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Introduction: The applicability of protein drugs is confined by protein degradation and rapid elimination. PEGylation of polypeptides improves protein stability by sterically obstructing the degradation by serum proteases and reduces renal clearance by the increased mass., Experimental Approach: We compared the antithrombotic activities of intact rhodostomin (Rn) and PEGylated rhodostomin (PRn) both in vitro and in vivo systems. In addition, the functional half-life in inhibiting platelet aggregation and the tendency in causing bleeding side effect were investigated., Results: Rn and PRn both potently inhibited human and mouse platelet aggregation induced by collagen, thrombin or ADP in vitro with a similar IC50 around 60-100nM. Rotational thromboelastometry assay indicated that PRn was more effective than Rn in preventing clot formation in human whole blood. In platelet-rich plasma from mice injected with Rn or PRn, the inhibitory effects on collagen-induced platelet aggregation were also comparable, but Rn caused higher bleeding tendency. In ferric chloride-induced arterial thrombosis, Rn and PRn significantly prolonged occlusion time at high dosage (0.2μg/g). However, PRn obviously prolonged the occlusion time even given at a lower dosage (0.06μg/g). The functional half-life assay revealed that PEGylation prolonged the in vivo half-life of Rn., Conclusions: PRn exhibits higher antithrombotic potency and longer half-life in vivo as compared with native Rn on a molar basis. In addition, PRn exhibits a better safety profile at an efficacious antithrombotic dose in vivo. Therefore, PEGylation may be one of the ideal options in modifying disintegrin derivatives as the safe therapeutic agents for integrin-related diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Clinical depression is a significant risk factor for cardiovascular diseases and confers an increased risk of mortality. Increased platelet reactivity may predispose depressed patients to cardiovascular diseases. The antidepressants selective serotonin reuptake inhibitors (SSRIs) have been found to have cardioprotective effects probably via the attenuation of platelet activation independently in addition to treatment of depression itself. However, the characters of the inhibitory effect of SSRIs on platelets remain largely unknown. Here we show that an SSRI, citalopram, specifically inhibited collagen-induced platelet aggregation. Citalopram, however, revealed only little inhibitory effect on platelet aggregation induced by thrombin, U46619, and ionomycin, and failed to inhibit reversible platelet aggregation induced by adenosine diphosphate with fibrinogen. Collagen-induced of αIIbβ3 integrin activation in platelets under a static condition was not influenced by citalopram. Citalopram inhibited convulxin-induced platelet aggregation and αIIbβ3 integrin activation. In the experiments with fibrinogen-induced aggregation in elastase-treated platelets, citalopram inhibited only collagen-induced αIIbβ3 activation but not the binding activities between activated αIIbβ3 integrin and fibrinogen. Moreover, citalopram inhibited α-granule and dense granule secretion from platelets in response to collagen, as determined by a reduced expression of P-selectin and adenosine triphosphate release, respectively. In addition, collagen-induced thromboxane A2 release in platelets was attenuated by citalopram pretreatment. These findings might specify the mechanisms of inhibitory effects of citalopram on collagen mediated platelet activation and aggregation, and further support the cardioprotective effect of SSRIs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We previously reported that the fibrinogen-binding segment (residues 221-550) of Staphylococcus aureus clumping factor A (ClfA), which binds to fibrinogen gamma chain C-terminus, exerted inhibitory effects on platelet aggregation and fibrin clot formation in vitro. Here, we further demonstrated the effectiveness of using ClfA221-550 to inhibit platelet-rich thrombus formation in vivo. Platelet-rich thrombi were formed in the mesenteric venules of fluorescein-loaded mice by filtered light illumination. It grew rapidly and ultimately resulted in the cessation of blood flow due to vessel occlusion. Given by intravenous bolus injection, ClfA221-550 delayed occlusive thrombi formation in a dose-dependent manner: 2-, 3- and 4.5-fold prolongations of vessel occlusion time were attained with 0.69, 6.9 and 34.5 mg/kg of ClfA221-550, respectively. Reduced fibrin clot formation at the late phase with plasmas, which were prepared from ClfA221-550-treated mice, was also dose-dependent. The suppression of fibrin formation ex vivo coincided with the delay of occlusive thrombus formation in vivo, suggesting that the antithrombotic effect of ClfA221-550 may result from the blockade of fibrinogen gamma chain C-terminal functions, in mediating platelet aggregation and fibrin clot formation. Administration of ClfA221-550 also lengthened the tail bleeding of mice; however, significant effect was achieved only with a higher dosage, namely 34.5 mg/kg. These results together showed that blockade of fibrinogen gamma chain C-terminus with ClfA221-550 preferentially affected platelet-rich thrombus formation rather than normal haemostasis, thus providing a rationale for selecting fibrinogen gamma chain C-terminus as a new target for thrombotic intervention.

Osteosarcoma is a very malignant bone tumor which has a high metastatic potential and usually lead to poor prognosis. The adhesion of tumor cells to the endothelium or extracellular matrix (ECM) is an essential step in the metastatic cascade. We investigated the effect of thrombin on the adhesion activity of the osteosarcoma cell line, ROS 17/2.8. Incubation with the low concentrations of thrombin (0.01-5 U/ml, 5 min to 24 h) elevated the adhesion activity of ROS 17/2.8 to both human umbilical vein endothelial cells (HUVEC) and extracellular matrix, with the peak effect at the concentration of 0.5 U/ml for 30 min at 37 degrees C. The ROS 17/2.8 cells responded to thrombin by a peak effect of increased adhesion to HUVEC (5.5 folds vs. control) and fibronectin (4.8 folds) after thrombin pretreatment (0.5 U/ml, 30 min, 37 degrees C). Pretreatment with monoclonal antibodies against beta3 integrins, including anti-alphavbeta3, 10E5 and 7E3, effectively antagonized the thrombin-enhanced cell adhesion activity, whereas anti-alpha3beta1 and anti-alpha5beta1 did not antagonize the enhanced cell adhesion. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, and synthetic peptide RGDS also blocked the thrombin-enhanced ROS 17/2.8 cell adhesion. This study demonstrated that thrombin enhanced the cell adhesion of ROS 17/2.8 cells to HUVEC or ECM through an upregulation of beta3 integrins, and rhodostomin was a strong inhibitor on thrombin-enhanced cell adhesion, either to HUVEC or fibronectin substratum.

The metastasis of tumor cells to bone involves migration, invasion and adhesion to bone. Breast and prostate cancer cells have predilection for spreading to bone. Snake venom-derived arginine-glycine-aspartic acid (RGD)-containing disintegrins (e.g. rhodostomin) have been demonstrated to inhibit cell adhesion. Here, we found that rhodostomin inhibited the adhesion of breast and prostate carcinoma cells to both unmineralized and mineralized bone extracellular matrices in a dose-dependent manner, without affecting the viability of tumor cells. In addition, rhodostomin also inhibited the migration and invasion of breast and prostate carcinoma cells. It specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin alphavbeta3, to tumor cells, but not those of other MoAbs against other integrin subunits such as alpha2, alpha3, alpha5 and beta1. As breast cancer cells MDA-MB-231 were locally injected into tibia in nude mice, histological examination of the tibia of control group revealed that most of the cancellous bone had been replaced by the breast cancer cells after 28 days' inoculation. In contrast, co-administration of trigramin with cancer cells markedly inhibited tumor growth and bone destruction. Taken together, disintegrins strongly inhibit the adhesion, migration, invasion of tumor cells and also tumor growth of human breast cancer cells in bone as well. Therefore, disintegrins may be developed as alternate therapy for bone metastasis of cancer cells.

Triflamp, a 28 kDa snake venom metalloproteinase purified from the venom of Trimeresurus flavoviridis, possesses the proteolytic activities toward P-selectin glycoprotein ligand-1 (PSGL-1), glycoprotein Ib (GPIb) and fibrinogen. In human whole blood preparation, however, triflamp (6 microg/ml) failed to cleave neutrophil PSGL-1 and platelet GPIb. Human alpha2-macroglobulin (alpha2M) was mainly responsible for the neutralization of the proteolytic effects of triflamp on PSGL-1, GPIb and fibrinogen. Human alpha2M neutralized triflamp at a stoichiometry about 1.1:1 (molar basis) determined by azocaseinolysis. SDS-PAGE analysis revealed that triflamp cleaved the bait-region of alpha2M. Western blot demonstrated that triflamp interacted with the C-terminal half-subunits of truncated alpha2M resulting in the formation of high-molecular-weight species of alpha2M-triflamp complexes. In the presence of competing nucleophile, 0.2 M methylamine, the proteolytic activity of triflamp was conserved. In vivo we found that mice neutrophils were resistant to the cleavage of PSGL-1 by triflamp. However, mouse PSGL-1 and GPIb were susceptible to be cleaved by triflamp in washed mouse neutrophil and platelet preparation, respectively. Similarly, mouse serum was also responsible for the inactivation of the proteolytic activity of triflamp. This study provides direct evidences for the reasonable explanation regarding the reduced proteolytic activity of triflamp toward its substrates in whole blood preparation and in vivo model.

The interaction of extracellular matrix with cells plays a key role in the regulation of cell adhesion, migration, proliferation as well as differentiation. Transformed cells express a different profile of adhesion molecules, which may mediate metastasis under specific matrix microenvironment. We here found that ROS 17/2.8 osteosarcoma cells and osteoblasts have different expression of alpha5 integrin, executing different fibronectin fibrillogenesis. As compared with ROS 17/2.8 cells, osteoblasts have higher expression of fibronectin, collagen, alpha5, beta1, alpha2 integrins and focal adhesion kinase as examined by immunostaining and flow cytometry. Crovidisin, a PIII snake venom metalloproteinase (SVMP) purified from venom of Crotalus viridis, exhibits collagen-binding activity and matrix metalloproteinase activity. Crovidisin selectively caused the detachment of ROS 17/2.8 osteosarcoma cells but not of primary cultured osteoblasts. On the other hand, triflavin, an RGD-dependent disintegrin purified from venom of Trimeresurus flavoviridis, did not cause the detachment of both osteoblasts and ROS 17/2.8 cells. Although ROS 17/2.8 cells detached from substratum after crovidisin treatment for 24 h, the loss of mitochondrial membrane potential was not observed unless a prolonged treatment for longer than 36 h. These results suggest that cultured primary rat osteoblasts and ROS 17/2.8 osteosarcoma cells possess different expression of integrins and matrix environment, and ROS 17/2.8 is much more susceptible to be detached by crovidisin. The matrix degradation by crovidisin may be responsible for the preferential detachment of ROS 17/2.8 osteosarcoma cells.

Trimucytin is a powerful platelet aggregation inducer isolated from the venom of Taiwan habu snake (Trimeresurus mucrosquamatus). In this study, we found that the snake venom protein, crovidisin, which prevents collagen-platelet interaction through its high-affinity binding to collagen, inhibited competitively trimucytin-induced aggregation of washed human platelets with a pA(2) value of 6.65. The ability of trimucytin in triggering platelet aggregation was suppressed by a monoclonal antibody (A2-IIE10) raised against the alpha2 subunit of alpha2beta1 integrin (glycoprotein Ia/IIa), indicating that platelet alpha2beta1 integrin plays a central role in trimucytin's platelet reactivity. Many studies have localized the major reactive site of alpha2beta1 integrin to the I-domain of alpha2 subunit. However, Escherichia coli-produced recombinant alpha2 I-domain (GST-alpha2 fusion protein) blocking collagen-induced platelet aggregation failed to inhibit aggregation of platelets in response to trimucytin. Based on these findings, it is concluded that the platelet reactivity of trimucytin is alpha2beta1 integrin-dependent, while the I-domain present in the alpha2 subunit is not involved. This novel snake venom protein would be useful for mapping the functional domain of alpha2beta1 integrin.

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.

A potent platelet aggregation inhibitor in the venom of Crotalus viridis snake was purified to homogeneity by gel filtration chromatography and reverse phase high-performance liquid chromatography. This purified principle, named crotavirin, is a single-chain polypeptide with a mol. wt of 9200 as estimated by SDS-polyacrylamide gel electrophoresis. It inhibited the aggregation of human washed platelets induced by collagen, thrombin and thomboxane analogue (U46619) with a similar IC50 (approximately 1.0 micrograms/ml, 0.11 microM). The binding of fluorescein isothiocyanate-conjugated crotavirin to platelets was abolished in the presence of divalent cation chelator, EDTA, indicating that divalent cation is essential for crotavirin's binding. A monoclonal antibody, 7E3, raised against platelet glycoprotein IIb-IIIa complex blocked the binding of fluorescein isothiocyanate-conjugated crotavirin to platelets, whereas the other monoclonal antibody against glycoprotein IIb-IIIa, 10E5, had no inhibitory effect. In addition, crotavirin inhibited in a concentration-dependent manner the binding of fluorescein isothiocyanate-conjugated rhodostomin, a member of the disintegrin family, to platelets. Its binding to platelets was blocked by disintegrins, e.g. trigramin and rhodostomin. It is concluded that crotavirin is a potent platelet aggregation inhibitor, which acts specifically on an epitope of glycoprotein IIb-IIIa, leading to the blockade of fibrinogen binding to glycoprotein IIb-IIIa and eventually the blockade of platelet aggregation.

A thrombin-like enzyme, grambin, was purified to homogeneity by gel filtration, affinity and ion-exchange chromatography from the venom of Trimeresurus gramineus. Its mol. wt was estimated to be 45,400 by SDS-PAGE under reduced conditions. The mass of neutral sugars in grambin is estimated to be 20.7% of total mass. Grambin's NH2-terminal ten amino acid residues show a high homology to other venom thrombin-like enzymes. It clots human fibrinogen with a specific activity of 220-250 NIH thrombin-equivalent units/mg protein. It preferentially releases fibrinopeptide A accompanied by a slow release of trace amounts of fibrinopeptide B as monitored by HPLC following enzyme treatment of fibrinogen. EDTA, aprotinin, hirudin and heparin did not affect the fibrinogen-clotting activity of grambin in purified human fibrinogen solution. Diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and leupeptin inhibited the clotting activity of grambin whereas iodoacetamide did not affect its activity, indicating that grambin is a serine protease rather than a cysteine protease. In addition, it caused defibrinogenation and showed a marked antiplatelet effect when administered intravenously to mice. It also significantly prolonged the time lapse of platelet-rich thrombus formation in the irradiated mesenteric venules of fluorescein sodium-treated mice. Therefore, grambin may be used as a therapeutic agent not only in treatment of venous thrombosis but also in prevention of arterial thrombosis.

Ancrod caused defibrinogenation and exhibited ex vivo antiplatelet activity in experimental rabbits. In this study, platelet thrombus was induced by irradiation of the mesenteric microvessels with filtered light in mice pretreated with fluorescein sodium intravenously. Ancrod (0.5-2 U/kg) dose-dependently, significantly prolonged the time lapse of inducing platelet plug formation in mesenteric venules when it was intravenously infused. At these doses, ancrod depleted plasma fibrinogen and displayed ex vivo antiplatelet aggregation induced by collagen. Ancrod (1 U/kg) prolonged the occlusion time about 2.1 folds (from control 103.2 +/- 17.0 to 211.2 +/- 26.3 seconds) with a duration longer than 60 min. On the other hand, PGI2 briefly prolonged the occlusion time about 1.5 folds when it was given by continuous infusion (250-500 ng/kg/min). Heparin (100-250 U/kg) had no significant effect in this model. Therefore, ancrod may be used as a therapeutic agent not only in treatment of venous thrombosis and possibly in prevention of arterial thrombosis.

Snake venoms can affect blood coagulation and platelet function in various ways. The physicochemical properties and the mechanisms of actions of the snake venom components affecting blood coagulation and platelet function are discussed.

Triflavin, an Arg-Gly-Asp (RGD)-containing peptide, purified from snake venom of Trimeresurus flavoviridis, inhibits human platelet aggregation through the blockade of fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex. In this report, we examined the effect of triflavin on tumor cells (human hepatoma J-5)-induced platelet aggregation (TCIPA) of heparinized platelet-rich plasma (PRP). ADP-scavenger agents, apyrase (10 U/ml) and creatine phosphate (5 mM)/creatine phosphokinase (5 U/ml) did not inhibit TCIPA while hirudin (5 U/ml) completely inhibited it. J-5 cells initially induced platelet aggregation, then blood coagulation occurred. J-5 cells concentration-dependently shortened the recalcification time of normal as well as Factor VIII, IX-deficient human plasmas, while it was inactive at shortening the recalcification time of Factor VII-deficient plasma, suggesting J-5 cells induced platelet aggregation through activation of extrinsic pathway, leading to thrombin formation as evidenced by the amidolytic activity on s-2238 by expressing tissue factor-like activity. Triflavin inhibited TCIPA in a dose-dependent manner (IC50, 0.02 microM). When compared on molar ratio, triflavin was approximately 30,000 times more potent than GRGDS (IC50, 0.58 mM). On the other hand, GRGES showed no significant effect on TCIPA, even its concentration was raised to 4 mM. Additionally, the monoclonal antibodies, raised against glycoprotein IIb/IIIa complex (i.e., 7E3 and 10 E5) inhibited J-5 TCIPA. In conclusion, we suggest the inhibitory effect of triflavin on J-5 TCIPA may be chiefly mediated by the binding of triflavin to the fibrinogen receptor associated with glycoprotein IIb/IIIa complex on platelet surface membrane.

Xanthyletin, xanthoxyletin, suberosin (all from Citrus grandis), aurapten (from Severinia huxifolia) and poncitrin (from Poncirus trifoliata) were isolated and their chemical structures were characterized to be coumarin compounds. All these coumarin compounds except xanthyletin inhibited the aggregation and ATP release of rabbit platelets induced by arachidonic acid, collagen, ADP, platelet-activating factor (PAF) or U46619 (a thromboxane A2 analog). Thrombin-induced ATP release, but not the aggregation, was also inhibited by these compounds. Xanthyletin inhibited only platelet aggregation induced by arachidonic acid and collagen, while poncitrin inhibited that caused by PAF more markedly than other coumarin compounds. The thromboxane B2 formation in washed platelets caused by arachidonic acid and collagen was suppressed by these coumarin compounds. The phosphoinositide breakdown caused by collagen and PAF was also inhibited by these compounds. They did not affect fibrinogen-induced aggregation of elastase-treated platelets. These antiplatelet actions were immediate, reversible by washout and independent on the incubation time (except suberosin). Antiaggregating effect was also studied by an electrical impedance method and the inhibitory effect of coumarin compounds on the whole blood and platelet-rich plasma was much less than that of platelet suspension in the aggregation induced by collagen, PAF and ADP. It is concluded that the antiplatelet actions of these coumarin compounds are due to the inhibition on thromboxane A2 formation and phosphoinositides breakdown.

HA-29, 2-[(3-methoxyphenyl)methyl]-pyrano[2,3-c]pyrazol-6(1H)-one, was investigated for its inhibitory mechanism of action in washed rabbit platelets. This compound inhibited the aggregation and ATP release of rabbit platelets induced by arachidonic acid and collagen in a concentration-dependent manner, without affecting those induced by ADP, PAF and thrombin. Prolongation of the incubation time of HA-29 with platelets did not cause further inhibition and the aggregability of the agent-treated platelets could be restored after washing of platelets. The concentration-response curve of U-46619-induced platelet aggregation was shifted to the right by HA-29 in a concentration-dependent manner, but the maximal aggregation was suppressed by HA-29. The pA2 and pA10 values of HA-29 on U-46619-induced platelet aggregation were 4.26 and 3.58, respectively, with a slope value of -1.4. The U-46619-induced aggregation was markedly disaggregated by HA-29 even it was added 5 min after U-46619. HA-29 inhibited the secondary aggregation and ATP release, but not the primary aggregation of human platelet-rich plasma induced by ADP and epinephrine. Thromboxane B2 formation caused by arachidonic acid, collagen and thrombin was markedly suppressed by HA-29. HA-29 also inhibited the formation of prostaglandin D2 caused by arachidonic acid. HA-29 inhibited almost completely the formation of inositol monophosphate caused by U-46619, but not that by collagen or thrombin. HA-29 did not affect U-46619-induced contraction of rat aorta. It is concluded that the antiplatelet effect of HA-29 is due to the inhibition of thromboxane A2 formation and blockade of thromboxane A2/prostaglandin endoperoxide receptor.

Denudatin B, an isomer of kadsurenone, was isolated from Magnolia fargesii. It inhibited the aggregation and ATP release of washed rabbit platelets caused by platelet-activating factor (PAF) in a concentration-dependent manner. The IC50 on PAF (2 ng/ml)-induced aggregation was about 10 micrograms/ml. High concentration of denudatin B (greater than 50 micrograms/ml) also inhibited the aggregation and ATP release of platelets caused by ADP, collagen, arachidonic acid and thrombin. However, shape change of platelets still existed. Prolongation of the incubation time with platelets could not cause further inhibition, and the aggregability of platelets could be restored after denudatin B was washed out from platelets. Thrombin-induced thromboxane B2 formation was almost completely suppressed. In the absence of extracellular calcium (EGTA 1 mM), ATP release caused by thrombin was inhibited. Thrombin-induced rise of the intracellular calcium concentration was suppressed by denudatin B, but not by BN52021 or kadsurenone. The generation of inositol phosphate in washed platelets caused by collagen, PAF and thrombin was also suppressed. The data indicate that PAF antagonist denudatin B has nonspecific antiplatelet action at high concentration by inhibiting phosphoinositides breakdown induced by collagen and thrombin.

The effects of four 1,4-naphthoquinone derivatives on the aggregation of rabbit platelets were examined. All the four 1,4-naphthoquinone derivatives inhibited the platelet aggregation of washed rabbit platelets induced by thrombin (0.1 U/ml) and the IC50 is: 2-chloro-3-methyl-1,4-naphthoquinone (CMN), 5 micrograms/ml; 3-methyl-5,8-dihydroxy-1,4-naphthoquinone, 13 micrograms/ml; 5,8-dihydroxy-1,4-naphthoquinone, 18 micrograms/ml; 3-methyl-1,4-naphthoquinone (vitamin K3), 53 micrograms/ml. CMN was the most potent in inhibiting the aggregation and release reaction induced by ADP, arachidonic acid, PAF, ionophore A23187, collagen and thrombin in a dose-dependent manner in washed platelets, platelet-rich-plasma and whole blood. The thromboxane B2 formation caused by collagen and ionophore A23187 was inhibited by CMN. However, the thromboxane B2 formation by arachidonic acid was markedly increased. The platelet inhibitory effect of CMN could not be antagonized either by raising the concentrations of extracellular Ca++ or by wash out. The phosphoinositides breakdown induced by thrombin was inhibited by CMN. Phospholipids (PE, PC, PI) could slightly antagonize the antiplatelet effect of CMN. It is concluded that the inhibitory effect of CMN on rabbit platelet aggregation may be due to the inhibition of phosphoinositides breakdown caused by the inducers.

By means of DEAE-Sephadex A-50 column chromatography and gel filtrations on Sephadex G-75, Sephacryl S-300 and Sephadex G-100, successively, a potent 5'-nucleotidase was purified from Trimeresurus gramineus venom. The venom 5'-nucleotidase is a single polypeptide chain and homogeneous as judged by SDS-polyacrylamide gel electrophoresis. It is a thermostable glycoprotein consisting of 589 amino acid residues. Its molecular weight was estimated to be 74,000 by SDS-polyacrylamide gel electrophoresis. It possessed nucleotidase activities toward adenosine monophosphate and adenosine diphosphate. The specific activities toward AMP and ADP were 504 +/- 28 and 101 +/- 8 micrograms Pi/min per mg, respectively. Pre-incubation of this venom's 5'-nucleotidase with ADP resulted in the cleavage of ADP and formation of adenosine. The 5'-nucleotidase activity was inhibited by EDTA. Both Zn2+ and Co2+/- reversed the inhibitory effect of EDTA. In rabbit platelet-rich plasma, it inhibited completely the ADP (2 x 10(-5) g/ml)-induced platelet aggregation. It also inhibited the platelet aggregations induced by sodium arachidonate (100 microM), collagen (20 micrograms/ml) and ionophore A-23187 (5 microM)-induced platelet aggregations were not affected significantly by this venom 5'-nucleotidase. In ADP-refractory platelet-rich plasma, the venom 5'-nucleotidase inhibited the platelet aggregations induced by collagen (20 micrograms/ml) or sodium arachidonate (100 microM). The venom 5'-nucleotidase showed a more pronounced inhibitory effect on sodium arachidonate-induced platelet aggregation than creatine phosphate/creatine phosphokinase and apyrase did. No lactate dehydrogenase was released by this venom 5'-nucleotidase, indicating that no platelet lysis occurred. It is concluded that removal of ADP, which is released by these platelet aggregation inducers, and the subsequent accumulation of adenosine are responsible for the inhibitory effect of the venom 5'-nucleotidase on platelet aggregations.

In addition to alpha-fibrinogenase (hemorrhagin I, HR1), a potent hemorrhagic principle (hemorrhagin II, HR2) was purified from Trimeresurus gramineus venom. It was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. HR2 was a single peptide chain containing 10% carbohydrate with a molecular weight of 81,500. It possessed 669 amino acid residues per molecule, while HR1 contained only 203 amino acid residues per molecule with a molecular weight of 23,500. Both hemorrhagins possessed proteolytic activities toward fibrinogen, casein and azocoll. However, the proteolytic activities of HR1 were much more potent than those of HR2. They were devoid of TAME-esterase and phospholipase A2 activities which were found in crude venom. beta-Mercaptoethanol and antivenin completely inhibited the hemorrhagic activities of HR1 and HR2, while epsilon-aminocaproic acid, trasylol, p-bromophenacyl bromide, phenylmethanesulfonyl fluoride and soybean trypsin inhibitor did not. EDTA completely inhibited the hemorrhagic, fibrinogenolytic and caseinolytic activities of HR1. EDTA also completely inhibited the caseinolytic and fibrinogenolytic activities of HR2, but only partially inhibited its hemorrhagic activity. Subsequent addition of Zn2+ (5 mM) reversed the EDTA-induced inhibitory effect on the hemorrhagic activity of HR1. However, ZN2+ did not reverse the EDTA-induced inhibitory effect on the HR2-induced hemorrhagic activity. These hemorrhagins were found to be ZN2+-containing metalloproteinases. Therefore, the hemorrhagic activity of HR1 seems to be related to its proteolytic activity while that of HR2 seems to be unrelated to its proteolytic activity.

The platelet aggregation inhibitor (PAI) purified from T. gramineus snake venom inhibited platelet aggregation even when it was added after platelet shape change had occurred. It inhibited the initial platelet aggregation phase in rabbit platelet-rich plasma (PRP), but not the second lytic phase induced by thimerosal (0.5 mM), a -SH reagent. In human platelet-rich plasma, the venom inhibitor also inhibited the platelet aggregation induced by ADP, collagen, epinephrine or ristocetin. The epinephrine-induced platelet aggregation was more susceptible to the venom inhibitor. The venom inhibitor (10 micrograms/ml) as well as N-ethylmaleimide (1.2 mM), a -SH reagent, completely inhibited the thrombin-induced clot retraction of PRP. It is concluded that PAI acts on a common step of platelet aggregation. It acts on membrane -SH containing macromolecule which directly mediates the platelet aggregation subsequent to platelet shape change.

By means of gel filtration on Sephadex G-75, DEAE-Sephadex A-50 column chromatography and three gel filtrations on Sephadex G-75, a potent platelet aggregation inhibitor was purified from Agkistrodon halys snake venom and shown to be a single peptide chain, as judged by SDS-polyacrylamide gel electrophoresis. The purified platelet aggregation inhibitor was an acidic protein with a molecular weight of 14,000 and possessed phospholipase A2 activity. Its inhibitory activity on platelet aggregation was heat stable (at 96 degrees C, 30 min) in an acidic medium (pH 5.5), while its phospholipase A enzymatic activity was heat labile under the same conditions. Its inhibitory activity on platelet aggregation induced by thrombin, sodium arachidonate, collagen or ionophore A-23187 was non-competitive and dose-dependent with a similar ID50 (approximately 11 micrograms/ml). It exerted its inhibitory action without pre-incubation with platelet suspension, however, its inhibitory effect could be moderately increased after longer incubation (30 min).

The platelet aggregation inhibitor purified from Agkistrodon halys snake venom inhibited rabbit platelet aggregations induced by thrombin, sodium arachidonate, collagen or ionophore A-23187. The IC50 was about 11 micrograms/ml in platelet aggregation regardless of which aggregation inducer was used. beta-Mercaptoethanol abolished both the phospholipase A enzymatic and platelet aggregation inhibitory activities of this venom inhibitor. p-Bromophenacyl bromide-treated venom inhibitor lost almost completely its phosphilipase A enzymatic activity, but retained its platelet aggregation inhibitory effect. In the presence of EGTA, the venom inhibitor still showed the same inhibitory activity on thrombin-, sodium arachidonate-, collagen- or ionophore A23187-induced platelet aggregations triggered by successive addition of Ca2+. The activation of platelet phospholipase A and the serotonin release reaction triggered by Ca2+ influx were unaffected by this venom inhibitor. It also inhibited the clot retraction of platelet-rich plasma. It is concluded that the inhibitory effect of the venom inhibitor on platelet aggregation is independent of its phospholipase A enzymatic activity. Its mode of action is different from those of other known platelet inhibitory drugs. This venom inhibitor possibly acts on a common step subsequent to platelet shape change, leading to inhibition of platelet aggregation.

Among all the purified components from A. acutus venom, including ADPase, 5'-nucleotidase, phospholipase A2 and fibrinogenases, only the venom ADPase (50-100 micrograms/ml) shows marked inhibitory action on ADP (10 microM)-, collagen (10 micrograms/ml)- and sodium arachidonate (100 microM)-induced platelet aggregations of rabbit platelet-rich plasma. The venom 5'-nucleotidase (100 micrograms/ml) inhibited ADP-induced platelet aggregation by 31 +/- 4% (n = 4, P less than 0.05). Fibrinogenolytic enzymes (fractions I and IX, 100 micrograms/ml) did not significantly inhibit platelet aggregation induced by ADP (10 microM), collagen (10 micrograms/ml) or sodium arachidonate (100 microM). However, when the fibrinogenase (fraction IX, 100 micrograms/ml) was preincubated with platelet-rich plasma for 30 min it inhibited collagen (20 micrograms/ml)- and ADP (10 microM)-induced platelet aggregations by 34 +/- 9% (n = 4, P less than 0.05) and 35 +/- 6% (n = 4, P less than 0.05), respectively. The phospholipase A2 (100 micrograms/ml) did not affect platelet aggregation. The venom ADPase is a single chain polypeptide with a molecular weight of 94,000. The specific ADPase activity is estimated to be 4.3 mu moles Pi/min/mg of protein. It also possesses phosphodiesterase and weak 5'-nucleotidase activities.

Eighteen proteases were isolated from six hemorrhagic venoms of snakes belonging to the families of Crotalidae and Viperidae. According to their actions, they are classified as thrombin-like enzymes, alpha-fibrinogenases, beta-fibrinogenases, Factor X activator, prothrombin activator, hemorrhagins and esterases. Thrombin-like enzymes, beta-fibrinogenases, hemorrhagins and esterase hydrolyzed Phe-Pip-Arg-pNA (S-2238, substrate for thrombin) more strongly than CBZ-Ile-Glu-Gly-Arg-pNA (S-2222, substrate for Factor Xa), CBZ-Phe-Val-Arg-pNA (B-7632) or CBZ-Pro-Phe-Arg-pNA (B-2133). Thrombin-like enzymes, beta-fibrinogenase and esterase hydrolyzed tosyl-L-arginine methyl ester and benzoyl-L-arginine ethyl ester. S-2238 is the most susceptible chromogenic substrate for most venom proteases. Thrombin-like enzymes degraded prothrombin molecule progressively down to prethrombin 2 while alpha- and beta-fibrinogenases degraded it only to prethrombin 1. Factor X activator of Vipera russelli venom and esterase of T. mucrosquamatus venom did not have any effect on prothrombin. Thus, the effects of venom proteases on prothrombin are not parallel to their amidolytic or esterolytic effects.

Effects of seven purified phospholipases A2 from the venoms of snakes (Naja naja atra, Trimeresurus mucrosquamatus and T. gramineus) and honey bee (Apis mellifera) on rabbit washed platelet suspension in the absence of bovine serum albumin have been studied. Only phospholipases A2 from N. n. atra, T. mucrosquamatus and A. mellifera venoms induced platelet aggregation with small amounts of 14C-serotonin release. They showed tachyphylaxis and also cross-tachyphylaxis in inducing platelet aggregation. The former two phospholipases A2 exhibited biphasic responses in which irreversible aggregations appeared at concentrations of 1-10 micrograms/ml. At higher concentrations, they elicited the reversible aggregation. Exogenous Ca2+ was essential to their activity. Indomethacin and EDTA completely abolished both phospholipase A2 induced platelet shape change and aggregation, while mepacrine, prostaglandin E1, verapamil and nitroprusside inhibited only the aggregation response. p-Bromophenacyl bromide-modified phospholipases A2, which almost completely lost enzymatic activity, failed to induce platelet aggregation. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol inhibited the phospholipase A2-induced platelet aggregation. These phospholipases A2 induced thromboxane B2 formation which was inhibited by EDTA and indomethacin, but not by prostaglandin E1. Pre-treatment of platelet suspension with phospholipase A2 from N. n. atra or A. mellifera venom (50 micrograms/ml) inhibited platelet aggregation induced by sodium arachidonate or collagen, but not that induced by thrombin or ionophore A-23187. Exogenous sodium arachidonate or lysophosphatidylcholine also showed unaltered inhibitory spectrum on platelet aggregation. It is concluded that phospholipases A2 induce platelet aggregation by virtue of their enzymatic activity, cleaving the membrane phospholipids resulting in arachidonic acid release and formation of thromboxane A2. On the other hand, the cleaved products, lysophosphatidylcholine, arachidonic acid or arachidonate metabolites (via lipoxygenase pathway) may be responsible for anti-platelet activity.

A potent platelet aggregation inducer was purified from Calloselasma rhodostoma snake venom by Sephadex G-75, CM-Sephadex C-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight estimated to be 28,160 +/- 1280. It was devoid of phospholipase A2, fibrino(geno)lytic and thrombin-like activities. The venom inducer elicited platelet aggregation and the serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its platelet activation. Creatine phosphate/creatine phosphokinase and indomethacin did not inhibit the venom inducer-induced aggregation and release reaction. Mepacrine and verapamil preferentially inhibited aggregation, while PGE1 completely blocked both aggregation and release reaction. It is concluded that the venom inducer activates platelets through the activation of endogenous phospholipase A2 or C, leading to intracellular calcium mobilization, but independent of the ADP release reaction or thromboxane A2 formation.

Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon rhodostoma (Malayan pit viper) snake venom was separated into eleven fractions. Fraction II had fibrinogenolytic activity, and when further purified by gel filtration was homogeneous, as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis. It had a single peptide chain with a molecular weight of 25,360 and an isoelectric point greater than 10. The fibrinogenolytic activity was completely destroyed after heating for 30 min at 60 degrees C at pH 5.6, 7.4 or 8.8. This enzyme cleaved specifically the alpha(A) chain of monomeric fibrinogen, without cleaving the beta(B) chain or gamma chain. The specific fibrinogenolytic activity was 51 mg fibrinogen/min per mg protein. This enzyme showed proteolytic activities toward fibrinogen, fibrin and casein, but was devoid of phospholipase A and tosyl-L-arginine methylester esterase activities which are found in the crude venom. The fibrinogenolytic activity was inhibited by EDTA and cysteine, but not by epsilon-aminocaproic acid.

Magnolol and honokiol are two position isomers isolated from the bark of Magnolia officinalis. Both inhibited the aggregation and ATP release of rabbit platelet-rich plasma induced by collagen and arachidonic acid without affecting that induced by ADP, PAF or thrombin. Aggregation of washed platelets was more markedly inhibited than that of platelet-rich plasma, while the aggregation of whole blood was least affected by both inhibitors. Thromboxane B2 formation caused by collagen, arachidonic acid or thrombin was in each case inhibited by magnolol and honokiol. The rise of intracellular calcium caused by arachidonic acid or collagen was also suppressed by both agents. Collagen-induced intracellular calcium increase in the presence of indomethacin was suppressed by magnolol. It is concluded that the antiplatelet effect of magnolol and honokiol is due to an inhibitory effect on thromboxane formation and also an inhibition of intracellular calcium mobilization.