Pierre Savi

Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.

The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6−/− mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6−/− mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.

Role of P2Y1 purinoceptor in ADP-induced platelet activation

ADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP‐induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [33P]2‐MeS‐ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [33P]2‐MeS‐ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti‐ADP agent. A3P5PS specifically inhibited 2‐MeS‐ADP‐induced shape change and calcium increase but did not affect adenylyl cyclase down‐regulation. 2‐MeS‐ADP‐induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non‐aggregating compound, therefore suggesting that P2Y1‐mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP‐induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.

Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel

The thienopyridine clopidogrel is not active in vitro and must be administered i.v. or orally, suggesting that metabolism is necessary for activity. To verify whether the effect after i.v. administration was consecutive to recycling by hepatic bile secretion of clopidogrel or its metabolite(s) in the digestive tract, a catheter was implanted in the choledocus of rats, preventing bile and pancreatic secretions from being excreted into the digestive tractus. Two hours after clopidogrel administration (10 mg/kg, i.v.), blood was withdrawn and platelet-rich plasma aggregation was measured after the addition of 5 microM ADP. Clopidogrel treatment was equally efficient for sham-operated and catheterized animals (% inhibition of platelet aggregation: 76% and 59%, respectively) suggesting that the i.v. effect of clopidogrel was independent of re-absorption of biliary-excreted products and consequently that enteric metabolism is not necessary for activity. The antiaggregating activity of clopidogrel in rats before and after functional hepatectomy by a porto-jugular shunt was then studied. A great difference between treated animals was observed 30 min after i.v. administration of 25 mg/kg of clopidogrel. Per cent inhibition of platelet aggregation was 76% and 6% (P less than 0.001) for sham-operated and hepatectomized animals, respectively. Similar results were obtained after intraduodenal administration of clopidogrel, showing that the treatment was completely ineffective in hepatectomized animals. In isolated, blood-perfused rat livers, clopidogrel inhibited ADP-induced platelet aggregation, thereby supporting the theory that the activity of clopidogrel is highly dependent on hepatic metabolism.

An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.

Much discussion has concerned the central role of ADP in platelet aggregation. We now describe a patient (M.L.) with an inherited bleeding disorder whose specific feature is that ADP induces a limited and rapidly reversible platelet aggregation even at high doses. Platelet shape change and other hemostatic parameters were unmodified. A receptor defect was indicated, for, while epinephrine normally lowered cAMP levels of PGE1-treated (M.L.) platelets, ADP was without effect. The binding of [3H]2-methylthio-ADP decreased from 836 +/- 126 molecules/platelet for normals to 30 +/- 17 molecules/platelet for the patient. Flow cytometry confirmed that ADP induced a much lower fibrinogen binding to (M.L.) platelets. Nonetheless, the binding in whole blood of activation-dependent monoclonal antibodies showed that some activation of GP IIb-IIIa complexes by ADP was occurring. Platelets of a patient with type I Glanzmanns thrombasthenia bound [3H]2-methylthio-ADP and responded normally to ADP in the presence of PGE1. Electron microscopy showed that ADP-induced aggregates of (M. L.) platelets were composed of loosely bound shape-changed platelets with few contact points. Thus this receptor defect has a direct influence on the capacity of platelets to bind to each other in response to ADP.

Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors

The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.

The mitogenic effect of H2O2 for vascular smooth muscle cells is mediated by an increase of the affinity of basic fibroblast growth factor for its receptor

Increased generation of active oxygen species such as hydrogen peroxide (H2O2) may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In this work, we showed that H2O2 was a potent mitogen for growth‐arrested cultured human aortic smooth muscle cells (SMC), stimulating an increase in cell number at 10 nM to 100 μM concentration. This effect was inhibited in a dose‐dependent manner by catalase, deferoxamine, dimethylthiourea or probucol showing that it was dependent on the oxidative activity of H2O2. H2O2‐induced SMC proliferation was strongly and specifically inhibited by a neutralizing monoclonal antibody directed against basic fibroblast growth factor (bFGF) but was not due to increased expression of bFGF or the bFGF receptor‐1 (FGFR‐1) by SMC. H2O2 strongly increased the affinity of bFGF for its receptor‐1 at the surface of the SMC, therefore showing that the mitogenic effect of H2O2 might occur through a direct effect on the bFGF receptor.

IL‐4 inhibits LPS‐, IL‐1β‐ and TNFα‐induced expression of tissue factor in endothelial cells and monocytes

Inflammatory mediators such as endotoxin, interleukin‐1β(IL‐1β) and tumor necrosis factors‐α (TNF‐α) dose‐dependently increased the expression of tissue factor on the surface of cultured bovine aortic endothelial cells (ABAE), human umbilical vein endothelial cells (HUVEC) and human monocytes. In ABAE, endotoxin‐, IL‐1β‐ and TNFα‐induced tissue factor expression was suppressed by interleukin‐4 (IL‐4) which also neutralized the pyrogenic effect of endotoxin in HUVEC and monocytes. IL‐4 did not alter TNF‐α‐induced procoagulant changes in HUVEC and monocytes but strongly protected the monocyte surface against IL‐1β‐induced procoagulant changes.

Ultrastructural Studies of Platelet Aggregates From Human Subjects Receiving Clopidogrel and From a Patient With an Inherited Defect of an ADP-Dependent Pathway of Platelet Activation

Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti-ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.

IL‐4 and IL‐13 exhibit comparable abilities to reduce pyrogen‐induced expression of procoagulant activity in endothelial cells and monocytes

Endotoxin (LPS), interleukin‐1β (IL‐1) and tumor necrosis factor‐α (TNF) increased the expression of tissue factor, a membrane‐anchored glycoprotein that initiates blood coagulation on the surface of cultured bovine aortic endothelial cells (ABAE) and human monocytes. These compounds simultaneously reduced the amount of thrombomodulin on the endothelial cell surface, further contributing to the procoagulant activity of the endothelium or monocytes. On endothelial cells and monocytes, interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13), a newly described lymphokine, both strongly inhibited LPS‐induced tissue factor expression, a similar activity also being obtained with regard to the pyrogenic effects of IL‐1 or TNF. When measured in parallel, IL‐4 and IL‐13 counteracted thrombomodulin down‐regulation induced by LPS, IL‐1 or TNF in endothelial cells. These results therefore show that both IL‐4 and IL‐13 protect the endothelial and the monocyte surface against inflammatory mediator‐induced procoagulant changes.

Clopidogrel: An Antithrombotic Drug Acting on the ADP-dependent Activation Pathway of Human Platelets

The aim of the study was to determine the effect of clopidogrel on adenosine diphosphate (ADP)- induced platelet activation in human volunteers. Platelets from human volunteers before and after a 7-day treatment with clopidogrel (75 mg/kg), were tested for their sensi tivity to ADP by measuring ADP-induced aggregation, adenylyl cyclase downregulation, and [3H]-2-MeS-ADP binding. Platelet membrane glycoprotein (GP IIb-IIIa; GP Ib, GMP-140) expression was measured by flow cy tometry using fluorescent-labeled antibodies or fibrino gen. After oral administration to human volunteers (75 mg/day for 7 days), clopidogrel, a novel ADP-selective antiplatelet agent, inhibited ADP-induced aggregation of platelets ex vivo. This effect was irreversible in nature, and no activity could be detected in the plasma of treated subjects. Although clopidogrel did not modify ADP- induced shape change, it prevented the inhibitory effect of ADP (but not that of epinephrine) on the prostoglandin- E 1 (PGE1)-induced increase in platelet cAMP. The num ber of binding sites for [ 3H]-2-MeS-ADP, a stable ana logue of ADP that labels ADP binding sites linked to the inhibition of stimulated adenylyl cyclase, was reduced from 525 ± 62 sites/cell in the controls to 32 ± 5 sites/cell after treatment with clopidogrel (p < 0.001). This effect occurred with no consistent change in the binding affinity of [3H]-2-MeS-ADP, indicating that inhibition of platelet functions by clopidogrel was mainly due to a selective and irreversible reduction of ADP binding sites on plate lets. Flow cytometry experiments showed that clopi dogrel selectively inhibited ADP-inducing binding of fi brinogen to platelets. This effect occurred through a ma jor reduction of the ADP-induced activation of the GP IIb-IIIa complex. These findings therefore indicate that clopidogrel downregulates platelet responses via a selec tive and direct interaction with the ADP receptors, me diating the inhibition of stimulated adenylyl cyclase ac tivity in human platelets.