A New Animal Model of Thrombophilia Confirms that High Plasma Factor VIII Levels Are Thrombogenic

108

130

The relationship between low-density lipoprotein receptor-related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophagespecific deficiency of LRP1 (macLRP1 ؊ ) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1 ؊ mice compared with control mice (1.6 ؎ 0.4 vs 1.0 ؎ 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1 ؊ mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1 ؊ mice did not result in further rise of VWF levels (0.1 ؎ 0.2 U/mL), in contrast to macLRP1 ؉ mice (rise in VWF, 0.8 ؎ 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWFcoated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm 2 , implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving ␤2-integrins that binds both VWF and LRP1 also is implicated because inhibition of ␤2-integrins led to increased VWF levels in control (rise, 0.19 ؎ 0.16 U/mL) but not in macLRP1 ؊ mice (0.08 ؎ 0.15 U/mL). (Blood. 2012; 119(9):2126-2134) Introductionvon Willebrand factor (VWF) is a hemostatic protein, the physiologic relevance of which is illustrated by the severe bleeding tendency associated with its functional deficiency. The contribution of VWF to hemostasis is 2-fold: (1) VWF is essential for the recruitment of platelets to the damaged vessel wall, particularly under conditions of arterial shear; and (2) VWF functions as a carrier protein for factor VIII (FVIII), a protein cofactor critical to the coagulation system.Whereas biosynthesis and secretion of VWF have been subject of study for more than 35 years, it is only in the last decade that clearance mechanisms of VWF have gained attention. 1,2 This has led to the discovery that increased clearance of VWF may explain part of the reduced VWF levels in von Willebrand disease (VWD). [3][4][5][6] This seems to be most prominent in case of VWDtype 1, although VWD-type 2 variants also are associated with reduced survival of the mutated VWF molecules. 2,7,8 The increased attention is further related to the development of novel therapeutic FVIII concentrates that are used in the treatment of hemophilia A. Given that FVIII circulates in a tight complex with VWF, it is already known that VWF is a major determinant of FVIII clearance. Indeed, the half-life of FVIII is considerably reduced in patients lacking VWF antigen or in those in which VWF is unable to bind FVIII correctly (VWD-type 2N). 9,10 Moreover, preinfusion VWF levels are positively correlated with FVIII half-life. 11,12 To this end, we have recently demonstrate...

Section: Clearance Of Fviii and Vwf Is Modulated After Inactivation Omentioning

confidence: 91%

Macrophage LRP1 contributes to the clearance of von Willebrand factor

Rastegarlari

Pegon

Casari

et al. 2012

108

130

“…13,14 In coagulation, VWF can have a carrier function for factor VIII (FVIII) and supply this factor to a growing thrombus. 15,16 Other authors propose a coagulation-stimulating role of GpIb by interaction with factor VIIa or thrombin 17,18 or a function of GpIb in fibrin binding to ␣ IIb ␤ 3 , VWF, or thrombin. [19][20][21] Hence, the GpIb-V-IX complex seems to be a central component in the interaction of platelets with VWF, thrombin and the coagulation end-product, fibrin.…”

Section: Introductionmentioning

confidence: 99%

Key role of glycoprotein Ib/V/IX and von Willebrand factor in platelet activation-dependent fibrin formation at low shear flow

Cosemans¹,

Schols

Stefanini

et al. 2011

A microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250 ؊1 ) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca 2؉ , morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by ␣ IIb ␤ 3 blockage, but it was greatly delayed and IntroductionPlatelets have prominent and divergent roles in the stimulation of blood coagulation. In vivo and in vitro evidence indicates that platelet-associated tissue factor triggers the extrinsic coagulation pathway. 1,2 In the presence of collagen, platelets contribute to the intrinsic, factor XII-dependent pathway of coagulation. 3,4 Furthermore, platelets stimulated by combinations of potent platelet agonists produce large amounts of thrombin because of exposure of phosphatidylserine (PS). This negatively charged phospholipid provides a surface on which the tenase and prothrombinase complexes assemble to produce factor Xa and thrombin, respectively. 5,6 Such platelets have been termed procoagulant or coated platelets; the latter designation refers to their property to assemble a protein coat consisting of granule-derived proteins, coagulation factors, fibrin(ogen), and von Willebrand factor (VWF). 7,8 Under static conditions, the procoagulant platelet response is suppressed by ␣ IIb ␤ 3 antagonists, 9,10 suggesting that this integrin has a regulating role in the coagulation process.The glycoprotein Ib-V-IX (GpIb-V-IX) complex, expressed at more than 20 000 copies per platelet, can act as a ligand for VWF and various coagulation factors. Quantitative or qualitative abnormalities either in the GpIb-V-IX complex (in Bernard-Soulier syndrome) or in VWF (in von Willebrand disease [VWD]) are accompanied by impaired hemostasis and increased bleeding risk. 11,12 Although this points to a general role of GpIb-V-IX in platelet function, the signaling consequences of VWF-GpIb-V-IX interaction have mostly been studied under flow at high arterial shear rates. 11 However, there is limited evidence for a role of VWF and GpIb in the coagulation process in the absence of high-shear flow. It has been reported that VWF in the presence of fibrin stimulates GpIb-dependent thrombin generation under semistatic conditions. 13,14 In coagulation, VWF can have a carrier function for factor VIII (FVIII) and supply this factor to a growing thrombus. 15,16 Other authors propose a coagulation-stimulating role of GpIb by interaction with factor VIIa or thrombin 17,18 or a function of GpIb in fibrin binding to ␣ IIb ␤ 3 , VWF, or thrombin. [19][20][21] Hence, the GpIb-V-IX complex seems to be a central component in the interaction of platelets with VWF, thrombin and the coagulation end-product, fibrin. However, it is unclear whether the GpIb-V-IX complex merely acts as an assembling component of these plasma factors or also has an active role in ...

“…This finding is in agreement with previous reports that GPIb of murine platelets can interact with human vWF. 22,23 Platelet aggregation study Platelet aggregation was monitored by measuring light transmission with the use of an AG-10 aggregation analyzer (Kowa, Tokyo, Japan). The instrument was calibrated with either the washed platelet suspension or platelet-rich plasma (PRP) for zero light transmission and with buffer or platelet-poor plasma (PPP) for 100% transmission, respectively.…”

mentioning

confidence: 99%

Role of Fc receptor γ-chain in platelet glycoprotein Ib–mediated signaling

Suzuki‐Inoue

Satoh

et al. 2001

117

126

Introductionvon Willebrand factor (vWF) is a multimeric protein that mediates platelet adhesion to exposed subendothelium at sites of vascular injury. The adhesive property of vWF is tightly regulated so that plasma vWF does not normally interact with circulating platelets. However, after vWF is activated by binding to damaged vessel walls, it serves to bridge the constituents of subendothelium to glycoprotein Ib (GPIb) on the membrane of circulating platelets. 1,2 Although much is known about the molecular basis of the interaction between vWF and GPIb, little has been clarified about the intracellular signal transduction pathway in GPIb-mediated platelet activation.Events related to protein-tyrosine phosphorylation have emerged as important signals mediated by GPIb. [3][4][5][6][7] GPIb-mediated platelet activation in response to vWF plus botrocetin, shear stress, or vWF from patients with von Willebrand disease type IIb induces tyrosine phosphorylation of multiple proteins, [3][4][5] suggesting that the binding of vWF to GPIb causes the activation of tyrosine kinases. Studies have shown that the activation of Syk and Src and their association can be induced by GPIb stimulation. [6][7][8][9][10] To date, there is no report showing that tyrosine residues within the GPIb molecule can be phosphorylated, nor has GPIb itself intrinsic kinase activity. Thus, how clustering of GPIb induced by vWF mediates the activation of tyrosine kinases such as Src and Syk has become an important issue. Because Syk is activated by engagement of its tandem SH2 domains with phosphorylated tyrosine residues in proteins containing the immunoreceptor tyrosine-based activation motif (ITAM), 11 it is of interest whether ITAM-containing transmembrane molecules are also involved in GPIb signaling. Two ITAM-containing proteins have been identified in platelets, the low-affinity receptor for immunoglobulin (Ig) G, Fc␥RIIA, and the Fc receptor ␥-chain (FcR ␥-chain). Of particular note is the finding that Fc␥RIIA is physically associated with GPIb and that it mediates some signaling events. 12,13 However, the level of Fc␥RIIA tyrosine phosphorylation induced by GPIb clustering is much lower than that inducible by Fc␥RIIA clustering. Further, murine platelets lacking Fc␥RIIA on their membrane appear to have normal responses to GPIbrelated signals. Thus, to what extent Fc␥RIIA contributes to the GPIb signaling pathway remains to be determined.The other ITAM-containing protein, FcR ␥-chain, has been recognized for its role in signaling related to the high-affinity receptors for IgE (Fc⑀RI) 14 and IgG (Fc␥RI), 15 and the low-affinity IgG receptor (Fc␥RIII). 16 In platelets, FcR ␥-chain is colocalized with GPVI and is critical for GPVI-mediated platelet activation. 17,18 Clustering and activation of GPVI induces tyrosine phosphorylation of FcR ␥-chain ITAM, 19 which then facilitates the recruitment of Syk, resulting in its activation. 20 Moreover, FcR ␥-chain is required for tyrosine phosphorylation of phospholipase C ␥2 (PLC␥2). 20 Recently, Falati...