Citrated Platelet Rich Plasma Research Articles

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. © 1996 Wiley-Liss, Inc.

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 beta-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 alpha-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 beta-thromboglobulin (beta TG), and expression on the platelet surface of the alpha-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 +/- 8.3% of beta TG and 54.1 +/- 4.6% of 14C-serotonin were released (mean +/- 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 beta TG 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 FPRCH2CI (PPACK), only 4.7 +/- 0.9% release of beta TG 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 microM), 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 microM), 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 alpha-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP.

Apyrase and anti-platelet activities from the salivary glands of the bed bug Cimex lectularius

Apyrase activity was detected in the salivary gland of Cimex lectularius. Kinetic, HPLC chromatography, isoelectric focusing gel electrophoresis and thermal sensitivity assays indicated that the enzymatic activity is the result of a true apyrase and not the result of independent or combined different enzyme activities. The apyrase activity was dependent on calcium and not on magnesium, manganese or zinc, and was inhibited in the presence of EDTA. The pH optimum for both, ATPase and ADPase activities was 8.5. A high specific activity of Cimex salivary apyrase for hydrolysis of both ADP and ATP was found. Cimex salivary gland apyrase activity decreased by 43 percent right after insects fed on blood as compared with insects before feeding. The apparent molecular mass of apyrase activity as determined by HPLC molecular sieving chromatography was 79 600 daltons. Additionally, a peak showing apyrase activity was detected with a molecular mass of 21 000. However, the small molecular mass activity shifted to a high molecular mass activity when the salivary gland homogenate was prepared in more alkaline buffer and frozen, thus suggesting that the high molecular weight form of Cimex apyrase may result from aggregation of active small units. The saliva of Cimex lectularius inhibited platelet aggregation induced by ADP; one salivary gland pair was sufficient to completely inhibit the aggregation of 0.1 ml of citrated platelet rich plasma induced by 5 μM ADP. The inhibitor was shown to be a component different than nitric oxide. Altogether the data suggest that Cimex salivary apyrase can act to prevent an important step on hemostasis, platelet aggregation, and thus play an important role during the insect feeding to overcome the vertebrate host hemostatic defense mechanism.

Cyclosporine-induced detachment of vascular endothelial cells initiates the intrinsic coagulation system in plasma and whole blood

Cyclosporine A (CsA) is supposed to alter the metabolism of vascular endothelial cells, leading to a prothrombotic state. We examined by which mechanism human umbilical vein endothelial cells (HUVECs) treated with CsA would promote coagulation in human plasma and in whole blood. Treatment of HUVECs with CsA at concentrations clinically used led to dose-dependent cell detachment, with the subsequent exposure of the highly procoagulant connective tissue. As determined by scanning electron microscopy, cell counting of detached and adherent cells, and antigenic measurement of collagen exposure, HUVECs treated with 0.4 μg/ml CsA (or more) for 4 days exhibited significant amounts of subendothelial areas. On CsA-treated HUVEC monolayers, the clotting time of recalcified citrated plateletrich plasma (PRP), but not platelet-poor plasma (PPP), was dose-dependently shortened. Likewise, the onset of thrombin generation was significantly earlier. Except at a high concentration of 8.0 μg/ml CsA, there was no procoagulant effect when PPP was used. To investigate CsA-treated HUVECs in whole blood, cells were cultivated on globular microcarriers and were incubated with nonanticoagulated whole blood. When untreated cells were used, generation of factor Xa, thrombin, and kallikrein was completely suppressed for 30 minutes. HUVEC beads treated with 0.4 and 0.8 μg/ml CsA, however, led to a dose-dependent generation of all three coagulation factors, with peak values at 2.5 to 5 minutes. Extrinsic activation was excluded, since CsA treatment did not induce tissue factor activity in HUVECs. Furthermore, the thrombomodulin activity of HUVECs was not altered by CsA. In conclusion, treatment of HUVECs with CsA for 4 days at concentrations clinically used leads to the exposure of subendothelial areas that induce activation of the intrinsic coagulation in recalcified PRP and nonanticoagulated whole blood.

Agonist-Induced Release of Neuropeptide Y by Platelets

Neuropeptide Y (NPY) is a multi-functional peptide localized in a number of tissues besides the brain, and its expression is high in strains of mice with autoimmune disease. Functionally, along with its neurotransmitter role, NPY has multiple effects on both vascular tissues and tissues involved in immune responses. Previous studies demonstrated that immunoreactive NPY (i-NPY) exists in large quantities in platelets and bone marrow of some species, and suggested its release during platelet aggregation by collagen. Because NPY has been hypothesized to have a role in a number of pathophysiological states in which platelets are involved, including immune responses, hypertension and vascular smooth muscle proliferation, we sought to further characterize platelet i-NPY content and release by a diverse group of physiological platelet agonists. Platelet rich plasma and gel filtered platelets were prepared from citrated whole blood of male Sprague Dawley rats. Platelet content and concentration of i-NPY were quantified by RIA. Platelet aggregation responses (turbidometric method) and release of i-NPY were measured in rat platelet preparations stimulated by a thromboxane agonist (U44069), collagen, thrombin and ADP. All agents induced dose-dependent aggregation, although ADP and U44069 were weak agonists in citrated platelet rich plasma, ADP only inducing primary aggregation. Total i-NPY content in platelet rich plasma of Sprague-Dawley rats was 32.1±3.8 pmol/ml; more than 20 pmol/ml was released into supernatant of aggregating platelets stimulated with high dose thrombin or collagen during secondary, irreversible aggregation. Although U44069 and ADP both stimulated i-NPY release in a dose-dependent manner, release was substantially lower than with the other agonists. We conclude that i-NPY release from rat platelets is associated mainly with secondary, irreversible aggregation, and can be produced by a variety of platelet stimulating agents as part of the platelet release reaction. The present study and other recent studies demonstrate wide variability in platelet i-NPY content between and within species. Investigation of the genetic regulation of i-NPY content in platelets could offer new insights into the relationship between NPY and pathophysiology of the immune and cardiovascular systems.

Streptokinase and APSAC inhibit platelet aggregation in vitro by fibrinogenolysis: Effect of plasma fibrinogen degradation products X and E

Summary Streptokinase (SK) in vitro can both activate and inhibit platelets. Inhibition has been ascribed either to proteolysis of membrane glycoproteins (GPs) or to the inhibitory effect of fibrinogen degradation products (FgDPs). Addition of a low concentration of SK (300 U/ml) to human citrated platelet rich plasma (cPRP) has been shown to lead to an aggregation defect, related to fibrinogenolysis, without discernible changes in the glycoprotein IIb/IIIa complex. In the present study, similar results were obtained with SK (300–5000 U/ml), and the acylated plasminogen streptokinase activator complex (APSAC) (0.01 U/ml). The aggregation defects appeared to be related to the extent of fibrinogenolysis. The knowledge of the FgDPs profile might help in the understanding of this aggregation defect. We have chosen to generate the FgDPs in plasma by activating plasminogen with SK 300 U/ml, 15 min at 37°C. Fragments X and E were separated by gel filtration and identified by electrophoresis and immunoblot. The effects of the fractions containing fragments X and E on aggregation were studied using human washed platelets resuspended in Tyrode buffer, 2 mM Ca++, 1 mM Mg++, after stimulation with ADP (10−5 mol/l). The results (expressed as percentage of the response obtained with intact fibrinogen) were: 72%±18 (p In conclusion, our data confirm and extend previous observations on the mechanisms of platelet aggregation inhibition by SK or APSAC. The main factor of platelet inhibition in plasma appears to be the FgDP E.

Original Article: Comparison of A23187 vs Ionomycin-induced Responses and Cytosolic Calcium Increases in Aequorin-loaded Human Platelets. Evidence for Ionophore-specific Differences in Intracellular Calcium Release

The dose-dependent induction of platelet aggregation, dense granule secretion and thromboxane formation by the divalent cation ionophores A23187 and ionomycin were compared in citrated platelet rich plasma (PRP), and measured simultaneously with the increases in cytosolic Ca(2+) levels in aequorin-loaded gel-filtered platelet (GFP). In citrated PRP, both ionophores induced similar extents of aggregation at comparable concentrations, whereas A23187 induced somewhat greater extents of secretion, and substantially greater extents of thromboxane B(2) (TxB(2)) formation, than ionomycin. When 5 mM EDTA or EGTA was added to PRP secretion and TxB(2) formation occurred only with A23187; ionomycin was inactive at all concentrations tested, up to 100 pM. In aequorin-loaded GFP containing 1 mM Ca(2+), 1 mM EGTA or 2 mM EDTA, ionomycin as well as A23187 induced these platelet responses, but the concentration dose-response curve for ionomycin was shifted to the right by approximately one order of magnitude relative to that for A23187. Simultaneous measurements of the cytosolic Ca(2+) ([Ca(2+)](1)) increases induced by the two ionophores showed that the increases produced by ionomycin were consistently 2040% less than those induced by A23187 at all ionophore concentrations tested. Analysis of the extents of secretion and TxB, formation obtained in EDTA- or EGTA-containing systems as a function of the [Ca(2+)](1) increases suggested that the data for both ionophores were described by the same [Ca(2+)](1) dose-response curve, indicating that the decreased extents of these responses seen with ionomycin vs A23187 were due primarily, if not solely, to the lower [Ca(2+)], increases produced by ionomycin. Since ionomycin is theoretically capable of transporting twice as much divalent cation as A23187, these findings in platelets, together with similar findings in certain other cell systems, provide evidence that factors associated with the intracellular environment may differentially affect the abilities of A23187 and ionomycin to induce cellular responses and, more specifically, to release intracellular Ca(2+) stores.

In vitro model to test the thrombogenicity of coronary stents

Thrombotic occlusion is a major complication limiting the application of stents in coronary arteries. In an in vitro model we investigated the thrombogenicity of different stent materials and several medical regimens to prevent thrombotic occlusion. Experiments were conducted in a closed system of silicon tubing with circulating citrated platelet rich plasma of healthy volunteers (n=7) and of patients (n=7 for each condition). Patients were either treated with phenprocoumon or with high or low dose heparin in combination with aspirin alone (100 mg) or aspirin (990 mg) plus dipyridamole (225 mg). After placement of tantalum wire stents into the system platelet aggregates were visible after 13.5 ± 3.0 min, and occlusion occurred after 15.0 ± 3.5 min. Similarly, with implanted stainless steel stents aggregation was seen after 13.0 ± 3.5 min and thrombosis occurred after 14.5 ± 3.5 min (p<0.001 vs control without stent). Microscopic examination revealed combined platelet fibrin thrombi occluding the lumen. Platelet components predominately covered stent wires, particularly at crossing points. In all experiments high-dose heparin prevented platelet aggregate formation and stent occlusion independently of additional aspirin or aspirin plus dipyridamole; perfusion time > 60 min (p<0.001 vs no heparin). Low-dose heparin could not prevent clotting. With aspirin alone aggregates were visible after 16.0 ± 4.0 min and clotting occurred after 23.0 ± 5.0 min. In combination with dipyridamole aggregates were visible after 15.5 ± 5.0 min and clotting after 21.0 ± 4.0 min (NS vs aspirin alone). Phenprocoumon prevented platelet aggregate formation and stent occlusion; perfusion time > 60 min. Thus, in our in vitro model both platelet aggregate formation and coagulation play an important role for stent occlusion. High-dose heparin and phenprocoumon prevented both. Low-dose heparin was less effective and could not prevent clotting even in combination with aspirin and dipyridamole.

Activation of human blood platelets induced by nocloprost, a stable prostaglandin E2 derivative.

Nocloprost, a 9 beta-chloro-16,16-dimethyl derivative of prostaglandin E2 (PGE2), belongs to the gastric cytoprotective agents that are used in the therapy of gastric ulcer. Since methylated derivatives of PGE2 are known to have proaggregatory effects the influence on platelets was studied. In platelet-rich citrated plasma, nocloprost (> 0.1 mumol/l) caused aggregation with a biphasic course at higher concentrations. Aggregation induced by nocloprost (1 mumol/l) corresponded to that induced by adenosine diphosphate (ADP) (5 mumol/l). Activation of platelets by nocloprost was accompanied by formation of thromboxane A2 and an increase in cytosolic calcium in Indo 1-loaded platelets. At 0.1 mumol/l it potentiated aggregation induced by low concentrations of ADP or adrenaline. The effect of nocloprost on platelets was blocked by iloprost, daltroban and indomethacin. PGE2, which was studied for comparison, at 0.1-1.0 mumol/l inhibited aggregation induced by 1 mumol/l nocloprost. The concentrations of nocloprost required for therapeutic use as antiulcer agent were lower by three orders of magnitude than those which induce human platelet aggregation.

Factors Affecting the Observation of a Synergistic Response in Platelet Aggregation Induced by Pairs of Excitatory Agonists

When aggregation is measured as the disappearance of single platelets synergistic interaction between excitatory agonist pairs can be observed using washed platelets in a modified Tyrode's medium or platelet-rich plasma anticoagulated with hirudin; but not using citrated platelet-rich plasma. For aggregation induced by the ADP/adrenaline agonist pair, both the observation of synergistic interaction and the sensitivity of the platelets to these agonists, is a function of extracellular [Ca(2+)]. Synergistic interaction and reduced sensitivity to the individual agonists, especially adrenaline, is observed when extracellular [Ca(2+)] > 100 µM. The data suggest that lower affinity binding of Ca(2+) to the glycoprotein IIb/IIIa complex may modulate platelet sensitivity to these excitatory agonists. The conditions used to resuspend the platelets also influences the nature of the response to the ADP/adrenaline agonist pair and the sensitivity of the platelets to these agonists. A synergistic response and/or reduced sensitivity to ADP is observed on resuspension in modified Tyrode's medium but does not occur on resuspension in citrated plasma or in plasma anticoagulated with hirudin. The factor responsible for enhancing sensitivity, and hence abolishing the synergistic response, is a species of low molecular weight (M(r) less than 25 KDa). It is neither citrate nor Ca(2+).

A Defect of Platelet Aggregation Associated With an Abnormal Distribution of Glycoprotein IIb-IIIa Complexes Within the Platelet: The Cause of a Lifelong Bleeding Disorder

Abstract A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate- polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.

A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.

Characterization of rat platelet serotonin receptors with tryptamine agonists and the antagonists: Ketanserin and SCH 23390

Current literature suggests that the platelet 5-HT2 receptor, thought to be the only active platelet serotonin (5-HT) receptor, may be both heterogeneous and not the sole 5-HT receptor subtype on platelet membranes. The present studies used more selective tryptamine agonists, and the antagonists ketanserin and SCH 23390 to characterize rat platelet 5-HT receptors in vitro. The present studies also addressed anticoagulant effects (citrate versus heparin) on platelet 5-HT aggregation in the rat. 5-HT was less potent at enhancing ADP-induced aggregation in heparinized rat platelet rich plasma (PRP) as compared to citrated PRP. However, potency and maximum aggregation to ADP were greater in heparinized platelets. In citrated rat PRP, the selective tryptamine agonists, 5-carboxamidotryptamine (5-CT) and 2-CH 3-5-HT, produced little change in the baseline ADP-induced aggregation and induced platelet shape change only in higher concentrations ( >1 μM). In contrast, α-CH 3-5-HT-induced shape change and enhancement of ADP aggregation were superimposable with that of 5-HT itself suggesting 5-HT 2 receptor activation. The antagonists, ketanserin and SCH 23390, inhibited 5-HT enhancement of ADP-induced aggregation with affinity constants consistent with the presence of 5-HT 2 receptors as well. Studies with heparinized rat PRP did not unmask activity to 5-CT or 2-CH 3-5-HT. Thus, although reports of multiple platelet 5-HT receptors exist, the only detectable, functional 5-HT receptor to enhance aggregation in rat platelets was probably of the 5-HT 2 type.

Relative importance of thrombin compared with plasmin-mediated platelet activation in response to plasminogen activation with streptokinase.

Platelet activation occurs in vivo during pharmacologic thrombolysis and may contribute to recurrent thrombosis. Plasmin does not directly activate platelets except at high concentrations; thus, the mechanisms for platelet activation during thrombolysis remain undefined. Increases in thrombin activity also occur in patients treated with fibrinolytic agents and may contribute to activation of platelets. We have shown that one mechanism for increased thrombin activity is activation of the coagulation system by plasmin. In the present study we sought to determine whether activation of platelets in response to pharmacologic activation of plasminogen in plasma is due primarily to plasmin or mediated by increased thrombin activity. Platelet-rich citrated plasma (PRP) was recalcified and incubated with 1,000 IU/ml of streptokinase or 1.0 caseinolytic units/ml of plasmin. Concentrations of fibrinopeptide A, a marker of thrombin activity, increased markedly over 10 minutes in plasma incubated with streptokinase or plasmin, but not in PRP incubated without plasminogen activator. Platelet activation characterized by the secretion of 14C-serotonin occurred within 2-4 minutes after thrombin activity increased. In stirred recalcified PRP, platelet aggregation was accelerated from 3.6 +/- 0.5 to 2.5 +/- 0.3 minutes (p less than 0.01) when incubated with 1,000 IU/ml of streptokinase. Leupeptin and aprotinin, inhibitors of plasmin activity, markedly attenuated platelet activation in response to pharmacologic activation of plasminogen. However, inhibition of thrombin with heparin, hirudin, or D-Phe-D-Pro-L-Arg-chloromethylketone was more effective in inhibiting the acceleration of platelet activation induced by plasminogen activation, despite the elaboration of plasmin activity. Activation of platelets during coronary thrombolysis may be due in part to increased procoagulant activity induced by plasminogen activation as well as other factors that promote platelet activation in vivo.

The Effect of Agonists and Antagonists of Platelet Aggregation on von Willebrand Factor-Mediated Platelet Agglutination

Ristocetin-induced platelet agglutination (RIPA) in EDTA-treated citrated platelet-rich plasma was reduced to 49 +/- 11% by 1.25 microM ADP, 41 +/- 14% by 1 microM A23187, and 26 +/- 7% by 0.1 microgram/ml platelet activating factor (PAF). The effect of 5-110 microM epinephrine was not dose-dependent, but varied between donors, with RIPA from 56-100% of the control. The inhibitory effects of these agonists were not altered by prior treatment of platelets with aspirin. Prior addition of 200 microM ATP (an ADP receptor antagonist acting at both high and low affinity ADP receptors) prevented the inhibitory action of ADP but not that of A23187 or PAF, suggesting that the inhibitory actions of the latter are not mediated by released ADP. As 700 microM 8-bromoadenosine 5-diphosphate (an ADP receptor antagonist acting mainly at the high affinity receptor) did not prevent ADP-induced inhibition of RIPA, interaction of ADP with the low affinity receptor is presumably responsible for its inhibitory action. As A23187, but not phorbol myristate acetate (0.1 microM) inhibited RIPA, an increase in intracellular calcium ions rather than direct stimulation of protein kinase C appears to mediate agonist-induced inhibition. Cytochalasin B (10.5-21 microM), dibucaine (0.5-1 mM), and prostaglandin E1 (25 nM), added before or after the agonist, prevented or reversed ADP-, A23187-, and PAF-induced inhibition of RIPA, suggesting that the state of the platelet cytoskeleton affects inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hydrostatic pressure and inert gases on platelet aggregation in vitro

A novel cuvette was used to subject citrated platelet-rich plasma (PRP) to high hydrostatic pressure with negligible contamination by He (used for compression of the apparatus). Aggregation was induced at pressure by ADP and quantified turbidimetrically. The maximum degree of aggregation (MDA) was reduced from a control level of 82.2 to 53.6% by exposure to 101 ATA. Because decompression bubbles did not form, aggregation was also measured immediately after a compression cycle. After exposure to 101 ATA hydrostatic pressure, platelets responded normally to ADP at 1 ATA. In a matching apparatus, PRP was equilibrated with high partial pressures of inert gases. Normal physiological plasma Po2 and pH were maintained during equilibration. N2O (5 ATA) reduced the MDA from 86.5 (control) to 58.1%. N2 (51 ATA) reduced the MDA from 74.7 (control) to 51.6%, and 101 ATA Pn2 reduced the MDA from 78.0 (control) to 32.3%. He (100 ATA) reduced the MDA from 83.6 to 38.6%. It was concluded that platelet aggregation was relatively sensitive to hydrostatic pressure and less sensitive to inert gases than predicted from their anesthetic potency ratios.

Endothelin does not affect aggregation of human platelets

Endothelin (ET-1) is a recently discovered endothelial-derived peptide with pronounced vasoconstrictor activity. The present study addressed whether ET-1, in analogy with several other vasoactive agents, can induce or modulate aggregation of human platelets in vitro. Venous blood from healthy donors was collected in citrate or heparin and platelet-rich plasma (PRP) was prepared. Portions of the PRP were added to drugs, and platelet aggregation was recorded according to Born & Cross (1963). ET-1 added to the PRP (final concentrations 1-100 nM) did not induce aggregation of platelets, either in citrate- or heparin-containing plasma. Adenosine-diphosphate (0.5-2 microM) or thrombin (0.1-0.4 NIH units ml-1) induced dose-dependent aggregation of platelets in citrate- or heparin-containing PRP; such aggregation was, however, not affected by ET-1 (1-100 microM) either. We conclude that ET-1, in contrast to other endothelial-derived vasoactive agents, lacks direct effect on platelet aggregation in vitro.

Observations on human platelet aggregation in native whole blood: Synergism and sensitivity to aggregating agents in vitro

Threshold sensitivity levels, as well as the synergistic effects of agonists for blood platelets have been evaluated by independence aggregometry using whole blood without anticoagulation. Platelet aggregation was studied using adenosine diphosphate (ADP), epinephrine, and thrombin. Threshold responses to ADP, epinephrine, and thrombin were observed at lower concentrations in diluted native whole blood (NWB) than reported values for citrated whole blood, and appear to be similar to those of citrated platelet rich plasma (PRP). Potentiation of aggregation occurred when both ADP and epinephrine, or epinephrine and thrombin but not ADP and thrombin were present in combination. Synergism between agonists thus is similar but not identical to that of PRP, and NWB appears to provide a sensitive system with a more physiologic milieu than other in vitro aggregation systems.

Mechanism of Rabbit Platelet Agglutination Induced by Acidic Mucopolysaccharide Extracted from Stichopus japonicus Selenka

The effects of acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus Selenka) (SJAMP) on rabbit platelets were studied. Using citrated platelet-rich plasma (PRP), washed platelets, and formaldehyde fixed platelets from 10 New Zealand white rabbits, we investigated the effects of platelet inhibitors and various plasma and its fractions on SJAMP-induced agglutination. It was found that the tracing of platelet agglutination induced by SJAMP showed a single phase without a lag period. The lowest concentration of SJAMP required for the agglutination of rabbit platelets was approximately 2 micrograms/ml, and the magnitude of agglutination induced by SJAMP was concentration dependent. In 8 out of 10 rabbits, the platelets in PRP were agglutinated by 10 micrograms/ml of SJAMP. Platelet inhibitors, such as aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose and EDTA did not inhibit the agglutination induced by SJAMP. Washed rabbit platelets were not agglutinated by SJAMP even though the concentration of SJAMP was raised up to 50 micrograms/ml. When rabbit plasma, serum, or 50-60% ammonium sulfate saturated plasma fraction was added to the reaction mixture, agglutination of washed platelets by SJAMP was recovered completely. But human plasma or fibrinogen did not have any effect on the reactivity of washed rabbit platelets to SJAMP. From these data we conclude that the SJAMP-induced rabbit platelet agglutination is independent of energy metabolism but requires plasma cofactor(s) other than fibrinogen. The plasma cofactor is present in 50-60% ammonium sulfate saturated plasma fraction.

Aggregation of Human Platelets by Acidic Mucopolysaccharide Extracted from Stichopus japonicus Selenka

The acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus Selenka) (SJAMP) has been shown to cause platelets to aggregate. Using citrated platelet-rich plasma (PRP), washed platelets and formaldehyde-fixed platelets from humans, we investigated the effects of platelet inhibitors and various plasmas and their fractions on SJAMP-induced platelet aggregation. It was found that the lowest concentration of SJAMP required for the aggregation of human platelets was about 0.4 micrograms/ml and the magnitude of aggregation induced by SJAMP was concentration dependent. The platelets were aggregated by SJAMP at 10 micrograms/ml in 25 out of 28 (89%) normal subjects tested. Platelet inhibitors such as PGE1, aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose and EDTA inhibited by 70 to 100% the aggregation induced by SJAMP. Washed platelets alone were not aggregated by SJAMP. In the presence of fibrinogen, washed platelets were aggregated by SJAMP but formaldehyde-fixed platelets were not. These data indicate that the SJAMP-induced human platelet aggregation requires extracellular calcium, fibrinogen, and energy metabolism. The second phase of aggregation is dependent upon the release of ADP, and cyclooxygenase pathway.