Roles of coagulation factor XIII in the functions of blood platelets

Activated blood coagulation factor XIII (FXIIIa) is a transglutaminase that stabilises fibrin clots and associates with platelets. In the present study, the role of factor XIII (FXIII) in modulating physiological platelet functional responses including adhesion, signal transduction and spreading were examined. Under static conditions, platelets adhered to surface-immobilised plasma-purified and recombinant human FXIII leading to the formation of filopodia and lamellipodia. Adhesion to FXIIIa was mediated through integrin-dependent mechanisms, since it was abolished by treatment with RGDS. Moreover, platelet adhesion to FXIIIa was reduced partially, but significantly by either the specific integrin dnbPs antagonist tirofiban or the selective avp3-blocking antibody LM609, and abolished when used in combination. However, spreading was exclusively mediated by OubPs since it was ablated by tirofiban, but unaffected by LM609. Importantly, FXIIIa-mediated platelet accrual was preserved under venous and arterial flow conditions where both integrins played essential roles. Under these conditions, platelet adhesion to immobilised activated FXIII (FXIIIa) was apparent at a shearrate of 300s"1, significantly reduced at 800s"1, but absent above 1000s"1. These platelet-FXIII interactions occurred independently of FXIII transglutaminase and protein disulfide isomerase activities. However, platelet adhesion and spreading were abolished by the Src family inhibitor PP1 indicating a tyrosine kinase-dependent mechanism. Consistent with this, FXIIIa stimulated tyrosine-phosphorylation of several proteins including Syk, SLP-76 and PLCv2 but not LAT, in adherent platelets. FXIIIa immobilised rapidly on collagen, and enhanced collagen-induced thrombus formation at a shear rate of 800s"1 . When co-immobilised with fibrinogen and vWF, the coagulation factor also accentuated platelet accrual by these key platelet adhesive proteins also at arterial shear. These data provide evidence that FXIIIa supports platelet adhesion under flow and potentiates the thrombogenic effects of established platelet ligands, suggesting a novel role for FXIIIa in enhancing platelet-dependent haemostasis

Acute hypertriglyceridemia induces platelet hyperactivity that is not attenuated by insulin in polycystic ovary syndrome.

Atherothrombosis is associated with platelet hyperactivity. Hypertriglyceridemia and insulin resistance (IR) are features of polycystic ovary syndrome (PCOS). The effect of induced hypertriglyceridemia on IR and platelet function was examined in young women with PCOS. Following overnight fasting, 13 PCOS and 12 healthy women were infused with saline or 20% intralipid for 5 hours on separate days. Insulin sensitivity was measured using a hyperinsulinemic euglycaemic clamp in the final 2 hours of each infusion. Platelet responses to adenosine diphosphate (ADP) and prostacyclin (PGI2) were measured by flow cytometric analysis of platelet fibrinogen binding and P-selectin expression using whole blood taken during each infusion (at 2 hours) and at the end of each clamp. Lipid infusion increased triglycerides and reduced insulin sensitivity in both controls (median, interquartile range ) (5.25 [3.3, 6.48] versus 2.60 [0.88, 3.88] mg kg(-1) min(-1), P<0.001) and PCOS (3.15 [2.94, 3.85] versus 1.06 [0.72, 1.43] mg kg(-1) min(-1), P<0.001). Platelet activation by ADP was enhanced and ability to suppress platelet activation by PGI2 diminished during lipid infusion in both groups when compared to saline. Importantly, insulin infusion decreased lipid-induced platelet hyperactivity by decreasing their response to 1 μmol/L ADP (78.7% [67.9, 82.3] versus 62.8% [51.8, 73.3], P=0.02) and increasing sensitivity to 0.01 μmol/L PGI2 (67.6% [39.5, 83.8] versus 40.9% [23.8, 60.9], P=0.01) in controls, but not in PCOS. Acute hypertriglyceridemia induced IR, and increased platelet activation in both groups that was not reversed by insulin in PCOS subjects compared to controls. This suggests that platelet hyperactivity induced by acute hypertriglyceridemia and IR could contribute athero-thrombotic risk. www.isrctn.org. Unique Identifier: ISRCTN42448814

Roles of coagulation factor XIII in the functions of blood platelets

Activated blood coagulation factor XIII (FXIIIa) is a transglutaminase that stabilises fibrin clots and associates with platelets. In the present study, the role of factor XIII (FXIII) in modulating physiological platelet functional responses including adhesion, signal transduction and spreading were examined. Under static conditions, platelets adhered to surface-immobilised plasma-purified and recombinant human FXIII leading to the formation of filopodia and lamellipodia. Adhesion to FXIIIa was mediated through integrin-dependent mechanisms, since it was abolished by treatment with RGDS. Moreover, platelet adhesion to FXIIIa was reduced partially, but significantly by either the specific integrin dnbPs antagonist tirofiban or the selective avp3-blocking antibody LM609, and abolished when used in combination. However, spreading was exclusively mediated by OubPs since it was ablated by tirofiban, but unaffected by LM609. Importantly, FXIIIa-mediated platelet accrual was preserved under venous and arterial flow conditions where both integrins played essential roles. Under these conditions, platelet adhesion to immobilised activated FXIII (FXIIIa) was apparent at a shear rate of 300s"1, significantly reduced at 800s"1, but absent above 1000s"1. These platelet-FXIII interactions occurred independently of FXIII transglutaminase and protein disulfide isomerase activities. However, platelet adhesion and spreading were abolished by the Src family inhibitor PP1 indicating a tyrosine kinase-dependent mechanism. Consistent with this, FXIIIa stimulated tyrosine-phosphorylation of several proteins including Syk, SLP-76 and PLCv2 but not LAT, in adherent platelets. FXIIIa immobilised rapidly on collagen, and enhanced collagen-induced thrombus formation at a shear rate of 800s"1 . When co-immobilised with fibrinogen and vWF, the coagulation factor also accentuated platelet accrual by these key platelet adhesive proteins also at arterial shear. These data provide evidence that FXIIIa supports platelet adhesion under flow and potentiates the thrombogenic effects of established platelet ligands, suggesting a novel role for FXIIIa in enhancing platelet-dependent haemostasis

Thrombospondin-1 induces platelet activation through CD36-dependent inhibition of the cAMP/protein kinase A signaling cascade

Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet function at sites of vascular injury. Here we show that thrombospondin-1 (TSP-1) prevents cAMP/protein kinase A (PKA) signaling through a CD36-dependent mechanism. Prostaglandin E 1 (PGE 1 ) induced a robust inhibition of both platelet aggregation and platelet arrest under physiologic conditions of flow. Exogenous TSP-1 reduced significantly PGE 1 -mediated inhibition of both platelet aggregation and platelet arrest. TSP-1 prevented PGE 1 -stimulated cAMP accrual and phosphorylation of PKA substrates, through a mechanism requiring phosphodiesterase3A. TSP-1 also inhibited VASP phosphorylation stimulated by the nonhydrolyzable cAMP analog, 8-bromocAMP, indicating that it may regulate cAMPmediated activation of PKA. The inhibitory effect of TSP-1 on cAMP signaling could be reproduced with a peptide possessing a CD36 binding sequence of TSP-1, while the effects of TSP-1 were prevented by a CD36 blocking antibody. TSP-1 and the CD36 binding peptide induced phosphorylation of Src kinases, p38 and JNK. More-over, inhibition of Src kinases blocked TSP-1-mediated regulation of cAMP concentrations and the phosphorylation of VASP, indicating that TSP-1 modulated the cAMP/PKA signaling events through a tyrosine kinase-dependent pathway downstream of CD36. These data reveal a new role for TSP-1 in promoting platelet aggregation through modulation of the cAMP-PKA signaling pathway. © 2010 by The American Society of Hematology

Oxidised low-density lipoproteins induce rapid platelet activation and shape change through tyrosine kinase and Rho kinase-signaling pathways

Oxidized low-density lipoproteins (oxLDL) generated in the hyperlipidemic state may contribute to unregulated platelet activation during thrombosis. Although the ability of oxLDL to activate platelets is established, the underlying signaling mechanisms remain obscure. Weshow that oxLDL stimulate platelet activation through phosphorylation of the regulatory light chains of the contractile protein myosin IIa (MLC). oxLDL, but not native LDL, induced shape change, spreading, and phosphorylation of MLC (serine 19) through a pathway that was ablated under conditions that blocked CD36 ligation or inhibited Src kinases, suggesting a tyrosine kinase–dependent mechanism. Consistent with this, oxLDL induced tyrosine phosphorylation of a number of proteins including Syk and phospholipase C g2. Inhibition of Syk, Ca21 mobilization, and MLC kinase (MLCK) only partially inhibited MLC phosphorylation, suggesting the presence of a second pathway. oxLDL activated RhoA and RhoA kinase (ROCK) to induce inhibitory phosphorylation of MLC phosphatase (MLCP). Moreover, inhibition of Src kinases prevented the activation of RhoA and ROCK, indicating that oxLDL regulates contractile signaling through a tyrosine kinase–dependent pathway that induces MLC phosphorylation through the dual activation of MLCK and inhibition of MLCP. These data reveal new signaling events downstream of CD36 that are critical in promoting platelet aggregation by oxLDL

cAMP signaling regulates platelet myosin light chain (MLC) phosphorylation and shape change through targeting the RhoA-Rho kinase-MLC phosphatase signaling pathway

Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet shape change through unknown mechanisms. We examined the effects of cAMP signaling on platelet contractile machinery. Prostaglandin E1 (PGE1)-mediated inhibition of thrombinstimulated shape change was accompanied by diminished phosphorylation of myosin light chain (MLC). Since thrombin stimulates phospho-MLC through RhoA/Rhoassociated, coiled-coil containing protein kinase (ROCK)-dependent inhibition of MLC phosphatase (MLCP), we examined the effects of cAMP on this pathway. Thrombin stimulated the membrane localization of RhoA and the formation of a signaling complex of RhoA/ROCK2/myosin phosphatase-targeting subunit 1 (MYPT1). This resulted in ROCK-mediated phosphorylation of MYPT1 on threonine 853 (thr853), the disassociation of the catalytic subunit protein phosphatase 1δ (PP1d) from MYPT1 and inhibition of basal MLCP activity. Treatment of platelets with PGE1 prevented thrombin-induced phospho-MYPT1-thr853 in a protein kinase A (PKA)-dependent manner. Examination of the molecular mechanisms revealed that PGE1 induced the phosphorylation of RhoA on serine188 through a pathway requiring cAMP and PKA. This event inhibited the membrane relocalization of RhoA, prevented the association of RhoA with ROCK2 and MYPT1, attenuated the dissociation of PP1δ from MYPT1, and thereby restored basal MLCP activity leading to a decrease in phospho-MLC. These data reveal a new mechanism by which the cAMP-PKA signaling pathway regulates platelet function

Acute Hypertriglyceridemia Induces Platelet Hyperactivity That is Not Attenuated by Insulin in Polycystic Ovary Syndrome

BACKGROUND: Atherothrombosis is associated with platelet hyperactivity. Hypertriglyceridemia and insulin resistance (IR) are features of polycystic ovary syndrome (PCOS). The effect of induced hypertriglyceridemia on IR and platelet function was examined in young women with PCOS. METHODS AND RESULTS: Following overnight fasting, 13 PCOS and 12 healthy women were infused with saline or 20% intralipid for 5 hours on separate days. Insulin sensitivity was measured using a hyperinsulinemic euglycaemic clamp in the final 2 hours of each infusion. Platelet responses to adenosine diphosphate (ADP) and prostacyclin (PGI(2)) were measured by flow cytometric analysis of platelet fibrinogen binding and P‐selectin expression using whole blood taken during each infusion (at 2 hours) and at the end of each clamp. Lipid infusion increased triglycerides and reduced insulin sensitivity in both controls (median, interquartile range ) (5.25 [3.3, 6.48] versus 2.60 [0.88, 3.88] mg kg(−1) min(−1), P<0.001) and PCOS (3.15 [2.94, 3.85] versus 1.06 [0.72, 1.43] mg kg(−1) min(−1), P<0.001). Platelet activation by ADP was enhanced and ability to suppress platelet activation by PGI(2) diminished during lipid infusion in both groups when compared to saline. Importantly, insulin infusion decreased lipid‐induced platelet hyperactivity by decreasing their response to 1 μmol/L ADP (78.7% [67.9, 82.3] versus 62.8% [51.8, 73.3], P=0.02) and increasing sensitivity to 0.01 μmol/L PGI(2) (67.6% [39.5, 83.8] versus 40.9% [23.8, 60.9], P=0.01) in controls, but not in PCOS. CONCLUSION: Acute hypertriglyceridemia induced IR, and increased platelet activation in both groups that was not reversed by insulin in PCOS subjects compared to controls. This suggests that platelet hyperactivity induced by acute hypertriglyceridemia and IR could contribute athero‐thrombotic risk. CLINICAL TRIAL REGISTRATION: URL: www.isrctn.org. Unique Identifier: ISRCTN42448814

Oxidized LDL activates blood platelets through CD36/NOX2–mediated inhibition of the cGMP/protein kinase G signaling cascade

Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in dyslipidemic disorders. Although oxLDL stimulates activatory signaling, it is unclear how these events drive accelerated thrombosis. Here, we describe a mechanism for oxLDL- mediated platelet hyperactivity that requires generation of reactive oxygen species (ROS). Under arterial flow, oxLDL triggered sustained generation of platelet intracellular ROS, which was blocked by CD36 inhibitors, mimicked by CD36-specific oxidized phospholipids, and ablated in CD362/2 murine platelets. oxLDL-induced ROS generation was blocked by the reduced NAD phosphate oxidase 2 (NOX2) inhibitor, gp91ds-tat, and absent in NOX22/2 mice. The synthesis of ROS by oxLDL/CD36 required Src-family kinases and protein kinase C (PKC)-dependent phosphorylation and activation of NOX2. In functional assays, oxLDL abolished guanosine 39,59-cyclic monophosphate (cGMP)- mediated signaling and inhibited platelet aggregation and arrest under flow. This was prevented by either pharmacologic inhibition of NOX2 in human platelets or genetic ablation of NOX2 in murine platelets. Platelets from hyperlipidemic mice were also found to have a diminished sensitivity to cGMP when tested ex vivo, a phenotype that was corrected by infusion of gp91ds-tat into the mice. This study demonstrates that oxLDL and hyperlipidemia stimulate the generation of NOX2-derived ROS through a CD36-PKC pathway and may promote platelet hyperactivity through modulation of cGMP signaling

Thrombospondin-1 induces platelet activation through CD36-dependent inhibition of the cAMP/protein kinase A signaling cascade

Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet function at sites of vascular injury. Here we show that thrombospondin-1 (TSP-1) prevents cAMP/protein kinase A (PKA) signaling through a CD36-dependent mechanism. Prostaglandin E(1) (PGE(1)) induced a robust inhibition of both platelet aggregation and platelet arrest under physiologic conditions of flow. Exogenous TSP-1 reduced significantly PGE(1)-mediated inhibition of both platelet aggregation and platelet arrest. TSP-1 prevented PGE(1)-stimulated cAMP accrual and phosphorylation of PKA substrates, through a mechanism requiring phosphodiesterase3A. TSP-1 also inhibited VASP phosphorylation stimulated by the nonhydrolyzable cAMP analog, 8-bromo-cAMP, indicating that it may regulate cAMP-mediated activation of PKA. The inhibitory effect of TSP-1 on cAMP signaling could be reproduced with a peptide possessing a CD36 binding sequence of TSP-1, while the effects of TSP-1 were prevented by a CD36 blocking antibody. TSP-1 and the CD36 binding peptide induced phosphorylation of Src kinases, p38 and JNK. Moreover, inhibition of Src kinases blocked TSP-1-mediated regulation of cAMP concentrations and the phosphorylation of VASP, indicating that TSP-1 modulated the cAMP/PKA signaling events through a tyrosine kinase-dependent pathway downstream of CD36. These data reveal a new role for TSP-1 in promoting platelet aggregation through modulation of the cAMP-PKA signaling pathway