Study on the Mechanism of the Adrenaline-Evoked Procoagulant Response in Human Platelets.

Adrenaline has recently been found to trigger phosphatidylserine (PS) exposure on blood platelets, resulting in amplification of the coagulation process, but the mechanism is only fragmentarily established. Using a panel of platelet receptors' antagonists and modulators of signaling pathways, we evaluated the importance of these in adrenaline-evoked PS exposure by flow cytometry. Calcium and sodium ion influx into platelet cytosol, after adrenaline treatment, was examined by fluorimetric measurements. We found a strong reduction in PS exposure after blocking of sodium and calcium ion influx via Na+/H+ exchanger (NHE) and Na+/Ca2+ exchanger (NCX), respectively. ADP receptor antagonists produced a moderate inhibitory effect. Substantial limitation of PS exposure was observed in the presence of GPIIb/IIIa antagonist, phosphoinositide-3 kinase (PI3-K) inhibitors, or prostaglandin E1, a cyclic adenosine monophosphate (cAMP)-elevating agent. We demonstrated that adrenaline may develop a procoagulant response in human platelets with the substantial role of ion exchangers (NHE and NCX), secreted ADP, GPIIb/IIIa-dependent outside-in signaling, and PI3-K. Inhibition of the above mechanisms and increasing cytosolic cAMP seem to be the most efficient procedures to control adrenaline-evoked PS exposure in human platelets.

Natural Polyphenols May Normalize Hypochlorous Acid-Evoked Hemostatic Abnormalities in Human Blood.

During pathogen invasion, activated neutrophils secrete myeloperoxidase (MPO), which generates high local concentrations of hypochlorous acid (HOCl), a strong antimicrobial agent. Prolonged or uncontrolled HOCl production may, however, affect hemostasis, manifesting in inhibition of platelet aggregation and thrombus formation and in elevated fibrin density and attenuated fibrinolysis. In this report, we investigated whether three plant-derived polyphenols with well-known antioxidant properties, i.e., quercetin (Que), epigallocatechin gallate (EGCG), and resveratrol (Resv), at concentrations not affecting platelet responses per se, may normalize particular aspects of hemostasis disturbed by HOCl. Specifically, Que (5-25 µM) and EGCG (10-25 µM) abolished HOCl-evoked inhibition of platelet aggregation (assessed by an optical method), while the simultaneous incubation of platelet-rich plasma with Resv (10-25 µM) enhanced the inhibitory effect of HOCl. A similar effect was observed in the case of thrombus formation under flow conditions, evaluated in whole blood by confocal microscope. When plasma samples were incubated with HOCl, a notably higher density of fibrin (recorded by confocal microscope) was detected, an effect that was efficiently normalized by Que (5-25 µM), EGCG (10-25 µM), and Resv (5-25 µM) and which corresponded with the normalization of the HOCl-evoked prolongation of fibrinolysis, measured in plasma by a turbidimetric method. In conclusion, this report indicates that supplementation with Que and EGCG may be helpful in the normalization of hemostatic abnormalities during inflammatory states associated with elevated HOCl production, while the presence of Resv enhances the inhibitory action of HOCl towards platelets.

Multidirectional Effects of Tormentil Extract on Hemostasis in Experimental Diabetes.

In our previous study, we showed that ellagitannin- and procyanidin-rich tormentil extract (TE) decreased experimental arterial thrombosis in normoglycemic rats through platelet inhibition. TE also slightly increased coagulation and attenuated fibrinolysis; however, these effects did not nullify the antithrombotic effect of TE. The present study aimed to assess whether TE exerts antithrombotic activity in streptozotocin (STZ)-induced diabetes, which is characterized by pre-existing increased coagulation and impaired fibrinolysis, in vivo and ex vivo thrombosis assays. TE (100, 200, or 400 mg/kg, p. o.) was administered for 14 days to STZ-induced diabetic rats and mice. TE at 100 mg/kg dose decreased the thrombus area in the mice model of laser-induced thrombosis through its potent antiplatelet effect. However, TE at 200 mg/kg dose increased thrombus weight in electrically induced arterial thrombosis in rats. The prothrombotic effect could be due to increased coagulation and attenuated fibrinolysis. TE at 400 mg/kg dose also improved vascular functions, which was mainly reflected as an increase in the arterial blood flow, bleeding time prolongation, and thickening of the arterial wall. However, TE at 400 mg/kg dose did not exert antithrombotic effect. Summarizing, the present results show that TE may exert multidirectional effects on hemostasis in STZ-induced diabetic rats and mice. TE inhibited platelet activity and improved endothelial functions, but it also showed unfavorable effects by increasing the activity of the coagulation system and by inhibiting fibrinolysis. These contrasting effects could be the reason for model-specific influence of TE on the thrombotic process in STZ-induced diabetes.

Adrenaline May Contribute to Prothrombotic Condition via Augmentation of Platelet Procoagulant Response, Enhancement of Fibrin Formation, and Attenuation of Fibrinolysis.

Background: Adrenaline is believed to play a role in thrombosis and hemostasis. The complex effect of its clinically relevant concentrations on thrombus formation, coagulation and fibrinolysis in human blood has never been specifically studied. Methods: Confocal microscopy was used to study thrombus formation under flow, exposure of phosphatidylserine (PS) in adhered platelets, to evaluate clots density, and to measure kinetics of fibrin formation and external fibrinolysis under flow. Flow cytometry was utilized to assess PS exposure in non-adhered platelets. Kinetics of clot formation and internal fibrinolysis was evaluated by thromboelastometry. Platelet aggregation was measured by optical aggremometry. Kinetics of clot retraction was assessed by using digital camera. Results: We found that adrenaline (1-10 nM) is able to enhance platelet activation evoked by subthreshold collagen (150 ng/ml), resulting in augmentation of platelet aggregation, thrombus formation under arterial flow conditions, platelet PS exposure, and formation of platelet-fibrin clots. The development of platelet procoagulant response evoked by adrenaline + low collagen was associated with the formation of denser platelet-fibrin clots and the decrease in rate of fibrinolysis despite whether lysis was initiated inside (internal fibrinolysis) or outside the clot (external fibrinolysis). The above phenomena were abolished by the α2-adrenergic receptor antagonist, rauwolscine. Adrenaline-collagen synergism, expressed as PS exposure, was significantly reduced by cyclooxygenase inhibitor (acetylsalicic acid), GPIIb/IIIa receptor blocker (tirofiban), and P2Y12 receptor antagonist (PSB 0739). Conclusion: Clinically relevant concentrations of adrenaline may significantly augment responses of human platelets in the presence of subthreshold concentrations of collagen, which should be considered during therapies involving adrenaline infusion. Routinely used antiplatelet drugs may reduce the prothrombotic state evoked by adrenaline-collagen synergism.

HAuCl4, Putative General Aquaporins Blocker, Reduces Platelet Spreading, Filopodia Formation, Procoagulant Response, and Thrombus Formation Under Flow.

Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated. Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo. Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10-100 µM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 µM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25-200 µM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1-10 mg/kg). Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.

New approaches for the assessment of platelet activation status in thrombus under flow condition using confocal microscopy.

The goal of the study was the assessment of heterogeneous platelet activation status in thrombus. In a ferric(III) chloride (FeCl3) thrombosis (intravital) model of C57BL/6 J mice, the area of irreversibly activated (phosphatidylserine (PS)-positive) platelets was assessed after 1-s exposure of a vessel to FeCl3. In a laser-induced thrombosis (intravital) model of GFP mice, the area of the thrombus composed of PS-negative platelets was evaluated. The ratio of the area of PECAM-1 to the area of the thrombus was used as a marker to assess the activity of PS-negative platelets. In the in vitro flow chamber model, the thrombus area (PS-negative and PS-positive platelets) and the platelet activation index (ratio of the area of PS-positive platelets to the area of thrombus) were determined. To assess platelet activation status with these models, acetylsalicylic acid (ASA) and iloprost (Ilo) were used. In the FeCl3 thrombosis, ASA (10 mg/kg, 100 mg/kg) decreased the area of PS-positive platelets. In the laser thrombosis, ASA (10 mg/kg) decreased the thrombus area, but the decrease in platelet activity was evident even at 3 mg/kg by an increased PECAM-1/thrombus ratio. In the flow chamber, ASA (0.02 mg/ml, 0.2 mg/ml) equally decreased the platelet activation index, whereas only at 0.2 mg/ml, it decreased the thrombus area. Ilo (3.6 ng/ml, 36 ng/ml) decreased the thrombus area but at 36 ng/ml increased the platelet activation index. We showed that intravital models and flow chamber provide a detailed assessment of platelet activation status and the mechanism of drug action.

Rapid effects of aldosterone on platelets, coagulation, and fibrinolysis lead to experimental thrombosis augmentation.

An increase in aldosterone levels positively correlates with an increased risk of acute cardiovascular thrombotic events. The aim of the study was to determine the mechanism of action of prothrombotic aldosterone focusing on the rapid effects of the hormone on platelets, coagulation, and fibrinolysis. A wide panel of advanced ex vivo and in vitro techniques was used for the evaluation of coagulation and fibrinolysis in aldosterone-treated rats. Additionally, two experimental mice models of thrombosis, which allowed for the intravital observation of the first stage of thrombus formation in real time, were used. Acute administration of aldosterone in rats increased the density of fibrin net and platelet aggregates in clots as well as reduced fibrinolysis. These effects were observed within 10 min and were partially suppressed by eplerenone. Moreover, acute administration of aldosterone in mice enhanced platelet accumulation at the site of endothelial injury induced by laser and increased the area of irreversibly activated platelets in FeCl3-induced thrombus. These results demonstrate that aldosterone acutely affects platelets, coagulation, and fibrinolysis, leading to an enhanced thrombosis. The aldosterone effects were mediated partially via a mineralocorticoid receptor. The mechanism seems to involve non-genomic signaling since the effects were observed within a few minutes of aldosterone administration.

The myeloperoxidase product, hypochlorous acid, reduces thrombus formation under flow and attenuates clot retraction and fibrinolysis in human blood.

Hypochlorite (HOCl), a strong oxidant and antimicrobial agent, has been proposed to be associated with hemostatic abnormalities during inflammatory response. However, its complex impact on hemostasis is not completely understood. In this report we studied the effect of clinically relevant (micromolar) HOCl concentrations on thrombus formation under flow, kinetics of platelet-fibrin clot formation, its architecture, retraction, and lysis. We found that HOCl (up to 500 µM) did not affect kinetics of coagulation measured in whole blood. HOCl (500-1000 µM) markedly diminished thrombus formation under flow. Clot retraction rate was reduced by HOCl dose-dependently (50-500 µM). HOCl (125-500 µM) inhibited fibrinolysis in whole blood and in platelet-depleted plasma, dose-dependently. Activity of plasmin was reduced by HOCl at concentrations started from 500 µM. HOCl (up to 500 µM) did not reduce plasminogen binding to fibrin under flow. HOCl (125-500 µM) modulated architecture of fibrin- and platelet-fibrin clots towards structures made of thin and densely packed fibers. Exposure of pure fibrinogen to HOCl (10-1000 µM) resulted in formation of dityrosine and was associated with altered fibrin structure derived from such modified fibrinogen. HOCl-altered fibrin net structure was not related with modulation of platelet procoagulant response, thrombin generation, and factor XIII activity. We conclude that, in human blood, clinically relevant HOCl concentrations may inhibit thrombus formation under flow, clot retraction and fibrinolysis. Fibrinolysis and clot retraction seem to be the most sensitive to HOCl-evoked inhibition. HOCl-modified fibrinogen and altered clot structure associated with it are likely to be primary sources of attenuated fibrinolysis.

Aquaporins in human platelets: intracellular localization and possible role in granule and lysosome secretion.

This study was undertaken to establish the presence and the role of aquaporins (AQPs) in human platelets. Immunodetection with polyclonal antibodies and fluorescent microscopy suggest the presence of AQP isoforms - 0-7 and 9-12 - localized (in resting platelets) in the plasma membrane and in the dense and alpha granules. In thrombin- or monensin-treated platelets, the granules' AQPs become visible in the whole cell body, indicating the granules' swelling. In our studies on the role of AQPs in platelet responses we used tetrachloroauric acid (HAuCl4), a classical water channel blocker. We found that 10-100 µM of Au(III) inhibited the hypotonicity-, monensin (simulating the action of Na+/H+ exchanger)-, and collagen-evoked platelet swelling and reduced tritiated water uptake by platelets treated by collagen or monensin, indicating its ability to block water channels in these cells. HAuCl4, at the concentrations reducing water influx, did not induce cell lysis, alter the plasma membrane shape or the -SH group content. The inhibitor also failed to affect Na+ and Cl--related osmotic gradient formation and protein kinase D2 phosphorylation. In platelets activated by threshold concentrations of collagen, the thrombin receptor activating peptide, ADP, calcium ionophore A23187, phorbol ester and arachidonic acid, HAuCl4 (100 µM) completely inhibited secretion of ATP from dense granules but failed to reduce platelet aggregation. In collagen-stimulated platelets, HAuCl4 (10-100 µM) reduced secretion from dense and alpha granules, as well as lysosomes, in a dose-dependent manner. We conclude that human platelets possess numerous AQPs subtypes localized in the plasma and granule membranes. AQP-mediated water fluxes may be crucial for platelet volume regulation as well as secretion from dense and alpha granules and lysosomes.