Fas-FasL expression and myocardial cell apoptosis in patients with viral myocarditis.

The aim of the current study was to investigate Fas and FasL expression and myocardial cell apoptosis in viral myocarditis patients. Human heart specimens were selected from patients who were autopsied between February 2012 and February 2015; of these, 25 patients were diagnosed with viral myocarditis. Another 15 cases with no diagnosis of myocarditis were selected for the control group. All tissue specimens were divided into two parts, one for reverse transcription-polymerase chain reaction analysis and the other for immunohistochemical and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. In situ detection of apoptosis was performed by the TUNEL method, which revealed that myocardial cells from the viral myocarditis group exhibited significant apoptosis, whereas no apoptotic cells were observed in the control group. The number of cells staining positive for Fas and FasL protein in the viral myocarditis group was significantly higher than that in the control group (P < 0.05). There was also a correlation between Fas and FasL protein expression levels and scores (r = 0.92, P < 0.05). The mRNA expression of Fas and FasL was significantly higher in the viral myocarditis group than in the control group (P < 0.05). In conclusion, the Fas-FasL system may be involved in the pathogenesis of viral myocarditis. Furthermore, cytotoxic T lymphocytes may mediate cardiac muscle cells apoptosis via Fas-FasL signaling, and thus participate in the pathogenesis of viral myocarditis.

Isolation and characterization of mesenchymal stromal cells from human anterior cruciate ligament.

BACKGROUND: The anterior cruciate ligament (ACL) is one of the most commonly injured ligaments of the knee. Because the torn ACL is always discarded during ACL reconstruction, it may be a potential source for isolating mesenchymal stromal cells (MSC). METHODS: To characterize MSC from human ACL, cells were enzymatically released from the ACL of adult human donors and seeded in plastic dishes with serial passages at confluence. At different passages, ACL-derived cells were subjected to in vitro assays to investigate their multilineage potential. Upon treatment, the phenotypes of the cell cultures were analyzed by histo- and immunohistochemistry and semi-quantitative reverse transcription-polymerase chain reaction for the expression of lineage-specific genes. RESULTS: Six independent cell lines from individual donors showed diversity in multilineage potential. Interestingly, five of the six lines displayed adipogenic potential, four had osteogenic and adipogenic potential, and only one cell line was tripotent. Both bone marrow (BM)- and ACL-derived MSC expressed marker genes for ligament fibroblasts, whereas the mRNA levels of collagen I and III were more abundant in ACL-derived MSC. DISCUSSION: Our study demonstrates that human MSC can be isolated from ACL with diversity in the potential to form bone, fat and cartilage and an increase as compared to BM MSC, in the potential to form ligament fibroblasts.

An αIIb ß3 antagonist prevents thrombosis without causing Fc receptor γ-chain IIa-mediated thrombocytopenia.

Essentials FcγRIIa-mediated thrombocytopenia is associated with drug-dependent antibodies (DDAbs). We investigated the correlation between αIIb ß3 binding epitopes and induction of DDAbs. An FcγRIIa-transgenic mouse model was used to evaluate thrombocytopenia among anti-thrombotics. An antithrombotic with binding motif toward αIIb ß-propeller domain has less bleeding tendency. SUMMARY: Background Thrombocytopenia, a common side effect of Arg-Gly-Asp-mimetic antiplatelet drugs, is associated with drug-dependent antibodies (DDAbs) that recognize conformation-altered integrin αIIb ß3 . Objective To explore the correlation between αIIb ß3 binding epitopes and induction of DDAb binding to conformation-altered αIIb ß3 , we examined whether two purified disintegrins, TMV-2 and TMV-7, with distinct binding motifs have different effects on induction of αIIb ß3 conformational change and platelet aggregation in the presence of AP2, an IgG1 inhibitory mAb raised against αIIb ß3 . Methods We investigated the possible mechanisms of intrinsic platelet activation of TMV-2 and TMV-7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ-chain IIa (FcγRIIa) transgenic mice. Results TMV-7 has a binding motif that recognizes the αIIb ß-propeller domain of αIIb ß3 , unlike that of TMV-2. TMV-7 neither primed the platelets to bind ligand, nor caused a conformational change of αIIb ß3 as identified with the ligand-induced binding site mAb AP5. In contrast to eptifibatide and TMV-2, cotreatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and the downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV-7 did not impair hemostatic capacity. Conclusions TMV-7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested αIIb ß3 antagonists, and may offer advantages as a therapeutic agent with a better safety profile.

Long-term follow-up of Dupuytren disease after injection of triamcinolone acetonide in Chinese patients in Taiwan.

Injection of triamcinolone acetonide is a non-operative treatment for early-stage Dupuytren disease in Caucasians, but its effectiveness in non-Caucasians is unclear. We report averaged 5-year follow-up results of 37 patients (49 affected hands) with early-stage Dupuytren disease for patients in Taiwan (non-Caucasian) who received a single dose of 5 mg triamcinolone acetonide injection into nodules monthly for 3 months. Using ultrasound, we recorded no progression of sizes of the modules following injection after 6 months. After an average 5-year follow-up, two patients with three hands (6%) experienced reactivation of the treated nodules. None required surgical intervention. Ultrasound examination showed that sizes of the treated Dupuytren nodules decreased significantly by 40% 6 months after injection and 56% at the final follow-up. We conclude that in these Chinese patients in Taiwan with early Dupuytren nodules, triamcinolone acetonide injection was effective in reducing the size of the Dupuytren nodules and maintaining long-term durable control of the nodular growth. LEVEL OF EVIDENCE: III.

ARHGEF10 knockout inhibits platelet aggregation and protects mice from thrombus formation.

Essentials ARHGEF10 single-nucleotide polymorphism provides risk of ischemic and atherothrombotic stroke. The role of ARHGEF10 in platelet function was examined using ARHGEF10 knockout mice. ARHGEF10 deficiency inhibits platelet function and arterial thrombus formation. ARHGEF10 knockout protects mice from stroke-induced infarction. SUMMARY: Background ARHGEF10, a member of the Rho guanine nucleotide exchange factor (GEF) family, stimulates Rho GTPases. Rho GTPases have been reported to regulate a variety of cellular behaviors, such as cell polarity, cytoskeletal organization, and gene transcription. ARHGEF10 single-nucleotide polymorphisms are linked to the risk of ischemic stroke. However, the role of ARHGEF10 in platelet function remains unknown. Objective To examine the role of ARHGEF10 in platelet function. Methods ARHGEF10-/- were generated. We examined the in vitro and in vivo effects of ARHGEF10 knockout on platelet function and arterial thrombosis formation. Results ARHGEF10-/- mice had normal platelet counts, but showed altered aggregation in response to thrombin, collagen, ADP, protease-activated receptor-4 peptide, and U46619 stimulation. ARHGEF10 knockout influenced platelet spreading on fibrinogen-coated surfaces, and caused the platelets to show less lamellipodia-like extension than wild-type platelets. ARHGEF10 knockout also inhibited platelet clot retraction induced by thrombin stimulation. ARHGEF10 knockout resulted in prolonged tail bleeding time and inhibited the stable thrombus formation induced by FeCl3 in the carotid artery. Conclusions ARHGEF10 serves as an important regulator in platelet shape change, spreading, and aggregation. Moreover, ARHGEF10 also plays an important role in arterial thrombosis formation.

Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom.

By means of DEAE-Sephadex A-50 Column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of alpha- (Fraction 1) and beta-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of alpha- and beta-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha-fibrinogenase and beta-fibrinogenase were pH greater than 10 and 4.5, respectively. The optimal pH of alpha-fibrinogenase was approx. 7.4 and that of beta-fibrinogenase was approx. 9.0. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.4, 7.4 and 9.0, while that of beta-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity alpha-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of beta-fibrinogenase was 9 mg fibrinogen/min per mg protein. alpha-Fibrinogenase cleaved specifically the alpha(A) chain of monomeric fibrinogen without cleaving the beta(B) chain and gamma-chain. beta-fibrinogenase preferentially cleaved the beta(B) chain, and the alpha(A) chain was also partially cleaved by beta-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 14 times that of crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethanesulfonylfluoride. alpha- and beta-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen.

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom.

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable malonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E1 completely blocked both aggregoserpentin-induced aggregation and release reaction. Furthermore, platelet aggregoserpentin lowered basal and prostaglandin E1-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A2, resulting in the formation of platelet activating factor, but not of prostaglandins.

Potent platelet aggregation inhibitor from Trimeresurus gramineus snake venom.

Using DEAE-Sephadex A-50 column chromatography and gel filtration, a potent platelet aggregation inhibitor from Trimeresurus gramineus venom was purified. It was an acidic phospholipase A, rich in aspartic acid, glutamic acid and half-cystine, with an isoelectric point of 3.6. At a concentration of 10 micrograms/ml, the purified inhibitor showed a marked inhibitory effect on platelet aggregations induced by adenosine diphosphate, collagen, sodium arachidonate and ionophore A-23187 in rabbit platelet-rich plasma, washed platelet suspension, as well as in thrombin-degranulated platelet suspension. The ID50 of this venom inhibitor was about 2.5-5 micrograms/ml in platelet aggregations induced by all these aggregation inducers. The action of this inhibitor could be partially antagonized by phosphatidylethanolamine. High concentration of Ca2+ (5 mM) did not reverse the inhibitory action even in the presence of ionophore A-23187. The [14C]serotonin release induced by sodium arachidonate and thrombin was unaffected. Malonic dialdehyde formation induced by these aggregation inducers remained unchanged. Basal and prostaglandin E1-stimulated cAMP levels were not altered by this inhibitor. No lactate dehydrogenase was released even at a concentration of 62.5 micrograms/ml. Polylysine-induced platelet agglutination was not affected. beta-Mercaptoethanol inactivated both its phospholipase A enzymatic and platelet inhibitory activities, while p-bromophenacyl bromide only inactivated the former activity. The possibility of acting on a common final step of platelet aggregation, i.e. the intercellular adhesion between the activated platelets, was proposed.

Effect on human platelet aggregation of phospholipase A2 purified from Heloderma horridum (beaded lizard) venom.

By means of gel filtration, ionic exchange chromatography and DEAE-column HPLC, an acidic phospholipase A2 (PLA2) was purified from beaded lizard (Heloderma horridum) venom. The purified PLA is a single-chain polypeptide, consisting of about 163 amino acid residues with a molecular mass of 19,000 Da as calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analysis. HHV-PLA showed a rather specific inhibitory effect on platelet aggregation induced by U46619 and epinephrine in human platelet-rich plasma in a dose- and time-dependent manner, whereas it had little effect on collagen- and ADP-induced aggregation. ATP-release reaction induced by various agonists were dose- and time-dependently inhibited by HHV-PLA, even though platelet aggregation was apparently not affected in human washed platelets. When HHV-PLA was chemically modified with p-bromophenacyl bromide, both of its enzymatic activity and antiplatelet activity were lost. Furthermore, exogenous lysophosphatidylcholine and HHV-PLA treated phosphatidylcholine inhibited platelet aggregation induced by U46619 in human washed platelets. In conclusion, PLA enzyme from H. horridum venom inhibits exclusively U46619- or thromboxane-induced platelet aggregation of human platelet-rich plasma probably by virtue of their PLA enzymatic activity on plasma phospholipids, converting phospholipids (e.g., phosphatidylcholine) into lysophospholipids, which in turn interfere with the coupling of TXA2 receptor and its signalling transduction system.

Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom.

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon acutus venom was separated into twelve fractions. The fibrinolytic activity was concentrated in Fraction 9. This fraction was rechromatographed on Sephadex G-75 three times and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.44 S was obtained by ultracentrifugation, the molecular weight of which was estimated to be 24 100, and the isoelectric point 3.8. The specific activity was four times higher than that of crude venom. The optimal pH value on fibrinolysis was 7.4. In addition to fibrinolytic activity, the purified principle also had fibrinogenolytic and caseinolytic activities. The purified fibrinolytic principle had a specific action on the a(A) chain subunit of fibrinogen, leaving the beta(B) chain and the gamma chain unaffected.

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom.

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.

The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by alpha v beta 3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C.

The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did not cause morphologic alteration of HUVEC by itself at concentrations ranging from 0.01 to 10 U/ml. However, when 0.02 U/ml of ancrod was added to cultured HUVEC monolayers in the presence of citrated plasma, it caused pronounced morphologic change of HUVEC after 6-10 h incubation period. Gly-Pro-Arg-Pro (4 mg/ml), an inhibitor of fibrin polymerization, prevented the morphologic alteration, indicating that the morphologic alteration was caused by the polymerized fibrin. The morphologic change of HUVEC caused by ancrod-generated fibrin was not observed in the presence of an intracellular calcium mobilization inhibitor TMB-8 (50 microM), and the morphologic alteration was also less pronounced with BAPTA(15 microM)-loaded HUVECs and HUVECs pretreated with EGTA (1.2 mM). Ancrod (in Medium 199) itself did not stimulate phosphoinositide breakdown of HUVEC. However, when ancrod was present in plasma, it caused an increase of [3H]IP1 of HUVECs preloaded with [3H]myoinositol. This IP1 increment was inhibited by Gly-Pro-Arg-Pro. The increase of IP1 was significantly inhibited by the pretreatment of monoclonal antibodies 23C6 and 7E3 directed against alpha v beta 3 integrin. Neomycin (1 mM) and pertussis toxin (100 ng/ml), but not aspirin or mepacrine, blocked this enhanced phosphoinositide breakdown. The morphologic change was also prevented by the monoclonal antibodies, 23C6 and 7E3. These results suggest that both intra- and extra-cellular calcium participate in the event of morphologic change of HUVEC caused by ancrod-generated fibrin, and the morphologic change is mediated, at least in part, by fibrin binding to integrin alpha v beta 3 on HUVECs, causing the subsequent activation of the endogenous G-protein coupled phospholipase C.

Vasodilatory action mechanisms of apigenin isolated from Apium graveolens in rat thoracic aorta.

The effect of apigenin, isolated from Apium graveolens, on the contraction of rat thoracic aorta was studied. Apigenin inhibited the contraction of aortic rings caused by cumulative concentrations of calcium (0.03-3 mM) in high potassium (60 mM) medium, with an IC50 of about 48 microM. After pretreatment it also inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contraction in a concentration (35-140 microM)-dependent manner with an IC50 of 63 microM. At the plateau of NE-induced tonic contraction, addition of apigenin caused relaxation. This relaxing effect of apigenin was not antagonized by indomethacin (20 microM) or methylene blue (50 microM), and still existed in endothelial denuded rat aorta or in the presence of nifedipine (2-100 microM). Neither cAMP nor cGMP levels were changed by apigenin. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine in the Ca(2+)-free solution were unaffected by apigenin. 45Ca2+ influx caused by either NE or K+ was inhibited by apigenin concentration-dependently. It is concluded that apigenin relaxes rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage- and receptor-operated calcium channels.

Mechanism of action of the antiplatelet peptide, arietin, from Bitis arietans venom.

Arietin, an Arg-Gly-Asp containing peptide from venom of Bitis arietans, inhibited aggregation of platelets stimulated by a variety of agonists with a similar IC50, 1.3-2.7.10(-7) M. It blocked aggregation through the interference of fibrinogen binding to fibrinogen receptors on platelet surface. In this paper, we further demonstrated that arietin had no significant effect on the intracellular mobilization of Ca2+ in Quin2-AM-loaded platelets stimulated by thrombin. It inhibited 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50, 1.1.10(-7) M). 125I-arietin bound to unstimulated, ADP-stimulated and elastase-treated platelets in a saturable manner and its Kd values were estimated to be 3.4.10(-7), 3.4.10(-8) and 6.5.10(-8) M, respectively, while the corresponding binding sites were 46,904, 48,958 and 34,817 per platelet, respectively. Arg-Gly-Asp-Ser (RGDS) inhibited 125I-arietin binding to ADP-stimulated platelets in a competitive manner. RGD-containing peptides, including trigramin and rhodostomin, EDTA and monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex, inhibited 125I-arietin binding to ADP-stimulated platelets, indicating that the binding sites of arietin appear to be located at or near glycoprotein IIb-IIIa complex. In conclusion, arietin and other RGD-containing trigramin-like peptides preferentially bind to the fibrinogen receptors associated with glycoprotein IIb-IIIa complex of the activated platelets, thus leading to the blockade of fibrinogen binding to its receptors and subsequent aggregation. The presence of RGD of arietin is essential for the expression of its biological activity. Its binding sites are overlapped with those of trigramin, rhodostomin and the monoclonal antibody, 7E3.

A novel alpha-type fibrinogenase from Agkistrodon rhodostoma snake venom.

By means of CM-Sephadex C-50 column chromatography, gel-filtration on sephadex G-75 and Sephacryl S-200 columns, a purified fibrinogenase, kistomin, was obtained from venom of Agkistrodon rhodostoma. It was a single peptide-chain with a molecular mass of about 21,800 Da containing about 202 amino-acid residues as revealed by amino acid analysis. Kistomin preferentially cleaved A alpha- and subsequently the gamma-chain of fibrinogen, leaving the B beta-chain unaffected. Its fibrinogenolytic activity was estimated to be 36.6 +/- 4.5 mg/min per mg protein and was inhibited by the pretreatment of EDTA, suggesting that it is a metalloproteinase. Its fibrinogenolytic activity in platelet-poor plasma is much less potent as compared to that in purified fibrinogen solution. It inhibited ristocetin-induced aggregation of human platelets in a dose-dependent manner in the presence of von Willebrand factor.

A new short chain RGD-containing disintegrin, accutin, inhibits the common pathway of human platelet aggregation.

A new short-chain disintegrin, accutin, was purified from the Formosan Agkistrodon acutus venom by using of gel filtration, ion exchanger and reverse phase HPLC. The homogeneous protein is a 47-residue polypeptide with a molecular mass of 5241 Da containing an Arg-Gly-Asp sequence and seven cysteinyl residues at positions highly homologous to other disintegrins. Accutin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, thrombin or the thromboxane analogue U46619 in platelet suspension with IC50 values of 66-267 nM. It was also active in inhibiting platelet aggregation of platelet-rich plasma. However, accutin apparently did not affect the shape change caused by these agonists. Accutin also inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner. Furthermore, accutin dose-dependently inhibited the binding reaction of fluorescein isothiocyanate (FITC)-conjugated arietin, a member of the disintegrin family, to human platelets. In addition, the binding of FITC-conjugated accutin to platelets was almost completely blocked by a monoclonal antibody, 7E3, raised against the platelet glycoprotein IIb/IIIa complex. On the other hand, accutin as well as other disintegrins, rhodostomin and arietin, exhibited an inhibitory effect on 7E3 binding toward platelets and endothelial cells in a dose-dependent manner. It is concluded that accutin, a new platelet aggregation inhibitor belonging to the short-chain disintegrin family, acts specifically on a binding epitope of GPIIb/IIIa overlapping with that of 7E3, leading to the blockade of fibrinogen binding to its receptor.

Triwaglerin: a potent platelet aggregation inducer purified from Trimeresurus wagleri snake venom.

Trimeresurus wagleri venom is the most potent inducer of platelet aggregation among the seven Trimeresurus snake venoms tested. By means of CM-Sephadex C-50 column chromatography, T. wagleri venom was separated into 19 fractions. Fraction XVI possessed the strongest aggregating activity and was further purified by Sephadex G-75 and on heparin-agarose columns, and finally Triwaglerin, with a molecular weight of 68000, was obtained. Its aggregating and ATP-releasing activity was dose-dependent and 10-times more potent than the crude venom. Triwaglerin was devoid of any of the enzymatic activities possessed by the crude venom. Triwaglerin-induced aggregation was not affected by indomethacin, creatine phosphate/creatine phosphokinase (CP/CPK), platelet-activating factor (PAF) antagonists, verapamil or heparin, but was inhibited completely by mepacrine, imipramine and forskolin and markedly by tetracaine and sodium nitroprusside. Thromboxane B2 formation caused by Triwaglerin was suppressed by mepacrine, imipramine and indomethacin. R59022 and TMB-8 caused a synergistic inhibitory effect against Triwaglerin-induced aggregation. These data suggest that Triwaglerin activates platelets in a unique action which is independent of formation of thromboxane A2 and PAF, or release of ADP.

Antiplatelet protease, kistomin, selectively cleaves human platelet glycoprotein Ib.

Kistomin, a metalloprotease purified from venom of Calloselasma rhodostoma, dose- and time-dependently prolonged the latent period of aggregation and inhibited ATP secretion of human washed platelets stimulated by thrombin. It inhibited aggregation induced by low concentrations of thrombin (< or = 0.2 U/ml) whereas it had only slight effect on aggregation induced by high concentrations of thrombin (> or = 0.5 U/ml). Meanwhile it also inhibited ristocetin-induced platelet aggregation in a dose- and time-dependent manner. It significantly inhibited cytosolic calcium rise of Quin 2--loaded platelets, completely blocked thromboxane B2 formation, and blocked [3H]inositol phosphates formation of [3H]myoinositol loaded platelets stimulated by 0.1 U/ml of thrombin. Kistomin inhibited significantly thromboxane but not [3H]inositol phosphates formation of platelets stimulated by a high concentration of thrombin (1 U/ml). Incubation of platelets with kistomin resulted in a selective cleavage of platelet membrane glycoprotein Ib as revealed by SDS/PAGE stained by periodic acid/Schiff reagent. These results suggested that thrombin activates platelets at least through two receptors/or effectors-mediated events. In addition to glycoprotein Ib, other surface membrane component(s) (e.g., the seven transmembrane domain thrombin receptor) may also be important in regulating the biochemical events of human platelets in response to thrombin. However, the extent and rate of platelet aggregation stimulated by low concentrations of thrombin ( < or = 0.2 U/ml) are closely related with the intactness of glycoprotein Ib.

Characterization of integrin expression and regulation on SW-480 human colon adenocarcinoma cells and the effect of rhodostomin on basal and upregulated tumor cell adhesion.

Integrins are a superfamily of cell surface glycoproteins that mediate cell-extracellular matrix (ECM) and cell-cell adhesion. Immunofluorescence microscopy and flow cytometric analysis using anti-integrin mAbs as the primary binding ligands demonstrated that the platelet integrin receptor alpha IIb beta 3, as well as alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1, are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against alpha IIb beta 3 and alpha 5 beta 1 inhibited unstimulated basal adhesion to fibronectin by approximately 30% and 40%, respectively. The surface immunoreactivity of tumor cells for alpha IIb beta 3 was enhanced by pretreatment (5 min) with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid, 12-hydroxyeicosatetraenoic acid (12-HETE) in a dose- and time-dependent manner. SW-480 cells possess a large intracellular pool of alpha IIb beta 3, from which the receptor complex translocates to the cell surface following pretreatment with TPA or 12(S)-HETE. This pretreatment enhances adhesion to fibronectin, which is mediated exclusively by alpha IIb beta 3 integrins. Staurosporine was found to block alpha IIb beta 3 up-regulation and enhanced-adhesion. TPA and 12(S)-HETE also facilitated the redistribution of alpha IIb beta 3 during the enhanced-spreading process. Rhodostomin, an Arg-Gly-Asp- (RGD) containing antiplatelet snake venom peptide, was about 400-times more potent than RGDS at inhibiting control, TPA- or 12(S)-HETE-enhanced adhesion of SW-480 cells to fibronectin. The binding of mAbs against alpha IIb beta 3, alpha v beta 3 and alpha 5 beta 1 was inhibited by pretreatment with rhodostomin, suggesting that rhodostomin binds via its RGD sequence to multiple integrin receptors (i.e., alpha IIb beta 3, alpha v beta 3, alpha 5 beta 1) expressed on the SW-480 cell surface, inhibiting cell adhesion to ECM.

Purification and characterization of an antiplatelet peptide, arietin, from Bitis arietans venom.

By means of Fractogel TSK-50, CM-Sephadex C-50 column chromatography, gel filtrations on Sephadex G-75 and Sephacryl S-200 columns and reverse-phase HPLC, an antiplatelet peptide, arietin, was purified from venom of Bitis arietans. Arietin was shown to be an Arg-Gly-Asp-containing peptide with a NH2-terminus, Ser-Pro-Pro-Val-Cys-Gly-Asn-Lys- (Mr 8500). Arietin dose-dependently inhibited aggregation of human platelet suspension stimulated by ADP, thrombin, collagen and U46619 with IC50, 1.3-2.7.10(-7) M, while it had no effect on the initial shape changes and only slightly affected ATP release of platelets caused by thrombin and collagen. Arietin also blocked platelet aggregation in platelet-rich plasma and whole blood, and inhibited thrombin-induced clot retraction of platelet-rich plasma. Furthermore, arietin (6.5.10(-8) M) completely blocked the fibrinogen-induced aggregation of elastase-treated platelets, indicating that arietin interferes with the fibrinogen binding to fibrinogen receptors on platelet membranes. In conclusion, arietin, an Arg-Gly-Asp-containing peptide, inhibits platelet aggregation probably through the blockade of fibrinogen binding to the activated platelets.