Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.

Protein disulfide isomerase (PDI) catalyzes the oxidation reduction and isomerization of disulfide bonds. We have previously identified an important role for extracellular PDI during thrombus formation in vivo. Here, we show that endothelial cells are a critical cellular source of secreted PDI, important for fibrin generation and platelet accumulation in vivo. Functional PDI is rapidly secreted from human umbilical vein endothelial cells in culture upon activation with thrombin or after laser-induced stimulation. PDI is localized in different cellular compartments in activated and quiescent endothelial cells, and is redistributed to the plasma membrane after cell activation. In vivo studies using intravital microscopy show that PDI appears rapidly after laser-induced vessel wall injury, before the appearance of the platelet thrombus. If platelet thrombus formation is inhibited by the infusion of eptifibatide into the circulation, PDI is IntroductionA considerable body of evidence implicates the oxidation state of labile disulfide bonds in critical hemostatic proteins in regulating the process of thrombus formation. 1 The oxidation state of these bonds is regulated by an enzyme(s) of the thiol isomerase family. Thiol isomerases, including protein disulfide isomerase (PDI), while containing endoplasmic reticulum retention signals, are found extracellularly. Among the cells that secrete PDI and display the enzyme on their surface are platelets and endothelial cells. [2][3][4][5][6][7] The importance of thiol-disulfide balance for platelet function has long been recognized. For example, reduced glutathione and cysteine inhibit platelet aggregation induced by several agonists, while dithiothreitol and ␤-mercaptoethanol promote aggregation. 4 PDI likely plays an important role in maintaining this balance. The levels of both PDI and ERp5, another member of the PDI family, on the platelet surface increase significantly upon agonist stimulation. 4,6 PDI has been implicated in ␣ IIb ␤ 3 and ␣ 2 ␤ 1 activity, 8,9 and glycoprotein Ib ␣ expresses one or more free thiols on the activated platelet surface, but not on resting platelets. 4 Inhibitory anti-PDI antibodies or bacitracin, a nonspecific inhibitor of thiol isomerases, inhibit platelet activation in vitro, suggesting that ␣ IIb ␤ 3 -dependent platelet aggregation and secretion require thiol isomerases. 10 PDI may play a role in the de-encryption of tissue factor. [11][12][13] In contrast, there is less information to support potential roles of extracellular thiol isomerases in the function of endothelial cells. Endothelial cells in culture secrete PDI, which then is bound to the cell surface. 5 A novel thiol isomerase that appears to be endothelial cell specific, EndoPDI or ERp46, has been reported. 14 Recent evidence indicates that the protein disulfide isomerases, ERp46 and ERp57, are present in endothelial cell plasma membrane preparations. 15 Endothelial cells in culture secrete an activity that reduces the size of very large multimers of von Willebrand factor (VW...

Laser-induced vessel wall injury leads to rapid thrombus formation in an animal thrombosis model. The target of laser injury is the endothelium. We monitored calcium mobilization to assess activation of the laser-targeted cells. Infusion of Fluo-4 AM, a calcium-sensitive fluorochrome, into the mouse circulation resulted in dye uptake in the endothelium and circulating hematopoietic cells. Laser injury in mice treated with eptifibatide to inhibit platelet accumulation resulted in rapid calcium mobilization within the endothelium. Calcium mobilization correlated with the secretion of lysosomal-associated membrane protein 1, a marker of endothelium activation. In the absence of eptifibatide, endothelium activation preceded platelet accumu-lation. Laser activation of human umbilical vein endothelial cells loaded with Fluo-4 resulted in a rapid increase in calcium mobilization associated cell fluorescence similar to that induced by adenosine diphosphate (10μM) or thrombin (1 U/mL). Laser activation of human umbilical vein endothelial cells in the presence of corn trypsin inhibitor treated human plasma devoid of platelets and cell microparticles led to fibrin for-mation that was inhibited by an inhibitory monoclonal anti–tissue factor antibody. Thus laser injury leads to rapid endothelial cell activation. The laser activated endothelial cells can support formation of tenase and prothrombinase and may be a source of activated tissue factor as well.

How this redox system is trafficked to and regulated at the cell surface, the identity of extracellular substrates, why so many thiol isomerases are required, and which thiol isomerase functions are necessary are critical unanswered questions in understanding the role of thiol isomerases in thrombus formation.

Signaling by bone morphogenetic proteins (BMPs) plays a central role in early embryonic patterning, organogenesis, and homeostasis in a broad range of species. Chordin, an extracellular antagonist of BMP signaling, is thought to readily diffuse in tissues, thus forming gradients of BMP inhibition that result in reciprocal gradients of BMP signaling. The latter determine cell fates along the embryonic dorsoventral axis. The secreted protein Twisted Gastrulation (TSG) is thought to help shape BMP signaling gradients by acting as a cofactor that enhances Chordin inhibition of BMP signaling. Here, we demonstrate that mammalian Chordin binds heparin with an affinity similar to that of factors known to functionally interact with heparan sulfate proteoglycans (HSPGs) in tissues. We further demonstrate that Chordin binding in mouse embryonic tissues was dependent upon its interaction with cellsurface HSPGs and that Chordin bound to cell-surface HSPGs (e.g. syndecans), but not to basement membranes containing the HSPG perlecan. Surprisingly, mammalian TSG did not bind heparin unless prebound to Chordin and/or BMP-4, although Drosophila TSG has been reported to bind heparin on its own. Results are also presented that indicate that Chordin-HSPG interactions strongly potentiate the antagonism of BMP signaling by Chordin and are necessary for the retention and uptake of Chordin by cells. These data and others regarding Chordin diffusion have implications for the paradigm of how Chordin is thought to regulate BMP signaling in the extracellular space and how gradients of BMP signaling are formed. Transforming growth factor-␤ (TGF-␤)1 -like bone morphogenetic proteins (BMPs) were first isolated from osteogenic extracts of bone, and some BMPs are capable of inducing ectopic bone formation (1-3). In addition to roles in bone and cartilage formation, BMPs are involved in morphogenesis of various soft tissues and organs during development and play central roles in dorsoventral patterning in early embryogenesis (4). Dorsoventral patterning in Xenopus requires BMP-2, -4, and -7 for specification of ventral mesodermal and ectodermal cell fates (5-7), whereas specification of dorsal mesoderm (Spemann's organizer) and neuroectoderm requires inhibition of BMP signaling. Dorsal inhibition of BMP signaling is achieved by secreted BMP antagonists such as Chordin and Noggin, which are expressed in Spemann's organizer (for a review, see Ref. 8).In Drosophila, dorsoventral patterning of the embryonic axis is mediated by the BMP-2/4 homolog Decapentaplegic (DPP) (9) and the BMP-7 homolog Screw (10), whereas Chordin is represented by the homolog Short Gastrulation (SOG) (11,12). Drosophila homologs do not exist for the other vertebrate extracellular antagonists of BMP signaling, such as Noggin and the Dan family of BMP inhibitors (13). In Drosophila, DPP is expressed dorsally, whereas SOG is expressed ventrolaterally. Secreted SOG is thought to inhibit signaling by binding DPP in an extracellular latent complex (14), and it appears that SOG diff...

This study was carried out to explore the effect of piperine-containing food in altering the pharmacokinetics of phenytoin, an anti-epileptic drug with a narrow therapeutic index. A preliminary pharmacokinetic study was carried out in mice by administering phenytoin (10 mg) orally, with or without piperine (0.6 mg). Subsequently, oral pharmacokinetics of phenytoin was carried out in six healthy volunteers in a crossover design. Phenytoin tablet (300 mg) was given 30 minutes after ingestion of a soup (melahu rasam) with or without black pepper. A further study of intavenous pharmacokinetics of phenytoin (1 mg) in rats with or without oral pretreatment with piperine (10 mg) was also conducted. The phenytoin concentration in the serum was analyzed by HPLC. The study showed a significant increase in the kinetic estimates of Ka, AUC(0-10) and AUC(0-infinity) in the piperine-fed mice. Similarly, in human volunteers piperine increased Ka, AUC(0-48), AUC(0-infinity), and delayed elimination of phenytoin. Intravenous phenytoin in the oral piperine-treated rat group showed a significant alteration in the elimination phase indicating its metabolic blockade. The significance of this finding in epileptic patients maintained on phenytoin therapy requires further investigation. This study may also have implications in the case of other drugs having a low therapeutic index.

Key Points Hermansky-Pudlak syndrome exhibits impaired granule exocytosis and PDI secretion that contribute to its associated bleeding disorder. Endothelial cells deficient in HPS6 show defective secretion of granules, including Weibel-Palade bodies.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (β6 Glu → Val) on the β-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.