Requirement Of Fibrinogen And Calcium For Inter-Platelet Bridges In Platelet Aggregation: A Hypothesis

Fibrinogen and calcium are required for the aggregation of platelets stimulated by ADP or pre-treated with proteolytic enzymes. Specific platelet surface fibrinogen binding sites (receptors) are exposed after platelets are stimulated by ADP or pre-treated with Chymotrypsin or pronase. It has previously been shown in our laboratory that an intact, symmetrical fibrinogen molecule is essential for fibrinogen binding and fibrinogen-induced aggregation of both ADP-stimulated and proteolytically-treated platelets. Here we propose that the mechanism by which fibrinogen and calcium aggregate platelets is by forming inter-platelet bridges linking the fibrinogen receptors of adjacent platelets together. In support of this proposition are the following new lines of evidence: 1) The fibrinogen-induced aggregations of ADP-stfiliulated or proteolytically-treated platelets are inhibited by high concentrations of fibrinogen (Ki=2.6 and 8.5 × 10 5M, respectively). The fibrinogen binding sites on adjacent platelets, at these concentrations, would be saturated by fibrinogen and therefore no inter-platelet fibrinogen bridges could be formed to hold the platelets together. 2) ADP-stimulated or chymotrypsin-treated platelets aggregated by fibrinogen are deaggregated by Chymotrypsin or pronase and this deaggregation coincides with the loss of 125I-fibrinogen from the platelet surface. 3) Preincubation of platelets with EDTA results in inhibition of both platelet aggregation and 125I-fibrinogen binding. Following the aggregations of ADP-stimulated or of chymotrypsin-treated platelets by fibrinogen, the addition of EDTA to the platelet aggregates results in both their deaggregation and their loss of bound 125I-fibrinogen. Thus it appears that divalent cations, especially calcium, are essential for the formation of fibrinogen-linked platelet aggregates.

Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

10.1055/s-0038-1661464 ◽

1986 ◽

Vol 55 (01) ◽

pp. 136-142 ◽

Cited By ~ 4

Author(s):

K J Kao ◽

David M Shaut ◽

Paul A Klein

Keyword(s):

Platelet Aggregation ◽

Binding Sites ◽

Inhibitory Effect ◽

Activated Platelets ◽

Low Concentrations ◽

Platelet Binding ◽

Functional Involvement ◽

Thrombin Activation ◽

Platelet Aggregates ◽

High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

PLATELET ACTIVATION INDUCED BY A MONOCLONAL ANTIBODY AGAINST THE PLATELET GP Ilb/IIIa COMPLEX

10.1055/s-0038-1643514 ◽

1987 ◽

Author(s):

P W Modderman ◽

J G Huisman ◽

J A van Mourik ◽

A E G Kr ◽

v d Borne

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Platelet Aggregation ◽

Medical Research ◽

Platelet Activation ◽

Dense Granules ◽

Fibrinogen Binding ◽

Platelet Release ◽

Induced Aggregation ◽

A receptor for fibrinogen on the platelet GP Ila/lIIb complex is induced by ADP, thrombin and other agonists. To study functional domains on GP Ilb/IIIa, the effects of anti-GP Ilb/IIIa monoclonal antibodies (Mab’s) on platelet function were determined. One of these Mab’s, 6C9, induced platelet aggregation. The antibody binds to the intact GP Ilb/IIIa complex only, not to free GP lib or free GP Ilia. Its epitope is different from that of C17, a Mab that inhibits ADP-induced fibrinogen binding and platelet aggregation. 6C9 induces fibrinogen-mediated aggregation rather than agglutination since 6C9-induced platelet interactions were blocked by treatments that also inhibited the effects of ADP etc., without inhibiting binding of 6C9 itself. 6C9 induces binding of 125I-fibrinogen (35.000 ± 7.300 molecules/platelet, Kd = 1.3 ± 0.4 µM) to unstirred platelets. Binding of fibrinogen was 60 to 80% inhibited by apyrase, which indicates that 6C9-induced fibrinogen binding is largely mediated via ADP released from platelets. In addition, 6C9 induced aggregation of platelets in the absence of extracellular fibrinogen. Mediation of this process by platelet fibrinogen or other a-granule proteins, released upon activation by 6C9, was implicated by the finding that aggregation of washed platelets, but not of platelets to which fibrinogen was added, could be blocked by PGI2. Platelet release was also assessed directly by measuring β-thromboglobulin (α-granules) and (14C) serotonin (dense granules) in the medium of unstirred platelets incubated with 6C9. F(ab')2 fragments of 6C9 only aggregated platelets in the presence of fibrinogen and did not release (14C) serotonin. Moreover, release induced by intact 6C9 was inhibited by anti-GP Ilb/IIIa Mab C17 but not by C17 F(ab’)2, although the latter inhibited ADP-induced platelet aggregation. These data indicate that binding of antibodies to specific sites on GP Ilb/IIIa may induce Fc-dependent platelet activation.This study was supported by the Foundation for Medical Research MEDIGON (grant no. 900-526-057.

Proteolysis of the platelet surface: dissociation of shape change from aggregation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1986.250.4.h550 ◽

1986 ◽

Vol 250 (4) ◽

pp. H550-H557

Author(s):

E. Kornecki ◽

Y. H. Ehrlich ◽

D. H. Hardwick ◽

R. H. Lenox

Keyword(s):

Adenylate Cyclase ◽

Binding Sites ◽

Prostaglandin E1 ◽

Direct Evidence ◽

Shape Change ◽

Adenylate Cyclase System ◽

Platelet Surface ◽

Fibrinogen Binding ◽

Induced Aggregation ◽

Platelet Shape

Stimulation of intact platelets by ADP results in a shape change followed by aggregation in the presence of fibrinogen. ADP was found to induce a shape change in chymotrypsin-treated platelets that was similar in extent and initial velocity to that of intact (untreated) platelets. Scanning-electron microscopy verified an ADP-induced shape change in chymotrypsin-treated platelets. This shape change could be completely blocked by stimulators of platelet adenylate cyclase (forskolin, prostaglandin E1, and prostacyclin). On the other hand, the aggregation of chymotrypsin-treated platelets by fibrinogen was not dependent on the presence of ADP and could not be blocked by forskolin, prostaglandin E1, or prostacyclin, even though the levels of cyclic AMP (cAMP) formed in chymotrypsin-treated platelets were comparable to levels that completely inhibited the ADP-induced aggregation of intact platelets. This lack of inhibition of platelet aggregation was not due to degradation of the adenylate cyclase or prostaglandin receptors, since chymotrypsin-treated platelets were found to have a functional adenylate cyclase system that could be stimulated by forskolin, prostaglandin E1, and prostacyclin and inhibited by ADP and epinephrine, similar to that of intact platelets. These results provide direct evidence that cAMP does not interact with fibrinogen binding sites once they have become permanently exposed on the surface of platelets. Pretreatment of platelets with chymotrypsin therefore appears to be a useful tool that allows for the dissociation of platelet shape change from aggregation, without inhibiting either response.

Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Blood ◽

10.1182/blood.v75.5.1081.bloodjournal7551081 ◽

1990 ◽

Vol 75 (5) ◽

pp. 1081-1086 ◽

Cited By ~ 1

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.

Binding of Aggregated Immunoglobulins to the Human Platelet Fc Receptors: A Mechanism of Platelet to Platelet Bridging

10.1055/s-0038-1646038 ◽

1987 ◽

Vol 58 (04) ◽

pp. 966-970 ◽

Cited By ~ 3

Author(s):

Anwar Parbtani ◽

William F Clark ◽

Anita Caveney ◽

Bruce Reid

Keyword(s):

Platelet Aggregation ◽

Fc Receptors ◽

Human Platelets ◽

Minimum Concentration ◽

Release Reaction ◽

Aggregation Response ◽

Aggregated Immunoglobulins ◽

Platelet Aggregates ◽

Induced Aggregation ◽

SummaryAggregated immunoglobulins react with human platelets by occupying the Fc receptors present on their surface, inducing aggregation and the release reaction. We studied the effect of heat aggregated gammaglobulins (HAGG) on ADP-induced aggregation of platelets. We used the minimum concentration of ADP required to induce a reversible aggregation of platelets without any substantial amount of serotonin (14C 5HT) release. EDTA (5 mM) added at the peak of platelet aggregation resulted in rapid deaggrcgation ot these platelets. However, incubation of platelets with HAGG at a dose that did not by itself induce any aggregation or release reaction, followed by ADP addition resulted in an irreversible platelet aggregation of greater magnitude accompanied by a substantial release of 14C-5HT. The addition of EDTA at the peak of platelet aggregation failed to deaggregate these platelets. To determine whether the augmented aggregation response and the inhibition of deaggiega-tion was due to HAGG or a consequence of platelet release products, we used thrombin-degranulated platelets. The augmented aggregation response and the inhibition of deaggregation due to HAGG and ADP could be demonstrated using these platelets. To confirm that the binding of HAGG to the platelet Pc receptors was responsible for these observations, we incubated platelets with an excess of Fc fragments of IgG prior to the addition of HAGG and ADP. This abolished the aggregation response observed previously. From this study we conclude that interplatelet bridging by HAGG renders the platelets hyperag-gregable and appears to be a mechanism involved in maintaining platelet aggregates.

Epinephrine-Induced Aggregation of Rabbit Platelets Refractory to ADP

10.1055/s-0038-1661316 ◽

1985 ◽

Vol 53 (03) ◽

pp. 366-371 ◽

Cited By ~ 4

Author(s):

C Lalau Keraly ◽

R L Kinlough-Rathbone ◽

J F Mustard

Keyword(s):

Platelet Aggregation ◽

Adrenergic Receptor ◽

Fibrinogen Binding ◽

Rabbit Platelets ◽

Induced Aggregation ◽

SummaryThe mechanisms involved in platelet aggregation induced by epinephrine are unclear. Although epinephrine does not aggregate washed rabbit platelets, platelets made refractory to ADP will aggregate in response to epinephrine in the presence of ADP. We have examined whether the mechanism(s) by which epinephrine induces aggregation of refractory platelets involves fibrinogen binding and Ca2+ association. With normal platelets, ADP causes aggregation, fibrinogen binding and Ca2+ association in a medium containing 0.2 mM 45Ca2+. After 3 min of incubation with ADP, fibrinogen dissociates from platelets, but 45Ca2+ does not. Epinephrine alone does not cause aggregation, fibrinogen binding or 45Ca2+ association. Platelets that are refractory to ADP do not aggregate and bind fibrinogen upon addition of ADP, but aggregate and bind fibrinogen in response to epinephrine, provided ADP is still present. These effects of epinephrine are mediated by the α-adrenergic receptor since they are blocked by phentolamine or verapamil and potentiated by propranolol. However, epinephrine-induced aggregation of platelets refractory to ADP does not involve further detectable increase in the amount of 45Ca2+ associated with the platelets.

Pharmacology Of 12-Deoxyphorbol Phenyl Acetate (12-DOPP) Induced Human Platelet Aggregation

10.1055/s-0038-1653262 ◽

1981 ◽

Author(s):

J Westwick ◽

E M Williamson ◽

F J Evans ◽

V V Kakkor

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Divalent Cations ◽

Dienoic Acid ◽

Free Radical Scavengers ◽

Phenyl Acetate ◽

Lipoxygenase Inhibitor ◽

Human Platelet Aggregation ◽

Induced Aggregation ◽

12-DOPP (0.1 to 3.6µM) induced human platelet aggregation which was dependent upon the presence of divalent cations, intracellular level of C-AMP and an intact microtubular system in common with other aggregating agents. However, the small amount of platelet secretion and thromboxane (Tx) B2 synthesis did not contribute to 12-DOPP induced platelet aggregation as neither the Tx/endoperoxide antagonists pinane A2 (0.001-0.004mM) and trimethoquinone (0.01-0.1mM), the Tx synthesis inhibitors clotrimazole (0.1 to 0.8mM) and 9, 11, aza-prosta-5-13 dienoic acid (0.002-0.1) nor the cyclo-oxygenase inhibitor indomethacin (0.03-0.1mM) inhibited 12-DOPP induced aggregation. Furthermore the free radical scavengers aminopyrine (0.2-2.0mM), thioanisole (0.2-2.0mM) and butylated hydroxy toluene (0.07-1.4mM); the lipoxygenase inhibitor phenidone (0.5mM) and the leucotriene B and C antagonist FPL55712 (0.005-0.06mM) failed to modify 12-DOPP induced aggregation.However compounds which are thought to act as phospholipase inhibitors bromophenacyl bromide (0.3mM), mepracrine (0.20mM) and propanolol (0.2mM) were found to be effective inhibitors of 12-DOPP induced aggregation as well as the so- called calmodulin antagonists imipramine (0.12mM), desmethy- 1imipramine (0.033mM), promethazine (0.1mM) and trifluoperazine (0.35mM).The aggregation induced by 12-DOPP involves a direct effect upon platelets followed by the release of unknown substances probably phospholipids, which induce further aggregation of platelets.

Granulocyte-platelet interactions and platelet fibrinogen receptor exposure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1988.255.3.h651 ◽

1988 ◽

Vol 255 (3) ◽

pp. H651-H658 ◽

Cited By ~ 2

Author(s):

E. Kornecki ◽

Y. H. Ehrlich ◽

R. Egbring ◽

M. Gramse ◽

R. Seitz ◽

...

Keyword(s):

Platelet Aggregation ◽

Binding Sites ◽

Human Platelets ◽

Granulocyte Elastase ◽

Fibrinogen Receptor ◽

Fibrinogen Binding ◽

Low Concentrations ◽

Number Of Binding Sites ◽

Proteolytic Action ◽

We have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase (0.5–1 microgram/ml). Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. Experiments performed by immunoblotting with use of polyclonal and monoclonal antibodies directed to GPIIIa showed that pretreatment of human platelets with granulocyte elastase resulted in the appearance of an additional proteolytic derivative of GPIIIa migrating with an apparent molecular mass of 120 kDa in a nonreduced system. GPIIIa appears to be the preferred substrate of elastase, since GPIIb was not degraded by human granulocyte elastase. We conclude that 1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and 2) human granulocyte elastase exposes active fibrinogen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen.

Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Blood ◽

10.1182/blood.v75.5.1081.1081 ◽

1990 ◽

Vol 75 (5) ◽

pp. 1081-1086 ◽

Cited By ~ 64

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

Abstract 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.

Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

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.