Pierre H. Mangin

Mechanisms underlying FeCl3-induced arterial thrombosis.

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

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.

The future of glycoprotein VI as an antithrombotic target.

Summary.u2002 The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided.

Preserved functional and biochemical characteristics of platelet components prepared with amotosalen and ultraviolet A for pathogen inactivation

Platelet concentrate (PC) functionality decreases during storage. This is referred to as the storage lesion. Pathogen inactivation may accelerate or induce lesions, potentially accounting for reduced viability. Our aim was to characterize functional and biochemical properties of platelets (PLTs) from photochemically treated buffy‐coat PCs (PCT‐PCs) compared to those from conventional PCs.

Blood coagulation factors bound to procoagulant platelets are concentrated in their cap structures to promote clotting

Binding of coagulation factors to phosphatidylserine (PS)-exposing procoagulant-activated platelets followed by formation of the membrane-dependent enzyme complexes is critical for blood coagulation. Procoagulant platelets formed upon strong platelet stimulation, usually with thrombin plus collagen, are large balloons with a small (∼1 μm radius) cap-like convex region that is enriched with adhesive proteins. Spatial distribution of blood coagulation factors on the surface of procoagulant platelets was investigated using confocal microscopy. All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin V were distributed nonhomogeneously: they were primarily localized in the cap, where their mean concentration was by at least an order of magnitude, higher than on the balloon. Assembly of intrinsic tenase on liposomes with various PS densities while keeping the PS content constant demonstrated that such enrichment can accelerate this reaction by 2 orders of magnitude. The mechanisms of such acceleration were investigated using a 3-dimensional computer simulation model of intrinsic tenase based on these data. Transmission electron microscopy and focal ion beam-scanning electron microscopy with Annexin V immunogold-labeling revealed a complex organization of the caps. In platelet thrombi formed in whole blood on collagen under arterial shear conditions, ubiquitous caps with increased Annexin V, FX, and FXa binding were observed, indicating relevance of this mechanism for surface-attached platelets under physiological flow. These results reveal an essential heterogeneity in the surface distribution of major coagulation factors on the surface of procoagulant platelets and suggest its importance in promoting membrane-dependent coagulation reactions.

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

Summary.u2002 Background:u2002Binding 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:u2002The 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:u2002Our 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.

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.

CD9 negatively regulates integrin αIIbβ3 activation and could thus prevent excessive platelet recruitment at sites of vascular injury

Platelet integrin aIIbb3 plays a central role in hemostasis and thrombosis by supporting platelet adhesion and aggregation at sites of vascular injury [1]. Although, in resting platelets, integrin aIIbb3 does not support binding of its main ligand, soluble fibrinogen, platelet activation results in a conformational change of integrin aIIbb3 that gives it high affinity for fibrinogen. Upon binding of fibrinogen, outside-in signaling leads to intracellular cytoskeletal reorganization, clot retraction, and aggregate stabilization [1–3]. Two members of the tetraspanin superfamily, CD151 and TSSC6, have been reported to physically link integrin aIIbb3 and to regulate its outside-in signaling. Mice deficient in CD151 (CD151) and TSSC6 (TSSC6) present bleeding defects that have been proposed to result from impaired integrin outside-in signaling [4,5]. In addition, TSSC6 mice were protected against experimental arterial thrombosis [4]. CD9, another member of the tetraspanin superfamily, is ubiquitously expressed. This molecule interacts with b1 and b3 integrins, and has been implicated in many cellular functions, including adhesion, apoptosis, tumor metastasis, and fertilization [6,7]. After integrin aIIbb3, CD9 is the secondmost strongly expressed platelet surface protein, with approximately 40 000 copies per platelet [8]. Several studies have reported a physical link between CD9 and integrin aIIbb3 [9–11]. However, despite early reports of proaggregatory effects of anti-CD9 monoclonal antibodies (mAbs), which depend on FcRcIIA signaling, the function of CD9 in platelets remains largely unknown [12]. To investigate the role of platelet CD9 in hemostasis and thrombosis, and its ability to regulate integrin aIIbb3 functions, we performed studies in CD9-deficient (CD9) mice [13]. Hemostasis was evaluated in a tail bleeding assay. The bleeding time was not significantly different between CD9 (median 81 s, n = 15) and wild-type (median 105 s, n = 15; P = 0.24)mice,despitea tendency fora shorterbleeding time in the knockout strain (Fig. 1A). A decreased bleeding tendency was alsoobservedonanalysis of the volumeof blood lost,which was smaller in CD9 (mean 120 ± 63 lL at 30 min, n = 15) than in wild-type (mean 191 ± 68 lL at 30 min, n = 15; P = 0.45) mice (data not shown). These results indicated that CD9 does not play a critical role in primary hemostasis. This behavior differs from that reported in CD151 or TSSC6 deficiency, both of which lead to increased bleeding [4,14]. TheeffectofCD9deficiencyonplateletactivationwasassessed in vitro, first by measuring levels of exposure of P-selectin, a marker of a-granule exocytosis. P-selectin increases were comparable to those innormalmice in response toarangeofagonists, including thrombin, the thromboxane A2 analog U46619, the protease-activated receptor-4agonist peptideAYPGKF,and the glycoprotein VI agonist convulxin (Fig. S1A). In addition, no defect in the rate or extent of platelet aggregationwasobserved in response to ADP (5 lmol L), thrombin (0.1 U mL), U46619 (2 lmol L), type I fibrillar collagen (1 lg mL), or AYPGKF (500 lmol L) (Fig. S1B). Despite normal aggregation, CD9 platelets nevertheless displayed statistically significant increased integrin aIIbb3 activation, as revealed by their capacity to bind fibrinogen, which was increased by 43.7% (n = 3; P = 0.0008), 37% (n = 4; P = 0.031) and 26.4% (n = 4; P = 0.034) in response to ADP (10 lmol L), Collagen-related peptide (CRP) (10 lg mL), and thrombin (0.25 U mL), respectively (Fig. 1B). This increased fibrinogen binding was not due to higher levels of integrin aIIbb3 at the platelet surface, as demonstrated by flow cytometrywith an antiaIIbb3 mAb (data not shown). These results suggested that CD9 could act as a repressor of integrin aIIbb3 inside-out signaling, in contrast to CD151 and TSSC6, which have been proposed to upregulateoutside-insignaling [4,5].AnindicationthatCD9does not participate in outside-in signalingwas the normal capacity of CD9 platelets to retract a fibrin clot (Fig. S2A). Furthermore, thrombin-induced tyrosine phosphorylation of the intracellular domain of integrin b3, which is amore directmeasure of outsidein signaling, was also similar in CD9 and wild-type cells (Fig. S2B) [15]. The above in vitro experiments suggested that CD9 regulates integrin aIIbb3 differently from CD151 and TSSC6, in that it restrains inside-out signaling and that CD9 deficiency may have opposite effects on thrombosis. Correspondence: Pierre Mangin, INSERM UMR_S 949, EFS-Alsace, 10, rue Spielmann, Strasbourg Cedex, F-67065, France. Tel.: +33 388 21 25 25; fax: +33 388 21 25 21. E-mail: pierre.mangin@efs-alsace.fr

Immobilized fibrinogen activates human platelets through glycoprotein VI

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

The role of extracellular matrix stiffness in megakaryocyte and platelet development and function

The extracellular matrix (ECM) is a key acellular structure in constant remodeling to provide tissue cohesion and rigidity. Deregulation of the balance between matrix deposition, degradation, and crosslinking results in fibrosis. Bone marrow fibrosis (BMF) is associated with several malignant and nonmalignant pathologies severely affecting blood cell production. BMF results from abnormal deposition of collagen fibers and enhanced lysyl oxidase‐mediated ECM crosslinking within the marrow, thereby increasing marrow stiffness. Bone marrow stiffness has been recently recognized as an important regulator of blood cell development, notably by modifying the fate and differentiation process of hematopoietic or mesenchymal stem cells. This review surveys the different components of the ECM and their influence on stem cell development, with a focus on the impact of the ECM composition and stiffness on the megakaryocytic lineage in health and disease. Megakaryocyte maturation and the biogenesis of their progeny, the platelets, are thought to respond to environmental mechanical forces through a number of mechanosensors, including integrins and mechanosensitive ion channels, reviewed here.