Adsorption of plasma proteins and adhesion of platelets onto novel polyetherurethaneureas?relationship between denaturation of adsorbed proteins and platelet adhesion

Platelets adhere to collagen fibers, undergo the release reaction and aggregate, soluble collagen, however, does not cause release or aggregation. Platelets, however, recognize soluble collagen since adhesion (but not aggregation) occurs when a soluble collagen coated surface is exposed to washed rabbit platelets in Tyrode and in competitive experiments preincubation of platelets with soluble collagen reduces the number adhering to a collagen surface. The latter effect is concentration dependent; preincubation with 100 μg/ml of soluble collagen reduces platelet adhesion by 70%.Similar competitive experiments were performed to examine the features on collagen responsible for platelet adhesion. Preincubation of platelets with a variety of soluble collagens, denatured soluble collagen, pepsin- and periodate-treated soluble collagen and cyanogen bromide peptides all decrease the ability of platelets to bind to a collagen surface. Similar effects arise moreover with the synthetic copolypeptides (Gly-Pro-Ala-Gly-Pro-Pro)n,(Gly-Pro-Pro)n and (Pro2Gly)n. In fact, the homopolymers, polyproline and polyhydroxyproline (but not proline or hydroxyproline) as well give effects similar to those of the collagen materials. The effect is rather specific since preincubation with plasma proteins and synthetic polypeptides based on other major amino acids present in collagen, viz. polyalanine, polyglutamic acid, polyaspartic acid, polyarginine and polylysine do not inhibit the binding of platelets. It would therefore appear that proline and hydroxyproline sequences incorporated in a macromolecular chain are the important, if perhaps not the only determinants involved in platelet recognition of collagen.Soluble collagen coated surfaces were used in this study, however, similar effects of the test material on platelet adhesion to collagen fibers could be demonstrated.

Download Full-text

Amino acid Sequence of a Peptide from Type III Collagen Involved in Platelet Adhesion

10.1055/s-0039-1684659 ◽

1979 ◽

Author(s):

F. Fauvel ◽

Y.J. Legrand ◽

H. Bentz ◽

G. Pignaud ◽

K. Kühn ◽

...

Keyword(s):

Vessel Wall ◽

Cyanogen Bromide ◽

Platelet Adhesion ◽

Type Iii Collagen ◽

Salt Precipitation ◽

Type Iii ◽

Imino Acid ◽

Precipitation Type ◽

Adhesion Of Platelets

The probable importance of the role of type III collagen in the initiation of thrombosis is due to its localization in the subendothelial layers of the vessel wall.The adhesion of platelets to type III has been therefore quantified by a method based on the filtration of non-adhesive 14C 5HT-labe11ed platelets through a Sepharose 2B column.Type III collagen was purified from calf skin by pepsin extraction and salt precipitation. Type III collagen was cleaved by cyanogen bromide and the adhesion induced by the resulting peptides was measured. The activity was attached to the central alpha l(III) CB4 peptide which was further cleaved by hydroxy lamine, chymotrypsin and trypsin. An adhesive potency was linked to three fragments (HA 1 obtained by hydroxylamine, C2 by chymotrypsin and T2 by trypsin) which possess a common portion of 9 amino-acids, localized in the central part of the alpha l(III) CB4 peptide and of the entire alpha 1(III) Chains, which probably represents the active part of type III collagen. Its activity could be due to a particular sterical conformation linked to the presence of 3 imino acid residues in the sequence Gly-Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys of this fragment.

Download Full-text

Thrombospondin inhibits adhesion of platelets to glass and protein- covered substrata

Blood ◽

10.1182/blood.v71.4.1096.1096 ◽

1988 ◽

Vol 71 (4) ◽

pp. 1096-1099 ◽

Cited By ~ 18

Author(s):

J Lahav

Keyword(s):

Platelet Adhesion ◽

Binding Capacity ◽

Inhibitory Effect ◽

Adsorbed Protein ◽

Platelet Binding ◽

Platelet Spreading ◽

Glass Surfaces ◽

Adhesion Of Platelets

Abstract Glass and protein-covered surfaces when treated with the platelet- secreted glycoprotein thrombospondin lose their capacity to bind unstimulated platelets. In comparison to the number that bind to fibronectin-covered glass surfaces, less than 3% bind to thrombospondin- covered glass surfaces. When the fibronectin-covered surface is incubated with thrombospondin, it loses 87% of its binding capacity for platelets. The inhibitory effect of thrombospondin on platelet binding increases with increasing amounts of the adsorbed protein and reaches maximal values at 65% saturation of the adsorption of thrombospondin to the surface. Platelet spreading on the surface is also completely inhibited by thrombospondin. These data suggest that thrombospondin is nonthrombogenic and can modulate platelet adhesion to the subendothelium.

Download Full-text

Physicochemical Studies on Artificial Surface/Protein/Platelet Interactions

10.1055/s-0039-1689328 ◽

1975 ◽

Author(s):

E. Nyilas ◽

T.-H. Chiu ◽

W. A. Morton ◽

D. M. Lederman ◽

G. A. Herzlinger ◽

...

Keyword(s):

Conformational Changes ◽

Plasma Proteins ◽

Platelet Adhesion ◽

Surface Protein ◽

Surface Proteins ◽

Molecular Architecture ◽

Number Density ◽

Protein Layer ◽

Artificial Surface ◽

Lag Period

To highlight the mechanisms of artificial surface/protein/platelet interactions, results obtained by various methods have been integrated to elucidate some of the correlations between phenomena which occur at the macromolecular level and subsequently influence those at the cellular level, such as platelet adhesion. Microcinematographic evidence obtained under the controlled conditions of the Stagnation Point Flow Experiment (SPFE) indicate that, even on glass, platelet adhesion commences only after 30-60 sees of exposure to native blood. This lag period is consistent with diffusion kinetics predicting the arrival of plasma proteins should overhwelmingly precede that of the cellular components. During the lag period, native plasma proteins collide with the artificial surface and, in most cases, adsorb with surface-induced conformational changes. The energy for altering the secondary protein structure is supplied by the heat of adsorption. The extent of adsorption and structural alterations depend upon both the type of protein and the molecular architecture of the artificial surface, viz., the number density and orientation of polar, H-bonding, etc. groups accessible to proteins. Using microparticulate glass (< μ dia.) and a microcalorimeter sensitive to ±0.00001° C in 100 ml of sample volume, serum albumin was found to adsorb, release heat, and desorb in a conformationally altered state. In contrast, γ-(7S)-globulin and fibrinogen underwent irreversible multilayer attachment releasing (1.0-1.7) χ 103 Kcal/mole of protein adsorbed directly to the glass surface. Proteins in the second, etc. sorbed layers released much smaller heats. The electrophoretic mobility of the same particles coated with varying amounts of the same proteins confirmed that the relatively greatest conformational change occurred in the protein layer directly attached to the artificial surface. On homologous Nylons exposed under identical hemodynamic conditions in the SPFE, the surface number density of platelets remaining adherent at points of identical shear was proportionate to the polar force contribution of those surfaces. These results indicate that the protein layer which settles first, is acting as a “proportional transformer” mediating the effects of artificial surfaces onto platelets.

Download Full-text

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

10.1055/s-0038-1652578 ◽

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.

Download Full-text

Effect of Sulphinpyrazone (SP) Aspirin (ASA) and Dipyridamole (DP) on Plateletvessel Wall Interaction after Oral Administration to Rabbits

10.1055/s-0039-1684734 ◽

1979 ◽

Author(s):

J.A. Davies ◽

V.C. Menys

Keyword(s):

Clinical Trials ◽

Platelet Aggregation ◽

Vessel Wall ◽

Platelet Adhesion ◽

Ex Vivo ◽

Inhibitory Effect ◽

Inhibitory Effects ◽

Human Dose ◽

Wall Interaction ◽

Adhesion Of Platelets

Clinical trials of anti-platelet drugs have suggested that they may be useful in the prevention of thrombotic disease. While such drugs inhibit platelet function, those which act on cyclooxygenase also reduce PGI2 synthesis and may interfere with tne natural antithrombotic properties of the vessel wall. We studied the effects of SP, ASA and OP ex vivo on the platelet-vessel wall interaction. Rabbits were dosed by mouth with drug (at about twice the weight-adjusted human dose) or placebo for 5 days, then exsanguinated and aortas removed. Washed platelets prepared from the blood were labelled with 51Cr. and their adhesion to everted aortapr epared from treated or control rabbits was measured in a perfusion device. PGI2-like activity in aortic rings was assayed by its inhibitory effect on platelet aggregation to ADP. Adhesion of platelets to aort as from SP- treated rabbits was i ncreased (p < 0.025), PGI2 - like activity was partially inhibited, but over all adhesion of SP-treated platelets to aor tas f rom SP-treated animals reduced by 30% (p < 0.02). Adhesion to aortas of ASA- treated rabbits was sliahtly inc r ea=-.ed (p > 0 . 1) , PGI 2 - l ike act ivi ty abolished , and no overall reduc tion in platelet adhesion seen. DP had no effecton adhesion or PGI-like activity. These results support the evidence that cyclo-oxygenase inhibitors reduce the inherent resistance of the vessel wall to platelet adhesion. However with SP, inhibitory effects on platelets appear to be more important.

Download Full-text

Fibrinogen Coating Density Affects the Conformation of Immobilized Fibrinogen: Implications for Platelet Adhesion and Spreading

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615072 ◽

1998 ◽

Vol 79 (04) ◽

pp. 824-831 ◽

Cited By ~ 36

Author(s):

Keith Moskowitz ◽

Bohdan Kudryk ◽

Barry Coller

Keyword(s):

Monoclonal Antibodies ◽

Platelet Adhesion ◽

Linear Increase ◽

Significant Decline ◽

High Density ◽

Fibrinogen Concentration ◽

Low Density ◽

Biphasic Response ◽

Adhesion Of Platelets

SummaryAdhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/ IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 μg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 μg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for γ 1-78 and 95-265, respectively, suggesting that the fibrinogen γ-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 μg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at >30 μg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.

Download Full-text

Interaction of human platelets with laminin and identification of the 67 kDa laminin receptor on platelets

Biochemical Journal ◽

10.1042/bj2740535 ◽

1991 ◽

Vol 274 (2) ◽

pp. 535-542 ◽

Cited By ~ 24

Author(s):

N N Tandon ◽

E A Holland ◽

U Kralisz ◽

H K Kleinman ◽

F A Robey ◽

...

Keyword(s):

Platelet Adhesion ◽

Polyclonal Antibodies ◽

Human Platelets ◽

Binding Domain ◽

Laminin Receptor ◽

Adhesion Assay ◽

Major Band ◽

High Affinity ◽

Fab Fragments ◽

Adhesion Of Platelets

A microtitre adhesion assay has been developed to define parameters affecting the adherence of washed platelets to laminin. Adherence was optimally supported by Mg2+ and was inhibited by Ca2+ and by anti-laminin Fab fragments, but significant adhesion (75-90% of control) was found both in heparinized plasma containing physiological levels of bivalent cations and in plasma anti-coagulated with EGTA. Adherence was unaffected by platelet activation with ADP but was decreased by 50% by treatment with alpha-thrombin (1 unit/ml, 5 min). Adherence was unaffected by monospecific polyclonal antibodies to glycoprotein (GP) Ib and GPIV, and was normal with platelets from two patients with Glanzmann's thrombasthaenia, indicating that GPIb, the GPIIb/IIIa complex and GPIV are not involved in platelet-laminin interaction. Affinity chromatography of Triton-solubilized membranes on laminin-Sepharose followed by elution with 0.2 M-glycine/HCl (pH 2.85) identified a major band with a molecular mass of 67 kDa in the reduced and of 53 kDa in the unreduced form. This protein gave a positive reaction on Western blotting with a monospecific polyclonal antibody raised against the high-affinity laminin receptor isolated from human breast carcinoma tissue. The adhesion of platelets to laminin was inhibited by two monoclonal IgM antibodies specific to the LR-1 domain of the 67 kDa receptor. The binding protein was surface-oriented, as shown by flow cytofluorimetry and by the fact that it could be iodinated in intact platelets, but it was not labelled by the periodate-borotritide procedure, suggesting that it did not contain terminal sialic acid. The laminin-derived peptides Tyr-Ile-Gly-Ser-Arg and Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH2, which constitute a complementary binding domain in laminin for the 67 kDa receptor, themselves supported platelet adhesion, bound to the receptor and inhibited the adhesion of platelets to laminin. In addition, Fab fragments of anti-Tyr-Ile-Gly-Ser-Arg antibody inhibited platelet adhesion to laminin. These results demonstrate that the high-affinity 67 kDa laminin receptor previously identified in a range of normal and transformed cells and its complementary Tyr-Ile-Gly-Ser-Arg binding domain play an important role in the interaction of platelets with laminin.

Download Full-text