Prevention by Suloctidil of Spontaneous Platelet Aggregates in Rats

Platelet Aggregates ◽

5 Ht Uptake ◽

Rat Platelets

Atherosclerosis and cerebrovascular disease have been reported by Wexler and True (Circ. Res. 12: 659,1963) to-occur in retired breeder rats but not in virgin rats of the same age. We have discovered that retired breeder (RB) male rats have spontaneous circulating platelet aggregates (CPA) as determined by the method of Wu and Hoak (Lancet 2: 924, 1974). These CPA respond to therapy with clinically proven antiplatelet drugs. Suloctidil (S) [l-(4-isopropyl-thiophenyl)-2-n-octylamino propanol, Continential Pharma, Brussels] administered for 8 days at 100 mg/kg i. g. showed a significant reduction in CPA from 0.76+0.05 (S.E.M.) to 0.92 + 0.03 (where 1.0 indicates no CPA). In these same rats, the endogenous platelet 5-HT level was significantly (P<0.01) lowered to 0.88+0.04 (S. E.M.) μg/10 9 platelets compared to control values of 1.66+0.08 μg/10 9platelets. Plasma 5-HT levels were not altered. In vitro rat platelet 5-HT uptake is inhibited 50% by S at 6.4 χ 10 -6M in platelet-rich plasma. In rat platelets with labeled 5-HT pools, 5 min incubation with S at 10 -5M resulted in significantly (P<0.01) greater release at 10, 20 and 30 sec after the addition of 2U/ml of thrombin. This suggests that S may decrease uptake as well as increase release of platelet 5-HT. The reduction of endogenous platelet 5-HT levels by S may be related to its observed platelet aggregate prevention activity.

In Vivo Effect of Suloctidil as an Antiplatelet Agent

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646799 ◽

1979 ◽

Vol 41 (03) ◽

pp. 465-474 ◽

Cited By ~ 7

Author(s):

Marcia R Stelzer ◽

Thomas S Burns ◽

Robert N Saunders

Keyword(s):

Ex Vivo ◽

Antiplatelet Agent ◽

Ratio Method ◽

Platelet Aggregate ◽

Permanent Effect ◽

Platelet Aggregates ◽

5 Ht Uptake ◽

High Doses

SummaryThe relationship between the effects of suloctidil in vivo as an antiplatelet agent and in vitro as a modifier of platelet serotonin (5-HT) parameters was investigated. Suloctidil was found to be effective in reducing platelet aggregates formation in the retired breeder rat as determined using the platelet aggregate ratio method (PAR) with an ED50 of 16.1 mg/kg 24 hours post administration. In contrast to the hypothesis that 5-HT depletion is involved in the anti-aggregatory mechanism of suloctidil, no correlation was found between platelet 5- HT content and this antiplatelet activity. Reduction of platelet 5-HT content required multiple injections of high doses (100 mg/kg/day) of suloctidil. Suloctidil administration for 8 days at 100 mg/kg/day, which lowered platelet 5-HT content by 50%, resulted in no permanent effect on ex vivo platelet 5-HT uptake or thrombin-induced release, nor alteration in the plasma 5-HT level. However, these platelets exhibited a short-lived, significant increase in percent leakage of 5-HT after 30 minutes of incubation. Therefore, suloctidil treatment at high doses may with time result in platelet 5-HT depletion, however this effect is probably not related to the primary anti-aggregatory activity of the drug.

Do patients with thromboembolic disease have circulating platelet aggregates?

Blood ◽

10.1182/blood.v64.1.205.bloodjournal641205 ◽

1984 ◽

Vol 64 (1) ◽

pp. 205-209 ◽

Cited By ~ 1

Author(s):

FH Kohanna ◽

MH Smith ◽

EW Salzman

Keyword(s):

Platelet Rich Plasma ◽

Venous Blood ◽

Adenosine Diphosphate ◽

Normal Subjects ◽

Thromboembolic Disease ◽

Platelet Aggregates ◽

Lower Ratio

Reports of circulating platelet aggregates (ie, microemboli) in thromboembolism and other vascular disorders are based on a method (Wu and Hoak , 1974) in which venous blood is collected via scalp vein needle and tubing into either formaldehyde, which fixes aggregates, or EDTA, which disperses them. The ratio of platelet counts in platelet- rich plasma (PRP) from the two blood samples after centrifugation is interpreted as a measure of platelet aggregates in the circulation in vivo. We compared this standard Wu and Hoak technique with a modified one, in which blood was drawn directly into a syringe, and with a third method that avoided centrifugation by counting single platelets in whole blood. Both modified techniques could detect aggregates generated in vitro with adenosine diphosphate (ADP). In 12 normal subjects, the three methods were equivalent, but in 37 patients with thromboembolic disorders, the standard Wu and Hoak method gave a lower ratio than the other methods. Similar results were found in a subset of eight patients with myocardial infarction. Heparin treatment of patients did not influence the results. The data suggest that formation of platelet aggregates occurred during venipuncture. Platelets may be hyperactive in patients with thromboembolic disease and may form aggregates in vitro during collection, but the concept of chronic microembolism in such patients should be reassessed.

Do patients with thromboembolic disease have circulating platelet aggregates?

Blood ◽

10.1182/blood.v64.1.205.205 ◽

1984 ◽

Vol 64 (1) ◽

pp. 205-209 ◽

Cited By ~ 15

Author(s):

FH Kohanna ◽

MH Smith ◽

EW Salzman

Keyword(s):

Platelet Rich Plasma ◽

Venous Blood ◽

Adenosine Diphosphate ◽

Normal Subjects ◽

Thromboembolic Disease ◽

Platelet Aggregates ◽

Lower Ratio

Abstract Reports of circulating platelet aggregates (ie, microemboli) in thromboembolism and other vascular disorders are based on a method (Wu and Hoak , 1974) in which venous blood is collected via scalp vein needle and tubing into either formaldehyde, which fixes aggregates, or EDTA, which disperses them. The ratio of platelet counts in platelet- rich plasma (PRP) from the two blood samples after centrifugation is interpreted as a measure of platelet aggregates in the circulation in vivo. We compared this standard Wu and Hoak technique with a modified one, in which blood was drawn directly into a syringe, and with a third method that avoided centrifugation by counting single platelets in whole blood. Both modified techniques could detect aggregates generated in vitro with adenosine diphosphate (ADP). In 12 normal subjects, the three methods were equivalent, but in 37 patients with thromboembolic disorders, the standard Wu and Hoak method gave a lower ratio than the other methods. Similar results were found in a subset of eight patients with myocardial infarction. Heparin treatment of patients did not influence the results. The data suggest that formation of platelet aggregates occurred during venipuncture. Platelets may be hyperactive in patients with thromboembolic disease and may form aggregates in vitro during collection, but the concept of chronic microembolism in such patients should be reassessed.

A Modification of the Wu and Hoak Method for the Determination of Platelet Aggregates and Platelet Adhesion

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661319 ◽

1985 ◽

Vol 53 (03) ◽

pp. 381-385 ◽

Cited By ~ 15

Author(s):

Sudhir K Bowry ◽

Colin R M Prentice ◽

J M Courtney

Keyword(s):

Platelet Adhesion ◽

Platelet Rich Plasma ◽

Blood Collection ◽

Buffer System ◽

Modified Method ◽

Healthy Donors ◽

Platelet Aggregate ◽

SummaryThe Wu and Hoak method for determining circulating platelet aggregates has poor reproducibility; problems have been reported with the composition of the buffer systems, haemolysis, the effects of blood collection technique and a divergence of the platelet aggregate ratio in blood for healthy donors from the theoretical value of 1. Our investigations suggest that the original technique is highly operator-dependent, especially the collection of blood and the method of counting platelets after centrifugation. We describe an improved modification of the Wu and Hoak technique; a new buffer system has been developed and the proportion of blood in the buffered EDTA and buffered EDTA- formalin solutions has been altered to obtain platelet rich plasma. The platelet aggregate ratio (PAR) by this modified method for healthy donors in two different studies was 0.97 ± 0.02 and 0.98 ± 0.01 respectively. Finally, the principle of Wu and Hoak was used to measure accurately platelet adhesion, without the role of platelet-platelet interactions (aggregation). Platelet adhesion and aggregation were then used to evaluate the thrombogenicity of various artificial surfaces, including silicone rubber and polytetra- fluoroethylene (PTFE) vascular grafts.

Haemodynamic flow conditions at the initiation of high-shear platelet aggregation: a combined in vitro and cellular in silico study

Interface Focus ◽

10.1098/rsfs.2019.0126 ◽

2020 ◽

Vol 11 (1) ◽

pp. 20190126 ◽

Cited By ~ 1

Author(s):

B. J. M. van Rooij ◽

G. Závodszky ◽

A. G. Hoekstra ◽

D. N. Ku

Keyword(s):

Platelet Aggregation ◽

In Silico ◽

Platelet Rich Plasma ◽

High Shear ◽

Shear Rates ◽

Flow Simulations ◽

In Silico Study ◽

The influence of the flow environment on platelet aggregation is not fully understood in high-shear thrombosis. The objective of this study is to investigate the role of a high shear rate in initial platelet aggregation. The haemodynamic conditions in a microfluidic device are studied using cell-based blood flow simulations. The results are compared with in vitro platelet aggregation experiments performed with porcine whole blood (WB) and platelet-rich-plasma (PRP). We studied whether the cell-depleted layer in combination with high shear and high platelet flux can account for the distribution of platelet aggregates. High platelet fluxes at the wall were found in silico . In WB, the platelet flux was about twice as high as in PRP. Additionally, initial platelet aggregation and occlusion were observed in vitro in the stenotic region. In PRP, the position of the occlusive thrombus was located more downstream than in WB. Furthermore, the shear rates and stresses in cell-based and continuum simulations were studied. We found that a continuum simulation is a good approximation for PRP. For WB, it cannot predict the correct values near the wall.

The Induction of Platelet Aggregates in Healthy Subjects by Venousocclusion

10.1055/s-0039-1682681 ◽

1977 ◽

Author(s):

D.A. F. Chamone ◽

J. Vermylen

Keyword(s):

Healthy Subjects ◽

Light Transmission ◽

Diastolic Pressure ◽

Platelet Rich Plasma ◽

In Vivo Evaluation ◽

Modified Method ◽

Platelet Aggregates ◽

Set Up

Circulating platelet aggregates have been observed in various clinical conditions (Wu and Hoak, Lancet, 1974, ii, 924). Using a slightly modified method, we have found that platelet aggregates can be induced in vivo in healthy subjects.Nine volunteers (7 males, 2 females, age 23-38 years) were studied. Blood was drawn from an antecubital vein of one arm immediately before and of the other arm after twenty minutes of occlusion midway between systolic and diastolic pressure. The ratio of the platelet count in platelet-rich plasma (PRP) obtained from blood collected on forma lin-EDTA to that from blood collected on EDTA only was 0.934 + 0.028 (mean ± S.E .) before and 0.768 ± 0.033 after occlusion (p < 0.001 ). Spontaneous aggregation in PRP, measured as percent increase in light transmission during 10 minutes of stirring in the a gg re gome ter, was 4 .20 ± 1.17 before and 3 .80 + I .69 after occlusion (p > 0 .1).This system may help elucidate some of the mechanisms involved in the generation of circulating platelet aggregates. It may also constitute a simple set-up for the in vivo evaluation of drugs affecting platelet function.

PLATELET AGGREGATE-ASSOCIATED FIBRIN FORMATION AS A FUNCTION OF AGGREGATE SIZE AND HEPARIN CONCENTRATION

10.1055/s-0038-1644857 ◽

1987 ◽

Author(s):

L J Wurzinger ◽

K Herbst ◽

H Schmid-Schonbein

Keyword(s):

Whole Blood ◽

Aggregate Size ◽

Arterial Disease ◽

Amidolytic Activity ◽

Protein Assay ◽

Heparin Concentration ◽

Platelet Aggregate ◽

Minimum Number ◽

The in vitro observation that fibrin forms withina few minutes in the crevices and niches inside and on the surface of platelet aggregates (PA), preparedfrom heparinized (5 U/ml) blood is consistent with the doubtful efficiency of heparin in the treatment of occlusive arterial disease (Thrcmb. Haemost. 46: 666,1981). Release of heparin- neutralizing proteins into limited and largely disclosed plasma compartments between aggregated platelets was held responsible for this remarkable phenomenon. However, the minimum number of aggregated platelets necessary to overcome the heparin inhibition remained undetermined then.PRP prepared from whole blood ant^coagulated with 0.5, 1 and 5 U/ml of mucosal heparin (Liquemin ), was aggregated with 10 or 100 pM ADP for 2 min at 37°C. Single PAs of various dimensions were withdrawn, washed, and incubated with a chromogenic substrate (S-2238, Kabi AB) to measure their thrombin content. Subsequently the number of platelets contained in the PA was evaluated by assaying the protein content of the aggregates. Microscopic PAs, their mass being toosmall to be determined precisely by a protein assay, were isolated with a filter technique, their extension was documented on photomicrographs for later calculation of aggregate volume and platelet content, before they were incubated with S-2238. Aggregates toosmall to develop detectable amidolytic activity, were checked microscopically for fibrin formed.S 2238 amidolytic activity (thrombin) in heparinizedPRP samples evolved as a linear function of the logarithm of PA mass. For a given heparin concentration (in whole blood) the following lowerthreshold platelet numbers of aggregates were found sufficient to allow the formation of detectable quantities of thrombin:These results suggest a fatal role platelet aggregates of minute dimensions may well play as a nidus of coagulation in fully heparinized blood.

Increased Plasma Fibrinogen and Platelet-Aggregates in Type II Hyperlipoproteinaemia

10.1055/s-0039-1687455 ◽

1979 ◽

Author(s):

J.L.H.C. Third ◽

G.D.O. Lowe ◽

M.M. Drummond ◽

W.F. Bremner ◽

T.D.V. Lawrie ◽

...

Keyword(s):

Plasma Lipids ◽

Young Patients ◽

Adult Life ◽

Arterial Disease ◽

Arterial Injury ◽

Plasma Fibrinogen ◽

Type Ii ◽

Platelet Aggregate ◽

Plasma-fibrinogen and circulating platelet-aggregates (method of Wu and Hoak1) were measured in 21 patients with Type II hyperlipoproteinaeima and 21 matched control subjects. Patients with hyperlipoproteinaemia had increased levels of fibrinogen (3.5 g/l ± SEM 0.2 vs. 2.5 g/l±0.1, p(0.01) and platelet-aggregates (platelet aggregate ratio 0.71 vs. 0.65, p(0.01). Young patients with hyperlipoproteinaemia had prematurely high fibrinogen levels, and the normal fibrinogen rise durina adult life was abolished. There were no significant correlations between fibrinogen, platelet-aggregates, and plasma lipids (cholesterol, cholesterol fractions, or triglyceride). High librinogen and platelet-aggregate levels may play a part in the development of the premature arterial disease associated with Type II hyperlipoproteinaemia, or may be markers of arterial injury. 1Wu, K.K., Hoak, J.C.Lancet, 1974, ii, 924.

In Vitro Inhibition of Platelet Aggregatton by the Gelatin Plasma Expander Haemaccel.(R)

10.1055/s-0039-1687291 ◽

1979 ◽

Author(s):

I. Stibbie ◽

P.M. van der Plas ◽

G.L. Ong ◽

D.S. de Jong ◽

E. Krenning-Douma ◽

...

Keyword(s):

Heart Surgery ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Open Heart Surgery ◽

Open Heart ◽

Specific Mechanism ◽

Plasma Expander ◽

Platelet Aggregates ◽

In Vitro Inhibition

In a study concerning open-heart surgery we found that, platelet aggregates present in heparinised human blood disappeared immediately after addition of the gelatin plasma expander haemaccel. A study was therefore initiated of the effect of haemaccei on platelet appregation (Payton apprepometer, 4W RPM as controlled by stroboscope, 37°C, final platelet count 200-300 χ 109/1) and compared with the effect of bovine serum albumin (USA) and platelet poor plasma (PPP). Haemaccel powder was kindly supplied by Rehringwerke and contained 0.98% sodium, 0.015% calcium and no measurable potassium. 0.3 ml human platelet rich plasma (PRP) was nixed with 0.2 ml haemaccel (final concentrations 0-20 mg/ml) in Tyrode’s solution (2 mM Ca++. pH 7,4), Haemaccel inhibited apgrepation in both citrated and heparinised PRP induced by collagen, ADP or adrenalin, both in the presence or absence of indomethacin (90/μM), PPP (0.2 ml) and BSA (in Tyrode’s solution, final concentrations 0-16 mp/ml) were also inhihitinp, hut on a weipht hasis less than haemaccel. Different Ca++ concentrations in the Tyrode’s solution did not alter the inhibition by haemaccel. Final pH in aggregation mixtures varied by less than 0.10 for a given experiment. It is concluded that, under the conditions used, haemaccel and USA inhibit platelet appregation, probably by a non-specific mechanism.

Influence of Fibrinogen Split Products on Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654084 ◽

1967 ◽

Vol 17 (01/02) ◽

pp. 078-098 ◽

Cited By ~ 13

Author(s):

M. I Barnhart ◽

D. C Cress ◽

R. L Henry ◽

J. M Riddle

Keyword(s):

Platelet Aggregation ◽

Plasma Concentration ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Platelet Response ◽

Transient Nature ◽

Platelet Morphology ◽

SummaryBreakdown products of fibrinogen and fibrin can play a role in hemostasis and also may be of consequence in thrombosis. β2 fibrinogen derivative D is an electropositive terminal proteolysis product of fibrinolysis which has the ability to aggregate platelets. The normal plasma concentration of such nonclottable fibrinogen relatives is 0.2 mg/ml. During fibrinolysis this concentration may reach 5 mg/ml plasma. Addition of β 2 fibrinogen D (raising the plasma concentration 0.1 to 5 mg/ml) either in vivo or in vitro induced platelet aggregations. Moreover, alterations in platelet morphology occurred which were obvious by electron microscopy.Platelet depletion was a consistent response to the infusion of purified β2 fibrinogen D (8 to 55 mg/kg body weight) into dogs. Circulating platelets decreased as much as 85% but were only temporarily aggregated and reappeared in the circulation within 1 to 5 hrs. Small platelet aggregates circulated while large aggregates were trapped in the microcirculation. Thrombin was not responsible for the platelet aggregations as neither prothrombin nor clottable fibrinogen were changed significantly. The transient nature and morphological features of the platelet response according to microscopic criteria were prominent during and after infusion of β2 fibrinogen D.In vitro studies included 3 systems; washed platelets, platelet rich plasma and whole blood. Positive results were obtained with all, but platelets in whole blood were most responsive. The magnitude of platelet aggregation and morphology correlated with the concentration of β2 fibrinogen D. Platelet aggregation induced by ADP (10~5 mg/ml) was compared with that induced by β2 fibrinogen D (0.09 to 0.72 mg/ml). With either reagent aggregates were of dendritic forms. Combination of the 2 reagents was additive but did not further change the morphology. Additional factors seem necessary for development of viscous metamorphosis.