scholarly journals Calpain I remains intact and intracellular during platelet activation. Immunochemical measurements with monoclonal and polyclonal antibodies

1987 ◽  
Vol 246 (2) ◽  
pp. 481-488 ◽  
Author(s):  
J A Samis ◽  
G Zboril ◽  
J S Elce
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Large Subunit ◽  
Active Form ◽  
Cytoskeletal Proteins ◽  
Exterior Surface ◽  
Activated Platelets ◽  
Igg1 Monoclonal Antibody ◽  
Calpain Ii

As a step towards understanding the physiological function of calpain (Ca2+-activated neutral proteinase, EC 3.4.22.17) in blood platelets, and in view of some suggestions that calpain is transferred to the platelet external surface during platelet activation, the enzyme was studied with immunochemical methods in resting and thrombin-activated cells. (1) A mouse IgG1 monoclonal antibody was prepared which binds strongly only to the denatured large subunit of human calpain I, and weakly to that of human calpain II. A polyclonal antibody raised against rat calpain II was available which, apart from binding strongly to rat calpain II, binds to the large subunits of human calpain I and II about equally. (2) With these antibodies, it was found that calpain could be detected in fixed platelets in suspension only after permeabilization with 0.1% saponin, and could not be detected on the exterior surface of resting or of activated platelets, or in the supernatant media of these platelets. It was concluded that calpain is not significantly externalized during platelet activation. (3) Immunoblotting showed that conversion of the larger calpain I subunit from 80 kDa into 76-78 kDa occurred only when thrombin-activated platelets were stirred to permit aggregation, and did not occur during unstirred thrombin activation. Although an action of calpain in the 80 kDa form on possible platelet substrates such as cytoskeletal proteins cannot be excluded, calpain is certainly not present as the 76-78 kDa form, which is assumed to be its active form, until aggregation is initiated.

scholarly journals Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 720-725 ◽  
Author(s):  
D Bienz ◽  
W Schnippering ◽  
KJ Clemetson
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Thrombin Activation ◽  
Strong Candidate ◽  
Triton X ◽  
Kinetics Of

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

scholarly journals Human solCD39 inhibits injury-induced development of neointimal hyperplasia

2010 ◽  
Vol 103 (02) ◽  
pp. 426-435 ◽  
Author(s):  
Rosemary Kraemer ◽  
Hao Shen ◽  
Rita Upmacis ◽  
Aaron Marcus ◽  
Elgilda Musi ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Platelet Activation ◽  
Blood Platelets ◽  
Neointimal Hyperplasia ◽  
Endothelial Injury ◽  
Vascular Endothelial ◽  
Leukocyte Infiltration ◽  
Post Injury ◽  
Arterial Lumen ◽  
Activated Platelets

SummaryBlood platelets provide the initial response to vascular endothelial injury, becoming activated as they adhere to the injured site. Activated platelets recruit leukocytes, and initiate proliferation and migration of vascular smooth muscle cells (SMC) within the injured vessel wall, leading to development of neointimal hyperplasia. Endothelial CD39/NTPDase1 and recombinant solCD39 rapidly metabolise nucleotides, including stimulatory ADP released from activated platelets, thereby suppressing additional platelet reactivity. Using a murine model of vascular endothelial injury, we investigated whether circulating human solCD39 could reduce platelet activation and accumulation, thus abating leukocyte infiltration and neointimal formation following vascular damage. Intraperitoneally-administered solCD39 ADP -ase activity in plasma peaked 1 hour (h) post-injection, with an elimination half-life of 43 h. Accordingly, mice were administered solCD39 or saline 1 h prior to vessel injury, then either sacrificed 24 h post-injury or treated with solCD39 or saline (three times weekly) for an additional 18 days. Twenty-four hours post-injury, solCD39-treated mice displayed a reduction in platelet activation and recruitment, P-selectin expression, and leukocyte accumulation in the arterial lumen. Furthermore, repeated administration of solCD39 modulated the late stage of vascular injury by suppressing leukocyte deposition, macrophage infiltration and smooth muscle cell (SMC) proliferation/migration, resulting in abrogation of neointimal thickening. In contrast, injured femoral arteries of saline-injected mice exhibited massive platelet thrombus formation, marked P-selectin expression, and leukocyte infiltration. Pronounced neointimal growth with macrophage and SMC accretion was also observed (intimal-to-medial area ratio 1.56 ± 0.34 at 19 days). Thus, systemic administration of solCD39 profoundly affects injury-induced cellular responses, minimising platelet deposition and leukocyte recruitment, and suppressing neointimal hyperplasia.

scholarly journals Preparations from Selected Cucurbit Vegetables as Antiplatelet Agents – An in Vitro Study

2021 ◽  
Author(s):  
Agata Rolnik ◽  
Bartosz Skalski ◽  
Anna Stochmal ◽  
Beata Olas
Keyword(s):  
Platelet Activation ◽  
Cucurbita Pepo ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
In Vitro Study ◽  
Blood Platelet ◽  
Arachidonic Acid Metabolism ◽  
Platelet Activity ◽  
Activated Platelets

Abstract Increased blood platelet activation plays an important role in cardiovascular diseases (CVDs). Recent experiments indicate that certain fruits and vegetables, including onion, garlic, and beetroot, have anti-platelet potential and therefore may reduce the likelihood of CVDs. While vegetables from the Cucuritaceae family are known to exerting beneficial antioxidant and anti-inflammatory effects, their effects on blood platelet activation are poorly understood. Therefore, the aim of the present study was to determine the effect on platelet adhesion of preparations from selected cucurbits: pumpkin (Cucirbita pepo; fruit without seeds), zucchini (Cucurbita pepo convar. giromontina; fruit with seeds), cucumber (Cucumis sativus; fruit with seeds), white pattypan squash (Cucurbita pepo var. patisoniana; fruit without seeds) and yellow pattypan squash (Cucurbita pepo var. patisoniana, fruit without seeds). It also evaluates the activity of these preparations on enzymatic lipid peroxidation in thrombin-activated washed blood platelets by TBARS assay. The study also determines the anti-platelet and anticoagulant properties of these five cucurbit preparations in whole blood by flow cytometry and with the total thrombus-formation analysis system (T-TAS) and evaluates the cytotoxicity of the tested preparations against platelets based on LDH activity. The results indicate that the yellow Cucurbita pepo var. patisoniana preparation demonstrated stronger anti-platelet properties than the other tested preparations, reducing the adhesion of thrombin-activated platelets to collagen/fibrinogen, and inhibiting arachidonic acid metabolism and GPIIb/IIIa expression on 10 µM ADP-activated platelets. None of the preparations was found to cause platelet lysis. Our findings provide new information on the anti-platelet activity of the tested cucurbit preparations and their potential for treating CVDs associated with platelet hyperactivity.

scholarly journals Glycoprotein Ic-IIa functions as an activation-independent fibronectin receptor on human platelets.

1988 ◽  
Vol 106 (4) ◽  
pp. 1359-1364 ◽  
Author(s):  
R S Piotrowicz ◽  
R P Orchekowski ◽  
D J Nugent ◽  
K Y Yamada ◽  
T J Kunicki
Keyword(s):  
Platelet Activation ◽  
Chicken Embryo ◽  
Polyclonal Antibodies ◽  
Chicken Embryo Fibroblast ◽  
Band 3 ◽  
Human Platelets ◽  
Fibronectin Receptor ◽  
Activated Platelets ◽  
Coated Surfaces ◽  
Adhesion Of Platelets

Soluble fibronectin binds specifically to glycoprotein (GP) IIb-IIIa on thrombin-activated platelets, and this binding is not observed with platelets of patients with Glanzmann's thrombasthenia (GT) which lack GPIIb-IIIa. Here we report that GT platelets retain the ability to interact with fibronectin-coated surfaces. Adhesion to fibronectin does not require platelet activation and is inhibited by soluble fibronectin, antibodies specific for fibronectin, peptides containing the sequence Arg-Gly-Asp and polyclonal antibodies specific for band 3 of the chicken embryo fibroblast fibronectin receptor (anti-band 3). Using anti-band 3, we have purified a second fibronectin receptor from human platelets, a heterodimer composed of glycoproteins previously designated GPIc and GPIIa. The GPIc-IIa complex is found on both GT and normal platelets and appears to be identical to the GP138 kD-GP160 kD complex recently immunopurified by Giancotti et al. (1986. Exp. Cell Res. 163:47-62) and by Sonnenberg et al. (1987. J. Biol. Chem. 268:10376-10383). In this report, we provide the first evidence that GPIc-IIa actually mediates adhesion of platelets to fibronectin-coated surfaces. GPIc-IIa thus represents a second functional fibronectin receptor, distinct from GPIIb-IIIa, that is largely responsible for the adhesion of nonactivated platelets to fibronectin-coated surfaces.

Activation-dependent surface expression of gC1qR/p33 on human blood platelets

2003 ◽  
Vol 89 (02) ◽  
pp. 331-339 ◽  
Author(s):  
Tara Murphy ◽  
Berhane Ghebrehiwet ◽  
Ellinor Peerschke
Keyword(s):  
Amino Acids ◽  
Platelet Activation ◽  
Human Blood ◽  
Cultured Cells ◽  
Blood Platelets ◽  
Surface Membrane ◽  
Protein A ◽  
Activated Platelets ◽  
Terminal Amino ◽  
Human Blood Platelets

SummaryGC1qR/p33 (gC1qR) is expressed by a variety of somatic and cultured cells, including blood platelets. It interacts with several cellular, viral, bacterial, and plasma proteins, suggesting a potential role in thrombosis, inflammation, and infection. Considerable controversy has surrounded the surface membrane localization of gC1qR, however, since its cDNA sequence does not predict a traditional membrane-anchoring domain, and bears a typical mitochondrial targeting sequence. The present study examined gC1qR expression on resting and activated human blood platelets using flow cytometry and confocal microscopy with two monoclonal antibodies, 74.5.2 and 60.11, directed against gC1qR C-terminal amino acids 204-218, and N-terminal amino acids 76-93, respectively. Unstimulated platelets reacted minimally with either antibody. In contrast, platelet activation with TRAP, epinephrine, or ADP produced markedly increased gC1qR expression as reflected by 74.5.2 binding but not 60.11 binding. Platelet activation was verified using PAC-1 and anti CD 62 antibodies. Whereas PAC-1 binding to activated platelets could be reversed following platelet incubation with PGE1, 74.5.2 binding remained unchanged, suggesting the sustained expression of gC1qR following platelet stimulation. The data further demonstrate that detection of cell surface gC1qR may be dependent on antibody specificity. The ability of gC1qR to bind proteins involved in complement, coagulation, and kinin systems, as well as viral and bacterial pathogens including S. aureus protein A, supports the hypothesis that gC1qR expressed on activated platelets may contribute directly to thrombosis, inflammation, and endovascular infections.

scholarly journals Preparations from selected cucurbit vegetables as antiplatelet agents

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Agata Rolnik ◽  
Bartosz Skalski ◽  
Anna Stochmal ◽  
Beata Olas
Keyword(s):  
Platelet Activation ◽  
Cucurbita Pepo ◽  
Fruits And Vegetables ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
Antiplatelet Agents ◽  
Blood Platelet ◽  
Arachidonic Acid Metabolism ◽  
Platelet Activity ◽  
Activated Platelets

AbstractIncreased blood platelet activation plays an important role in cardiovascular diseases (CVDs). Recent experiments indicate that certain fruits and vegetables, including onion, garlic, and beetroot, have anti-platelet potential and therefore may reduce the likelihood of CVDs. While vegetables from the Cucuritaceae family are known to exerting beneficial antioxidant and anti-inflammatory effects, their effects on blood platelet activation are poorly understood. Therefore, the aim of the present study was to determine the effect on platelet adhesion of preparations from selected cucurbits: pumpkin (Cucurbita pepo; fruit without seeds), zucchini (Cucurbita pepo convar. giromontina; fruit with seeds), cucumber (Cucumis sativus; fruit with seeds), white pattypan squash (Cucurbita pepo var. patisoniana; fruit without seeds) and yellow pattypan squash (Cucurbita pepo var. patisoniana, fruit without seeds). It also evaluates the activity of these preparations on enzymatic lipid peroxidation in thrombin-activated washed blood platelets by TBARS assay. The study also determines the anti-platelet properties of these five cucurbit preparations in whole blood by flow cytometry and with the total thrombus-formation analysis system (T-TAS) and evaluates the cytotoxicity of the tested preparations against platelets based on LDH activity. The results indicate that the yellow Cucurbita pepo var. patisoniana preparation demonstrated stronger anti-platelet properties than the other tested preparations, reducing the adhesion of thrombin-activated platelets to collagen/fibrinogen, and inhibiting arachidonic acid metabolism and GPIIb/IIIa expression on 10 µM ADP-activated platelets. None of the preparations was found to cause platelet lysis. Our findings provide new information on the anti-platelet activity of the tested cucurbit preparations and their potential for treating CVDs associated with platelet hyperactivity.

scholarly journals Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 720-725 ◽  
Author(s):  
D Bienz ◽  
W Schnippering ◽  
KJ Clemetson
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Thrombin Activation ◽  
Strong Candidate ◽  
Triton X ◽  
Kinetics Of

Abstract Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

Intracellular Factor XIII Crosslinks Platelet Cytoskeletal Elements upon Platelet Activation

2002 ◽  
Vol 88 (08) ◽  
pp. 315-320 ◽  
Author(s):  
Katherine Serrano ◽  
Dana Devine
Keyword(s):  
Fluorescence Microscopy ◽  
Platelet Activation ◽  
Factor Xiii ◽  
Actin Polymerization ◽  
Cytoskeletal Proteins ◽  
Platelet Lysate ◽  
Homogeneous Distribution ◽  
Immunoblotting Analysis ◽  
Activated Platelets ◽  
Cytoskeletal Elements

SummaryThe platelet cytoplasm contains approximately half of the factor XIII (FXIII) transglutaminase content circulating in blood, yet the function of cytoplasmic FXIII is poorly understood. This study investigated functions of platelet FXIII in internal platelet processes by studying the interactions of FXIII with platelet cytoskeletal proteins. FXIII was present in cytoskeletal fractions of platelet lysate separated by centrifugation. When cytoskeletal fractions were immobilized on nitrocellulose membranes, thrombin-activated rFXIII (rFXIIIa*) or calcium iontreated rFXIII (rFXIIIa°) bound to some of these proteins, whereas untreated rFXIII did not. Utilizing fluorescence microscopy, an actin polymerization-dependent transient translocation of FXIII from a diffuse homogeneous distribution throughout the cytoplasm to the platelet periphery was observed upon platelet activation, suggesting an association with cytoskeletal proteins. Transglutaminase activity increased in cytoskeletal fractions of activated versus non-activated platelets. Immunoblotting analysis of platelet cytoskeletal fractions identified filamin and vinculin as being crosslinked upon platelet activation.

Inability of Probiotic Bacterial Strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 To Induce Human Platelet Aggregation In Vitro

2005 ◽  
Vol 68 (11) ◽  
pp. 2459-2464 ◽  
Author(s):  
J. S. ZHOU ◽  
K. J. RUTHERFURD ◽  
H. S. GILL
Keyword(s):  
Platelet Aggregation ◽  
Infective Endocarditis ◽  
Platelet Activation ◽  
Blood Platelets ◽  
Lactobacillus Rhamnosus ◽  
Contributory Factor ◽  
Bifidobacterium Lactis ◽  
Activated Platelets ◽  
Probiotic Strains

Platelet aggregation contributes to the pathogenesis of infective endocarditis, and aggregation of platelets induced by lactobacilli is thought to be an important contributory factor in the development and progression of Lactobacillus endocarditis. The main purpose of this study was to examine the effect of immunity-enhancing probiotic strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 on the activation and aggregation of human blood platelets. Whole blood samples from healthy individuals were incubated in vitro with HN001 or HN019 and subsequently labeled with platelet-specific monoclonal antibodies, fluorescein isothiocyanate–conjugated anti-CD41a (expressed on normal platelets), and phycoerythrin-streptavidin–conjugated anti-CD62p (expressed on activated platelets) before analysis by flow cytometry. Platelet-rich plasma was used to assist the gating of the platelet cluster. ADP and epinephrine were used as the physiological platelet activation agonists. Platelet aggregation–inducing strain Streptococcus sanguis 133-79 was used as a positive control strain. The mean fluorescence intensity of phycoerythrin and the percentage of platelets expressing the CD62p marker were used to assess the degree of platelet activation. The percentage of CD62p-positive platelets and the light scatter profiles of the agonist-activated platelets were used to identify the occurrence and degree of platelet aggregation. HN001 and HN019 had no effect on spontaneous platelet activation and aggregation; they also failed to exacerbate the platelet aggregation activity induced by ADP and epinephrine. Therefore, these test probiotic strains HN001 and HN019 are less likely to participate in the pathogenesis of infective endocarditis or other thrombotic disorders with regard to platelet aggregation factors.

scholarly journals Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets.

1993 ◽  
Vol 121 (6) ◽  
pp. 1329-1342 ◽  
Author(s):  
M E Bertagnolli ◽  
M C Beckerle
Keyword(s):  
Platelet Aggregation ◽  
Actin Cytoskeleton ◽  
Platelet Activation ◽  
Blood Platelets ◽  
Immunoblot Analysis ◽  
Activated Platelets ◽  
Extracellular Matrix Components ◽  
Thrombin Activation ◽  
Activation Response ◽  
Granule Secretion

Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) (also referred to as alpha IIb beta 3 integrin), becomes incorporated into the detergent-insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-IIIa is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, approximately 22% of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, alpha-actinin, or vinculin in the complex. We found that the cytoskeleton-associated GPIIb-IIIa is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPIIb-IIIa associated with the alpha-granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, alpha-granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPIIb-IIIa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPIIb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-IIIa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.

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