Serial measurement of surface expressions of CD63, P-selectin and CD40 ligand on platelets in atherosclerotic ischemic stroke. A possible role of CD40 ligand on platelets in atherosclerotic ischemic stroke

BACKGROUND: Platelet activation is a key step in the progression of atherosclerosis. The CD40 ligand (CD40L) on platelets may be a critical factor to develop the acute vascular events from atheroma. METHODS: To determine the role of CD40L on platelets in atherosclerotic ischemic stroke, we serially measured the expressions of CD63, P-selectin and CD40L on platelets in patients with atherosclerotic ischemic stroke (n = 25) and compared them with those in patients with asymptomatic carotid stenosis (n = 20) and in normal subjects (n = 24). RESULTS: The expressions of CD63 and P-selectin on platelets were significantly higher in patients with atherosclerotic ischemic stroke (n = 25) than in normal subjects (n = 24). The extents of surface expressions of CD63 and P-selectin on platelets showed no significant differences between atherosclerotic ischemic stroke and asymptomatic carotid stenosis. However, the CD40L expression on platelets was significantly higher in atherosclerotic ischemic stroke when compared to that in asymptomatic carotid stenosis. CONCLUSIONS: In our data, among the population with large artery atherosclerosis, the patients with symptomatic ischemic events showed a significantly elevated expression of CD40L on platelets compared to those without ischemic events. Therefore, the upregulation of CD40L on platelets may be a specific marker of platelet activation to provoke ischemic stroke from large artery atherosclerosis.     

Optical density variations and microscopic observations in the evaluation of platelet shape change and microaggregate formation

Addition of Ristocetin to formalin-fixed platelets suspended in diluted PPP induces a marked increase in turbidity which is not caused by platelet shape change but by the formation of microaggregates. If diluted PPP of patients with severe von Willebrand's disease is used, only an initial increase in turbidity and no further decrease is observed without any form variation of the platelets but again with formation of many microaggregates. In normal PRP diluted with buffered EDTA, ADP induces an increase in turbidity without further changes in optical density. Simultaneously platelets immediately change their shape with formation of pseudopodia and sphering but at the same time also microaggregates appear. Shape change and microaggregate formation can also be observed after the addition of Collagen to undiluted PRP which is followed by the formation of large aggregates and a decrease in optical density. Increase in optical density in undiluted PRP is not a specific indicator of platelet shape changes. Microaggregates can alone or partially be responsible for these changes. For the evaluation of platelet shape changes but also for the estimation of microaggregate formation microscopic methods are preferred.     

The state of platelets preserved in extracorporeal circulation with a glycoprotein IIb/IIIa inhibitor

INTRODUCTION: Temporary inhibition of platelet function during extracorporeal circulation (platelet anesthesia) can preserve platelet count. We hypothesized that platelet anesthesia with a glycoprotein IIb/IIIa inhibitor could preserve activated platelets. MATERIALS AND METHODS: Fresh human blood from donors was recirculated for 120 min in a simulated extracorporeal circuit. Heparin and FK633, a short-acting platelet glycoprotein IIb/IIIa inhibitor, were added to recirculated blood in one group (group F, n=5) whereas only heparin was used in controls (group C, n=5). Blood samples were obtained from the donors, and at 0, 5, 15, 30, 60, and 120 min of recirculation. Platelet counts, beta-thromboglobulin, thrombin-antithrombin complex, and aggregation to adenosine diphosphate were measured. Flow cytometry was performed for measurement of fibrinogen binding, platelet surface expression of P-selectin, and microparticles. RESULTS AND CONCLUSIONS: In the FK633 group, platelet counts were preserved and beta-thromboglobulin levels remained unchanged, whereas in group C, platelet counts decreased significantly and beta-thromboglobulin increased significantly from 30 and 60 min, respectively. FK633 inhibited platelet aggregation and fibrinogen binding to platelets throughout recirculation. A significant difference between groups with respect to microparticle parameters and thrombin-antithrombin complex levels was evident by 120 min. P-selectin expression increased at 0 min in both groups, and was preserved significantly at 5 min and reduced at 120 min in group F. Platelet counts were preserved by platelet anesthesia during recirculation without platelet activation. These results suggest that FK633 inhibits the amplification loop by reducing the binding of fibrinogen to glycoprotein IIb/IIIa and platelet aggregation.     

Abnormal aggregation accompanies abnormal platelet Ca2+ handling in arterial thrombosis

The resting levels of cytoplasmic Ca2+ (measured by Quin 2 fluorescence) and dense tubular Ca2+ (measured by chlorotetracycline, CTC, fluorescence) are shown to be higher in platelets from patients with arterial thrombosis than from normal donors. Turbidimetric studies of aggregation of diluted platelet-rich plasma (PRP) at 135 microM Ca2+ showed increased rates of aggregation for patients relative to normal controls. For ADP-stimulated aggregation, increased maximal rates (Vmax) and decreased doses for half-maximal rates were observed. With collagen-stimulated aggregation, patient samples showed only decreased ED50 values relative to normal controls. The changes in these values are linearly correlated with the elevation of resting dense tubular Ca2+ level determined by the calcium-CTC test carried out at 2 mM external Ca2+. For ADP-stimulated aggregation this relationship can be mimicked by pre-incubating normal platelets with subcritical concentrations of the Ca2+ ionophore A23187. These results suggest that elevated cytoplasmic and dense tubular Ca2+ in the "resting state" is a major factor in arterial thrombosis, rendering the platelet more sensitive to the stimulation by physiologic agents.     

Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

Introduction

Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition.

Methods

Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients.

Results

In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r² = 0.33 and r² = 0.34 respectively, p < 0.0015). In patients with a low platelet count (< 150 × 10⁹ platelets/L), both densities were increased relative to controls (82% and 33% respectively, p < 0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r² = 0.33 and r² = 0.29, p < 0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p < 0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p < 0.05).

Conclusions

There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count.     

Abciximab treatment in vitro after aspirin treatment in vivo has additive effects on platelet aggregation, ATP release, and P-selectin expression

To prevent arterial thrombosis, abciximab is administered together with aspirin. However, whether or not there are benefits to combine abciximab with aspirin is not yet well defined. Healthy volunteers were studied for the effect of aspirin+abciximab using sodium arachidonate and adenosine diphosphate (ADP) alone or in combination to induce platelet activation/aggregation. Abciximab produced complete inhibition of platelet aggregation induced with ADP but only 40% inhibition of aggregation induced by 0.75-mmol/l sodium arachidonate. Abciximab added in vitro to platelet-rich plasma (PRP) from platelets from aspirin-treated donors produced an almost complete inhibition of platelet aggregation. Aspirin, and abciximab alone, did not inhibit adenosine triphosphate (ATP) release as thoroughly as aspirin+abciximab did. Abciximab (3–5 μg/ml) produced inhibition of P-selectin expression induced with 5 (from 46.2±6.0% to 27.4±7.0%, P=.002) and 20-μmol/l ADP (from 53.1±8.1% to 35.1±11.0%, P=.019), but no effect was observed when 0.75-mmol/l sodium arachidonate was used (P=.721). Aspirin diminished P-selectin expression in sodium arachidonate-stimulated platelets (from 77.7±11.8% to 40.2±3.6%, P<.0001) in non-aspirinated and platelets from aspirin-treated donors, respectively. Abciximab (3, 4, and 5 μg/ml) added to platelets from aspirin-treated donors decreased P-selectin expression in platelets stimulated with sodium arachidonate from 40.2±8.6% to 25.6±11.5% (P=.027), to 20.5±3.5% (P<.0001), and to 22.5±1.8% (P<.0001). We concluded that the antiplatelet effect of abciximab is greatly increased by aspirin.     

An assessment of whole blood impedance aggregometry using blood from normal subjects and haemodialysis patients

Impedance aggregometry allows the measurement of platelet responses in whole blood as well as in PRP. The variability of haematocrit values encountered when applying this technique to haemodialysis patients prompted an investigation of the effects of red cells on platelet aggregation in whole blood. Collagen induced aggregation was measured in both PRP and whole blood from haemodialysis patients and healthy controls, Platelets from haemodialysis patients were less aggregable than those from the controls when tested in PRP, but more aggregable when tested in whole blood. Blood samples with a range of haematocrit values were prepared by mixing PRP and autologous red cells, and used to study the effect of haematocrit on platelet aggregation. In blood from control subjects aggregation rate was reduced by rising haematocrit but no reduction of maximum aggregation occurred until haematocrit exceeded 40%. In contrast uraemic platelets showed increased responses in the presence of red cells. In a limited cross-over study no significant difference was found in the effect on platelet aggregation of washed erythrocytes from uraemic and non-uraemic donors. It is concluded that red cell presence influences platelet aggregation by complex mechanisms during impedance aggregometry and that this effect must be considered when interpreting results.     

Assessing aspirin-induced attenuation of platelet reactivity by flow cytometry

INTRODUCTION: Certain patient subpopulations do not respond to antithrombotic effects of aspirin and different approaches have been proposed to detect and define this so-called aspirin resistance. In this study, a methodological and clinical evaluation of a flow cytometric method for the detection of aspirin-induced inhibition of platelet cyclooxygenase (COX) is presented. MATERIALS AND METHODS: Platelet CD62p-antigen (P-selectin) expression was determined by flow cytometry after incubating diluted platelet rich plasma (PRP) with arachidonic acid (ARA). After establishing the method's technical characteristics, it was used to investigate 114 individuals (70 patients with atherosclerotic vascular disease under long-term medication of 100 mg aspirin daily, 29 age-matched patients with vascular disease without anti-platelet medication and 15 healthy volunteers). Data were compared to those obtained by the PFA-100 platelet function analyzer. RESULTS: Imprecision was between 3.3% and 13%. Sample storage at room temperature increased baseline activity of platelets already after 2 h. After ARA stimulation, the proportion of CD62p-positive platelets was considerably lower in aspirin-treated patients than in controls (median [lower-upper quartile]: 4% [3-6] vs. 50% [29-68], p<0.001). Only one aspirin-treated patient (1.5%) showed normal reactivity to ARA. In contrast to flow cytometry, PFA-100 analysis yielded normal results in 17% of aspirin-treated patients. CONCLUSIONS: The presented flow cytometric method can be used to monitor aspirin-induced inhibition of platelet COX. Aspirin resistance defined as failure to inhibit platelet COX is a rare phenomenon suggesting that most cases of aspirin resistance detected using the PFA-100 are caused by COX-independent mechanisms.     

Kinetic parameters of platelet aggregation as an expression of platelet responsiveness

The kinetics of platelet aggregation induced by collagen and by ADP were studied. The maximum aggregation (deltaLTmax) and the ADP and collagen concentrations required to produce half-maximum aggregation (Kd) were determined using platelets obtained from normal individuals, individuals who ingested aspirin and individuals whose platelet-rich plasma (PRP) demonstrated spontaneous aggregation. The Kd and deltaLTmax for ADP-induced platelet aggregation were variable and markedly affected by the citrate concentration. Conversely, kinetic parameters of collagen-induced aggregation were more reproducible and less affected by citrate. The Kd for collagen in platelet aggregation was increased following aspirin ingestion and decreased in samples of PRP that demonstrated spontaneous aggregation. These results suggest that kinetic parameters of platelet aggregation may be useful to express the responsiveness of platelets.     

Human anti-heparin-platelet factor 4 antibodies are capable of activating primate platelets: towards the development of a HIT model in primates

In the first step to establish an animal model of heparin-induced thrombocytopenia (HIT) that is physiologically relevant to humans, studies were undertaken to determine the similarities or differences between human and non-human primate (Macaca mulatta) platelets in HIT assay systems. The collagen-, ADP-, and TRAP-induced platelet aggregation, and flow cytometric analysis of P-selectin expression and microparticle formation were similar for both species platelets (p>0.1, n=18 each). The classical HIT assays using platelet-rich plasma (PRP) as well as a flow cytometric assay revealed the activation/aggregation and serotonin release assay (SRA) profiles for both primate and human platelets were similar in response to human HIT positive sera. All assays were heparin concentration-dependent; heparin, at 0.1 U/mL, produced maximum and similar platelet activation/aggregation and SRA responses with both primate (76+/-7%, n=18) and human (68+/-11%, n=20; p>0.1) platelets. At concentrations > or =10 U/mL, heparin suppressed the platelet aggregation and SRA responses in both systems. Primate and human platelets displayed similar behavior to low molecular weight heparin and pentasaccahride in HIT assay systems. Immunoglobulins isolated from serum of patients with HIT caused activation/aggregation of human (65+/-18%, n=10 donors) and primate (79+/-12%, n=6 monkeys, p>0.08) platelets. Unlike human platelets, the primate platelets exhibited a more consistent aggregation/release response (15 out of 18 primate platelets reactive). In contrast, human donors showed wide variations in the activation/release response (4 out of 10 reactive). These observations suggest that primate platelets are activatable by anti-H-PF4 antibodies, and support the hypothesis that primates can be used to develop an animal model to study the pathogenesis of HIT.     

Responses of platelets to strains of streptococcus sanguis: findings in healthy subjects, Bernard-Soulier, Glanzmann's, and collagen-unresponsive patients

The ability of endocarditis and dental strains of Streptococcus sanguis to induce platelet aggregation in plasma (PRP) from normal subjects were examined and compared to responses of PRP with known platelet membrane glycoprotein (GP) and response defects. S. sanguis strains differed in their ability to induce normal PRPs to aggregate. Strains that induced PRP aggregation in more than 60% of donors were significantly faster agonists (mean lag times to onset of aggregation less than 6 min) than those strains inducing response in PRPs of fewer than 60% of donors. Platelets from patients with Bernard-Soulier syndrome aggregated in response to strains of S. sanguis. In contrast, platelets from patients with Glanzmann's thrombasthenia and from a patient with a specific defect in response to collagen were unresponsive to S. sanguis. These observations show that GPIb and V are not essential, but GPIIb-IIIa and GPIa are important in the platelet response mechanism to S. sanguis. Indeed, the data suggests that the platelet interaction mechanisms of S. sanguis and collagen may be similar.     

Differential effect of the GPIIb/IIIa antagonist orbofiban on human platelet aggregate formation in vitro

Platelet microaggregates have been demonstrated in the systemic circulation of patients with atherosclerotic cardiovascular diseases. Glycoprotein IIb/IIIa antagonists have been reported to play roles in platelet activation, which may be associated with pro-thrombotic events. We report the effect of the orally active GPIIb/IIIa antagonist, orbofiban, on human platelet microaggregate formation in vitro. Laser light scatter (LSS) technology was used to monitor small, medium and large platelet aggregates formed in platelet-rich plasma in response to ADP or thrombin receptor activating peptide (TRAP)-6. ADP at 0.5 microM induced only small platelet aggregates that were prevented by orbofiban in a concentration-dependent manner. Likewise, orbofiban dissolved small aggregates after their formation. In marked contrast, in the presence of strong agonist stimulation (20 microM ADP or 3 microM TRAP-6) which generated primarily large platelet aggregates, orbofiban blocked the large aggregates while significantly augmenting small aggregates by three- to sixfold. The data suggest that GPIIb/IIIa antagonists do not induce platelet microaggregation directly but may augment small platelet microaggregate formation indirectly at conditions of strong stimuli. The percentage of the microaggregate population expressing P-selectin remained the same in the presence of orbofiban, indicating that these small microaggregates remain activated, although the mean intensity of expression was diminished, possibly reflecting the reduced size of the particles or density of P-selectin molecules. In summary, an increase in small platelet microaggregates might have contributed to pro-thrombotic events in orbofiban-treated patients.     

Do extra-platelet sources contribute to the plasma level of thrombospondin?

Thrombospondin, a trimeric glycoprotein contained in the platelet alpha-granules, has been proposed as a marker of in vivo platelet activation. However, it is also synthesised by a range of other cells. The extraplatelet contribution to plasma levels of thrombospondin was therefore estimated by investigating the relationship between plasma thrombospondin levels and platelet count in samples from profoundly thrombocytopenic patients with marrow hypoplasia, using the platelet-specific alpha-granule protein beta-thromboglobulin as control. Serum concentrations of both proteins were highly correlated with platelet count, but while plasma beta-thromboglobulin levels and platelet count also correlated, there was no relationship between the number of platelets and thrombospondin concentrations in plasma. Serial sampling of patients recovering from bone marrow depression indicated that the plasma thrombospondin contributed by platelets is superimposed on a background concentration of at least 50 ng/ml probably derived from a non-platelet source, and plasma thrombospondin levels do not simply reflect platelet release.     

Pulsed Radiofrequency Neuromodulation Contributes to Activation of Platelet-Rich Plasma in In Vitro Conditions

ObjectivesRecent years have brought new developments in interventional chronic pain management, namely regenerative orthopedics utilizing platelet-rich plasma (PRP) as well as further evolution of pulsed radiofrequency neuromodulation (PRF). Both methods have been used separately. Here, we investigated whether PRF may potentiate the activation of platelets in PRP samples when both these techniques are combined together in in vitro conditions.Materials and MethodsStudies were performed on concentrated PRP samples (PRPs) obtained from acid citrate dextrose-treated blood taken from 11 healthy volunteers. PRPs were divided into four groups: 1) nonactivated PRP; 2) thrombin-activated PRP as a positive control for maximal platelets activation; 3) PRF-treated PRP exposed for 20 min to PRF energy generated by neurotherm radio frequency generator at 500 kHz, with a voltage of 40 V and maximal temperature of 42°C; and 4) a combination of groups 2 and 3.ResultsPRF-induced platelet activation measured by platelet factor 4 (PF4) and ATP release from PRPs was significantly higher compared to nonactivated PRPs, and similar to PF4 and ATP release from thrombin-activated PRPs. Thrombin activation did not potentiate PF4 release in PRF samples and even reduced ATP level. Additionally, PRF neither induced any platelet membrane damage measured by lactic dehydrogenase release from PRP nor modified any platelets viability or metabolism measured by MTT.ConclusionsWe confirmed that PRF may activate PRP without additional platelet activators. So, a combination of both methods PRF and PRP application may provide a more effective opportunity for tissue regeneration in dentistry, surgery, dermatology, or in orthopedics.     

Glycoprotein Ibalpha inhibition and ADP receptor antagonists, but not aspirin, reduce platelet thrombus formation in flowing blood exposed to atherosclerotic plaques

Anti-platelet drugs are used to prevent intra-arterial thrombus formation after rupture of atherosclerotic plaques. Until now, the inhibitory effect of present and future anti-platelet drugs such as aspirin, ADP receptor P2Y(1)/P2Y(12) antagonists and glycoprotein (GP) Ibalpha inhibitors on the interaction of platelets with human plaques is not known. To study those effects we obtained human atherosclerotic plaques by surgical endarterectomy. Plaques induced maximal platelet aggregation in hirudinized platelet-rich plasma (PRP) and blood that was effectively inhibited by aspirin, the P2Y(1) antagonist MRS2179 and the P2Y(12) antagonist AR-C69931MX, but not by GPIbalpha blockade with the mAB 6B4. Inhibition of platelet aggregation by MRS2179 was 74 +/- 37% and 68 +/- 20%, by AR-C69931MX 94 +/- 7% and 80 +/- 6%, and by aspirin 88 +/- 19% and 64 +/- 28%, in PRP and blood, respectively (mean +/- SD; n = 6-12 with plaques from 6 patients). The combination of both ADP receptor antagonists completely inhibited plaque-induced platelet aggregation in hirudinized PRP and blood. Under arterial flow conditions (1,500s(-1)), blockade of platelet GPIbalpha resulted in a strong decrease of plaque-stimulated platelet adhesion/aggregate formation of 77 +/- 5% (mean +/- SD; n = 4). Furthermore, MRS2179, AR-C69931MX and their combination reduced plaque-dependent platelet aggregate formation by 35 +/- 14%, 32 +/- 13% and 58 +/- 12% (mean +/- SD; n = 5), respectively. Aspirin was without significant effect. In conclusion, a GPIbalpha-blocking antibody, as well as P2Y(1) and P2Y(12) receptor antagonists, alone or in combination, reduce in contrast to aspirin human plaque-induced platelet thrombus formation under arterial flow. Although these new anti-platelet agents inhibit platelet thrombus formation after plaque rupture, more efficient platelet blockers are required.     

Differing susceptibilities of platelets from normal individuals and patients with von Willebrand's disease to attack by quinine- and quinidine-dependent antiplatelet antibodies

Reactivity of quinine- and quinidine-dependent antiplatelet antibodies has been compared in platelet-rich-plasma (PRP) from normal donors and from patients with von Willebrand's disease (vWd). One quinine-dependent antibody (Q.Ab) caused platelet aggregation and [14C] serotonin release with only 7 of 12 normal donors, while another Q.Ab and a quinidine-dependent antibody (Qd.Ab) caused aggregation and release with all 12. Drug-dependent IgG binding and PF 3 availability induced by the antibodies were, however, comparable in all donors. Differences in responsiveness were associated with platelets and not plasma. vWd platelets showed normal drug-dependent IgG binding, but decreased aggregation and serotonin release to most drug-dependent antibodies. Responsiveness was not restored by purified vWf:Ag, but, in one case, was corrected by normal plasma or cryoprecipitate. Drug-dependent binding of the Q.Ab which caused variable responsiveness in normals was to the same platelet antigens (GPIb and GPIIIa) in both normal and vWd platelets and did not require plasma components. Reduced PF 3 availability was seen with some antibodies in some vWd patients. Plasma from two of these patients inhibited aggregation of normal platelets to Q.Ab and one of these inhibited aggregation to ADP. Antiplatelet antibodies were detected in these two plasmas by ELISA. Thus some Q.Ab produce different responses with platelets from different donors. In vWd, reduced responsiveness to Q.Ab and Qd.Ab may result from production of inhibitory antiplatelet antibodies.     

Increased levels of platelet activation markers are positively associated with carotid wall thickness and other atherosclerotic risk factors in obese patients

The role of platelets in the development of atherosclerosis and obesity-related prothrombotic state is still under investigation. In this cross-sectional cohort study, we measured the levels of different platelet activation markers and evaluated their relationship with carotid intima-media thickness (IMT) along with other atherosclerotic risk factors in obese patients with or without atherosclerotic co-morbidities. We enrolled 154 obese patients, including 98 with either hypertension, type 2 diabetes mellitus or dyslipidaemia, 56 without these co-morbidities and 62 age- and sex-matched healthy controls. Platelet P-selectin expression and the number of platelet-derived microparticles (PMPs) were measured by flow cytometry; soluble P-selectin levels were analysed by ELISA and Thr715Pro P-selectin polymorphism was determined by PCR-RFLP. Carotid IMT was examined by ultrasonography. The levels of platelet activation parameters were significantly elevated in all obese subjects with increased carotid IMT compared to healthy controls. There was no effect of Thr715Pro genotype on soluble P-selectin levels in obese individuals contrary to normal subjects. Significant and positive association was revealed between carotid IMT and platelet P-selectin (p<0.0001), soluble P-selectin (p=0.039) and PMP (p=0.0001) levels. After adjusting for multiple variables, independent association was found between soluble P-selectin and fibrinogen (p=0.007), PMP levels and body mass index (p<0.0001) as well as platelet P-selectin and carotid IMT (p=0.012) plus plasminogen activator inhibitor-1 (p=0.009). In conclusion, P-selectin and PMP levels showed positive associations with abnormal carotid IMT and other risk factors in obesity suggesting a critical role of enhanced platelet reactivity in atherosclerotic wall alteration.     

Prolonged bleeding time in patients with lupus anticoagulant.

Platelet adhesion to collagen under flow conditions was studied in 18 patients with lupus anticoagulant, seven of which showed a prolonged bleeding time in the presence of a normal platelet count. The effect of patient plasma, IgG and purified anticardiolipin antibodies on platelet adhesion was also examined. We found a significant reduction of platelet adhesion in patients with lupus anticoagulant, which was more evident in patients with prolonged bleeding time. This platelet adhesion defect could be attributed to a plasma factor. In fact, patients' platelets regained normal adhesion when mixed with normal plasma, whereas controls' platelets showed abnormal adhesion in the presence of patient plasma. A causative role of antiphospholipid antibodies was demonstrated in experiments using purified immunoglobulins and anticardiolipin antibodies.     

Platelet aggregation: the use of optical density fluctuations to study microaggregate formation in platelet suspension

A new approach to the investigation of microaggregate formation kinetics in platelet suspension is described. The method is based on the analysis of light transmission fluctuations produced by the changes in the number of platelets in optical channel. The relative dispersion of such fluctuations (D) was used as a parameter to estimate the degree of platelet aggregation and to analyze the aggregation kinetics. It was found that a continuous recording of this parameter allows to detect microaggregate formation during spontaneous aggregation and aggregation induced by very low concentrations of ADP (0.05-0.15 uM) in platelet-rich citrate plasma. When 3-10 uM ADP was added to acid citrate-dextrose anticoagulated plasma at pH 6.5, microaggregate formation was detected by increase in D, whereas light transmission decreased by 15-20%. Analysis by scanning electron microscopy and the use of standard inhibitors of aggregation confirmed that in all these cases the microaggregate formation did take place.     

P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation

Introduction

Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology.

Methods and Results

Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and P-selectin-deficient (CD62P^−/−) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet α_IIbβ₃. Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P^−/− mice. In addition, CD62P^−/− mice exhibited thrombosis abnormalities without an α_IIbβ₃ activation defect.

Conclusions

This study demonstrates that the role of platelet P-selectin is not solely adhesive; its binding to PSGL-1 induces platelet activation that enhances platelet aggregation and thrombus formation. Therefore, targeting platelet P-selectin or its ligand PSGL-1 could provide a potential therapeutic approach in the management of thrombotic disorders.