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PubMed2003

Dynamics of GPIIb/IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy.

The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22 degrees C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of "vanguard" platelets, "follower" platelets attached to the spread-out vanguard platelets. Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets. Thus, thrombi formed as a concerted process rather than as sequential processes. Treatment of human platelets with monoclonal antibody (mAb) 7E3 (anti-GPIIb/IIIa (alphaIIbbeta3) + alphaVbeta3) or tirofiban (anti-GPIIb/IIIa) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi. Similar results were obtained with Glanzmann thrombasthenia platelets. Wild-type mouse platelets in the presence of mAb 1B5 (anti-GPIIb/IIIa) and platelets from beta3-null mice behaved like human platelets in the presence of 7E3 or tirofiban. Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model, but those obtained with 7E3- and tirofiban-treated human platelets, 1B5-treated mouse platelets, or beta3-null platelets demonstrated a more uniform deposition than predicted. Thus, in this model system, absence or blockade of GPIIb/IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition.

Animals↗

PubMed1993

Role of platelet membrane glycoproteins Ib/IX and IIb/IIIa, and of platelet alpha-granule proteins in platelet aggregation induced by human osteosarcoma cells.

We have previously shown that the platelet-aggregating activity of human MG-63 and HOS osteosarcoma cells depends at least in part upon tumor cell surface-associated thrombospondin, and suggested that platelet-osteosarcoma cell interactions could occur through interactions with specific platelet membrane receptors. In this study, the platelet-aggregating activity of MG-63 and HOS cells was studied by using a variety of platelet disorders. Both osteosarcoma cell lines induced a biphasic platelet aggregation response when added to normal platelet-rich plasma, while the second phase of aggregation was absent when added to gray platelets (deficiency in alpha-granule proteins) and to aspirin-treated platelets. Platelets from two unrelated patients with type I Glanzmann's thrombasthenia (deficiency in glycoprotein (GP) GPIIb/IIIa) did not aggregate at all with osteosarcoma cells. Using giant platelets from three patients with Bernard-Soulier syndrome (deficiency in GPIb/IX), the aggregation response induced by MG-63 and HOS cells was monophasic and reversible when compared to normal-sized platelets and to giant platelets from a patient with May-Hegglin anomaly (no membrane GP defect). Because GPIb serves as a receptor for von Willebrand factor during hemostasis, aggregation experiments were also conducted with the platelet-rich plasma of two patients with a low plasma von Willebrand factor concentration (type I von Willebrand's disease) before and after the infusion of deamino-D-arginine vasopressin. MG-63 and HOS cells induced biphasic platelet aggregation both before and after deamino-D-arginine vasopressin treatment, while the ristocetin-dependent binding of von Willebrand factor to platelets only occurred after deamino-D-arginine vasopressin treatment. Preincubation of normal platelet-rich plasma with monoclonal antibody SZ-2 directed against the von Willebrand binding domain of GPIb did not inhibit the platelet-aggregation activity of osteosarcoma cells, whereas anti-GPIb antibody SZ-2 did inhibit ristocetin-induced platelet agglutination. In addition, anti-GPIX antibodies did not affect platelet-osteosarcoma cell interactions. In conclusion, our data demonstrate that the first phase of the platelet-aggregating activity of human osteosarcoma cells is initiated by the interaction of these tumor cells with platelet membrane GPIIb/IIIa, whereas the second phase, even if plasma von Willebrand factor is deficient, involves platelet membrane GPIb and the participation of platelet alpha-granule proteins in membrane-mediated events, making aggregation irreversible.

Antibodies, Monoclonal↗

PubMed1986

The relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons.

The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In-labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high-density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6). Although a slight increase in the density of all platelets during platelet senescence is indicated by these studies, we conclude that platelet density heterogeneity is not primarily a consequence of age-related changes in platelet density.

Animals↗

PubMed1983

Survival of density subpopulations of rabbit platelets: use of 51Cr-or 111In-labeled platelets to measure survival of least dense and most dense platelets concurrently.

The origin of the density heterogeneity of platelets was studied by measuring the survival of density subpopulations of rabbit platelets separated by discontinuous Stractan density gradient centrifugation. When a total population of 51Cr-labeled platelets was injected into recipient rabbits, the relative specific radioactivity of the most dense platelets decreased rapidly. In contrast, that of the least dense platelets had not changed 24 hr after injection, and then decreased slowly. To distinguish between the possibilities that most dense platelets are cleared from the circulation more quickly than least dense platelets or that platelets decrease in density as they age in the circulation, the concurrent survival of least dense and most dense platelets, labeled with either 51Cr or 111In-labeled total platelet populations, determined concurrently in the same rabbits, were identical, calculated from 1 hr values as 100%. However, the 1-hr recovery of 111In-labeled platelets was slightly but significantly less than that of 51Cr-labeled platelets. Therefore, we studied the survival of 51Cr-labeled least dense and 111In-labeled most dense platelets as well as that of 111In-labeled least dense and 51Cr-labeled most dense platelets. Mean 1-hr recovery of least dense platelets, labeled with either isotope (78% +/- 7%, SD) was similar to that of most dense platelets, labeled with either isotope (77% +/- 8%; SD). Mean survival of least dense platelets was 47.3 +/- 18.7 hr (SD), which was significantly less than that of most dense platelets (76.1 +/- 21.6 hr; SD) (p less than 0.0025). These results indicate that platelets decrease in buoyant density as they age in the circulation and that most dense platelets are enriched in young platelets, and least dense in old. Thus, the events that affect platelets as they age in the circulation contribute to platelet density heterogeneity, although they may not be the sole cause of it.

Animals↗

PubMed2004

Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation.

Annexin A5↗

PubMed1993

Intact platelet membranes, not platelet-released microvesicles, support the procoagulant activity of adherent platelets.

The possibility that platelets release microvesicles on adherence to either von Willebrand factor (vWf) or collagen was examined by flow cytometry analysis of the supernatant above layers of adherent platelets. No microvesicle release was detected as a result of adherence to vWf or to collagen, a known platelet agonist. Approximately 8% of the total platelet mass was released as microvesicles after thrombin stimulation of the vWf- or collagen-adherent platelets. A larger portion of the vWf-adherent platelet membranes (approximately 21%) was released as microvesicles subsequent to platelet stimulation with the nonphysiological agonist calcium ionophore A23187. Calpeptin, a calpain inhibitor, had no effect on microvesicle release, suggesting that calpain proteolysis of platelet cytoskeletal proteins was not responsible for microvesicle shedding under the conditions studied. Examination of the vWf-adherent platelets by scanning electron microscopy showed that virtually no microvesicles bound to exposed vWf multimers. No microvesicle binding to the adherent platelets was observed, indicating that the majority of the microvesicles were shed from the platelet and vWf surface on platelet activation. The ability of the microvesicle population to support procoagulant activity was measured with a prothrombinase activity assay and was compared with the activity supported by the adherent platelet membranes. More than 85% of the total prothrombinase activity remained associated with the adherent platelet membranes, both for unstimulated platelets and platelets stimulated with physiological agonists. Furthermore, the residual activity found in the buffer fraction containing detached platelets and any released microvesicles could be attributed to the detached platelets. No activity could be attributed to the microvesicles, as thrombin stimulation of either vWf-or collagen-adherent platelets did not promote increased procoagulant activity relative to the unstimulated adherent platelets, even though microvesicle release was detected as a result of agonist addition. Neither full platelet activation nor microvesicle shedding played an essential role in generating procoagulant activity in the adherent platelet system.

Blood Coagulation↗

PubMed1987

Platelet enhancement of tumor cell adhesion to subendothelial matrix: role of platelet cytoskeleton and platelet membrane.

Platelet involvement during tumor cell adhesion to subendothelial matrix was examined in vitro. Platelets were subjected to thrombin stimulation and mechanical lysis and examined for their effects on tumor cell adhesion. These treatments altered the platelet ultrastructure and cytoskeletal integrity. Untreated washed rat platelets (WRP) exhibited extensive adhesion to and spreading on substrates and substantially enhanced tumor cell adhesion to the same substrates (i.e., 250% greater than tumor cells without platelets). Thrombin prestimulation of platelets limited platelet adhesion and spreading and platelet facilitation of tumor cell adhesion. Complete mechanical lysis disrupted both the platelet membrane and the cytoskeleton and eliminated the ability of platelets to adhere or to enhance tumor cell adhesion. Partially lysed platelets resembled membrane ghosts and facilitated tumor cell adhesion by a mechanism independent of spreading and cytoskeletal rearrangement. Fractionation studies indicated that platelet cytoskeletal components played a role in the adhesion process. Pretreatment of WRP with cytochalasin A or B dose dependently inhibited microfilament-mediated platelet spreading and platelet-enhanced tumor cell adhesion. Colchicine and vinblastine induced microtubule depolymerization, but they had no observable effect on platelet spreading or platelet-enhanced tumor cell adhesion. It was concluded that platelet-enhanced tumor cell adhesion to subendothelial matrix depends on an intact platelet cytoskeleton and on a platelet membrane component(s) and is mediated by surface contact between platelets and tumor cells. Furthermore, platelet-mediated tumor cell adhesion to subendothelial matrix may involve two mechanisms: one dependent on, and one independent of, platelet spreading and cytoskeletal rearrangement.

Animals↗

PubMed1999

Possible mechanisms of the altered platelet volume distribution in type 2 diabetes: does increased platelet activation contribute to platelet size heterogeneity?

A direct consequence of increased platelet sensitivity in diabetes mellitus might be augmented release of platelet granule contents, which, in turn, may lead to the formation of a platelet volume gradient, increased platelet turnover and reduced survival of platelets from diabetic individuals. In this study we addressed the question whether diabetes-induced and lipid fluidity-mediated changes in platelet receptor exposure and accessibility might be part of a general mechanism underlying the increased rate of platelet ageing and reduced platelet survival in diabetes. Diabetic individuals showed higher numbers of platelets of extreme dimensions: very small platelets and larger platelets were more frequent compared to controls ( P(chi(2))< 0.03). The shifts in platelet volume distributions were paralleled by decreased expression of the alpha subunit of glycoprotein Ib (by up to 17%, P < 0.01) in platelet membranes from diabetic patients, increased expression of P-selectin in thrombin-stimulated diabetic platelets (P< 0.02), an increased number of platelet microparticles in diabetic individuals (P< 0.05 or P< 0.03 for resting or stimulated platelets, respectively), and reduced platelet membrane fluidity (by 5.2 +/- 0.6%, P< 0.01). We suggest that the distinct bimodality of platelet distribution in diabetic patients might arise from accelerated thrombopoiesis in diabetic subjects, and this is supported by the demonstration of elevated fractions of reticulated (rich with residual RNA) platelets in diabetic patients (14.6 +/- 5.6% vs 8.1 + 2.1% p(u) < 0.025). Overall, our results point to a fluidity-mediated platelet hypersensitivity and accelerated rate of platelet production in subjects with type 2 diabetes mellitus, which results in a greater number of very large and hypersensitive younger platelets and a more abundant fraction of small exhausted platelets.

Journal Article↗

PubMed2003

Integration of proteomics and genomics in platelets: a profile of platelet proteins and platelet-specific genes.

Platelets, while anucleate, contain RNA, some of which is translated into protein upon activation. Hypothesising that the platelet proteome is reflected in the transcriptome, we identified 82 proteins secreted from activated platelets and compared these, as well as published proteomic data, to the transcriptional profile. We also compared the transcriptome of platelets to other tissues to identify platelet-specific genes and used ontology to determine gene categories over-represented in platelets. RNA was isolated from highly pure platelet preparations for hybridization to Affymetrix oligonucleotide arrays. We identified 2,928 distinct messages as being present in platelets. The platelet transcriptome was compared with the proteome by relating both to UniGene clusters. Platelet proteomic data correlated well with the transcriptome, with 69% of secreted proteins detectable at the mRNA level, and similar concordance was obtained using two published datasets. While many of the most abundant mRNAs are for known platelet proteins, messages were detected for proteins not previously reported in platelets. Some of these may represent residual megakaryocyte messages; however, proteomic analysis confirmed the expression of many previously unreported genes in platelets. Transcripts for well-described platelet proteins are among the most platelet-specific messages. Ontological categories related to signal transduction, receptors, ion channels, and membranes are over-represented in platelets, while categories involved in protein synthesis are depleted. Despite the absence of gene transcription, the platelet proteome is mirrored in the transcriptome. Conversely, transcriptional analysis predicts the presence of novel proteins in the platelet. Transcriptional analysis is relevant to platelet biology, providing insights into platelet function and the mechanisms of platelet disorders.

Blood Platelets↗

PubMed1983

Platelet kinetics with indium-111 platelets: comparison with chromium-51 platelets.

The application of 111In-oxine to platelet labeling has contributed to the understanding of platelet kinetics along three lines: 1. It allows the measurement of new parameters of splenic function, such as the intrasplenic platelet transit time, which has shed new light on the physiology of splenic blood cell handling. 2. It facilitates the measurement of platelet life span in conditions, such as ITP, in which 51Cr may undergo undesirable elution from the platelet as a result of platelet-antibody interaction. 3. It allows the determination of the fate of platelets, that is, the site of platelet destruction in conditions in which reduced platelet life span is associated with abnormal platelet consumption, as a result of either premature destruction of "abnormal" platelets by the RE system, or the consumption (or destruction) of normal platelets after their interaction with an abnormal vasculature. Future research using 111In platelets may yield further valuable information on the control as well as the significance of intrasplenic platelet pooling, on the role of platelets in the development of chronic vascular lesions, and on the sites of platelet destruction in ITP. With regard to the latter, methods will have to be developed for harvesting sufficient platelets representative of the total circulating platelet population from severely thrombocytopenic patients for autologous platelet labeling. This would avoid the use of homologous platelets, which is likely to be responsible for some of the contradictory data relating to the use of radiolabeled platelet studies for the prediction of the response of patients with ITP to splenectomy.

Blood Flow Velocity↗

PubMed1997

Reduced platelet aggregation after fluvastatin therapy is associated with altered platelet lipid composition and drug binding to the platelets.

AIMS: High plasma cholesterol concentration and increased platelet activity are two major risk factors for atherosclerosis. Lovastatin, the lipophilic drug was shown to inhibit platelet aggregation whereas pravastatin, the hydrophilic drug had no such effect. Analysis of the effect of fluvastatin which is both a lipophilic and hydrophilic drug, on platelet aggregation was the goal of the present study. METHODS: Fluvastatin 40 mg daily was administered to 25 patients with hypercholesterolaemia for up to 24 weeks. Normal subjects acted as controls. The influence of fluvastatin on plasma lipids and on platelet aggregation and fluidity was studied. The direct effect of fluvastatin on platelets was compared with that of other statins. RESULTS: Fluvastatin therapy (40 mg day (-1) for a period of 4 weeks) in hypercholesterolaemic patients resulted in a 23% and 29% reduction in plasma levels of total cholesterol and LDL-cholesterol respectively. Platelet cholesterol/phospholipids molar ratio was reduced by 26% and platelet aggregation was significantly (P<0.02) reduced by 10% after 4 weeks of fluvastatin treatment. On continuing fluvastatin therapy for additional 20 weeks, no further decrement in plasma LDL cholesterol levels or in platelet cholesterol/phospholipid ratio were noted. However, platelet aggregation was further significantly (P<0.01) reduced by up to 15%. Incubation of platelets with increasing concentrations of fluvastatin or lovastatin, demonstrated a dose-dependent reduction in platelet aggregation, whereas pravastatin showed no effect. This inhibitory effect of fluvastatin or lovastatin on platelet aggregation (up to 34% or 22% respectively at a concentration of 1 microg statin ml (-1) was found both in platelet rich plasma and in washed platelet suspensions. Fluvastatin and lovastatin (but not pravastatin), seem to share similar platelet binding sites, as non labelled fluvastatin or lovastatin were able to displace [3H]-labeled-fluvastatin from its binding sites on platelets. CONCLUSIONS: Fluvastatin therapy reduces platelet aggregation via a dual effect which involves its in vivo hypocholesterolaemic action on platelet cholesterol content, and also a direct effect of the drug binding to the platelets. The antiatherogenicity of fluvastatin may be related, in addition to its plasma cholesterol lowering ability, to its inhibitory effect on platelet activation.

Adult↗

PubMed2005

Factors affecting posttransfusion platelet increments, platelet refractoriness, and platelet transfusion intervals in thrombocytopenic patients.

A variety of patient and product-related factors influenced the outcome of 6379 transfusions given to 533 patients in the Trial to Reduce Alloimmunization to Platelets (TRAP). Responses measured were platelet increments, interval between platelet transfusions, and platelet refractoriness. Patient factors that improved platelet responses were splenectomy and increasing patient age. In contrast, at least 2 prior pregnancies, male gender, splenomegaly, bleeding, fever, infection, disseminated intravascular coagulation, increasing height and weight, lymphocytotoxic antibody positivity, an increasing number of platelet transfusions, or receiving heparin or amphotericin were associated with decreased posttransfusion platelet responses. Platelet factors that were associated with improved platelet responses were giving ABO-compatible platelets, platelets stored for 48 hours or less, and giving large doses of platelets while ultraviolet B (UV-B) or gamma irradiation decreased platelet responses. However, in alloimmunized lymphocytoxic antibody-positive patients, the immediate increment to UV-B-irradiated platelets was well maintained, whereas all other products showed substantial reductions. Refractoriness to platelet transfusions developed in 27% of the patients. Platelet refractoriness was associated with lymphocytotoxic antibody positivity, heparin administration, fever, bleeding, increasing number of platelet transfusions, increasing weight, at least 2 pregnancies, and male gender. The only factors that reduced platelet refractoriness rates were increasing the dose of platelets transfused or transfusing filtered apheresis platelets.

Blood Platelets↗

PubMed2001

Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction.

BACKGROUND: Platelet surface P-selectin is considered the "gold standard" marker of platelet activation. Degranulated, P-selectin-positive platelets, however, aggregate with leukocytes in vitro and rapidly lose surface P-selectin in vivo. METHODS AND RESULTS: Flow cytometric tracking of autologous, biotinylated platelets in baboons enabled us to directly demonstrate for the first time in vivo that (1) infused degranulated platelets very rapidly form circulating aggregates with monocytes and neutrophils, and (2) 30 minutes after infusion of the degranulated platelets, the percentage of circulating monocytes aggregated with infused platelets persist at high levels, whereas the percentage of circulating neutrophils aggregated with infused platelets and the platelet surface P-selectin of nonaggregated infused platelets return to baseline. We therefore performed 2 clinical studies in patients with acute coronary syndromes. First, after percutaneous coronary intervention (n=10), there was an increased number of circulating monocyte-platelet (and to a lesser extent, neutrophil-platelet) aggregates but not P-selectin-positive platelets. Second, of 93 patients presenting to an Emergency Department with chest pain, patients with acute myocardial infarction (AMI) (n=9) had more circulating monocyte-platelet aggregates (34.2+/-10.3% [mean+/-SEM]) than patients with no AMI (n=84, 19.3+/-1.4%, P<0.05) and normal control subjects (n=10, 11.5+/-0.8%, P<0.001). Circulating P-selectin-positive platelets, however, were not increased in chest pain patients with or without AMI. CONCLUSIONS: As demonstrated by 3 independent means (in vivo tracking of activated platelets in baboons, human coronary intervention, and human AMI), circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin.

Acute Disease↗

PubMed1998

Shear-induced platelet activation and platelet-neutrophil aggregate formation by equine platelets.

OBJECTIVES: To determine whether platelets become activated and form platelet-platelet or platelet-neutrophil aggregates, or both, when subjected to shear. SAMPLE POPULATION: Blood obtained from 3 Thoroughbreds. PROCEDURES: Blood, with PCV adjusted to 32 (low hematocrit) or 60 (high hematocrit)%, was subjected to shear rates of 11.25, 22.5, 45, 90, 225, and 750/s for 3 minutes by use of a cone-plate viscometer. Flow cytometric techniques were used to identify activated platelets, platelet-platelet aggregates, and platelet-neutrophil aggregates. RESULTS: Shear resulted in decreased platelet count, increased mean platelet volume, platelet activation, and formation of platelet-platelet and platelet-neutrophil aggregates. These changes occurred at lower shear rates in blood with high hematocrit. Platelet-neutrophil aggregate formation was inhibited by blocking P-selectin, but not CD11/CD18 receptors. CONCLUSIONS: Shear-induced platelet activation and aggregate formation occur at physiologic shear rates. CLINICAL RELEVANCE: Shear-induced platelet activation may explain the exercise-associated platelet-neutrophil aggregates observed in Thoroughbreds undergoing treadmill exercise.

Animals↗

PubMed2001

Leukocyte-platelet aggregation, platelet surface P-selectin, and platelet surface glycoprotein IIIa after percutaneous coronary intervention: Effects of dalteparin or unfractionated heparin in combination with abciximab.

BACKGROUND: Plaque disruption with resultant platelet activation and leukocyte-platelet aggregation is a pathophysiologic process common to both acute coronary syndromes and percutaneous coronary interventions. Unfractionated heparin is a standard antithrombotic therapy in patients with both acute coronary syndromes and in those undergoing percutaneous coronary interventions. Low-molecular-weight heparins have been reported to cause less platelet activation than unfractionated heparin. METHODS: Monocyte-platelet aggregates, neutrophil-platelet aggregates, platelet surface P-selectin, and platelet surface glycoprotein (GP) IIIa were measured serially by whole blood flow cytometry in 40 patients with unstable angina (randomly assigned to either unfractionated heparin 70 U/kg or the low-molecular-weight heparin dalteparin 60 IU/kg) undergoing coronary intervention with planned abciximab administration (in 2, one-half-dose boluses). Assays were performed at baseline, 5 minutes after administration of either type of heparin, 10 minutes after the first bolus of abciximab, 10 minutes after second bolus of abciximab, and 8 to 10 and 16 to 24 hours after administration of either heparin. RESULTS: No significant differences in clinical outcomes were observed between patients receiving either unfractionated heparin or dalteparin. The number of circulating P-selectin-positive platelets was increased by unfractionated heparin but not dalteparin, and abciximab reversed this increase. The number of circulating P-selectin-positive platelets was reduced below baseline levels in both treatment groups 8 to 10 and 16 to 24 hours after study drug administration. At 8 to 10 and 16 to 24 hours after administration of study drug, platelet degranulation in response to iso-thrombin receptor agonist peptide 1.5 mmol/L was significantly reduced by almost 50% (compared with immediately after study drug administration). Both unfractionated heparin and dalteparin significantly increased the numbers of circulating monocyte-platelet and neutrophil-platelet aggregates, which were subsequently reduced to baseline levels after administration of the second abciximab bolus and to below baseline at both 8 to 10 and 16 to 24 hours in all patients. After both unfractionated heparin and dalteparin administration, platelet surface GP IIIa expression was significantly increased compared with baseline at both 8 to 10 and 16 to 24 hours. CONCLUSIONS: Dalteparin in combination with abciximab in patients with unstable angina undergoing coronary intervention appears to be safe. Unfractionated heparin, but not dalteparin, degranulates platelets in patients with unstable angina. Both heparins increase the number of circulating monocyte-platelet and neutrophil-platelet aggregates. Abciximab therapy during coronary interventions rapidly reduces the number of degranulated platelets and leukocyte-platelet aggregates.

Abciximab↗

PubMed2002

Platelet-platelet and platelet-leukocyte interactions induced by outer membrane vesicles from N. meningitidis.

UNLABELLED: A large part of native meningococcal lipopolysaccharide (LPS), i.e., LPS integrated in the outer cell membrane, is released in the form of 'blebs' from surplus outer membrane material. In the present study we investigated the effects of purified outer membrane vesicles (OMVs) on blood platelet-platelet and platelet-leukocyte interactions. Citrated whole blood was stimulated in vitro with equal amounts (on a weight basis) of OMV-integrated LPS, purified LPS (P-LPS) from the same meningococcal strain and purified E. coli-LPS. The samples were analyzed by flow cytometry. Upon OMV stimulation platelet aggregation increased 2.1-fold, platelet degranulation 1.8-fold, (measured as CD62P expression), platelet binding to monocytes 2.6-fold, whereas platelet binding to granulocytes increased 2.8-fold. Also, the fraction of large heteroconjugates, i.e., large CD45-positive cell aggregates increased 15.7-fold compared to control. P-LPS and E. coli-LPS also significantly increased platelet aggregation and heteroconjugate formation but did not influence platelet degranulation and binding of platelets to leukocytes in whole blood. When using platelet-rich plasma (PRP), OMVs increased platelet aggregation 2.1-fold and CD62P expression 1.9-fold. P-LPS and E. coli-LPS also significantly increased platelet aggregation in PRP but did not influence platelet degranulation. None of the LPS preparations induced platelet microvesiculation, either in whole blood or in PRP. CONCLUSION: Meningococcal-derived OMVs as well as purified meningococcal LPS, contribute to increased platelet-platelet and platelet-leukocyte aggregation and may thus be of great importance in the development of microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.

Blood Platelets↗

PubMed1999

Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.

OBJECTIVE: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. METHODS: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. RESULTS: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. CONCLUSIONS: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.

Blood Platelets↗

PubMed1990

The platelet extinction coefficient measured by aggregometry is dependent on platelet composition rather than size: implications for studies of platelet heterogeneity and abnormalities.

Extinction (E) or light transmission measurements of platelet suspensions have been widely used to evaluate platelet structure and aggregation for platelet populations of varying mean platelet size (V). However, useful comparisons of platelet suspensions from donors having abnormally-sized platelets with those from healthy controls require a knowledge of the dependence of E on V. We have analyzed the extinction per platelet (e) as a function of the geometric scattering cross-section of equivalent spheres, V2/3, related to the apparent mean platelet optical efficiency, k. Such an analysis using data previously reported by Holme et al., for healthy controls and a variety of platelet-associated disorders showed e/V2/3 or k to be constant over an eight-fold variation in V. The outstanding exception was platelet suspensions from myeloproliferative disorder patients (MPD) which showed approximately 25% reduction in k values. When we analyzed e/V2/3 data for different-sized platelet subpopulations which were isolated by elutriation from healthy donors, we found that k doubled with a doubling in V from small to large platelets. It appears that the use of aggregometry devices with wide light acceptance angles yields k values for platelets which are insensitive to variations in V, but rather are sensitive to the expected variations in refractive index associated with distinct platelet internal composition and organization expected for different-sized normal subpopulations and "diseased" platelets as found in MPD. We relate these observations to photometric studies of platelet heterogeneity and of aggregation of platelets with different V and k values.

Adult↗