Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands fromHaemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C₈ reverse phase HPLC. The purified activity, named longicomin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggegation of platelets in plasma and of washed platelets (IC₅₀ was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar ca²⁺ level of platelets in response to collagen were completely eliminated by longicomin. Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicomin, indicating that longicomin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicornin, indicating that longicomin did not bind directly to collagen fibers. The identification and isolation of longicomin demonstrates the existence of a new type of platelet inhibitor that should be useful to better undentand the mechanism of collagen stimulation of platelets. Project supported by the National Natural Science Foundation of China (Grant No. 39170591).     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tickHaemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands fromHaemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C₈ reverse phase HPLC. The purified activity, named longicomin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggegation of platelets in plasma and of washed platelets (IC₅₀ was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar ca²⁺ level of platelets in response to collagen were completely eliminated by longicomin. Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicomin, indicating that longicomin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicornin, indicating that longicomin did not bind directly to collagen fibers. The identification and isolation of longicomin demonstrates the existence of a new type of platelet inhibitor that should be useful to better undentand the mechanism of collagen stimulation of platelets.     

A collagen-like immunodeterminant on the surface of Streptococcus sanguis induces platelet aggregation

The basis of similarities in the mechanism of human platelet aggregation induced by soluble collagen and the dental plaque bacterium Streptococcus sanguis was analyzed. Structural and functional comparisons were made by using molecular probes, including rabbit antibody fractions reactive with components on S. sanguis and a synthetic, collagen-like octapeptide mimicking segments from cyanogen bromide fragments 6 and 4 of types I and III collagen, respectively. When platelets were pretreated with tryptic peptides or class II antigen of S. sanguis or with the synthetic, collagen-like octapeptide, the onset of aggregation in response to S. sanguis and collagen was prolonged. When compared to other peptides of similar size and charge, the collagen-like peptide's action towards platelets was shown to be selective. Indeed, absorption of antiserum to S. sanguis cells with particulate type I collagen removed specificities directed at a single S. sanguis antigen. These observations suggested that a common platelet-interactive immunodeterminant on soluble types I and III collagens, particulate type I collagen, and S. sanguis cells was present. Selective inhibition by antibody was used to show structural similarities between the S. sanguis surface proteins and collagen. When either agonist was pretreated with anti-S. sanguis IgG or Fab fragments, the lag time to onset of platelet aggregation was increased. Greater increases in the lag time to aggregation was seen when S. sanguis cells or collagen were pretreated with anti-S. sanguis IgG or Fab fragments made relatively specific for the class II antigen. Neutralization of the platelet-interactive action of the octapeptide by anti-S. sanguis antibody fractions showed that the immunodeterminant common to S. sanguis and collagen triggered platelets in plasma to aggregate. Although the anti-S. sanguis antibodies could inhibit fibrillogenesis, this action was apparently independent of interactions with platelets. In contrast, S. sanguis could bind or adhere to platelets by different determinants. Our data suggest that platelets have at least two distinct sites that bind collagen or S. sanguis. One of these may be a common site for collagen and S. sanguis agonists.     

Evidence that a collagen derived octapeptide inhibits fibrinogen binding to platelets stimulated by collagen and not by ADP

A synthetic octapeptide derived from type III collagen which specifically inhibits the activation and aggregation of platelets by collagen without affecting their adhesion was assayed on the collagen and ADP dependent fibrinogen binding to platelets. With 20 micrograms/ml collagen, the octapeptide (6 mM) inhibited by 68% the fibrinogen binding: this inhibition was correlated (p less than 0.01) to a decrease in the velocity of aggregation, suggesting that the fibrinogen binding might influence this parameter. The octapeptide did not affect the ADP-induced platelet aggregation and fibrinogen binding. This indicates that the octapeptide does not inhibit the binding of fibrinogen to its receptor directly, but interferes with some step(s) preceding the collagen-induced expression of the fibrinogen receptor.     

Antiplatelet effects of clausine-D isolated from Clausena excavata

Clausine-D inhibited concentration-dependently the aggregation and release of washed rabbit platelets induced by arachidonic acid and collagen, without affecting those induced by U46619, PAF and thrombin. The IC₅₀ values of clausine-D on arachidonic acid-and collagen-induced platelet aggregation were calculated to be 9.0±1.1 and 58.9±0.9 μM, respectively. Thromboxane B₂ and prostaglandin D₂ formation in platelets caused by arachidonic acid were also suppressed. Clausine-D inhibited increased intracellular concentration of calcium in platelets caused by arachidonic acid and collagen, and also abolished the generation of inositol monophosphate caused by arachidonic acid, but not that by collagen U46619, PAF and thrombin. In human citrated platelet-rich plasma, clausine-D inhibited the secondary phase, but not the primary phase, of aggregation induced by epinephrine and ADP. These results indicate that the antiplatelet effect of clausine-D is due to inhibition of the formation of thromboxane A₂.     

Technetium-99m labeled 50H.19 antibody fragments: Interaction of the antibody with platelets

The monoclonal antibody 50H.19 recognized three antigens (M_r = 31-, 40-, 45-K) on normal and thromboasthenic platelets, but only one (M_r = 31-K) on Bernard-Soulier platelets. The intact antibody and its F(ab′)₂ fragments, had direct platelet-aggregating activity, and induced the platelet release reaction. The intact antibody potentiated platelet aggregation induced by platelet-activating factor or thrombin. Additions of indomethacin did not inhibit aggregation: addition of PGI₂, or a calcium channel blocker completely inhibited aggregation. A reduced amount of platelet-aggregating activity was observed with antibody fragments prepared for labeling with ^99mTc by pre-exposure to stannous ions, and herein used in biodistribution studies and elsewhere in thrombus imagining studies (J. Nucl. Med. 27: 1315; 1986). Antibody fragments radiolabeled with ^99mTc bound to isolated platelets and to clots containing platelets.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

Brazilin isolated from Caesalpinia sappan L. acts as a novel collagen receptor agonist in human platelets

Background

Brazilin, isolated from the heartwood of Caesalpinia sappan L., has been shown to possess multiple pharmacological properties.

Methods

In this study, platelet aggregation, flow cytometry, immunoblotting analysis, and electron spin resonance (ESR) spectrometry were used to investigate the effects of brazilin on platelet activation ex vivo. Moreover, fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used in in vivo study.

Results

We demonstrated that relatively low concentrations of brazilin (1 to 10 μM) potentiated platelet aggregation induced by collagen (0.1 μg/ml) in washed human platelets. Higher concentrations of brazilin (20 to 50 μM) directly triggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an antagonist of ADP). It was not inhibited by yohimbine (an antagonist of epinephrine), by SCH79797 (an antagonist of thrombin protease-activated receptor [PAR] 1), or by tcY-NH2 (an antagonist of PAR 4). Brazilin did not significantly affect FITC-triflavin binding to the integrin α_IIbβ₃ in platelet suspensions. Pretreatment of the platelets with caffeic acid phenethyl ester (an antagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies raised against collagen receptor glycoprotein VI and integrin α₂β₁, respectively, abolished platelet aggregation stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the phosphorylation of phospholipase C (PLC)γ2 and Lyn, which were significantly attenuated in the presence of JAQ1 and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. An in vivo mouse study showed that brazilin treatment (2 and 4 mg/kg) significantly shortened the occlusion time for platelet plug formation in mesenteric venules.

Conclusion

To the best of our knowledge, this study provides the first evidence that brazilin acts a novel collagen receptor agonist. Brazilin is a plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.     

Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47^phox, a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47^phox and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-α_IIbβ₃ activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.     

Comparison of the effects of inhibitors of cytochrome P-450-mediated reations on human platelet aggregation and arachidonic acid metabolism

Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactiors, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1?¹⁴C]thromboxane B₂ from [1?¹⁴C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation.     

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin

Background

We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin.

Principal Findings

Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE₁ induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg⁻¹) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen.

Conclusions

These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.     

Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow

Background

The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function.

Objectives

Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo.

Methods and Results

We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa.

Conclusions

Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.     

Pharmacological characterization of the biosynthesis of prostanoids and hydroxyeicosatetraenoic acids in human whole blood and platelets by targeted chiral lipidomics analysis

Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B₂ production; ASA selectively affected TXB₂ production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB₂ while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y₁₂ blocker) affected platelet aggregation associated with profound inhibition of TXB₂ generation. P2Y₁₂ receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.     

Interaction of human platelet membrane glycoproteins with collagen and lectins

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with ¹²⁵I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.     

Inhibition of epinephrine-induced platelet aggregation by a derivative of wheat germ agglutinin

A nonagglutinating derivative of wheat germ agglutinin has been prepared and used as a probe to explore the initial events in platelet activation. The lectin derivative had no effect on platelet aggregation by adenosine diphosphate, collagen, ristocetin, wheat germ agglutinin or trypsin but aggregation induced by epinephrine or thrombin was inhibited. Unlike thrombin, the inhibition of aggregation by the derivative could not be overcome by increasing the concentration of epinephrine. The derivative did not affect the binding of [³H]dihydroergocryptine to platelets. A 74,000 dalton protein isolated from platelet membranes by lectin affinity chromatography strongly inhibited platelet activation by thrombin but not by epinephrine. The receptors for thrombin and for epinephrine on platelets are different but they are closely linked.     

Platelet-reactive sites in collagens type I and type III. Evidence for separate adhesion and aggregatory sites.

The adhesion of human and rabbit platelets to collagens and collagen-derived fragments immobilized on plastic was investigated. Adhesion appeared to be independent of collagen conformation, since similar attachment occurred to collagen (type I) in monomeric form, as fibres or in denatured state. The adhesion of human platelets was stimulated to a variable degree by Mg2+, but rabbit platelet adhesion showed little if any dependence on this cation. Collagens type I, III, V and VI were all able to support adhesion, although that to collagen type V (native) was lower than that to the other collagens. Adhesion to a series of peptides derived from collagens I and III was measured. Attachment did not require the presence of peptides in triple-helical configuration. The extent of adhesion ranged from relatively high, as good as to the intact parent collagen molecule, to little if any adhesive activity beyond the non-specific (background) level. The existence of very different degrees of activity suggests that platelet adhesion is associated with specific structural sites in the collagen molecule. Adhesion in many instances was essentially in accord with the known platelet-aggregatory activity of individual peptides. However, two peptides, alpha 1(I)CB3 and alpha 1(III)CB1,8,10,2, exhibited good adhesive activity although possessing little if any aggregatory activity. Of particular interest, despite its near-total lack of aggregatory activity, adhesion to peptide alpha 1(I)CB3 was as good as that to the structurally homologous peptide alpha 1(III)CB4, in which is located a highly reactive aggregatory site. This implies that platelet adhesion to collagen may involve sites in the collagen molecule distinct from those more directly associated with aggregation.     

Simplagrin,a Platelet Aggregation Inhibitor from Simulium nigrimanum Salivary Glands Specifically Binds to the Von Willebrand Factor Receptor in Collagen and Inhibits Carotid Thrombus Formation In Vivo

Background

Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin.

Methods and Findings

Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2–15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα₂β₁ interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (K_D 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease.

Conclusion

Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods.     

Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase

Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5?5 μM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca²⁺]_i mobilization, thromboxane A₂ (TxA₂) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH^●) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA₂ cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.     

Prostaglandin D2 as a potential antithrombotic agent

Prostaglandin D₂ was found to be a potent inhibitor of platelet aggregation. Aggregation of human platelets by ADP, collagen and prostaglandin G₂ was inhibited more strongly by PGD₂ than by PGE₁. Although ADP-induced aggregation of rabbit platelets was inhibited more strongly by PGE₁ than by PGD₂ the latter prostaglandin gave a more long-lasting inhibitory effect on platelet aggregation following intravenous or oral administration. These results coupled with the finding that PGD₂ has less hypotensive effects on the cardiovascular system than PGE₁ suggest the possible use of PGD₂ as an antithrombotic agent.