Platelet Gelsolin Research Articles

Effect of ticagrelor and clopidogrel dual antiplatelet therapy on MPVLR, MAADP, and AA inhibition rate in acute coronary syndrome patients after percutaneous coronary intervention.

To explore the effects of ticagrelor and clopidogrel dual antiplatelet therapy on the mean platelet volume-to-lymphocyte ratio (MPVLR), maximum amplitude of adenosine diphosphate-induced platelet-fibrin clots (MAADP), and arachidonic acid (AA) inhibition rates in patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI). A total of 120 patients with ACS undergoing elective PCI in our hospital between March 2020 and November 2021 were recruited. Patients were divided into 2 groups using the random number table method, with 60 patients in each group. The control group received clopidogrel + aspirin dual antiplatelet therapy, while the study group received ticagrelor + aspirin dual antiplatelet therapy. MPVLR, MAADP, and AA inhibition rates were compared between the 2 groups. Platelet activation indices, platelet micro PNA-223, and platelet gelsolin levels were measured before and 4 weeks after PCI. Changes in cardiac function indices, bleeding rates, and major adverse cardiovascular events (MACE) were compared between groups. The MAADP score of the study group was lower than that of the control group 3 days after surgery (P < .05). Compared with before surgery, CD62p, CD63, miR-223, PAC-1, platelet membrane glycoprotein IIb/IIIa complex, and gelsolin levels markedly decreased in both groups 4 weeks after surgery (P < .05). The platelet activation index and platelet miR-223 and gelsolin levels were significantly lower in the study group than in the control group 4 weeks after surgery (P < .05). The overall platelet inhibition effect was significantly better in the study group than in the control group (P < .05). Compared with before surgery, the left ventricular ejection fraction and stroke volume were significantly increased, and the left ventricular end-diastolic volume and left ventricular end-diastolic diameter significantly decreased in both groups 4 weeks after surgery (P < .05). No significant differences were found between the 2 groups in terms of the incidence of bleeding events or MACE (P > .05). Ticagrelor is more effective than clopidogrel for platelet inhibition after PCI in patients with ACS and is worthy of clinical recommendation.

GW24-e2354 Effect of Xiongshao Capsule on the platelet activation level and platelet gelsolin in rat model of Acute Myocardial infarction in vivo

ObjectivesTo investigate the effects of active ingredients of Chuanxiong rhizome and red peony root (Chuangxiongol and paeoniflorin) on the platelet activation level and platelet gelsolin in rat model of acute...

Correlation between Platelet Gelsolin and Platelet Activation Level in Acute Myocardial Infarction Rats and Intervention Effect of Effective Components of Chuanxiong Rhizome and Red Peony Root

The biological role of platelet gelsolin in platelet activation of acute myocardial infarction is not defined. In order to provide a potential new antiplatelet target for Chinese medicine and to elucidate the contribution of Xiongshao capsule, the effective components of Chuanxiong rhizome and red peony root, in this study, we randomly allocated Sprague Dawley rats to left anterior descending coronary artery ligation or sham surgery and different drug prophylaxis as control. We found that gelsolin is highly expressed in platelet rich plasma and lowly expressed in platelet poor plasma, accompanied by the high platelet activation level in model rats; plasma actin filaments and mean fluorescence intensity (MFI) of platelet calcium ion increased and plasma vitamin D binding protein decreased in model rats. Xiongshao capsule could inhibit the gelsolin expression in platelet rich plasma and ischemic heart tissue simultaneously and reduce the level of plasma F-actin and MFI of platelet calcium ion. Our study concludes that platelet gelsolin is an important contributor to platelet activation, and platelet gelsolin inhibition may form a novel target for antiplatelet therapy. Xiongshao capsule may be a promising Chinese medicine drug for antiplatelet and aspirin-like cardioprotection effect.

Correlation between platelet gelsolin levels and different types of coronary heart disease

Gelsolin is an important cytoskeletal protein of platelets and studies have shown a close relationship between gelsolin and cardiovascular disease. However, the role of gelsolin in the development of coronary heart disease (CHD) is unclear. In this study, we record the distribution of gelsolin in human platelets and plasma and its association with different types of CHD. This study included 114 cases, with 33 stable angina pectoris (SAP) cases, 81 acute coronary syndrome (ACS) cases—composed of 39 unstable angina pectoris (UAP) and 42 acute myocardial infarction (AMI) cases, and 31 healthy control participants. Gelsolin concentration in platelet rich plasma (PRP) and platelet poor plasma (PPP), actin filament (F-actin) and Gc-globulin of PPP were determined by enzyme-linked immunoadsorbent assay (ELISA). The fluorescence intensity of CD62p and cytoplasmic calcium ([Ca2+]i) in human platelets measured by flow cytometry. We also used turbidimetry to detect the platelet aggregation rate (PAR). We analyzed the correlation between platelet gelsolin concentration and CD62p or plasma F-actin levels among each different patient group. Compared with the control group, the gelsolin level in PRP of UAP and AMI groups increased significantly (P<0.01), while the gelsolin level in PPP of all the three patient groups decreased markedly (P<0.01), and the CD62p, PAR, [Ca2+]i of platelets, F-actin and Gc-globulin of the UAP and AMI groups increased significantly (P<0.01). Compared with the SAP group, the gelsolin level in PRP, the PAR, [Ca2+]i of platelets and CD62p of other two groups increased significantly (P<0.01), F-actin of the AMI group increased markedly (P<0.01). Platelet cytoskeleton protein dynamics vary among the different types of CHD. Platelet gelsolin levels are markedly increased and accompanied by increased platelet activity, F-actin and [Ca2+]i of ACS patients, while gelsolin levels in PPP are markedly lower. Abnormally increased platelet gelsolin levels show high positive correlation with the level of platelet activity. Therefore, platelet gelsolin might be a novel molecular marker and/or a new potential therapeutic target of anti-platelet therapy of ACS.

Correlation between platelet gelsolin level and different types of coronary heart disease

PurposeIn this study, we observe the distribution of gelsolin in human platelet and plasma and its association with different types of coronary heart disease (CHD).MethodsOne hundred and fourteen cases were...

Research on the correlation between platelet gelsolin and blood-stasis syndrome of coronary heart disease

To study the distribution of gelsolin in human platelet and plasma, and the association with blood-stasis syndrome (BSS) of coronary heart disease (CHD). Sixty patients with CHD (30 in BSS group and 30 in non-BSS group) and 30 healthy subjects (control group) were included in this study. The classification of the syndrome was based on clinical symptoms and signs. Gelsolin concentration in platelet rich plasma (PRP), platelet poor plasma (PPP), filamentous actin (F-actin) and group-specific component globulin (Gc-globulin) of PPP were determined by enzyme-linked immunosorbent assay (ELISA). The fluorescence intensity of CD62p and cytoplasmic calcium ([Ca(2+)](i)) in human platelets of patients and healthy persons was measured with flow cytometry. Compared with the control group, gelsolin in PRP of the BSS group increased significantly (P<0.01), while that in PPP of the BSS and non-BSS groups decreased markedly (P<0.05), the CD62p, [Ca(2+)](i) of platelet, F-actin, and Gc-globulin of the BSS and non-BSS groups increased significantly (P<0.01). Compared with the non-BSS group, the gelsolin concentration in PRP of BSS group increased significantly (P<0.01), the [Ca(2+)](i) of platelet of the BSS group increased markedly (P<0.01), while the F-actin and Gc-globulin of the BSS group had no statistical defference (P>0.05). Gelsolin concentration in PRP was increased and accompanied by the elevated [Ca(2+)](i) of platelet in CHD with BSS, while gelsolin in PPP were lowered markedly. We speculate that plasma gelsolin may clear F-actin from circulation, thus resulting in depletion of plasma gelsolin significantly. This, in addition to the increased calcium influx of platelets, may lead to the gelsolin abnormal expression on platelets during the process of BSS in CHD. Therefore, platelet gelsolin may serve as a new potential biomarker and a therapeutic target of BSS in CHD.

Studies on Platelet Storage Biomarkers: Effect of Different Protein Extraction Buffers on Platelet Gelsolin and B-Actin Profiling

Platelet concentrates (PCs) are one of the blood cell components that play a major role in transfusion medicine. The shelf-life of PC is 5–7 days at 22°C. During storage PCs undergo certain morphological changes as well as physiological changes such as loosing of its surface glycoproteins, and reduced aggregation response to its agonists, which are collectively referred to as “storage lesions”. To date there is no in vitro “gold standard” that unequivocally serves as a surrogate marker to predict PC quality in vivo following transfusion. A comprehensive survey of reports in this field clearly indicate that there is some confusion with regards to the determination of which platelet protein or a panel of proteins could serve as potential markers associated with PC storage. Following a careful review of all available information, we recognized that perhaps one core issue to the observed discrepancies could be the buffer system that is being utilized by individual laboratories in extracting proteins in profiling protein alterations in PCs during storage. To address this, in the present study we analyzed two platelet proteins, b-actin and gelsolin-related 40 kDa protein. Profiles of these two proteins were previously reported by others to be altered during PC storage and hence implicated to be useful as biomarkers of platelet storage. By employing 3 different protein extraction buffer systems to isolate proteins from platelets at three storage intervals (Table 1), our results indicate thatb-actin and gelsolin 40 kDa protein levels do not appear to change with time in platelet storage and therefore may not be useful as platelet storage markers andBuffer systems do make a difference in extracting different platelet proteins for analysis, for example 40 kDa gelsolin was not extractable in buffer 3 (Table 1), suggesting that one should be careful in selecting a buffer system for platelet proteomics to have meaningful results.Same level of 40 kDa GelsolinSame level of b-actinBuffer systemDay 0Day 5Day 16Day 0Day 5Day 16Buffer 1Not identifiedYesYesYesYesYesBuffer 2Not identifiedYesYesYesYesYesBuffer 3Not identifiedNot identifiedNot identifiedYesYesYesThe findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Isolation and characterization of platelet gelsolin

Isolation and characterization of platelet gelsolin

Anti-gelsolin antibody for detection of plasma and platelet gelsolin

Anti-gelsolin antibody for detection of plasma and platelet gelsolin

Gelsolin is Ca 2+-sensitive regulator of actomyosin system in platelet

Effects of gelsolin on the actomyosin system in platelet have been studied. MgATPase activity of platelet actomyosin is enhanced up to two folds by 200 nM of platelet gelsolin in the presence, but not in the absence of Ca ion. The half maximum enhancement is observed at the concentration of Ca2+ around 10(-5) M. The effect of gelsolin to enhance the ATPase activity of actomyosin is potentiated by tropomyosin, which is a Ca2+-insensitive actomyosin enhancer. The results indicate that gelsolin may control the activity of actomyosin system in platelets.

Preparation and characterization of pig plasma and platelet gelsolins.

Pig plasma gelsolin has been prepared by a revised method involving poly(ethylene glycol) precipitation, chromatography on CM-cellulose and affinity chromatography on actin-Sepharose. Pig platelet gelsolin has been prepared by chromatography on DEAE-cellulose and actin-Sepharose. Partial chemical and proteolytic cleavage shows that the two proteins are closely related in their fragmentation patterns. The amino acid sequences are identical at the N-terminus of the platelet protein, but the plasma protein has an additional nine residues on the N-terminal side of the common sequence. Calcium binding studies show that the plasma protein has similar calcium binding properties to both macrophage and platelet gelsolins.

Gelsolin inhibits nucleotide exchange from actin.

The effects of platelet gelsolin on the state and exchangeability of the nucleotide bound to skeletal muscle actin monomer have been investigated. In the presence of Ca2+, a stable ternary complex consisting of two actins and one gelsolin is formed. Removal of Ca2+ from this species results in the formation of a highly stable binary gelsolin-actin complex. The interaction of gelsolin with actin monomer has no effect on the virtually negligible [less than 0.01 mol of Pi X h-1 X (mol of actin)-1] intrinsic ATPase activity of actin monomer (in the absence of Mg2+). A single molecule of ATP is bound to the binary complex while two molecules of ATP are bound to the actins within the ternary complex. The ATP within the binary complex is nonexchangeable, and only one of the two ATP molecules in the ternary complex is exchangeable. In the latter case the rate constant for this nucleotide exchange is decreased compared to that for free actin monomer. These results demonstrate the nonequivalence of actin monomers within the ternary complex. The involvement of these oligomeric complexes of gelsolin and actin in the expression of the activity(ies) of gelsolin is discussed.

Definition of an N-terminal actin-binding domain and a C-terminal Ca2+ regulatory domain in human brevin.

Brevin is a Ca2+-modulated actin-associated protein that will sever F-actin and cap barbed filament ends. Limited proteolysis with chymotrypsin or subtilisin cleaves the molecule approximately in half. Cleavage is approximately 10-fold more rapid in Ca2+ than in EGTA. The two fragments are readily separated from each other and from undigested brevin by high pressure liquid chromatography on a DEAE resin. A 40,000-mol-wt fragment from the N-terminal is not retained by DEAE, while a 45,000-mol-wt C-terminal fragment binds more tightly than brevin. The N-terminal fragment retains approximately 10% of the nucleation activity, caps barbed ends, and retains 50% of the total severing activity defined by dilution induced depolymerization of pyrenyl actin, but, in contrast to brevin, none of these functions are affected by Ca2+. Fluorescent actin binding studies and gel-filtration demonstrate that the 40,000-mol-wt fragment binds two actin monomers. The 45,000-mol-wt C-terminal fragment has no severing, nucleating, or capping activity. Cross-reaction with two monoclonal antibodies against two specific Ca2+-induced conformations of human platelet gelsolin suggest that both Ca2+ binding sites are located on the carboxyl half of the brevin molecule. One epitope, defined as the rapidly exchanging Ca2+ binding site in the gelsolin-actin complex, is lost when a 20,000-mol-wt fragment is cleaved from the carboxyl terminal. The second epitope, related to the poorly exchanging Ca2+ binding site in the complex, is nearer the middle of the brevin molecule.

Immuno-identification of Ca2+-induced conformational changes in human gelsolin and brevin.

Gelsolin is a 90,000-mol-wt protein with two actin and two high affinity calcium-binding sites that can form complexes with Ca2+ ions and monomeric actin. These complexes will nucleate filament growth and cap the barbed end of filaments, but will not fragment F-actin. Uncomplexed gelsolin severs F-actin. (Bryan, J., and L. M. Coluccio, 1985, J. Cell Biol., 101:1236-1244). These associations with actin are modulated by Ca2+. We have purified and characterized monoclonal antibodies that recognize Ca2+-induced conformational changes in human platelet gelsolin (G) and human plasma brevin (B), a closely related protein. Two hybridomas, 8G5 and 4F8, were adapted to growth in serum-free medium. 8G5 was found to secrete an IgG; 4F8 secretes an IgA. On immunoblots, both antibodies gave a strong reaction if Ca2+ was present, but gave barely detectable reactions if EGTA was used. 8G5 IgG-Sepharose columns retained gelsolin (as GCa2) or brevin (as BCa2) in 0.1 mM CaCl2 containing buffers, but released these molecules when eluted with 4 mM EGTA. 8G5 IgG-Sepharose columns also retained gelsolin-actin-Ca2+ complexes, as GA1Ca2 or higher oligomers from platelet extracts containing 0.1 mM CaCl2. Elution with 4 mM EGTA released material that gel filtration showed to be the EGTA-stable 130,000-mol-wt gelsolin-actin complex, GA1Ca1. The results demonstrate that the 8G5 IgG recognizes a conformation of gelsolin or brevin induced by binding of an easily exchangeable Ca2+ ion. Actin is not required for this conformational change, and the antibody discriminates, for example, GCa2 from G and GCa1. A 4F8 IgA-Sepharose column retained brevin or gelsolin in 0.1 mM CaCl2-containing buffers, but, like the 8G5 IgG, released these molecules when eluted with 4 mM EGTA. The 4F8 IgA column also retained gelsolin or brevin-actin-Ca2+ complexes, for example, as BA1Ca2, or higher oligomers, in 0.1 mM CaCl2. No protein was recovered, however, upon elution with 4 mM EGTA, but elution with 0.1 M glycine-HCl, pH 2.8, released bound brevin or gelsolin and actin. Similarly, preformed brevin-actin-Ca2+ complex, equilibrated with EGTA, was retained by 4F8 IgA-Sepharose. The results demonstrate that the 4F8 IgA recognizes a conformation of gelsolin or brevin that is maintained and presumably induced by binding of a nonexchangeable Ca2+ ion that is trapped in the complex.

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes.

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F-actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.

Gelsolin, a Ca2+-dependent actin-binding protein in a hamster insulin-secreting cell line.

Using a gel overlay technique we have previously described a 90,000-mol wt actin-binding protein in a number of hormone-secreting tissues and tentatively identified this protein as gelsolin. Gelsolin is a protein that cuts or solates cross-linked actin filaments and can also serve as a nucleating site for actin polymerization. The objective of this study was to isolate this protein from a hamster insulin-secreting (HIT) cell line and compare the immunologic properties and peptide maps of purified rabbit macrophage gelsolin, human platelet gelsolin, and the HIT cell 90,000-mol wt protein. DNase I-Sepharose retained the HIT cell actin-binding proteins in 1 mM CaCl2; some of the 90,000-mol wt protein could then be eluted with 1 mM EGTA. The remaining actin-binding proteins were eluted using a buffer containing SDS. The EGTA peak fractions contained two major protein bands of Mr = 90,000 and 42,000, which suggested that a 90,000-mol wt-actin complex was eluted from the DNase I-Sepharose column. Specific antibodies to the human platelet and rabbit macrophage gelsolins bound to the 90,000-mol wt bands in the eluates, but did not crossreact with other actin-binding proteins. Indirect immunofluorescence using an anti-human platelet gelsolin antibody localized the 90,000-mol wt protein to stress fibers that were also stained with phalloidin, which suggested that gelsolin is associated with actin in vivo. Tryptic peptide maps of all three radioiodinated gelsolins were virtually indistinguishable. Thus, gelsolin is a highly conserved gene product found in at least three diverse cell types, an insulin-secreting beta cell line, macrophages, and platelets, and may link a transient increase in Ca2+ cellular levels with changes in actin polymerization and/or the gel-sol state of these cells.

Isolation and characterization of human blood platelet gelsolin

A 90-kDa protein-actin stable complex was purified from blood platelets by a short and efficient procedure giving at the same time actin used in polymerization assays. 90-kDa protein free of actin was prepared from the complex by 8 M ureau treatment and renaturation. By its molecular mass, immunological cross-reactivity with macrophage gelsolin and its effect on G- and F-actin the 90-kDa protein appears as the platelet gelsolin.

Actin-gelsolin interactions. Evidence for two actin-binding sites.

We have used a fluorescence enhancement of actin labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-actin) to study the interactions between rabbit skeletal muscle G-actin and either purified platelet gelsolin or a 130-kDa binary complex of platelet actin and gelsolin that is stable in EGTA and can be purified from human platelets. We have delineated four binding reactions. The exchange of Mg2+ for Ca2+ on the divalent cation-binding site of NBD-actin gives a small fluorescence increase. Binding of monomeric NBD-actin to the binary complex results in a 2.5-fold increase in the emission at 530 nm in the presence of Ca2+ and a 2-fold increase in the presence of EGTA. Titration experiments show that, under nonpolymerizing conditions, one additional actin is bound to the 130-kDa species to form a ternary complex. This binding is Ca2+-sensitive. Purified gelsolin does not appear to bind to NBD-actin in the presence of EGTA, as determined by fluorescence enhancement, gel filtration, or sedimentation measurements, but the addition of Ca2+ promotes rapid binding with a 1.6-1.7-fold enhancement of the emission intensity. A comparison of the relative fluorescence yields/NBD-actin molecule for a binary complex of gelsolin and one NBD-actin, a ternary complex of gelsolin and two NBD-actin molecules, and a ternary complex with an unlabeled actin in the EGTA-stable site and an NBD-actin in the second site indicates that the first NBD-actin, in the EGTA-stable site, does not give a fluorescence increase on binding but the second one does. Finally, we have demonstrated that one molecule of 45Ca2+ is "trapped" when the binary complex is formed and cannot be removed by EGTA. A summary model for these reactions is presented that indicates the interaction between actin and gelsolin is not a freely reversible Ca2+-controlled reaction.

Platelet activation induces the formation of a stable gelsolin-actin complex from monomeric gelsolin.

We have studied the interactions between gelsolin and actin in crude extracts from activated and unactivated platelets and in mixtures of purified platelet gelsolin and muscle actin. Extracts were prepared using 10 mM EGTA from human platelets treated either with 100 microM aspirin and 2.5 mM tetracaine to retard activation or with the calcium ionophore A23187 to effect activation. The extracts were fractionated by gel filtration on Sephadex G-150 or by sedimentation on sucrose gradients and then analyzed using anti-gelsolin immunoblots and actin filament nucleation assays. The nucleation activity in both extracts was associated with gelsolin. The activity in the extracts from unactivated platelets sedimented with an S value of 5.2 and had an Mr = 90,000. The activity in the extracts prepared with EGTA from activated platelets sedimented at 6.8 S and had an Mr = 130,000. We have shown previously that the Mr = 130,000 species is an EGTA-stable binary complex of one actin and one gelsolin. Transient exposure of the extracts from unactivated platelets to 100 microM Ca2+ and subsequent fractionation in EGTA-containing buffers demonstrated that the formation of the binary complex occurs in the presence of Ca2+. Fractionation in the presence of 100 microM Ca2+ demonstrated higher order complexes including a ternary complex with a sedimentation constant of 8.2 S and an Mr = 165,000. Sedimentation and gel filtration experiments using purified platelet gelsolin and rabbit skeletal muscle actin demonstrated that formation of the EGTA-stable binary complex required Ca2+. At least one additional actin is bound to the binary complex in the presence of Ca2+, but is not sufficiently stable to be purified when EGTA is added. The results suggest that gelsolin exists either as a monomer or perhaps as a weak complex with actin in unactivated platelets but complexes tightly with actin during the transient Ca2+ rise that occurs during activation.

Cytochalasin D and platelet gelsolin accelerate actin polymer formation. A model for regulation of the extent of actin polymer formation in vivo.

Cytochalasin D and platelet gelsolin accelerate actin polymer formation. A model for regulation of the extent of actin polymer formation in vivo.