Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4.

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.

Tetraspanin CD9 is required for microparticle release from coated-platelets.

CD9, a member of the tetraspanin superfamily, is the third most abundant protein on the platelet surface, but its function remains unknown. In this report, we demonstrate that CD9 is required for the release of microparticles from coated-platelets. Coated-platelets are formed as a result of dual agonist activation with collagen and thrombin, and each coated-platelet releases 15-25 microparticles averaging 0.4 microm in diameter. We report here that four separate monoclonal antibodies against CD9 inhibited microparticle release from coated-platelets by 72-102% with an IC(50) of approximately 500 ng/mL for ALB6 and SN4. In addition, the anti-alpha(IIb)beta(3) monoclonal antibody AP2 also inhibited microparticle release although additional anti-alpha(IIb)beta(3) monoclonals did not. These data support participation of the tetraspanin CD9, together with the integrin alpha(IIb)beta(3), in the membrane vesiculation process associated with platelet microparticle release.

Characterization of a streptococcal cholesterol-dependent cytolysin with a lewis y and b specific lectin domain.

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that often exhibit distinct structural changes that modify their pore-forming activity. A soluble platelet aggregation factor from Streptococcus mitis (Sm-hPAF) was characterized and shown to be a functional CDC with an amino-terminal fucose-binding lectin domain. Sm-hPAF, or lectinolysin (LLY) as renamed herein, is most closely related to CDCs from Streptococcus intermedius (ILY) and Streptococcus pneumoniae (pneumolysin or PLY). The LLY gene was identified in strains of S. mitis, S. pneumoniae, and Streptococcus pseudopneumoniae. LLY induces pore-dependent changes in the light scattering properties of the platelets that mimic those induced by platelet aggregation but does not induce platelet aggregation. LLY monomers form the typical large homooligomeric membrane pore complex observed for the CDCs. The pore-forming activity of LLY on platelets is modulated by the amino-terminal lectin domain, a structure that is not present in other CDCs. Glycan microarray analysis showed the lectin domain is specific for difucosylated glycans within Lewis b (Le (b)) and Lewis y (Le (y)) antigens. The glycan-binding site is occluded in the soluble monomer of LLY but is apparently exposed after cell binding, since it significantly increases LLY pore-forming activity in a glycan-dependent manner. Hence, LLY represents a new class of CDC whose pore-forming mechanism is modulated by a glycan-binding domain.

Role of mitochondrial permeability transition pore in coated-platelet formation.

OBJECTIVE: Coated-platelets are a subset of cells observed during costimulation of platelets with collagen and thrombin. Important characteristics of coated-platelets include retention of multiple alpha-granule proteins and expression of phosphatidylserine on the cell surface. The mitochondrial permeability transition pore (MPTP) is a key step in apoptosis and is suggested to be involved in some forms of platelet activation. The objective of this study was to examine the role of MPTP in the synthesis of coated-platelets. METHODS AND RESULTS: Flow cytometric analysis of coated-platelet production was used to examine the impact of pharmacological effectors of MPTP formation. Cyclosporin A, coenzyme Q, and bongkrekic acid all inhibit MPTP formation as well as production of coated-platelets. Phenylarsine oxide and diamide, both potentiators of MPTP formation, stimulate coated-platelet synthesis. Atractyloside, another inducer of MPTP formation, does not affect the percentage of coated-platelets synthesized; however, it does increase the level of phosphatidylserine exposed on the surface of coated-platelets. CONCLUSIONS: These findings indicate that MPTP formation is an integral event in the synthesis of coated-platelets. Although the exact function of the MPTP remains to be determined, these data support a growing body of evidence that apoptosis-associated events are vital components of the platelet activation process. Formation of coated-platelets involves a complex set of activation events initiated by dual agonist activation. The mitochondrial permeability transition pore (MPTP) is a key intermediate in apoptosis and has been suggested to impact platelet activation. This report demonstrates that MPTP formation is essential to production of coated-platelets.

Active tissue factor pathway inhibitor is expressed on the surface of coated platelets.

The incorporation of blood-borne forms of tissue factor (TF) into a growing blood clot is necessary for normal fibrin generation and stabilization of the blood clot. Tissue factor pathway inhibitor (TFPI) is the primary physiologic inhibitor of tissue factor and is present within platelets. Expression of TFPI on the platelet surface may be the optimal location for it to abrogate blood-borne TF activity that incorporates within the blood clot, balancing the need for adequate hemostasis while preventing development of occlusive thrombosis. TFPI is produced by megakaryocytes but is not expressed on the platelet surface. Activation of platelets with thrombin receptor activation peptide does not cause release or surface expression of TFPI, demonstrating that TFPI is not stored within platelet alpha granules. TFPI is expressed on the platelet surface following dual-agonist activation with convulxin plus thrombin to produce coated platelets. In association with its expression on the surface of coated platelets TFPI is also released in microvesicles or as a soluble protein.

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface.

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.