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Biomedical subjects

Mark L Kahn

Publications and source records attributed to Mark L Kahn.

At least 19 recordsLinked to original sources
PubMed2006

Structure-function analysis reveals discrete beta3 integrin inside-out and outside-in signaling pathways in platelets.

A unique aspect of integrin receptor function is the transmission of bidirectional signals. In platelets alphaIIbbeta3 integrins require "inside-out" signals to bind fibrinogen and form thrombi. Following ligand binding, alphaIIbbeta3 integrins generate "outside-in" signals that contribute to thrombus stability. Because integrin cytoplasmic tails are short and lack enzymatic activity, bidirectional signals are believed to be mediated by interactions with intracellular proteins, but the molecular basis for integrin signal transduction remains poorly understood. In the present study we have used retroviral vectors to express alphaIIbbeta3 integrins with mutant beta3 tails in mouse platelets and test mechanisms of bidirectional signaling. Using this approach we identify mutations (eg, beta3Y747A) that confer loss of signaling in both directions and others (eg, beta3T762A) that confer a selective loss of outside-in signals. These results reveal the presence of discrete bidirectional signaling pathways controlled by integrin beta subunits in platelets and describe a high-throughput means of further investigating these pathways in vivo.

Animals↗
PubMed2006

Novel platelet and vascular roles for immunoreceptor signaling.

The immunoreceptor signaling pathway has classically been defined by its role in mediating intracellular signals downstream of immune receptors on circulating cells, but recent studies have revealed new and unexpected roles for this pathway in vascular biology. In platelets the immunoreceptor signaling pathway is coupled to 2 structurally distinct platelet collagen receptors, glycoprotein VI and integrin alpha2beta1, and is required for the activation of platelets after exposure to vessel wall collagen during plaque rupture. During vascular development immunoreceptor signaling is required for proper formation of the lymphatic system, a role that has revealed the contribution of hematopoietic endothelial progenitors to that process. In conjunction with the identification of new biological roles in vascular cell types, new molecular mechanisms of activating this signaling pathway have been discovered, including activation by integrins and immunoreceptor tyrosine activation motifs (ITAMs) on receptors that do not function as part of the immune response. Here we discuss some of these recent findings and their implications for vascular biology and the treatment of human vascular diseases.

Animals↗
PubMed2006

Structural basis for platelet collagen responses by the immune-type receptor glycoprotein VI.

Activation of circulating platelets by exposed vessel wall collagen is a primary step in the pathogenesis of heart attack and stroke, and drugs to block platelet activation have successfully reduced cardiovascular morbidity and mortality. In humans and mice, collagen activation of platelets is mediated by glycoprotein VI (GPVI), a receptor that is homologous to immune receptors but bears little sequence similarity to known matrix protein adhesion receptors. Here we present the crystal structure of the collagen-binding domain of human GPVI and characterize its interaction with a collagen-related peptide. Like related immune receptors, GPVI contains 2 immunoglobulin-like domains arranged in a perpendicular orientation. Significantly, GPVI forms a back-to-back dimer in the crystal, an arrangement that could explain data previously obtained from cell-surface GPVI inhibition studies. Docking algorithms identify 2 parallel grooves on the GPVI dimer surface as collagen-binding sites, and the orientation and spacing of these grooves precisely match the dimensions of an intact collagen fiber. These findings provide a structural basis for the ability of an immune-type receptor to generate signaling responses to collagen and for the development of GPVI inhibitors as new therapies for human cardiovascular disease.

Binding Sites↗
PubMed2006

In vivo beta1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines.

Integrins are heterodimeric adhesion receptors associated with bidirectional signaling. In vitro studies support a role for the binding of evolutionarily conserved tyrosine motifs (NPxY) in the beta integrin cytoplasmic tail to phosphotyrosine-binding (PTB) domain-containing proteins, an interaction proposed to be dynamically regulated by tyrosine phosphorylation. Here we show that replacement of both beta1 integrin cytoplasmic tyrosines with alanines, resulting in the loss of all PTB domain interaction, causes complete loss of beta1 integrin function in vivo. In contrast, replacement of beta1 integrin cytoplasmic tyrosines with phenylalanines, a mutation that prevents tyrosine phosphorylation, conserves in vivo integrin function. These results have important implications for the molecular mechanism and regulation of integrin function.

Amino Acid Motifs↗
PubMed2006

Activation-independent, antibody-mediated removal of GPVI from circulating human platelets: development of a novel NOD/SCID mouse model to evaluate the in vivo effectiveness of anti-human platelet agents.

GPVI is a 62-kDa membrane glycoprotein expressed in noncovalent association with the Fc receptor gamma chain on human and murine platelets and serves as the major activating receptor for collagen. GPVI-specific antibodies have the capacity to specifically deplete GPVI from mouse and human platelets in vivo, rendering them unresponsive to collagen and GPVI-specific agonists. Such antibodies do not remove GPVI from noncirculating platelets in vitro, however, making it difficult to evaluate their antithrombotic potential and mechanism of action, particularly in human platelets. We devised a model system in which human platelets are introduced into the retroorbital plexus of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, allowed to circulate, and evaluated for the effects of GPVI-specific murine monoclonal antibodies (mAbs) on platelet survival and function. GPVI-specific mAbs triggered depletion of GPVI from human, but not murine, platelets. Soluble truncated human GPVI appeared concomitantly in mouse plasma. GPVI-depleted human platelets had markedly diminished responses to GPVI-specific agonists and unexpectedly exhibited somewhat depressed responses to G-protein-coupled agonists. The ability to evaluate in living mice the in vivo function and survival of circulating human platelets may prove valuable for determining mechanisms of antibody-mediated platelet passivation and aid in the development of novel anti-platelet therapeutics.

Animals↗
PubMed2006

Syk and Slp-76 mutant mice reveal a cell-autonomous hematopoietic cell contribution to vascular development.

Developmental studies support a common origin for blood and endothelial cells, while studies of adult angiogenic responses suggest that the hematopoietic system can be a source of endothelial cells later in life. Whether hematopoietic tissue is a source of endothelial cells during normal vascular development is unknown. Mouse embryos lacking the signaling proteins Syk and Slp-76 develop abnormal blood-lymphatic endothelial connections. Here we demonstrate that expression of GFPSlp-76 in a subset of hematopoietic cells rescues this phenotype, and that deficient cells confer focal vascular phenotypes in chimeric embryos consistent with a cell-autonomous mechanism. Endogenous Syk and Slp-76, as well as transgenic GFPSlp-76, are expressed in circulating cells previously proposed to be endothelial precursors, supporting a causal role for these cells. These studies provide genetic evidence for hematopoietic contribution to vascular development and suggest that hematopoietic tissue can provide a source of vascular endothelial progenitor cells throughout life.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Klf2 is an essential regulator of vascular hemodynamic forces in vivo.

Hemodynamic responses that control blood pressure and the distribution of blood flow to different organs are essential for survival. Shear forces generated by blood flow regulate hemodynamic responses, but the molecular and genetic basis for such regulation is not known. The transcription factor KLF2 is activated by fluid shear stress in cultured endothelial cells, where it regulates a large number of vasoactive endothelial genes. Here, we show that Klf2 expression during development mirrors the rise of fluid shear forces, and that endothelial loss of Klf2 results in lethal embryonic heart failure due to a high-cardiac-output state. Klf2 deficiency does not result in anemia or structural vascular defects, and it can be rescued by administration of phenylephrine, a catecholamine that raises vessel tone. These findings identify Klf2 as an essential hemodynamic regulator in vivo and suggest that hemodynamic regulation in response to fluid shear stress is required for cardiovascular development and function.

Anemia↗
PubMed2006

Evidence for the requirement of ITAM domains but not SLP-76/Gads interaction for integrin signaling in hematopoietic cells.

Syk tyrosine kinase and Src homology 2 (SH2) domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) are signaling mediators activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing immunoreceptors and integrins. While the signaling cascades descending from integrins are similar to immunoreceptors, the mechanism of Syk activation and SLP-76 recruitment remains unclear. We used an in vivo structure-function approach to study the requirements for the domains of Syk and SLP-76 in immunoreceptor and integrin signaling. We found that both SH2 domains and the kinase domain of Syk are required for immunoreceptor-dependent signaling and cellular response via integrins. While the Gads-binding domain of SLP-76 is needed for immunoreceptor signaling, it appears dispensable for integrin signaling. Syk and SLP-76 also are required for initiating and/or maintaining separation between the blood and lymphatic vasculature. Therefore, we correlated the signaling requirement of the various domains of Syk and SLP-76 to their requirement in regulating vascular separation. Our data suggest ITAMs are required in Syk-dependent integrin signaling, demonstrate the separation of the structural features of SLP-76 to selectively support immunoreceptor versus integrin signaling, and provide evidence that the essential domains of SLP-76 for ITAM signals are those which most efficiently support separation between lymphatic and blood vessels.

Adaptor Proteins, Signal Transducing↗
PubMed2005

GPVI and alpha2beta1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow.

The roles of the 2 major platelet-collagen receptors, glycoprotein VI (GPVI) and integrin alpha2beta1, have been intensely investigated using a variety of methods over the past decade. In the present study, we have used pharmacologic and genetic approaches to study human and mouse platelet adhesion to collagen under flow conditions. Our studies demonstrate that both GPVI and integrin alpha2beta1 play significant roles for platelet adhesion to collagen under flow and that the loss of both receptors completely ablates this response. Intracellular signaling mediated by the cytoplasmic adaptor Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) but not by the transmembrane adaptor linker for activation of T cells (LAT) is critical for platelet adhesion to collagen under flow. In addition, reduced GPVI receptor density results in severe defects in platelet adhesion to collagen under flow. Defective adhesion to collagen under flow is associated with prolonged tail-bleeding times in mice lacking one or both collagen receptors. These studies establish platelet-collagen responses under physiologic flow as the consequence of a close partnership between 2 structurally distinct receptors and suggest that both receptors play significant hemostatic roles in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Density of platelet collagen receptors glycoprotein VI and alpha2beta1 and prior myocardial infarction in human subjects, a pilot study.

BACKGROUND: Prior studies evaluating whether platelet collagen receptors affect risk of myocardial infarction (MI) have used common polymorphisms thought to affect receptor density, and have yielded conflicting results. Our objective in the current pilot study was to obtain preliminary information on the relationship between platelet collagen receptor density and prior MI. MATERIAL/METHODS: We measured glycoprotein VI (GPVI) and integrin alpha2beta1 density by flow cytometry using FITC-conjugated monoclonal antibodies in consecutive subjects from an outpatient cardiology practice. We used a case-control design to match 77 patients with prior MI to 77 control patients. Matching was performed on the basis of traditional risk factors for MI. RESULTS: The mean GPVI density was 14% higher in those with prior MI (P = 0.098), and the alpha2beta1 density was 6% higher in those with prior MI (P = 0.49). A GPVI or alpha2beta1 density within the lowest decile was associated with a lower MI prevalence (OR = 6.3, 95% CI: 1.3-29.2 P = 0.009). CONCLUSIONS: Our preliminary data suggests that platelet collagen receptor density, when measured directly, may be related to prior myocardial infarction. An appropriately sized clinical study using this methodology is warranted to further investigate whether collagen receptor density serves as a novel risk factor for myocardial infarction.

Aged↗
PubMed2005

Glycoprotein VI is associated with GPIb-IX-V on the membrane of resting and activated platelets.

The platelet collagen receptor, glycoprotein (GP)VI, initiates platelet aggregation at low shear stress while GPIb-IX-V, which binds von Willebrand factor, elicits platelet aggregation under high shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIb alpha-specific monoclonal antibody, SZ2. Immunoprecipitation of GPIb-IX with anti-GPIb alpha from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblotting with anti-GPVI demonstrated association between GPIb-IX and GPVI. This association was maintained when platelets were activated by thrombin. Pre-treatment of platelets with methyl-beta-cyclodextrin to disrupt lipid rafts did not affect association in resting platelets under these conditions of detergent lysis. The association is also independent of cytoskeletal attachment, since it was unaffected by treatment with N-ethylmaleimide or DNaseI, which dissociate GPIb-IX from filamin and the actin-containing cytoskeleton, respectively. Finally, the association involves an interaction between the ectodomains of GPIb alpha and GPVI, since soluble fragments of GPIb alpha (glycocalicin) and GPVI are co-precipitated from the platelet supernatant under conditions where GPVI is shed. A contribution of GPIb-IX-V to GPVI-induced platelet responses, and vice versa, therefore warrants further investigation.

Binding Sites↗
PubMed2004

Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase.

Thrombosis can be initiated when activated platelets adhere to injured blood vessels via the interaction of subendothelial collagen with its platelet receptor, glycoprotein (GP) VI. Here we observed that incubation of platelets with convulxin, collagen, or collagen-related peptide (CRP) resulted in GPVI signaling-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble fragment of GPVI as revealed by immunoblotting. Ethylenediaminetetraacetic acid (EDTA) or GM6001 (a metalloproteinase inhibitor with broad specificity) prevented this loss. In other receptor systems, calmodulin binding to membrane-proximal cytoplasmic sequences regulates metalloproteinase-mediated ectodomain shedding. In this regard, we have previously shown that calmodulin binds to a positively charged, membrane-proximal sequence within the cytoplasmic tail of GPVI. Incubation of platelets with calmodulin inhibitor W7 (150 microM) resulted in a time-dependent loss of GPVI from the platelet surface. Both EDTA and GM6001 prevented this loss. Surface plasmon resonance demonstrated that W7 specifically blocked the association of calmodulin with an immobilized synthetic peptide corresponding to the calmodulin-binding sequence of GPVI. These findings suggest that disruption of calmodulin binding to receptor cytoplasmic tails by agonist binding to the receptor triggers metalloproteinase-mediated loss of GPVI from the platelet surface. This process may represent a potential mechanism to regulate GPVI-dependent platelet adhesion.

Amino Acid Sequence↗
PubMed2004

Anti-GPVI-associated ITP: an acquired platelet disorder caused by autoantibody-mediated clearance of the GPVI/FcRgamma-chain complex from the human platelet surface.

Platelet glycoprotein (GP) VI is a 62-kDa membrane glycoprotein that exists on both human and murine platelets in a noncovalent complex with the Fc receptor (FcR) gamma chain. The GPVI/FcRgamma-chain complex serves as the major activating receptor for collagen, as evidenced by observations that platelets genetically deficient in GPVI or the FcRgamma chain are highly refractory to collagen-induced platelet activation. Recently, several different rat anti-murine GPVI monoclonal antibodies, termed JAQs 1, 2, and 3, were produced that had the unique property of "immunodepleting" GPVI from the murine platelet surface and rendering it unresponsive to collagen or GPVI-specific agonists like convulxin or collagen-related peptide (CRP). Herein, we describe a patient with a mild bleeding disorder and a moderately reduced platelet count whose platelets fail to become activated in response to collagen or CRP and inefficiently adhere to and form thrombi on immobilized collagen under conditions of arterial shear. Although the amount of GPVI platelet mRNA and the nucleotide sequence of the GPVI gene were found to be normal, both GPVI and the FcRgamma chain were nearly absent from the platelet surface and were markedly reduced in wholeplatelet detergent lysates. Patient plasma contained an autoantibody that bound specifically to GPVI-positive, normal platelets, and cleared soluble GPVI from the plasma, suggesting that the patient suffers from a rare form of idiopathic thrombocytopenic purpura caused by a GPVI-specific autoantibody that mediates clearance of the GPVI/FcRgamma-chain complex from the platelet surface. Since antibody-induced GPVI shedding now has been demonstrated in both humans and mice, these studies may provide a rationale for developing therapeutic reagents that induce temporary depletion of GPVI for the treatment of clinical thrombosis.

Adult↗
PubMed2004

Platelet receptor glycoprotein VI-mediated adhesion to type I collagen under hydrodynamic flow.

Glycoprotein VI (GPVI) is a platelet receptor that directly binds collagen. It has been shown by expressing GPVI in rat basophilic leukemia (RBL-2H3) cells that GPVI mediates adhesion to type I collagen under static conditions. However, the ability of GPVI to secure adhesion to collagen type I under flow has not been measured. We studied the interaction of GPVI and type I collagen under hydrodynamic flow using RBL-2H3 cells transfected with the GPVI receptor. We found that GPVI-expressing RBL-2H3 cells adhere to collagen under flow, significantly more so than non-GPVI-expressing RBL-2H3 cells. Inhibition of GPVI by the 11A12 anti-GPVI antibody significantly blocks adhesion to collagen, indicating that GPVI specifically interacts with collagen. Probing the role of signaling in GPVI binding to collagen, we used mutants of GPVI and observed that signal transduction did not inhibit adhesion. To test the correlation between receptor expression and adhesion, we tested three GPVI-expressing RBL-2H3 cell lines (A, B, and C) with different levels of receptor expression. At a single shear rate, the level of adhesion increases monotonically with surface expression. The results, using this model cell line, indicate that GPVI is capable of mediating adhesion to collagen under shear, in a density-dependent fashion that is independent of GPVI signaling.

Animals↗
PubMed2004

Platelet-collagen responses: molecular basis and therapeutic promise.

Exposure of circulating blood to collagen after vessel injury has been recognized as a primary event in arterial thrombosis for many years but an understanding of the molecular basis of this response has only recently materialized. Platelet collagen interactions are initiated indirectly by interaction of platelet glycoprotein Ib (GPIb) with collagen-bound von Willebrand Factor (vWF). Slowed, rolling platelets firmly adhere following the generation of platelet activating signals in a process initiated by the platelet collagen receptor glycoprotein VI (GPVI). The contribution of the first platelet collagen receptor identified, integrin alpha (2)beta (1), remains controversial. alpha (2)beta (1) is not required for platelet responses to collagen under many experimental conditions but GPVI and other intracellular signals may activate the integrin, which is likely to play a later, secondary role. In vivo studies of arterial thrombosis using genetically modified mice suggest that blockade of platelet-collagen interactions may be a highly effective means of inhibiting arterial thrombosis. Recent studies have generated a more complete model of the molecular basis of platelet-collagen responses and provided both the means and incentive to create novel therapeutic agents aimed at blocking this process to treat human atherothrombotic diseases such as myocardial infarction and stroke.

Animals↗
PubMed2004

The endothelial cell-specific antibody PAL-E identifies a secreted form of vimentin in the blood vasculature.

During mammalian vascular development, endothelial cells form a complex array of vessels that differ markedly in structure and function, but the molecular basis for this vascular complexity is poorly understood. Recent insights into endothelial diversity have come from the identification of molecular markers expressed on distinct endothelial cell populations. One such marker, the PAL-E antibody, has been used for almost 20 years to distinguish blood and lymphatic vessels, but the identity of the protein recognized by PAL-E has been unknown. In the present study we have used protein purification and tandem mass spectrometry analysis of tryptic peptides to identify the PAL-E antigen as a secreted form of vimentin. Vimentin has been well characterized as an intracellular intermediate filament protein expressed broadly in mesenchymal cells. In contrast, PAL-E-reactive vimentin is secreted extracellularly, its synthesis is restricted to a distinct population of blood endothelial cells and activated macrophages, and PAL-E-reactive vimentin is found in circulating human blood. PAL-E-reactive vimentin does not arise from an endothelial cell-specific mRNA transcript but is the product of cell-specific posttranslational modification. The PAL-E antibody therefore defines secretion of vimentin as a molecular distinction among endothelial cells and exposes a novel, extracellular role for vimentin in the blood vasculature.

Amino Acid Sequence↗