission process.     

Modification of Ca2+, Mg2+-ATPase and F-actin distribution in hepatocytes of cyclosporine A treated rats. Effect of soyabean lecithin and triacylglycerol

Two oligodeoxynucleotide (oligodN) binding proteins of 100-110 kDa on plasma membranes of human cell lines were recently identified by us. These two proteins seemed to play a role in oligodN uptake. In this study, the impact of the chain length and the sequence of the oligodN on the interaction with those two proteins was investigated. Chain length of oligodN was an important determinant, but not the sole determinant for the interaction. Binding affinity of oligodNs was determined predominantly by base composition, where pyrimidine bases but not purine bases were required in the sequence to retain high affinity. The binding kinetics of the homopolymers of deoxycytidine (dC21) and deoxythymidine (dT21) suggests that the proteins may have different binding sites, with one site preferring thymine bases and the other cytosine bases. Moreover, some additional plasma membrane proteins were identified, with an apparent molecular mass ranging from 40 to 58 kDa, which could bind thymine bases but not cytosine bases.     

Contribution of platelet microparticle formation and granule secretion to the transmembrane migration of phosphatidylserine

Activation of human platelets by complement proteins, C5b-9, thrombin plus collagen, or a Ca2+ ionophore results in surface exposure of phosphatidylserine (PS), accompanied by the expression of membrane catalytic activity for the tenase (VIIaIXa) and prothrombinase (VaXa) coagulation enzyme complexes. The mechanism underlying this surface exposure of PS upon platelet activation remains unresolved. Using fluorescent derivatives of PS (NBD-PS), we have investigated how the transmembrane migration of PS is related to microvesiculation of the platelet plasma membrane and to fusion of storage granules with the plasma membrane. Gel-filtered platelets were incubated with NBD-PS, allowing 90 +/- 10% of the incorporated NBD-PS to accumulate into the inner leaflet of the plasma membrane. Migration of NBD-PS from the inner leaflet to the plasma membrane surface was monitored by time-based flow cytometry, and correlated with the appearance of platelet microparticles and alpha-granule secretion. Platelet activation by C5b-9 or the Ca2+ ionophore, A23187, increased surface exposure of NBD-PS, due to acceleration of the apparent rate of migration from inner to outer plasma membrane leaflets. The onset of this accelerated migration of NBD-PS to the surface coincided with the onset of plasma membrane vesiculation, and the NBD-PS that partitioned into the membrane of the shed microparticle was also rapidly exposed to the surface (t1/2 < 2 min). In addition to a temporal correlation, microparticle formation and the surface exposure of inner leaflet NBD-PS showed a similar requirement for Ca2+. These results demonstrate that agonist-induced microvesiculation of the platelet plasma membrane is accompanied by accelerated migration of a PS analogue from the inner leaflet to the surface of the shed microparticle membrane, suggesting the mechanism by which induction of platelet microparticle formation exposes catalytic surface for tenase and prothrombinase assembly.