Platelet Glycoprotein Ibalpha Research Articles

Novel Causal Plasma Proteins for Hypothyroidism: A Large-scale Plasma Proteome Mendelian Randomization Analysis.

Although several risk proteins for hypothyroidism have been reported in recent years, many more plasma proteins have not been tested. To determine potential mechanisms and novel causal plasma proteins for hypothyroidism using Mendelian randomization (MR). A large-scale plasma proteome MR analysis was conducted using protein quantitative trait loci (pQTLs) for 2297 plasma proteins. We classified pQTLs into 4 different groups. MR analyses were conducted within the 4 groups simultaneously. Significant proteins were discovered and validated in 2 different cohorts. Colocalization analysis and enrichment analysis were conducted using proteins found with MR. Thirty-one proteins were identified in the discovery cohort. Among them, 13 were validated in the validation cohort. Nine of the 13 proteins are risk factors (ISG15, Fc receptor-like protein 2, tumor necrosis factor ligand superfamily member 14, Rab-2A, FcRL3, thrombomodulin, interferon [IFN]-lambda-1, platelet glycoprotein Ib alpha chain, IL-7RA) for hypothyroidism, whereas others are protective proteins (protein O-glucosyltransferase 1 [POGLUT1], tumor necrosis factor ligand superfamily, 3-hydroxyisobutyryl-CoA hydrolase, transferrin receptor protein 1). Among the significant proteins, POGLUT1 strongly colocalized with expression quantitative trait loci from whole blood (posterior probability of colocalization [PP4] = 0.978) and the thyroid (PP4 = 0.978). Two different trans-pQTLs (rs2111485 PP4 = 0.998; rs35103715 PP4 = 0.998) for IFN-lambda-1 strongly colocalized with hypothyroidism in different chromosomes. Thirteen various proteins were identified and validated to be associated with hypothyroidism using univariable MR. We reinforced and expanded the effect of IFN on hypothyroidism. Several proteins identified in this study could explain part of the association between the coagulation system and hypothyroidism. Our study broadens the causal proteins for hypothyroidism and provides the relationships between plasma proteins and hypothyroidism. The proteins identified in this study can be used as early screening biomarkers for hypothyroidism.

Comparative transcriptomics analyses and revealing candidate networks and genes involved in lordosis of the Yunlong grouper (Epinephelus moara ♀ × Epinephelus lanceolatus ♂)

Grouper is an economically important fish in China. However, it exhibits a high frequency of skeletal abnormalities, particularly vertebral deformities. The molecular mechanisms underlying fish vertebral deformities are still poorly understood. In this study, a HiSeq™ 4000 platform (Illumina) was used to analyze the transcriptomic profiles of the brain, pituitary, and vertebrae from normal fish (NF) and fish with lordosis (LF) of Yunlong grouper. A total of 87,888 unigenes were assembled with lengths that varied from 201 to 28,922 bp and a N50 length of 2670 bp. A total of 36,268 unigenes were functionally annotated by BLAST alignments. A total of 2875 significantly differentially expressed genes (DEGs) were identified between the NF group and the LF group, including 706 upregulated unigenes and 2169 downregulated unigenes in LF. GO and KEGG pathway enrichment analyses showed that DNA binding, transmembrane receptor activity, cytokine receptor interaction, neuroactive ligand-receptor interaction, calcium signaling pathway and ECM-receptor interaction HIF-1 signaling pathway, and mineral absorption may be involved in the formation of vertebral deformities. Furthermore, weighted gene co-expression network analyses, including three modules (turquoise, yellow, and blue), significantly positively corrected with vertebral deformities. A network map that included these three modules enabled the identification of a series of hub genes, including claudin-22-like (cldn22), fibronectin type III domain-containing protein 1 isoform X2 (fndc1l2), E3 ubiquitin-protein ligase NRDP1-like (rnf41), and Catenin alpha-2 (ctnna1). We found that the levels of most genes in the blue module were closely related to the expression of parvalbumin, thymic CPV3-like isoform X2 (ocm), platelet glycoprotein Ib alpha chain (gp1ba), and matrix metalloproteinase-9 (mmp9), suggesting that this module is associated with skeletal development. Some uncharacterized genes associated with known bone-related genes, including Unigene0067643, Unigene0056862, and Unigene0059867, were detected by a weighted gene co-expression network analysis. A detailed functional investigation of these networks and genes will further improve our understanding of the molecular mechanisms that underlie the formation of lordosis in fish.

Synergic effect of GPIBA and von Willebrand factor in pathogenesis of deep vein thrombosis.

In cardiovascular disease, deep vein thrombosis is one of the vital symptoms causing pulmonary thromboembolism. However, the pathogenesis of deep vein thrombosis is still not clear. One of the critical factors leading to deep vein thrombosis is the platelet aggregation that is mediated by a set of key genes including platelet membrane protein coded by platelet glycoprotein Ib alpha chain (GPIBA). Deep vein thrombosis model was established according to the previous protocol, and venous blood and thrombi were collected for further analysis. The dynamic changes of GPIBA and coagulation factor, von Willebrand factor, were observed in deep vein thrombosis models. Meanwhile, critical proteins participating in adhesion and binding of platelets such as epithelial membrane protein 2 (EMP2), vascular cell adhesion protein 1 (VCAM1), immunoreceptor tyrosine-based activation motif 1 (ITAM1), integrin subunit alpha M (ITGAM), or fibronectin were also differentially expressed in deep vein thrombosis models. Application of heparin could reverse these dynamic changes in deep vein thrombosis models. Thus, we explained the potential synergic role of GPIBA and von Willebrand factor in regulating the occurrence of deep vein thrombosis and provide therapeutic target against cardiovascular disease.

Lack of LRP8 triggers the formation of aortic dissection in angiotensin II infused mice

Factor XI-thrombin amplification loop depends on platelets and monocytes and augments angiotensin II (AngII) driven vascular injury in experimental hypertension in mice. Low-density lipoprotein receptor-related protein 8 (LRP8) plays an important role in apolipoprotein E homeostasis and was also found to be important in endothelial barrier stabilization. Moreover, LRP8 is able to form a complex with the platelet glycoprotein Ib alpha (GPIbalpha) which is the main receptor for von Willebrand factor. To explore the role of LRP8 and GPIbalpha in the development of hypertension related to platelet-dependent thrombin generation. We infused LRP8 KO mice with AngII for either 1 or 4 weeks using osmotic minipumps. Blood pressure was recorded using tail cuff measurement. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging (IVM). Histology of aortas was performed with sirius red staining. Infusion of AngII for 1 week in LRP8 KO mice altered endothelial dependent and independent vascular relaxation to the same extend as in control mice. Vascular rolling and adherence to the carotid endothelium, visualized by IVM was similar between LRP8 KO and control mice infused with AngII. There were no significant differences in AngII-induced blood pressure increase between the LRP8 KO and control mice. Unexpectedly, LRP8 KO mice demonstrated a drastically increased mortality rate in response to AngII infusion. In the 4 weeks AngII infusion experiments, 80% of the LRP8 KO mice died within the first 10 days of AngII infusion due to aortic dissection complicated by hemorrhage. LRP8 receptor dysfunction lead to the formation of aortic dissection in AngII infused mice. Further studies are needed to better characterize the role of immune cells and platelets in the development of these dissections.

Plasma-derived Extracellular Vesicles Contain Predictive Biomarkers and Potential Therapeutic Targets for Myocardial Ischemic (MI) Injury

Myocardial infarction (MI) triggers a potent inflammatory response via the release of circulatory mediators, including extracellular vesicles (EVs) by damaged cardiac cells, necessary for myocardial healing. Timely repression of inflammatory response are critical to prevent and minimize cardiac tissue injuries, nonetheless, progression in this aspect remains challenging. The ability of EVs to trigger a functional response upon delivery of carried bioactive cargos, have made them clinically attractive diagnostic biomarkers and vectors for therapeutic interventions. Using label-free quantitative proteomics approach, we compared the protein cargo of plasma EVs between patients with MI and from patients with stable angina (NMI). We report, for the first time, the proteomics profiling on 252 EV proteins that were modulated with >1.2-fold after MI. We identified six up-regulated biomarkers with potential for clinical applications; these reflected post-infarct pathways of complement activation (Complement C1q subcomponent subunit A (C1QA), 3.23-fold change, p = 0.012; Complement C5 (C5), 1.27-fold change, p = 0.087), lipoprotein metabolism (Apoliporotein D (APOD), 1.86-fold change, p = 0.033; Apolipoprotein C-III (APOCC3), 2.63-fold change, p = 0.029) and platelet activation (Platelet glycoprotein Ib alpha chain (GP1BA), 9.18-fold change, p < 0.0001; Platelet basic protein (PPBP), 4.72-fold change, p = 0.027). The data have been deposited to the ProteomeXchange with identifier PXD002950. This novel biomarker panel was validated in 43 patients using antibody-based assays (C1QA (p = 0.005); C5 (p = 0.0047), APOD (p = 0.0267); APOC3 (p = 0.0064); GP1BA (p = 0.0031); PPBP (p = 0.0465)). We further present that EV-derived fibrinogen components were paradoxically down-regulated in MI, suggesting that a compensatory mechanism may suppress post-infarct coagulation pathways, indicating potential for therapeutic targeting of this mechanism in MI. Taken together, these data demonstrated that plasma EVs contain novel diagnostic biomarkers and therapeutic targets that can be further developed for clinical use to benefit patients with coronary artery diseases (CADs).

Genetic deletion of platelet glycoprotein Ib alpha but not its extracellular domain protects from atherosclerosis.

The pathogenesis of atherosclerosis involves the interplay of haematopoietic, stromal and endothelial cells. Platelet interactions with endothelium and leukocytes are pivotal for atherosclerosis promotion. Glycoprotein (GP) Ibα is the ligand-binding subunit of the platelet GPIb-IX-V receptor complex; its deficiency causes the Bernard-Soulier syndrome (BSS), characterised by absent platelet GPIb-IX-V, macrothrombocytopenia and bleeding. We designed this study to determine the role of platelet GPIbα in the pathogenesis of atherosclerosis using two unique knockout models. Ldlr-/- mice were reconstituted with wild-type (wt), GPIbα-/- (lacks GPIbα) or chimeric IL-4R/GPIbα-Tg (lacks GPIbα extracellular domain) bone marrow and assayed for atherosclerosis development after feeding with pro-atherogenic "western diet". Here, we report that Ldlr-/-mice reconstituted with GPIbα-/- bone marrow developed less atherosclerosis compared to wt controls; accompanied by augmented accumulation of pro-inflammatory CD11b+ and CD11c+ myeloid cells, reduced oxLDL uptake and decreased TNF and IL 12p35 gene expression in the aortas. Flow cytometry and live cell imaging in whole blood-perfused microfluidic chambers revealed reduced platelet-monocyte aggregates in GPIbα-/- mice, which resulted in decreased monocyte activation. Interestingly, Ldlr-/-mice reconstituted with IL-4R/GPIbα-Tg bone marrow, producing less abnormal platelets, showed atherosclerotic lesions similar to wt mice. Platelet interaction with blood monocytes and accumulation of myeloid cells in the aortas were also essentially unaltered. Moreover, only complete GPIbα ablation altered platelet microparticles and CCL5 chemokine production. Thus, atherosclerosis reduction in mice lacking GPIbα may not result from the defective GPIbα-ligand binding, but more likely is a consequence of functional defects of GPIbα-/- platelets and reduced blood platelet counts.

Mapping Paratope on Antithrombotic Antibody 6B4 to Epitope on Platelet Glycoprotein Ibalpha via Molecular Dynamic Simulations

Binding of platelet receptor glycoprotein Ibα (GPIbα) to the A1 domain of von Willebrand factor (vWF) is a critical step in both physiologic hemostasis and pathologic thrombosis, for initiating platelet adhesion to subendothelium of blood vessels at sites of vascular injury. Gain-of-function mutations in GPIbα contribute to an abnormally high-affinity binding of platelets to vWF and can lead to thrombosis, an accurate complication causing heart attack and stroke. Of various antithrombotic monoclonal antibodies (mAbs) targeting human GPIbα, 6B4 is a potent one to inhibit the interaction between GPIbα and vWF-A1 under static and flow conditions. Mapping paratope to epitope with mutagenesis experiments, a traditional route in researches of these antithrombotic mAbs, is usually expensive and time-consuming. Here, we suggested a novel computational procedure, which combines with homology modeling, rigid body docking, free and steered molecular dynamics (MD) simulations, to identify key paratope residues on 6B4 and their partners on GPIbα, with hypothesis that the stable hydrogen bonds and salt bridges are the important linkers between paratope and epitope residues. Based on a best constructed model of 6B4 bound with GPIbα, the survival ratios and rupture times of all detected hydrogen bonds and salt bridges in binding site were examined via free and steered MD simulations and regarded as indices of thermal and mechanical stabilizations of the bonds, respectively. Five principal paratope residues with their partners were predicted with their high survival ratios and/or long rupture times of involved hydrogen bonds, or with their hydrogen bond stabilization indices ranked in top 5. Exciting, the present results were in good agreement with previous mutagenesis experiment data, meaning a wide application prospect of our novel computational procedure on researches of molecular of basis of ligand-receptor interactions, various antithrombotic mAbs and other antibodies as well as theoretically design of biomolecular drugs.

Six Novel Mutations Identified in the Glycoproteins Ib Alpha, Ib Beta and IX Genes Among Twenty-Two Unrelated Patients with Bernard-Soulier Syndrome in Brazil

Abstract 1156▪▪This icon denotes a clinically relevant abstract Introduction:Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder, caused by mutations within the glycoprotein (GP) Ib alpha, GP Ib beta and GP IX genes that encode three of four subunits of the platelet GP Ib-V-IX adhesion receptor. In the present study we evaluated the mutations involved in the diagnosis of BSS from twenty-two unrelated patients. Patients and Methods: All patients were followed in two large bleeding disorder reference centers in Brazil. The diagnosis of BSS was established based on the presence of mucocutaneous bleeding and macrotrombocytopenia, and was confirmed by platelet ristocetin aggregation and flow cytometry for the platelet GP Ib alpha (CD 42b), GP Ib beta (CD 42c), and GP IX (CD 42a). Available first- and second-degree relatives were also contacted for clinical, laboratory and molecular evaluation. Genomic DNA from all index cases was used for sequence analysis of the three genes, GP1BA, GP1BB and GP9, and the results were confirmed in relatives when available. Results: Twenty-two unrelated patients with the confirmed diagnosis of BSS were enrolled in this study. Among twenty-two index cases, twenty-one had one or two mutations identified, including six novel mutations. We also identified two mutations that have been previously reported, GP1BA C209S (Simsek, et al., 1994), and GP9 A140T (Wang, et al., 2004). The six novel mutations correspond to conserved regions, and they consist of two mutations in the GP1BA (L51R, and L99P), three in GP1BB (M-25A, L72R, and L112P), and one in GP9 (P52Q). One of these novel mutations, the GP1BB L112P was observed in twelve of twenty-two unrelated cases. PCR-restriction fragment length analysis of genomic DNA from a hundred normal unrelated controls were performed to evaluate the presence of GP1BA L99P and GP1BB L112P mutations by using the AlwN I and Alu I restriction enzymes, respectively. All controls were negative for both mutations. Interestingly, when we analyzed the relatives of the index cases indentified with the mutations GP1BA L99P and GP1BB M-25A, we found evidence of mild macrotrombocytopenia in the heterozygote carriers of these mutations. The relatives heterozygous for GP1BB L112P showed normal platelet count and morphology. However, in three index cases with mild macrotrombocytopenia, GP1BB L112P in heterozygosity was the only mutation identified. Furthermore, site-directed mutagenesis was carried out to evaluate the expression of the novel mutations GP1BA L51R, and GP1BA L99P. Compared to the GP1BA wild-type, both mutations were only minimally expressed in CHO bIX cells, which stably express GP Ib beta and GP IX. Conclusions: We identified in this study eight distinct mutations among twenty-two unrelated SBS patients, including six novel mutations. The GP1BB L112P mutation was found in twelve of the twenty-two index cases, suggesting that this could be due to a founder effect. Identifying such a frequent mutation in this population of BSS patients will be helpful for genetic diagnosis of this condition in Brazil. Furthermore these mutations significantly add to the mutation database of BSS and will inevitably provide insights into the function of GP Ib-V-IX. Disclosures:No relevant conflicts of interest to declare.

Destabilization of the A1 Domain in von Willebrand Factor Dissociates the A1A2A3 Tri-domain and Provokes Spontaneous Binding to Glycoprotein Ibα and Platelet Activation under Shear Stress

This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ibalpha (GPIbalpha). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIbalpha without ristocetin with an apparent K(D) of 85 +/- 34 nm, comparable with that of WT (36 +/- 12 nm) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s(-1) shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s(-1), platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIbalpha and alphaIIbbeta3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.

Engineering and Characterization of a Chimeric Anti-platelet Glycoprotein Ibα Monoclonal Antibody and Preparation of Its Fab Fragment

Glycoprotein Ibalpha (GPIbalpha) is a platelet-specific membrane protein. It mediates platelet adhesion to collagen exposed at the vascular injury site by binding to von Willebrand factor (VWF) in plasma. This process is crucial for arterial thrombus formation. Blocking interaction between GPIbalpha and VWF may prevent platelet adhesion and thrombus formation. We previously generated a high affinity monoclonal antibody against human platelet GPIbalpha, SZ2, which inhibits both ristocetin- and botrocetin-induced platelet aggregation in vitro. To convert SZ2 into mouse/human chimeric antibody for anti-platelet therapy in humans, in this study, we constructed a mouse/human chimeric antibody derived from the hybridoma cells producing murine antibody against platelet glycoprotein Ibalpha, conducted its expression in dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells, and prepared its chimeric Fab fragment. Results from ELISA and Western blot analysis showed that the chimeric antibody was secreted from the cells and that the heavy and light chains were assembled correctly. Flow cytometry analysis confirmed specific binding of the chimeric antibody to the GPIb-expressing CHO cells. In vitro functional studies revealed that the chimeric antibody and its Fab fragment prevented platelet adhesion to VWF under high shear stress and inhibited ristocetin-induced platelet aggregation in a dose-dependent manner. These results demonstrated that the chimeric antibody was successfully engineered and suggested that the Fab fragment of chimeric antibody against GPIbalpha is a promising therapeutic antibody more suitable for prevention and treatment of human arterial thrombosis.

Inhibition of Platelet GPIbα and Promotion of Melanoma Metastasis

Platelet glycoprotein Ibalpha (GPIb alpha) is part of the receptor complex GPIb-V-IX, which has a critical role in hemostasis, especially through interactions with the subendothelial von Willebrand factor. As there is accumulating evidence for a contribution of platelet receptors to hematogenous tumor metastasis, GPIb alpha is an interesting molecule to study in this context. We have investigated the effect of GPIb alpha inhibition by monovalent Fab fragments on experimental pulmonary metastasis in a syngeneic mouse model using C57BL/6 mice and B16F10 melanoma cells. The early fate of green fluorescent protein (GFP)-transfected melanoma cells under GPIb alpha blockade was also assessed, as was the effect of GPIb alpha inhibition on pulmonary metastasis in mice lacking P-selectin. Surprisingly and, to our knowledge previously unreported, GPIb alpha inhibition led to a significant increase in pulmonary metastasis, and assessment of the early fate of circulating GFP-labeled B16F10 showed improved survival and pulmonary arrest of tumor cells shortly after GPIb alpha inhibition, indicating that inhibition of a platelet protein can, in some cases, promote metastasis of a malignant tumor. In contrast, GPIb alpha blockade in P-selectin-deficient mice had no enhancing effect on metastasis, suggesting the involvement of GPIb alpha in the initial, P-selectin-dependent steps of metastasis. These findings suggest that GPIb alpha contributes to the control of tumor metastasis, in addition to its role in hemostasis.

Diagnosis of platelet-type von Willebrand disease by flow cytometry

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder which is due to a mutation in the gene encoding for platelet glycoprotein Ibalpha (GPIbalpha) resulting in enhanced affinity for von Willebrand factor (VWF). PT-VWD is often mistakenly diagnosed as type 2B VWD for the similarities between these two conditions. We characterized a new case of PT-VWD and evaluated the usefulness of a flow cytometric assay in the differential diagnosis between PT-VWD (n=1) and type 2B VWD (n=4). The flow cytometric assay was able to highlight the increased affinity of VWF for GPIbalpha as much as did RIPA and to differentiate the two diseases through mixing tests. Genetic analysis revealed a heterozygous point mutation in codon 239 of the GPIbalpha gene leading to a methionine to valine substitution (M239V). Flow cytometry represents a useful tool for the diagnosis of PT-VWD.

Calpain Regulates ADAM17-Mediated Platelet Glycoprotein Ibalpha Ectodomain Shedding.

Abstract 2996Poster Board II-974 Introduction:The interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbalpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbalpha shedding. Calpain, the calcium-dependent neutral protease, has been thought to play a key role in GPIbalpha shedding, whereas it still remains unclear the role of calpain in ADAM17-mediated GPIbalpha shedding. Methods:Washed platelets isolated from vein blood of healthy donors were pre-incubated with various reagents. GPIbalpha fragments were detected by Western blot with anti- GPIbalpha C- or N-terminal antibodies. GPIbalpha surface expression was determined by flow cytometry with anti-GPIbalpha N-terminal antibody. Results:Western blot and flow cytometry results showed that cell permeable calpain inhibitors, MDL 28170, calpain inhibit I and calpain inhibitor II, significantly reduced calmodulin inhibitor W7- and calcium ionophore A23187-induced GPIbalpha shedding. Dibucaine induced GPIbalpha shedding was potently inhibited by calpain inhibitors. W7 and A23187 induced talin cleavage was abolished by calpain inhibitors. Calpain inhibitors had no obvious effect on N-ethylmaleimide-induced GPIbalpha shedding. In order to investigate the role of protein kinase C (PKC) in calpain-mediated GPIbalpha shedding, calpain inhibitors or PKC inhibitor (bisindolylmaleimide I hydrochloride, BIM) were incubated with platelets respectively, and then platelets were further incubated with PKC activator phorbol-ester (PMA) or dibucaine. PMA-induced or dibucaine-mediated GPIbalpha shedding did not inhibit by calpain inhibitors or PKC inhibitor respectively, indicating that PKC is not involved in calpain-mediated GPIbalpha shedding. In addition, two CHO cell lines expressing wild-type GPIb-IX and GPIb-IX mutant with truncated GPIbalpha lacking filamin A binding site were used to test the role of filamin A in calpain-regulated GPIbalpha shedding. Dibucaine-induced GPIbalpha shedding was inhibited by MDL28170 in both of the cell lines. Conclusion:The data indicate that calpain plays key roles in ADAM17-mediated GPIbalpha ectodomain shedding and the calpain-regualted GPIbalpha shedding is independent of PKC activity or filamin A cleavage. Disclosures:No relevant conflicts of interest to declare.

The Effects of Hyperthermia On Platelet Physiology.

Abstract 2422Poster Board II-399 Introduction:In some patients with fever or hyperthermia, hemorrhage is a significant pathological feature, which incurs severe or even fatal consequence. Although the mechanisms of hemorrhage in patients with hyperthermia or fever have been though to be complex, whether there is an association between hemorrhage and hyperthermia or fever is not well understood. Platelets play a central role in maintaining integrity of endothelium and biological hemostasis. Platelet count obviously reduces in most of patients with dengue fever or heatstroke, and reduced platelet aggregations have been found in patients with hemorrhage fever, raising the possibility that fever or hyperthermia incurs reduction of platelet count or function leading to hemorrhage. Methods and Results:To explore the effect of hyperthermia on platelet function, platelet-rich plasma (PRP) was isolated and incubated at hypothermia (22 degrees C), normothermia (37 degrees C) or hyperthermia (40 and 42 degrees C) for 1 or 2 hours, and then induced to aggregation by ADP. Platelet aggregation was significantly reduced with the growth of temperature in a time-dependent manner. To exclude the possible interference from plasma proteins, and to further investigate the effects of hyperthermia on platelet function, alpha-thrombin induced platelet aggregations were examined in washed platelets incubated at different temperatures for 1 or 2 hours. Washed platelets presented normal aggregation response to alpha-thrombin in platelets incubated at hypothermia, whereas alpha-thrombin induced platelet aggregations reduced gradually with the increase of temperature and time duration. Next, we investigated whether the reductions of platelet aggregation were resulted from platelet apoptosis under high temperature conditions. Hyperthermia gradually induced apoptotic events in platelets with the increase of temperature and time duration, including depolarization of mitochondrial inner transmembrane potential (ΔΨm), gelsolin cleavage, and phosphatidylserine (PS) exposure. Furthermore, hyperthermia incurs platelet glycoprotein (GP) Ibalpha ectodomain shedding. Conclusions:These results indicate that hyperthermia induces platelet apoptosis, thus suggesting the possible reason why platelet count or function was reduced in some patients with fever or hyperthermia. These findings not only have important implications for the pathogenesis of hemorrhage in some fever or hyperthermia-related diseases, but also suggest that attentions should be paid on platelet apoptosis under relative high temperature conditions, such as during hyperthermia therapy or platelet storage. Disclosures:No relevant conflicts of interest to declare.

Functional association of phosphoinositide-3-kinase with platelet glycoprotein Ibalpha, the major ligand-binding subunit of the glycoprotein Ib-IX-V complex.

The adhesion receptor glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet-mediated tumor metastasis. The cytoplasmic C-terminal domain of the ligand-binding GPIbalpha subunit contains binding sites for filamin (residues 561-572, critically Phe568/Trp570), 14-3-3zeta (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide-3-kinase (PI3-kinase) regulatory subunit, p85. We previously showed that, as compared with wild-type receptor, deleting the contiguous sequence 580-590 or 591-610, but not upstream sequences, of GPIbalpha expressed as a GPIb-IX complex in Chinese hamster ovary cells inhibited VWF-dependent Akt phosphorylation, which is used as a read-out for PI3-kinase activity. Pulldown experiments using glutathione-S-transferase (GST)-p85 or GST-14-3-3zeta constructs, and competitive inhibitors of 14-3-3zeta binding, suggested an independent association of 14-3-3zeta and PI3-kinase with GPIbalpha. The objective of this study was to analyze a further panel of GPIbalpha deletion mutations within residues 580-610. We identified a novel deletion mutant, Delta591-595, that uniquely disrupts 14-3-3zeta binding but retains the functional p85/PI3-kinase association. Deletion of other sequences within the 580-610 region were less discriminatory, and either partially affected p85/PI3-kinase and 14-3-3zeta binding (Delta580-585, Delta586-590, Delta596-600, Delta601-605), or strongly inhibited binding of both proteins (Delta606-610). Together, these findings have significant implications for interpreting the functional role of p85 and/or 14-3-3zeta in GPIb-dependent signaling or platelet functional studies involving truncation of the C-terminal residues in cell-based assays and mouse models. The Delta591-595 mutation provides another strategy for determining the function of GPIbalpha-associated 14-3-3zeta by selective disruption of 14-3-3zeta but not p85/PI3-kinase binding.

Factor XI Binding to Platelets

Platelets are crucial in supporting coagulation reactions. The classical view is that platelets bind the vitamin K-dependent factors (VII, IX, X, II, protein C, S, and Z) via their gamma carboxyglutamic acid (gla) residues, and in this way localize coagulation factor complexes to the platelet membrane.1 For binding of gla-containing proteins to the platelet surface to occur, the platelet needs to be activated after which negatively charged phospholipids, including phosphatidylserine, are translocated from the inner to the outer leaflet of the platelet membrane. Exposure of negatively charged lipids enables a Ca2+-dependent interaction of gla-containing proteins with the negatively charged platelet membrane. See accompanying article on page 1602 In addition to the gla-containing coagulation factors, multiple coagulation factors lacking a gla-domain, including thrombin,2 factor XI(a),3 factor XII(a),4 and high-molecular-weight kininogen5 interact with platelets, and the interaction of all these proteins has been shown to be (in part) mediated by glycoprotein Ibα (GPIbα). Recent work has demonstrated that also some of the gla-containing coagulation factors, factor VII(a),6 factor IX(a),7 and both zymogen and activated protein C8 interact with GPIbα. These findings indicate that localization of coagulation factors to the platelet surface is much more complicated than anticipated by the model in which the interaction with negatively charged phospholipids was considered required and sufficient for gla-containing proteins to bind to platelets. Indeed, it has been demonstrated that the thrombin-generating capacity of activated platelets from different …

Shear‐regulated uptake of nanoparticles by endothelial cells and development of endothelial‐targeting nanoparticles

The purpose of this research project was to develop nanoparticles with improved targeting, adhesion, and cellular uptake to activated or inflamed endothelial cells (ECs) under physiological flow conditions. Our hypothesis is that by mimicking platelet binding to activated ECs through the interaction between platelet glycoprotein Ibalpha (GP Ibalpha) and P-selectin on activated endothelial cells, GP Ibalpha-conjugated nanoparticles could exhibit increased targeting and higher cellular uptake in injured or activated endothelial cells under physiological flow conditions. To test this hypothesis, fluorescent-carboxylated polystyrene nanoparticles were selected for the study as a model particle because of its narrow size distribution as a "proof-of-concept." Using confocal microscopy, fluorescent measurements, and protein assays, cellular uptake properties were characterized for these polystyrene nanoparticles. The study also found that conjugation of 100-nm polystyrene nanoparticles with glycocalicin (the extracellular segment of GP Ibalpha) significantly increased the particle adhesion on P-selectin-coated surfaces and cellular uptake of nanoparticles by activated endothelial cells under physiological flow conditions. The results demonstrate that these novel endothelial-targeting nanoparticles could be the first step toward developing a targeted and sustained drug delivery system that can improve shear-regulated particle adhesion and cellular uptake.

Staphylococcal Superantigen-Like 5 Activates Platelets, and Supports Platelet Adhesion Under Flow Conditions, Which Is Mediated by GPIb- Alpha and Alpha-IIb-Beta-3

Introduction - Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus Aureus and has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Since PSGL-1 shows close homology to platelet glycoprotein Ib-alpha (GPIb), and as platelets have been shown to play an important role in neutrophil chemotaxis, we investigated whether SSL5 also interacts with platelets.Results - When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, full and irreversible aggregation was observed even in the absence of exogenous fibrinogen. Proteolysis of the extracellular part of GPIb-alpha or the addition of dRGDW abrogated platelet aggregation. SSL5 was shown to interact with glycocalicin, a soluble GPIb-alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3zeta to the actin cytoskeleton. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s−1, large stable platelet aggregates were observed. Platelet adhesion was substantially reduced by proteolysis of GPIb-alpha or addition of dRGDW. In addition, real-time video analysis showed that initial tethering of the platelets was abrogated by proteolysis of GPIb-alpha. Furthermore, when whole citrated blood was perfused over immobilized SSL5, real-time video analysis revealed that platelets activated with the PAR-1 activating peptide SFLLRN made transient contacts with the immobilized SSL5, although no permanent contacts were observed. Finally, SSL5 was shown to interact with endothelial cell matrix (ECM) from cultured human umbilical vein endothelial cells. The interaction of SSL5 with ECM enhanced aggregation of platelets from whole blood to this ECM.Discussion - Here, we show that SSL5 is capable of activation, adhesion and aggregation of platelets, in which both GPIb-alpha and alpha-IIb-beta-3 play a role. We hypothesize that platelet activation by SSL5 could be important in colonization of the vascular bed and evasion of the immune system.

P-selectin Glycoprotein Ligand-1 Decameric Repeats Regulate Selectin-dependent Rolling under Flow Conditions

P-selectin glycoprotein ligand-1 (PSGL-1) interacts with selectins to support leukocyte rolling along vascular wall. L- and P-selectin bind to N-terminal tyrosine sulfate residues and to core-2 O-glycans attached to Thr-57, whereas tyrosine sulfation is not required for E-selectin binding. PSGL-1 extracellular domain contains decameric repeats, which extend L- and P-selectin binding sites far above the plasma membrane. We hypothesized that decamers may play a role in regulating PSGL-1 interactions with selectins. Chinese hamster ovary cells expressing wild-type PSGL-1 or PSGL-1 molecules exhibiting deletion or substitution of decamers with the tandem repeats of platelet glycoprotein Ibalpha were compared in their ability to roll on selectins and to bind soluble L- or P-selectin. Deletion of decamers abrogated soluble L-selectin binding and cell rolling on L-selectin, whereas their substitution partially reversed these diminutions. P-selectin-dependent interactions with PSGL-1 were less affected by decamer deletion. Videomicroscopy analysis showed that decamers are required to stabilize L-selectin-dependent rolling. Importantly, adhesion assays performed on recombinant decamers demonstrated that they directly bind to E-selectin and promote slow rolling. Our results indicate that the role of decamers is to extend PSGL-1 N terminus far above the cell surface to support and stabilize leukocyte rolling on L- or P-selectin. In addition, they function as a cell adhesion receptor, which supports approximately 80% of E-selectin-dependent rolling.

Platelet adhesion: a game of catch and release

The interaction of circulating platelets with the vessel wall involves a process of cell catch and release, regulating cell rolling, skipping, or firm adhesion and leading to thrombus formation in flowing blood. In this regard, the interaction of platelet glycoprotein Ibalpha (GPIbalpha) with its adhesive ligand, vWF, is activated by shear force and critical for platelet adhesion to the vessel wall. In this issue of the JCI, Yago and colleagues show how gain-of-function mutations in the GPIbalpha-binding vWF A1 domain disrupt intramolecular interactions within WT vWF A1 that regulate binding to GPIbalpha and flow-enhanced platelet rolling and adhesion (see the related article beginning on page 3195). Together, these studies reveal molecular mechanisms regulating GPIbalpha-vWF bond formation and platelet adhesion under shear stress.