Contact- and agonist-regulated microvesiculation of human platelets

SummaryAfter exposure to an agonist, platelets are activated and become aggregated. They also shed membrane microparticles that participate in the pathogenesis of thrombosis, hyper-coagulation and inflammation. However, microvesiculation can potentially disrupt the integrity of platelet aggregation by shedding the membrane receptors and phosphatidylserine critical for forming and stabilising a platelet clot. We tested the hypothesis that adhesion and microvesiculation are functions of different subsets of platelets at the time of haemostasis by real-time monitoring of agonist-induced morphological changes and microvesiculation of human platelets. We identified two types of platelets that are adherent to fibrinogen: a high density bubble shape (HDBS) and low-density spread shape (LDSS). Adenosine diphosphate (ADP) predominantly induced HDBS platelets to vesiculate, whereas LDSS platelets were highly resistant to such vesiculation. Thrombin-receptor activating peptide (TRAP) stabilised platelets against microvesiculation by promoting a rapid HDBS-to-LDSS morphological transition. These activities of ADP and TRAP were reversed for platelets in suspension, independent of an engagement integrin αIIbβ 3. As the result of membrane contact, LDSS platelets inhibited the microvesiculation of HDBS platelets in response to ADP. Aspirin and clopidogrel inhibited ADP-induced microvesiculation through different mechanisms. These results suggest that platelet aggregation and microvesiculation occur in different subsets of platelets and are differently regulated by agonists, platelet-platelets and platelet-fibrinogen interactions.

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The small proteoglycan decorin supports adhesion and activation of human platelets

Blood ◽

10.1182/blood.v100.5.1707.h81702001707_1707_1714 ◽

2002 ◽

Vol 100 (5) ◽

pp. 1707-1714 ◽

Cited By ~ 36

Author(s):

Gianni Guidetti ◽

Alessandra Bertoni ◽

Manuela Viola ◽

Enrica Tira ◽

Cesare Balduini ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Platelet Adhesion ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Membrane Receptors ◽

Thrombin Receptor ◽

Membrane Glycoprotein ◽

Intracellular Proteins ◽

Subendothelial Matrix ◽

Small Proteoglycan

Decorin is a small leucine-rich proteoglycan able to interact with several molecules of the subendothelial matrix, such as collagen and fibronectin. In this work, we investigated the ability of purified decorin to support adhesion of human platelets. We found that gel-filtered platelets were actually able to interact with immobilized decorin. Platelet adhesion to decorin was time dependent, required the presence of Mg2+ ions, and was totally mediated by the protein core of the proteoglycan. Platelet stimulation with either adenosine diphosphate (ADP) or a thrombin receptor–activating peptide significantly increased interaction of these cells with the proteoglycan. Upon adhesion to immobilized decorin a number of platelet proteins were found to become tyrosine-phosphorylated. By immunoprecipitation experiments with specific antibodies, the tyrosine phosphorylation of the tyrosine kinase Syk and the phospholipase Cγ2 (PLCγ2) isozyme was demonstrated in decorin-adherent platelets. Interaction of platelets with decorin was selectively prevented by 2 different antibodies against membrane integrin α2β1, but not by a number of antibodies against other membrane receptors. In addition, integrin α2β1, purified from platelet membranes, was able to specifically interact with immobilized decorin. Finally, purified decorin bound to Sepharose beads could precipitate integrin α2β1 from a platelet membrane glycoprotein preparation. Therefore, these results demonstrate that human platelets can bind to immobilized decorin through integrin α2β1, and that this interaction results in the tyrosine phosphorylation of intracellular proteins.

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Lipoprotein Platelet Interaction: The Effect On Platelet Cholesterol Content And Platelet Function

10.1055/s-0038-1652564 ◽

1981 ◽

Author(s):

M Aviram ◽

J G Brook

Keyword(s):

Platelet Aggregation ◽

Cholesterol Metabolism ◽

Cholesterol Content ◽

Human Platelets ◽

Low Density Lipoproteins ◽

Membrane Receptors ◽

High Density Lipoproteins ◽

Low Density ◽

Very Low Density Lipoproteins ◽

Opposing Effects

Intracellular cholesterol metabolism is regulated by membrane receptors which selectively bind plasma low density lipoproteins (LDL), the major extracellular source of cholesterol. Human platelets, unlike other cells, are unable to synthesize cholesterol, but bind LDL with specificity. We have shown that very low density lipoproteins (VLDL) and LDL, both of which contain apolipoprotein B compete with 125I-LDL for the platelet binding sites, while high density lipoproteins (HDL) is only able to compete to a limit extent and this by virtue of its apolipoprotein E content. Platelet uptake of both LDL and HDL is saturable at physiologic concentrations of these lipoproteins. The lipoproteins are internalized by the platelet but degradation occurs only to a limited extent. Incubation of LDL AND HDL with gel-filtered platelet results in significant changes in the platelet cholesterol content. IDL ( 1 mg protein/ml) increases cholesterol content by 15% whereas the same concentration of HDL causes a 5% reduction. In the presence of thrombin LDL enhances platelet aggregation by 300% whereas HDL decreases aggregation by 50%.We have thus shown that the lipoprotein platelet interaction alfects both platelet cholesterol content and also platelet aggregation. LDL and HDL have opposing effects and this again highlights their different roles in the atherogenic process.

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Protease-activated receptors 1 and 4 do not stimulate Gi signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of Gisignaling

Blood ◽

10.1182/blood.v99.10.3629 ◽

2002 ◽

Vol 99 (10) ◽

pp. 3629-3636 ◽

Cited By ~ 126

Author(s):

Soochong Kim ◽

Carolyn Foster ◽

Anna Lecchi ◽

Todd M. Quinton ◽

Dina M. Prosser ◽

...

Keyword(s):

Platelet Aggregation ◽

Adenylyl Cyclase ◽

Signaling Pathways ◽

Human Platelet ◽

Adenosine Diphosphate ◽

Human Platelets ◽

P2y12 Receptor ◽

General Mechanism ◽

Thrombin Receptor ◽

Human Platelet Aggregation

Thrombin is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. Thrombin activates platelets mainly through protease-activated receptor 1 (PAR1), PAR4, and glycoprotein Ib. Because adenosine diphosphate and thromboxane A2 have been shown to cause platelet aggregation by concomitant signaling through Gq and Gipathways, we investigated whether coactivation of Gq and Gi signaling pathways is the general mechanism by which PAR1 and PAR4 agonists also activate platelet fibrinogen receptor (αIIbβ3). A PAR1-activating peptide, SFLLRN, and PAR4-activating peptides GYPGKF and AYPGKF, caused inhibition of stimulated adenylyl cyclase in human platelets but not in the presence of either Ro 31-8220, a protein kinase C selective inhibitor that abolishes secretion, or AR-C66096, a P2Y12 receptor–selective antagonist; α-thrombin–induced inhibition of adenylyl cyclase was also blocked by Ro 31-8220 or AR-C66096. In platelets from a P2Y12 receptor–defective patient, α-thrombin, SFLLRN, and GYPGKF also failed to inhibit adenylyl cyclase. In platelets from mice lacking the P2Y12 receptor, neither α-thrombin nor AYPGKF caused inhibition of adenylyl cyclase. Furthermore, AR-C66096 caused a rightward shift of human platelet aggregation induced by the lower concentrations of α-thrombin and AYPGKF but had no effect at higher concentrations. Similar results were obtained with platelets from mice deficient in the P2Y12. We conclude that (1)thrombin- and thrombin receptor-activating peptide–induced inhibition of adenylyl cyclase in platelets depends exclusively on secreted adenosine diphosphate that stimulates Gi signaling pathways and (2) thrombin and thrombin receptor-activating peptides cause platelet aggregation independently of Gi signaling.

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Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642389 ◽

1994 ◽

Vol 71 (01) ◽

pp. 091-094 ◽

Cited By ~ 15

Author(s):

M Cattaneo ◽

B Akkawat ◽

R L Kinlough-Rathbone ◽

M A Packham ◽

C Cimminiello ◽

...

Keyword(s):

Cardiovascular Disease ◽

Platelet Aggregation ◽

Prostaglandin E1 ◽

Human Platelet ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Before And After ◽

Normal Human ◽

Platelet Aggregates ◽

Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

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Collagen-Induced Platelet Aggregation: -Evidence Against the Essential Role of Platelet Adenosine Diphosphate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657015 ◽

1979 ◽

Vol 42 (04) ◽

pp. 1193-1206 ◽

Cited By ~ 9

Author(s):

Barbara Nunn

Keyword(s):

Platelet Aggregation ◽

Time Course ◽

Essential Role ◽

Atp Release ◽

Adenosine Diphosphate ◽

Human Platelets ◽

High Doses ◽

Induced Aggregation

SummaryThe hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 μM ADP, were not inhibited by 500 pM adenosine, a concentration that greatly reduced the effect of 300 μM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

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Opposite Effect of Lysine on Platelet Aggregation Induced by Arachidonate and by Other Aggregants

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657221 ◽

1982 ◽

Vol 48 (01) ◽

pp. 078-083 ◽

Cited By ~ 3

Author(s):

C Ts'ao ◽

S J Hart ◽

D V Krajewski ◽

P G Sorensen

Keyword(s):

Platelet Aggregation ◽

Secondary Phase ◽

Aminocaproic Acid ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Human Platelets ◽

Primary Phase ◽

High Doses ◽

The Many ◽

Induced Aggregation

SummaryEarlier, we found that ε-aminocaproic acid (EACA) inhibited human platelet aggregation induced by adenosine diphosphate (ADP) and collagen, but not aggregation by arachidonic acid (AA). Since EACA is structurally similar to lysine, yet these two agents exhibit vast difference in their antifibrinolytic activities, we chose to study the effect of lysine on platelet aggregation. We used L-lysine-HCl in these studies because of its high solubility in aqueous solutions while causing no change in pH when added to human plasma. With lysine, we repeatedly found inhibition of ADP-, collagen- and ristocetin-induced aggregation, but potentiation of AA-induced aggregation. Both the inhibitory and potentiation effects were dose-dependent. Low doses of lysine inhibited the secondary phase of aggregation; high doses of it also inhibited the primary phase of aggregation. Potentiation of AA-induced aggregation was accompanied by increased release of serotonin and formation of malondialdehyde. These effects were not confined to human platelets; rat platelets were similarly affected. Platelets, exposed to lysine and then washed and resuspended in an artificial medium not containing lysine, remained hypersensitive to AA, but no longer showed decreased aggregation by collagen. Comparing the effects of lysine with equimolar concentrations of sucrose, EACA, and α-amino-n-butyric acid, we attribute the potent inhibitory effect of lysine to either the excess positive charge or H+ and C1− ions. The -NH2 group on the α-carbon on lysine appears to be the determining factor for the potentiation effect; the effect seems to be exerted on the cyclooxygenase level of AA metabolism. Lysine and other chemicals with platelet-affecting properties similar to lysine may be used as a tool for the study of the many aspects of a platelet aggregation reaction.

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Interactions Between Blood Platelets and Vascular Endothelium in vitro Studies

10.1055/s-0039-1684685 ◽

1979 ◽

Author(s):

M.A. Gimbrone ◽

K.D. Curwen ◽

R. I. Handin

Keyword(s):

Platelet Aggregation ◽

Vascular Endothelium ◽

Potent Inhibitor ◽

Platelet Reactivity ◽

Blood Platelets ◽

Primary Cultures ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Platelet Adherence

Endothelial cells (EC) can actively influence the hemostatic response at sites of vascular injury through multiple mechanisms. For example, EC can degrade adenosine diphosphate, release plasminogen activator, and synthesize prostacyclin (PGI2), a potent inhibitor of platelet aggregation. We have examined whether PGI2 also might account for the normal lack of platelet adherence to the uninjured EC surface. In a monolayer adherence assay, radiolabeled human platelets in citrated plasma showed minimal interaction with primary cultures of human EC (<1 platelet adhering per cell). Platelets from aspirin-treated and untreated donors behaved similarly. However, aspirin pretreatment of EC consistently resulted in ~2-fold increases in platelet adherence which could be completely abolished by exogenous PGI2 (0.5–1.0 μg/ml). SV40-transformed human EC (SVHEC), which are deficient in PGI2 production compared to primary EC, showed 10-30 times more platelet adherence. Exogenous PGI2 produced a dose - related (.001-1.0 μg/ml) decrease in platelet adherence to SVHEC but did not result in the basal levels observed with normal EC monolayers. These data suggest that : 1) In addition to its effects on platelet aggregation, PGI2 can influence platelet endothelial cell interactions; 2) The increased platelet reactivity of transformed EC is associated with, but not completely attributable, to decreased PGI2 production; and 3) Factors other than PGI2 may play a role in the thromboresistance of normal vascular endothelium.

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Aprotinin: is it prothrombotic?

Perfusion ◽

10.1177/026765910101600510 ◽

2001 ◽

Vol 16 (5) ◽

pp. 401-409 ◽

Cited By ~ 3

Author(s):

M Poullis ◽

R C Landis ◽

K M Taylor

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Mechanism Of Action ◽

Adenosine Diphosphate ◽

Platelet Membrane ◽

Calcium Flux ◽

Thrombin Receptor ◽

Proteolytic Activation ◽

Agonist Peptide ◽

Receptor Family

Controversy continues as to whether aprotinin (Trasylol) is prothrombotic. The recent discovery of the thrombin receptor family, known as the protease-activated receptor family (PAR) has been essential in aiding our understanding of the mechanism of action of aprotinin. Our results show that aprotinin has no effect on platelet aggregation induced by adrenaline, adenosine diphosphate, phorbol-12-myristate-13-acetate, collagen or PAR 1 agonist peptide. However, aprotinin inhibits thrombin-induced platelet activation as assessed by macroaggregation, microaggregation and platelet membrane calcium flux. Aprotinin inhibits proteolytic activation of platelets, but platelets can still be activated by non-proteolytic mechanisms.

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Fibrinogen-independent aggregation and deaggregation of human platelets: studies in two afibrinogenemic patients

Blood ◽

10.1182/blood.v70.1.221.221 ◽

1987 ◽

Vol 70 (1) ◽

pp. 221-226 ◽

Cited By ~ 25

Author(s):

M Cattaneo ◽

RL Kinlough-Rathbone ◽

A Lecchi ◽

C Bevilacqua ◽

MA Packham ◽

...

Keyword(s):

Platelet Aggregation ◽

Plasma Proteins ◽

Platelet Rich Plasma ◽

Platelet Activating Factor ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Human Fibrinogen ◽

Control Subjects ◽

Platelet Aggregates ◽

And Control

Abstract Platelets from two afibrinogenemic patients were used to determine whether fibrinogen is essential for platelet aggregation and to examine whether released fibrinogen contributes to the stabilization of platelet aggregates when platelets have been induced to aggregate and release their granule contents by stimulation with thrombin. The addition of adenosine diphosphate (ADP) to platelet-rich plasma (PRP) or to suspensions of washed platelets from the afibrinogenemic patients caused the formation of small aggregates, which was either not inhibited or only slightly inhibited by the F(ab')2 fragments of an antibody to fibrinogen but was inhibited by an antibody (10E5) to glycoprotein IIb/IIIa. Thus there is a component of ADP-induced platelet aggregation that is not dependent on fibrinogen or other plasma proteins but is dependent on glycoprotein IIb/IIIa. There was little difference in the extent of aggregation and the release of granule contents of normal and afibrinogenemic platelets in response to the release-inducing agents collagen, platelet-activating factor (PAF), sodium arachidonate, or thrombin. With normal or afibrinogenemic platelets, aggregation by thrombin (0.2 U/mL or higher) was not inhibited by the F(ab')2 fragments of an antibody to human fibrinogen. Deaggregation by combinations of inhibitors of platelets aggregated by 1 U/mL thrombin showed no difference between platelets from afibrinogenemic and control subjects, indicating that released fibrinogen does not make a major contribution to the stabilization of platelet aggregates formed by thrombin stimulation.

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Interspecies Comparison of Platelet Aggregation, LIBS Expression and Clot Retraction: Observed Differences in GPIIb-IIIa Functional Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649981 ◽

1995 ◽

Vol 74 (06) ◽

pp. 1551-1556 ◽

Cited By ~ 25

Author(s):

Lisa K Jennings ◽

Melanie M White ◽

Timothy D Mandrell

Keyword(s):

Platelet Aggregation ◽

Conformational Changes ◽

Low Dose ◽

Species Differences ◽

Receptor Occupancy ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Clot Retraction ◽

Fibrinogen Receptor ◽

Functional Aspects

SummaryWe examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species’ platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.

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