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PubMed1987

A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.

We found a novel platelet aggregating factor in a patient with steroid-responsive immune thrombocytopenic purpura that is associated with defective collagen-induced platelet functions. The aggregating factor and platelet functions were analyzed. The patient, a 58-year-old female, had purpura and prolonged bleeding time despite adequate platelet counts (greater than 140,000/microL) after steroid therapy. The patient's platelets responded normally to all agonists except collagen. Platelet adhesion to collagen fibrils was decreased. The patient's plasma induced irreversible aggregation and ATP release in normal platelet-rich plasma (PRP). This platelet aggregating factor was found in F(ab')2 fragments of the patient's IgG, which caused thromboxane B2 synthesis, elevation of cytoplasmic Ca2+ levels, and phosphorylation of 40 kDa protein in normal platelets. Platelet aggregation by the patient's IgG was inhibited by prostacyclin, dibutyryl cAMP, diltiazem, disodium ethylenediaminetetraacetate, and antimycin A plus iodoacetate, but ADP scavengers, cyclo-oxygenase inhibitors, and heparin had little or no effect. The aggregating activity of the patient's IgG absorbed to and eluted from normal platelets. The patient's Fab fragments did not induce platelet aggregation in eight of ten normal PRP but specifically inhibited aggregation induced by collagen and by the patient's IgG. The major component of an immunoprecipitate made with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a 62 kDa mol wt, while no such precipitate appeared in extracts of the patient's platelets. These results indicated that platelet aggregation by the patient's IgG was induced by the reaction of an antibody with a specific antigen on the normal platelet membrane through stimulus-response coupling. This antigen may be a collagen receptor on the platelet, most likely a polypeptide of 62 kDa under reducing condition. The defect of collagen-induced aggregation of the patient's platelets seemed to be due to alteration of the membrane protein related to this putative collagen receptor.

Autoimmune Diseases↗
PubMed1998

Membrane lipid fluidity of blood platelets: a common denominator that underlies the opposing actions of various agents that affect platelet activation in whole blood.

Membrane lipid fluidity (MLF) is thought to play a crucial role in signal transduction and is believed to affect the responsiveness of blood platelets. In a recent study it was demonstrated that EDTA, used as the blood anticoagulant, brought about a significant increase in expression of GMP-140 antigen, and this effect was accompanied by a significant increase in platelet MLF. Moreover, this spontaneous EDTA-driven platelet activation was vastly attenuated in the presence of tissue-type plasminogen activator, which is also known to affect platelet MLF. The hypothesis was raised that the modulation of platelet membrane fluidity by EDTA might underlie platelet spontaneous activation in the presence of EDTA. To further explore the possible molecular mechanism(s) of the EDTA-dependent triggering of signal transduction pathway(s) in human blood platelets, we monitored the extent of spontaneous platelet activation in the presence of EDTA and selected platelet membrane 'fluidizers' and 'rigidizers'. A reduction in the EDTA-dependent platelet release and activation was noted, not only in the presence of rt-PA (by over 50%, P < 0.001), which acted as a rigidizer of platelet membrane fluidity (ESR h+1/h0 ratios of 5-DOXYL-Ste and 12-DOXYL-Ste decreased by 6.2%, P<< 0.0001, and 3.8%, P < 0.02, respectively), but also in the presence of other modulators of MLF, regardless of their fluidizing or rigidizing effects. Both rigidizers (procaine and lidocaine, 5-DOXYL-Ste h+1/h0 reduced by up to 6.5%, 12-DOXYL-Ste h+1/h0- by up to 4.5%, P < 0.02 or less) and fluidizers (benzyl alcohol, ethanol, 12-DOXYL-Ste h+1/h0 increased by 17.8% and 6.1%, respectively, P <<0.0001) of platelet membranes significantly depressed platelet activation (respectively, down to 1.1%, 7.7%, 6.7% and 8.5% vs control EDTA 22.9% of CD62-positive platelets). We suggest that EDTA induces alterations in membrane glycoprotein structure and affect MLF by altering lipid-protein interactions, and thus triggers signal transduction in the course of platelet activation. The resulting displacements in platelet membrane proteins, dislocation of membrane components and/or distortion of lipid-protein interactions could generate an 'outside-in' signalling that is mediated by the altered platelet MLF. Overall, it is likely that interference with the structure and conformation of selected domains of platelet membrane proteins might be the crucial mechanism by which EDTA leads to exaggerated activation of platelets in whole blood.

Journal Article↗
PubMed1997

Differences between contrast media in the inhibition of platelet activation by specific platelet agonists.

RATIONALE AND OBJECTIVES: The authors evaluated the ability of three x-ray contrast agents--a nonionic monomeric agent (iohexol), a nonionic dimeric agent (iodixanol), and an ionic dimeric agent (ioxaglate)--to either directly activate platelets or inhibit a platelet agonist from activating platelets. METHODS: Fluorescence spectroscopy was used to detect the effect of contrast media on platelet activation. In this method, the platelet is first exposed to a fluorescent probe, which is de-esterified and trapped to Fluo-3 within the platelet. In the presence of calcium, the fluorescence emission from Fluo-3 is increased 80-fold. Thus, the increase in the free platelet calcium associated with platelet activation can be used to indicate platelet activation. RESULTS: None of the agents were shown to directly activate platelets. However, wide differences in the ability of contrast media to inhibit platelet activation by a specific agonist were observed. Activation of platelets by epinephrine or arachidonic acid was not affected by any of the three contrast media studied. All three agents partially inhibited collagen activation of platelets, with ioxaglate the more potent inhibitor. Ioxaglate was the only agent to inhibit thrombin activation of platelets. Inhibition of adenosine diphosphate platelet activation was more extensive with ioxaglate than with iodixanol; iohexol produced no inhibition at all. CONCLUSION: Direct activation of platelets by contrast media was not observed. Of greater importance is the finding that ionic contrast media, but not nonionic contrast media, inhibit thrombin activation of platelets by binding to the anion-binding exosite I, thus preventing thrombin from binding to and activating the platelet.

Arachidonic Acid↗
PubMed2004

[Comparison of platelet activators used in slide platelet aggregation test].

The study was purposed to explore the suitable platelet activators to be used in slide platelet aggregation test. Experiments were as follows: (1) to detect the intensity and time in 15 healthy donors' platelet aggregation tests induced by cationic propyl gallate (c-PG) and the usual platelet activators: ADP, collagen, epinephrine, arachidonic acid and ristocentin, respectively; (2) to detect the time in platelet aggregation tests of 15 healthy donors induced by c-PG and the above usual platelet activators respectively after addition of PGI2, cAMP or EDTA; (3) to detect the time in 15 healthy donors' platelet aggregation tests induced by c-PG after addition of heparin; (4) to detect the intensity and time of platelet aggregation induced by c-PG at the platelet count of (240-15) x 10(9)/L, (5) to detect the time of platelet aggregation induced by c-PG in eight patients each of whom had taken 100 mg aspirin per day for five days. The results showed that (1) c-PG reduced the strongest intensity of platelet aggregation and the time taken was appropriate, (2) c-PG was the most effective activator to reveal the inhibitive effect on platelet by PGI2, cAMP or EDTA, (3) 0.5 - 3 U/ml heparin did not significantly change the platelet aggregation induced by c-PG, (4) 15 healthy donors' platelet aggregation induced by c-PG displayed clearly on the slide until the platelet count below 30 x 10(9)/L, (5) The platelet aggregation time induced by c-PG was significantly prolonged in eight patients who had taken aspirin. In conclusion, compared to the usual platelet activators, c-PG has remarkable potential advantages when used in slide platelet aggregation test.

Cyclic AMP↗
PubMed1987

Effect of monoclonal antibodies against von Willebrand factor and platelet glycoproteins IIb/IIIa on the platelet retention test.

The platelet retention test provides a measure of the number of platelets retained in a column of glass beads and is one of the few in vitro platelet function tests that is abnormal in von Willebrand's disease (vWd). In a two-stage test, 1 mL of blood (designated A) was passed through the column, followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which platelet retention was measured. With normal blood as A and B, retention is very high in all 5 mL of blood B. In the first stage, platelets adhere to the glass beads; this requires fibrinogen but not von Willebrand factor (vWf). The platelet-platelet adhesion in the second stage requires vWf, is dependent on release of ADP, and fails to occur if thrombasthenic platelets are tested. Retention was normal when blood from a patient with afibrinogenemia was used as blood B. We have now used monoclonal antibodies to elucidate further the mechanism of platelet retention. Five antibodies to different epitopes on vWf essentially abolished retention in the one-stage test and in the second stage of the two-stage test, but had no effect on the first stage. Thus, the entire vWf molecule must be free of antibody to function in the platelet-platelet adhesion of the second stage of this test. Binding of the antigen-antibody complex to the platelet Fc receptor was not responsible, as Fab and F(ab')2 fragments of one of the antibodies were as effective as intact antibody, and as neither heat-aggregated IgG nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2 fragments of 6D1, an antibody to platelet GP Ib that prevents binding of vWf to platelets, also inhibited the second phase of retention. An antibody that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8) inhibited both the first and second stages of retention, whereas LJ-P5, an antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused slight inhibition of retention when normal or afibrinogenemic blood was used as blood B and was reported to cause only partial inhibition of ADP-induced platelet aggregation in this afibrinogenemic patient. The results suggest that vWf is altered during rapid passage of blood through the glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which then binds the altered vWf or fibrinogen, either of which can induce platelet aggregation (platelet-platelet adhesion) and thus retention in the column.

Antibodies, Monoclonal↗
PubMed1981

Interaction of platelet plasma membranes with thrombin-activated platelets.

This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin-activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

Blood Platelets↗
PubMed1997

Local delivery of platelets with encapsulated iloprost to balloon injured pig carotid arteries: effect on platelet deposition and neointima formation.

Local delivery of a drug to the arterial wall during angioplasty is an approach which might reduce the incidence of occlusive events such as thrombosis and restenosis, without the risk of systemic side effects. By exploiting their natural primary haemostatic properties, platelets, with encapsulated drugs, can be targeted to a vessel wall injury site and act as a depot for sustained release. The platelet plasma membrane can be reversibly permeabilised by high voltage, short duration electrical pulses (electroporation). Drugs will diffuse into porated platelets and become trapped on resealing. We have studied the effects of autologous platelets, electroloaded with the stable prostacyclin analogue, iloprost on platelet deposition and neointima formation in a pig carotid angioplasty model. Iloprost loaded or control platelets were delivered locally and immediately to the balloon injured site using a double balloon delivery catheter. Acute platelet deposition was measured using 111-Indium, and neointima formation at 21 days post angioplasty was assessed by morphometric analysis. In pigs treated with iloprost loaded platelets, platelet deposition on the artery at 2 hours post injury was dramatically reduced (to approximately monolayer coverage), when compared with arteries from pigs treated with control platelets. In pigs with deeply injured arteries, i.e. with extensively ruptured internal elastic lamina (IEL), platelet deposition was reduced by 88% compared with control arteries (118 +/- 20 x 10(6)/cm vs. 14 +/- 2 x 10(6)/cm, means +/- SI, 2P < 0.001). In minimally injured arteries (IEL intact) a 65% reduction in platelet deposition was observed (55 +/- 24 x 10(6)/cm vs. 19 +/- 3 x 10(6)/cm. 2P < 0.002). A high concentration of free iloprost, delivered to the angioplasty site, with control platelets, had far less effect on platelet deposition, substantiating the advantage of platelet encapsulation. At 21 days post injury, morphometry of the carotid arteries after treatment with iloprost loaded platelets showed significant reductions in intimal area and intimal/medial ratios in minimally injured vessels (P < 0.05) as compared with vessels from pigs treated with control platelets. With deeply injured vessels, the mean differences (control vs. treated) for the same morphometric parameters were not significant. This novel approach of electro-encapsulating drugs within autologous platelets, and using them as highly biocompatible and biodegradable drug targeting vehicles might, with the appropriate choice of encapsulated agent, have potential for reducing the incidence of occlusion after angioplasty and thrombolysis procedures.

Animals↗
PubMed1997

C3H mice have larger spleens, lower platelet counts, and shorter platelet lifespans than C57BL mice: an animal model for the study of hypersplenism.

Several studies have shown that C3H mice have a modal megakaryocyte ploidy class of 32N as opposed to the 16N found in all other mouse strains tested. In addition to higher megakaryocyte DNA content, C3H mice are characterized by greater 35S-sulfate incorporation into platelets but somewhat lower platelet counts than C57BL mice. The aim of this study was to determine the etiology of mild thrombocytopenia in C3H mice. Several studies have indicated that an increase in platelet 35S incorporation parallels increased megakaryocyte ploidy. Thus the higher 35S incorporation by C3H mouse platelets is likely related to their higher megakaryocyte ploidy, but these differences do not explain the lower platelet counts observed in C3H mice. To elucidate the paradox of relatively low platelet counts in C3H mice despite elevated platelet 35S incorporation, we performed a hematological evaluation (including assessment of platelet counts and lifespans) and measured (relative) spleen size in C3H and C57BL mice. Our data confirm earlier reports of significantly lower platelet counts in C3H mice (p < 0.0005) compared with C57BL mice. We also found that C3H mice have larger spleens (p < 0.0005) than C57BL mice. Other groups of C3H and C57BL mice were splenectomized to determine the role of the spleen in decreasing platelet counts in C3H mice compared with C57BL mice. C3H mice had an average platelet lifespan of 4.8 days, which was significantly shorter (p < 0.01) than the 5.7-day platelet lifespan observed in normal C57BL mice. Platelet lifespans for splenectomized C3H mice were significantly (p < 0.005) longer than those found in intact C3H control mice, but splenectomy of C57BL mice had no effect on their platelet lifespans (p < 0.40). The lengthened platelet lifespans of splenectomized C3H mice indicate that the spleens of C3H mice were sequestering or destroying platelets at a faster rate than the spleens of C57BL mice. These data lead to the hypothesis that the relatively large spleens of C3H mice produce mild thrombocytopenia with increased platelet production (shorter platelet lifespan) compared with spleens of C57BL mice.

Animals↗
PubMed1996

Exogenous diacylglycerols synergize with PAF with human platelets, but inhibit PAF-induced responses of rabbit platelets.

To investigate whether diacylglycerol (DAG) has a role in reversible platelet aggregation induced by low concentrations of platelet-activating factor (PAF), we attempted to use the DAG kinase inhibitor, R59022, to prevent rapid conversion of DAG to phosphatidic acid. However, we found that R59022 inhibited the binding of [3H]PAF to human and rabbit platelets and to rabbit platelet membranes. We then investigated whether exogenous, cell-penetrating DAGs (1,2-dihexanoyl-sn-glycerol (DHG) and 1-oleoyl-2-acetyl-sn-glycerol (OAG)) act synergistically with low concentrations of PAF that alone induce only reversible aggregation. Platelets were isolated and labeled with [14C]serotonin. DHG (25-75 microM) caused slow, weak aggregation and some release of [14C]serotonin with human, but not rabbit, platelets. OAG (25-75 microM) did not aggregate either species' platelets. Phosphorylation of pleckstrin by DHG was more transient in rabbit platelets than previously observed with human platelets. Both DHG and OAG synergistically potentiated PAF-induced aggregation of human platelets, but, paradoxically, concurrently inhibited the PAF-induced increase in intracellular Ca2+ ([Ca2+]i): potentiation decreased upon incubation with DAGs before PAF addition. In contrast, DHG strongly inhibited PAF-induced aggregation of rabbit platelets; inhibition decreased upon preincubation. OAG, added with PAF, slightly potentiated aggregation of rabbit platelets: upon preincubation, OAG progressively inhibited. Effects of DHG and OAG on PAF-induced increases in [Ca2+]i in rabbit platelets followed a similar pattern; thus, with rabbit platelets, inhibition of the [Ca2+]i increase may at least partially account for inhibition of PAF-induced aggregation by exogenous DAGs. Results with human platelets are consistent with stimulation of protein kinase C by DAGs, and then metabolism of DAGs and/or negative feedback by DAGs, but results with rabbit platelets indicate both an unexpected species difference and a difference between the effects of DHG and OAG on PAF-induced platelet aggregation.

Animals↗
PubMed2004

Moderate-intensity exercise suppresses platelet activation and polymorphonuclear leukocyte interaction with surface-adherent platelets under shear flow in men.

The reciprocal modulation of platelet and polymorphonuclear leukocyte (PMN) activities is important in the pathogenesis of thrombosis and inflammation. This study investigated how moderate exercise affects shear-induced platelet activation and subsequent PMNs interaction with platelet-related thrombi under shear flow. Sixteen sedentary healthy men engaged in moderate exercise (about 60% VO(2max)) on a bicycle ergometer. Platelet activation, PMNs interaction with surface-adherent platelets, and PMN-dependent inhibition of platelet activation under shear flow were measured both before and immediately after exercise. The results of this study can be summarized as follows: (1) moderate exercise was associated with lower extents of shear-induced platelet adhesion and aggregation, binding of von Willbrand factor (vWF) to platelets, and glyco-protein IIb/IIIa activation and P-selectin expression on platelet than at rest; (2) the velocity and percentage of rolling PMNs increased while the number of PMNs remaining bound to surface-adherent platelets decreased after moderate exercise; (3) although treating the PMNs with oxidized-low density lipoprotein (Ox-LDL) enhanced PMNs interaction with surface-adherent platelets, moderate exercise suppressed the enhancement of platelet-PMN interaction by Ox-LDL; (4) moderate exercise decreased platelet [Ca (2+)](i) elevation induced by ADP and platelet [Ca(2+)](i) levels mediated by PMN and Ox-LDL-treated PMN; and (5) plasma and PMN-derived nitric oxide metabolites and plasma vWF antigen and activity increased after moderate exercise, whereas plasma and platelet-derived soluble P-selectin levels remained unchanged in response to exercise. Therefore, we conclude that moderate-intensity exercise suppresses shear-induced platelet activation and subsequent PMNs adhesion to platelets deposited at sites of vascular injury under flow, thereby reducing the risks of vascular thrombosis and inflammation.

Blood Platelets↗
PubMed1999

Post-DDAVP thrombocytopenia in type 2B von Willebrand disease is not associated with platelet consumption: failure to demonstrate glycocalicin increase or platelet activation.

Thrombocytopenia is frequently reported in type 2B von Willebrand disease (vWD), and thought to be related to the abnormally high affinity of 2B von Willebrand factor (vWF) for platelet GPIb-IX. To gain an insight into the nature of this thrombocytopenia, we measured plasma glycocalicin (GC) levels (as a marker of platelet turnover), and platelet surface expression of the alpha granule protein P-selectin (as a marker of platelet activation) in 9 patients with type 2B vWD before, and in 4 patients also following the infusion of 1-desamino-8-d-arginine vasopressin (DDAVP). Three patients presented a persistent decrease of platelet counts in the resting condition. GC levels were within the normal range, regardless of the platelet counts, in all but one patient who presented, on the other hand, a normal platelet count. Moreover, platelets expressed normal amounts of P-selectin on their surface, regardless of platelet counts. These findings suggest that the thrombocytopenia observed in type 2B vWD is not due to platelet activation and subsequent consumption in circulation. Despite a significant, albeit transient, decrease in platelet count, DDAVP did not induce an increase in plasma GC levels, nor enhance P-selectin expression. These observations indicate that the acute post-DDAVP thrombocytopenia in type 2B vWD is not related to platelet activation and consumption. We advance that the post-DDAVP 2B vWF is hemostatically more active, and able to induce agglutination but not aggregation of circulating platelets. This would explain both the prompt recovery of basal platelet counts after the post-DDAVP decrease, and the lack of reported thrombotic complications in this disorder. Therefore, even though 2B vWF is characterized by an enhanced affinity for the platelet surface, its binding to platelet GPIb-IX in the soluble phase is not able to induce true platelet aggregation: vWF thus appears to be mainly an adhesive protein, rather than an aggregating agent.

Adult↗
PubMed1989

Platelet size affects both micro- and macro-aggregation: contributions of platelet number, volume fraction and cell surface.

The relationship between platelet size (v), platelet number (N0), platelet volume fraction (phi) and platelet aggregation was evaluated in human platelet subpopulations separated on the basis of volume using counterflow centrifugation. The original platelet population and three size-dependent platelet fractions were concentrated and resuspended in autologous citrated platelet-poor plasma at varying N0. Micro-aggregation (PA) and macro-aggregation (TA) were determined respectively by electronic particle counting and light transmission (turbidimetry). At similar N0, large platelets were about two-fold more sensitive and more rapidly recruited into both micro (PA) and macro (TA) aggregates in response to ADP than the small platelets (v were respectively 7.3 +/- 0.2 and 4.2 +/- 0.2 fl, each 16 +/- 4% of the total population). At volume fractions favouring the small platelets, however, the above differences persisted for PA, but not for TA. Similar size-dependent differences were observed for two other receptor-mediated platelet activators, namely a stable thromboxane A2 analogue (U46619) and platelet activating factor (PAF), but not for ristocetin-induced agglutination. Increasing platelet size appears to optimize platelet aggregation due to (i) the simple geometric advantage of large-sized platelets associated with any given N0, seen for both PA and TA, and (ii) intrinsic differences in size-dependent subpopulations which favour more efficient platelet membrane surface changes associated with receptor-mediated micro-aggregation (PA), for which a likely model is proposed.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2002

Phosphatidylserine exposure in platelet concentrates during the storage period: differences between the platelets collected with different cell separators.

BACKGROUND AND OBJECTIVES: Platelet alterations occur during the production and storage of platelet concentrates, the so called "storage lesion". We studied the platelet alterations during the storage period in apheresis concentrates, employing flow cytometry for phosphatidylserine (PS) detection on platelets during the five days of storage. MATERIAL AND METHODS: Twenty-seven single donor platelet concentrates harvested with the Cobe Trima, Baxter Amicus, or Haemonetics MCS+ were analyzed for PS exposure by flow cytometry on the day of production (day 1) and on days 3 and 5 of storage. Furthermore PS expression was analyzed in platelet donors' blood samples withdrawn before plateletpheresis. RESULTS: PS expression on platelets gave the following median values: in blood donors before apheresis it was 1.12% (0.13-1.78) in platelets concentrates on the first day (2 h after apheresis) 2.06% (0.66-15.2), the third day 6.57% (1.98-51.13) and the fifth day 23.04% (3.86-80.23). All differences between median values of PS expression in blood samples before apheresis, and platelets concentrates on days 1, 3 and 5 of storage, are statistically significant. The expression of PS in platelet concentrates was analyzed in relation to the blood cell separator used for the collection procedure and showed the following results: on day 1 the median values of PS in platelet concentrates collected with the three different blood cell separators, Trima, Cobe and MCS, did not show statistically significant differences. On day 3, the platelets concentrates collected with the Trima and with the MCS showed differences that were statistically significant. Those were respectively 10.59% (4.56-51.13) and 3.53% (1.98-12.61), p = 0.005. The PS expression in platelet concentrates collected with the Trima and MCS showed differences that are also statistically significant on day 5 at respectively 32.4% (9.61-80.23) and 8.57% (3.86-48.42), p = 0.005. CONCLUSIONS: PS exposure in platelet concentrates on days 3 and 5 rise to levels that could compromise the quality of the platelet units. Improvements in standardized platelet quality controls, and in platelet collection systems are required to reduce the storage lesions in platelets concentrates.

Adult↗
PubMed2001

Vimentin exposed on activated platelets and platelet microparticles localizes vitronectin and plasminogen activator inhibitor complexes on their surface.

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), is found in plasma and platelets. PAI-1 circulates in complex with vitronectin (Vn), an interaction that stabilizes PAI-1 in its active conform. In this study, we examined the binding of platelet-derived Vn and PAI-1 to the surface of isolated platelets. Flow cytometry indicate that, like P-selectin, PAI-1, and Vn are found on the surface of thrombin- or calcium ionophore-activated platelets and platelet microparticles. The binding of PAI-1 to the activated platelet surface is Vn-dependent. Vn mediates the binding of PAI-1 to platelet surfaces through a high affinity (K(d) of 80 nm) binding interaction with the NH(2) terminus of vimentin, and this Vn-binding domain is expressed on the surface of activated platelets and platelet microparticles. Immunological and functional assays indicate that only -5% of the total PAI-1 in platelet releasates is functionally active, and it co-precipitates with Vn, and the vimentin-enriched cytoskeleton fraction of activated platelet debris. The remaining platelet PAI-1 is inactive, and does not associate with the cytoskeletal debris of activated platelets. Confocal microscopic analysis of platelet-rich plasma clots confirm the co-localization of PAI-1 with Vn and vimentin on the surface of activated platelets, and platelet microparticles. These findings suggest that platelet vimentin may regulate fibrinolysis in plasma and thrombi by binding platelet-derived Vn.PAI-1 complexes.

Animals↗
PubMed1976

A comparison of platelet size, latelet count, and platelet 35S incorporation as assays for thrombopoietin.

Average platelet size, platelet count, and 35S-incorporation into platelets were compared as methods for the measurement of thrombopoietin-stimulated thrombopoiesis. In mice injected with rabbit anti-mouse platelet serum (RAMPS) average platelet size was shown to be increased as mice were recovering from thrombocytopenia. Also, 35S-measurements on platelets of these mice showed significant increases in cpm/average platelet 2-4 days after RAMPS treatment. Significant increases in 35S-incorporation into the total circulating mass of platelets were found on days 3-4. In normal mice or mice in rebound-thrombocytosis injected with thrombopoietin, platelet size remained unchanged, whereas the platelet cound and 35S-incorporation into platelets were shown to be significantly increased. Moreover, a dose-response experiment in mice pretreated with RAMPS showed a slight increase in platelet count as the dose of TSF was increased, but platelet sizes were unaltered. The % 35S-incorporation into platelets showed a significant linear dose-response, i.e. as the dose of thrombopoietin was increased, as increase in % 35S-incorporation into platelets was observed. These data indicated that of the three indirect measurements of thrombopoietin, the % 35S-incorporation into mouse platelets was the most sensitive, followed by platelet counting; the least sensitive measurement of thrombopoiesis was change in platelet size.

Animals↗
PubMed1999

Activated protein C resistance: effect of platelet activation, platelet-derived microparticles, and atherogenic lipoproteins.

Plasma and platelet factor Va represent different substrates for activated protein C (APC). In this study, we have measured platelet-dependent APC resistance and the effect of aspirin and a platelet glycoprotein IIbIIIa antagonist (GR144053F) on this phenomenon. In platelet rich plasma (PRP), progressive APC resistance was observed with increasing platelet activation. APC sensitivity ratios of 1.8, 1.7, and 1.4 were observed after platelet activation with thrombin receptor activating peptide (TRAP), collagen, and A23187, respectively. Ultracentrifugation at 77,000g for 1 hour abolished APC resistance indicating that the phenotype is associated exclusively with the platelet membrane. APC resistance was not observed in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles or purified human plasma lipoproteins. APC resistance was observed in the presence of platelet-derived microparticles, but to a lesser degree than that in the presence of activated platelets. The platelet-dependent APC resistance phenotype was also observed when endogenous APC was generated by Protac (American Diagnostica, Inc, Greenwich, CT). In vitro inhibition of platelet activation with aspirin had no effect, but the fibrinogen receptor antagonist, GR144053F, inhibited platelet-dependent APC resistance. These results indicate that platelet activation results in an APC-resistant phenotype comparable to that observed in the plasma of patients with factor V gene mutations affecting critical APC cleavage sites. This suggests that platelet activation at the site of endothelial damage downregulates a critical natural anticoagulant mechanism. The antithrombotic effect of aspirin may be due to an indirect effect on platelet-dependent APC resistance with reduced platelet retention within a developing thrombus. The more potent antithrombotic effect of glycoprotein IIbIIIa antagonists may in addition be the result of reduced platelet factor Va expression and modulation of the platelet-dependent APC resistance phenotype.

Activated Protein C Resistance↗
PubMed1984

Platelet concentration and platelet volume distribution in healthy cats.

An electronic particle counter and size analysis system were used to determine platelet count, mean platelet volume (MPV), platelet volume heterogeneity (SD of platelet volumes), and platelet mass values in feline platelet-rich plasma (PRP) prepared by brief centrifugation of blood. Electronic platelet counts correlated well with manual counts done by phase microscopy over a platelet count range of 5,000/microliters to 840,000/microliters (r = 0.93). Variability in manual and electronic counts was not significantly different. However, due to differential stratification of platelet concentration in PRP, electronic counts on aspirated, mixed PRP were significantly less variable (P less than 0.01). Reference values, determined on blood from 25 healthy adult cats, were platelet count: 296,000 to 850,000/microliters; MPV: 11.0 to 18.1 fl; platelet volume heterogeneity: 5.8 to 10.6 fl, and platelet mass: 4.0 to 11.4 microliters/ml of blood. There was a direct relationship between MPV and platelet volume heterogeneity and an inverse relationship between MPV and platelet count. Platelet concentration and size were not constant in different PRP layers. Aliquots of PRP near the top had a significantly lower platelet concentration (P less than 0.001) and lower MPV (P less than 0.001) than did aliquots from near the bottom of the PRP layer. During the 4 hours after sample collection, there was a progressive decrease in platelet count and progressive increase in MPV, but the changes were not significant. When platelets were incubated in counting fluid, there was a slight increase in MPV by 1.5 hours. By 2.5 hours, there was a significant increase in MPV (P less than 0.05).

Animals↗
PubMed1994

Role of platelet GpIIb/IIIa receptors in the modulation of platelet plasminogen activator inhibitors type-1 (PAI-1) release.

This study was undertaken to determine the role of platelet glycoprotein (GP) IIb/IIIa receptors in the modulation of plasminogen activator type-1 (PAI-1) release from human platelets as compared to other platelet functions. To address this issue, the effect of various agonists on human platelet aggregation, [125I]fibrinogen binding and the release of PAI-1 was examined in normal and Glanzmann's thrombasthenic (GT) platelets. In control subjects, maximum platelet aggregation and PAI-1 secretion were observed within 5 min in response to the different agonists including thrombin, collagen, adenosine diphosphate (ADP), and arachidonic acid. Agonist-induced platelet GpIIb/IIIa receptor activation was confirmed by [125I]fibrinogen binding analysis. In contrast, platelets from GT subjects demonstrated a lack of fibrinogen binding and a lack of an aggregatory response to all agonists tested except to the GPIb- mediated aggregation induced by ristocetin. However, GT platelets demonstrated normal responsiveness in secreting PAI-1 in response to the various agonists. Similarly, when platelet GpIIb/IIIa receptors were blocked in normal platelets by the tripeptide Arg-Gly-Asp (RGD) or the tetrapeptide Arg-Gly-Asp-Ser (RGDS) at 10(-3) M, agonist-induced platelet aggregation and fibrinogen binding were blocked, but platelet PAI-1 release was not blocked. Furthermore, flow cytometric analysis using dual fluorescence markers for the platelet GPIIb/IIIa membrane receptors (FITC-labeled cyclic RGD analog, XL086) and for the alpha granule (PE-monoclonal antibody for P-selectin) demonstrated a dissociation between the platelet GPIIb/IIIa receptors and granular secretion. These results suggest a lack of a role for platelet GpIIb/IIIa receptors in the modulation of platelet PAI-1 release.

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