M protein, a classical bacterial virulence determinant, forms complexes with fibrinogen that induce vascular leakage

Increased vascular permeability is a key feature of inflammatory conditions. In severe infections, leakage of plasma from the vasculature induces a life-threatening hypotension. Streptococcus pyogenes, a major human bacterial pathogen, causes a toxic shock syndrome (STSS) characterized by excessive plasma leakage and multi-organ failure. Here we find that M protein, released from the streptococcal surface, forms complexes with fibrinogen, which by binding to beta2 integrins of neutrophils, activate these cells. As a result, neutrophils release heparin binding protein, an inflammatory mediator inducing vascular leakage. In mice, injection of M protein or subcutaneous infection with S. pyogenes causes severe pulmonary damage characterized by leakage of plasma and blood cells. These lesions were prevented by treatment with a beta2 integrin antagonist. In addition, M protein/fibrinogen complexes were identified in tissue biopsies from a patient with necrotizing fasciitis and STSS, further underlining the pathogenic significance of such complexes in severe streptococcal infections.     

Platelet Activation and Thrombus Formation over IgG Immune Complexes Requires Integrin αIIbβ3 and Lyn Kinase

IgG immune complexes contribute to the etiology and pathogenesis of numerous autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus, rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Patients suffering from immune complex-related disorders are known to be susceptible to platelet-mediated thrombotic events. Though the role of the Fc receptor, FcγRIIa, in initiating platelet activation is well understood, the role of the major platelet adhesion receptor, integrin αIIbβ3, in amplifying platelet activation and mediating adhesion and aggregation downstream of encountering IgG immune complexes is poorly understood. The goal of this investigation was to gain a better understanding of the relative roles of these two receptor systems in immune complex-mediated thrombotic complications. Human platelets, and mouse platelets genetically engineered to differentially express FcγRIIa and αIIbβ3, were allowed to interact with IgG-coated surfaces under both static and flow conditions, and their ability to spread and form thrombi evaluated in the presence and absence of clinically-used fibrinogen receptor antagonists. Although binding of IgG immune complexes to FcγRIIa was sufficient for platelet adhesion and initial signal transduction events, platelet spreading and thrombus formation over IgG-coated surfaces showed an absolute requirement for αIIbβ3 and its ligands. Tyrosine kinases Lyn and Syk were found to play key roles in IgG-induced platelet activation events. Taken together, our data suggest a complex functional interplay between FcγRIIa, Lyn, and αIIbβ3 in immune complex-induced platelet activation. Future studies may be warranted to determine whether patients suffering from immune complex disorders might benefit from treatment with anti-αIIbβ3-directed therapeutics.     

Roles for fibrinogen, immunoglobulin and complement in platelet activation promoted by Staphylococcus aureus clumping factor A

Staphylococcus aureus is an important cause of infective endocarditis (IE) in patients without a history of prior heart valve damage. The ability to stimulate the activation of resting platelets and their subsequent aggregation is regarded as an important virulence factor of bacteria that cause IE. Clumping factor A is the dominant surface protein responsible for platelet activation by S. aureus cells in the stationary phase of growth. This study used Lactococcus lactis as a surrogate host to study the mechanism of ClfA-promoted platelet activation. Expression of ClfA from a nisin-inducible promoter demonstrated that a minimum level of surface-expressed ClfA was required. Using platelets that were purified from plasma, the requirement for both bound fibrinogen and immunoglobulin was demonstrated. The immunoglobulin G (IgG) requirement is consistent with the potent inhibition of platelet activation by a monoclonal antibody specific for the platelet FcgammaRIIa receptor. Furthermore the IgG must contain antibodies specific for the ClfA A domain. A model is proposed whereby bacterial cells armed with a sufficient number of surface-bound fibrinogen molecules can engage resting platelet glycoprotein GPIIb/IIIa, aided by bound IgG molecules, which encourages the clustering of FcgammaRIIa receptors. This can trigger activation of signal transduction leading to activation of GPIIb/IIIa and aggregation of platelets. In addition, analysis of a mutant of ClfA totally lacking the ability to bind fibrinogen revealed a second, although less efficient, mechanism of platelet activation. The fibrinogen-independent pathway required IgG and complement deposition to trigger platelet aggregation.     

Antibodies against a surface protein of Streptococcus pyogenes promote a pathological inflammatory response

Streptococcal toxic shock syndrome (STSS) caused by Streptococcus pyogenes is a clinical condition with a high mortality rate despite modern intensive care. A key feature of STSS is excessive plasma leakage leading to hypovolemic hypotension, disturbed microcirculation and multiorgan failure. Previous work has identified a virulence mechanism in STSS where M1 protein of S. pyogenes forms complexes with fibrinogen that activate neutrophils to release heparin-binding protein (HBP), an inducer of vascular leakage. Here, we report a marked inter-individual difference in the response to M1 protein-induced HBP release, a difference found to be related to IgG antibodies directed against the central region of the M1 protein. To elicit massive HBP release, such antibodies need to be part of the M1 protein-fibrinogen complexes. The data add a novel aspect to bacterial pathogenesis where antibodies contribute to the severity of disease by promoting a pathologic inflammatory response.     

Antibacterial activity of the contact and complement systems is blocked by SIC, a protein secreted by Streptococcus pyogenes

Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype.     

Fluid shear regulates the kinetics and receptor specificity of Staphylococcus aureus binding to activated platelets

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1beta. Peak adhesion efficiency occurs at low shear (100 s(-1)) and decreases with increasing shear. The molecular interaction of platelet alpha(IIb)beta(3) with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (相似文献   

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Plant food delphinidin-3-glucoside significantly inhibits platelet activation and thrombosis: novel protective roles against cardiovascular diseases

Delphinidin-3-glucoside (Dp-3-g) is one of the predominant bioactive compounds of anthocyanins in many plant foods. Although several anthocyanin compounds have been reported to be protective against cardiovascular diseases (CVDs), the direct effect of anthocyanins on platelets, the key players in atherothrombosis, has not been studied. The roles of Dp-3-g in platelet function are completely unknown. The present study investigated the effects of Dp-3-g on platelet activation and several thrombosis models in vitro and in vivo. We found that Dp-3-g significantly inhibited human and murine platelet aggregation in both platelet-rich plasma and purified platelets. It also markedly reduced thrombus growth in human and murine blood in perfusion chambers at both low and high shear rates. Using intravital microscopy, we observed that Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for vessel occlusion. Dp-3-g also significantly inhibited thrombus growth in a carotid artery thrombosis model. To elucidate the mechanisms, we examined platelet activation markers via flow cytometry and found that Dp-3-g significantly inhibited the expression of P-selectin, CD63, CD40L, which reflect platelet α- and δ-granule release, and cytosol protein secretion, respectively. We further demonstrated that Dp-3-g downregulated the expression of active integrin αIIbβ3 on platelets, and attenuated fibrinogen binding to platelets following agonist treatment, without interfering with the direct interaction between fibrinogen and integrin αIIbβ3. We found that Dp-3-g reduced phosphorylation of adenosine monophosphate-activated protein kinase, which may contribute to the observed inhibitory effects on platelet activation. Thus, Dp-3-g significantly inhibits platelet activation and attenuates thrombus growth at both arterial and venous shear stresses, which likely contributes to its protective roles against thrombosis and CVDs.     

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin-binding proteins FnBPA and FnBPB are major platelet-activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen-binding A domain or the fibronectin-binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA-promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV-3) specific for the FcgammaRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcgammaRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV-3 suggesting that the IgG-FcgammaRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.     

Modifying murine von Willebrand factor A1 domain for in vivo assessment of human platelet therapies

The A1 domain of von Willebrand factor (VWF-A1) plays a crucial role in hemostasis and thrombosis by initiating platelet adhesion at sites of arterial injury through interactions with the platelet receptor glycoprotein Ib alpha (GPIbalpha). Here we report that murine VWF-A1 supports limited binding of human platelets. However, atomic models of GPIbalpha-VWF-A1 complexes identified an electrostatic 'hot-spot' that, when mutated in murine VWF-A1, switches its binding specificity from mouse to human GPIbalpha. Furthermore, mice expressing this mutant VWF-A1 display a bleeding phenotype that can be corrected by infusion of human platelets. Mechanistically, human platelets correct the phenotype by forming occlusive thrombi, an event that can be abrogated by blockade of GPIbalpha or by the preadministration of inhibitors of platelet activation or adhesion (clopidogrel (Plavix) and abciximab (ReoPro), respectively). Thus, by modifying a protein interface, we have generated a potential biological platform for preclinical screening of antithrombotics that specifically target human platelets.     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.     

Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.     

Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium.

The biochemical responses of intact human platelets to the monoclonal antibody (mAb) AG-1 were investigated. AG-1 is a murine IgG mAb that recognizes a series of platelet membrane glycoproteins (Gp) from M(r) 21,000 to 29,000, one of which is the M(r) 24,000 (p24) receptor for anti-CD9 mAbs. AG-1 causes platelet aggregation and secretion. Platelets binding AG-1 demonstrate a dose- and time-dependent breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), production of diacylglycerol, and generation of phosphatidic acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intracellular calcium, and fibrinogen binding. Platelet PA generation and PKC activation in response to AG-1 are inhibited by mAbs to platelet GpIIb-IIIa or by extracellular EGTA, but not by a mAb to platelet GpIb or by inhibiting platelet Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride. Platelet cytoplasmic free calcium ([Ca2+]i) is elevated in response to AG-1, and this elevation is inhibited by mAbs to GpIIb-IIIa, an RGDS peptide or by chelating extracellular calcium. These results suggest that AG-1 binding to a unique platelet-surface glycoprotein initiates platelet responses through the activation of PIP2-specific phospholipase C, and that this occurs through a signal pathway that is dependent on GpIIb-IIIa and extracellular calcium.     

Effect of synthetic peptides corresponding to residues 313-332 of the alphaIIb subunit on platelet activation and fibrinogen binding to alphaIIbbeta3.

The platelet integrin receptor alphaIIbbeta3 plays a critical role in thrombosis and haemostasis by mediating interactions between platelets and several ligands but primarily fibrinogen. It has been shown previously that the YMESRADR KLAEVGRVYLFL (313-332) sequence of the alphaIIb subunit plays an important role in platelet activation, fibrinogen binding and alphaIIbbeta3-mediated outside-in signalling. Furthermore, we recently showed that the 20-residue peptide (20-mer) alphaIIb 313-332, is a potent inhibitor of platelet aggregation and fibrinogen binding to alphaIIbbeta3, interacting with fibrinogen rather than the receptor. In an effort to determine the sequence and the minimum length required for the biological activity of the above 20-mer, we synthesized seven octapeptides, each overlapping by six residues, covering the entire sequence and studied their effect on platelet activation as well as fibrinogen binding to activated platelets. We show for the first time that octapeptides containing the RAD sequence are capable of inhibiting platelet aggregation and secretion as well as fibrinogen binding to the activated alphaIIbbeta3, possibly interacting with the ligand rather than the receptor. This suggests that the RAD sequence, common to all the inhibitory peptides, is critical for their biological activity. However, the presence of the YMES sequence, adjacent to RAD, significantly increases the peptide's biological potency. The development of such inhibitors derived from the 313-332 region of the alphaIIb subunit may be advantageous against the RGD-like antagonists as they could inhibit platelet activation without interacting with alphaIIbbeta3, thus failing to further induce alphaIIbbeta3-mediated outside-in signalling.     

Significance of the laboratory diagnosis of hypercoagulability

Alterations characteristic of the hypercoagulation syndrome such as changes in fibrinogen content, in FDP concentration and platelet counts as well as the presence of fibrinogen complexes were demonstrated by laboratory findings in 175 patients with severe diseases and disturbances of haemostasis. Twenty per cent of the patients showed no clinical signs of disturbances of haemostasis, in 32.5 per cent pronounced venous thrombosis occurred, bleeding complications arised in 28 per cent and microthrombosis developed in 20 per cent. The different findings in the individual groups are assumed to be due not only to haemostatic factors but also to other ones.     

Pertussis toxin can activate human platelets. Comparative effects of holotoxin and its ADP-ribosylating S1 subunit

The activation of phospholipase C in human platelets is coupled to agonist receptors via guanine nucleotide-binding protein(s), and prior treatment of permeabilized platelets with GTP gamma S, GDP beta S, or pertussis toxin modifies platelet responses to agonists. Pertussis toxin is thought to act primarily as an uncoupler of Gi from cell receptors due to its ADP-ribosylating activity. However, we have found that pertussis toxin by itself can act as an agonist for intact or permeabilized platelets. Though believed to lack receptors for pertussis toxin, intact platelets, when incubated with the toxin (5-20 micrograms/ml), undergo aggregation and accumulate inositol trisphosphate and phosphatidic acid. Treatment of platelets with aspirin, incubation in the presence of creatine phosphate/creatine phosphokinase, or omission of Ca2+ and fibrinogen do not affect toxin-mediated phospholipase C activation. These effects are not observed with the ADP-ribosylating S1 monomer of toxin in intact or permeabilized platelets. Further, modification of the holotoxin with N-ethylmaleimide eliminates the toxin's ADP-ribosylating activity but does not affect its promotion of platelet aggregation and phospholipase C activation. Therefore, the activating effect of holotoxin is separable from its ADP-ribosylating activity and does not depend either upon cyclooxygenase or the ADP that may be released during platelet activation. Given the combined potentially stimulatory and inhibitory effects of pertussis holotoxin, we suggest caution in interpretation of results with this material.     

Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.     

Effect of cerebrocrast on the function of human platelets and release of the arachidonic acid from plasma membrane

Diabetes mellitus (DM) is accompanied by several cardiovascular complications such as coronary artery disease, atherosclerosis, hypertension, cerebral and myocardial infarction, etc. DM induces the alteration of platelet functions including activation, hyperaggregation, adhesiveness, and formation of thrombi. Release of AA from phospholipids of the PM, synthesis of TxA(2),PGE(2), activity of PLA(2), and PLC are increased in the platelets of the DM patients. Stimulation of PLA(2) activity and accumulation of bioactive metabolites such as AA, its oxygenated derivatives, prostaglandins and PAF can evoke glucose production, also. In this study we explored the effect of the 1,4-dihydropyridine compound cerebrocrast at a low concentration (10(-6)-10(-8)M) on the level of intracellular calcium in unstimulated human platelets and those stimulated with thrombin as well as release of [(3)H] AA from phospholipids of platelet PM. Cerebrocrast at a concentration of 10(-6) M decreased the basal level of intracellular calcium concentration (platelets were loaded with Fura-2) in unstimulated as well as in thrombin stimulated platelets. Cerebrocrast at concentrations of 10(-6), 10(-7), 10(-8) M inhibited release of [(3)H] AA from phospholipids of platelet PM. We conclude that blockade of human platelet activation with cerebrocrast can prevent aggregation, adhesion and formation of thrombi. The inhibition of [(3)H] AA release from phospholipids of platelet PM can prevent formation of eicosanoids such as TxA(2), PGG(2), and PGH(2) plus AA oxygenated derivatives. These effects of cerebrocrast are very significant in the treatment of DM-evoked cardiovascular complications.     

Proteolysis leads to the appearance of the long form of beta3-endonexin in human platelets

After vessel injury, platelets adhere to the subendothelial matrix. Platelet adhesion leads to activation of the platelet integrin alpha(IIb)beta3, which then binds to fibrinogen, leading to platelet aggregation. It has been shown that a beta3-integrin binding protein, beta3-endonexin, can activate the integrin alpha(IIb)beta3 expressed in transfected CHO cells. Several isoforms of beta3-endonexin are known but it is not clear which isoforms are expressed in platelets and what role they may play during haemostasis. Here, we show that the long form of beta3-endonexin (EN-L) can be detected in platelet lysates several hours after thrombus formation, after long-term storage of platelets and after glucose deprivation. After subcellular fractionation, EN-L is found in the detergent insoluble fraction suggesting that it might be associated with the cytoskeleton. EN-L generation is temperature and Ca++ dependent and requires physiological salt concentrations. Proteolysis is responsible for the appearance of EN-L since a calpain inhibitor prevents its formation and the addition of calpain to platelet lysates induces its formation. The appearance of EN-L seems to be linked to apoptotic events occurring during long-term storage of platelets and, possibly, during late steps of haemostasis after thrombus formation.