Platelet membrane receptor glycoprotein IIb/IIIa (gpiibiiia) is a receptor detected on platelets. Adenosine diphosphate (ADP) activates gpiibiiia and P2Y12, causing platelet aggregation and thrombus stabilization during blood loss. Chitosan biomaterials were found to promote surface induced hemostasis and were capable of activating blood coagulation cascades by enhancing platelet aggregation. Our current findings show that the activation of the gpiibiiia complex and the major ADP receptor P2Y12 is required for platelet aggregation to reach hemostasis following the adherence of various concentrations of chitosan biomaterials [7% N,O-carboxymethylchitosan (NO-CMC) with 0.45 mL collagen, 8% NO-CMC, oligochitosan (O-C), and oligochitosan 53 (O-C 53)]. We studied gpiibiiia and P2Y12 through flow cytometric analysis and western blotting techniques. The highest expression of gpiibiiia was observed with Lyostypt (74.3 ± 7.82%), followed by O-C (65.5 ± 7.17%). Lyostypt and O-C resulted in gpiibiiia expression increases of 29.2% and 13.9%, respectively, compared with blood alone. Western blot analysis revealed that only O-C 53 upregulated the expression of P2Y12 (1.12 ± 0.03-fold) compared with blood alone. Our findings suggest that the regulation of gpiibiiia and P2Y12 levels could be clinically useful to activate platelets to reach hemostasis. Further, we show that the novel oligochitosan is able to induce the increased expression of gpiibiiia and P2Y12, thus accelerating platelet aggregation in vitro.

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

1. Matrix metalloproteinase-2 (MMP-2) plays a role in agonist- and tumour cell-induced platelet aggregation (TCIPA). 2. MMP-2 is synthesized as a proenzyme and is activated at the cell surface by membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14). 3. The significance of tumour cell-associated MT1-MMP for TCIPA was investigated using human breast carcinoma MCF7 cells stably coexpressing the integrin alphavbeta3 with MT1-MMP, cells expressing alphavbeta3 alone and mock-transfected cells. 4. Western blot and zymography confirmed that alphavbeta3/MT1-MMP cells expressed MT1-MMP and efficiently processed proMMP-2 to MMP-2. 5. Aggregometry, phase-contrast and transmission electron microscopy and flow cytometry were used to characterize TCIPA induced by MCF7 cell lines. 6. The aggregating potency of cells was: alphavbeta3/MT1-MMP >alphavbeta3=mock cells, as shown by aggregometry and phase-contrast microscopy. 7. Electron microscopy revealed close, membrane-membrane interactions between activated platelets and alphavbeta3/MT1-MMP cells during TCIPA. 8. Inhibition of MMP-2 with the neutralizing anti-MMP-2 antibody (5 microg ml(-1)) and o-phenanthroline (100 microm) reduced aggregation induced by alphavbeta3/MT1-MMP cells. 9. TCIPA induced by alphavbeta3/MT1-MMP cells was also reduced by inhibiting the generation and actions of ADP with apyrase (250 microg ml(-1)) and 2-methylthio-AMP (2-MeSAMP) (30 microm), but not N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) (30 microm). 10. Flow cytometry demonstrated that TCIPA enhanced expression of glycoprotein (GP) Ib and IIb/IIIa receptors not only on platelets but also on breast cancer cells. 11. Thus, (a) human breast carcinoma cell surface-associated MT1-MMP, via activating proMMP-2, stimulates TCIPA; (b) ADP amplifies the effects of MMPs via stimulation of P2Y(12) receptors and (c) both tumour- and platelet-derived GPIb and GPIIb/IIIa are involved in the aggregatory effects of MT1-MMP.

Glycoprotein IIb/IIIa (αIIb/β3) is involved in platelet adhesion, and triggers a series of intracellular signaling cascades, leading to platelet shape change, granule secretion, and clot retraction. In this study, we evaluated the effect of ginsenoside Ro (G-Ro) on the binding of fibrinogen to αIIb/β3.

The impact of upregulation of platelet membrane glycoprotein (GP)IIb/IIIa and P-selectin on the onset of arterial thrombosis, venous thrombosis, and cancer encourages to hypothesize that dual inhibitor of GPIIb/IIIa and P-selectin receptors should simultaneously inhibit arterial thrombosis, block venous thrombosis, and slow tumor growth.

New nanomaterials intended for systemic administration have raised concerns regarding their biocompatibility and hemocompatibility. Quantum dots (QD) nanoparticles have been used for diagnostics, and recent work suggests their use for in vivo molecular and cellular imaging. However, the hemocompatibility of QDs and their constituent components has not been fully elucidated. In the present study, comprehensive investigation of QD-platelet interactions is presented. These interactions were shown using transmission electron microscopy. The effects of QDs on platelet function were investigated using light aggregometry, quartz crystal microbalance with dissipation, flow cytometry, and gelatin zymography. Platelet morphology was also analyzed by phase-contrast, immunofluorescence, atomic-force and transmission electron microscopy. We show that the QDs bind to platelet plasma membrane with the resultant upregulation of glycoprotein IIb/IIIa and P-selectin receptors, and release of matrix metalloproteinase-2. These findings unravel for the first time the mechanism of functional response of platelets to ultrasmall QDs in vitro.

The interaction of fibrinogen with platelet membrane glycoprotein (GP) IIb/IIIa complex is inhibited by the RGD peptides and the peptides corresponding to a sequence unique to fibrinogen in the carboxyl-terminal domain of the gamma chain. The present study was designed to examine the effects of these synthetic peptides on fibrinogen binding to thrombin-activated platelets. The order of potency of these peptides was as follows: RGDS = RGDF > GQQHHLGGAKQAGDV (G15). The RGES peptide had no inhibitory activity. The inhibitory effects of the peptides were synergistic when fibrinogen binding was measured in the mixture of the RGD peptide and the gamma chain peptide. By contrast, the inhibitory effect was additive when the binding was measured in the presence of the RGDS peptide and the RGDF peptide. Thrombin-induced platelet aggregation was also inhibited by these synthetic peptides. The order of potency was the same as that of the inhibition binding assay. These results provide evidence that the RGD sequences present at two different sites in the alpha chain and the gamma chain sequence inhibit fibrinogen binding to the GPIIb/IIIa complex, are mutually exclusive, and are in close spatial proximity in the folded molecule. We conclude that the presence of the RGD sequence and the gamma chain sequence involved in fibrinogen binding domains increases the affinity of fibrinogen to activated platelets.

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.