Role of Internalization in Platelet Activation Induced by Collagen Fibers – Differential Effects of Aspirin, Cytochalasin D, and Prostaglandin E1

1991 ◽  
Vol 66 (06) ◽  
pp. 708-714 ◽  
Author(s):  
Andreas Ruf ◽  
Heinrich Patscheke ◽  
Eberhard Morgenstern
Keyword(s):  
Prostaglandin E1 ◽  
Close Contact ◽  
Shape Change ◽  
Human Platelets ◽  
Serotonin Release ◽  
Collagen Fibers ◽  
Cytochalasin D ◽  
Collagen Membrane ◽  
Computer Assisted ◽  
Initial Contact

SummaryWashed human platelets were stimulated with fibrillar collagen and platelet aggregation was prevented by non-stirring conditions. In these samples, electron microscopy revealed three fractions of platelets: 1) a majority without contacts to the collagen fibers, 2) with focal contacts to collagen, and 3) a small fraction of platelets with internalized collagen. All platelets had undergone shape change, and exhibited an internal contraction and granule release. However, only those with internalized collagen were completely degranulated. The internalized collagen was found to be in close contact to the contractile sphere in the platelet center, as it was demonstrable with computer assisted 3-D reconstruction from serial sections. Aspirin inhibited neither the adhesion to collagen nor its internalization by internal contraction. Also it did not impair the shape change and degranulation in the platelet fractions that internalized collagen. However, aspirin blocked the shape change and internal contraction of the other platelets and inhibited the [3H]serotonin release. Cytochalasin D 0.1 uM also suppressed the internalization of collagen, the shape change, the formation of a contractile gel, the degranulation, and the [3H]serotonin release in all platelets, whereas the number of platelets that adhered to collagen remained unchanged. The same effects were produced by prostaglandin E1. If the platelets were stimulated with the TXA2 mimetic, U46619, cytochalasin D at 0.1 εM had no effect; but at 20 εM it strongly enhanced the degranulation and the [3H]serotonin release, although the platelets remained discoid. It is concluded that collagen triggers a focal activation of an adherent platelet at the site of its initial contact to collagen. This subthreshold activation proceeds to degranulation and TXA2 formation via a feedback cascade which involves internal contraction, internalization of collagen and multiplication of the collagen membrane contacts.

Physiological Effects of Nonimmune Platelet Associated Immunoglobulin G

1981 ◽  
Vol 45 (01) ◽  
pp. 027-033 ◽  
Author(s):  
K Sugiura ◽  
M Steiner ◽  
M Baldini
Keyword(s):  
Shape Change ◽  
Fluorescein Isothiocyanate ◽  
Recovery Phase ◽  
Human Platelets ◽  
Serotonin Release ◽  
Platelet Volume ◽  
Igg Antibody ◽  
Heterologous Antiserum ◽  
Igg Binding ◽  
Series Of Experiments

SummaryThe function of nonimmune IgG associated with platelets is unknown. In a series of experiments we have investigated this problem, relating amount of platelet-associated IgG (PAIgG) to platelet volume, serotonin release, adherence of platelets to monocytes and platelet senescence. Most of these studies were performed with human platelets. Platelets freed of preexisting PAIgG by incubation at 22° C were incubated with IgG in a series of concentrations ranging from 0.4 — 27.0 X10-6 M. The IgG preparations used were demonstrably free of aggregated forms of the protein. The amount of PAIgG bound to platelets was determined by the use of fluorescein isothiocyanate-conjugated anti-IgG antibody (F-anti-IgG antibody) which was quantified in a fluorospectrophotometer. Newly bound IgG was assayed similarly by the use of F-IgG. A dose-dependent increase in platelet volume was associated with the binding of nonimmune IgG by platelets. The process which leveled off at an IgG concentration of 1.2 —1.5 X10-5 M was almost fully reversible and was not due to platelet shape change or aggregation. Release of serotonin from IgG-treated platelets was relatively small but to the extent that it occurred was positively related to the IgG concentration to which platelets were exposed. Adherence to autologous monocytes studied quantitatively by the use of formaldehyde-fixed cells was also positively related to the amount of IgG on the platelets. Normal or IgG-defident serum had a potent inhibitory (noncompetitive) action on the binding of F-IgG and F-anti-human IgG antibody to human platelets. Cohorts of platelets prepared in rabbits during the recovery phase of immunological thrombocytopenia induced by injection of heterologous antiserum, showed an age-dependent increase of PAIgG and of IgG binding. These results suggest that PAIgG plays a role in the clearance of senescent platelets.

scholarly journals Part of the activating cross-linked immunoglobulin G is internalized by human platelets to sites not accessible for enzymatic digestion

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 12-18 ◽  
Author(s):  
MO Spycher ◽  
UE Nydegger
Keyword(s):  
Immunoglobulin G ◽  
Prostaglandin E1 ◽  
Cytochalasin B ◽  
Enzymatic Digestion ◽  
Human Platelets ◽  
Serotonin Release ◽  
Antimycin A ◽  
Energy Dependent ◽  
Increased Susceptibility ◽  
Pronase Treatment

Abstract The differential uptake of tritium-labeled immunoglobulin G (IgG) cross- linked with bisdiazonium-benzidine (BDB) (3H-BDB-IgG) by washed, pooled human platelets to sites inaccessible to pronase digestion was tested. Up to 52% of the 3H-BDB-IgG associated with platelets at 37 degrees C resisted pronase treatment, whereas only 23% of the cross-linked IgG associated with platelets at 4 degrees C, or at 37 degrees C but in the presence of deoxyglucose/antimycin A, remained refractory to pronase. This effect was not due to platelet agglutination. Pronase resistance reached a maximum after a 60-minute incubation period at 37 degrees C. With increasing 3H-BDB-IgG input, both the total cross-linked IgG associated with platelets and the fraction resistant to pronase digestion approached saturation at 4 degrees C, but not at 37 degrees C. The proportion of 3H-BDB-IgG bound to platelets at 4 degrees C that was resistant to pronase treatment increased by 13% within five minutes of warming the platelets to 37 degrees C. Pretreatment of platelets with 10 mmol/L acetylsalicylic acid (or 10 mumol/L prostaglandin E1) prior to the addition of 3H-BDB-IgG led to a 74% (95%) inhibition of the 3H-BDB-IgG-induced 14C-serotonin release, but to only a 44% (49%) inhibition of pronase-digestible bound ligand. In contrast, pretreatment with 10 mumol/L cytochalasin B led to a mere 17% reduction of 14C-serotonin release, whereas acquisition of resistance to pronase digestion by the bound 3H-BDB-IgG was inhibited by 90%. Incubation of platelets at 37 degrees C with 3H-BDB-IgG and removal of unbound material prior to the addition of prostaglandin E1 or deoxyglucose/antimycin A had little effect on the susceptibility of platelet-associated 3H-BDB-IgG to pronase, whereas the addition of cytochalasin B to 3H-BDB-IgG-treated platelets resulted in greatly increased susceptibility of the platelet-associated ligand to pronase. Thus, after binding, 3H-BDB-IgG becomes transferred in an energy- dependent process to pronase-resistant cellular sites, most likely to the open canalicular system.

scholarly journals Effects of Lectins on Blood Platelets from Various Species

1977 ◽  
Author(s):  
O. Tangen ◽  
B. Karlstam ◽  
S. Bygdeman
Keyword(s):  
Shape Change ◽  
Blood Platelets ◽  
Human Platelets ◽  
Serotonin Release ◽  
Variable Degree ◽  
Con A ◽  
Platelet Release ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Platelet Shape

Earlier it has been shown that different lectins induce a variable degree of aggregation of platelets. The present study confirmed previous data and demonstrated that wheat germ agglutinin (WGA) was very active, 1eucoagglutinin had about a tenth of the activity of WGA on a concentration basis, and Con A had a weak aggregating effect on human gel filtered platelets (GFP). Soy bean lectin did not aggregate human GFP.The fact that adenosine inhibited WGA- and leucoagglutinin-induced aggregation that WGA and Con A caused serotonin release, and that the aggregation- curves indicated platelet shape change are indications that the lectins influenced glycosyl moieties involving one or more molecules relevant to release and aggregation reaction.GFP were markedly more responsive to the lectins than platelets in plasma, probably due to interfering glycosyl groups amongst the plasma constituents.Platelets from man, rabbit, rat, cow and pig reacted differently towards the lectins, human platelets being the most reactive and bovine and porcine platelets being almost unreactive. These results pose intriguing questions regarding the glycosyl content of platelet membranes in different species and their relation to platelet release and aggregation.

An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets

2015 ◽  
Vol 114 (08) ◽  
pp. 313-324 ◽  
Author(s):  
Isabel Sánchez Guiu ◽  
Irene Martínez-Martinez ◽  
Constantino Martínez ◽  
José Navarro-Fernandez ◽  
Faustino Garcia-Candel ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Human Platelets ◽  
Serotonin Release ◽  
Platelet Glycoprotein ◽  
Causative Role ◽  
Temperature Dependent ◽  
Activation Markers

SummaryPlatelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donorcitrated platelet-rich plasma (PRP), but not in PRP from Glanzmann’s thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, 14C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

IRREVERSIBLE BLOCKADE OF THE THROMBOXANE A2/PROSTAGLANDIN H2 RECEPTOR OF HUMAN PLATELETS BY AZIDO-BSP

1987 ◽  
Author(s):  
H Zehender ◽  
E C Witte ◽  
K Stegemeier ◽  
A Patscheke
Keyword(s):  
Shape Change ◽  
Thromboxane A2 ◽  
High Specificity ◽  
Short Wave ◽  
Human Platelets ◽  
Serotonin Release ◽  
Irreversible Inhibition ◽  
High Concentrations ◽  
Very High ◽  
Platelet Shape

Azido-BSP (4-[2-(4-azido-benzenesulphonylamino)-ethyl]phen-oxyacetic acid) is a photolabile derivative of the competitive thromboxane A2 /prostaglandin H2 (TXA2/PGH2) receptor antagonist sulotroban (=BM 13.177). If protected from short wave light, azido-BSP reversibly inhibited the platelet shape change induced by the PGH2 analogue U 46619 but notthe shape change induced by ADP or PAF. Schild analysis revealed an apparent KD=0.2 μM with washed platelets. The irreversible inhibition requiredirradiation of the platelet suspensionwith UVlight (254 nm) for 5 minutes in the presenceof azido-BSP. After this treatment,the platelets were washed twice and used forplatelet function tests. Treatment with 0.5 μM of azido-BSP suppressed the U 46619(10 μM)-induced (3H)serotonin release and 1 μM of azido-BSP was necessary to block the U 46619(2 μM)-inducedaggregation.The platelet shape change induced by U 46619 (0.01μM) was only partially inhibited, even at very high concentrations (50μM) of the antagonist.This suggests that a small portion of the TXA2/PGH2 receptors could not be blocked bythe photoaffinity treatment with azido-BSP. After treatment with 1 μM azido-BSP, the shape change stimulated by ADP or PAF was not reduced. This indicates a high specificity of thephotoaffinity ligand for the TXA2/PGH2 receptor. It is concluded that UV irradiation of azido-BSP generates anitrene intermediate that covalently links to the majority of the TXA2/PGH2 receptors. Azido-BSP provides a specific tool for tagging and subsequent purification of the TXA2/PGH2 receptor of platelets.(Supported by the Deutsche Forschungsgemeinschaft, Grant Pa263).

scholarly journals Part of the activating cross-linked immunoglobulin G is internalized by human platelets to sites not accessible for enzymatic digestion

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 12-18
Author(s):  
MO Spycher ◽  
UE Nydegger
Keyword(s):  
Immunoglobulin G ◽  
Prostaglandin E1 ◽  
Cytochalasin B ◽  
Enzymatic Digestion ◽  
Human Platelets ◽  
Serotonin Release ◽  
Antimycin A ◽  
Energy Dependent ◽  
Increased Susceptibility ◽  
Pronase Treatment

The differential uptake of tritium-labeled immunoglobulin G (IgG) cross- linked with bisdiazonium-benzidine (BDB) (3H-BDB-IgG) by washed, pooled human platelets to sites inaccessible to pronase digestion was tested. Up to 52% of the 3H-BDB-IgG associated with platelets at 37 degrees C resisted pronase treatment, whereas only 23% of the cross-linked IgG associated with platelets at 4 degrees C, or at 37 degrees C but in the presence of deoxyglucose/antimycin A, remained refractory to pronase. This effect was not due to platelet agglutination. Pronase resistance reached a maximum after a 60-minute incubation period at 37 degrees C. With increasing 3H-BDB-IgG input, both the total cross-linked IgG associated with platelets and the fraction resistant to pronase digestion approached saturation at 4 degrees C, but not at 37 degrees C. The proportion of 3H-BDB-IgG bound to platelets at 4 degrees C that was resistant to pronase treatment increased by 13% within five minutes of warming the platelets to 37 degrees C. Pretreatment of platelets with 10 mmol/L acetylsalicylic acid (or 10 mumol/L prostaglandin E1) prior to the addition of 3H-BDB-IgG led to a 74% (95%) inhibition of the 3H-BDB-IgG-induced 14C-serotonin release, but to only a 44% (49%) inhibition of pronase-digestible bound ligand. In contrast, pretreatment with 10 mumol/L cytochalasin B led to a mere 17% reduction of 14C-serotonin release, whereas acquisition of resistance to pronase digestion by the bound 3H-BDB-IgG was inhibited by 90%. Incubation of platelets at 37 degrees C with 3H-BDB-IgG and removal of unbound material prior to the addition of prostaglandin E1 or deoxyglucose/antimycin A had little effect on the susceptibility of platelet-associated 3H-BDB-IgG to pronase, whereas the addition of cytochalasin B to 3H-BDB-IgG-treated platelets resulted in greatly increased susceptibility of the platelet-associated ligand to pronase. Thus, after binding, 3H-BDB-IgG becomes transferred in an energy- dependent process to pronase-resistant cellular sites, most likely to the open canalicular system.

The Role Of Membrane-Bound Tubulin In Platelet Functions

1981 ◽  
Author(s):  
Y Ikeda ◽  
M Handa ◽  
Y Yoshii ◽  
M Imai ◽  
K Sugiura ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Shape Change ◽  
Human Platelets ◽  
Binding Activity ◽  
Serotonin Release ◽  
Platelet Membranes ◽  
Coomassie Blue ◽  
Tubulin Subunit ◽  
Membrane Bound

Evidence has been presented which suggests the existence of tubulin, subunit protein of microtubules, as an integral part of plasma membrane of certain cells. We have investigated whether tubulin is also a constituent of platelet plasma membrane or not, and, if so, what the functional significance is? Platelet membranes isolated by glycerol lysis technique according to the method of Barber and Jamieson retained colchicine-binding activity, 6.2 ± 1.4 n mol colchicine per 100 mg platelet membranes. Colchicine-binding activity of platelet membranes was not decreased after membranes were washed 3 times, indicating that colchicinebinding activity of membranes is not due to contamination of loosely bound cytoplasmic soluble tubulin. On SDS-poly- acrylamide gel electrophoresis, platelet membranes revealed Coomassie blue stained band of molecular weight 55,000, which comigrated with purified cytoplasmic tubulin isolated from human platelets by two successive cycles of temperature -dependent polymerization depolymerization as described previously(Ikeda & Steiner, J. Biol. Chem. 251:6135, 1976). Monospecific antibody against platelet tubulin was prepared in rabbits by injecting soluble tubulin at weekly intervals for 4 weeks. Platelets preincubated with anti-tubulin F(ab’)2 fragment showed reduced platelet aggregation and shape change induced by collagen, but not by ADP or epinephrine. Collagen-induced release of 14C-serotonin was also inhibited by anti-tubulin F(ab’)2 fragment while ADP- or epinephrine-induced serotonin release was not inhibited(collagen 2μg/ml:45.6% of control, ADP 10μM:92.0% of control, epinephrine 4μg/ml: 98.0% of control).Our results suggest that membrane-associated tubulin may play important roles in collagen-platelet interactions.

Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

1994 ◽  
Vol 71 (01) ◽  
pp. 091-094 ◽  
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.

Conditions Affecting the Responses of Human Platelets to Epinephrine

1988 ◽  
Vol 60 (02) ◽  
pp. 209-216 ◽  
Author(s):  
Chantal Lalau Keraly ◽  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Hidenori Suzuki ◽  
J Fraser Mustard
Keyword(s):  
Platelet Rich Plasma ◽  
Shape Change ◽  
Human Platelets ◽  
Dense Granule ◽  
Primary Phase ◽  
Lag Phase ◽  
Acid Citrate Dextrose ◽  
Two Phases ◽  
Citrate Dextrose ◽  
Thromboxane Formation

SummaryConditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-pheny- lalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin- apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 μM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albu- min-apyrase solution containing approximately 20 μM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate- dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13.177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur. Thus thromboxane A2 formation is not necessary for the formation of these small aggregates, but is required for extensive aggregation and release. As with other weak agonists, the close platelet-to-platelet contact in the low Ca2+ medium appears to be necessary for full secondary aggregation. Omission of fibrinogen from the low Ca2+ medium prevented both primary and secondary aggregation in response to epinephrine. An antibody (10E5) to the glycoprotein Ilb/IIIa complex was completely inhibitory in the presence of fibrinogen. Thus the response of human platelets to epinephrine is influenced by the concentration of Ca2+ and the presence of fibrinogen in the medium in which they are suspended.

Sign in / Sign up

Export Citation Format

Share Document