F. Lanza

Mechanisms underlying FeCl3-induced arterial thrombosis.

See also Brill A. A ride with ferric chloride. This issue, pp 776–8.

Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization

Summary.  Background: We previously described a model of laser‐induced thrombosis in mesenteric arterioles with superficial and deep levels of injury producing a transient thrombus resolving within 2 min and a larger almost occlusive thrombus, respectively. Both types of lesion were sensitive to platelet GPIIb‐IIIa and P2Y12 inhibition, whereas only deep injuries were sensitive to thrombin blockade. Objective: The aim of the present study was to use histologic methods and electron and intravital microscopy to characterize the lesions and thrombi and to extend our knowledge of the sensitivity of this model to genetic and pharmacologic inhibition. Results: A superficial injury was found to detach the endothelial cells and expose a collagen III‐ and IV‐rich subendothelium where platelets could adhere. Tissue factor and fibrin were not detected. Deeper penetration of the external elastic lamina occurred in deep injuries, with exposure of collagen I, III and IV. Here the thrombus was composed of platelets exhibiting a decreasing gradient of degranulation from the deepest lesion area to the surface. Fibrin was found close to the most activated platelets. Consistently, glycoprotein VI (GPVI)‐collagen and GPIb–von Willebrand factor (VWF) interactions were found to be critical in superficial injuries. After deep lesion, thrombus formation was modestly reduced in GPVI‐immunodepleted mice and still strongly inhibited in VWF−/− mice. Combined hirudin infusion and GPVI depletion further inhibited thrombosis after deep injury. Conclusions: This study confirms the feasibility of inducing arterial thrombosis with distinct levels of severity and establishes the central roles of collagen and VWF in thrombus formation after superficial injury. Collagen, VWF and thrombin all appear to contribute to thrombosis after deep arterial lesion.

Spectrum of the Mutations in Bernard–Soulier Syndrome

Bernard–Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb‐IX‐V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbβ), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.

Integrin α6β1 Is the Main Receptor for Vascular Laminins and Plays a Role in Platelet Adhesion, Activation, and Arterial Thrombosis

Background— Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (&agr;1&bgr;1&ggr;1) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the &agr;2, &agr;4, or &agr;5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown. Methods and Results— Using an in vitro flow assay, we show that laminin-411 (&agr;4&bgr;1&ggr;1), laminin-511 (&agr;5&bgr;1&ggr;1), and laminin-521 (&agr;5&bgr;2&ggr;1), but not laminin-211 (&agr;2&bgr;1&ggr;1), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin &agr;6&bgr;1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by &agr;5-containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin &agr;6. Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. &agr;6&bgr;1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis. Conclusions— This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin &agr;6&bgr;1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice

See also Ravid K. The value of a native milieu: mutated non‐muscle myosin IIA does lead to thrombocytopenia. This issue, pp 2241–2.

Myosin IIA is critical for organelle distribution and F-actin organization in megakaryocytes and platelets

During proplatelet formation, a relatively homogeneous content of organelles is transported from the megakaryocyte (MK) to the nascent platelets along microtubule tracks. We found that platelets from Myh9(-/-) mice and a MYH9-RD patient were heterogeneous in their organelle content (granules and mitochondria). In addition, Myh9(-/-) MKs have an abnormal cytoplasmic clustering of organelles, suggesting that the platelet defect originates in the MKs. Myosin is not involved in the latest stage of organelle traffic along microtubular tracks in the proplatelet shafts as shown by confocal observations of proplatelet buds. By contrast, it is required for the earlier distribution of organelles within the large MK preplatelet fragments shed into the sinusoid circulation before terminal proplatelet remodeling. We show here that F-actin is abnormally clustered in the cytoplasm of Myh9(-/-) MKs and actin polymerization is impaired in platelets. Myosin IIA is required for normal granule motility and positioning within MKs, mechanisms that may be dependent on organelle traveling and tethering onto F-actin cytoskeleton tracks. Altogether, our results indicate that the distribution of organelles within platelets critically depends on a homogeneous organelle distribution within MKs and preplatelet fragments, which requires myosin IIA.

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

Summary.  Background: Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib–IX complex initiates a signaling cascade leading to integrin αIIbβ3 activation, a key process in hemostasis and thrombosis. Interaction of 14‐3‐3ζ with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. Objective: The aim of our study was to determine whether other members of the 14‐3‐3 family bind to GPIb–IX. Results: In this study, western blot analyses showed that platelets also contain the 14‐3‐3β, 14‐3‐3γ, 14‐3‐3ε, 14‐3‐3η and 14‐3‐3θ isoforms, but lack 14‐3‐3σ. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14‐3‐3 isoforms expressed in platelets, including, as previously reported, 14‐3‐3ζ, bind to GPIb–IX. In addition, their interaction was found to critically require the same GPIbα domains (580–590 and 605–610) already identified as essential for 14‐3‐3ζ binding, in agreement with the conservation of the sequence of the I‐helix among these different isoforms. Pull‐down experiments indicated that all six 14‐3‐3 isoforms present in platelets bind to GPIbβ. In contrast, deletion or mutation of the GPIbβ intracytoplasmic tail did not affect the interaction of GPIb–IX with the 14‐3‐3 isoforms, questioning the importance of this domain. Conclusions: Our study suggests that, to inhibit GPIb‐induced integrin αIIbβ3 activation, a more appropriate strategy than inhibiting individual 14‐3‐3 isoforms would be to target the 14‐3‐3‐binding motif on GPIb or, alternatively, the conserved 14‐3‐3 I‐helix.

Broader expression of the mouse platelet factor 4‐cre transgene beyond the megakaryocyte lineage

Transgenic mice expressing cre recombinase under the control of the platelet factor 4 (Pf4) promoter, in the context of a 100‐kb bacterial artificial chromosome, have become a valuable tool with which to study genetic modifications in the platelet lineage. However, the specificity of cre expression has recently been questioned, and the time of its onset during megakaryopoiesis remains unknown.

Targeting Platelet GPIbβ Reduces Platelet Adhesion, GPIb Signaling and Thrombin Generation and Prevents Arterial Thrombosis

Objective—The glycoprotein (GP) Ib-V-IX complex regulates the adhesion, activation, and procoagulant activity of platelets. We previously reported that RAM.1, a rat monoclonal antibody directed against the extracellular domain of mouse GPIb&bgr;, diminished adhesion of platelets and chinese hamster ovary cells transfected with the human GPIb-IX complex to von Willebrand factor under flow conditions. Here, we further evaluated the functional importance of GPIb&bgr; by studying the impact of RAM.1 on GPIb-mediated platelet responses and in vitro and in vivo thrombus formation. Approach and Results—We show that RAM.1 dramatically reduced GPIb-mediated filopodia extension of chinese hamster ovary GPIb-IX cells after adhesion to von Willebrand factor. RAM.1 also reduced filopodia extension and GPIb-mediated Ca2+ signaling after adhesion of mouse platelets to von Willebrand factor. RAM.1 inhibited thrombin generation in platelet-rich plasma without impairing phosphatidylserine exposure. In addition, RAM.1 reduced thrombus formation after perfusion of mouse whole blood over collagen in a shear-dependent manner. This effect was confirmed in vivo, because injection of F(ab)′2 fragments of RAM.1 diminished thrombus formation induced by laser beam injury of mesenteric arterioles and forceps injury of the abdominal aorta. In contrast, RAM.1 F(ab)′2 did not prolong the tail-bleeding time or increase the volume of blood lost. Conclusions—These findings are the first evidence that targeting a subunit other than GPIb&agr; can lead to an antithrombotic effect via the GPIb-V-IX complex. This could represent an alternative way to reduce thrombus formation with a minor impact on hemostasis.

Inhibition of adhesive and signaling functions of the platelet GPIb‐V‐IX complex by a cell penetrating GPIbα peptide

Summary.  Background: Interaction between the platelet glycoprotein (GP)Ib‐V‐IX complex and von Willebrand factor (VWF) is critical for initiating platelet‐vessel wall contacts, particularly under high shear conditions. This interaction also plays an important role in initiating platelet activation through the generation of intracellular signals resulting in platelet shape change and integrin αIIbβ3 activation. Objective: A cell‐penetrating peptide strategy was used to study the role of the intracellular domain of the GPIbα subunit in VWF/GPIb‐V‐IX‐dependent adhesion and activation. Methods: Peptides of 11–13 amino acids, covering the 557–610 region, were coupled to a nine‐arginine permeating tag (R9) and the effects of their cell entry on VWF‐dependent responses were analyzed. Results: The R9α557 peptide corresponding to the 557–569 segment reduced platelet agglutination in response to VWF, while the other peptides had no effect. The decreased platelet agglutination appeared to be an indirect consequence of adenosine diphosphate release as a normal response was restored by apyrase or a P2Y1 receptor antagonist. A more direct effect of R9α557 on GPIb VWF‐dependent functions was observed in adhesion studies on a VWF matrix, where it decreased platelet adhesion and profoundly inhibited filopodia formation. In addition, cell adhesion was reduced and shape change absent when Chinese hamster ovary cells expressing the GPIb‐IX complex were incubated with R9α557. Conclusion: This study performed in intact platelets suggests a functional role of the 557–569 domain of GPIbα in controlling VWF‐dependent adhesion and signaling.