Thrombin Stimulated Platelet Accumulation on Protein Coated Glass Capillaries: Role of Adhesive Platelet α-Granule Proteins

1989 ◽  
Vol 62 (03) ◽  
pp. 989-995 ◽  
Author(s):  
Juliette N Mulvihill ◽  
J Andrew Davies ◽  
Florence Toti ◽  
Jean-Marie Freyssinet ◽  
Jean-Pierre Cazenave
Keyword(s):  
Human Platelets ◽  
Capillary Perfusion ◽  
Coated Glass ◽  
Bovine Collagen ◽  
Glass Capillaries ◽  
Human Thrombin ◽  
Platelet Accumulation ◽  
Granule Proteins ◽  
Von Willebrand

SummaryThe generation of trace amounts of thrombin at artificial surfaces in contact with blood is likely to be a contributing factor in thrombosis on biomaterials. Using an in vitro capillary perfusion system, platelet accumulation on glass surfaces, uncoated or precoated with purified bovine collagen or human plasma proteins, was determined in the presence or absence of preadsorbed purified human thrombin. Static adsorption for 15 min at 22° C from solutions of thrombin 100 NIH units (33 μg)/ml gave surface concentrations in the range 0.019-0.101 μg/cm2. Protein coated capillaries, thrombin treated or untreated, were perfused for 2 min at 37° C with suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer containing 40% washed red blood cells, under conditions of controlled, non pulsatile laminar flow (50 s−1 or 2,000 s−1). Platelet accumulation was increased in the presence of surface adsorbed thrombin on uncoated and albumin or fibrinogen coated glass but little affected on fibronectin or collagen coated glass. On von Willebrand factor (vWF) coated glass, thrombin enhancement was observed only at high shear forces. In experiments using antibodies against human platelet α-granule proteins, thrombin stimulated platelet deposition in uncoated glass capillaries was inhibited at 2,000 s−1 by anti-vWF and to a lesser extent by anti-fibrinogen but not by antithrombospondin antibodies.

The Use of Monoclonal Antibodies to Human Platelet Membrane Glycoprotein IIb-IIIa to Quantitate Platelet Deposition on Artificial Surfaces

1987 ◽  
Vol 58 (02) ◽  
pp. 724-731 ◽  
Author(s):  
Juliette N Mulvihill ◽  
Han G Huisman ◽  
Jean-Pierre Cazenave ◽  
Jan A van Mourik ◽  
Willem G van Aken
Keyword(s):  
Whole Blood ◽  
Human Albumin ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Bovine Collagen ◽  
Glass Capillaries ◽  
Platelet Deposition ◽  
A New Technique ◽  
Albumin Adsorption

SummaryA new technique is described to quantitate platelet deposition in vitro on artifical surfaces, based on a surface phase radioimmunoassay using the monoclonal antibody 6C9, directed specifically against the membrane glycoprotein complex IIb-IIIa of human platelets. Results correlate in linear fashion with those obtained using 111Indium labeled platelets. The method offers particular advantages for the measurement of platelet deposition in whole blood, since platelet separation, washing and labeling procedures are eliminated, together with the ensuing possible selection of platelet populations. In vitro perfusion is performed in glass capillaries of precisely defined diameter (0.80 or 0.56 mm i.d.). Blood flow is laminar and accurately controlled over wall shear rates ranging from venous to capillary (50-4,000 s-1). Using glass capillaries precoated with purified human albumin or fibrinogen or bovine collagen, platelet deposition from suspensions of washed human platelets in Tyrode's-albumin buffer in the presence of a 40% hematocrit is (platelets/mm2): 11,000 (albumin), 78,000 (fibrinogen) and 306,000 (collagen) after 5 min perfusion at 2,000 s-1. In heparin, citrate or hirudin anticoagulated whole blood, surfaces are passivated, probably by albumin adsorption from plasma (platelets/mm2): 400 (albumin), 3,600 (fibrinogen) and 48,000 (collagen) after 5 min perfusion in the presence of 13 mM citrate.

PLATELET DEPOSITION ON ARTIFICIAL SURFACES: EFFECT OF PROTEIN PRECOATING, ENDOTHELIAL CELL COVERAGE AND SHEAR RATE

1987 ◽  
Author(s):  
A Poot ◽  
A Dekker ◽  
T Beugeling ◽  
A Bantjes ◽  
W G Van Aken
Keyword(s):  
Endothelial Cell ◽  
Shear Rate ◽  
Capillary Tube ◽  
Capillary Perfusion ◽  
Perfusate Flow ◽  
Platelet Deposition ◽  
Artificial Surface ◽  
Platelet Spreading ◽  
Von Willebrand

In the present study the in vitro capillary perfusion system according to Cazenave was used. This system consists of a capillary tube connected to a syringe containg the perfusate which consisted of washed human platalet (111 In-labeled or native) and washed red cells in Ca2+ /Mg2+ -containing Tyrode-albumin buffer. Perfusate flow is controlled by a syringe pump. Polyethylene tubes (PE, 0.75 mm ID, 25 cm long) were precoated with purified human von Willebrand factor (vWF), fibrinogen (Fb), fibronectin (Fn), immunoglobulin G (IgG), albumin (HSA) or plasma. Compared with uncoated PE, platelet deposition increased after precoating with vWF, Fb or Fn, and decreased by pre adsorbed IgG, HSA or plasma. Platelet deposition was positively correlated with shear rate only on surfaces precoated with vWF, Fb or Fn. Scanning electron microscopy showed platelet aggregates on IgG—coated PE, whereas on all other surfaces single adherent platelets were observed. Complete platelet spreading was only observed after precoating with Fn. In contrast with PE coated with vWF, Fb or Fn, platelet adhesion on uncoated PE did not increase further after 5 minutes of perfusion probably due to passivation of the surface by albumin present in the perfusate. This could be overcome by addition of Fb to the perfusate. Perfusates prepared in this way were used for studying the effect of human endothelial cell (HEC) coverage on platelet deposition. HEC were seeded at different densities in PE tubes precoated with partially purified Fn. Platelet deposition decreased with 2increasing HEC coverage, and was negligible at 60,000 HEC/cm . Our results indicate the applicability of this perfusion model for the in vitro testing of artificial surface thrombogenicity.

Microfibrils (MF) Platelet Interaction: Requirement Of Von Willebrand Factor In Platelet Adhesion To A Placenta Microfibrillar Component (PMC)

1981 ◽  
Author(s):  
F Fauvel ◽  
Y J Legrand ◽  
N Gutman ◽  
J P Muh ◽  
G Tobelem ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Human Artery ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets ◽  
Aggregation Of Platelets

It has been shown that collagenase resistant arterial microfibrils (MF) are able to interact with platelets and therefore represents, besides collagen, a second thrombogenic structure in the vessel wall. In vitro observation using a PMC purified from the villosities of human placenta by a mechanical non denaturing procedure confirm this interaction between platelets and MF. PMC was homogenous under electron microscope (feltwork of MF with a mean diameter of 120 – 130 A) and was glycoproteic in nature. PMC were able to induce an aggregation of human platelets only if the platelets were in plasma. The role of Von Willebrand factor (F VIII/WF) as a cofactor of the aggregation of platelets by MF has been postulated from the fact that twice washed platelets from normal subject resuspended in PPP obtained from a severe Von Willebrand deficient patient were not aggregated by the PMC. Furthermore, aggregation was restored after resuspension of the same platelets in the PPP of the same patient 30 and 120 minutes after perfusion of cryoprecipitate (40 units F VIII/RA per kg).F VIII/WF mediates platelet adhesion after binding to subendothelium of human artery. Our observation strongly supports the idea that MF are the subendothelial components to which F VIII/WF binds, thus promoting an adhesion of platelets.

scholarly journals Activation of Human Platelets by the Membrane-Expressed A1 Domain of von Willebrand Factor

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4425-4437 ◽  
Author(s):  
Jan Schulte am Esch ◽  
Miguel A. Cruz ◽  
Jonathan B. Siegel ◽  
Josef Anrather ◽  
Simon C. Robson
Keyword(s):  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Human Platelets ◽  
Pathological Process ◽  
Xenograft Rejection ◽  
Modifying Factors ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractPlatelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF–A1-domains to interact with human platelets in vitro. Both human and porcine vWF–A1-domains expressed as glycosyl phosphatidylinositol–linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.

Hydrogen sulphide reduces the activity of human endothelial cells

2020 ◽  
pp. 1-11
Author(s):  
Eberhard Grambow ◽  
Gina Klee ◽  
Wentao Xie ◽  
Clemens Schafmayer ◽  
Brigitte Vollmar
Keyword(s):  
Endothelial Cells ◽  
Thrombus Formation ◽  
Umbilical Vein ◽  
Protein S ◽  
Coagulation Factors ◽  
Human Platelets ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Biotin Switch

INTRODUCTION: The volatile endogenous mediator hydrogen sulfide (H2S) is known to impair thrombus formation by affecting the activity of human platelets. Beside platelets and coagulation factors the endothelium is crucial during thrombogenesis. OBJECTIVE: This study evaluates the effect of the H2S donor GYY4137 (GYY) on human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Flow cytometry of resting, stimulated or GYY-treated and subsequently stimulated HUVECs was performed to analyse the expression of E-selectin, ICAM-1 and VCAM-1. To study a potential reversibility of the GYY action, E-selectin expression was further assessed on HUVECs that were stimulated 24 h after GYY exposure. A WST-1 assay was performed to study toxic effects of the H2S donor. By using the biotin switch assay, protein S-sulfhydration of GYY-exposed HUVECs was assessed. Further on, the effects of GYY on HUVEC migration and von Willebrand factor (vWF) secretion were assessed. RESULTS: GYY treatment significantly reduced the expression of E-selectin and ICAM-1 but not of VCAM-1. When HUVECs were stimulated 24 h after GYY treatment, E-selectin expression was no longer affected. The WST-1 assay revealed no effects of GYY on endothelial cell viability. Furthermore, GYY impaired endothelial migration, reduced vWF secretion and increased protein S-sulfhydration. CONCLUSIONS: Summarizing, GYY dose dependently and reversibly reduces the activity of endothelial cells.

Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4486-4493 ◽  
Author(s):  
Gregor Theilmeier ◽  
Carine Michiels ◽  
Erik Spaepen ◽  
Ingrid Vreys ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Perfusion Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n = 7 and 5, respectively). In vitro, endothelial VWF–platelet glycoprotein (GP) Ib and platelet P-selectin– endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIbα, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation

2012 ◽  
Vol 107 (06) ◽  
pp. 1107-1121 ◽  
Author(s):  
Thomas Vanassche ◽  
Alexandre Kauskot ◽  
Jan Verhaegen ◽  
Willy Peetermans ◽  
Joanne van Ryn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Platelet Aggregation ◽  
Typical Feature ◽  
Human Platelets ◽  
Fibrin Scaffold ◽  
Immunoglobulin Binding ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryInteractions of Staphylococcus aureus (S. aureus) and platelets play an important role in the pathogenesis of intravascular infections such as infective endocarditis (IE). A typical feature of S. aureus is the ability to generate thrombin activity through the secretion of two prothrombin activating molecules, staphylocoagulase and von Willebrand factor-binding protein (vWbp), which bind to human prothrombin to form the enzymatically active staphylothrombin complex. The role of staphylothrombin in the interaction between S. aureus and platelets has not yet been studied. We found that in contrast with thrombin, staphylothrombin did not directly activate human platelets. However, the staphylothrombin-mediated conversion of fibrinogen to fibrin initiated platelet aggregation and secondary activation and facilitated S. aureus-platelet interactions. Both the genetic absence of staphylocoagulase and vWbp and pharmacological inhibition of staphylothrombin increased the lag time to aggregation, and reduced platelet trapping by S. aureus in high shear stress conditions. The combined inhibition of staphylothrombin and immunoglobulin binding to platelets completely abolished the ability of S. aureus to aggregate platelets in vitro. In conclusion, although staphylothrombin did not directly activate platelets, the formation of a fibrin scaffold facilitated bacteria-platelet interaction, and the inhibition of staphylothrombin resulted in a reduced activation of platelets by S. aureus.

Inhibition of Calmodulin Triggers Apoptosis Events in Human Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3003-3003
Author(s):  
Zhicheng Wang ◽  
Suping Li ◽  
Guanglei Liu ◽  
Quanwei Shi ◽  
Rong Yan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Eukaryotic Cell ◽  
Human Platelets ◽  
Platelet Apoptosis ◽  
Potent Antagonist ◽  
Von Willebrand

Abstract Abstract 3003 Poster Board II-980 Calmodulin (CaM) is a calcium-sensing protein ubiquitously expressed in every eukaryotic cell type regulating biological processes such as cell proliferation, vesicular fusion, fertilization and apoptosis. CaM antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. Tamoxifen (TMX), a potent antagonist of CaM, has been in the center of management of hormone-sensitive breast cancer, and also represents the best example of chemo-prevention to reduce the incidence of invasive breast cancer. Furthermore, TMX is potentially useful in treatment of other kinds of cancer. However, TMX has some severe side effects, one of which is thrombocytopenia. Up to now, the pathogenesis of thrombocytopenia still remains unclear. In platelets, CaM has been found to bind directly to cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet function. However, it is still unclear whether CaM antagonists, especially TMX, induce platelet apoptosis. Here, we show that CaM antagonists TMX and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) dose-dependently induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, gelsolin cleavage and phosphatidylserine (PS) exposure. CaM antagonist did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP- and botrocetin-induced platelet aggregation and platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonist. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis, which suggests a possible pathogenesis of thrombocytopenia in some patients treated with CaM antagonist drugs, and also may present as a novel mechanism for platelet clearance and dysfunction in vivo or in vitro. The elevation of the cytosolic Ca2+ level may involve in the regulation of CaM antagonist-induced platelet apoptosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activation of Human Platelets by the Membrane-Expressed A1 Domain of von Willebrand Factor

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4425-4437
Author(s):  
Jan Schulte am Esch ◽  
Miguel A. Cruz ◽  
Jonathan B. Siegel ◽  
Josef Anrather ◽  
Simon C. Robson
Keyword(s):  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Human Platelets ◽  
Pathological Process ◽  
Xenograft Rejection ◽  
Modifying Factors ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF–A1-domains to interact with human platelets in vitro. Both human and porcine vWF–A1-domains expressed as glycosyl phosphatidylinositol–linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.

scholarly journals Antimicrobial Peptides from Human Platelets

2002 ◽  
Vol 70 (12) ◽  
pp. 6524-6533 ◽  
Author(s):  
Yi-Quan Tang ◽  
Michael R. Yeaman ◽  
Michael E. Selsted
Keyword(s):  
Antimicrobial Peptides ◽  
High Performance ◽  
Antimicrobial Activities ◽  
Reversed Phase ◽  
Human Platelets ◽  
Platelet Factor ◽  
E Coli ◽  
Mediators Of Inflammation ◽  
Human Thrombin

ABSTRACT Platelets share structural and functional similarities with granulocytes known to participate in antimicrobial host defense. To evaluate the potential antimicrobial activities of platelet proteins, normal human platelets were stimulated with human thrombin in vitro. Components of the stimulated-platelet supernatants were purified to homogeneity by reversed-phase high-performance liquid chromatography. Purified peptides with inhibitory activity against Escherichia coli ML35 in an agar diffusion antimicrobial assay were characterized by mass spectrometry, amino acid analysis, and sequence determination. These analyses enabled the identification of seven thrombin-releasable antimicrobial peptides from human platelets: platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin β-4 (Tβ-4), fibrinopeptide B (FP-B), and fibrinopeptide A (FP-A). With the exception of FP-A and FP-B, all peptides were also purified from acid extracts of nonstimulated platelets. The in vitro antimicrobial activities of the seven released peptides were further tested against bacteria (E. coli and Staphylococcus aureus) and fungi (Candida albicans and Cryptococcus neoformans). Each peptide exerted activity against at least two organisms. Generally, the peptides were more potent against bacteria than fungi, activity was greater at acidic pHs, and antimicrobial activities were dose dependent. Exceptions to these observations were observed with PF-4, which displayed a bimodal dose-response relationship in microbicidal assays, and Tβ-4, which had greater activity at alkaline pHs. At concentrations at which they were individually sublethal, PF-4 and CTAP-3 exerted synergistic microbicidal activity against E. coli. Collectively, these findings suggest a direct antimicrobial role for platelets as they are activated to release peptides in response to trauma or mediators of inflammation.

Sign in / Sign up

Export Citation Format

Share Document