Comparison of platelet aggregability and P-selectin surface expression on platelets isolated by different methods

Three methods commonly used for isolation of blood platelets from plasma were compared. Platelets were isolated by: 1) a washing method; 2) a method of metrizamide-gradient centrifugation; 3) a modified method of gel-filtration. The last method employed BSA-Sepharose gel instead of routinely used Sepharose gel saturated with BSA. BSA-Sepharose gel was prepared by covalent binding of thermally deactivated BSA to CNBr-activated Sepharose 2B. In contrast to platelets isolated by the other methods, an aggregability of the gel-filtered platelets and control platelets in plasma, both activated with ADP, were comparable. When expression of P-selectin on the surface of freshly isolated platelets was examined, the gel-filtered platelets exhibited the same extent of fluorescence signal as platelets in the citrated blood, whereas platelets isolated by the other methods exhibited twice the extent of the signal. The methods involving the centrifugation process cause a low but a significant platelet activation.     

Multicolor flow cytometry for evaluation of platelet surface antigens and activation markers

Introduction

Flow cytometry allows the analysis of multiple antigens in a single tube at a single cell level. We present a rapid and sensitive two tube flow cytometric protocol for the detection of multiple platelet antigens and activation markers gated on a pure platelet population.

Materials and methods

The presence of platelet specific antigens was analyzed in citrated whole blood of normal platelets and from patients diagnosed with platelet abnormalities. Quiescent platelets as well as stimulated platelets were analyzed using a gating strategy based on ubiquitously expressed platelet membrane markers.A ubiquitously expressed platelet marker was combined with antibodies against the activated alpha2b-beta3 (PAC-1), Lysosomal Activated Membrane Protein (CD63) and P-selectin (CD62P).

Results

We were able to detect the platelet antigens CD36, CD41, CD42a, CD42b and CD61 in one single tube. Our approach allowed the single tube determination of PAC-1, CD63 and CD62P after activation of platelets by thrombin, collagen, ADP and PAR-1, and determination of platelet abnormalities.

Conclusions

Our two tube multi-parameter screening protocol is suited for the analysis of platelet antigens expressed on quiescent and activated platelets and allows the detection of aberrancies as found in blood of patients with thrombocytopathy such as Glanzmann Thrombasthenia, storage pool disease with diminished granule content and patients treated with clopidogrel and acetylsalicylic acid.     

A hitherto undescribed defect of platelet coagulant activity in polycythaemia vera and essential thrombocythaemia

A recently described platelet coagulant activity (factor X activating activity) was studied in two patients with essential thrombocythaemia and ten with polycythaemia vera. It was markedly reduced in the five patients with bleeding tendency, and also reduced in one patient with a history of peripheral ischaemia. It was either normal or increased in patients with no haemostatic complications. In the two subjects with lowest activity (five and thirteen percent) and most serious bleeding, chemotherapy resulted in normalisation of the platelet count and of platelet coagulant activity and in disappearance of the bleeding episodes. Our results suggest an association between bleeding tendency and reduced platelet coagulant activity. These findings may be of relevance to 1) the pathogenesis of abnormal bleeding in patients with myeloproliferative disorders and 2) the understanding of the role of platelet coagulant activities other than platelet factor 3 in disturbed haemostasis.     

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.     

Thrombin receptor occupancy modulates aggregation efficiency and platelet surface expression of vWF and thrombospondin at low thrombin concentrations.

Previous studies evaluating requirements for occupancy of thrombin receptors in normal platelet secretion and aggregation, using the thrombin antagonists hirudin and PPACK (D-Phe-Pro-Arg-chloromethylketone), have suggested that at low thrombin activating concentrations (0.025-0.13 U/ml), occupancy was required only in the first 45-60 s following activation. In our study, we differentiate between thrombin receptor occupancy requirements for surface expression of secreted adhesive proteins, for activation of GPIIb-IIIa receptors, and for aggregation of washed platelets (WP) in laminar shear flow. Platelets activated with 0.05 U/ml thrombin for 10 min to allow maximal secretion (hereafter referred to as "pre-activated platelets"), then sheared, showed a 50-70% decrease in platelet counts after 60 s of shear. Treatment of pre-activated platelets with hirudin or PPACK produced a 65% reduction of capture efficiencies, alphaG (reflecting experimental/theoretical initial rates of aggregation), as well as a 30-40% decrease in the surface expression of von Willebrand factor (vWF) and thrombospondin (TSP). However, alpha-granule membrane P-selectin expression and numbers of activated GPIIb-IIIa receptors were comparable for treated and non-treated platelets. No significant difference in any of the parameters tested was observed when platelets were similarly pre-activated with 0.2 U/ml thrombin, due to treatment with thrombin antagonists. Binding of soluble FITC-vWF (GRGDSP-sensitive) to pre-activated, thrombin antagonist treated platelets, was greatly reduced (> or =80%). Soluble Fg was shown to bind to antagonist-treated pre-activated platelets, but could not significantly enhance platelet aggregation. Although occupancy of thrombin receptors by catalytically active thrombin is required transiently for secretion and activation of platelets, there is a further requirement for thrombin occupancy at low thrombin concentrations, for optimizing initial rates of platelet aggregation, surface expression of vWF and TSP, and activated GPIIb-IIIa ligand recognition.     

A novel enzyme immunoassay for plasma thrombospondin. Comparison with beta-thromboglobulin as platelet activation marker in vitro and in vivo

A novel enzyme immunoassay for plasma thrombospondin (TSP) based on commercially available monoclonal antibodies was established. The following conditions for correct collection and preservation of blood samples were required: venipuncture directly into a vacutainer containing citrate, theophylline, adenosine and dipyridamole, storage on ice, and separation of plasma within 30 minutes. Thereafter, the plasma TSP concentration remained constant at room temperature and after five times of freezing and thawing. Both inter- and intraassay variation coefficients were 5%. The lower detection limit was 20 microg/L. Median TSP concentration among 40 healthy blood donors was 43 microg/L, slightly lower than previously published. The assay is valid, reliable, and has certain advantages compared with previously published methods. TSP and beta-thromboglobulin (BTG) were then compared as platelet activation and biocompatibility markers in vivo: 23 patients undergoing cardiopulmonary bypass (CPB); and in vitro: effect of coating polyvinyl chloride with heparin. The kinetic patterns of TSP and BTG were markedly different in vivo but virtually identical in vitro, explained by different in vivo clearance mechanisms during CPB. We conclude that BTG is superior to TSP for evaluation of platelet activation during in vivo CPB, whereas TSP and BTG are virtually identical as markers in vitro.     

Increased expression of platelet P-selectin and formation of platelet-leukocyte aggregates in blood from patients treated with unfractionated heparin plus eptifibatide compared with bivalirudin

INTRODUCTION: Platelet-leukocyte aggregates have been implicated in atherogenesis. This study was designed to determine the influence in vivo of a direct thrombin inhibitor, bivalirudin, compared with unfractionated heparin (UFH) plus the GP IIb-IIIa inhibitor eptifibatide (E) on platelet reactivity, the formation of platelet-leukocyte aggregates, and leukocyte activation. MATERIALS AND METHODS: Blood was taken before and after percutaneous coronary intervention (PCI) from 60 patients randomized to UFH+E (n=26) or bivalirudin (n=34). Platelet function and the formation in vivo of platelet-monocyte aggregates (PMA) and platelet-neutrophil aggregates (PNA) were assessed with the use of flow cytometry. Myeloperoxidase (MPO) elaborated during leukocyte activation was measured by ELISA. RESULTS: Compared with those treated with bivalirudin, patients treated with UFH+E exhibited a 45% decrease in the capacity of platelets to bind fibrinogen (p=0.006) but a 2-fold increase in platelet surface expression of P-selectin (p=0.04) in samples taken from the coronary ostium before PCI. Platelet-leukocyte aggregation in vivo was greater (PMA=2-fold, p=0.04; PNA=3-fold, p=0.006) with UFH+E as was the concentration in blood of MPO (1.5-fold, p=0.007). CONCLUSIONS: Increased platelet surface expression of P-selectin, augmented platelet-leukocyte aggregation in vivo, and consequent activation of leukocytes was seen before PCI in blood from patients treated with UFH+E compared with bivalirudin. Benefits associated with decreased platelet aggregation when PCI is performed with UFH plus GP IIb-IIIa inhibition may be partially offset by increased platelet-leukocyte aggregation.     

PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets

We have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-1 and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1(-/-) compared to wild-type mice. The leukocyte integrin alphaMbeta2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte-neutrophil elastase, known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium andWeibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet.     

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.     

Glycoproteins IIb and IIIa and platelet thrombospondin in a liposome model of platelet aggregation

Platelet membrane glycoproteins IIb and IIIa and platelet thrombospondin were incorporated onto phosphatidylcholine liposomes, by freeze thawing and sonication. Protein orientation on the liposomes was confirmed by susceptibility to neuraminidase cleavage and binding to lentil lectin-Sepharose (GPIIb-IIIa liposomes) and to heparin-Sepharose (thrombospondin liposomes). Glycoproteins IIb-IIIa bound 125I-fibrinogen with Kd of 7.5 X 10(-7)M. Binding was reversible and calcium-dependent. IIb-IIIa liposomes underwent fibrinogen-dependent aggregation in the presence of 10 mM CaCl2. Maximal aggregate formation was observed with a combination of IIb-IIIa liposomes and thrombospondin liposomes. This aggregation was partially inhibited by preincubation with monoclonal antibodies to the IIb-IIIa complex. Addition of EDTA caused complete reversal of aggregates. Thrombospondin liposomes also underwent fibrinogen and calcium dependent aggregation, however, this aggregation was less than that observed with the GPIIb-IIIa liposomes. Maximal aggregate formation was observed with a mixture of IIb-IIIa liposomes and thrombospondin liposomes. These studies demonstrate that GPIIb-IIIa and thrombospondin can be incorporated into phospholipid vesicles with preservation of function. Direct evidence is provided to demonstrate that glycoprotein IIb and IIIa and fibrinogen are sufficient for platelet aggregation and to demonstrate that thrombospondin may also contribute to platelet aggregation.     

Influences of fixatives on flow cytometric measurements of platelet P-selectin expression and fibrinogen binding

Sample fixation is an important issue in flow cytometric platelet assays. However, previous reports were less than consistent regarding the influence of sample fixation on the assays. We evaluated the effects of formaldehyde and paraformaldehyde fixation on platelet P-selectin expression and fibrinogen binding using whole-blood flow cytometry and a Coulter EPICS XL-MCL cytometer. Fluorescent-labeled whole-blood samples were diluted with HEPES-buffered saline or fixed with formaldehyde (0.2, 0.5, and 1. 0%) or paraformaldehyde (0.5, 1.0, and 2.0%). Platelet P-selectin expression was 1.1+/-0.3% and 39.6+/-13.7% in unfixed resting and 10(-5) M ADP stimulated samples, respectively. Resting P-selectin expression was not significantly altered by 0.2 or 0.5% formaldehyde fixation, but was slightly decreased by 1.0% formaldehyde fixation or PFA fixation. Formaldehyde fixation caused small increases of P-selectin expression in ADP-stimulated samples. Compared to platelet fibrinogen binding of unfixed resting (4.5+/-2.1%) and ADP-stimulated (56.7+/-22.6%) samples, formaldehyde or paraformaldehyde fixation had no significant influence on resting samples, but mildly increased fibrinogen binding in stimulated samples. Unfixed samples were stable for 2 h. Fixed samples were generally stable for at least 6 h, but not thereafter. Thus, formaldehyde and paraformaldehyde have mild but complex influences on platelet P-selectin expression and fibrinogen binding measurements. To evaluate the stabilities of unfixed and fixed samples, samples were analyzed after different durations (0, 1, 2, 4, 6, 12, and 24 h) of storage at 4 degrees C in the dark. The results suggest that sample manipulation without fixation may be used when the samples are analyzed within 2 h, and that fixation with 0.5-1.0% formaldehyde or paraformaldehyde seems to be preferable when sample analysis is delayed. Effects of fixation should be carefully evaluated when establishing flow cytometric platelet assays in every laboratory.     

Quantitation of platelet fibrinogen and thrombospondin in Glanzmann's thrombasthenia by electroimmunoassay

Fibrinogen and thrombospondin are major constituents of human platelet -granules and contribute to cell-cell interactions following their release. Glanzmann's thrombasthenia is characterized by the absence of platelet aggregation and reduced levels of GP IIb-IIIa complexes and platelet fibrinogen. The level of thrombospondin is thought to be normal but has not so far been quantified. Using an electroimmunoassay method adapted from Laurell, we have measured fibrinogen and thrombospondin in platelet extracts of four patients with classical Glanzmann's thrombasthenia and two variants with abnormal platelet aggregation associated with subnormal levels of GP IIb-IIIa complexes. Triton X-100 lysates were prepared in the presence of leupeptin or EDTA to avoid endogenous calcium-dependent protease activation during the solubilization procedure. Platelet fibrinogen was not detected in one patient with type I Glanzmann's thrombasthenia ; it was reduced to 5–10% of normal values in two other type I patients and to 65% of normal values in one type II patient. It was normal in patient R.P., a variant of Glanzmann's thrombasthenia with 60% of GP IIb-IIIa complexes but decreased in patient A.P. a newly described variant with 35% of GP IIb-IIIa complexes. These findings support a role for GP IIb-IIIa complexes in the packaging of fibrinogen into -granules. Normal or subnormal amounts of thrombospondin were measured in thrombasthenic platelets. Patient A.P., who was investigated on two different occasions, demonstrated variable levels of thrombospondin. This underlines the need for quantifying this protein when evaluating its expression in this disorder.     

Increased platelet activation in the chronic phase after cerebral ischemia and intracerebral hemorrhage

BACKGROUND AND PURPOSE: Enhanced thromboxane (TX) biosynthesis has previously been reported in the acute phase after ischemic stroke. We investigated whether enhanced urinary excretion of 11-dehydro-TXB2, a noninvasive index of platelet activation, was present in the chronic phase after a transient ischemic attack (TIA) or stroke, including intracerebral hemorrhage. METHODS: We obtained a single urinary sample from 92 patients between 3 and 9 months after onset of stroke or TIA. The urinary excretion of the major enzymatic metabolite of TXA2, 11-dehydro-TXB2, was measured by a previously validated radioimmunoassay. The excretion rates were compared with those of 20 control patients with nonvascular neurological diseases. RESULTS: Urinary 11-dehydro-TXB2 averaged 294+/-139, 413+/-419, and 557+/-432 pmol/mmol creatinine for patients with TIA, ischemic stroke, and intracerebral hemorrhage, respectively; the values were higher in all subgroups (P<0.01) than that in control patients (119+/-66 pmol/mmol). Increased 11-dehydro-TXB2 excretion was present in 59% of all patients, in 60% (P<0.001) of patients with TIA, in 56% (P<0.001) of patients with ischemic stroke, and in 73% (P<0.001) of patients with intracerebral hemorrhage. Atrial fibrillation, no aspirin use, and severity of symptoms at follow-up contributed independently to the level of 11-dehydro-TXB2 excretion in a multiple linear regression analysis. CONCLUSIONS: Platelet activation is often present in patients in the chronic phase after stroke, including those with intracerebral hemorrhage. Persistent platelet activation, which is associated with atrial fibrillation and poor stroke outcome, can be substantially suppressed by aspirin treatment.