The Fibrinogen γ Chain Dodecapeptide Inhibits Agonist-induced Aggregation of Rabbit Platelets and Fibrinogen Binding to Rabbit Glycoprotein IIb-IIIa

1999 ◽  
Vol 82 (12) ◽  
pp. 1680-1686 ◽  
Author(s):  
Marian Packham ◽  
Desirée Taylor ◽  
Erik Yeo ◽  
Cynthia Gemmell ◽  
Sonali Patil ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Dependent Manner ◽  
Affinity Matrix ◽  
Human Fibrinogen ◽  
Fibrinogen Binding ◽  
Rabbit Platelets ◽  
Induced Aggregation ◽  
Dose Dependent

SummaryThe HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the γ chains and the RGD sequences in the Aα chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen γ chain sequence. Proteins of ∼135 kDa and ∼95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.

Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

1976 ◽  
Vol 36 (02) ◽  
pp. 376-387 ◽  
Author(s):  
Teruhiko Umetsu ◽  
Kazuko Sanai ◽  
Tadakatsu Kato
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Rabbit Platelets ◽  
Β Blocker ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

MECHANISMS OF PLATELET AGGREGATION BY ACIDIC MUCOPOLYSACCHARIDE EXTRACTED FROM STICHOPUS JAPONICUS SELENKA IN HUMANS AND RABBITS

1987 ◽  
Author(s):  
J Z Li ◽  
E C-Y Lian
Keyword(s):  
Platelet Aggregation ◽  
Ammonium Sulfate ◽  
Human Platelets ◽  
Platelet Inhibitors ◽  
Rabbit Plasma ◽  
Human Fibrinogen ◽  
Stichopus Japonicus ◽  
Saturated Ammonium Sulfate ◽  
Rabbit Platelets ◽  
Induced Aggregation

It has been reported that acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus selenka) (SJAMP) induced the aggregation of human and animal platelets by an unknown mechanism, using platelet-rich plasma (prp) and washed human and rabbit platelets we studied the effects of storage, platelet inhibitors, and various plasmas and their fractions on SJAMP-induced platelet aggregation. we found that the lowest concentrations of SJAMP required for aggregation of human and rabbit platelets were 0.4 and 2 ug/ml respectively. The reactivity of human platelets to SJAMP decreased with time after drawing of blood; rabbit platelets did not show this phenomenon. Platelet inhibitors such as aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose, and EDTA inhibited by 50 to 100% the aggregation of human platelets induced by SJAMP; but these inhibitors had no effect on SJAMP-induced aggregation of rabbit platelets. Washed human and rabbit platelets were not aggregated by SJAMP. The aggregation of washed human platelets by SJAMP was restored completely by human or rabbit plasma, by human fibrinogen, or by 0 to 30% saturated ammonium sulfate fraction but not by serum. The aggregation of rabbit platelets by SJAMP could only be restored by rabbit plasma or serum, or by 50 to 60% saturated ammonium sulfate fraction. The data indicate that the mechanisms of aggregation of human and rabbit platelets by SJAMP are different. THe SJAMP-induced human platelet aggregation is dependent upon metabolism, release of ADP and the cyclooxygenase pathway requiring fibrinogen and Ca++. The aggregation of rabbit platelets induced by SJAMP is independent of metabolism, release of ADP and cyclooxygenase pathway, and does not require fibrinogen and Ca++, but needs certain protein(s) in the 50 to 60% saturated ammonium sulfate fraction of rabbit plasma.

The In Vitro Effect of Ticlopidine on Fibrinogen and Factor VIII Binding to Human Platelets

1981 ◽  
Vol 46 (03) ◽  
pp. 590-592 ◽  
Author(s):  
Helen Lee ◽  
R C Paton ◽  
C Ruan ◽  
J P Caen
Keyword(s):  
Factor Viii ◽  
Antiplatelet Agent ◽  
Human Platelets ◽  
Dependent Manner ◽  
Fibrinogen Binding ◽  
The Mean ◽  
In Vitro Effects ◽  
Vitro Effect ◽  
Induced Aggregation

SummaryThe mode of action of the antiplatelet agent ticlopidine is not yet fully understood. Its multiple effects on platelet function include prolongation of the bleeding time, reduction in primary and secondary Waves of ADP-induced aggregation and inhibition of collagen and thrombin-induced aggregation. We have studied the in vitro effects of ticlopidine on fibrinogen binding induced by ADP and adrenaline as well as factor VIII/vWF binding induced by ristocetin. 125I-fibrinogen binding was measured in suspensions of freshly washed normal platelets stimulated by 10 μM ADP or 10 μM adrenaline. The binding of 125I-factor VIII/vWF in the presence of 1 mg/ml ristocetin was measured in both washed and paraformaldehyde-fixed platelets. Ticlopidine at final concentrations of 200, 100, 50 and 25 μM inhibited both ADP and adrenaline-induced fibrinogen binding in a dose-dependent manner. The mean % inhibition of ADP-induced fibrinogen binding was 82, 73, 42 and 32 respectively. The mean % inhibition of adrenaline induced fibrinogen binding was 86, 82, 60 and 35 respectively. In contrast, the factor VIII/vWF binding was unaffected by ticlopidine at all concentrations except at 200 μM using fresh platelets where a slight inhibition (19%) was observed.These results suggest that ticlopidine either inhibits platelet activation and consequently fibrinogen binding, or inhibits the binding directly, presumably by having an effect on the specific configuration of the platelet membrane required for normal fibrinogen binding.

The In Vitro Effect Of Ticlopidine On Fibrinogen And Factor VIII Binding To Human Platelets

1981 ◽  
Author(s):  
H Lee ◽  
R C Paton ◽  
C Ruan
Keyword(s):  
Factor Viii ◽  
Human Platelets ◽  
Dependent Manner ◽  
Prolonged Bleeding Time ◽  
Fibrinogen Binding ◽  
The Mean ◽  
In Vitro Effects ◽  
Vitro Effect ◽  
Induced Aggregation

Ticlopidine is an anti-aggregating drug whose mode of action is not yet fully understood. Its multiple effects on platelet function include prolonged bleeding time, reduction in primary and secondary waves of ADP-induced aggregation and inhibition of collagen and thrombin- induced aggregation. We have studied the in vitro effects of ticlopidine on fibrinogen binding induced by ADP and adrenaline as well as Factor VIII/vWF binding induced by ristocetin. 125I fibrinogen binding was measured in suspensions of freshly-washed normal platelets stimulated by 10 μM ADP or 10 μM adrenaline. The binding of 125I-Factor VIII/vWF in the presence of 1 mg/ml ristocetin was measured in both washed and paraformaldehyde-fixed platelets. Ticlopidine at final concentrations of 200, 100, 50 and 25 μM inhibited both ADP and adrenaline-induced fibrinogen binding in a dose-dependent manner. The mean % inhibition of ADP-induced fibrinogen binding was 82, 73, 42 and 32 respectively. The mean % inhibition of adrenaline-induced fibrinogen binding was 86, 82, 60 and 35 respectively. In contrast, the Factor VIII/vWF binding was unaffected by ticlopidine at all concentrations except at 200 μM using fresh platelets where a slight inhibition (19 %) was observed.These results suggest that ticlopidine either inhibits platelet activation and consequently fibrinogen binding, or inhibits the binding directly, presumably by having an effect on the specific configuration of the platelet membrane required for normal fibrinogen binding.

Antithrombotic Activity of SR 46349, a Novel, Potent and Selective 5-HT2 Receptor Antagonist

1993 ◽  
Vol 69 (03) ◽  
pp. 262-267 ◽  
Author(s):  
J M Herbert ◽  
A Bernat ◽  
G Barthelemy ◽  
F Dol ◽  
M Rinaldi
Keyword(s):  
Oral Administration ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Venous Shunt ◽  
Induced Aggregation ◽  
Dose Dependent

SummarySR 46349 (trans-4-[(3Z)3-(2-dimethylaminoethyl)oxyimino-3(2-fluorophenyl)propen-1-yl] phenol, hemifumarate) is the first member of a newly-developed 5-HT2 antagonist series. SR 46349 potently inhibited serotonin-induced aggregation of rabbit and human platelets (IC50 = 1 and 3.9 nM respectively) but had no effect on the action of other platelet aggregating agents. SR 46349 was 118 and 25 times more potent than ketanserin against 5-HT + epinephrine-induced aggregation of rabbit and human platelets respectively.A single per os administration of SR 46349 (1 mg/kg) resulted in a strong inhibition of 5-HT + epinephrine-induced platelet aggregation in the rabbit as measured ex vivo (67% inhibition, 6 h after the administration). Intravenous or oral administration of SR 46346 inhibited in a dose-dependent manner venous thrombosis induced by ligature of the jugular vein of rabbits whose blood was made hypercoagulable by i.v. administration of tissue thromboplastin. The doses of SR 46349 which inhibited 50% of thrombus formation were 1.5 ± 0.8 mg/kg and 17 ± 0.5 mg/kg after i.v. or oral administration respectively. When given i.v. to rabbits, SR 46349 exhibited a dose-dependent antithrombotic effect in an arterio-venous shunt model. Significant increase of the bleeding time was observed after the i.v. administration of 5 mg/kg of SR 46349 (3-fold increase). In dogs, SR 46349 inhibited cyclic coronary artery blood flow variations, complete abolition of CFVs being achieved after the i.v. administration of 0.5 mg/kg.In conclusion, SR 46349 is a highly potent, selective antagonist of serotonin in vitro and is to be considered as a potent, orally active antithrombotic agent.

Factors Influencing the Deaggregation of Human and Rabbit Platelets

1983 ◽  
Vol 49 (03) ◽  
pp. 162-167 ◽  
Author(s):  
R L Kinlough-Rathbone ◽  
J F Mustard ◽  
D W Perry ◽  
E Dejana ◽  
J-P Cazenave ◽  
...  
Keyword(s):  
Human Platelets ◽  
Release Reaction ◽  
Factors Influencing ◽  
Platelet Adherence ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
Induced Aggregation

SummaryThe mechanisms involved in platelet deaggregation are unclear. Washed platelets from rabbits or humans aggregated by ADP can be deaggregated by EDTA or PGI2 if the release reaction has not occurred; during deaggregation 125I-fibrinogen dissociates from the platelets. Human platelets suspended in a medium without calcium undergo the release reaction during ADP-induced aggregation; EDTA, PGE, or PGI2 do not deaggregate these platelets although EDTA displaces much of the 125I-fibrinogen that associates with them during aggregation. Rabbit platelets aggregated by low concentrations of releaseinducing stimuli (sodium arachidonate, collagen or thrombin) can be deaggregated by EDTA, PGI2 or PGE1 and 125I-fibrinogen dissociates from them; with high concentrations of collagen or thrombin, deaggregation and dissociation of l25I-fibrinogen is slower. Human platelets that have undergone the release reaction in response to thrombin, collagen or a combination of sodium arachidonate and ADP are not readily deaggregated by EDTA or PGE1. Since aggregation and fibrinogen binding involving the glycoprotein IIb/IIIa complex are readily reversed by EDTA, and since Ca2+ is required for thrombospondin binding to activated platelets, there may be a third type of platelet-platelet adherence that is not disrupted by EDTA; this type of binding plays a greater role with human than with rabbit platelets.

Effect of Amino Sugars on Platelet Aggregation and on Fibrinogen Binding

1984 ◽  
Vol 52 (01) ◽  
pp. 075-080 ◽  
Author(s):  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Inhibitory Effect ◽  
Human Platelets ◽  
Primary Amines ◽  
Amino Sugars ◽  
Ionophore A23187 ◽  
Release Reaction ◽  
Fibrinogen Binding ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
Induced Aggregation

SummaryThe amino sugars glucosamine, galactosamine and man- nosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction.The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectinlike activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.

scholarly journals Platelet Aggregation Induced by Serotype Polysaccharides from Streptococcus mutans

2004 ◽  
Vol 72 (5) ◽  
pp. 2605-2617 ◽  
Author(s):  
Jean-San Chia ◽  
Ya-Lin Lin ◽  
Huei-Ting Lien ◽  
Jen-Yang Chen
Keyword(s):  
Cell Wall ◽  
Platelet Aggregation ◽  
Streptococcus Mutans ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Dependent Manner ◽  
Wild Type ◽  
Cell Wall Polysaccharides ◽  
Induce Platelet Aggregation ◽  
Rabbit Platelets

ABSTRACT Platelet aggregation plays an important role in the pathogenesis of infective endocarditis induced by viridans streptococci or staphylococci. Aggregation induced in vitro involves direct binding of bacteria to platelets through multiple surface components. Using platelet aggregometry, we demonstrated in this study that two Streptococcus mutans laboratory strains, GS-5 and Xc, and two clinical isolates could aggregate platelets in an irreversible manner in rabbit platelet-rich plasma preparations. The aggregation was partially inhibited by prostaglandin I2 (PGI2) in a dose-dependent manner. Whole bacteria and heated bacterial cell wall extracts were able to induce aggregation. Cell wall polysaccharides extracted from the wild-type Xc strain, containing serotype-specific polysaccharides which are composed of rhamnose-glucose polymers (RGPs), could induce platelet aggregation in the presence of plasma. Aggregation induced by the serotype-specific RGP-deficient mutant Xc24R was reduced by 50% compared to the wild-type strain Xc. In addition, cell wall polysaccharides extracted from Xc24R failed to induce platelet aggregation. The Xc strain, but not the Xc24R mutant, could induce platelet aggregation when preincubated with plasma. Both Xc and Xc24R failed to induce platelets to aggregate in plasma depleted of immunoglobulin G (IgG), but aggregation was restored by replenishment of anti-serotype c IgG. Analysis by flow cytometry showed that S. mutans RGPs could bind directly to rabbit and human platelets. Furthermore, cell wall polysaccharides extracted from the Xc, but not the Xc24R, strain could induce pseudopod formation of both rabbit and human platelets in the absence of plasma. Distinct from the aggregation of rabbit platelets, bacterium-triggered aggregation of human platelets required a prolonged lag phase and could be blocked completely by PGI2. RGPs also trigger aggregation of human platelets in a donor-dependent manner, either as a transient and reversible or a complete and irreversible response. These results indicated that serotype-specific RGPs, a soluble product of S. mutans, could directly bind to and activate platelets from both rabbit and human. In the presence of plasma containing IgG specific to RGPs, RGPs could trigger aggregation of both human and rabbit platelets, but the degree of aggregation in human platelets depends on the donors.

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.

scholarly journals Pathways of Thrombin-Induced Aggregation and Release with Human and Rabbit Platelets

1977 ◽  
Author(s):  
R.L. Kinlough-Rathbone ◽  
D.W. Perry ◽  
M.A. Packham ◽  
J.F. Mustard
Keyword(s):  
Platelet Aggregation ◽  
Direct Effect ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
The Third ◽  
Rabbit Platelets ◽  
Induced Aggregation

There are at least 3 mechanisms involved in thrombin-induced aggregation and release: (1) released ADP, (2) formation of thromboxane A2 and (3) a third mechanism(s). We have examined whether the third pathway is due to formation or release of a substance from platelets which affects other platelets. Washed human platelets were exposed to thrombin (2.5 u/ml) for 15 min at 37°C in the presence of indomethacin to block thromboxane A2 formation. Platelets were removed by centrifugation and the thrombin neutralized with hirudin or DFP. Addition of the superna te to washed human platelets prelabeled with 14C-serotonin caused platelet aggregation but release did not occur. Treatment of the supernate with apyrase, CP/CPK or dialysis abolished aggregation, indicating that the material was ADP. Thus, the mechanism by which thrombin induces aggregation and release with human platelets in the presence of agents which destroy ADP and block the formation of thromboxane A2 is a direct effect of thrombin on platelets and does not involve a substance freed from platelets. In contrast, when washed rabbit platelets were treated with thrombin in the presence of indomethacin and the released ADP was removed, material remained in the supernate which caused aggregation and release from washed rabbit platelets but was without effect on washed human platelets. The activity of this material (MW > 10,000) was not abolished by dialysis or boiling. Therefore rabbit platelets differ from human platelets because they have a mechanism in addition to released ADP, thromboxane A2 and the direct effect of thrombin on platelets that can cause aggregation and release.

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