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

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 ◽  
Platelet Aggregates

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heparin (Liquemin ), was aggregated with 10 or 100 pM ADP for 2 min at 37°C. Single PAs of various dimensions were withdrawn, washed, and incubated with a chromogenic substrate (S-2238, Kabi AB) to measure their thrombin content.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.

scholarly journals Automated Analysis of Platelet Aggregation on Cultured Endothelium in a Microfluidic Chip Perfused with Human Whole Blood

Micromachines ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 781 ◽  
Author(s):  
Albers ◽  
Passier ◽  
van den Berg ◽  
van der Meer
Keyword(s):  
Image Analysis ◽  
Whole Blood ◽  
Microfluidic Channel ◽  
Aggregate Size ◽  
Blood Perfusion ◽  
Automated Analysis ◽  
Microfluidic Channels ◽  
Size Distributions ◽  
Platelet Aggregate ◽  
Platelet Aggregates

Organ-on-a-chip models with incorporated vasculature are becoming more popular to study platelet biology. A large variety of image analysis techniques are currently used to determine platelet coverage, ranging from manually setting thresholds to scoring platelet aggregates. In this communication, an automated methodology is introduced, which corrects misalignment of a microfluidic channel, automatically defines regions of interest and utilizes a triangle threshold to determine platelet coverages and platelet aggregate size distributions. A comparison between the automated methodology and manual identification of platelet aggregates shows a high accuracy of the triangle methodology. Furthermore, the image analysis methodology can determine platelet coverages and platelet size distributions in microfluidic channels lined with either untreated or activated endothelium used for whole blood perfusion, proving the robustness of the method.

In Vivo Effect of Suloctidil as an Antiplatelet Agent

1979 ◽  
Vol 41 (03) ◽  
pp. 465-474 ◽  
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.

Increased Plasma Fibrinogen and Platelet-Aggregates in Type II Hyperlipoproteinaemia

1979 ◽  
Vol 42 (05) ◽  
pp. 1503-1507 ◽  
Author(s):  
G D O Lowe ◽  
Maureen M Drummond ◽  
Jane L H C Third ◽  
W F Bremner ◽  
C D Forbes ◽  
...  
Keyword(s):  
Plasma Lipids ◽  
Young Patients ◽  
Adult Life ◽  
Arterial Disease ◽  
Arterial Injury ◽  
Plasma Fibrinogen ◽  
Type Ii ◽  
Familial Type ◽  
Platelet Aggregate ◽  
Platelet Aggregates

SummaryPlasma fibrinogen and platelet-aggregates (method of Wu and Hoak) were measured in 21 patients with familial Type II hyperlipoproteinaemia and 21 matched control subjects. Patients with hyperlipoproteinaemia had increased levels of fibrinogen and platelet- aggregates (p<0.01). Young patients with hyperlipoproteinaemia had prematurely high fibrinogen levels, and the normal rise in fibrinogen during adult life was abolished. There were no statistically significant correlations within the patient group between fibrinogen, platelet-aggregates, and plasma lipids. High fibrinogen 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.

Determination of Platelet Aggregate Size in vitro Using Digital Microscopy

2013 ◽  
Vol 47 (3) ◽  
pp. 121-125 ◽  
Author(s):  
V. A. Doubrovski ◽  
I. V. Zabenkov ◽  
S. O. Torbin ◽  
V. I. Eremin ◽  
O. E. Tsareva
Keyword(s):  
Aggregate Size ◽  
Digital Microscopy ◽  
Platelet Aggregate

Increased Plasma Fibrinogen and Platelet-Aggregates in Type II Hyperlipoproteinaemia

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 ◽  
Circulating Platelet Aggregates ◽  
Platelet Aggregates

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.

Regulation of High Shear Rate Induced von Willebrand Factor Mediated Platelet Thrombi by ADAMTS-13 .

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 516-516
Author(s):  
Roberta Donadelli ◽  
Jennifer N. Orje ◽  
Miriam Galbusera ◽  
Giuseppe Remuzzi ◽  
Zaverio M. Ruggeri
Keyword(s):  
Shear Stress ◽  
Blood Cells ◽  
Metal Ion ◽  
Aggregate Size ◽  
Shear Rates ◽  
Platelet Aggregate ◽  
Platelet Aggregates ◽  
Platelet Thrombi ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS-13 is a metalloproteinase that cleaves von Willebrand factor (VWF) at the peptide bond Tyr842-Met843 within the A2 domain of the mature subunit, thus contributing to the regulation of multimers size in the circulation. Cleavage is effective on newly released VWF bound to the surface of endothelial cells, but the extent to which the protease acts on circulating VWF or limits the adhesive properties of multimers during thrombus formation remains unclear. To begin to address these questions, we have established a real-time videomicroscopy technique to visualize the formation of platelet aggregates mediated by the binding of VWF A1 domain to the platelet membrane glycoprotein (GP) Ibα. In this process, single platelets first adhere to surface-immobilized VWF, and then bind soluble VWF from plasma and aggregate through a mechanism dependent on shear rate above a critical threshold. Platelet aggregates formed promptly when whole human blood containing the thrombin inhibitor D-phenyl alanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride as an anticoagulant (80 μM) was perfused over immobilized VWF at wall shear rates above 10,000 s−1. The size of aggregates increased during the first 5 min of perfusion, then started to decrease and was less than 30% of maximum after 10 min of perfusion. When the metal ion chelator, EDTA (5mM), was added to the blood before perfusion, the size of aggregates was larger than in control blood and showed no decrease over a 10 min period. A similar result was obtained when plasma was removed and washed blood cells were suspended in a buffer containing 20 μg/ml purified VWF multimers, suggesting that a metal ion dependent plasma protease was responsible for the time-dependent reduction of platelet aggregate size. To evaluate this hypothesis, recombinant human ADAMTS-13 purified from the culture medium of stably transfected D. melanogaster cells was added to washed blood cells resuspended in buffer/VWF, and the suspension was perfused over immobilized VWF at shear rates above 10,000 s−1. In this case, VWF-mediated thrombi started to form but began to dissipate within 3 min. After 6 min, the size of thrombi was 30% or less of that seen in the absence of ADAMTS-13. When the latter was added to washed blood cells resuspended in buffer/VWF after 5 min of perfusion, when platelet aggregates had reached their maximum dimensions, a reduction in size to 30% or less of maximum occurred within 5 min. Pre-incubation of ADAMTS-13 with the blood cell/VWF suspension before perfusion did not accelerate or enhance the reduction of platelet aggregate size, indicating that the enzyme likely acts only under flow conditions and/or after VWF multimers are bound to platelet GP Ibα and exposed to shear stress. ADAMTS-13 also had no significant effect on the size of platelet thrombi formed onto collagen type I fibrils at shear rates as high as 6,000 s−1. These findings suggest that ADAMTS-13 provides a selective mechanism to regulate the size of platelet thrombi, but the effect may be limited to conditions under which the cohesion between platelets depends mainly on VWF binding induced by pathologically elevated shear stress.

Influence of Fibrinogen Split Products on Platelets

1967 ◽  
Vol 17 (01/02) ◽  
pp. 078-098 ◽  
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 ◽  
Platelet Aggregates

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.

Investigating Platelet Interactions in Sickle Cell Disease Using a Novel Multi-Shear "Endothelialized" Microfluidic System

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4155-4155
Author(s):  
Margo Renee Rollins ◽  
Byungwook Ahn ◽  
Yumiko Sakurai ◽  
Jordan C Ciciliano ◽  
Wilbur A Lam
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell ◽  
Whole Blood ◽  
Blood Cells ◽  
Platelet Adhesion ◽  
Blood Samples ◽  
Shear Rates ◽  
Healthy Control ◽  
Platelet Aggregates

Abstract Sickle Cell Disease (SCD) is an inherited disorder of the β-globin chain of hemoglobin, in which a single point mutation leads to decreased deformability of red blood cells (RBCs) and increased cellular adhesion to endothelium. The effect of this mutation on RBCs has been well characterized, and the interplay of endothelial cells, RBCs, and white blood cells (WBCs) have also been well characterized. However, few studies have specifically investigated how platelets interact with endothelial cells and other blood cells in the context of SCD and the role these cell fragments may have in vaso-occlusion. To that end, we utilized microfluidic technology previously developed in our lab to perform a “real time” in vitro analyses of platelet-endothelial cell interactions in SCD patient samples. This “microvasculature-on-a-chip” enables the visualization of blood cell-endothelial cell interactions under a controlled hemodynamic environment (Tsai et al, JCI, 2012). As shear stress can trigger platelet activation, we further modified and optimized our standard microfluidic devices to encompass 3 different physiologic shear rates. Our device features microchannels 50µm in diameter with human umbilical vein endothelial cells (HUVEC) confluently lining the channels; there are 12 channels in each device, grouped in 3 sets of 4 channels with graduating shear rates spanning 3 orders of magnitude (Figure 1). Our initial experiments were performed under normoxic conditions allowing characterization of platelet-endothelial interactions in an “arterial” in vitro environment. Whole blood samples were obtained from 3 patient populations: patients with HgbSS SCD on hydroxyurea (HgbSS+HU), patients with HgbSS SCD not on hydroxyurea (HgbSS-no HU), and normal healthy controls. Over 30 minutes, whole blood stained with fluorescently labeled CD41 to identify platelets and Hoeschst to identify HUVEC nuclei was perfused at a rate of 1.5µl/minute under videomicroscopy. Accumulation of platelets on the endothelialized channels and platelet aggregates were quantified based on anti-CD41 fluorescence. Within 1 minute of perfusion, HgbSS-no HU whole blood samples exhibited extensive platelet aggregates at 1 and 10 dyne/cm2 (Figure 2); this phenomenon did not occur under any of the shear conditions in blood samples from Hgb SS+HU or healthy control samples. In HgbSS-no HU blood samples, some of these “thrombi”-like aggregates were stable under flow, increased in size, and persisted for the remainder of the 30 minute experiments. In contrast, mild, uniform, platelet adhesion slowly developed at high shear conditions in Hgb SS+HU with fewer platelet aggregates forming as compared to patients with HgbSS- no HU. Healthy control samples did not exhibit this platelet aggregation. There appears to be an attenuating effect of hydroxyurea on platelets that prevents platelet clumping from occuring as frequently under various shear conditions that is not present in the Hgb SS-no HU samples (Figure 3). In conclusion, using our novel in vitro system, we have demonstrated the platelets from Hgb SS-no HU patients have a significantly increased propensity to adhere, aggregate, and accumulate in endothelialized microvasculature-sized microchannels. Interestingly, this effect appears to be attenuated in blood samples from Hgb SS+HU patients and not present in healthy controls, demonstrating that hydroxyurea appears to be an important modifier of this phenomenon. Experiments investigating the underlying mechanisms of this phenomenon, the effects of deoxygenation and the potential role of platelets in vaso-occlusion, the effects of sickle cell platelet adhesion/aggregation on endothelial function, and how hydroxyurea may or may not affect any or all of these parameters, are all currently ongoing. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Intercellular calcium communication regulates platelet aggregation and thrombus growth

2003 ◽  
Vol 160 (7) ◽  
pp. 1151-1161 ◽  
Author(s):  
Warwick S. Nesbitt ◽  
Simon Giuliano ◽  
Suhasini Kulkarni ◽  
Sacha M. Dopheide ◽  
Ian S. Harper ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Purinergic Receptor ◽  
Cytosolic Calcium ◽  
Calcium Flux ◽  
Aggregate Formation ◽  
Signaling Mechanism ◽  
Activated Platelets ◽  
Platelet Aggregate ◽  
Platelet Aggregates

The ability of platelets to form stable adhesion contacts with other activated platelets (platelet cohesion or aggregation) at sites of vascular injury is essential for hemostasis and thrombosis. In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet–platelet adhesion contacts under the influence of flow. An examination of platelet calcium flux during platelet aggregate formation in vitro demonstrated a key role for intercellular calcium communication (ICC) in regulating the recruitment of translocating platelets into developing aggregates. We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin αIIbβ3 and the recently cloned ADP purinergic receptor P2Y12. Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth.

scholarly journals Antiaggregatory activity of new 1-ethylxanthine cyclohexylammonium salt in vitro

2013 ◽  
Vol 94 (5) ◽  
pp. 692-695
Author(s):  
F Kh Kamilov ◽  
G A Timirkhanova ◽  
A I Samorodova ◽  
A V Samorodov ◽  
F A Khaliullin ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Platelet Aggregation ◽  
Aggregate Size ◽  
Adenosine Diphosphate ◽  
Biochemical Effect ◽  
Platelet Aggregate ◽  
Antiaggregatory Activity ◽  
The Impact ◽  
Induced Aggregation

Aim. To study the biochemical effect on hemostasis of a new cyclohexilammonium salt of 2-[1-ethyl-3-methyl-7(dioxotiethanyl-3)xantinyl-8-thio]acetic acid in vitro. Methods. Thromboelastography was performed using the citrate blood samples of healthy male donors. Global hemostatic effect, fibrinogen and platelet function, fibrinolysis and clot strength, and stability were analyzed at thromboelastography. The impact of firstly synthesized xantine derivative and pentoxifylline on the functional activity of platelets in vitro was studied using a laser analyzer of platelet aggregation. Adenosine diphosphate, collagen, epinephrine and ristocetin induced clotting were registered. General clotting characteristics, maximal aggregation values, maximal aggregation speed, mean platelet aggregate size, activity of platelet-derived factor 3, level of platelet-derived factor 4 were measured. Release of platelet-derived factors 3 and 4 at platelet aggregation were assessed after adenosinediphosphate-induced aggregation and centrifugation. Results. Cyclohexylammonium salt of 2-[1-ethyl-3-methyl-7-(dioxotiethanyl-3)xantinyl-8-thio]acetic acid in vitro showed antiaggregatory activity that exceeds such of pentoxifylline. It has been revealed that the second platelet aggregation wave, that is induced by small dose of adenosinediphosphate, is absent in the presence of the new cyclohexylammonium salt, lag-period in collagen-induced platelet aggregation elongates, and availability and release of platelet-derived factors 3 and 4 decreases. Conclusion. The research findings show potentially high antiaggregatory activity of 2-[1-ethyl-3-methyl-7-(dioxotiethanyl-3)xantinyl-8-thio]acetic acid cyclohexylammonium salt as an inhibitor of platelet release reaction.

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