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PubMed1996

PAICA: a method for characterizing platelet-associated antibodies--its application to the study of idiopathic thrombocytopenic purpura and to the detection of platelet-bound c7E3.

In idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA ("Platelet-Associated IgG Characterization Assay"), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient's own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA ("Monoclonal Antibody-specific Immobilization of Platelet Antigens"). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP IIb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP IIb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.

Adult↗
PubMed1998

Intravascular and total body platelet equilibrium in healthy volunteers and in thrombocytopenic patients transfused with single donor platelets.

Instrument platelet counts used in corrected count increment (CCI) and percent platelet recovery (PPR) formulas presume the transfused platelets are in equilibrium during the first hour after platelet transfusion. The timing of the pre-transfusion count affects CCI results, and we postulate that timing of CCI post transfusion affects CCI results. Platelet equilibrium using indium-111 platelet transfusions has not been reported. Platelet redistribution was studied in 16 healthy volunteers and 12 thrombocytopenic patients by generally infusing less than 72-hr stored single-donor platelets along with an aliquot of indium-111-labeled platelets by intravenous push. Counts were measured at 10, 15, 20, 60, and 120 min, and 24, 48, 72 hr along with continuous body scanning for 2 hr in healthy volunteers, and static organ scanning in patients and volunteers. Results indicated transfused platelets do not reach intravascular equilibrium for 60 min post-infusion and that the 10-min count cannot detect platelet refractoriness. However, total body equilibrium varies considerably between normal volunteers and thrombocytopenic patients. It is recommended to continue with the 1-hr post transfusion count.

Antigens, Human Platelet↗
PubMed1989

Measuring platelet function with platelet shape change, an early event in aggregation.

Platelet shape change precedes aggregation and is sensitive to significantly less stimulant. In this study, differences in percent light transmission (%T) through stirred suspensions of spheroid or discoid platelets are exploited for studying the response of platelets to Platelet Activating Factor (PAF). Changes in %T between stirred and non-stirred suspensions of unstimulated platelets in Platelet Rich Plasma (PRP) varied with the starting concentration. Aggregation was blocked by the presence of citrate while activation by ADP was minimized by apyrase. However, phosphatidic acid formation, ATP release and minimal serotonin release occurred. A shape change parameter (SCP) was defined which varied with the concentration of PAF. Its dependence on PAF was shifted to lower concentration with washed platelets. The dose-response curves for the SCP were found to be virtually identical for both sheep and human platelets. Human platelets were then used to correlate morphology, as determined by phase contrast microscopy, with spectrophotometric data in the form of the SCP. A linear regression analysis of SCP and percent activation revealed a virtual 1:1 correspondence with a slope of 1.066 and an intercept of 0.009 with the standard error of the estimate equal to 0.053. The correlation coefficient was 0.990, making it significant at p less than 0.001. This method provides an alternative to platelet aggregation assays as a convenient, sensitive and reliable measure of platelet activation.

Adenosine Triphosphate↗
PubMed1999

Increased platelet sensitivity toward platelet inhibitors during physical exercise in patients with coronary artery disease.

Generalized atherosclerosis and coronary artery disease (CAD) are associated with endothelial dysfunction and during acute myocardial ischemia platelet activation has been reported. Activated platelets exert activated fibrinogen receptors (GP IIb/IIIa) and express CD 62p being regarded as reliable marker for platelet activation. Patients with angiographically proven CAD performed a bicycle exercise test until the onset of angina or ST-segment depression. We studied the ischemia-induced alterations in fibrinogen binding to activated platelet GP IIb/IIIa receptors and CD 62p expression. Therefore, the basal fibrinogen binding to GP IIb/IIIa and CD 62p expression and the thrombin-concentration for half-maximal platelet activation before and after exercise testing were determined. Additionally, inhibition of thrombin-induced platelet activation by increasing concentrations of the prostacyclin-analog iloprost and the NO-donor SIN-1 was examined. In patients with CAD, a significantly reduced basal activation and a highly significant reduction in sensitivity towards thrombin was measured. The thrombin-induced expression of GP IIb/IIIa and CD 62p was significantly diminished in patients with CAD after physical exercise and their platelets were significantly more sensitive towards the inhibitory effects of iloprost and SIN-1. These data demonstrate a significant reduction in platelet activation in response to physical exercise in patients with CAD and advanced atherosclerosis. Despite exercise induced myocardial ischemia as evidenced by angina and ECG-changes, the platelets are not generally activated, as it could be expected. Thus, patients with myocardial ischemia experienced a reduced platelet activity and enhanced sensitivity towards prostacyclin (PGI2) and nitric oxide, probably due to an augmented release of endogenous platelet inhibitory mediators.

Blood Platelets↗
PubMedDate not supplied

Oxygen consumption, platelet aggregation and release reactions in platelets freeze-preserved with dimethylsulfoxide.

Platelets were frozen with 4% or 5% DMSO at an overall rate of 2 to 3 C per minute and were stored at -80 C for as long as 10 months. They were washed with DMSO-plasma and acid-citrate-dextrose (ACD) solutions and were stored in 30 ml of autologous plasma at room temperature for about three hours before transfusion. Measurements were made of oxygen consumption, platelet aggregation and release reaction, platelet factor-3 and-4 activities, and platelet response to hypotonic stress. Platelet basal and latex stimulated oxygen consumption were found to be significantly impaired; platelet aggregation response to ADP, epinephrine, and collagen were decreased; platelet ATP and ADP content and release reactions were decreased; platelet antiheparin activity (platelet factor-4 level) was decreased; and the platelet response to hypotonic stress was impaired. What the results of these in vitro tests mean in relation to in vivo survival and hemostatic function of preserved platelets was not established.

Adenosine Diphosphate↗
PubMed1999

Platelet concentrates prepared and stored under currently optimal conditions: minor impact on platelet adhesive and cohesive functions after storage.

BACKGROUND: The effect on platelets of two standard methods of platelet concentrate (PC) preparation was studied by flow cytometry. The findings were correlated with those obtained in an experimental in vitro perfusion model. STUDY DESIGN AND METHODS: PCs were prepared from whole blood by the platelet-rich plasma (PRP) or buffy coat (BC) method and placed on a flatbed platelet agitator at 22 degrees C for up to 5 days. Platelet glycoproteins (GP)lbalpha, GPIIb/IIIa, and GPIV, p-selectin and lysosomal integral membrane protein, and the binding of von Willebrand factor, fibrinogen, fibronectin, and coagulation factor Va were measured with the corresponding specific conjugated antibodies. Perfusions were carried out in an annular chamber with citrated blood depleted of platelets and white cells by filtration, to which samples from PCs were added. RESULTS: PRP-PC production provoked intense platelet activation. In contrast, in BC-derived PCs, platelet activation was milder, and only a significant increase in bound fibrinogen was seen. After 1 day of storage, differences between the methods that had been observed immediately after separation had almost disappeared. During the remaining storage period, increases in activation-dependent antigens and in procoagulant activity were measured. Of the studied platelet GPs, only GPIIIb/ IIIa decreased by 25 percent in PRP-PCs. Differences in covered surface were not significant in perfusion studies performed on Day 0 and after 5 days of storage in PRP-PCs (26.8 +/- 6.9 vs. 20.5 +/- 5.8) or BC-PCs (23.8 +/- 11 vs. 24.8 +/- 10.2). CONCLUSION: Platelet activation occurred during the separation and storage of PCs prepared by both methods, and it was higher in PRP-PCs only in samples obtained immediately after preparation. Despite these changes, platelet adhesive and cohesive functions were similar in both types of PCs and remained basically unchanged after storage.

Blood Platelets↗
PubMed2002

Determination of platelet aggregation inhibition during percutaneous coronary intervention with the platelet function analyzer PFA-100.

BACKGROUND: A simple device to rapidly evaluate platelet function may aid in optimizing glycoprotein IIb/IIIa inhibition during percutaneous coronary intervention (PCI). We prospectively studied platelet function in 250 patients receiving abciximab or eptifibatide during PCI. METHODS AND RESULTS: The platelet function analyzer PFA-100 (Dade-Behring, Deerfield, Ill) measures platelet function by determining the time to occlusion of an aperture in a biochemically active membrane as whole blood flows under high shear conditions. Platelet aggregation causes aperture occlusion, and results are reported as a closure time (CT). All patients received either abciximab or eptifibatide, along with aspirin and heparin; patients undergoing stent implantation received aspirin and a thienopyridine postprocedure. The CT was measured at baseline and 10 minutes, 4 hours, 12 hours (abciximab-only), and 24 hours after the bolus. Profound inhibition was exhibited in most patients shortly after the platelet inhibitor bolus and during the course of therapy. We observed recovery of platelet function 12 hours after discontinuation of abciximab, with a high degree of interpatient variability, and ongoing profound platelet inhibition 4 to 6 hours after the discontinuation of eptifibatide. Among patients treated with abciximab, patients who were obese recovered from platelet inhibition sooner than patients who were not obese, whereas patients who were elderly had delayed recovery compared with patients who were not elderly. Failure to achieve maximal platelet inhibition (nonclosure) at 10 minutes indicated a possible association with adverse clinical events at the 6-month follow-up examination (60% vs 20%). CONCLUSIONS: PFA-100 is a rapid simple assay used as a means of assessing inhibition of platelet aggregation during PCI performed with glycoprotein IIb/IIIa inhibition. Failure to achieve nonclosure early after the initiation of abciximab therapy warrants further investigation because there may be an association with adverse cardiac events at 6-month follow-up.

Abciximab↗
PubMed1989

The effects of platelet-activating factor and platelet-derived compounds on bovine luteal cell progesterone production.

This study was conducted to characterize bovine platelets with respect to serotonin (5-HT) concentration and platelet-activating factor (PAF)-activation and to examine the in vitro effects of PAF and platelet-derived compounds on bovine luteal progesterone (P4) production. The concentration of 5-HT in platelets, as determined by high-performance liquid chromatography, was 538.8 +/- 40.83 ng/1 x 10(8) platelets. Based on a circulating platelet concentration range of 2.3 x 10(8) 5.8 x 10(8) platelets/ml, the circulating concentration of 5-HT would be approximately 1239-3125 ng/ml of blood. Bovine platelets were found to aggregate in response to PAF (1-40 ng/0.5 ml), with maximal aggregation occurring at 20-40 ng/0.5 ml. Coincubation of luteal cells with platelets (1 x 10(7)-4 x 10(8] enhanced luteal P4 production (p less than 0.05). Addition of the 5-HT receptor antagonist mianserin blocked the platelet-induced increases in P4 (p less than 0.05). Preincubation of platelets with indomethacin did not alter the production of P4 (p greater than 0.05), nor did the addition of propranolol (p greater than 0.05). Platelet-derived growth factor at 8 and 16 ng/ml enhanced basal P4 production (p less than 0.05) but had no effect on the responsiveness of luteal cells to luteinizing hormone (LH) (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Morphine-potentiated agonist-induced platelet aggregation through alpha2-adrenoceptors in human platelets.

Morphine dose-dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and ATP release stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Furthermore, morphine (1 and 5 microM) markedly potentiated collagen (1 microg/ml) evoked an increase of intracellular Ca2+ mobilization in fura 2-AM loading human platelets. Morphine (1 and 5 microM) did not influence the binding of fluorescein isothiocyanate-triflavin to platelet glycoprotein IIb/IIIa complex. Yohimbine (0.1 microM), a specific alpha2-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by collagen. Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E1 (10 microM)-induced cAMP formation in human platelets, and yohimbine (0.1 microM) significantly reversed the inhibition of cAMP by morphine (0.6 and 1 microM) in this study. Morphine (1 and 5 microM) significantly potentiated thromboxane B2 formation stimulated by collagen in human platelets, and yohimbine also reversed this effect of morphine in this study. In addition, morphine (1 and 5 microM) did not significantly affect nitrate production in human platelets. Morphine may exert its potentiation in platelet aggregation by binding to alpha2-adrenoceptors in human platelets, which leads to reduced cAMP formation and subsequently to increased intracellular Ca2+ mobilization; this, in turn, is followed by increased thromboxane A formation and finally potentiates platelet aggregation and ATP release.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2000

Flow cytometric monitoring of glycoprotein IIb/IIIa blockade and platelet function in patients with acute myocardial infarction receiving reteplase, abciximab, and ticlopidine: continuous platelet inhibition by the combination of abciximab and ticlopidine.

BACKGROUND: Improvement of thrombolysis may be achieved by concomitant strong platelet inhibition. To monitor platelet function in patients with myocardial infarction (n=46) who were treated with the fibrinolytic agent reteplase, the glycoprotein (GP) IIb/IIIa blocker abciximab, and the ADP receptor antagonist ticlopidine, we developed a flow cytometric assay. METHODS AND RESULTS: Binding of abciximab to platelets was directly monitored as the percentage of platelets stained by a goat anti-mouse antibody. Blood drawn 10 minutes and 2 hours after the start of therapy with reteplase and abciximab and during the 12-hour infusion of abciximab demonstrated a maximal blockade of GP IIb/IIIa (10 minutes, 86.2+/-10.3%; 12 hours, 85.8+/-7.1%). Starting at 24 hours, abciximab binding gradually decreased (24 hours, 74.6+/-16.2%; 48 hours, 66.8+/-14.9%; 72 hours, 60.5+/-16.7%; 96 hours, 49.4+/-17.8%; 120 hours, 35.8+/-16. 4%; and 144 hours, 29.9+/-15.3%). Binding of a chicken anti-fibrinogen antibody to platelets, indicating the level of functional blockade of GP IIb/IIIa, was inversely correlated with the binding of abciximab (r=-0.72, P:<0.0001). In blood drawn at 10 minutes, platelet aggregation was maximally inhibited but recovered within 48 hours even if the majority of GP IIb/IIIa receptors were still blocked by abciximab. Reteplase did not influence abciximab binding and did not activate platelets, as measured by P-selectin expression, fibrinogen binding, and platelet aggregation. Platelet inhibition that was achieved during the first 24 hours by abciximab was directly maintained by additional treatment with ticlopidine. CONCLUSIONS: Flow cytometric monitoring of platelet function allows differentiation of the effects of reteplase, abciximab, and ticlopidine. The combination of abciximab and ticlopidine is an attractive therapeutic strategy that provides a fast and continuous platelet inhibition.

Abciximab↗
PubMed1998

Activation of platelets in platelet-rich plasma by rotablation is speed-dependent and can be inhibited by abciximab (c7E3 Fab; ReoPro).

BACKGROUND: Rotational atherectomy with the Rotablator catheter has improved percutaneous treatment of certain coronary atherosclerotic lesions, but the "no-reflow" phenomenon remains a serious complication. Because platelet activation by rotablation may contribute to the no-reflow phenomenon, we developed an in vitro system to test the effect of rotablation on platelets in the absence or presence of platelet GP IIb/IIIa receptor blockade with abciximab. METHODS AND RESULTS: Platelet-rich plasma (PRP) was prepared from 28 healthy human volunteers. PRP was divided into 4 samples: (1) no treatment, (2) 6D1 (anti-GP Ib), (3) c7E3 Fab (anti-GP IIb/IIIa+alpha(v)beta3), and (4) c7E3 Fab+6D1. Samples were pumped through a flow chamber containing a 2.5-mm burr rotating at various speeds and then placed in an aggregometer. PRP samples tested in the absence of antibody underwent more rapid and extensive aggregation when rotablated at 150000 and 180000 rpm compared with 0 rpm (P<0.001 at both speeds). Preincubation of platelets with c7E3 Fab decreased the slope of aggregation at each rotablation speed, with 98%, 79%, and 71% reductions at 70000, 150000, and 180000 rpm, respectively (P=0.09 for 70000 and P<0.001 for both 150000 and 180000 rpm). Preincubation of platelets with 6D1 did not decrease the slope of aggregation at any rotablation speed (P>0.5, P=0.99, and P=0.091 for 70000, 150000, and 180000 rpm). Platelet ATP release, a marker of granule release and cell damage, was markedly increased at 180000 rpm (P=0.002 compared with 0 rpm in the control group). Electron microscopy revealed extensive rotablation-induced platelet damage at 150000 and 180000 rpm, and leakage of LDH confirmed platelet lysis at these speeds (P=0.002 and P<0.001 compared with 0 rpm). CONCLUSIONS: High-speed rotablation induces platelet activation of PRP, leading to aggregation; pretreating PRP with abciximab decreases the aggregation. These data suggest that pretreatment of patients with abciximab may decrease rotablation-induced platelet aggregation during rotational atherectomy.

Abciximab↗
PubMed1989

Occupancy of platelet receptors for platelet-activating factor in patients with septicemia.

The possible involvement of platelet-activating factor (PAF) in the pathogenesis of endotoxemia, was investigated by using a binding assay to patients' platelets, complemented with the extraction and chemical characterization of PAF obtained from patients' platelets. Platelets from 12 human volunteers had 281 +/- 63 freely accessible high affinity binding sites (PAF-receptors) per platelet; whereas this number was of 49 +/- 37 PAF-receptors per platelet, n = 14 samples, P less than 0.01, in a group of 13 patients with positive blood culture. A group of patients with respiratory or cardiovascular disturbances and negative blood culture had 253 +/- 74, accessible receptors per platelet (n = 19 samples from 16 patients, P less than 0.01 as compared to septic patients, which was not significantly different when compared to control individuals). Patients with sepsis possessed significant amounts of PAF associated to their platelets, whereas this mediator could not be isolated from platelets of patients with respiratory or cardiovascular disturbances and negative blood culture, nor from platelets of control individuals. PAF was also assayed in whole blood samples and found at high concentrations in sepsis patients. These data indicate that occupancy of PAF receptors in combination with high amounts of platelet-associated PAF, is a common finding in patients with sepsis.

Adult↗
PubMed1999

Interaction between platelet-aggregating response and vasoconstrictive response to platelet activating factor.

It is very important to know under medical treatment which kinds of platelet agonists participate in abnormal platelet-blood vessel interactions. The present study, focusing on platelet activating factor (PAF) was undertaken in an attempt to investigate its action on platelet aggregating response and vasocontractile response to noradrenaline (NA-R). We used autologous platelets and isolated perfused arterial segments from Japanese white rabbits. Firstly, typical tracings of platelet aggregating response to PAF were increased in a dose-dependent fashion, which remained constant and long-lasting. Secondly, noradrenaline (NA) at 5 to 25 ng elicited an initially augmented response in the presence of platelet rich plasma (PRP) with PAF, followed by gradually attenuated responses. Based on the light transmission intensity, platelet aggregation did not seem to be directly or strictly linked to vasocontractile response. Pretreatment with either dibutyryl cyclic AMP (DBcAMP) or indomethacin (IM, a cyclo-oxygenase inhibitor) clearly caused reductions in NA-R as well as platelet aggregation in the presence of PRP with collagen, whereas platelet aggregation and NA-R in the presence of PRP with PAF were scarcely influenced by pretreatment with either DBcAMP or IM. Thus, it seems reasonable to conclude that, in contrast to the response to collagen, platelet aggregation response to PAF was almost indifferent to the adenylate cyclase-cyclic AMP system and the cyclo-oxygenase metabolic pathway.

Animals↗
PubMed1992

Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate.

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

The use of inhibitors of platelet activation or protease activity in platelet concentrates stored for transfusion.

During storage of platelet concentrates the platelets show signs of activation, and extracellular protease activity becomes evident in the plasma. The consequences of platelet activation and plasma protease activity are potentially detrimental to the preservation of platelet function in vitro. The earlier use of prostaglandins during preparation of platelet concentrates to increase the harvest of platelets from whole blood did little to improve their shelf-life. Other compounds that sustain elevated cyclic AMP levels or that directly inhibit platelet agonists provide more effective inhibition of platelet activation during storage. Also, the inclusion of general or specific protease inhibitors appears to improve platelet preservation over extended storage periods. These studies demonstrate the possibility of prolonging the shelf-life of platelet concentrates stored at 22 degrees C through the addition of non-toxic formulations of inhibitors of platelet activation and protease activity.

Alprostadil↗
PubMed1992

[Platelet adhesion and adhesive proteins--how to measure the platelet adhesiveness].

We examined, platelet adhesive function in vivo by comparing the platelet retention rate (PRR) with other laboratory tests. In 71 patients, PRR did not correlate with bleeding time (BT), ristocetin-induced platelet agglutination (RIPA), vWF:AG, vWF:RCo, FVIII:C, fibrinogen, platelets counts or platelet aggregation tests. Among them, the coincidence rate of PRR with BT seems relatively better than that of other tests. In patients with Bernard-Soulier syndrome (BSS), PRR, RIPA and high shear-induced platelet aggregation (SIPA), measured using a cone-plate type instrument, were decreased, while platelet adhesion in a static system and SIPA at a low shear rate were normal. In patients with Glanzmann's thrombasthenia (TA), PRR and SIPA were decreased at both high and low shear rates, while RIPA and platelet adhesion were normal in a static system. In patients with deficient glycoprotein Ia/IIa complex which is one of the putative receptors for collagen, platelet adhesion in a static system is markedly decreased. In patients with von Willebrand disease, the pattern of platelet adhesion tests was similar to that of BSS patients, while in patients with afibrinogenemia, it was similar to that of TA patients except for SIPA at a high shear rate. The various experiments using monoclonal antibodies support these abnormalities. These findings, suggest that PRR is a suitable screening test, but further rapid and easy tests will be necessary to diagnose the defects of platelet adhesion at various shear rates.

Bleeding Time↗
PubMed1992

Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation.

We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.

Antibodies↗
PubMed1993

Evaluation of whole blood flow cytometric detection of platelet bound fibrinogen on normal subjects and patients with activated platelets.

Activated platelets can be detected by measuring platelet-bound fibrinogen in a whole blood, flow cytometric assay, using a fluorescently-conjugated polyclonal antibody. Fibrinogen binding to unstimulated platelets from normal subjects was low in this assay, as was expression of the CD63 antigen. Single cell counting of samples prepared for flow cytometric analysis showed platelet aggregates do not form during the assay procedure. Immune complexes were not seen, and fibrinogen binding to the platelets was unaffected by the CD32 MAb, IV.3. Artefactual activation of the unfixed samples could be minimised by control of phlebotomy, time and temperature of incubation. Variations in platelet count in the range 140-430 x 10(9) 1(-1) and in plasma fibrinogen in the range 2-6 g 1(-1) did not affect the assay results. Comparison of fibrinogen binding with expression of CD63 antigen on normal platelets, stimulated with agonists in vitro, demonstrated that fibrinogen binding detects an earlier stage of platelet activation. Platelet bound fibrinogen was shown to be sensitive in detecting small numbers of activated platelets in clinical samples in twelve patients on intensive care, four undergoing haemofiltration. The patients had a significantly higher median percentage of circulating platelets with bound fibrinogen (p < 0.005), but fibrinogen binding was significantly lower (p < 0.02) in response to 10(-5) M ADP, compared to twelve age-matched normal controls.

Adult↗