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.

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.

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.

Applying ATP bioluminescence to design and evaluate a successful new intensive care unit cleaning programme.

This was a two-phase prospective intervention study in the cardiology intensive care unit (CICU) and medical intensive care unit (MICU) and of a public 1800-bed medical centre in Taiwan. In phase I, cleaning efficacy was monitored by ATP bioluminescence after daily morning cleaning, and only 43.9% of 221 tested surfaces passed. The baseline data were used to define an intervention consisting of a new cleaning protocol as well as a new education/training programme. In phase II, following the intervention, 88.1% of 270 surfaces were found to be clean. The combined infection rate in the CICU and MICU showed a statistically significant decrease of 49.7%.

PPemd26, an anthraquinone derivative, suppresses angiogenesis via inhibiting VEGFR2 signalling.

BACKGROUND AND PURPOSE: Angiogenesis contributes to coronary heart disease, immune disorders and numerous malignancies. VEGF-A and its receptors (VEGFRs) play a pivotal role in regulating angiogenesis. In an effort to discover more effective inhibitors of tumour angiogenesis, we have analysed the actions of a novel anthraquinone derivative, PPemd26, and explored its anti-angiogenic mechanisms. EXPERIMENTAL APPROACH: The effects of PPemd26 were evaluated in vitro using HUVEC cultures to assess proliferation, migration, invasion and tube formation. Immunoblotting was used to analyse phosphorylation of signalling kinases. Effects in vivo were assayed using Matrigel plug and xenograft mouse models. KEY RESULTS: PPemd26 significantly inhibited VEGF-A-induced proliferation, migration, invasion and tube formation of HUVECs. PPemd26 also attenuated VEGF-A-induced microvessel sprouting from rat aortic rings ex vivo and suppressed formation of new blood vessels in implanted Matrigel plugs in models of angiogenesis in vivo. In addition, PPemd26 inhibited VEGF-A-induced phosphorylation of VEGFR2 and its downstream protein kinases including Akt, focal adhesion kinase, ERK and Src. Furthermore, systemic administration of PPemd26 suppressed the growth of s.c. xenografts of human colon carcinoma in vivo. Histochemical analysis of the xenografts revealed a marked reduction in stainingfor the vascular marker CD31 and proliferation marker Ki-67. CONCLUSIONS AND IMPLICATIONS: This study provides evidence that PPemd26 suppressed tumour angiogenesis through inhibiting VEGFR2 signalling pathways, suggesting that PPemd26 is a potential drug candidate for developing anti-angiogenic agents for the treatment of cancer and angiogenesis-related diseases.

Inhibitory effects of polypeptides derived from a snake venom C-type lectin, aggretin, on tumor cell-induced platelet aggregation.

BACKGROUND AND OBJECTIVES: Podoplanin, a transmembrane sialoglycoprotein, is expressed by lymphatic endothelial cells and many tumor cells, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. A recent study found that C-type lectin-like receptor 2 (CLEC-2) is a physiologic receptor for podoplanin. Previous studies showed that aggretin, a snake venom-derived protein, activates platelets by targeting platelet CLEC-2. We hypothesized that the C-terminal fragment of aggretin may bind to platelet CLEC-2 and displace podoplanin, in turn exerting antitumor metastatic effects. METHODS AND RESULTS: Aggretin α-chain C-terminus (residues 106-136; AACT) prolonged the lag phase of platelet aggregation induced by aggretin in human washed platelets, indicating that AACT may target the binding site of CLEC-2. HepG2 cells, which are podoplanin-expressing hepatoma cells, induced platelet aggregation with a lag phase. Pretreatment with AACT inhibited platelet aggregation and prolonged the lag phase induced by HepG2 cells. This inhibitory effect was also found with another hepatocarcinoma cell line, HuH-7. AACT inhibited the interaction between HuH-7 cells and platelets, and a specific binding assay demonstrated that CLEC-2 was the binding site for AACT on platelets. In addition, the invasive ability of HepG2 cells was abolished by AACT in a chick embryo chorioallantoic membrane model. Furthermore, formation of lung metastases after intravenous administration of HuH-7 cells was significantly reduced when mice were treated with AACT. CONCLUSIONS: AACT interacts with CLEC-2 of platelets, leading to interference with platelet aggregation and the subsequent metastatic potential of tumor cells. These results suggest that aggretin AACT is a potential candidate for the treatment of tumor metastasis through CLEC-2 blockade.

Improvements in endotoxemic syndromes using a disintegrin, rhodostomin, through integrin αvß3-dependent pathway.

BACKGROUND AND OBJECTIVES: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg-Gly-Asp-containing snake venom disintegrin, on lipopolysaccharide (LPS)-activated phagocytes in vitro and LPS-induced endotoxemia in vivo. METHODS AND RESULTS: Rn inhibited adhesion, migration, cytokine production and mitogen-activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti-αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP-1, Rn bound to LPS-activated THP-1 and specifically blocked anti-αvß3 mAb binding to THP-1. Binding assays proved that integrin αvß3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS-induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS-induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor-α (TNF-a), interleukin (IL)-6, -1ß and -10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS-induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. CONCLUSIONS: Rn may interact with αvß3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS-induced endotoxemia may be attributed to its anti-inflammation activities in vivo.

A novel mechanism of cytokine release in phagocytes induced by aggretin, a snake venom C-type lectin protein, through CLEC-2 ligation.

BACKGROUND: Macrophages are major immune cells and play an important role in modulating homeostasis and the immune defense mechanism. In inflammatory responses to the infection of pathogens, macrophages are activated, producing various inflammatory mediators. Snake venom C-type lectin proteins (snaclecs) have diverse targets, including platelet GPVI, GPIb, integrin α2ß1 or CLEC-2 expressed in platelets, endothelial cells or myeloid cells. METHODS: In this study, murine macrophages (RAW 264.7 cells) and human monocytes (THP-1) were treated with different snaclecs, including aggretin, gramicetin, trowaglerix and convulxin, in the absence or presence of LPS for 24 h. RESULTS: The production of cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in supernatants was measured by ELISA. Aggretin increased the production of TNF-α and IL-6 in both RAW264.7 and THP-1 cells; however, the other snaclecs did not. Aggretin induced extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) tyrosine phosphorylation of RAW264.7 cells. Pretreatments with inhibitor of ERK, JNK, p38 or NF-κB abolished cytokine release caused by aggretin. Aggretin bound to THP-1 cells in a concentration-dependent manner and it displaced the CLEC-2 mAb binding to THP-1 cells and the immobilized aggretin selectively bound to CLEC-2 of both platelets and THP-1 cell lysates. Furthermore, aggretin elevated the plasma level of IL-6 in ICR mice as it was administered intramuscularly. CONCLUSION: These results indicate that aggretin may induce cytokine TNF-α/IL-6 release via interacting with CLEC-2 receptor and the subsequent MAPK and NF-κB activation in monocytes/macrophages.

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.

A snake venom metalloproteinase, kistomin, cleaves platelet glycoprotein VI and impairs platelet functions.

BACKGROUND AND OBJECTIVES: Injuries to the vessel wall and subsequent exposure of the matrix of the subendothelial layer resulted in thrombus formation. Platelet glycoprotein (GP) Ib and VI play a crucial role in matrix-induced activation and aggregation of platelets. METHODS AND RESULTS: In the present study, we reported that the GPIb-cleaving snake venom metalloproteinase (SVMP), kistomin, inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin (CVX, a GPVI agonist) and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased platelet GPVI but not integrin alpha2beta1 and alphaIIbbeta3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25- and 35-kDa fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu180 to Asn249 as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE205/A206TA and NKV218/F219TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin. CONCLUSIONS: We provided here the first evidence that a P-I snake venom metalloproteinase, kistomin, inhibits the interaction between collagen and platelet GPVI through its proteolytic activity on GPVI, thus providing an alternative strategy for developing new anti-thrombotic agents.

The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding.

BACKGROUND: C-type lectin proteins (CLPs) have diverse targets including platelet GPIb, GPVI and integrin alpha(2)beta(1), and affect platelet function in a various way. In this study, we characterized a huge, heterodimeric venom protein, trowaglerix, which belongs to the CLP family. METHODS: We purified a potent platelet-aggregation inducer, trowaglerix, from the crude venom of Tropidolaemus wagleri. Biotinylated trowaglerix was used for binding assays, and immunoblotting was used to investigate the signal transduction involved. RESULTS: Two distinct subunits of trowaglerix with similar masses of around 16 kDa were eluted by high-performance liquid chromatography after reduction and alkylation. Trowaglerix induced platelet aggregation of washed human platelets and platelet-rich plasma (PRP) in a concentration-dependent manner. Biotinylated trowaglerix specifically bound to platelet membrane GPVI, but not to GPIb or alpha(2) integrin. Treatment with trowaglerix induced GPVI loss in human platelets in vitro and impaired the platelet aggregation of mouse PRP ex vivo in response to collagen but not in response to adenosine diphosphate (ADP). However, GM6001, a matrix metalloproteinase (MMP) inhibitor, inhibited trowaglerix-induced GPVI cleavage and restored the platelet responsiveness of PRP to collagen. CONCLUSIONS: Trowaglerix activates platelets through specific binding to GPVI, leading to kinases-dependent exposure of functional alpha(IIb)beta(3) and platelet aggregation, and also induces MMP-dependent GPVI shedding from platelets.

A novel tetrameric venom protein, agglucetin from Agkistrodon acutus, acts as a glycoprotein Ib agonist.

A novel platelet agglutination inducer, agglucetin, was purified from the Formosan Agkistrodon acutus snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked alpha1, alpha2, beta1 and beta2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like glycoprotein (GP) Ib binding proteins. Functional studies showed that agglucetin. in the absence of von Willebrand factor (vWF), dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca+2 mobilization and thromboxane B, formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, or indomethacin exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb. It is concluded that agglucetin, acts specifically on an epitope of platelet membrane GP Ib overlapping with that of API, causing platelet agglutination in a Ca+2- and GP IIb/IIIa-independent manner.

The role of high-resolution ultrasonography in management of calcific tendonitis of the rotator cuff.

This article predicts the possibility of resorption of the calcific plaques in the shoulder using high-resolution ultrasonography (HRUS) and color Doppler ultrasound (CDUS), and evaluates the therapeutic effect of US-guided fine-needle multiple punctures of the calcific plaque. A total of 100 patients with calcific tendenosis were divided into 3 groups: In group 1, patients having chronic shoulder pain received conservative treatment; in group 2, patients having acute exacerbation of shoulder pain also received conservative treatment; and group 3 patients received US-guided fine-needle multiple punctures or aspiration. In CDUS, all images were classified as grade 0 (no color flow signals), grade 1 (weak spotty color flow signal), grade 2 (few rod-like color flow signals), grade 3 (many rod-like or linear color flow signals). In the follow-up study, marked improvement of patients' clinical condition with more than 50% size reduction of calcific plaque was defined as an effective treatment. There was no significant difference between group 1 and group 3 (p = 0.558) in CDUS, but there was a significant difference between group 1 and group 2 (p = 0.000), and group 2 and group 3 (p = 0.000) on the basis of classification of grade < 1 and grade > or = 1. There was also significant difference in the follow-up result of effective management between group 1 and group 3 (p = 0.000), and group 1 and group 2 (p = 0.000). In conclusion, HRUS with CDUS proved to be a good modality in evaluating the possibility of resorption of shoulder calcification and, if CDUS > or = grade 1 in calcific tendonitis, we highly recommend conservative treatment with regular follow-up. On the other hand, if CDUS < grade 1, fine-needle repeated puncture could be considered as an effective alternative treatment.

Modulation of protein kinase A activation by fibronectin matrix proteins at developing neuromuscular synapses in Xenopus laevis cell cultures.

Extracellular matrix proteins, such as fibronectin, laminin, and collagen, have been implicated in a wide variety of cellular properties, which include cell adhesion, migration, differentiation, and proliferation. In this study, we investigated the modulation of protein kinase A (PKA) activity by matrix proteins at developing motoneurons. The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus laevis embryos. Spontaneous synaptic currents (SSC) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (V(h) = -60 to approximately -65 mV). Bath application of agents, which directly or indirectly activate PKA, such as forskolin (20 microM), dibutyryl cAMP (DBcAMP) (1 mM), isoproterenol (10 microM), or albuterol (10 microM), significantly increased SSC frequency in cultures grown on fibronectin (FN)-coated substratum, but not on laminin- or collagen-coated glasses. The evoked synaptic currents increased in response to forskolin in neurons grown on FN substratum. Triflavin, an Arg-Gly-Asp-dependent disintegrin, inhibited potentiating action of isoproterenol in neurons grown on FN substratum, suggesting that integrin is involved in the potentiation of the PKA pathway in the regulation of acetylcholine (ACh) release. There is collaboration of neurotrophic factors and the FN matrix in regulating synaptic transmission in response to DBcAMP. Chronic treatment with neurotrophic factors, such as ciliary neurotrophic factor (150 ng/ml), glial cell line-derived neurotrophic factor (30 ng/ml), or neurotrophin-3 (50 ng/ml), enhanced the SSC-increasing action of DBcAMP in neurons grown on FN-coated glasses. These results suggest that the FN matrix potentiates synaptic transmission in response to PKA activation. Neurotrophic factors may collaborate with FN to regulate spontaneous ACh secretion at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular synapses.

Aggretin, a C-type lectin protein, induces platelet aggregation via integrin alpha(2)beta(1) and GPIb in a phosphatidylinositol 3-kinase independent pathway.

Aggretin purified from Calloselasma rhodostoma venom was previously identified as alpha(2)beta(1) agonist in triggering platelet aggregation, and exists as a heterodimer sharing a great homologous sequence to GPIb binding proteins. We show here that binding to GPIb is also required in aggregation-inducing activity of aggretin. A2-IIE10, an anti-integrin alpha(2) monoclonal antibody, delayed platelet aggregation while agkistin, a GPIb antagonist, only slightly inhibited platelet aggregation caused by aggretin. However, the aggretin-induced platelet aggregation was completely abolished by a combination of A2-IIE10 and agkistin. Either A2-IIE10 or agkistin significantly inhibited the binding of FITC-aggretin toward fixed platelets. Aggretin and collagen induced a similar signal transduction in platelets involving a time-dependent tyrosine phosphorylation of p125(FAK) and PLCgamma2, but aggretin caused a much-delayed tyrosine-phosphorylation of PI 3-kinase compared with collagen. LY294002, a PI 3-kinase inhibitor, showed a significant inhibitory effect on collagen, but not aggretin-stimulated platelet aggregation. These findings indicate aggretin induces platelet aggregation via binding of alpha(2)beta(1) and GPIb, causing phosphorylation of p125(FAK) and PLCgamma2 leading to platelet activation without the involvement of PI 3-kinase activation.

Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells.

Apoptosis, a programmed, physiological mode of cell death, is important in tissue homoeostasis. Here we report that a new metalloproteinase, graminelysin I, purified from Trimeresurus gramineus venom, induced apoptosis of human endothelial cells as examined by electrophoresis and flow cytometry. Graminelysin I contains only a metalloproteinase domain. It is a single-chain proteinase with a molecular mass of 27020 Da. cDNA sequence analysis revealed that the disintegrin-like and cysteine-rich domains of the putative precursor protein of graminelysin I are likely to be processed post-translationally, producing the proteinase domain (graminelysin I). Graminelysin I cleaved the alpha chain of fibrinogen preferentially and cleaved the beta chain either on longer incubation or at higher concentration. Graminelysin I inhibited the adhesion of human umbilical-vein endothelial cells (HUVECs) to immobilized fibrinogen and induced HUVECs detachment in a dose-dependent manner. These effects on HUVECs were abolished when graminelysin I was pretreated with EDTA. However, graminelysin I did not inhibit the adhesion of HUVECs to immobilized collagen. HUVECs were susceptible to death after treatment with graminelysin I when they were cultured on immobilized fibrinogen. In contrast, HUVECs were rather resistant to treatment with graminelysin I if they were cultured on immobilized collagen. Furthermore, graminelysin I induced apoptosis of HUVECs in a dose-dependent manner. Similarly, its apoptosis-inducing activity was blocked if it was treated with EDTA. These results suggest that the catalytic activity of graminelysin I on matrix proteins contributes to its apoptosis-inducing activity.

Rhodostomin, a snake venom disintegrin, inhibits angiogenesis elicited by basic fibroblast growth factor and suppresses tumor growth by a selective alpha(v)beta(3) blockade of endothelial cells.

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, although angiogenic factor and integrin-extracellular matrix interaction modulate this process. We report here that a snake venom-derived disintegrin, rhodostomin, inhibited distinct steps in angiogenesis elicited by basic fibroblast growth factor (bFGF), and also suppressed in vivo murine melanoma tumor growth. Rhodostomin dose-dependently inhibited bFGF-induced human umbilical vein endothelial cell (HUVEC) proliferation as examined by cell number count, metabolic activity, and BrdU incorporation assays with submicromolar IC(50) values. However, it apparently did not affect the viability of murine B16F10 melanoma cells, even up to 50 microM. Rhodostomin also inhibited HUVEC migration and invasion evoked by bFGF, and tube formation of bFGF-treated HUVECs in Matrigel. Moreover, rhodostomin selectively inhibited bFGF-, but not vascular endothelial growth factor-associated angiogenesis in the chick chorioallantoic membrane model. Furthermore, rhodostomin blocked both bFGF- and B16F10-induced neovascularization in murine Matrigel plug model and suppressed the growth of subcutaneously inoculated B16F10 solid tumor, leading to a prolonged survival of the rhodostomin-treated C57BL/6 mice. The antiangiogenic effect of rhodostomin on bFGF-treated HUVECs is related to the integrin alpha(v)beta(3) blockade, as evidenced by its selective inhibition on the binding of 7E3, a monoclonal antibody (mAb) raised against alpha(v)beta(3,) but not that of P1F6, an alpha(v)beta(5) mAb toward both naive and bFGF-primed HUVECs. Moreover, 7E3 specifically blocked fluorescein isothiocyanate-conjugated rhodostomin binding to HUVEC, whereas P1F6 and anti-integrin alpha(2), alpha(3), alpha(4), or alpha(5) mAbs did not.

Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist.

1. Agkistin, purified from the snake venom of Formosan Agkistrodon acutus, belongs to the family of C-type lectin GPIb binding proteins. It is a heterodimeric molecule, consisting of alpha- (16.5 kDa) and beta- (15.5 kDa) subunits with a molecular mass of 32,512 Daltons examined by SDS - PAGE and mass spectrometry. 2. In vitro, agkistin concentration-dependently inhibited ristocetin-induced human platelet agglutination and aggregation in the presence of vWF. It also inhibited TXA2 formation and prolonged the latent period in triggering aggregation by a low concentration of thrombin (0.03 u x ml(-1)). 3. 125I-agkistin specifically bound to unactivated human platelets in a saturable manner with a KD value of 223+/-10.6 nM. This binding reaction was rapid and reversible. Monoclonal antibodies, AP1 and 6D1 raised against platelet GPIb, almost completely blocked 125I-agkistin binding to platelets. However, monoclonal antibody 7E3 raised against GPIIb/IIIa complex, trigramin, a GPIIb/IIIa antagonist, ADP and EDTA did not affect 125I-agkistin binding reaction. 4. Agkistin (250 microg x kg(-1)) significantly prolonged the bleeding time and induced transient thrombocytopenia of mice when given intravenously. Furthermore, it markedly inhibited platelet plug formation in irradiated mesenteric venules of fluorescein-treated mice in vivo. 5. In conclusion, agkistin inhibits ristocetin induced platelet aggregation mainly through its specific binding to platelet GPIb, thereby blocking the interaction between GPIb and vWF. In addition, agkistin exhibits antithrombotic activity in vivo.

Viper venom components affecting angiogenesis.

Angiogenesis is a complex process consisting of the proliferation, migration and differentiation of endothelial cells, and it is essential for the progression of malignant solid tumors. In this report, we examine the effects of disintegrins (e.g. rhodostomin and accutin) and glycoprotein-lb-binding proteins (e.g. agkistin) on each step in angiogenesis using in vitro and in vivo models. Rhodostomin (but not agkistin) inhibited the viability of human umbilical vein endothelial cells (HUVECs) and capillary tube formation of HUVECs. Rhodostomin also inhibited HUVEC migration and invasion evoked by basic fibroblast growth factor (bFGF). In in vivo studies, rhodostomin inhibited bFGF-, but not vascular-endothelial-growth-factor (VEGF)- associated angiogenesis in chick chorioallantoic membrane model, blocked both bFGF and B16F10 melanoma cell-induced neovascularization, and suppressed the growth of subcutaneously inoculated B16F10 solid tumor, leading to a prolonged survival of the C57BL/6 mice treated with rhodostomin. The antiangiogenic effects of rhodostomin on bFGF-treated HUVECs may be mainly related to the blockade of the interaction of endothelial alpha(v)beta(3) and extracellular matrix.