Johan W. M. Heemskerk

Glycoprotein VI but not α2β1 integrin is essential for platelet interaction with collagen

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post‐traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin α2β1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP‐mediated loss of β1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of β1‐null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, β1‐null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s−1) and high (1000 s−1) shear flow conditions, probably through binding of αIIbβ3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet‐collagen interactions by activating different adhesive receptors, including α2β1 integrin, which strengthens adhesion without being essential.

Nebivolol: A Third-Generation beta-Blocker That Augments Vascular Nitric Oxide Release : Endothelial beta(2)-Adrenergic Receptor-Mediated Nitric Oxide Production.

BACKGROUND Nebivolol is a beta(1)-selective adrenergic receptor antagonist with proposed nitric oxide (NO)-mediated vasodilating properties in humans. In this study, we explored whether nebivolol indeed induces NO production and, if so, by what mechanism. We hypothesized that not nebivolol itself but rather its metabolites augment NO production. METHODS AND RESULTS Mouse thoracic aorta segments were bathed in an organ chamber. Administration of nebivolol did not affect NO production. When nebivolol was allowed to metabolize in vivo in mice, addition of plasma of these mice caused a sustained 2-fold increase in NO release. Interestingly, coadministration of a selective beta(2)-adrenergic receptor antagonist (butoxamine) prevented the response. Immunohistochemistry and Western blot analysis demonstrated the presence of beta(2)- but not beta(1)-adrenergic receptors on endothelial cells. In the absence of calcium, metabolized nebivolol failed to increase NO production, suggesting a role for calcium-dependent NO synthase. With digital fluorescence imaging, a rapid and sustained rise in endothelial cytosolic free Ca(2+) concentration was observed after administration of metabolized nebivolol, which also was abrogated by butoxamine pretreatment. CONCLUSIONS In vivo metabolized nebivolol increases vascular NO production. This phenomenon involves endothelial beta(2)-adrenergic receptor ligation, with a subsequent rise in endothelial free [Ca(2+)](i) and endothelial NO synthase-dependent NO production. This may be an important mechanism underlying the nebivolol-induced, NO-mediated arterial dilation in humans.

Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis

CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l(-/-)) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l(-/-) platelets into Apoe(-/-) mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l(-/-) platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l(-/-) platelets in contrast to Cd40l(+/+) platelets did not promote lesion formation. Remarkably, injection of Cd40l(+/+), but not Cd40l(-/-), platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l(-/-) platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions.

Platelet Inhibition by Insulin Is Absent in Type 2 Diabetes Mellitus

Objective—ADP-induced P2y12 signaling is crucial for formation and stabilization of an arterial thrombus. We demonstrated recently in platelets from healthy subjects that insulin interferes with Ca2+ increases induced by ADP-P2y1 contact through blockade of the G-protein Gi, and thereby with P2y12-mediated suppression of cAMP. Methods and Results—Here we show in patients with type 2 diabetes mellitus (DM2) that platelets have lost responsiveness to insulin leading to increased adhesion, aggregation, and procoagulant activity on contact with collagen. Using Ser473 phosphorylation of protein kinase B as output for insulin signaling, a 2-fold increase is found in insulin-stimulated normal platelets, but in DM platelets there is no significant response. In addition, DM2 platelets show increased P2y12-mediated suppression of cAMP and decreased P2y12 inhibition by the receptor antagonist AR-C69931MX. Conclusion—The loss of responsiveness to insulin together with increased signaling through P2y12 might explain the hyperactivity of platelets in patients with DM2.

Dual role of collagen in factor XII–dependent thrombus formation

In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cgamma2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCgamma2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

Platelet-based coagulation: different populations, different functions

Summary.  Platelets in a thrombus interact with (anti)coagulation factors and support blood coagulation. In the concept of cell‐based control of coagulation, three different roles of platelets can be distinguished: control of thrombin generation, support of fibrin formation, and regulation of fibrin clot retraction. Here, we postulate that different populations of platelets with distinct surface properties are involved in these coagulant functions. Platelets with elevated Ca2+ and exposed phosphatidylserine control thrombin and fibrin generation, while platelets with activated αIIbβ3 regulate clot retraction. We review how coagulation factor binding depends on the platelet activation state. Furthermore, we discuss the ligands, platelet receptors and downstream intracellular signaling pathways implicated in these coagulant functions. These insights lead to an adapted model of platelet‐based coagulation.

PKCα regulates platelet granule secretion and thrombus formation in mice

Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCalpha and PKCbeta and novel isoforms PKCdelta and PKCtheta. Here, we used a genetic approach to definitively demonstrate the role played by PKCalpha in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca-/- mice, and PKCalpha was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca-/- platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCalpha expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca-/- mice. In summary, the effects of PKCalpha ablation on thrombus formation and granule secretion may implicate PKCalpha as a drug target for antithrombotic therapy.

Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1.

In the absence of PLD1, platelets do not form stable aggregates under high shear conditions. Aggregation Regulation When damage occurs to the endothelium lining a blood vessel and exposes the underlying extracellular matrix, platelets adhere to the site of injury and aggregate to stop blood loss. However, aggregated platelets can cause ischemia if they occlude the vessel, thus creating the need for therapies that can limit platelet aggregation without increasing blood loss. Elvers et al. found that platelets from mice deficient in phospholipase D1 (PLD1) showed reduced activation of αIIbβ3 integrin, a major adhesion receptor, and did not form stable aggregates when experiencing high shear forces (such as those found in small arterioles). PLD1 deficiency conferred protection against thrombosis and cerebral ischemia in vivo, an effect that was seen with Pld1−/− mice and wild-type mice transplanted with bone marrow from Pld1−/− mice. PLD1 deficiency did not, however, increase blood loss after tail wounding. Thus, PLD1 could be a potential therapeutic target to prevent or treat stroke or other ischemic conditions. Platelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesive receptor, αIIbβ3 integrin, from a low- to high-affinity state for its ligands, a process that enables adhesion and aggregation. In response to stimulation of heterotrimeric guanosine triphosphate–binding protein or immunoreceptor tyrosine-based activation motif–coupled receptors, phospholipases cleave membrane phospholipids to generate lipid and soluble second messengers. An essential role in platelet activation has been established for phospholipase C (PLC) but not for PLD and its product phosphatidic acid. Here, we report that platelets from Pld1−/− mice displayed impaired αIIbβ3 integrin activation in response to major agonists and defective glycoprotein Ib–dependent aggregate formation under high shear conditions. These defects resulted in protection from thrombosis and ischemic brain infarction without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.

Mildly Oxidized Low Density Lipoprotein Induces Contraction of Human Endothelial Cells through Activation of Rho/Rho Kinase and Inhibition of Myosin Light Chain Phosphatase

Mildly oxidized low density lipoprotein (mox-LDL) is critically involved in the early atherogenic responses of the endothelium and increases endothelial permeability through an unknown signal pathway. Here we show that (i) exposure of confluent human endothelial cells (HUVEC) to mox-LDL but not to native LDL induces the formation of actin stress fibers and intercellular gaps within minutes, leading to an increase in endothelial permeability; (ii) mox-LDL induces a transient decrease in myosin light chain (MLC) phosphatase that is paralleled by an increase in MLC phosphorylation; (iii) phosphorylated MLC stimulated by mox-LDL is incorporated into stress fibers; (iv) cytoskeletal rearrangements and MLC phosphorylation are inhibited by C3 transferase from Clostridium botulinum, a specific Rho inhibitor, and Y-27632, an inhibitor of Rho kinase; and (v) mox-LDL does not increase intracellular Ca2+concentration. Our data indicate that mox-LDL induces endothelial cell contraction through activation of Rho and its effector Rho kinase which inhibits MLC phosphatase and phosphorylates MLC. We suggest that inhibition of this novel cell signaling pathway of mox-LDL could be relevant for the prevention of atherosclerosis.

Atherosclerotic geometries exacerbate pathological thrombus formation poststenosis in a von Willebrand factor-dependent manner

Rupture of a vulnerable atherosclerotic plaque causes thrombus formation and precipitates cardiovascular diseases. In addition to the thrombogenic content of a plaque, also the hemodynamic microenvironment plays a major role in thrombus formation. How the altered hemodynamics around a plaque promote pathological thrombus formation is not well understood. In this study, we provide evidence that plaque geometries result in fluid mechanical conditions that promote platelet aggregation and thrombus formation by increased accumulation and activity of von Willebrand factor (vWF) at poststenotic sites. Resonant-scanning multiphoton microscopy revealed that in vivo arterial stenosis of a damaged carotid artery markedly increased platelet aggregate formation in the stenotic outlet region. Complementary in vitro studies using microfluidic stenotic chambers, designed to mimic the flow conditions in a stenotic artery, showed enhanced platelet aggregation in the stenotic outlet region at 60–80% channel occlusion over a range of input wall shear rates. The poststenotic thrombus formation was critically dependent on bloodborne vWF and autocrine platelet stimulation. In stenotic chambers containing endothelial cells, flow provoked increased endothelial vWF secretion in the stenotic outlet region, contributing to exacerbated platelet aggregation. Taken together, this study identifies a role for the shear-sensitive protein vWF in transducing hemodynamic forces that are present around a stenosis to a prothrombogenic microenvironment resulting in spatially confined and exacerbated platelet aggregation in the stenosis outlet region. The developed stenotic microfluidic chamber offers a realistic platform for in vitro evaluation of shear-dependent thrombus formation in the setting of atherosclerosis.