Platelet Membrane Receptor Proteolysis: Implications for Platelet Function

The activities of adhesion and signaling receptors in platelets are controlled by several mechanisms. An important way of regulation is provided by proteolytic cleavage of several of these receptors, leading to either a gain or a loss of platelet function. The proteases involved are of different origins and types: (i) present as precursor in plasma, (ii) secreted into the plasma by activated platelets or other blood cells, or (iii) intracellularly activated and cleaving cytosolic receptor domains. We provide a comprehensive overview of the proteases acting on the platelet membrane. We describe how these are activated, which are their target proteins, and how their proteolytic activity modulates platelet functions. The review focuses on coagulation-related proteases, plasmin, matrix metalloproteinases, ADAM(TS) isoforms, cathepsins, caspases, and calpains. We also describe how the proteolytic activities are determined by different platelet populations in a thrombus and conversely how proteolysis contributes to the formation of such populations.

Glycoprotein IIb/IIIa and P2Y12Induction by Oligochitosan Accelerates Platelet Aggregation

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 P2Y12is 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 P2Y12through 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 P2Y12levels 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 aggregationin vitro.

Streptococcus mitis Phage-Encoded Adhesins Mediate Attachment to α2-8-Linked Sialic Acid Residues on Platelet Membrane Gangliosides

ABSTRACT The direct binding of bacteria to human platelets contributes to the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 is mediated in part by two bacteriophage-encoded proteins, PblA and PblB. However, the platelet membrane receptor for these adhesins has been unknown. In this study, we demonstrate that these proteins mediate attachment of bacterial cells to sialylated gangliosides on the platelet cell surface. Desialylation of human platelet monolayers reduced adherence of SF100, whereas treatment of the platelets with N- or O-glycanases did not affect platelet binding. Treatment of platelets with sialidases having different linkage specificities showed that removal of α2-8-linked sialic acids resulted in a marked reduction in bacterial binding. Preincubation of SF100 with ganglioside GD3, a glycolipid containing α2-8-linked sialic acids that is present on platelet membranes, blocked subsequent binding of this strain to these cells. In contrast, GD3 had no effect on the residual binding of platelets by strain PS344, an isogenic ΔpblA ΔpblB mutant. Preincubating platelets with specific monoclonal antibodies to ganglioside GD3 also inhibited binding of SF100 to platelets, but again, they had no effect on binding by PS344. When the direct binding of S. mitis strains SF100 and PS344 to immobilized gangliosides was tested, binding of PS344 to GD3 was reduced by 70% compared to the parent strain. These results indicated that platelet binding by SF100 is mediated by the interaction of PblA and PblB with α2-8-linked sialic acids on ganglioside GD3.

Inhibition of thrombin-induced platelet aggregation using human single-chain Fv antibodies specific for TREM-like transcript-1

SummaryTREM-like transcript-1 (TLT-1) is a novel platelet membrane receptor, which has been recently characterized in mice. TLT-1 is expressed exclusively in platelets and megakaryocytes, and its expression is dramatically upregulated upon platelet activation, suggesting that it plays a unique role in hemostasis and/or thrombosis. In this study we identified and characterized highly specific human monoclonal antibodies that bind to TLT-1 by screening a naïve library of single chain Fv fragments (scFvs) displayed on filamentous phage (Thomlinson I library). These scFvs detected plate-bound TLT-1, captured soluble TLT-1, and readily reacted with cell-bound TLT-1 on transfectants and primary human platelets. Most importantly, anti-TLT-1 scFvs inhibited thrombin-mediated human platelet aggregation. This inhibition was specific for thrombin-induced aggregation and was reversible with higher doses of agonist. These data are the first to demonstrate a biological role for TLT-1 and its potential as a therapeutic target. The human scFvs isolated in this study may represent novel anti-platelet therapeutic agents.

Clumping Factor A Mediates Binding ofStaphylococcus aureus to Human Platelets

ABSTRACT The direct binding of bacteria to platelets may be an important virulence mechanism in the pathogenesis of infective endocarditis. We have previously described Staphylococcus aureus strain PS12, a Tn551-derived mutant of strain ISP479, with reduced ability to bind human platelets in vitro. When tested in an animal model of endocarditis, the PS12 strain was less virulent than its parental strain, as measured by bacterial densities in endocardial vegetations and incidence of systemic embolization. We have now characterized the gene disrupted in PS12 and its function in platelet binding. DNA sequencing, Southern blotting, and PCR analysis indicate that PS12 contained two Tn551 insertions within the clumping factor A (ClfA) locus (clfA). The first copy was upstream from the clfA start codon and appeared to have no effect on ClfA production. The second insertion was within the region encoding the serine aspartate repeat of ClfA and resulted in the production of a truncated ClfA protein that was secreted from the cell. A purified, recombinant form of the ClfA A region, encompassing amino acids 40 through 559, significantly reduced the binding of ISP479C to human platelets by 44% (P = 0.0001). Immunoprecipitation of recombinant ClfA that had been incubated with solubilized platelet membranes coprecipitated a 118-kDa platelet membrane protein. This protein does not appear to be glycoprotein IIb. These results indicate that platelet binding by S. aureus is mediated in part by the direct binding of ClfA to a novel 118-kDa platelet membrane receptor.

Arteriosclerosis and the promise of GPIIb/IIIa inhibitors in stroke

Ischemic mechanisms in patients with brain and heart attacks have been studied for more than 150 years. Antiplatelets agents did show benefit in secondary prevention. Aspirin is the most common antiaggregant in clinical use today. However, the benefit produced by the "best" antiplatelet regimen in stroke prevention is lower than 40%. The adherence of circulating platelets to the subendothelium is mediated by glycoprotein (GP) residing on the cell's surface. GPIIb/IIIa is the most important platelet membrane receptor that mediates the process of platelet aggregation, and thrombus formation. Thus, new drugs that block the GPIIb/IIIa receptor have recently emerged. Clinical trials using these agents have shown effectiveness in acute coronary syndromes. However, the absence of studies in cerebrovascular disease and the potential hemorrhagic complications questioned their use in stroke prevention. We review the clinical trials using the new GPIIb/IIIa agents in myocardial ischemia, and consider the potential implications for cerebrovascular disease.