IS THE ENDOTHELIAL EXTRACELLULAR MATRIX THROMBOGENIC OR THROMBORESISTANT? EFFECT OF PREPARATION AND 13-HODE LEVELS

1987 ◽  
Author(s):  
M R Buchanan ◽  
E Bastida ◽  
J Aznar-Salatti ◽  
P de Groot
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cell ◽  
Cellulose Acetate ◽  
Surface Area ◽  
Whole Blood ◽  
Hplc Analysis ◽  
Platelet Adhesion ◽  
Flow Conditions ◽  
Hydroxyoctadecadienoic Acid ◽  
Static Conditions

It is generally thought that the extracellular matrix (ECM) is thrombogenic.However,one of us (MRB) has reported that the ECM is thromboresistant,and postulated that this was due to the release of endothelial cell (EC) 13-hydroxyoctadecadienoic acid (13-HODE) into the ECM. To test this possibility, we measured platelet adhesion (PLT ADH) onto cultured ECs and their ECMs exposed by 3 methods. We also extracted the ECMs for HPLC analysis of 13-HODE.PLT ADH was expressed as i)adhesion of 3H-adenine labelled platelets/mm2 of ECs or ECMs under static conditions, and ii) % surface^ area coverage measured morphometrically following 5"perfusion with citrated whole blood at 1300 sec-1 in the flat chamber.ECMs were prepared by removing the EC monolayers by freeze thawing , cellulose acetate stripping or NH4OH treatment. PLT ADH to ECs under static and flow conditions were 4700±240/mm2 and 0.1%, respectively, and were associated with 12,6± 1 pg of 13-HODE/mm2 of EC surface (M+SEM). Removal of the ECs by freeze thawing or stripping, resulted in a 18% and 25% increase in PLT ADH to the ECM,under static and flow conditions respectively, and a 80% decrease in ECM associated 13-HODE level. Removal of the EC by NH4OH resulted in a 380% and 770% increase in PLT ADH to the ECM in static and flow conditions. 13-HODE was undetectable.These data support the hypothesis that 13-HODE released from ECs influences the ECM thrombogenecity, and indicate that the residual amounts of components present in the ECMs following EC removal is influenced by the method of ECM preparation.

scholarly journals Fibulin-1 mediates platelet adhesion via a bridge of fibrinogen

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2569-2577 ◽  
Author(s):  
S Godyna ◽  
M Diaz-Ricart ◽  
WS Argraves
Keyword(s):  
Extracellular Matrix ◽  
Vascular Injury ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Solid Phase ◽  
Blood Perfusion ◽  
General Mechanism ◽  
Binding Assays ◽  
Solid Phase Binding ◽  
Coated Surfaces

Fibulin-1 is a component of the extracellular matrix that surrounds vascular smooth muscle. This observation, along with the recent finding that fibulin-1 can bind fibrinogen (J Biol Chem 270:19458, 1995), prompted investigation into the potential role of fibulin-1 as a thrombogenic agent. In perfusion chamber assays, platelets in whole blood under flow conditions attached and spread on surfaces coated with fibulin-1. This adhesion was completely blocked by fibulin-1 antibodies. Platelets free of plasma did not attach to fibulin-1 coated surfaces; however, with the addition of fibrinogen, platelet adhesion to fibulin-1 took place. When detergent extracts of platelets were subjected to fibulin-1-Sepharose affinity chromatography, the integrin alpha IIb beta 3 was selected. Solid phase binding assays using purified components showed that integrin alpha IIb beta 3 could not bind directly to fibulin-1 but in the presence of fibrinogen the integrin bound to fibulin-1-coated surfaces. Monoclonal alpha IIb beta 3 antibodies capable of blocking its interaction with fibrinogen completely blocked platelet adhesion to fibulin-1 in both whole blood perfusion and static adhesion assays. The results show that fibulin-1 can support platelet attachment via a bridge of fibrinogen to the platelet integrin alpha IIb beta 3. When fibroblast monolayers containing extracellular matrix-incorporated fibulin-1 were used as adhesion substrates, platelet adhesion in the presence of fibrinogen could be inhibited by 30% using antibodies to fibulin-1. Following vascular injury, fibulin-1 present in the extracellular matrix of the vessel wall may therefore interact with plasma fibrinogen and promote platelet adhesion, leading to the formation of a platelet plug. Thus, fibulin-1 joins the list of matrix proteins including collagens I and IV and fibronectin that mediate platelet adhesion via a plasma protein bridge. This bridging phenomenon may represent a general mechanism by which platelets interact with exposed subendothelial matrices following vascular injury.

Development Of a Novel Method To Assess Neonatal Platelet Function

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4740-4740
Author(s):  
Kristina M. Haley ◽  
Michael Recht ◽  
Owen J.T. McCarty
Keyword(s):  
Cord Blood ◽  
Platelet Function ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Platelet Response ◽  
Primary Hemostasis ◽  
Platelet Aggregometry ◽  
Age Dependent ◽  
Static Conditions

Background The hemostatic system is developmentally regulated, resulting in qualitative and quantitative differences in the mediators of primary and secondary hemostasis as well as fibrinolysis. Age-dependent values of pro- and anti-coagulant proteins have been determined. However, the task of defining age-dependent normal values of neonatal platelet function has been met with challenges owing to difficulties in obtaining adequate blood volumes for functional assays and inconsistent results amongst varying testing methods. In order to overcome many of these challenges, cord blood is often used as a source of neonatal platelets. Platelet aggregometry comparing adult and cord blood derived platelets has demonstrated a near lack of platelet response to epinephrine, collagen, and thromboxane in cord blood samples. In contrast, other studies of platelet function, such as flow cytometry, have failed to demonstrate this phenotypic difference. Assays of primary hemostasis reveal that neonatal blood mediates primary hemostasis as effectively as adult blood. In order to overcome the challenges associated with studying neonatal platelets, we have developed a novel platelet function assay employing small volumes of blood obtained directly from the neonate in order to assess platelet adhesion, activation, and aggregation simultaneously. Methods Eight-well slide chambers were coated with either fibrillar collagen or fibrinogen and allowed to adsorb at room temperature for one hour. Blood was obtained from healthy adult controls via venipuncture and neonatal samples via heelstick into sodium citrate. The blood was separated into two 200 µl aliquots, and TRAP (Thrombin Receptor Activating Peptide: 30 mM) was added to one aliquot. 100 µl of plain whole blood was added to both a collagen and a fibrinogen coated well and 100 µ of whole blood plus TRAP was added to a fibrinogen coated well. The samples were then incubated at 37°C for 30 minutes. Non-adherent cells were washed three times with modified HEPES-Tyrode buffer. FITC-P-selectin was then added (10 µg/ml), and the samples were incubated at 37 oC for 10 minutes and subsequently washed. Samples were imaged with differential interference contrast (DIC) and fluorescence microscopy on a Zeiss Axiovert 200 M microscope. Results Platelet adhesion, activation, and aggregation were assessed for 3 neonatal samples and 3 adult control samples. Both adult and neonatal platelets adhered to fibrinogen and collagen equally. Exposure to collagen and fibrinogen (+/- TRAP) resulted in alpha granule release and P-selectin expression in both neonatal and adult platelets. In addition, both adult and neonatal platelets were observed to undergo the characteristic cytoskeletal changes that result in platelet spreading on fibrinogen (+/- TRAP) and collagen surfaces. Both neonatal and adult platelets were observed to form platelet aggregates on both surfaces under static conditions. (Figure 1) Conclusions We have successfully developed a novel platelet function assay using small volumes of whole blood to assess three key platelet functions: adhesion, activation, and aggregation. This is the first study to demonstrate that neonatal platelets spread on adhesive and extracellular matrix proteins and suggests that neonatal platelets contain the cytoskeletal machinery necessary to undergo this change in platelet formation. This assay fills a critical need in clinical pediatric hematology where efforts to diagnose and treat neonatal platelet dysfunction are often met with technical challenges related to conventional platelet function assays. Disclosures: No relevant conflicts of interest to declare.

Whole blood platelet deposition on extracellular matrix under flow conditions in preterm neonatal sepsis

2002 ◽  
Vol 161 (5) ◽  
pp. 270-274 ◽  
Author(s):  
Yaron Finkelstein ◽  
Boris Shenkman ◽  
Lea Sirota ◽  
Tali H. Vishne ◽  
Rima Dardik ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Whole Blood ◽  
Neonatal Sepsis ◽  
Blood Platelet ◽  
Flow Conditions ◽  
Platelet Deposition

scholarly journals Towards Biohybrid Lung Development—Fibronectin-Coating Bestows Hemocompatibility of Gas Exchange Hollow Fiber Membranes by Improving Flow-Resistant Endothelialization

Membranes ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 35
Author(s):  
Michael Pflaum ◽  
Sophie Jurmann ◽  
Katherina Katsirntaki ◽  
Marisa Mälzer ◽  
Axel Haverich ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Lung Disease ◽  
Hollow Fiber ◽  
Lung Development ◽  
Hollow Fiber Membrane ◽  
Cell Loss ◽  
Flow Conditions ◽  
Alternative Treatment Option ◽  
Static Conditions

To provide an alternative treatment option for patients with end-stage lung disease, we aim for biohybrid lung development (BHL) based on hollow fiber membrane (HFM) technology used in extracorporeal membrane oxygenators. For long-term BHL application, complete hemocompatibility of all blood-contacting surfaces is indispensable and can be achieved by their endothelialization. Indeed, albumin/heparin (AH) coated HFM enables initial endothelialization, but as inexplicable cell loss under flow conditions was seen, we assessed an alternative HFM coating using fibronectin (FN). Therefore, endothelial cell (EC) adherence and viability on both coated HFM were analyzed by fluorescence-based staining. Functional leukocyte and thrombocyte adhesion assays were performed to evaluate hemocompatibility, also in comparison to blood plasma coated HFM as a clinically relevant control. To assess monolayer resistance and EC behavior under clinically relevant flow conditions, a mock circulation setup was established, which also facilitates imitation of lung-disease specific blood gas settings. Besides quantification of flow-associated cell loss, endothelial responses towards external stimuli, like flow exposure or TNFα stimulation, were analyzed by qRT-PCR, focusing on inflammation, thrombus formation and extracellular matrix production. Under static conditions, both coated HFM enabled the generation of a viable, confluent, non-inflammatory and anti-thrombogenic monolayer. However, by means of homogenous FN coating, cell retention and physiologic gene regulation towards an improved hemocompatible-and extracellular matrix producing phenotype, was significantly superior compared to the inhomogeneous AH coating. In summary, our adaptable in-house FN coating secures the endothelial requirements for long-term BHL application and may promote monolayer establishment on all other blood contacting surfaces of the BHL (e.g., cannulae).

scholarly journals Platelet adhesion to cyanogen-bromide fragments of collagen alpha 1(I) under flow conditions

Blood ◽  
1993 ◽  
Vol 82 (10) ◽  
pp. 3029-3033 ◽  
Author(s):  
EU Saelman ◽  
LF Horton ◽  
MJ Barnes ◽  
HR Gralnick ◽  
KM Hese ◽  
...  
Keyword(s):  
Cyanogen Bromide ◽  
Platelet Adhesion ◽  
Collagen Type I ◽  
Type I ◽  
Flow Conditions ◽  
Human Collagen ◽  
Collagen Fragment ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

Adhesive Domains in the Collagen III Fragment α1(III)CB4 that Support α2β1- and von Willebrand Factor-mediated Platelet Adhesion under Flow Conditions

1999 ◽  
Vol 82 (09) ◽  
pp. 1137-1144 ◽  
Author(s):  
Martin IJsseldijk ◽  
Glenda Heijnen-Snyder ◽  
Eric Huizinga ◽  
Laurence Morton ◽  
C. Graham Knight ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Platelet Adhesion ◽  
Recognition Site ◽  
Flow Conditions ◽  
Binding Studies ◽  
Collagen Iii ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummarySeven overlapping peptides derived from the bovine α1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin α2β1-recognition site has been assigned within this fragment to residues 522-528 of the collagen α1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both α2β1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solidphase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*-GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the α1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the α2 subunit of integrin α2β1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the α2β1-recognition site in this locality in α1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the α1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of α2β1-, vWF- and rLAPP-binding sites all in close proximity in α1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

KINETIC BEHAVIOUR OF VON WILLEBRAND FACTOR AND FIBRONECTIN EXPLAINS DEPENDENCE OF PLATELET ADHESION ON PHYSICAL PARAMETERS

1987 ◽  
Author(s):  
Hans H F I van Breugel ◽  
Philip G de Groot ◽  
Jan J Sixma
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Physical Parameters ◽  
Flow Conditions ◽  
Binding Studies ◽  
Platelet Suspension ◽  
Coated Surface ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

To study the kinetics of the contribution of von Willebrand Factor (vWF) and fibronectin (FN) in platelet adhesion we developed a method with which we can perform binding studies of platelets to these purified proteins under static and flow conditions. Glass coverslips were incubated for one hour with vWF (50 (jg/ml) or FN (300 pg/ml) in saline and were perfused with washed platelets (resuspended in human albumin solution) in the flat perfusion chamber as developed by Sakariassen (J.Lab.Clin.Med. 102, 522-535, 1983). Static conditions were achieved by incubating the coated coverslips with the platelet suspension.In this system, adhesion of platelets to FN coated coverslips strongly decreased at shear rates above 300 /s. The adhesion to this surface could be inhibited with antibodies against platelet glycoprotein Ilbllla and against lb, under static and under flow conditions.Adhesion to vWF coated surfaces increased with increasing shear rate and ultimately reached a plateau at about 800 /s. Adhesion to a vWF coated surface could be totally inhibited by anti GP-Ib and only partially by GP-IIbllla.When after perfusion of a FN coated surface with platelets, the same surface was perfused with a platelet free perfusate, the coverage of platelets on this surface decreased. No decrease in platelet coverage was found when this experiment was performed with a vWF coated coverslip.From these results we conclude that platelets bind to FN at a high rate and with a low affinity, while they bind slowly but with a high affinity to vWF, probablyvia similar platelet receptors.

scholarly journals Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin- dependent platelet adhesion under flow conditions

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 82-88
Author(s):  
PF Nievelstein ◽  
JJ Sixma
Keyword(s):  
Platelet Adhesion ◽  
Blood Platelets ◽  
Attachment Site ◽  
Type I ◽  
Flow Conditions ◽  
Shear Rates ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Static Conditions ◽  
Induced Aggregation

Previous studies have indicated that activated blood platelets interact with fibronectin through binding of fibronectin to the glycoprotein IIb- IIIa complex (GPIIb-IIIa). The cell attachment site of fibronectin with its crucial arg-gly-asp(-ser) [RGD(S)]sequence is involved in these bindings. We studied the importance of these interactions for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a nonreactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin-induced aggregation and adhesion under static conditions at 0.1 mmol/L. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mmol/L had a significant inhibitory effect at 1,500 s-1, but not at the lower shear rates of 800 and 300 s-1 where platelet adhesion is also fibronectin dependent. On the matrix of cultured human umbilical vein endothelial cells, 1 mmol/L GRGDSP had no effect on platelet adhesion. The relation between GPIIb- IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann's thrombasthenia and monoclonal antibodies to GPIIb-IIIa using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann's thrombasthenia showed a similar inhibition of adhesion in the presence of fibronectin-free plasma after the ECMs had been preincubated with antifibronectin F(ab')2 fragments. Incubation of platelets with anti-GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed even though separate experiments had shown that these anti-GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin-activated platelets. These data suggest the existence of another binding system for the interaction of platelets with fibronectin that may only appear when fibronectin is present on a surface.

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