The Syk-kinase inhibitor R406 impairs platelet activation and monocyte tissue factor expression triggered by heparin-PF4 complex directed antibodies.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a rare but severe complication of heparin therapy in which immunoglobulin G (IgG) antibodies against the platelet factor 4-heparin complex activate platelets through the FcγRIIA receptor. Clustering of FcγRIIA initiates signaling cascades involving tyrosine kinases including the spleen tyrosine kinase (Syk). Moreover, besides the critical role of platelets, the expression of tissue factor (TF) by human monocytes triggered by HIT antibodies has been shown to contribute to the hypercoagulability and the thrombotic complications in HIT patients. OBJECTIVES: We investigated the effect of R406, a small molecule inhibitor of Syk developed as a potential treatment of autoimmune diseases, allergic disorders and B-cell related hematological malignancies, on FcγRIIA-mediated platelet activation. To further assess the potential activity of Syk inhibitors in HIT treatment, the effect of R406 was also evaluated on HIT antibodies-induced expression of TF and procoagulant activity of monocytic cells. RESULTS: We show that R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross-linking by specific antibodies or by sera from HIT patients. Syk inhibition efficiently prevents FcγRIIA-induced LAT phosphorylation and activation of phosphoinositide 3-kinase, Akt, phospholipase Cγ2 and p38 MAP-kinase. As a consequence, FcγRIIA-induced platelet aggregation, granule secretion and microparticles production are strongly inhibited by R406. Moreover, the Syk inhibitor efficiently impairs the expression of TF and the procoagulant activity of human monocytes triggered by HIT antibodies. CONCLUSION: Syk inhibitors may be of therapeutic interest in the treatment of HIT by reducing HIT antibodies-mediated platelet activation and monocyte procoagulant activity.

Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow.

BACKGROUND: Microparticles (MPs) released by activated or apoptotic cells increase in number in the blood of subjects with vascular or metabolic diseases and may contribute to thrombotic complications. OBJECTIVES: In this study, we investigated whether MPs promoted platelet recruitment to endothelial cells in flow conditions, and by which mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) grown in microslide perfusion chambers were exposed to MPs prepared in vitro from HUVECs, monocytes or platelets. RESULTS: Videomicroscopy of DIOC-labelled blood perfused at arterial rate on human umbilical vein ECs demonstrated that, irrespective of their cell origin, MPs promoted the formation of platelet strings at the surface of HUVECs. This platelet/endothelial cell interaction was dependent on von Willebrand factor (VWF) expression at the HUVEC surface and involved Glycoprotein Ib and P-selectin. Interestingly, HUVECs internalized MPs within a few hours through a process involving anionic phospholipids, lactadherin and αvß3 integrin. This uptake generated the production of reactive oxygen species via the xanthine/xanthine oxidase system (inhibited by allopurinol and the ROCK inhibitor Y-27632) and the NADPH oxidase (inhibited by SOD). Reactive oxygen species appeared essential for VWF expression at the endothelial cell surface and subsequent platelet/endothelial cell interaction under flow. The pathophysiological relevance of this process is underlined by the fact that circulating MPs from Type I diabetic patients induced platelet/endothelial cell interaction under flow, with an intensity correlated with the severity of the vasculopathy.

Inhibition of tissue factor synthesis by disruption of ERK kinases and PKC signaling pathways in human vascular SMCs.

Tissue Factor (TF), the receptor for plasma VII/VIIa and the initiator of blood coagulation, is inducible in vascular smooth muscle cells (SMCs) by growth factors and bacterial lysopolysaccharides (LPS) and is expressed in vivo after vascular injury. As TF expression is a determinant of the thrombogenicity of vascular lesions, we investigated the signal pathways involved in this process. Human vascular SMCs were obtained from normal arteries and made quiescent by serum deprivation. Baseline TF antigen and activity were up-regulated by various agonists: fetal calf serum (FCS), LPS, and platelet derived growth factor (PDGF) being the most effective but with different kinetics. TF expression induced by LPS was transient with a maximum 6 h after stimulation and returned to baseline levels after 24 h whereas TF expression induced by serum or PDGF was sustained for at least 24 h. Rapid and transient activation of Extracellular signal-Regulated Kinase (ERK) was observed after stimulation by PDGF and FCS, but not by LPS. The role of ERK, Ras and protein kinase C activities were investigated using specific inhibitors, PD 98059, manumycin A and calphostin C respectively. For TF induction by LPS, PKC activity was required and the ERK/Ras pathway was not involved. In contrast, the effect of PDGF was strictly ERK and Ras dependent, but partially prevented by PKC inhibitors. TF induction by FCS was ERK dependent but partially Ras and PKC dependent. In conclusion, TF expression appears to be a non-specific response of SMCs to numerous stimuli through multiple signal pathways which differ according to the inducing agent.

Effects of lysophosphatidic acid on proliferation and cytosolic Ca++ of human adult vascular smooth muscle cells in culture.

Lysophosphatidic acid (LPA) is a lipid mediator generated by activated platelets and having various effects on numerous cell types. We investigated some effects of 1-oleyl LPA on vascular smooth muscle cells cultured from adult human normal arteries. At micromolar concentrations, LPA induced a mitogenic effect ([3H]-thymidine incorporation and cell proliferation) on quiescent cells, without an additional growth factor being required. This effect was equipotent to that of 10% fetal calf serum, and it was accompanied by early (5 minutes) and late (1-3 hours) phosphorylation of mitogenactivated protein kinase. LPA inhibited cell migration through collagen coated membranes, with or without platelet-derived growth factor BB as chemoattractant. LPA induced a typical biphasic Ca2+ signal response made up of a rapid first phase due to Ca2+ release from intracellular stores followed by a second wave due to external Ca2+ influx. These findings support the proposal that LPA released from activated platelets is a mediator for smooth muscle cell response at the site of vessel injury in humans.

Effects of heparin and related sulfated polysaccharides on tissue factor expression induced by mitogenic and non-mitogenic factors in human vascular smooth muscle cells.

Smooth muscle cells (SMCs) of the intima are generally quiescent and non proliferative. Their proliferation due to different stimulations occurs in myointimal hyperplasia and is regularly present in atherogenesis or after transluminal angioplasty leading to vascular occlusive stenosis. In the course of these pathologies, the Tissue Factor (TF) synthesis was upregulated and rapidly expressed at the membrane of the SMCs. Heparin is known to inhibit SMCs proliferation induced by FCS. We evaluated the inhibitory effect of heparin on the expression of TF induced by various mitogenic (FCS, PDGF-BB and EGF) and non-mitogenic (bacterial LPS) agents. Inhibition by heparin of SMCs proliferation induced by the same agonists was also determined. Quiescent human vascular SMCs from normal adult arteries were treated for 1 h by heparin and related sulfated polysaccharides before stimulation by the agonists. All the agonists up-regulated the expression of TF antigen and activity. TF expression induced by the growth factors was inhibited by heparin (IC 50: 10-30 microg/ml), and other sulfated polysaccharides (IC 50: 1-5 microg/ml). SMCs proliferation, late activation of the extracellular signal-regulated kinases (ERK1/2), and PKC activity were inhibited by heparin (IC 50: 30-50 microg/ml) in SMCs stimulated by FCS but not in SMCs treated by PDGF or EGF. In contrast, heparin had no effect on LPS-induced TF expression nor on LPS-induced PKC activation. These results indicate that, besides its well known effect on SMC proliferation, heparin displays an inhibitory effect on cell mediated blood clotting processes through regulation of the TF expression.

Up-regulation of tissue factor expression by platelet-derived growth factor in human vascular smooth muscle cells in culture--role of mitogen-activated protein kinase and effects of intracellular cyclic AMP.

Human vascular smooth muscle cells (SMCs) isolated from normal adult arteries were investigated for the expression of Tissue Factor (TF) procoagulant activity at their membrane surface and TF antigen in lysed cells. In growing conditions, SMCs expressed high levels of TF activity and antigen. When cells were made quiescent by 72 h of subculture in serum-poor medium, these levels fell to about one third of the initial values. Platelet-derived growth factor BB (PDGF) up-regulated in a dose-dependent manner TF expression with a significant increase (1.8 fold) within five hours. PD 98059, a specific inhibitor of mitogen-activated protein kinase (MAPK) pathway involved in cell response to PDGF, also prevented TF up-regulation. Short-term treatment by Pentoxifylline and dibutyryl cAMP also completely prevented induced TF up-regulation, but remained without effect on baseline levels of quiescent, unstimulated cells. At their effective concentrations, pentoxifylline and dibutyryl cAMP also inhibited the activation of MAPK induced by PDGF. The rapid induction of TF upon growth factor stimulation may be important in circumstances where SMCs proliferate, such as vascular events or vessel development.