Perry Dw

Preparation of Suspensions of Washed Platelets from Humans

Summary: Methods have been developed for the preparation of suspensions of washed platelets from humans. Heparin is used in the washing fluids to prevent: thrombin generation and apyrase is used to prevent adenine nucleotide accumulation. Platelets suspended in Eagles tissue culture medium containing albumin were more responsive to ADP than platelets in Tyrodes‐albumin solution. Addition of fibrinogen is required for maximum sensitivity to ADP‐induced aggregation. These platelets can be stored for 4 hr or more at 37°C in the presence of apyrase and maintain their ability to aggregate upon the addition of low concentrations of ADP.

Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: Direct measurement of percent release of β‐thromboglobulin and assessment by flow cytometry of P‐selectin expression

Contrary to a recent report [Rinder et al.: Blood 82:505, 1993], aspirin does inhibit the release of α‐granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP‐induced aggregation in citrated platelet‐rich plasma (PRP). Measurements were: percent release of 14C‐serotonin from prelabeled platelets, radio‐immunoassay of β‐thromboglobulin (βTG), and expression on the platelet surface of the α‐granule constituent, P‐selectin, by flow cytometry. During the second phase of ADP‐induced aggregation, 69.0 ± 8.3% of βTG and 54.1 ± 4.6% of 14C‐serotonin were released (means ± SEM, n = 13); aspirin treatment reduced these values to 6.0 ± 1.2 and 1.0 ± 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 ± 1.7% release of βTG and 2.1 ± 0.4% release of 14C‐serotonin; these low values were not appreciably affected by aspirin. During ADP‐induced primary aggregation in PRP anticoagulated with FPRCH2Cl (PPACK), only 4.7 ± 0.9% release of βTG and no detectable release of 14C‐serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 μM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2–10 μM), the mean fluorescence intensity due to P‐selectin was <14% of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P‐selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiological Ca2??? concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P‐selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both α‐granule and dense granule contents during ADP‐induced secondary aggregation in citrated PRP.

Hypersensitivity to ADP of platelets from diabetic rats associated with enhanced fibrinogen binding

Abstract. Platelets from diabetic humans and animals are hypersensitive to ADP. The hypersensitivity to ADP of platelets from diabetic rats occurs independently of activation of the arachidonate pathway or release of dense granule contents. During platelet aggregation by ADP, fibrinogen binds to its receptor on platelets. We examined if the hypersensitivity to ADP of platelets from diabetic rats is associated with enhanced early binding of fibrinogen to its receptor on these platelets. Fibrinogen association with platelets from rats with spontaneous or streptozotocin‐induced diabetes was significantly greater 10 s or 1 min after addition of ADP (10μm) than with platelets from their corresponding control rats. Since enhanced fibrinogen association occurred with platelets from insulin‐treated rats with spontaneous diabetes, and from rats with streptozotocin‐induced diabetes that did not receive insulin, the enhanced fibrinogen binding is likely due to the diabetic state rather than to the administration of insulin or the mechanism responsible for the diabetes. Therefore, enhanced early fibrinogen association with platelets from diabetic rats is associated with their hypersensitivity to ADP.

Effects of cathepsin G pretreatment of platelets on their subsequent responses to aggregating agents

Cathepsin G, a proteolytic enzyme from activated leukocytes, can interact with platelets during inflammation and thrombosis. Platelets that have been exposed to cathepsin G in thrombi may recirculate if they are freed during fibrinolysis. To determine whether some of the subsequent functions of such platelets would be impaired, we investigated the responses of cathepsin G-pretreated platelets to agonists that they would encounter in the circulation. Suspensions of washed human platelets were labeled with [14C]serotonin and resuspended in Tyrode-albumin solution (with 2 mM Ca2+ and apyrase). After 15 minute incubation with 400 nM cathepsin G at 37 degrees C, 52+/-3% of [14C]serotonin had been released, and glycoprotein Ib was degraded. The platelets were washed and resuspended in fresh medium to remove cathepsin G and released materials. Ristocetin-induced agglutination was abolished, indicating that the binding site for von Willebrand Factor on glycoprotein Ib had been removed. Aggregation and release of residual [14C]serotonin in response to 0.1-1.0 U/mL thrombin was blocked or greatly reduced by the cathepsin G pretreatment. This inhibition is probably largely due to cleavage by cathepsin G of some of the protease-activated receptors at the C-terminal side of Ser42 so that the tethered ligand is lost. Pretreatment with cathepsin G did not affect responses to ADP or a low concentration of platelet-activating factor in the presence of fibrinogen, indicating that receptors for these agonists were unaffected and that the function of the fibrinogen receptor, GPIIb/IIIa was unchanged. Responses to cathepsin G, the thrombin receptor-activating peptide SFLLRN, collagen, or the thromboxane A2 mimetic U46619 were partially inhibited, even in the presence of added fibrinogen. Platelet adhesion to a collagen-coated surface was 51+/-7% inhibited, which may indicate cleavage of a collagen receptor or receptors; this may partly account for strong inhibition of collagen-induced aggregation and release of granule contents; additionally, as shown by inhibition of responses to U46619, the function of the thromboxane A2 receptor may be compromised. Thus, although cathepsin G activates platelets, if they recirculate after interaction with it, their subsequent adhesion to damaged vessel walls, aggregation, and release of granule contents induced by thrombin and collagen will be diminished.

Responses to aggregating agents after cleavage of GPIb of human platelets by the O-sialoglycoprotein endoprotease from Pasteurella haemolytica- potential surrogates for Bernard-Soulier platelets?

Most proteolytic enzymes that cleave glycoprotein lb (GPlb) also cleave other glycoproteins or receptors on the surface of platelets. We have used an O-sialoglycoprotein endoprotease from Pasteurella haemolytica that selectively cleaves the heavily O-glycosylated GPlb, but does not cleave N-linked glycoproteins or unglycosylated proteins. Isolated, [14C]serotonin-labeled platelets in Tyrode-albumin solution were incubated with 10 microg/mL endoprotease for 60 minutes at 37 degrees C. These platelets did not release [14C]serotonin, had no detectable GPIb, and were unresponsive to ristocetin/von Willebrand factor. Compared with control platelets, aggregation and release of [14C]serotonin by the endoprotease-pretreated platelets were inhibited in response to low concentrations of thrombin, SFLLRN (the PAR-1-activating peptide), collagen, and U46619 (a thromboxane A(2) mimetic); aggregates were smaller in size. The presence of fibrinogen overcame the inhibition of responses induced by SFLLRN, collagen, and U46619. With fibrinogen, primary ADP-induced aggregation was scarcely affected by pretreatment with the endoprotease. Thus, the PAR-1 receptor for thrombin, and receptors for collagen, thromboxane A(2), fibrinogen (GPIIb/IIIa), and ADP appear to function normally on the endoprotease-pretreated platelets. Since only GPIb is cleaved by the endoprotease, these platelets seem to provide potential surrogates for Bernard-Soulier syndrome platelets for further studies of platelet functions in this condition.

Role of Secreted Adenosine Diphosphate in the Synergistic Effects of Cathepsin G on Human Platelets

BRIEF COMMUNICATION Role of Secreted Adenosine Diphosphate in the Synergistic Effects of Cathepsin G on Human Platelets R.L. Kinlough-Rathbone1, D.W. Perry1, M.L. Rand2,3 and M.A. Packham3 1Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; 2Division of Haematology/Oncology, The Hospital for Sick Children, Toronto, Ontario, Canada; 3Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

Rabbit lung macrophages stimulate platelets in vitro as observed by density-gradient centrifugation and transmission electron microscopy

Both platelets and macrophages play a role in the pathogenesis of atherosclerosis. To examine whether they may interact and, if they do, to elucidate the mechanisms of such an interaction, suspensions of the two cell types from rabbits were mixed together, then subjected to Stractan density-gradient centrifugation and transmission electron microscopy. Suspensions of only one cell type served as controls. When otherwise unstimulated platelets and macrophages came into contact with each other, the platelets became less dense. Ultrastructurally, the platelets underwent shape changes without losing their granules, and were often arranged around the macrophages like a rosette. The processes of the macrophages became elongated. ADP caused a similar shift in platelet density and, when the cell types were together, increased this shift. With ADP the rosetting was abolished, but platelet aggregates were found to be in superficial contact with the macrophages. With thrombin the contact between the platelet aggregates and macrophages was close. Addition of platelet antagonists showed that the shift in platelet density and the rosetting upon contact with macrophages are dependent on divalent cations. Neither ADP, nor thrombin, nor PAF seem to be involved in the reactions.