Vasopressin acts on platelets to generate procoagulant activity.

The aim of our study was to examine whether arginine vasopressin (AVP) is able to evoke in human platelets a procoagulant response due to activation of an Na+/H+ exchanger. It was found that treatment of platelets with AVP (20-100 nmol/l) results in generation of a weak calcium signal, activation of Na+/H+ exchanger, aggregation, and development of a procoagulant response. The AVP-evoked procoagulant response was dose and time dependent, weaker than that produced by collagen or monensin (mimics Na+/H+ exchanger), and less pronounced following the inhibition of Na+/H+ exchanger by 5-(N-ethyl-N-isopropyl) amiloride or genistein. Flow cytometry studies reveal that in-vitro platelet treatment with AVP results in an unimodal left shift in the forward and side scatter of the entire platelet population, indicating morphological changes on the plasma membrane. The shift was dose related, weaker than that evoked by collagen, similar to that produced by monensin and strongly reduced in the presence of 5-(N-ethyl-N-isopropyl) amiloride or genistein. Using flow cytometry, we demonstrated enhanced expression of phosphatidylserine on the AVP-treated platelets. AVP-evoked phosphatidylserine exposure was dose dependent, inhibited by 5-(N-ethyl-N-isopropyl) amiloride or genistein and weaker than that produced by collagen. AVP in a dose-dependent manner produced a rise in platelet volume. The swelling was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, and its kinetics was similar to that observed in the presence of monensin. We conclude that prolonged treatment of platelets with AVP results in a procoagulant response, which may occur as a consequence of Na+ influx mediated by Na+/H+ exchanger.

The involvement of Na+/K(+)-ATPase in the development of platelet procoagulant response.

In circulation, platelets may come into contact with both exogenous (cardiac glycoside treatment) and endogenously produced inhibitors of Na+/K(+)-ATPase. We examined whether blocking of platelet Na+/K(+)-ATPase by ouabain results in generation of procoagulant activity. It was shown that an in vitro treatment of platelets with ouabain (20-200 microM for 20 to 60 min) is associated with an intracellular accumulation of sodium ([Na+](i)), generation of a weak calcium signal, and expression of procoagulant activity. The ouabain-induced procoagulant response was dose- and time-related, less pronounced than that evoked by collagen and similar to that produced by gramicidin, not affected by EDTA or aspirin, and strongly reduced in the absence of extracellular Na+ or by hyperosmolality. Flow cytometry studies revealed that ouabain treatment results in a unimodal left shift in the forward and side scatter of the entire platelet population indicating morphological changes of the plasma membrane. The shift was dose related, weaker than that evoked by collagen and similar to that produced by gramicidin. Ouabain-treated platelets express phosphatidylserine (PS). The ouabain-evoked PS expression was dose- and time-dependent, weaker than that produced by collagen and similar to that evoked by gramicidin. Electronic cell sizing measurements showed a dose-dependent increase in mean platelet volume upon treatment with ouabain. Hypoosmotically-evoked platelet swelling resulted in the appearance of procoagulant activity. Thromboelastography measurements indicate that, in whole blood, nanomolar (50-1000 nM, 15 min) concentrations of ouabain significantly accelerate the rate of clot formation initiated by contact and high extracellular concentration of calcium. We conclude that inefficiently operating platelet Na+/K(+)-ATPase results in a rise in [Na+](i). An increase in [Na+](i) and the swelling associated with it may produce PS exposure and a rise in membrane curvature leading to the generation of a procoagulant activity.

Cyclosporine enhances platelet procoagulant activity.

BACKGROUND: Clinical use of cyclosporine (CsA) was suggested to be associated with an increased risk of thromboembolic complications. The molecular mechanisms underlying these effects remain unresolved. METHODS: We tested the hypothesis that CsA may produce platelet procoagulant activity due to its interaction with the platelet plasma membrane. To verify this hypothesis the possible relationship between platelet morphology, exposure to platelet phosphatidylserine (PS) and platelet procoagulant activity (measured as phospholipid-dependent thrombin generation) was studied. RESULTS: It was found that CsA (1-100 microg/ml) potentiates collagen-evoked platelet procoagulant response. Platelets treated in vitro with CsA (20-200 microg/ml 20-60 min) expressed procoagulant activity. The CsA-induced platelet procoagulant response was both dose- and time-related and weaker than that produced by collagen. Flow cytometry studies revealed that CsA treatment results in a left shift (decrease) in the forward and side scatter of the entire platelet population. The shift was unimodal, dose-dependent and less pronounced than that elicited by collagen. Using flow cytometry and fluorescein isothiocyanate-labelled annexin V as a probe for PS, we demonstrated an increased binding of this marker to a CsA-treated platelet population. CsA-evoked PS-expression was dose- and time-dependent and smaller than that produced by collagen. CsA, at concentrations similar to those affecting platelet procoagulant response, released lactate dehydrogenase from platelets. CONCLUSIONS: These observations indicate that the thrombogenic properties of CsA may result from the alteration of lipid organization in platelet plasma membrane, leading to externalization of PS and accelerated thrombin generation.

The role of Na+/H+ exchanger in serotonin secretion from porcine blood platelets.

This study was undertaken to evaluate whether a link exists between the activation of protein kinase C (PKC), operation of Na(+)/H(+) exchanger (NHE), cell swelling and serotonin (5-HT) secretion in porcine platelets. Activation of platelets by thrombin or phorbol 12-myristate 13-acetate (PMA), a PKC activator, initiated a rapid rise in the activity of Na(+)/H(+) exchanger and secretion of 5-HT. Both thrombin- and PMA-evoked activation of Na(+)/H(+) exchanger was less pronounced in the presence of ethyl-isopropyl-amiloride (EIPA), an NHE inhibitor, and by GF 109203X, a PKC inhibitor. Monensin (simulating the action of NHE) caused a dose-dependent release of 5-HT that was not abolished by GF 109203X or EGTA. Lack of Na(+) in the suspending medium reduced thrombin-, PMA-, and monensin-evoked 5-HT secretion. GF 109203X nearly completely inhibited 5-HT release induced by PMA-, partly that induced by thrombin, and had no effect on 5-HT release induced by monensin. EIPA partly inhibited 5-HT release induced by thrombin and nearly totally that evoked by PMA. Electronic cell sizing measurements showed an increase in mean platelet volume upon treatment of cells with monensin, PMA or thrombin. The PMA- and thrombin-evoked rise in mean platelet volume was strongly reduced in the presence of EIPA. As judged by optical swelling assay monensin and PMA produced a rapid rise in platelet volume. The swelling elicited by PMA was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Hypoosmotically evoked platelet swelling did not affect platelet aggregation but significantly potentiated thrombin-evoked release of 5-HT and ATP. Taken together, these results show that in porcine platelets PKC may promote 5-HT secretion through the activation of NHE. It is hypothesized that enhanced Na(+)/H(+) antiport may result in a rise in cell membrane tension (due to cell swelling) which in turn facilitates fusion of secretory granules with the plasma membrane leading to 5-HT secretion.

Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response.

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.

Nitric oxide and platelet energy metabolism.

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.

The effect of some epsilon-aminocaproic acid derivatives on platelet responses.

epsilon-Aminocaproic acid (EACA) is a synthetic low molecular drug with antifibrinolytic activity. However, treatment with this drug can be incidentally associated with an increased thrombotic tendency. The aim of the present work was to test synthetic EACA derivatives for their antiplatelet activities. We investigated the effect of three EACA derivatives with antifibrinolytic activity: I. epsilon-aminocaproyl-L-leucine hydrochloride (HCl*H-EACA-L-Leu-OH), II. epsilon-aminocaproyl-L-(S-benzyl)-cysteine hydrochloride (HCl*H-EACA-L-Cys(S-Bzl)-OH) and III. epsilon-aminocaproyl-L-norleucine (H-EACA-L-Nle-OH) on platelet responses (aggregation and adhesion) and on their integrity. It was found that: 1. as judged by LDH release test, none of the tested compounds, up to 20 mM, was toxic to platelets, 2. in comparison with EACA, all the synthetic derivatives inhibited much stronger the ADP- and collagen-induced aggregation of platelets suspended in plasma (platelet rich plasma) and aggregation of these cells in whole blood, 3. EACA and its derivatives exerted a similar inhibitory effect on the thrombin-induced adhesion of platelets to fibrinogen-coated surfaces. Since platelet activation and blood coagulation are tightly associated processes, the antiplatelet properties of EACA derivatives are expected to indicate reduced thrombotic properties of these derivatives compared to EACA.

[Morphologic parameters of blood platelets and activity NA+/H+ (NHE-1) exchanger in normal pregnancy and pregnancy complicated with preeclampsia]. / Parametry morfologiczne plytek krwi i aktywnosc wymieniacza Na+/H+ (NHE-1) u ciezarnych zdrowych i z preeklampsja.

UNLABELLED: The aim of the study was the evaluation of number and morphologic parameters of blood platelets and sodium-proton platelet exchangers in pregnant women in the 3rd trimester of pregnancy, healthy (155) and with preeclampsia (40). RESULTS: Blood platelet number was significantly lower in patients with preeclampsia than in healthy subjects (195,000 vs 222,000). Mean platelet volume was significantly higher in patients with preeclampsia (9.5 fl vs 8.6 fl). The activity of Na+/H+ was slightly higher in pregnants with preeclampsia.

Platelet sodium-proton exchanger and phospholipid-dependent procoagulant activity in patients with type 2 diabetes.

Platelet Na(+)/H(+) exchanger (NHE) activity, phospholipid-dependent thrombin generation, and platelet factor 3 (PF3) availability were measured in 83 type 2 diabetics and in 40 age- and sex-matched healthy subjects. Na(+)/H(+) exchanger activity was significantly increased in diabetic patients in comparison to the controls (kappa = 4.29 +/- 0.71 x 10(-3) x s(-1) v 3.21 +/- 0.64 x 10(-3) x s(-1), P <.00001). However, there was no significant difference between subjects with (kappa = 4.28 +/- 0.75 x 10(-3) x s(-1)) and without (kappa = 4.26 +/- 0.32 x10(-3) x s(-1)) arterial hypertension, as well as between patients with normo- and microalbuminuria or overt proteinuria (kappa = 4.26 +/- 0.58 x 10(-3) x s(-1), kappa = 4.47 +/- 0.93 x 10(-3) x s(-1) and kappa = 4.07 +/- 0.38 x10(-3) x s(-1), respectively). Comparatively high NHE activity was observed in the group of patients with hemoglobin A(1c) (HbA(1c)) less than 7.5%. Multiple regression analysis revealed that the factors independently related to platelet Na(+)/H(+) exchanger activity were: total PF3 activity (beta = 0.77, P =.011) and triglyceride (TG) concentration (beta = 0.44, P =.039). Phospholipid-dependent thrombin generation and PF3 availability were also enhanced in all plasma fractions of diabetic patients, especially in platelet-poor plasma (PPP) and platelet-free plasma (PFP) (P <.0001 and P <.00001, respectively). There was a positive correlation between NHE activity and thrombin generation, as well as with PF3 availability in all plasma fractions. Our results suggest that enhanced platelet Na(+)/H(+) exchanger activity associated with raised phospholipid-dependent procoagulant activity may increase the risk of vascular damage in type 2 diabetic patients.

[Factors influencing platelet procoagulant activity in type II diabetic patients. The role of sodium-proton exchange]. / Czynniki regulujace aktywnosc prokoagulacyjna plytek krwi u pacjentów z cukrzyca typu 2. Rola antyporterów sodowo-protonowych.

The aim of our study was the estimation of platelet sodium-proton exchanger activity and platelet pro-coagulant activity, expressed as the availability of platelet factor 3 (PF3) and thrombin generation, in 83 type 2 diabetic patients (mean age 56.7 +/- 7.8 years) and 40 healthy subjects (mean age 54.4 +/- 6.2 years). Thrombin generation was measured in platelet rich plasma, using a chromogenic substrate S-2238. The availability of PF3 was estimated in platelet rich plasma, platelet poor plasma and platelet filtrated plasma, to assess procoagulant activity connected with platelets and cell derived microparticles, shedding upon activation (according to Jy and Horstman). The activity of platelet Na+/H+ exchanger was measured using an optical swelling assay. We found that the activity of PF3 and phospholipid dependent thrombin generation were significantly higher in diabetic patients, irrespective of their vascular complications and metabolic control. The highest increase of PF3 activity was observed in platelet poor (p < 0.0001) and platelet filtrated plasma (p < 0.000001). Na+/H+ exchange rate was significantly higher in diabetic patients in comparison to the controls (4.29 +/- 0.71 x 10(-3)/s vs 3.21 +/- 0.64 x 10(-3)/s, p < 0.00001). There was also a positive correlation between Na+/H+ exchanger activity and PF3 activity in all plasma fractions. Our results suggest that increased thrombin generation, enhanced platelet Na+/H+ exchanger activity and raised PF3 availability, connected mainly with cell derived microparticles, may enhance the risk of vascular damage in type 2 diabetic patients.

[Sodium-proton exchanges and platelet procoagulant activity in type 1 diabetic patients]. / Antyportery sodowo-protonowe i aktywnosc prokoagulacyjna plytek krwi u chorych na cukrzyce typu 1.

Platelet sodium-proton exchange rate and phospholipid dependent procoagulant activity were measured in 31 type 1 diabetics (mean age 32.3 +/- 10.1 years) and 35 healthy subjects (mean age 35.4 +/- 9.4 years). The activity of platelet Na+/H+ exchanger was measured in platelet rich plasma, using an optical swelling assay, according to Rosskopf et al. Platelet procoagulant activity was measured in platelet rich plasma, platelet poor plasma and platelet/microparticles filtrated plasma, using Russell's viper venom (according to Jy and Horstman) and calibrated with ship L-alpha-phosphatidylethanolamine. We found that Na+/H+ exchange rate was significantly higher in diabetic patients in comparison to the controls (p = 0.0009). There was also a positive correlation between the activity of Na+/H+ exchanger and phospholipid dependent procoagulant activity in all plasma fractions. We did not find a significant association between Na+/H+ exchanger activity and metabolic parameters studied, however in patients with HbA1c level > 7.5% higher Na+/H+ exchange rates were noted. Total procoagulant activity did not rise significantly in diabetic patients, but was markedly higher in platelet poor and platelet filtrated plasma. It was supposed that it originated from platelet derived microparticles, enriched in phospholipids. Our results suggest that an increased platelet Na+/H+ exchange rate and raised procoagulant activity connected with platelet microparticles may enhance the risk of vascular damage in type 1 diabetic patients.

The involvement of the Na(+)/H(+) exchanger in the formation of microvesicles by porcine platelets.

Activated platelets release microvesicles, which express procoagulant activity. The mechanism by which vesicles are formed is not entirely clear. This study was undertaken to determine whether a link exists between the operation of the plasma membrane Na(+)/H(+) exchanger (NHE) and vesiculation. It was found, that platelets treated with NHE-simulating monensin and the sodium influx-inducing gramicidin (without concomitant H+ efflux) produced vesicles demonstrating procoagulant activity. Alkalinization of platelet cytosol by NH4Cl failed to evoke vesicle release. Collagen and phorbol ester (PMA)-evoked vesiculation was diminished in the presence of 5-(N-ethyl-N-isopropyl amiloride) (EIPA, inhibitor of NHE) or GF 109203X (inhibitor of protein kinase C). Vesicle formation induced by collagen, PMA, and the calcium ionophore A23187 was less pronounced in the absence of external Na+. In comparison with collagen, thrombin was a stronger inducer of vesiculation. Platelets stimulated by thrombin, collagen, and PMA accumulated 22Na+, a phenomenon inhibited in the presence of EIPA. Collagen-evoked vesicle formation started with aggregation but culminated after its completion. The data indicate a significant contribution of the Na(+)/H(+) exchanger in the formation of microvesicles by porcine platelets.