Harshal Nandurkar

Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation

Extracellular nucleotides play an important role in thrombosis and inflammation, triggering a range of effects such as platelet activation and recruitment, endothelial cell activation, and vasoconstriction. CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase), converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inflammatory mediator adenosine. Deletion of CD39 renders mice exquisitely sensitive to vascular injury, and CD39-null cardiac xenografts show reduced survival. Conversely, upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inflammation. In this study we examined the consequences of transgenic expression of human CD39 (hCD39) in mice. Importantly, these mice displayed no overt spontaneous bleeding tendency under normal circumstances. The hCD39 transgenic mice did, however, exhibit impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism. Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived longer in a mouse cardiac transplant model of vascular rejection. These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates.

The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

Ibrutinib inhibits collagen-mediated but not ADP-mediated platelet aggregation

The BTK (Bruton’s tyrosine kinase) inhibitor ibrutinib is associated with an increased risk of bleeding. A previous study reported defects in collagen- and adenosine diphosphate (ADP)-dependent platelet responses when ibrutinib was added ex vivo to patient samples. Whereas the collagen defect is expected given the central role of BTK in glycoprotein VI signaling, the ADP defect lacks a mechanistic explanation. In order to determine the real-life consequences of BTK platelet blockade, we performed light transmission aggregometry in 23 patients receiving ibrutinib treatment. All patients had reductions in collagen-mediated platelet aggregation, with a significant association between the degree of inhibition and the occurrence of clinical bleeding or bruising (P=0.044). This collagen defect was reversible on drug cessation. In contrast to the previous ex vivo report, we found no in vivo ADP defects in subjects receiving standard doses of ibrutinib. These results establish platelet light transmission aggregometry as a method for gauging, at least qualitatively, the severity of platelet impairment in patients receiving ibrutinib treatment.

Identification of myotubularin as the lipid phosphatase catalytic subunit associated with the 3-phosphatase adapter protein, 3-PAP

Myotubularin is a dual-specific phosphatase that dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol (3,5)-bisphosphate. Mutations in myotubularin result in the human disease X-linked myotubular myopathy, characterized by persistence of muscle fibers that retain an immature phenotype. We have previously reported the identification of the 3-phosphatase adapter protein (3-PAP), a catalytically inactive member of the myotubularin gene family, which coprecipitates lipid phosphatidylinositol 3-phosphate-3-phosphatase activity from lysates of human platelets. We have now identified myotubularin as the catalytically active 3-phosphatase subunit interacting with 3-PAP. A 65-kDa polypeptide, coprecipitating with endogenous 3-PAP, was purified from SDS/PAGE, subjected to trypsin digestion, and analyzed by collision-induced dissociation tandem MS. Three peptides derived from human myotubularin were identified. Association between 3-PAP and myotubularin was confirmed by reciprocal coimmunoprecipitation of both endogenous and recombinant proteins expressed in K562 cells. Recombinant myotubularin localized to the plasma membrane, causing extensive filopodia formation. However, coexpression of 3-PAP with myotubularin led to attenuation of the plasma membrane phenotype, associated with myotubularin relocalization to the cytosol. Collectively these studies indicate 3-PAP functions as an “adapter” for myotubularin, regulating myotubularin intracellular location and thereby altering the phenotype resulting from myotubularin overexpression.

New oral anticoagulants: a practical guide on prescription, laboratory testing and peri-procedural/bleeding management

New oral anticoagulants (NOAC) are becoming available as alternatives to warfarin to prevent systemic embolism in patients with non‐valvular atrial fibrillation and for the treatment and prevention of venous thromboembolism. An in‐depth understanding of their pharmacology is invaluable for appropriate prescription and optimal management of patients receiving these drugs should unexpected complications (such as bleeding) occur, or the patient requires urgent surgery. The Australasian Society of Thrombosis and Haemostasis has set out to inform physicians on the use of the different NOAC based on current available evidence focusing on: (i) selection of the most suitable patient groups to receive NOAC, (ii) laboratory measurements of NOAC in appropriate circumstances and (iii) management of patients taking NOAC in the perioperative period, and strategies to manage bleeding complications or ‘reverse’ the anticoagulant effects for urgent invasive procedures.

Transgenic Overexpression of CD39 Protects Against Renal Ischemia-Reperfusion and Transplant Vascular Injury

The vascular ectonucleotidases CD39[ENTPD1 (ectonucleoside triphosphate diphosphohydrolase‐1), EC 3.6.1.5] and CD73[EC 3.1.3.5] generate adenosine from extracellular nucleotides. CD39 activity is critical in determining the response to ischemia‐reperfusion injury (IRI), and CD39 null mice exhibit heightened sensitivity to renal IRI. Adenosine has multiple mechanisms of action in the vasculature including direct endothelial protection, antiinflammatory and antithrombotic effects and is protective in several models of IRI. Mice transgenic for human CD39 (hCD39) have increased capacity to generate adenosine. We therefore hypothesized that hCD39 transgenic mice would be protected from renal IRI. The overexpression of hCD39 conferred protection in a model of warm renal IRI, with reduced histological injury, less apoptosis and preserved serum creatinine and urea levels. Benefit was abrogated by pretreatment with an adenosine A2A receptor antagonist. Adoptive transfer experiments showed that expression of hCD39 on either the vasculature or circulating cells mitigated IRI. Furthermore, hCD39 transgenic kidneys transplanted into syngeneic recipients after prolonged cold storage performed significantly better and exhibited less histological injury than wild‐type control grafts. Thus, systemic or local strategies to promote adenosine generation and signaling may have beneficial effects on warm and cold renal IRI, with implications for therapeutic application in clinical renal transplantation.

Pig Thrombomodulin Binds Human Thrombin but Is a Poor Cofactor for Activation of Human Protein C and TAFI

Incompatibility between pig thrombomodulin (TM) and primate thrombin is thought to be an important factor in the development of microvascular thrombosis in rejecting pig‐to‐primate xenografts. To examine this interaction at the molecular level, we cloned pig TM and measured its ability to bind human thrombin and act as a cofactor for the activation of human protein C and TAFI. The 579‐residue pig TM protein showed approximately 69% sequence identity to human TM. Within the EGF domains necessary for binding of thrombin (EGF56), protein C (EGF4) and TAFI (EGF3), all of the amino acids previously identified as critical for the function of human TM, with the exception of Glu‐408 in EGF5, were conserved in pig TM. Comparison of transfected cells expressing pig or human TM demonstrated that both proteins bound human thrombin and inhibited its procoagulant activity. However, pig TM was a poor cofactor for the activation of human protein C and TAFI, with domain swapping showing that EGF5 was the most important determinant of compatibility. Thus, while pig TM may be capable of binding thrombin generated in the vicinity of xenograft endothelium, its failure to promote the activation of human protein C remains a significant problem.

FHL3 Is an Actin-binding Protein That Regulates α-Actinin-mediated Actin Bundling FHL3 LOCALIZES TO ACTIN STRESS FIBERS AND ENHANCES CELL SPREADING AND STRESS FIBER DISASSEMBLY

Four and a half LIM domain (FHL) proteins are members of the LIM protein superfamily. Several FHL proteins function as co-activators of CREM/CREB transcription factors and the androgen receptor. FHL3 is highly expressed in skeletal muscle, but its function is unknown. FHL3 localized to the nucleus in C2C12 myoblasts and, following integrin engagement, exited the nucleus and localized to actin stress fibers and focal adhesions. In mature skeletal muscle FHL3 was found at the Z-line. Actin was identified as a potential FHL3 binding partner in yeast two-hybrid screening of a skeletal muscle library. FHL3 complexed with actin both in vitro and in vivo as shown by glutathione S-transferase pull-down assays and co-immunoprecipitation of recombinant and endogenous proteins. FHL3 promoted cell spreading and when overexpressed in spread C2C12 cells disrupted actin stress fibers. Increased FHL3 expression was detected in highly motile cells migrating into an artificial wound, compared with non-motile cells. The molecular mechanism by which FHL3 induced actin stress fiber disassembly was demonstrated by low speed actin co-sedimentation assays and electron microscopy. FHL3 inhibited α-actinin-mediated actin bundling. These studies reveal FHL3 as a significant regulator of actin cytoskeletal dynamics in skeletal myoblasts.

The Inositol Polyphosphate 5-Phosphatases and the Apurinic/Apyrimidinic Base Excision Repair Endonucleases Share a Common Mechanism for Catalysis

Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized. These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking. The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases. PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores. A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases. Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity. We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity. Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.

Thrombocytopenia and kidney disease in mice with a mutation in the C1galt1 gene

An N-ethyl-N-nitrosourea mutagenesis screen in mice was performed to isolate regulators of circulating platelet number. We report here recessive thrombocytopenia and kidney disease in plt1 mice, which is the result of a severe but partial loss-of-function mutation in the gene encoding glycoprotein-N-acetylgalactosamine-3-β-galactosyltransferase (C1GalT1), an enzyme essential for the synthesis of extended mucin-type O-glycans. Platelet half-life and basic hemostatic parameters were unaffected in plt1/plt1 mice, and the thrombocytopenia and kidney disease were not attenuated on a lymphocyte-deficient rag1-null background. gpIbα and podocalyxin were found to be major underglycosylated proteins in plt1/plt1 platelets and the kidney, respectively, implying that these are key targets for C1GalT1, appropriate glycosylation of which is essential for platelet production and kidney function. Compromised C1GalT1 activity has been associated with immune-mediated diseases in humans, most notably Tn syndrome and IgA nephropathy. The disease in plt1/plt1 mice suggests that, in addition to immune-mediated effects, intrinsic C1Gal-T1 deficiency in megakaryocytes and the kidney may contribute to pathology.