scholarly journals Correlation between fibrinogen binding to human platelets and platelet aggregability

Blood ◽  
1980 ◽  
Vol 55 (5) ◽  
pp. 841-847 ◽  
Author(s):  
EI Peerschke ◽  
MB Zucker ◽  
RA Grant ◽  
JJ Egan ◽  
MM Johnson
Keyword(s):  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Fibrinogen Concentration ◽  
Platelet Aggregability ◽  
Single Receptor ◽  
Fibrinogen Binding ◽  
Fibrinogen Molecule ◽  
Bernard Soulier Syndrome

Fibrinogen is essential for aggregating platelets with adenosine diphosphate (ADP) and was recently shown to bind to platelets stimulated with ADP. The present work confirms the specific and saturable nature of the platelet-fibrinogen interaction. Binding of 125iodine-labeled fibrinogen to human gel-filtered platelts was maximal at 1 min, and the receptors were saturated when the fibrinogen concentration in the suspending medium approached 0.8 mg/ml. Assuming that one fibrinogen molecule interacts with a single receptor, experiments with 9 normal donors revealed the presence of 12,896 +/- 2456 receptors per platelet. Much of the bound material dissociated from platelets after incubation with apyrase or EDTA. Binding was markedly inhibited at pH 6.5, in the presence of EDTA, and with platelets from 3 thrombasthenic patients but not with those from a patient with the Bernard-Soulier syndrome. Fibrinogen binding was also virtually absent with platelets that had been incubated with EDTA for 8 min at 37 degrees C and pH 7.8. These platelets could not aggregate when mixed with ADP and adequate CaCl2 and fibrinogen, although they could still change their shape. Thus, ADP-induced binding of fibrinogen correlates with platelet aggregability.

scholarly journals Correlation between fibrinogen binding to human platelets and platelet aggregability

Blood ◽  
1980 ◽  
Vol 55 (5) ◽  
pp. 841-847 ◽  
Author(s):  
EI Peerschke ◽  
MB Zucker ◽  
RA Grant ◽  
JJ Egan ◽  
MM Johnson
Keyword(s):  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Fibrinogen Concentration ◽  
Platelet Aggregability ◽  
Single Receptor ◽  
Fibrinogen Binding ◽  
Fibrinogen Molecule ◽  
Bernard Soulier Syndrome

Abstract Fibrinogen is essential for aggregating platelets with adenosine diphosphate (ADP) and was recently shown to bind to platelets stimulated with ADP. The present work confirms the specific and saturable nature of the platelet-fibrinogen interaction. Binding of 125iodine-labeled fibrinogen to human gel-filtered platelts was maximal at 1 min, and the receptors were saturated when the fibrinogen concentration in the suspending medium approached 0.8 mg/ml. Assuming that one fibrinogen molecule interacts with a single receptor, experiments with 9 normal donors revealed the presence of 12,896 +/- 2456 receptors per platelet. Much of the bound material dissociated from platelets after incubation with apyrase or EDTA. Binding was markedly inhibited at pH 6.5, in the presence of EDTA, and with platelets from 3 thrombasthenic patients but not with those from a patient with the Bernard-Soulier syndrome. Fibrinogen binding was also virtually absent with platelets that had been incubated with EDTA for 8 min at 37 degrees C and pH 7.8. These platelets could not aggregate when mixed with ADP and adequate CaCl2 and fibrinogen, although they could still change their shape. Thus, ADP-induced binding of fibrinogen correlates with platelet aggregability.

Adenosine Diphosphate-Induced Aggregation of Human Platelets in Flow Through Tubes: III. Shear and Extrinsic Fibrinogen-Dependent Effects

1994 ◽  
Vol 71 (01) ◽  
pp. 078-090 ◽  
Author(s):  
H L Goldsmith ◽  
M M Frojmovic ◽  
Susan Braovac ◽  
Fiona McIntosh ◽  
T Wong
Keyword(s):  
Shear Rate ◽  
Single Cells ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Size Analysis ◽  
Fibrinogen Concentration ◽  
Human Fibrinogen ◽  
Shear Rates ◽  
Rate Dependent ◽  
Induced Aggregation

SummaryThe effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23°C was studied using a previously described double infusion technique and resistive particle counter size analysis (1). Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 × 105 μl−1; (17)] with [fibrinogen] from 0 to 1.2μM, the, rate and extent of aggregation with 0.7 μM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, Ḡ, = 41.9, 335 and 1,335 s−1. As measured by the decrease in singlet concentration, aggregation at 1.2 μM fibrinogen increased with increasing Ḡ up to 1,335 s1, in contrast to that previously reported in citratcd plasma, in which aggregation reached a maximum at Ḡ = 335 s−1. Without added fibrinogen, there was no aggregation at Ḡ = 41.9 s1; at Ḡ = 335 s1, there was significant aggregation but with an initial lag time, aggregation increasing further at Ḡ = 1,335 s−1. Without added fibrinogen, aggregation was abolished at all Ḡ upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab’)2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37°C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab’)2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of the stable prostacyclin derivative, ZK 36 374, and resuspension in Tyrodes-albumin at 5 × 104 μl−1, aggregated with 2 and 5 μM ADP at Ḡ = 335 and 1,335 s−1 in the absence of added fibrinogen. We therefore postulate that a protein such as von Willebrand factor, secreted during platelet isolation or in flow at sufficiently high shear rates, may yield the observed shear-rate dependent aggregation without fibrinogen.

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2648-2656 ◽  
Author(s):  
Juan A. Rosado ◽  
Else M. Y. Meijer ◽  
Karly Hamulyak ◽  
Irena Novakova ◽  
Johan W. M. Heemskerk ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

scholarly journals Participation of ADP in the binding of fibrinogen to thrombin- stimulated platelets

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 553-555 ◽  
Author(s):  
EF Plow ◽  
GA Marguerie
Keyword(s):  
Creatine Phosphokinase ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Fibrinogen Binding ◽  
Platelet Secretion

Abstract Thrombin and adenosine diphosphate (ADP) supported the binding of 125I- fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.

scholarly journals Immunocytochemical localization of fibrinogen on washed human platelets. Lack of requirement for fibrinogen during adenosine diphosphate-induced responses and enhanced fibrinogen binding in a medium with low calcium levels

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 850-860 ◽  
Author(s):  
H Suzuki ◽  
RL Kinlough-Rathbone ◽  
MA Packham ◽  
K Tanoue ◽  
H Yamazaki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Induced Responses ◽  
Immunocytochemical Localization ◽  
Platelet Surface ◽  
Gold Particles ◽  
Fibrinogen Binding ◽  
Large Numbers ◽  
Lowicryl K4m

Abstract The association of fibrinogen with washed human platelets was examined by immunocytochemistry during aggregation induced by adenosine diphosphate (ADP) and during deaggregation. The platelets were suspended either in a medium containing 2 mmol/L Ca2+ or in a medium containing no added Ca2+ (20 mumol/L Ca2+). Platelets were fixed at several times during aggregation and deaggregation, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. To determine whether the method detected fibrinogen associated with the platelets, the platelets were pretreated with chymotrypsin (10 U/mL) and aggregated by fibrinogen; gold particles were apparent not only in the alpha granules but on the platelet surface and between adherent platelets as well. In the medium with 2 mmol/L Ca2+, ADP caused extensive aggregation of normal platelets in the presence of fibrinogen (0.4 mg/mL), and gold particles were evident between the adherent platelets and on the platelet surface; when the platelets deaggregated, gold was no longer present on the surface. In a medium without added Ca2+, ADP caused extensive aggregation in the presence of fibrinogen, and large numbers of gold particles were on the platelet surface and even more between adherent platelets. In this medium, the platelets did not deaggregate, and by five minutes, the granules appeared to be swollen or fused. In the absence of external fibrinogen, ADP caused the formation of small aggregates, and fibrinogen was not detected between adherent platelets. Thus, the association of fibrinogen with the platelet surface enhances platelet aggregation but is not essential for the ADP-induced formation of small aggregates. The association of fibrinogen with platelets is greater under conditions in which platelets release their granule contents and do not deaggregate because both endogenous and exogenous fibrinogen take part in aggregation.

scholarly journals Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

Blood ◽  
2009 ◽  
Vol 113 (4) ◽  
pp. 902-910 ◽  
Author(s):  
Ramesh B. Basani ◽  
Hua Zhu ◽  
Michael A. Thornton ◽  
Cinque S. Soto ◽  
William F. DeGrado ◽  
...  
Keyword(s):  
Point Mutations ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Rgd Motif ◽  
Fibrinogen Binding ◽  
Evolutionary Response ◽  
Arginine Glycine Aspartic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Compared with human platelets, rodent platelets are less responsive to peptides and peptidomimetics containing an arginine-glycine-aspartic acid (RGD) motif. Using chimeric human-rat αIIbβ3 molecules, we found that this difference in Arg-Gly-Asp-Ser (RGDS) sensitivity was the result of amino acid substitutions at residues 157, 159, and 162 in the W3:4-1 loop and an Asp-His replacement at residue 232 in the W4:4-1 loop of the αIIb β propeller. Introducing the entire rat W3:4-1 and W4:4-1 loops into human αIIbβ3 also decreased the inhibitory effect of the disintegrins, echistatin and eristostatin, and the αIIbβ3 antagonists, tirofiban and eptifibatide, on fibrinogen binding, whereas the specific point mutations did not. This suggests that RGDS interacts with αIIb in a different manner than with these small molecules. None of these species-based substitutions affected the ability of αIIbβ3 to interact with RGD-containing macromolecules. Thus, human von Willebrand factor contains an RGD motif and binds equally well to adenosine diphosphate-stimulated human and rodent platelets, implying that other motifs are responsible for maintaining ligand binding affinity. Many venoms contain RGD-based toxins. Our data suggest that these species amino acids differences in the αIIb β-propeller represent an evolutionary response by rodents to maintain hemostasis while concurrently protecting against RGD-containing toxins.

scholarly journals Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1081-1086 ◽  
Author(s):  
M Cattaneo ◽  
MT Canciani ◽  
A Lecchi ◽  
RL Kinlough-Rathbone ◽  
MA Packham ◽  
...  
Keyword(s):  
Binding Sites ◽  
Creatine Phosphokinase ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
Fibrinogen Binding ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Selective Impairment

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta- thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.

The FcR γ Chain Potentiates Outside-In Integrin Signaling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 215-215 ◽  
Author(s):  
Brian Boylan ◽  
Debra K. Newman ◽  
Peter J. Newman
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Second Messengers ◽  
Chain Complex ◽  
Adenosine Diphosphate ◽  
Adaptor Proteins ◽  
Immunoblot Analysis ◽  
Human Platelets ◽  
Rgd Peptides ◽  
Fibrinogen Binding

Abstract The FcRγ chain is a 12 kDa homodimer that exists in the platelet plasma membrane non-covalently associated with platelet membrane glycoprotein (GP)VI. Interaction of laminin or collagen fibers in the vessel wall with GPVI results in rapid transmission of activation signals into the cell - a process that is dependent on Src family kinase-mediated phosphorylation of Immunoreceptor tyrosine-based activation motifs (ITAMs) within the cytoplasmic domain of FcRγ. Tyrosine phosphorylated FcRγ, in turn, recruits the tyrosine kinase Syk, which then initiates a signal amplification pathway involving assembly of a series of adaptor proteins, activation of phospholipase Cγ2, and generation of second messengers that facilitate granule secretion, activation of the platelet integrins, and platelet aggregation. We and others have recently shown that loss of the GPVI/FcRγ chain complex from the surface of either human or murine platelets renders them unresponsive to collagen and laminin. Unexpectedly, GPVI/FcRγ-chain-depleted platelets also exhibited moderately reduced reactivity to adenosine diphosphate (ADP) and thrombin, which activate platelets by binding to the G protein coupled receptors (GPCRs) P2Y1, P2Y12, PAR1, and PAR4, respectively. Because GPCR-mediated platelet activation has not previously been thought to involve signals emanating from the GPVI/FcRγ-chain complex, we sought to determine whether the FcRγ chain might function to amplify platelet activation responses following GPCR-mediated signaling. Human platelets were exposed to ADP, thrombin or the TXA2-mimetic, U46619, allowed to aggregate, and detergent lysed. Phosphotyrosine immunoblot analysis revealed that the FcRγ chain became rapidly phosphorylated on its ITAMs during platelet aggregation induced by either thrombin or U46619. Interestingly, FcRγ chain phosphorylation was completely inhibited when platelets were activated in the presence of RGD peptides, which allow platelet activation and granule release, but prevent fibrinogen binding to the major platelet integrin, GPIIb-IIIa. The observation that the FcRγ chain becomes tyrosine phosphorylated following GPCR signaling in an RGD-inhibitable manner suggests that integrin-associated kinases might be capable of phosphorylating FcRγ, either directly or indirectly, downstream of ligand binding, though evidence for this remains to be determined. Taken together, these dataprovide support for the notion that the FcRγ chain functions as a general amplifier of platelet activation,helps to explain why GPVI-depleted platelets exhibit blunted responses to agonists specific for GPCRs, andmay lead to important new insights into the mechanism of action of contemplated GPVI-based therapeutics.

scholarly journals ADP and epinephrine-induced release of platelet fibrinogen

Blood ◽  
1981 ◽  
Vol 58 (4) ◽  
pp. 797-802 ◽  
Author(s):  
KL Kaplan ◽  
MJ Dauzier ◽  
S Rose
Keyword(s):  
Platelet Aggregation ◽  
Bovine Serum Albumin ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Dense Granule ◽  
Fibrinogen Concentration ◽  
Platelet Factor ◽  
Granule Proteins ◽  
Induced Aggregation

Abstract Human platelets gel-filtered into Tyrode's buffer containing 1 mM Mg++ and 0.35% bovine serum albumin were studied to determine whether they would undergo biphasic aggregation and release of alpha-granule proteins in response to adenosine diphosphate (ADP) or epinephrine without addition of exogenous fibrinogen. Fibrinogen concentration in the supernatant of unaggregated gel-filtered platelets was less than 1 pmole/ml. With addition of ADP or epinephrine, biphasic aggregation was seen, with release of platelet fibrinogen, beta-thromboglobulin, and platelet factor 4. Fibrinogen concentration in the supernatant after aggregation ranged from 15 to 70 pmole/ml. Release of the alpha-granule proteins by epinephrine was coincidental with release of the dense granule adenine nucleotides. Aggregation and alpha-granule protein release by both ADP and epinephrine were inhibited by added Ca++ at 1-- 2 mM. The ability of gel-filtered platelets to undergo ADP- and epinephrine-induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen may be able to fulfill the requirement for fibrinogen in ADP- and epinephrine-induced platelet aggregation and release.

scholarly journals Characterization of the gamma chain platelet binding site on fibrinogen fragment D

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2643-2648 ◽  
Author(s):  
NE Kirschbaum ◽  
MW Mosesson ◽  
DL Amrani
Keyword(s):  
Platelet Aggregation ◽  
Binding Site ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Gamma Chain ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Chain Sequence ◽  
Carboxy Terminal

Abstract Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400–411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400–411 peptide in inhibiting ADP- induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.

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