Mitochondrial ATP generation in stimulated platelets is essential for granule secretion but dispensable for aggregation and procoagulant activity

Abstract Abstract 2023 Platelets activated by physiological agonists such as thrombin and collagen shed procoagulant microparticles (MPs) and externalize the procoagulant phospholipid phosphatidylserine (PS), both of which are critical to hemostasis and play an important role in inflammation. To date, the signaling mechanisms that regulate agonist-induced MP formation and PS exposure in platelets remain unclear. In this study, we demonstrate that the small GTPases Rac1 and RhoA play important roles in regulating the procoagulant activity of platelets. Rac1 null (-/-) mouse platelets or human platelets treated with the Rac1 inhibitor NSC23766 (NSC) displayed a significant defect in MP formation and PS exposure induced by various agonists. Furthermore, Rac1-/- platelets and NSC-treated human platelets displayed a defect in procoagulant activity as demonstrated by a prolonged coagulation time following recalcification of citrated PRP. The stimulatory role of Rac1 in platelet MP formation and PS exposure is distinct from the known function of Rac1 in facilitating platelet granule secretion and secretion-dependent amplification of platelet aggregation, because supplementation of the granule content ADP rescued the defect in platelet aggregation caused by Rac1 inhibition, but failed to rescue the defect in MP formation caused by Rac1 inhibition. In contrast to Rac1, RhoA plays an inhibitory role in regulating platelet procoagulant activity, because treatment of platelets with the Rho inhibitor C3-toxin (C3) significantly enhanced agonist-induced MP formation, PS exposure, and procoagulant activity. The enhancing effect of C3 on platelet procoagulant activity is not caused by an overall enhancement of platelet activation because C3 significantly inhibited platelet secretion and aggregation. Thus, our data demonstrates that while Rac1 and RhoA both play important stimulatory roles in platelet granule secretion and aggregation, they play opposing roles in MP formation and PS exposure in platelets. Rac1 is important for stimulating platelet MP formation, PS exposure, and procoagulant activity, which is antagonized by RhoA. Disclosures: No relevant conflicts of interest to declare.

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The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646459 ◽

1991 ◽

Vol 66 (05) ◽

pp. 559-564 ◽

Cited By ~ 4

Author(s):

Jerome M Teitel

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Factor Viia ◽

Factor Xa ◽

Procoagulant Activity ◽

Tissue Factor Expression ◽

Prothrombin Complex Concentrates ◽

Cell Tissue ◽

Factor Expression ◽

Necrosis Factor

SummaryAn experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.

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Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647943 ◽

1976 ◽

Vol 35 (01) ◽

pp. 186-190 ◽

Cited By ~ 6

Author(s):

Eugen A. Beck ◽

Peter Bachmann ◽

Peter Barbier ◽

Miha Furlan

Keyword(s):

Ionic Strength ◽

Factor Viii ◽

Gel Filtration ◽

High Ionic Strength ◽

Procoagulant Activity ◽

Carrier Protein ◽

Protease Inhibition ◽

Functional Sites ◽

Molecular Weight Peak ◽

Von Willebrand

SummaryAccording to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW~ 2 × 106) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37°C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macro molecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

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Prolonged Expression of Procoagulant Activity of Human Platelets Degranulated by Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649855 ◽

1995 ◽

Vol 74 (03) ◽

pp. 958-961 ◽

Cited By ~ 6

Author(s):

Raelene L Kinlough-Rathbone ◽

Dennis W Perry

Keyword(s):

Catalytic Activity ◽

Thrombus Formation ◽

Annexin V ◽

Human Platelets ◽

Procoagulant Activity ◽

Prothrombinase Complex ◽

Arterial Thrombus ◽

Flowing Blood

SummaryPlatelets are exposed to thrombin when they take part in arterial thrombus formation, and they may return to the circulation when they are freed by fibrinolysis and dislodged by flowing blood. Thrombin causes the expression of procoagulant activity on platelets, and if this activity persists, the recirculating platelets may contribute to subsequent thrombosis. We have developed techniques to degranulate human platelets by treatment with thrombin, and recover them as single, discrete platelets that aggregate in response to both weak and strong agonists. In the present study we examined the duration of procoagulant activity on the surface of thrombin-degranulated platelets by two methods: a prothrombinase assay, and the binding of 125I-labeled annexin. Control platelets generated 0.9 ± 0.4 U thrombin per 107 platelets in 15 min. Suspensions of thrombin-degranulated platelets formed 5.4 ± 0.1 U thrombin per 107 platelets in this time. Binding of 125I-annexin V was also greater with thrombin-treated platelets than with control platelets (controls: 1.7 ±0.1 ng annexin/107 platelets; thrombin-degranulated platelets: 6.8 ± 0.2 ng annexin/107 platelets). With thrombin-degranulated platelets, increased procoagulant activity and annexin binding persisted for at least 4 h after degranulation and resuspension, indicating that the catalytic activity for the prothrombinase complex is not reversed during this time. These platelets maintained their ability to aggregate for 4 h, even in response to the weak agonist, ADP. Thus, platelets that have taken part in thrombus formation and returned to the circulation may contribute to the promotion of further thrombotic events because of the persistence of procoagulant activity on their surface.

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Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650577 ◽

1996 ◽

Vol 76 (03) ◽

pp. 322-327 ◽

Cited By ~ 73

Author(s):

Dominique Helley ◽

Amiram Eldor ◽

Robert Girot ◽

Rolande Ducrocq ◽

Marie-Claude Guillin ◽

...

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Annexin V ◽

Mean Value ◽

Procoagulant Activity ◽

Dependent Manner ◽

Cell Disease ◽

Homozygous Sickle Cell Disease

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

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Rôle of Thrombin in Prothrombin Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655588 ◽

1964 ◽

Vol 12 (02) ◽

pp. 484-488

Author(s):

W. H Seegers ◽

H Schröer ◽

D Heene

Keyword(s):

Partial Thromboplastin Time ◽

Procoagulant Activity ◽

Autoprothrombin C ◽

Generation Test ◽

Prothrombin Activation

SummaryThe partial thromboplastin time and purified thrombin were used to demonstrate the procoagulant power of thrombin. Only 0.007 μg of thrombin could be detected in prothrombin activation. Traces of thrombin and autoprothrombin C can fully account for the generation of procoagulant activity in the thromboplastin generation test. Inactivation of these two activities by antithrombin explains the disappearance of the procoagulant power in that test, so that there now remains no valid demonstration of the existence of plasma thromboplastin or of anti-plasma thromboplastin.

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Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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Trombodynamics Method in the Assessment of Coagulation Parameters in Psychopharmacotherapy of Endogenous Mental Disorders

Psychiatry ◽

10.30629/2618-6667-2020-18-4-16-25 ◽

2020 ◽

Vol 18 (4) ◽

pp. 16-25

Author(s):

N. S. Karpova ◽

O. S. Brusov ◽

I. V. Oleichik ◽

M. I. Faktor ◽

N. S. Levchenko ◽

...

Keyword(s):

Mental Disorders ◽

Response To Therapy ◽

Procoagulant Activity ◽

Coagulation Activity ◽

Continuous Type ◽

Chronic Neuroinflammation ◽

Psychopharmacological Treatment ◽

Before And After ◽

First Time ◽

Schizotypal Disorder

Background: currently, it has been proven that the pathogenesis of endogenous mental disorders is associated with the process of neuroinflammation in the brain of patients. It is also known that chronic neuroinflammation, accompanied by a violation the permeability of the blood-brain barrier. It is accompanied by the activation of platelets that generate procoagulant microparticles, which leads to a disturbance of the hemostasis system, causing an increase in blood clotting in patients. Objective: to investigate the dynamics of procoagulant activity of blood in patients with endogenous mental disorders before and after psychopharmacotherapy.Patients and methods: the study included 185 patients aged 16 to 64 years with the following mental disorders: schizophrenia with attack-like/attack-progressive/continuous type of course (F20.00–2), affective disease (F31.1–5; F32.0–3; F33.0–3), schizotypal disorder with affective fluctuations (F21.3–4). The thrombodynamic test (TD) was performed on T-2 Trombodynamis device according to the manufacturer’s instructions (Hemacore LLC, Moscow, Russia). All patients received standard pharmacotherapy according to their condition.Results: a significant decrease of procoagulant activity of spontaneous clots in the patients’ blood after psychopharmacological treatment is observed. Our data on the positive dynamics of changes in the values of TD test’s indicators in most of the examined patients suggest that a decrease in the coagulation activity of the patients’ blood as a result of treatment may be associated with the anti- inflammatory effect of antipsychotics and antidepressants.Conclusion: for the first time, it was shown that there is a positive dynamic in changing the values of the main parameters of the TD test in most patients with endogenous mental diseases. The results of TD tests can be the basis for monitoring the response to therapy.

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