Biomedical subjects

Karin M Hoffmeister

Publications and source records attributed to Karin M Hoffmeister.

9 recordsLinked to original sources

PubMed2005

The macrophage alphaMbeta2 integrin alphaM lectin domain mediates the phagocytosis of chilled platelets.

alpha(M)beta(2) integrin receptors on myeloid cells mediate the adhesion or uptake of diverse ligands. Ligand binding occurs in the alpha(M) chain, which is composed of an I domain and a lectin domain. The alpha(M) I domain binds iC3b, fibrinogen, intercellular adhesion molecule-1, and other ligands and mediates the adhesion of neutrophils to platelet glycoprotein Ibalpha (GPIbalpha). alpha(M)beta(2) also recognizes beta-GlcNAc residues on GPIbalpha that are clustered on platelets after cooling. The phagocytosis of chilled platelets could be reconstituted when Chinese hamster ovary cells were transfected with alpha(M)beta(2). Replacement of the I domain or the lectin domain of the alpha(M) chain with the corresponding domain from the alpha(X) chain (p150) revealed that the activity of the alpha(M)beta(2) integrin toward chilled platelets resides within the lectin domain and does not require the I domain. Additional evidences for this conclusion are: 1) Sf9 cells expressing solely the alpha(M) lectin domain bound chilled platelets, and 2) soluble recombinant alpha(M) lectin domain inhibited the phagocytosis of chilled platelets by alpha(M)beta(2)-expressing THP-1 cells, whereas I domain substrates showed no inhibitory effect. Therefore chilled platelets are removed from blood by an interaction between beta-GlcNAc residues on clustered GPIbalpha and the lectin domain of alpha(M) chain of the alpha(M)beta(2) integrin, distinguishing this interaction from those mediated by the alpha(M) I domain.

Animals↗

PubMed2004

Perturbed IFN-gamma-Jak-signal transducers and activators of transcription signaling in tuberous sclerosis mouse models: synergistic effects of rapamycin-IFN-gamma treatment.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by widespread development of hamartomas, which is caused by mutations in either TSC1 or TSC2. We demonstrate a dramatic decrease of IFN-gamma expression in tumors and mouse embryo fibroblast cell lines that lack either Tsc1 or Tsc2, which is reversed by rapamycin (mammalian target of rapamycin inhibitor) therapy. Increased signal transducers and activators of transcription (STAT) 1 expression and phosphorylation at Ser 727 and increased pSTAT3 Tyr705 levels also are seen in Tsc1 null and Tsc2 null cells and in tumors. Treatment of Tsc1 or Tsc2 null cells with IFN-gamma induces apoptosis, in contrast to control cell lines, with reduction in pSTAT3 Tyr705 levels and major increases in pSTAT1 Tyr701, bax, and caspase-1 and -9 levels. A combination of IFN-gamma and rapamycin is markedly synergistic in induction of apoptosis in Tsc1 or Tsc2 null cells because pSTAT3 Tyr705 phosphorylation is abolished completely and the other effects of IFN-gamma are maintained or enhanced. Rapamycin-IFN-gamma has unique potential therapeutic benefit for management of TSC tumors.

Animals↗

PubMed2003

Glycosylation restores survival of chilled blood platelets.

Cooling of blood platelets clusters the von Willebrand factor receptor complex. Macrophage alphaMbeta2 integrins bind to the GPIbalpha subunit of the clustered complex, resulting in rapid clearance of transfused, cooled platelets. This precludes refrigeration of platelets for transfusion, but the current practice of room temperature storage has major drawbacks. We document that alphaMbeta2 is a lectin that recognizes exposed beta-N-acetylglucosamine residues of N-linked glycans on GPIbalpha. Enzymatic galactosylation of chilled platelets blocks alphaMbeta2 recognition, prolonging the circulation of functional cooled platelets. Platelet-associated galactosyltransferase produces efficient galactosylation when uridine diphosphate-galactose is added, affording a potentially simple method for storing platelets in the cold.

Acetylglucosamine↗

PubMed2003

Metalloproteinase inhibitors improve the recovery and hemostatic function of in vitro-aged or -injured mouse platelets.

Platelet transfusions are a crucial component of support for patients with severe thrombocytopenia. Storage of platelet concentrates, however, is associated with a reduction in platelet posttransfusion recovery and hemostatic function. In this study, we established a model of mitochondrial injury that resembles platelet storage lesion. Mitochondrial injury, provoked by incubation of platelets with carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to reduced posttransfusion recovery in mice, an effect that directly correlated with the duration of treatment. Damaged platelets were characterized by shape change, disruption of membrane asymmetry, surface expression of P-selectin, and profound proteolysis of GPIbalpha. Using our model, we identified a key role for endogenous metalloproteinase(s) in platelet clearance, as their inhibition markedly improved posttransfusion recovery of both the mitochondria-injured and in vitro-aged mouse platelets. Metalloproteinase inhibition also prevented proteolysis of GPIbalpha on damaged platelets, thereby improving the hemostatic function of these cells in vivo. We propose that inhibition of metalloproteinase activity during storage could significantly improve the effectiveness of platelet transfusions. Surface expression of GPIbalpha might be a powerful marker to determine the quality of platelet concentrates, because it reflects metalloproteinase activity in vitro.

Animals↗

PubMed2003

Mechanisms and implications of platelet discoid shape.

The platelet marginal band consists of a single peripheral microtubule (MT) that is wound in 8 to 12 coils and maintains discoid cell shape. About 90% of beta-tubulin in the marginal band is of the divergent, megakaryocyte (MK)/platelet-restricted beta1 isoform. beta1-tubulin-null mice show reduced proplatelet formation, thrombocytopenia, and platelet spherocytosis. Here, we show that structural abnormalities in resting beta1-tubulin-/- platelets include frequent kinks and breaks in the marginal band. Platelets derived from mice lacking the transcription factor GATA1 show similar defects, probably as a direct consequence of absent beta1-tubulin. beta1-tubulin+/- platelets have normal ratios of beta-tubulin isotypes but the marginal band is half the normal thickness, which is sufficient to maintain elliptical cell shape. Thus, a threshold 50% or less of the normal amount of beta1-tubulin is required to preserve marginal band integrity and cell shape. beta1-tubulin-/- platelets have normal size and contents and show no defects in serotonin release or aggregation. Accordingly, the apparently isolated spherocytosis allows investigation of the role of discoid platelet shape in hemostasis. On agonist stimulation, the disorganized MTs in beta1-tubulin-/- platelets fail to condense into central rings and instead are dispersed in short bundles and linear arrays. Nevertheless, intravital microscopy and flow chamber studies demonstrate full functionality of these spherocytic platelets under physiologic shear conditions. Together, these findings highlight the essential requirements of the MK/platelet-restricted beta1-tubulin isoform in platelet structure and suggest that spherocytosis does not impair many aspects of platelet function.

Animals↗

PubMed2003

The clearance mechanism of chilled blood platelets.

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.

Animals↗

PubMed2003

Platelet moesin interacts with PECAM-1 (CD31).

Platelet activation results information of filopodia and cell spreading by extension of lamellae. Moesin is a member of the ezrin/radixin/moesin (ERM) family of proteins, which localize in cell extensions like filopodia and function as cross-linkers between the actin cytoskeleton and the plasma membrane. Here we investigated whether the adhesion molecule PECAM-1 (CD31) is a membrane-binding partner for moesin in platelets. Our data show that moesin co-immunoprecipitated with PECAM-1 in lysates from thrombin-stimulated, but not resting platelets. Furthermore, PECAM-1 co-localized with moesin at the cell periphery and in filopodia of glass-activated platelets. Our observations suggest that moesin may play a role in platelet adhesion, linking PECAM-1 with the actin cytoskeleton.

Actins↗

PubMed2002

Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts.

We investigated the effect of actin filament barbed end uncapping on Arp23 complex function both in vivo and in vitro. Arp23 complex redistributes rapidly and uniformly to the lamellar edge of activated wild-type platelets and fibroblasts but clusters in marginal actin filament clumps in gelsolin-null cells. Treatment of gelsolin-null platelets with the negative dominant N-WASp C-terminal CA domain has no effect on their residual actin nucleation activity, placing gelsolin actin filament severing, capping, and uncapping function upstream of Arp23 complex nucleation. Actin filaments capped by gelsolin or the gelsolin homolog CapG fail to enhance Arp23 complex nucleation in vitro, but uncapping of the barbed ends of these actin filaments restores their ability to potentiate Arp23 complex nucleation. We conclude that Arp23 complex contribution to actin filament nucleation in platelets and fibroblasts importantly requires free barbed ends generated by severing and uncapping.

Actin-Related Protein 2↗

PubMed2002

Normal Arp2/3 complex activation in platelets lacking WASp.

Arp2/3 complex is believed to induce de novo nucleation of actin filaments at the edge of motile cells downstream of WASp family proteins. In this study, the signaling pathways leading to Arp2/3 complex activation, actin assembly, and shape change were investigated in platelets isolated from patients with Wiskott-Aldrich Syndrome (WAS), that is, who lack WASp, and in WASp-deficient mouse platelets. WASp-deficient human and mouse platelets elaborate filopodia, spread lamellae, and assemble actin, identical to control WASp-expressing platelets. Human platelets contain 2 microM Arp2/3 complex, or 8600 molecules/cell. Arp2/3 complex redistributes to the edge of the lamellae and to the Triton X-100-insoluble actin cytoskeleton of activated WASp-deficient platelets. Furthermore, the C-terminal CA domain of N-WASp, which sequesters Arp2/3 complex, inhibits by half the actin nucleation capacity of octylglucoside-permeabilized and activated WAS platelets, similar to its effect in WASp-expressing cells. Along with WASp, platelets express WAVE-2 as a physiologic activator of Arp2/3 complex and a small amount of N-WASp. Taken together, our findings show that platelets activate Arp2/3 complex, assemble actin, and change shape in the absence of WASp, indicating a more specialized role for WASp in these cells.

Actin-Related Protein 2↗