Human platelet activation by C3a and C3a des-arg.

C3a liberated from C3 by treatment with C3 convertase (or by trypsin) induced aggregation of gel-filtered human platelets and stimulated serotonin release. At concentrations of 10(-10) M to 8 X 10(-12) M, C3a induced aggregation when added alone to platelets. However, at lower concentrations (2 X 10(-12) M) C3a did not aggregate platelets directly but exhibited highly significant synergism (two-way analysis of variance P less than 0.0001) with ADP in mediating platelet aggregation and release of serotonin. Removal of the C-terminus arginine from C3a abolished anaphylotoxin activity but did not affect the platelet-stimulating activity of the peptide. C3a and C3a des-arg were equally reactive in mediating platelet aggregation and release of serotonin. Further C3a and C3a des-arg exhibited synergism with ADP of equal significance in both aggregation and the release reaction. The concentrations of C3a required for the platelet-stimulating activity involve relatively small number of molecules per platelet (4,000-10,000 for the synergistic reaction with ADP). These data suggest the possibility of a C3a (C3a des-arg) receptor on human platelets. This premise is strengthened by the demonstration ultrastructurally of C3a on the platelet membrane subsequent to C3a stimulation.

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Thrombin Interaction with Human Platelets Potentiation of Thrombin-induced Aggregation and Release by Inactivated Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647686 ◽

1974 ◽

Vol 32 (01) ◽

pp. 207-215 ◽

Cited By ~ 20

Author(s):

David R. Phillips

Keyword(s):

Platelet Aggregation ◽

Binding Sites ◽

Human Platelet ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Induce Platelet Aggregation ◽

Release Reaction ◽

Proteolytic Site ◽

Induced Aggregation ◽

Inhibited Enzyme

SummaryThe possibility that thrombin acts on platelets by a mechanism other than proteolysis was investigated. The proteolytic site of thrombin was modified with phenylmethylsulfonyl fluoride (PMSF). This modified enzyme did not induce platelet aggregation or the platelet release reaction. Platelets were then incubated with the inactivated enzyme (PMS-thrombin) and later with active thrombin. In this sequence of incubation, PMS-thrombin enhanced not only platelet aggregation induced by active thrombin but also the thrombin-induced release reaction. Preincubation with PMS-thrombin was essential for this enhancement as the inhibited enzyme did not affect aggregation if added after active thrombin. The effect of PMS-thrombin was limited to thrombin-induced reactions of the platelet. The inhibited enzyme had no effect on aggregation induced by adenosine diphosphate or collagen, or on thrombininduced coagulation of fibrinogen. These results suggest (1) that both proteolytic and binding sites for thrombin are present on the human platelet plasma membrane ; and (2) that interaction of thrombin with the binding site potentiates the activity of the proteolytic site.

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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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Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function

Blood ◽

10.1182/blood.v51.6.1065.1065 ◽

1978 ◽

Vol 51 (6) ◽

pp. 1065-1071 ◽

Cited By ~ 48

Author(s):

S Levy-Toledano ◽

G Tobelem ◽

C Legrand ◽

R Bredoux ◽

L Degos ◽

...

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Shape Change ◽

Antigenic Site ◽

Human Platelets ◽

Clot Retraction ◽

Igg Antibody ◽

Normal Human ◽

Induced Aggregation ◽

Bovine Factor

Abstract In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF- induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.

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Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽

10.1182/blood.v82.9.2704.2704 ◽

1993 ◽

Vol 82 (9) ◽

pp. 2704-2713 ◽

Cited By ~ 53

Author(s):

R Vezza ◽

R Roberti ◽

GG Nenci ◽

P Gresele

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Prostaglandin E2 ◽

Platelet Activation ◽

Human Platelets ◽

Platelet Surface ◽

Kinase C ◽

Activated Platelets ◽

Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

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Studies on the Effect of Platelet Inhibitors on Platelet Adhesion to Collagen and Collagen-Induced Human Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661310 ◽

1985 ◽

Vol 53 (03) ◽

pp. 337-342 ◽

Cited By ~ 7

Author(s):

S Krishnamurthi ◽

V V Kakkar

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Adhesion ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Antiplatelet Agents ◽

Calmodulin Antagonist ◽

Release Reaction ◽

Low Concentrations

SummaryThe effect of pyridoxal 5’-phosphate (PALP) and trifluoperazine (TFPZ), the calmodulin antagonist, on in vitro platelet adhesion to collagen and collagen-induced platelet activation was studied using platelet-rich-plasma (PRP) or washed platelets (WPL). Platelet aggregation and [14C]-5HT release induced by “threshold” or low concentrations of collagen (0.6 μg/ ml) in PRP were completely abolished by PALP (24 mM), TFPZ (250 μM) as well as indomethacin (10 μM). At higher concentrations of collagen (10–15 μg/ml) in PRP and WPL, the use of stirred and unstirred platelets treated with collagen enabled a distinction to be made between aggregation and adhesion- mediated release reaction. Platelet aggregation and the aggregation-mediated release reaction induced by these concentrations of collagen in stirred platelets were completely abolished by PALP, TFPZ and indomethacin although neither adhesion to collagen nor the adhesion-mediated release reaction of unstirred platelets was significantly affected by these inhibitors. Interestingly, both adhesion and the adhesion-mediated release reaction were abolished by concentrations of PALP 10–40 fold higher than those required to abolish aggregation. Collagen-induced platelet aggregation, but not platelet adhesion, was inhibited in resuspended platelets pretreated with PALP and NaBH4 indicating a separation in the membrane sites involved in aggregation and adhesion. The results further emphasize the distinction between adhesion and aggregation-mediated events with regards to collagen with the latter being more susceptible to inhibition by antiplatelet agents such as PALP and TFPZ.

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Human platelet aggregation by murine monoclonal antiplatelet antibodies is subtype-dependent

Blood ◽

10.1182/blood.v81.7.1792.1792 ◽

1993 ◽

Vol 81 (7) ◽

pp. 1792-1800 ◽

Cited By ~ 28

Author(s):

S De Reys ◽

C Blom ◽

B Lepoudre ◽

PJ Declerck ◽

M De Ley ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Platelet Aggregation ◽

Platelet Activation ◽

Human Platelet ◽

Human Platelets ◽

Antigen Recognition ◽

Metabolic Inhibitors ◽

Antigen Binding ◽

Human Platelet Aggregation ◽

Murine Monoclonal Antibodies

Abstract Twenty murine monoclonal antibodies (MoAbs) generated against different human platelet antigens induced clumping of human platelets in plasma and buffer. Whereas one MoAb could agglutinate platelets, clumping for 19 MoAbs was blocked by metabolic inhibitors, indicating that these induce platelet activation. Fifteen MoAbs were of IgG1, two of IgG2a, and two of IgG2b subtype. F(ab')2 fragments of these did not evoke an aggregatory response, but specifically inhibited aggregations by and binding of their respective intact MoAbs to platelets. Single-platelet counting technology indicated that the MoAbs bind through their antigen- binding and Fc domains mainly to the surface of the same platelet, rather than cause interplatelet-binding. Despite these similarities, the mechanism of action was nevertheless subtype-dependent. Aggregation induced by all IgG1 antibodies could consistently be prevented by blocking the Fc gamma II-receptor, whereas aggregations induced by all IgG2 antibodies still occurred with blocked Fc-receptor, provided functional complement was present. We therefore conclude that platelet activation by MoAb-binding is initiated by antigen recognition followed by an Fc domain-dependent step, which involves the Fc gamma II-receptor for IgG1-type MoAbs and complement-binding for IgG2-type MoAbs. Thus, antibodies of different subtypes can aggregate platelets via different pathways.

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Effect of anti-P1A1 antibody on human platelets. I. The role of complement

Blood ◽

10.1182/blood.v53.4.567.567 ◽

1979 ◽

Vol 53 (4) ◽

pp. 567-577 ◽

Cited By ~ 26

Author(s):

DB Cines ◽

AD Schreiber

Keyword(s):

Platelet Aggregation ◽

Complement Activation ◽

Platelet Rich Plasma ◽

Plasma Concentrations ◽

Human Platelets ◽

Serotonin Release ◽

Buffer System ◽

Induce Platelet Aggregation ◽

Platelet Surface ◽

Release Reaction

Abstract We studied the interaction of complement with human platelets. Complement was activated by IgG anti-P1A1 antibody obtained from 3 patients with the post-transfusion purpura syndrome. We used a heparin- plasma buffer system that permits complement activation and also preserves platelet function. With this system complement activation was efficient, and platelet immune alteration was extensive. Anti-P1A1 antibody was effective only in the presence of complement, in which case both platelet lysis and serotonin release (release reaction) in the absence of lysis were observed. Platelet lysis, as assessed by 51Cr loss, required 10-fold more antibody than was necessary to induce platelet aggregation and release of 14C-serotonin. This platelet release reaction required an intact classic complement sequence through C6. The extent of platelet serotonin release parallelled the depletion of C1 and C4 from platelet-rich plasma. Concentrations of antibody insufficient to induce platelet aggregation and serotonin release could still activate C1 and deposit increased C3 on the platelet surface. These studies demonstrated that complement activation by anti-P1A1 antibody can alter human platelets in a nonlytic system. Several phases of complement-mediated human platelet alteration are possible, depending on the concentration of anti-P1A1 antibody.

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Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5′-[β-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction

Biochemical Journal ◽

10.1042/bj2500209 ◽

1988 ◽

Vol 250 (1) ◽

pp. 209-214 ◽

Cited By ~ 18

Author(s):

S Krishnamurthi ◽

Y Patel ◽

V V Kakkar

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Platelet Activation ◽

Human Platelet ◽

Inhibitory Mechanism ◽

Inhibitory Effects ◽

Similar Concentration ◽

Granule Secretion ◽

Induced Aggregation ◽

Inhibition Of Thrombin

The effect of guanosine 5′-[beta−thio]diphosphate (GDP[beta S]), reported to be an antagonist of GTP at the G-protein-binding site, on human platelet activation was examined. GDP[beta S] (0.3-3 mM) had significant inhibitory effects on platelet aggregation and 5-hydroxytryptamine (5HT) secretion induced by thrombin, collagen, the thromboxane mimetic U46619 and 1,2-dioctanoylglycerol (diC8) in intact platelets, as well as in saponin-permeabilized platelets. Similar inhibitory effects in intact platelets were also observed with ATP (over similar concentration ranges) and GDP and GTP (at 2- and 10-fold higher concentrations respectively). All four nucleotides also inhibited ADP-induced platelet aggregation in indomethacin-treated platelets under conditions where no 5HT secretion occurred. Inhibition of thrombin-induced aggregation and secretion by GDP[beta S] and ATP in intact platelets was accompanied by a reduction in the thrombin-induced rise in intracellular Ca2+ levels and 45 kDa-protein phosphorylation. The results suggest that at least some of the effects of GDP[beta S] may be unrelated to inhibition of G-protein-GTP interaction, but, instead, may be mediated via an extracellular site, common to all the nucleotides tested and perhaps via inhibition of the effects of endogenous/released ADP. The usefulness of GDP[beta S] as a tool in studying G-protein-GTP interactions in platelets is thus questionable.

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Thromboxane A2-Stimulated Human Platelet Aggregation Is Potentiated By Epinephrine Acting VIA Alpha Adrenergic Receptors

10.1055/s-0038-1653296 ◽

1981 ◽

Author(s):

G J Johnson ◽

G H R Rao ◽

J G White

Keyword(s):

Platelet Aggregation ◽

Adrenergic Receptors ◽

Human Platelet ◽

Thromboxane A2 ◽

Human Platelets ◽

Adrenergic Agents ◽

Alpha Adrenergic Receptors ◽

Human Platelet Aggregation ◽

Induced Aggregation

Epinephrine (E) potentiates arachidonate (A)-induced aggregation of human platelets. A-insensitive dog platelets (AIP), that form thromboxane A2 (T) but do not aggregate when stirred with A alone, aggregate when exposed to E + A. Therefore, we studied the effect of E on T-stimu- lated human platelet aggregation. AIP stirred with A formed T which was confirmed by TLC. 1/100 to 1/200 volume of AIP was removed 30 sec. after A, and transferred to gel- filtered, aspirin-incubated human platelets. Recipient platelet aggregation was proportional to the volume of AIP transferred. The addition of the thromboxane synthetase inhibitor, Azo Analog I, abolished the aggregating activity of AIP. Transfer of an aliquot of AIP that was inadequate to aggregate human gel-filtered, aspirin-incubated platelets resulted in irreversible aggregation in the presence of ≥0.5nM E. E potentiated aggregation when added 3 min. before but not 3 min. after aliquot transfer. T-stimulated aggregation was abolished by the T-antagonist, 13 azapro- stenoic acid (APA), but E added after APA and before T restored aggregation. E potentiation of T-stimulated aggregation was abolished by prior exposure to equimolar yohimbine, dihydroergocryptine and phentolamine, agents that bind to alpha2 adrenergic receptors, but not by prazosin an alpha1 antagonist. Higher concentrations of E reversed the inhibitory effects of the alpha2 adrenergic agents. All of these agents in higher concentrations (1-100μM) also blocked aggregation induced by T alone. Therefore T-induced platelet aggregation is potentiated by E, in concentrations attained in vivo, by a mechanism linked to platelet alpha adrenergic receptors. Platelet alpha2 receptors have a close functional relationship to the postulated T receptor. E may initiate platelet aggregation in vivo when T is formed in quantities inadequate to alone induce aggregation.

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EFFECTS OF HUMAN ATRIAL NATRIURETIC PEPTIDES ON SECRETION REACTION IN HUMAN PLATELETS

10.1055/s-0038-1644873 ◽

1987 ◽

Author(s):

A Tanable ◽

Y Yatomi ◽

T Ohashi ◽

H Oka ◽

T Kariya ◽

...

Keyword(s):

Platelet Rich Plasma ◽

Atp Release ◽

Smooth Muscle Contraction ◽

Inhibitory Effect ◽

Human Platelets ◽

Serotonin Release ◽

Release Reaction ◽

Induced Aggregation ◽

Atrial Natriuretic

Human atrial natriuretic peptide (h-ANP) has vasodilating and natriuretic properties, and inhibits smooth muscle contraction, renal renin secretion and adrenal aldosterone release. Although Schiffrin has reported that human platelets have receptors for ANP, its effects in platelets are not established in vivo. We therefore investigated the influence of h-ANP on secretion reaction in human platelets. Eight healthy subjects, males, aged 20 to 24 years, donated blood for the study. Citrated platelet-rich plasma (PRP) was incubated with or without h-ANP at 37 C for 2.5 minutes. The samples of 0.5 ml PRP then used to measure ADP induced aggregation, ATP release reaction and C-serotonin release reaction. H-ANP, at concentration of 1x10 -6M, decreased ADP induced aggregation (after h-ANP: 77.4±9.7 % of control aggregation), and inhibited ATP release reaction (after h-ANP: 31.8±13.1%). Serotonin releasereaction induced by ADP was also inhibited ( control: 15.3±2.2%, after h-ANP: 8.3±0.5 %). The inhibitory effect of h-ANP on aggregation and secretion reaction was maximal by 3 minutes. These data suggest that h-ANP inhibits secretion reaction in human platelets.

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