Pressure-Induced Metabolic Activation of Platelets Demonstrated In Vitro by Determination of Malondialdehyde Formation

1984 ◽  
Vol 52 (01) ◽  
pp. 004-006 ◽  
Author(s):  
Alessandro Torsellini ◽  
Luca Doni ◽  
Wulf Palinski ◽  
Giovanni Guidi ◽  
Valeriano Lombardi
Keyword(s):  
Arachidonic Acid ◽  
Atmospheric Pressure ◽  
Platelet Rich Plasma ◽  
Metabolic Activation ◽  
Platelet Factor ◽  
Malondialdehyde Formation ◽  
Shape Changes

SummaryPlatelet rich plasma (PRP) exposed in vitro to 200 mm Hg above atmospheric pressure showed a significant increase in malondialdehyde (MDA) formation compared to PRP at atmospheric pressure. This difference is also evident when platelets are incubated with arachidonic acid.The increase of MDA demonstrates that the increased beta- thromboglobulin and platelet factor 4 in plasma and the shape changes of platelets after pressure stimulation in vitro that were described in a previous paper result from the release reaction.Pressure-induced effects in vivo are discussed

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

Platelet inhibition by aspirin is diminished in patients during carotid surgery: a form of transient aspirin resistance?

2004 ◽  
Vol 92 (07) ◽  
pp. 89-96 ◽  
Author(s):  
David Payne ◽  
Chris Jones ◽  
Paul Hayes ◽  
Sally Webster ◽  
A. Naylor ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Platelet Inhibition ◽  
Aspirin Resistance ◽  
Arachidonic Acid Metabolism ◽  
Significant Rise ◽  
Control Group

SummaryThe majority of patients who suffer peri-operative thromboembolic complication while undergoing vascular procedures do so despite taking aspirin. This study examined the antiplatelet effect of aspirin during surgery in patients undergoing carotid endarterectomy (CEA). Fifty patients undergoing CEA were standardised to 150 mg aspirin daily for ≥2 weeks. Platelet aggregation in response to arachidonic acid (AA) was measured in platelet rich plasma prepared from blood taken prior to, during, and at the end of surgery. Spontaneous platelet aggregation was also studied, as was the role of physiological agonists (ADP, collagen, thrombin, and epinephrine) in mediating the in vivo and in vitro responses to AA. Eighteen patients undergoing leg angioplasty, also on 150 mg aspirin, without general anaesthesia, served as a control group. In the CEA patients aggregation induced by AA (5 mM) increased significantly from 7.6 ± 5.5% pre-surgery to 50.8 ± 29.5% at the end of surgery (p <0.0001). Aggregation to AA was even greater in samples taken mid-surgery from a sub-set of patients (73.8 ± 7.2%; p = 0.0001), but fell to 45.9 ± 7.4% by the end of surgery. The increased aggregation in response to AA was not due to intra-operative release of physiological platelet agonists since addition of agents that block/neutralise the effects of ADP (apyrase; 4 µg/ml), thrombin (hirudin; 10 units/ml), or epinephrine (yohimbine; 10 µM/l) to the samples taken at the end of surgery did not block the increased aggregation.The patients undergoing angioplasty also showed a significant rise in the response to AA (5 mM), from 5.6 ± 5.5% pre-angioplasty to 32.4 ± 24.9% at the end of the procedure (p <0.0001), which fell significantly to 11.0 ± 8.1% 4 hours later. The antiplatelet activity of aspirin, mediated by blockade of platelet arachidonic acid metabolism, diminished significantly during surgery, but was partially restored by the end of the procedure without additional aspirin treatment.This rapidly inducible and transient effect may explain why some patients undergoing cardiovascular surgery remain at risk of peri-operative stroke and myocardial infarction.

HEPARIN DIRECTS THE INACTIVATION OF ANTITHROMBIN BY ELASTASE

1987 ◽  
Author(s):  
R E Jordan ◽  
J Kilpatrick ◽  
J Nelson ◽  
J O New gren ◽  
M A Fournel
Keyword(s):  
Alternative Explanation ◽  
Anticoagulant Activity ◽  
Molar Ratio ◽  
Heparin Binding ◽  
Apparent Contradiction ◽  
Platelet Factor ◽  
Complete Inactivation

In apparent contradiction to its anticoagulant activity, we have observed a previously undetected, and potentially opposing function for heparin: a distinct heparin-dependency for the in vitro inactivation of highly-purified human antithrombin by neutrophil elastase. Similar to its ability to accelerate antithrombin-mediated inhibition of coagulation enzymes, anticoagulantly-active heparin was also found to stimulate the rate of inactivation of antithrombin by the neutrophil enzyme.In the absence of heparin, or in the presence of the heparin antagonists platelet factor 4 or polybrene, little or no inactivation of antithrombin occurred. Catalytic amounts of heparin and elastase caused the complete inactivation of antithrombin (approximate molar ratio of 1:1:400 respectively) in 5-10 minutes. The loss of heparin binding affinity by the elastase-cleaved form of antithrombin permitted its separation from active antithrombin by heparin-agarose chromatography.The purified elastase-inactivated antithrombin was injected into rabbits for determination of its comparative clearance behavior. In contrast to intact, functional antithrombin (t 1/2 >30 hours) and the thrombin-antithrombin (T-AT) complex (t 1/2 previously shown to be minutes), elastase-inactivated antithrombin circulated for approximately 13 hours. This prolonged clearance relative to the T-AT complex may suggest an alternative explanation for the circulating, non-functional antithrombin observed in certain coagulopathic states. In summary, these results point to a potential and unexpected role for heparin in directing the inactivation of antithrombin and suggest a possible in vivo mechanism for neutralizing the usually non-thrombogenic nature of the vascular lining.

In Vitro and In Vivo Effects of Adrenaline and Phentolamine on Platelet Function

1978 ◽  
Vol 40 (02) ◽  
pp. 428-438 ◽  
Author(s):  
Oreste Ponari ◽  
Emilio Civardi ◽  
Alessandro Megha ◽  
Mario Pini ◽  
Raffaele Poti’ ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Threshold Concentration ◽  
Two Phase ◽  
Platelet Factor ◽  
Induce Platelet Aggregation ◽  
In Vivo Effects ◽  
In Vivo Administration

Summary In vitro and in vivo effects of adrenaline (ADR) on platelet aggregation, on platelet factor 3 (PF3) availability and on platelet factor 4 (PF4) release were studied in man. Inhibitory action of an alpha-blocker, phentolamine (PHEN) was investigated in the same conditions.The threshold concentration (TC) of ADR inducing the typical two-phase response in aggregation tests when added to platelet-rich plasma (PRP) varied in different pools of plasma, but always induced an evident PF4 release and increased PF3 availability. A further increase in both parameters was obtained with higher concentrations but without any significant dose/response correlation.Adding PHEN alone to PRP did not induce platelet aggregation or modify PF4 release induced by stirring, but it reduced PF3 availability. On the other hand, PHEN prevented the effects of ADR in different platelet tests, at appropriate concentrations.Intravenous infusion of ADR lowered the TC, and increased PF3 availability and PF4 release. In vivo administration of PHEN, in contrast, increased TC and reduced PF3 availability, while PF4 remained unchanged.

Effect of New Synthetic Heparin Mimetics on Whole Blood Thrombin Generation In Vivo and In Vitro in Rats

2002 ◽  
Vol 87 (02) ◽  
pp. 238-244 ◽  
Author(s):  
J.P. Hérault ◽  
A. Bernat ◽  
C. Gaich ◽  
J.M. Herbert
Keyword(s):  
Venous Thrombosis ◽  
Charge Density ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Lag Time ◽  
Drug Candidate ◽  
Platelet Factor

SummaryThe effect of new heparin mimetics (synthetic oligosaccharides) was studied in vitro with regard to thrombin generation (TG) in rat platelet rich plasma (PRP) and whole blood (WB) and in vivo on stasis-induced venous thrombosis in the rat.TG in PRP and in WB was highly dependent on platelet count and strongly influenced by the haematocrit. The peak of TG appeared to be significantly higher in WB than in PRP whereas the endogenous thrombin potential (ETP) was not significantly different under either condition.The effect of hirudin, the synthetic pentasaccharide SR90107/ Org31540 (SP) and heparin were measured on TG in PRP and WB. We then compared the effect of two new synthetic heparin mimetics (SR121903A and SanOrg123781) with potent and comparable antithrombin (AT) mediated activity against factor Xa and thrombin. These two compounds were made of a pentasaccharide with a high affinity to AT, prolonged at the non-reducing end by an oligosaccharide chain recognised by thrombin. In SR121903A, the charge density and charge distribution was analogous to that of heparin whereas in SanOrg123781 the charges were only located on the last 5 saccharides of the non-reducing end of the molecule. In PRP and in WB, SR121903A acted on the lag time and on the AUC whereas SanOrg123781 inhibited thrombin formation with no effect on the lag time. SanOrg123781 was more potent in inhibiting TG than SR121903A. This difference was due to the structures of the compounds that differed in their ability to be neutralised by platelet factor 4. The antithrombotic effect of the two compounds was examined in a venous thrombosis model in rats. We observed that SanOrg123781 was more active than SR121903A and heparin.Taken together, these results indicate that the activity of oligosaccharides is greatly influenced by the global charge density of the molecule and show that SanOrg123781 is a potent and promising antithrombotic drug candidate.

scholarly journals In vivo interaction of von Willebrand factor with platelets following cryoprecipitate transfusion in platelet-type von Willebrand's disease

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 226-230
Author(s):  
JL Miller ◽  
BD Boselli ◽  
JM Kupinski
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Platelet Factor ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Previous studies performed in vitro have indicated that platelets from patients with platelet-type von Willebrand's disease (vWD) have receptors for von Willebrand factor (vWF) already exposed on their surfaces and that the addition of purified vWF or cryoprecipitate to patient platelet-rich plasma under stirring conditions is capable of inducing platelet aggregation and secretion. The present work reports the results of the transfusion of cryoprecipitate in a patient with platelet-type vWD. It is shown that, while factor VIII-related antigen and ristocetin cofactor activities maintain elevated levels for up to 12 hr following transfusion, the highest molecular weight vWF multimers decline rapidly. The platelet count also declines, followed in turn by a rise in the plasma level of platelet factor 4. Shortening of the bleeding time occurs only very transiently. The results of this study provide direct evidence that, in patients with platelet-type vWD, an abnormal interaction of their platelets with plasma vWF occurs in vivo, resulting in the absence of high molecular weight vWF multimers, low platelet counts, and impaired hemostasis that are characteristic of this disease.

Effect of SR121566A, a Potent GP IIb-IIIa Antagonist on Platelet-mediated Thrombin Generation In Vitro and In Vivo

1998 ◽  
Vol 79 (02) ◽  
pp. 383-388 ◽  
Author(s):  
J. P. Herault ◽  
V. Peyrou ◽  
P. Savi ◽  
A. Bernat ◽  
J. M. Herbert
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Venous Thrombosis ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Venous Stasis ◽  
Lag Phase ◽  
Dependent Manner

SummaryThe effect of SR121566A, a new non-peptide GP IIb-IIIa antagonist was studied in vitro with regard to thrombin generation in platelet rich plasma and in vivo on stasis-induced venous thrombosis in the rabbit. SR121566A inhibited ADP-, arachidonic acid- and collagen-induced human platelet aggregation with IC50 values of 46 ± 7.5, 56 ± 6 and 42 ± 3 nM, respectively. In the same experimental conditions, SR121566A strongly inhibited thrombin generation triggered by low concentrations of tissue factor. SR121566A reduced in a dose-dependent manner both the area under the curve and the thrombin peak concentration but did not affect the lag phase (defined as the time until 10 nM thrombin was generated). Aspirin (100 µg/ml) did not affect thrombin generation.One hour after intravenous administration to rabbits, SR121566A exhibited a potent ex vivo inhibitory effect against ADP-, arachidonic acid- and collagen-induced platelet aggregation. The ID50 were 0.6 ± 0.25, 0.7 ± 0.08 and 0.13 ± 0.08 mg/kg, respectively. The ability of aspirin and SR121566A to affect venous stasis was determined in a stasis-induced venous thrombosis model in rabbits under high and low thrombogenic challenges. While aspirin was ineffective in both conditions, SR121566A significantly inhibited thrombus formation under low thrombogenic challenge demonstrating for the first time that a potent non-peptide platelet GP IIb-IIIa antagonist inhibits thrombin generation in vivo and exhibits a strong antithrombotic effect with regard to stasis-induced venous thrombosis. These results therefore confirm the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation but also emphasise the key role of platelets in the development of venous thrombosis, most likely through activation of the GP IIb-IIIa complex.

scholarly journals In vivo interaction of von Willebrand factor with platelets following cryoprecipitate transfusion in platelet-type von Willebrand's disease

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 226-230 ◽  
Author(s):  
JL Miller ◽  
BD Boselli ◽  
JM Kupinski
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Platelet Factor ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Previous studies performed in vitro have indicated that platelets from patients with platelet-type von Willebrand's disease (vWD) have receptors for von Willebrand factor (vWF) already exposed on their surfaces and that the addition of purified vWF or cryoprecipitate to patient platelet-rich plasma under stirring conditions is capable of inducing platelet aggregation and secretion. The present work reports the results of the transfusion of cryoprecipitate in a patient with platelet-type vWD. It is shown that, while factor VIII-related antigen and ristocetin cofactor activities maintain elevated levels for up to 12 hr following transfusion, the highest molecular weight vWF multimers decline rapidly. The platelet count also declines, followed in turn by a rise in the plasma level of platelet factor 4. Shortening of the bleeding time occurs only very transiently. The results of this study provide direct evidence that, in patients with platelet-type vWD, an abnormal interaction of their platelets with plasma vWF occurs in vivo, resulting in the absence of high molecular weight vWF multimers, low platelet counts, and impaired hemostasis that are characteristic of this disease.

scholarly journals Metabolism of Thrombopoietin (TPO) In Vivo: Determination of the Binding Dynamics for TPO in Mice

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 4063-4070 ◽  
Author(s):  
Eric Stefanich ◽  
Tauri Senn ◽  
Ramon Widmer ◽  
Christine Fratino ◽  
Gilbert-André Keller ◽  
...  
Keyword(s):  
Blood Cell ◽  
Platelet Rich Plasma ◽  
In Vivo Studies ◽  
In Vivo Metabolism ◽  
In Vivo Binding ◽  
Curve Fit ◽  
Degradation Pattern

AbstractPrevious in vivo studies have established that plasma thrombopoietin (TPO) levels are regulated by binding to c-Mpl on platelets and that, in vitro, platelets bind and degrade TPO. To determine if the in vivo metabolism of TPO was specific and saturable, we injected normal CD-1 mice IV with trace amounts of 125I-rmTPO with or without a saturating concentration of rmTPO. The amount of radioactivity present in the spleen, blood cell fraction, platelet fraction, tibia/fibula, and femur was significantly greater in the mice receiving 125I-rmTPO alone. Conversely, the amount of radioactivity present in the plasma was significantly greater in the mice receiving both 125I-rmTPO and rmTPO, thus suggesting the uptake of rmTPO by the spleen, platelets, and bone marrow in vivo was saturable. Platelet and spleen homogenates from animals receiving 125I-rmTPO alone showed a degradation pattern of 125I-rmTPO similar to that observed in vitro using mouse platelet rich plasma. To determine the in vivo binding dynamics for rmTPO, mice were injected with 125I-rmTPO alone or with increasing concentrations of rmTPO; spleen and blood cell-associated radioactivity was determined at 2 hours postinjection. A 4-parameter curve fit of the data indicated that the “in vivo binding affinity” for rmTPO was approximately 6.4 μg/kg. These data indicate that after a dose of approximately 6.4 μg/kg, 50% of all c-Mpl receptors will be saturated with rmTPO. Electron microscopy indicated that radioactivity was present bound to and within megakaryocytes and platelets in both sternum and spleen and platelets in circulation. Together these data demonstrate that in vivo, 125I-rmTPO is mainly metabolized by platelets and to a small extent by cells of the megakaryocyte lineage, via a specific and saturable mechanism.

Platelet Reactivity In Vitro in Relation to Thromboxane in Healthy Pregnancy

1996 ◽  
Vol 75 (02) ◽  
pp. 346-351 ◽  
Author(s):  
E H Horn ◽  
E Hardy ◽  
J Cooper ◽  
S Heptinstall ◽  
P C Rubin
Keyword(s):  
Arachidonic Acid ◽  
Pregnant Women ◽  
Biosynthetic Pathway ◽  
Platelet Reactivity ◽  
Platelet Rich Plasma ◽  
High Concentration ◽  
Increased Sensitivity ◽  
The Difference

SummaryThere is substantial evidence of increased platelet reactivity in vivo and in vitro during pregnancy. Previous in vitro studies suggest that platelets from pregnant women show increased sensitivity to agonists, the response to which has a thromboxane dependent component. The aim of this study was to determine whether this is due to increased activity of the thromboxane biosynthetic pathway or to increased platelet sensitivity to the effects of thromboxane. During pregnancy, platelets were more sensitive to the pro-aggregatory effects in vitro of the thromboxane mimetic U46619, in whole blood and in platelet rich plasma, compared to those from non-pregnant controls. The difference in extent of U46619-induced platelet aggregation between groups was abolished in the presence of a high concentration of the specific thromboxane antagonist ICI 192605, but not by prior incubation of blood with aspirin. Platelets from pregnant women were significantly less sensitive to inhibition of arachidonic acid induced activation by the thromboxane synthetase inhibitor dazmegrel, but there was no change in platelet cyclic AMP accumulation under these conditions. Arachidonic acid induced platelet thromboxane B2 production was similar in pregnant and non-pregnant subjects. In conclusion, platelets are more sensitive to the activating effects of thromboxane during pregnancy, but there is no change in the intrinsic reactivity of the thromboxane biosynthetic pathway.

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