scholarly journals Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms

1986 ◽  
Vol 240 (2) ◽  
pp. 403-412 ◽  
Author(s):  
E Kloprogge ◽  
J W Akkerman
Keyword(s):  
Platelet Activation ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
A Minor

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [(3H]arachidonate mobilization and thromboxane synthesis) and secretion [(14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.

Human platelets possess high-affinity binding sites for 3H-imipramine

1979 ◽  
Vol 58 (3) ◽  
pp. 347-348 ◽  
Author(s):  
Michael S. Briley ◽  
Rita Raisman ◽  
S.Z. Langer
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity

Detection Of High Affinity Fibrinogen Binding Sites On Human Platelets Stimulated By ADP: Comparison Of Two Methods Of Platelet Separation

1981 ◽  
Author(s):  
Stefan Niewiarowski ◽  
Thomas A Morinelli ◽  
Elizabeth Kornecki
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Gel Filtration ◽  
Human Platelets ◽  
Binding Studies ◽  
Test Analysis ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
Specific Receptors

Binding of fibrinogen to specific receptors on human platelets exposed by ADP results in platelet aggregation. There are controversial data regarding classes and number of fibrinogen receptors, the values range from one to two classes and 1,000-80,000 receptors per platelet as reported in the literature. We have studied the interaction of fibrinogen with a) platelets washed by differential centrifugation according to Mustard and colleagues (washed platelets - WP) and with b) gel-filtered platelets (GFP). Platelet aggregation was studied with 100 μM ADP and with various concentration of fibrinogen. Maximal velocities of aggregation for WP and GFP were 81 and 47 units per min, respectively, and the Km values for fibrinogen calculated from the rate of aggregation were 0.9 × 10-7M for WP and 5.8 × 10-7M for GFP. The level of platelet fibrinogen released into the suspension from WP and GFP amounted to 2.4 μg and 15.0 μg per 10 9 platelets/ml, respectively, as measured by the staphylococcal clumping test. Analysis of 125I-fibrinogen binding data by the method of Scatchard and Feldman revealed 1,300 high affinity receptors (KD 3.2 × 10-8M) and 80,000 low affinity receptors (KD 5.6 × 10-5M) for WP. The binding of 125I-fibrinogen to GFP was greatly diminished. The number of fibrinogen receptors exposed by ADP on GFP and their binding affinity are under investigation in our laboratory. In conclusion, GFP were less sensitive to fibrinogen than were WP as shown in the aggregation and 125I-fibrinogen binding studies. It appears that the method of platelet separation is critical for the assessment of fibrinogen binding. Platelet activation and release of intact platelet fibrinogen during gel-filtration may interfere with the detection of high affinity fibrinogen binding sites.

Demonstration of specific “high affinity” binding sites for [3H] imipramine on human platelets

Life Sciences ◽  
1980 ◽  
Vol 26 (12) ◽  
pp. 953-959 ◽  
Author(s):  
Steven M. Paul ◽  
Moshe Rehavi ◽  
Phil Skolnick ◽  
Frederick K. Goodwin
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity

Postnatal changes in the substance P receptor on rabbit gastric smooth muscle

1992 ◽  
Vol 262 (2) ◽  
pp. G291-G297
Author(s):  
P. E. Hyman ◽  
S. Kimura ◽  
T. Tomomasa ◽  
Q. X. Yuan ◽  
W. J. Snape ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Substance P ◽  
Binding Sites ◽  
Tissue Homogenate ◽  
Neurokinin A ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gastric Smooth Muscle

We used radioligand binding to tissue homogenates and isometric contraction of muscle strips to characterize the substance P (SP) receptor on gastric smooth muscle from 1- (newborn) and 7-day-old and 4- and 11-wk-old (weanling) rabbits. Scatchard analysis for newborns was curvilinear, suggesting the presence of multiple binding sites. In newborns the dissociation constant (Kd) of high-affinity binding site was 2.2 +/- 0.3 nM, and the maximum binding (Bmax) was 0.57 +/- 0.06 pmol/mg DNA. The number of high-affinity binding sites decreased with age, disappearing by 11 wk. The Kd for the low-affinity site was more than two orders of magnitude greater than that of the high-affinity site. In competitive binding studies with [3H]SP, the order of potency for the neurokinins was SP much greater than neurokinin A (NKA) greater than neurokinin B (NKB), suggesting that the high-affinity binding sites were NK-1 receptors. [125I]NKA is also bound to newborn tissue homogenate with high affinity. With [125I]NKA the order was NKA greater than SP greater than NKB, suggesting that NK-2 receptors were also present. In contraction studies, atropine and tetrodotoxin had no effect on tachykinin-stimulated contraction, suggesting solely myogenic tachykinin effects on this tissue. In newborn rabbits, the potency and efficacy of SP and NKA were similar. The half-maximal effective dose (ED50) of SP was nearly two orders of magnitude less in newborn rabbits than in weanlings; the potency of NKA did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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