. Background: Fucoidan, an antithrombotic polysaccharide, can induce endothelial colony-forming cells (ECFC) to adopt an angiogenic phenotype in vitro. Objectives: We evaluated the effect of fucoidan on vasculogenesis induced by ECFC in vivo. Methods: We used a murine hindlimb ischemia model to probe the synergic role of fucoidan-treatment and ECFC infusion during tissue repair. Results: We found that exposure of ECFC to fucoidan prior to their intravenous injection improved residual muscle blood flow and increased collateral vessel formation. Necrosis of ischemic tissue was significantly reduced on day 14, to 12.1% of the gastronecmius cross-sectional surface area compared with 40.1% in animals injected with untreated-ECFC. ECFC stimulation with fucoidan caused a rapid increase in cell adhesion to activated endothelium in flow conditions, and enhanced transendothelial extravasation. Fucoidan-stimulated ECFC were resistant to shear stresses of up to 21 dyn cm-2. Direct binding assays showed strong interaction of fucoidan with displaceable binding sites on the ECFC membrane. Bolus intramuscular administration of fucoidan 1 day after surgery reduces rhabdomyolysis. Mice injected with fucoidan (15 mg kg-1) had significantly lower mean serum creatine phosphokinase (CPK) activity than control animals. This CPK reduction was correlated with muscle preservation against necrosis (P < 0.001). Conclusions: Fucoidan greatly increases ECFC-mediated angiogenesis in vivo. Its angiogenic effect would be due in part to its transportation to the ischemic site and its release after displacement by proteoglycans present in the extracellular matrix. The use of ECFC and fucoidan together, will be an efficient angiogenesis strategy to provide therapeutic neovascularization.

BACKGROUND AND PURPOSENew antithrombotic agents with the potential to prevent atherothrombotic complications are being developed to target receptors on platelets and other cells involved in plaque growth. The aim of this study was to investigate the antiplatelet effects of F 16618, a new non-peptidic PAR1 (thrombin receptor) antagonist.EXPERIMENTAL APPROACHWe investigated the inhibitory effect of F 16618 on human platelet aggregation ex vivo, in whole blood and washed platelets, by using a multiple-electrode platelet aggregometer based on impedance and an optical aggregometer, respectively. Its effects on whole-blood haemostasis (clot parameters) were analysed with the ROTEM thromboelastometry device and the platelet function analyser PFA-100. A guinea-pig model of arterial thrombosis was used to investigate its effects on thrombus formation in vivo.KEY RESULTSF 16618 inhibited PAR1 agonist peptide (SFLLR-peptide)-induced washed platelet aggregation ex vivo. This effect was concentration-dependent and exhibited a competitive inhibition profile. Washed platelet aggregation, as well as P-selectin expression induced by thrombin, were significantly inhibited by 10 mu M F 16618. In whole-blood experiments, 20 mu M F 16618 inhibited SFLLR-induced platelet aggregation by 49%. In contrast, it had no effect on whole-blood haemostasis. In the guinea-pig model of carotid thrombosis, 0.32 mg.kg(-1) F 16618 doubled the occlusion time.CONCLUSIONS AND IMPLICATIONSF 16618 was shown to have strong antithrombotic activity in vivo and moderate antiplatelet effects ex vivo. As these effects were not associated with major effects on physiological haemostasis, this molecule is a good antiplatelet drug candidate for use either alone or in combination with current treatments.

Les progéniteurs endothéliaux circulants (PEC) possèdent des propriétés angiogéniques et la modulation de leur nombre et de leurs fonctions représente un axe de recherche en thérapie cellulaire des pathologies ischémiques. La VE-statine (Egfl7) est une protéine de découverte récente d'expression principalement endothéliale, et régulant la tubulogenèse, l'élastogenèse, l'intégrité vasculaire et la migration des cellules musculaires lisses. Toutefois, son rôle dans la vasculogenèse post-natale n'est pas parfaitement élucidé. Nous avons étudié le rôle de la VE-statine dans la biologie des PEC tardifs isolés à partir de sang adulte et de sang de cordon. Les modèles classiques d'angiogenèse in vitro (formation de pseudo-tubes en matrigel, test de prolifération au pNPP et test de migration par scratch test) et in vivo (modèle d'implant de matrigel sous cutané chez la souris C57/Bl6) ont été utilisés. La VE-statine a été produite en milieu conditionné (fibroblastes 3T3 transfectés avec la construction plasmidique pVE-statine- HA). L'inhibition de l'expression de la VE-statine dans les PEC a été réalisée par l'utilisation de siRNA par lipofection avec le kit Primefect (LONZA®). Nos résultats montrent que le milieu conditionné contenant la VE-statine inhibe la différenciation des PEC tardifs de 95 %, leur prolifération de 96 % et leur migration après blessure de 34 %. Inversement, l'inhibition de 90 % de l'expression du gène de la VE-statine avec un siRNA entraîne une augmentation de leur différenciation, prolifération et migration. L'ajout de VE-statine au basic Fibrobast Growth Factor dans un modèle pré-clinique d'implant de matrigel sous cutané chez la souris, induit une inhibition importante de la vascularisation des implants. Ces résultats suggèrent que la VE-statine est un régulateur négatif des propriétés des PEC et son inhibition pourrait être une cible pour augmenter l'expansion et le potentiel angiogénique des PEC.

Activation of the mitogen-activated protein (MAP) kinase pathway in nucleated cells is dependent on both growth factor receptors and integrins engaged in cell adhesion. Human platelets are an interesting model for studying cell adhesion and the involvement of integrin engagement on extracellular signal-regulated kinase (ERK) activation, independently from the nuclear-DNA signal pathway. Maximal phosphorylation and activity of ERK2 occurred late during thrombin-induced platelet aggregation (90 s and later), an alphaIIbbeta3 integrin-dependent event. Surprisingly, alphaIIbbeta3 inhibition by the RGDS ligand peptide, or (Fab')2 fragments of the AP-2 monoclonal antibody, resulted in a 2-fold enhancement in ERK2 phosphorylation and activity. A similar 2-fold enhancement of ERK2 activation was observed in thrombasthenic platelets which are defective in alphaIIbbeta3 and do not aggregate. This suggests that ERK2 activation in thrombin-induced platelet aggregation is dependent on thrombin rather than on alphaIIbbeta3 and is down-regulated by alphaIIbbeta3 engaged in ligand (fibrinogen) binding and/or aggregation. Finally, in the absence of stirring which allows fibrinogen binding to alphaIIbbeta3 but prevents aggregation, ERK2 was again overactivated. This overactivation appears to be consecutive to inhibition of aggregation itself and to alphaIIbbeta3 ligand binding. We conclude that in platelets, alphaIIbbeta3 engaged in aggregation down-regulates thrombin-induced ERK2 activation. To our knowledge, this is the first report of a down-regulation of the MAP kinase pathway by integrin engagement.

The stong inhibition of thrombin-induced platelet functions induced by okadaic acid is not correlated with the partial modification of pleckstrin phosphorylation, which remains still phosphorylated two min after stimulation, indicating that protein kinase C is not affected by okadaic acid. We then investigated the effect of okadaic acid on platelet lipid metabolism. Our data indicate that inhibition indeed strongly affects phosphatidic acid as well as phosphatidylinositol 3,4-bisphosphate synthesis at low concentrations of okadaic acid, and phosphatidylinositol 4,5-bisphosphate at higher concentrations. Since thrombin-induced tyrosine phosphorylations were completely inhibited in the presence of okadaic acid, as a consequence, phosphatidylinositol 3-kinase was no longer detected in antiphosphotyrosine immunoprecipitates, thus explaining the absence of phosphatidylinositol 3,4-bisphosphate synthesis. Finally, okadaic acid inhibited thrombin-induced fibrinogen binding, indicating that serine/threonine phosphatases may affect the inside-out signalling which regulates the αllbb3 integrin, downstream protein kinase C activation.

Phosphorylations induced by 2-MeS-ADP, a potent agonist of platelet ADP receptors, have been studied in rat platelets, and the effect of clopidogrel, a compound which inhibits platelet aggregation by selectively reducing the binding of ADP to its low affinity receptors on platelets, has been determined, 2-MeS-ADP induced platelet activation (shape change and aggregation) simultaneously with the phosphorylation of myosin light chain (P-20) and plekstrin (P-47). Phosphorylation of P-20 and P-47 was transient, a maximum being observed 10s after addition of the agonist when shape change reached its maximum. P-20 and P-47 phosphorylations were not strongly affected by clopidogrel treatment. Following stimulation of platelets with 2-MeS-ADP, several proteins were phosphorylated at tyrosine residues. Clopidogrel treatment inhibited the increase in phosphorylation of P-140, P-100, P-80/85, P-66 and P-55 concomitantly with the inhibition of platelet aggregation. However, clopidogrel did not interfere with the early phosphorylation of the P-80/85 kD doublet which occurs at the time of the shape change. P-80/85, identified by immunodetection as cortactin, could be involved in the reorganization of the cytoskeleton necessary for morphological changes.Thus, by using clopidogrel-treated rat platelets, we were able to determine some of the phosphorylations coupled either to clopidogrel-resistant high-affinity ADP receptors leading to shape change or to clopidogrel sensitive low-affinity ADP receptors coupled to the aggregation process.

Platelet signal transduction involves not only reversible phosphorylation of proteins on both tyrosine and serine/threonine residues, but also mechanisms of cross-talk to coordinate different pathways. We have, therefore, investigated the effect of okadaic acid, a potent inhibitor of serine/threonine protein phosphatases type 1 and type 2A (PP1 and PP2A), to better understand the interplay that must exist between serine/threonine and tyrosine phosphorylations during platelet activation. Okadaic acid drastically inhibits thrombin-induced platelet aggregation, secretion, and thromboxane synthesis. The inhibition is accompanied by a marked increase in the phosphorylation of at least 5 proteins (230, 210, 74, 57, and 50 to 52 kDa). However, protein kinase C activity is not modified because thrombin-and phorbol-12-myristate-13-acetate-induced phosphorylation of pleckstrin is still occurring, although slightly decreased. Inhibition of platelet function and extent of the phosphorylation of the 5 substrates in the presence of okadaic acid are concentration and time dependent, suggesting a relation between the accumulation of one or more phosphoproteins and the inhibitory effect of okadaic acid. Okadaic acid inhibits thrombin-induced tyrosine phosphorylation in a concentration-dependent manner. According to Brautigan and Pinault, the inhibition of protein phosphatases in kidney cells resulted in the activation of a 55-kDa-tyrosine phosphatase and the tyrosine phosphatase activity was synergistically increased when okadaic acid acted in concert with prostaglandin I2 (PGI2). Interestingly, in agreement with these results, the okadaic acid-induced phosphorylation of the 50-kDa substrate, which occurs without a cyclic adenosine monophosphate increase in platelets, has the same molecular weight as the platelet membrane tyrosine phosphatase isolated by Dawicki and Steiner. Furthermore, we also found that thrombin-induced tyrosine phosphorylation was markedly inhibited in the presence of low concentrations of both okadaic acid and PGI2, therefore explaining the synergistic inhibition of platelet aggregation and secretion. The results greatly support the notion of a cross-talk between stimulation of serine/threonine kinases (in response to inhibition of serine/threonine PP) and inhibition of tyrosine phosphorylations and emphasize the role of the 50-kDa substrate in regulating platelet activation.

1. We evaluated the effects of daltroban on (i) human platelet shape change and aggregation in vitro, and (ii) mean systemic and pulmonary arterial pressures (MAP and MPAP, respectively) as well as haematocrit, in anaesthetized, open-chest Sprague-Dawley rats, compared with those of a chemically distinct prostanoid thromboxane A2 (TxA2) receptor antagonist, SQ 29,548, and agonist, U-46619. 2. In human platelets in vitro, daltroban (10 nM-100 microM; n = 6 per group) concentration-dependently induced shape change, attaining at 50 microM, a maximum amplitude of 0.83 +/- 0.09 mV representing 46.4 +/- 4.8% of that evoked by U-46619 (1.78 +/- 0.20 mV at 0.2 microM; n = 9); and inhibited U-46619-induced platelet aggregation with an IC50 of 77 (41-161)nM. SQ 29,548 (10 nM-100 microM; n = 6 per group) failed to evoke any platelet shape change, but potently inhibited U-46619-induced platelet aggregation with an IC50 < 10 nM. 3. In anaesthetized rats in vivo, daltroban (10-2500 micrograms kg-1, i.v. infused over 2 min; n = 4-8 per group) produced a bell-shaped dose-response curve for MPAP and haematocrit, and evoked maximal increases of 12.7 +/- 2.1 mmHg and 5.8 +/- 1.5% at 80 micrograms kg-1 (n = 6) and 630 micrograms kg-1 (n = 8), respectively (both P < 0.05) with ED50s of 20 (16-29) and 217 (129-331) micrograms kg-1, respectively. By comparison, U-46619(0.16-20 microg kg-1, i.v.), induced dose-dependent increases in MPAP and haematocrit (25.4 +/- 1.0 mmHg and 16.1 +/- 2.9% at the highest dose; n = 12, both P<0.01), with ED50s of 1.8 (1.3-2.5) and 3.9(3.5- 5.4) microg kg- 1, respectively. Daltroban dose-dependently increased MAP with a maximum amplitude of 42.2 +/- 4.4 mmHg at a dose of 80 microg kg-1 [ED50 = 94 (64-125) microg kg-1], similar to that induced by U-46619 (41.3 +/- 9.6 mmHg) at a dose of 0.63 microg kg-1 [ED50= 0.22 (0.13-0.24) microg kg-1]. SQ 29,548(10-2500 microg kg-1, i.v.; n =4 per group) failed to modify significantly any of these parameters.4. Our results clearly demonstrate that daltroban, in a similar manner to the TxA2 analogue, U-46619,but unlike the TxA2 receptor antagonist, SQ 29,548, exhibits significant intrinsic activity in human platelets in vitro and in the rat vasculature in vivo, possibly through TxA2 receptor activation.

Summary KRDS, a tetrapeptide from human lactotransferrin, inhibits thrombin-induced platelet aggregation, secretion and thromboxane (TX) synthesis without interfering with phospholipase C (PLC) β activation, since in previous work we have shown that Ca2+ mobilization and phosphorylation of the myosin light chain kinase (20 kDa) and pleckstrin (47 kDa) were normal. However, the inhibition of arachido- nic acid-induced aggregation in the presence of KRDS is accompanied by normal TX synthesis suggesting that it does not interfere with the cyclooxygenase activity. To elucidate further the mechanisms of action of this peptide we tested its effect on U46619-induced platelet activation. KRDS inhibits U46619-induced platelet aggregation time- and dose-dependently without inhibiting the phosphorylation of pleckstrin. This suggests that the PLC pathway is not affected and that the inhibitory effect of KRDS is not due to an uncoupling of TXA2 from its receptor. In addition to the PLC pathway, protein tyrosine kinases play a major role in platelet signal transduction mechanisms. At least 7 tyrosine-phosphorylated proteins are detected upon stimulation of platelets by thrombin. KRDS strongly inhibits the tyrosine- phosphorylated substrates, in particular two 100-105 kDa substrates which are related to GP Ilb/IIIa activation and platelet aggregation. The absence of TX synthesis observed in the presence of KRDS could be due to the inactivation of cPLA2 since the latter needs tyrosine phosphorylation to be activated, thus explaining the inhibitory action of KRDS on platelet functions.

Pentosan polysulfate is a low-molecular-weight sulfated polysaccharide used as an antithrombotic drug. We present two patients who developed thrombocytopenia and venous thrombosis during treatment with pentosan polysulfate. The relationship between pentosan polysulfate and thrombocytopenia is supported by platelet aggregation and serotonin release tests. In the light of the literature and our two cases, it appears that pentosan polysulfate alone as standard heparin and low-molecular-weight heparin can induce thrombocytopenia and thrombosis. Platelet counts should therefore be periodically monitored during pentosan polysulfate treatment. In the case of pentosan polysulfate-induced thrombocytopenia, it seems that heparin or low-molecular-weight heparin should not be instituted during the acute phase even it platelet aggregation studies are negative, because of their low sensitivity. After remission of thrombocytopenia, whether or not glycoaminoglycans can be reinstituted, at least temporarily, after antibody had disappeared is still an open question. © 1994 Wiley-Liss, Inc.

Protein tyrosine kinase (PTK) blockers (tyrphostins) inhibit in a dose-dependent fashion thrombin-induced aggregation and serotonin release with IC50 values in the 10-35 microM concentration range. The inhibition of thrombin-induced aggregation correlates with their potency in inhibiting phosphorylation of proteins on tyrosine residues. Using metabolically 32P-labelled human platelets, it was found that the tyrphostins have no effect on the decrease in [32P]phosphatidylinositol bisphosphate but prevent the replenishment of [32P]polyphosphoinositide. Tyrphostins decreased [32P]phosphatidic acid production induced by thrombin, although never by more than 50%, and only delayed the peak of diacylglycerol, suggesting that phospholipase C was still activated. Tyrphostins inhibited the thrombin-elicited early phosphorylation of p43 and p20, substrates for protein kinase C (PKC) and myosin light chain kinase, respectively, at short times of activation. This inhibition, however, was overcome after 1 min of stimulation with thrombin. Tyrphostin AG213 also inhibited platelet aggregation and tyrosine protein phosphorylation induced by phorbol myristate acetate (PMA), but did not inhibit pleckstrin phosphorylation. These results suggest that thrombin induces the phosphorylation of proteins on tyrosine residues which most probably results in the activation of phosphoinositide kinases. The ability of tyrphostins to inhibit phosphorylation of p43 and p20 when induced by thrombin but not when induced by PMA confirms that PTKs may be involved subsequent to PKC activation.

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.