Molecular mechanisms of platelet exocytosis: insights into the "secrete" life of thrombocytes.
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Blood Platelet Disorders↗
Biomedical subjects
J Polgár
Publications and source records attributed to J Polgár.
A critical role for N-ethylmaleimide-sensitive fusion protein (NSF) in platelet granule secretion.
The molecular mechanisms that regulate membrane targeting/fusion during platelet granule secretion are not yet understood. N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements of a conserved molecular machinery for membrane targeting/fusion that have been detected in platelets. We examined whether NSF, an ATPase that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule secretion. Peptides that mimic NSF sequence motifs inhibited both alpha-granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar amino acids in a scrambled sequence inhibited platelet secretion. The peptides that inhibited platelet granule secretion also inhibited the human recombinant alpha-SNAP-stimulated ATPase activity of recombinant NSF. It was also found that anti-NSF antibodies, which inhibited recombinant alpha-SNAP-stimulated ATPase activity of NSF, inhibited platelet granule secretion in permeabilized cells. The inhibition by anti-NSF antibodies was abolished by the addition of recombinant NSF. These data provide the first functional evidence that NSF plays an important role in platelet granule secretion.
Amino Acid Sequence↗
Adenosine diphosphate (ADP) and ADP receptor play a major role in platelet activation/aggregation induced by sera from heparin-induced thrombocytopenia patients.
The molecular basis for heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, is not yet fully understood. We found that pretreatment of platelets with AR-C66096 (formerly FPL 66096), a specific platelet adenosine diphosphate (ADP) receptor antagonist, at a concentration of 100 to 200 nmol/L that blocked ADP-dependent platelet aggregation, resulted in complete loss of platelet aggregation responses to HIT sera. AR-C66096 also totally inhibited HIT serum-induced dense granule release, as judged by measurement of adenosine triphosphate (ATP) release. Apyrase, added to platelets at a concentration that had only minor effects on thrombin- or arachidonic acid-induced aggregation, also blocked completely HIT serum-induced platelet aggregation. Furthermore, AR-C66096 inhibited platelet aggregation and ATP release induced by cross-linking Fc gamma RIIA with specific antibodies. These data show that released ADP and the platelet ADP receptor play a pivotal role in HIT serum-induced platelet activation/aggregation. The thromboxane receptor inhibitor, Daltroban, had no effect on HIT serum-induced platelet activation whereas GPIIb-IIIa antagonists blocked platelet aggregation but had only a moderate effect on HIT serum-induced dense granule release. Pretreatment of platelets with chondroitinases but not with heparinases resulted in concentration dependent inhibition of HIT serum-induced platelet aggregation. These novel data relating to the mechanism of platelet activation induced by HIT sera suggest that the possibility should be examined that ADP receptor antagonists or compounds that inhibit ADP release may be effective as therapeutic agents for the prevention or treatment of complications associated with heparin therapy.
Adenosine Diphosphate↗
Snake venom C-type lectins as tools in platelet research.
The snake C-type lectins are a major group of proteins present in venoms that fold to a structure with similarities to classic C-type lectins. The loop that would be involved in calcium and sugar binding is truncated and heterodimers are linked by a disulphide bond and by swapping loop domains between the subunits. M any of these C-type lectins interact with platelet receptors to inhibit or induce platelet activation. The use of these C-type lectins to investigate platelet function is discussed and illustrated with specific examples.
Journal Article↗
Platelet glycoprotein Ia* is the processed form of multimerin--isolation and determination of N-terminal sequences of stored and released forms.
Glycoprotein Ia* (GPIa*), a very high molecular mass, platelet alpha-granule protein consisting of 167 kDa subunits disulphide-linked in a multimeric structure, was first described by Bienz and Clemetson in 1989 (J. Biol. Chem. 264, 507-514). In 1991 Hayward et al. (J. Biol. Chem. 266, 7114-7120) independently identified a platelet protein with multimeric structure. Despite strong similarities to GPIa* they concluded that it was a novel multimeric protein and named it first p-155 and later, multimerin. Multimerin has also been found in endothelial cells and has been cloned recently from an endothelial cell cDNA library. This has made it possible for us to clarify the relationship between GPIa* and multimerin. GPIa* was isolated from platelet releasate and the N-terminal sequence of 167 kDa and 155 kDa subunit species were determined. The N-terminal 15 amino acids of GPIa* were identical to the deduced amino acids 184-198 of endothelial multimerin. The N-terminal sequence of the 155 kDa protein was identical to the deduced amino acids 318-326 of multimerin. Thus, platelet GPIa* (167 kDa) is the main processed form of multimerin stored in platelet alpha-granules. The GPIa*/processed multimerin (167 kDa) still contains an RGDS sequence near its N-terminus as well as an EGF domain which may be involved in binding to the platelet surface after release. This sequence and domain are cleaved off in the p-155 form, described earlier as platelet multimerin, which is probably formed after release from alpha-granules.
Amino Acid Sequence↗
Human group II 14 kDa phospholipase A2 activates human platelets.
Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.
Arachidonic Acid↗
Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor.
Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma chain, phospholipase Cgamma2, p72(SYK), c-Cbl, and p36-38. However, unlike collagen, pp125(FAK) and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72(SYK), showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha2beta1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma chain and, by a mechanism previously described in T- and B-cells, activate p72(SYK) that is critical for downstream activation of platelets.
Amino Acid Sequence↗
Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper).
Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein.
Amino Acid Sequence↗
Asp-49 is not an absolute prerequisite for the enzymic activity of low-M(r) phospholipases A2: purification, characterization and computer modelling of an enzymically active Ser-49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper).
Several studies have shown that Asp-49 is the residue that controls calcium binding in, and so plays a critical role in the calcium-mediated activation of, low-M(r) group I-III phospholipases A2 (PLA2s). The present paper provides experimental evidence that Asp-49 is not an absolute prerequisite for the enzymic activity of PLA2s, and that proteins with amino acid(s) other than Asp at position 49 can exhibit significant phospholipase activity. The purification, complete amino acid sequence and characterization of ecarpholin S, a PLA2 from Echis carinatus sochureki (saw-scaled viper) venom, is described. This single-chain, 122-amino-acid, basic (pI 7.9) protein is a group II PLA2. Although Asp-49 is replaced by Ser and Tyr-28 by Phe (both of these positions being involved in the Ca(2+)-binding site of PLA2s), the lipolysis of soybean phosphatidylcholine and egg yolk in the presence of 10 mM CaCl2 was 1.5 times and 2.9 times greater respectively with ecarpholin S than with recombinant human group II PLA2. The Ca(2+)-dependencies of the enzymic activities of ecarpholin S and rPLA2 were found to be similar. Ecarpholin S added to washed platelets induced aggregation; the presence of Ca2+ was a prerequisite for this platelet-aggregating effect. Computer modelling of the Ca(2+)-binding site of Ser-49 PLA2 compared with the Asp-49 and Lys-49 forms, for which crystallographic data exist, shows that the Ca(2+)-binding site is sterically blocked by Lys-49 but not by Ser-49; in the latter, the Ser hydroxy group may replace the Asp carboxylate in stabilization of Ca2+ binding. Sequence comparisons of ecarpholin S and other low-M(r) PLA2s predicts the presence of a Ser-49 group in the protein family of low-M(r) PLA2s that is distinct from the Asp-49 and Lys-49 groups.
Amino Acid Sequence↗
Transformation of cellular factor XIII into an active zymogen transglutaminase in thrombin-stimulated platelets.
The cellular form of blood coagulation factor XIII (FXIII) is present in platelets, monocytes and macrophages. During long-term stimulation of platelets by thrombin cellular FXIII becomes activated and cross-links proteins, however, the mechanism of its activation has not been elucidated. It was shown that, contrary to plasma FXIII, the intracellular activation of platelet FXIII does not involve proteolysis. FXIII remained intact in thrombin-activated platelets, i.e., the activation peptide was not removed from the molecule. Part of the zymogen FXIII molecules, however, assumed an active configuration as was demonstrated both by the measurement of transglutaminase activity and by active-site-SH titration. These findings clearly indicate that during platelet activation, when intracellular Ca2+ concentration is raised, a slow non-proteolytic transformation of FXIII zymogen into an active transglutaminase occurs.
Blood Platelets↗
[Stress capacity and extension of total removable prostheses].
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Aged↗
Additional GPI-anchored glycoproteins on human platelets that are absent or deficient in paroxysmal nocturnal haemoglobinuria.
In order to detect novel glycophosphatidylinositol (GPI)-anchored platelet proteins, human platelets were incubated with PI-specific phospholipase C (PI-PLC) and the supernatant was analysed by PAGE and silver-staining for additional protein bands. PI-PLC treatment resulted in the appearance of at least two additional novel GPI-linked glycoproteins (GP), GP500 and GP175, in the supernatant. Their presence on the platelet plasma membrane surface was demonstrated by periodate/[3H]borohydride surface-labelling. Activation of platelets did not enhance the amount of GP500 and GP175 that could be cleaved by PI-PLC. In Triton X-114 phase partitioning of platelet membranes the membrane form of GP175, mfGP175, was in the Triton phase while mfGP500 was found in the water phase. Neither GP500 nor GP175 were present in the supernatant of surface-labelled platelets treated with PI-PLC from 4 patients, diagnosed as having paroxysmal nocturnal haemoglobinuria (PNH), but the supernatant from platelets from healthy volunteers treated the same way contained both.
Blood Platelets↗
Platelet factor XIII becomes active without the release of activation peptide during platelet activation.
The potentially active A subunit of factor XIII of blood coagulation has also been detected in platelets and monocytes/macrophages through the exact function of this cellular protransglutaminase has not yet been elucidated. In physiological conditions the first step in the activation of plasma factor XIII is the removal of an activation peptide from the N-terminal end of subunit A by thrombin. The A subunit then, in the presence of Ca2+, dissociates from the inhibitory B subunit and assumes an active conformation. Cellular factor XIII, which lacks B subunit, can be proteolytically activated in vitro by thrombin and the intracellular Ca2+ sensitive protease, calpain, in the same way as plasma factor XIII subunit A, and calpain has been suggested as the intracellular protease involved in the activation of cellular factor XIII in platelets. In the present experiments it was shown by SDS PAGE that during long-term stimulation of platelets with thrombin nondisulfide-crosslinked high M(r) protein polymers not penetrating the concentrating gel were formed. The lack of these polymers in thrombin-stimulated factor XIII deficient platelets clearly indicated that their formation in normal platelets was due to factor XIII that became active during platelet activation. However, no release of the activation peptide could be detected by Western blotting during this process. Similarly, no proteolytic cleavage of factor XIII was detectable when platelets were stimulated by Ca2+ ionophore through this stimulus activated calpain as it was clearly demonstrated by the breakdown of major intracellular calpain substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
Blood Platelets↗
Non-proteolytic activation of cellular protransglutaminase (placenta macrophage factor XIII).
Plasma Factor XIII is a zymogen (plasma protransglutaminase) with the tetrametric structure A2B2, whereas the cellular protransglutaminase, i.e. Factor XIII in the platelet and monocyte/macrophage, consists exclusively of A subunits (A2). It is generally accepted that at Ca2+ concentrations comparable with that in plasma the proteolytic removal of an N-terminal activation peptide is the prerequisite for the Ca2(+)-induced formation of a catalytically active configuration of subunit A. In this study it was demonstrated that at high concentrations NaCl or KCl induced a non-proteolytic activation of cellular (placental macrophage) but not plasma protransglutaminase. The activation depended on time and salt concentration, and Ca2+, in the range 0-20 mM, greatly enhanced the activation process. At 1.25 M-NaCl maximal activation occurred within 60 min in the presence of 2 mM-CaCl2, and even at physiological NaCl concentration a slow progressive activation could be observed in the presence of Ca2+. The specific activity of salt-activated Factor XIII was 1.5-2.0-fold higher than that obtained after thrombin activation. The non-proteolytic activation of cellular protransglutaminase was abolished by the addition of subunit B of plasma Factor XIII in stoichiometric amount, which suggests that (one of) the physiological function(s) of the B subunit in plasma Factor XIII is to prevent the slow spontaneous activation of A subunit that would occur in a plasmatic environment.
Calcium↗
Effect of human seminal plasma on the lytic activity of natural killer cells and presumptive identification of participant macromolecules.
Using the lytic activity of natural killer (NK) cells as an in vitro parameter, the immunoregulatory properties of human seminal plasma (SePl) and participant macromolecules have been investigated. Significant (P less than 0.05) suppression of NK cell activity by SePl and chromatographically separable fractions was demonstrated in association with high and low molecular weight (Mr) macromolecules. SePl suppression was retained after heating to 56 degrees C for 30 min, and appeared to function at the level of the effector, rather than target cell. Physicochemical characterization of high and low Mr fractions provided presumptive identification of the participation of transglutaminase and prostaglandins as the principal molecules contributory to SePl immunosuppression.
Humans↗
Platelet vinculin: a substrate of activated factor XIII.
In addition to plasma, Factor XIII of blood coagulation (FXIII) is also present in the cytosol of platelets, monocytes and macrophages. However, its intracellular function has not yet been revealed. Activated Factor XIII (FXIIIa) is a transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) of highly restricted substrate specificity with only a few known protein substrates. In this report, we showed that FXIIIa can link dansylcadaverine, radiolabelled histamine and putrescine to vinculin. Quantitative determinations revealed that in the vinculin molecule a single glutamine residue can serve as acyl donor for the incorporation of small-molecular-weight amines. Vinculin could not be crosslinked to another vinculin molecule. It could be covalently bound, however, to fibrinogen, which indicates that the acyl donor glutamine residue can be engaged in an epsilon-(gamma-glutamyl)lysyl crosslink formation. Since it has been shown that platelet actin and myosin, two main components of cytoskeleton, are also substrates for FXIIIa, and that vinculin is associated to the cytoskeleton during platelet activation, the involvement of FXIII in the stabilization of cytoskeleton at certain phases of cellular function is a likely possibility.
Animals↗
Thrombomodulin inhibits the activation of factor XIII by thrombin.
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Enzyme Activation↗
Factor XIII of blood coagulation in human monocytes.
The presence of Factor XIII subunit a was demonstrated in human monocytes by immunoperoxidase staining using specific antisera against Factor XIII and its subunits. This finding was verified by immunobiochemical techniques, as well. In an immunoblotting system after SDS polyacrylamide gel electrophoresis of denatured monocyte homogenate a protein band comigrating with Factor XIII subunit a showed positive reaction with antibodies against this subunit or whole Factor XIII. In contrast, no subunit b of Factor XIII could be detected by either of these methods in monocytes. Activity measurements were carried out by the dansylcadaverine incorporation assay in the absence and presence of anti-Factor XIII antibody with and without thrombin activation. The expression of transglutaminase activity required thrombin and was completely abolished in presence of anti- Factor XIII antibody, which clearly indicate that practically all the transglutaminase activity measured in monocytes comes from Factor XIII. Factor XIII of monocytes and macrophages might have a role in formation of focal fibrin thrombi as well as in organization of stable, fibrinolysis resistant fibrin clot at the site of inflammation or around tumor cells.
Acyltransferases↗