Isolation and characterization of the alpha and beta subunits of the platelet-activating glycoprotein from the venom of Crotalus durissus cascavella.

It was concluded in a previous paper that the high Mr platelet-activating glycoprotein isolated earlier from the venom of Crotalus durissus cascavella has an hexameric structure of the alpha 3 beta 3 type involving two distinct subunits. Data reported here demonstrate that these two subunits are separable from each other by ion exchange chromatography under denaturating conditions, have similar Mrs (alpha = 12,540 et beta = 13,770) and exist in a one to one ratio within the native molecule. Carbohydrate analysis indicated that they are both similarly glycosylated to a small extent. They have slightly different amino-acid compositions, a common N-terminal sequence up to the fifth residue and similar extinction coefficients at 280 nm. The native molecule has a calculated Mr of 78,930. Additional data demonstrated that convulxin from the venom of Crotalus durissus terrificus is the same platelet-activating agent as the presently described platelet-activating glycoprotein (PAG) from the venom of Crotalus durissus cascavella.

Subunit structure of a potent platelet-activating glycoprotein isolated from the venom of Crotalus durissus cascavella.

The potent platelet-activating factor isolated from the venom of Crotalus durissus cascavella is an acid-soluble multisubunit glycoprotein of Mr 72,000 built up of two types of subunits, alpha and beta, linked by disulphide bonds. The mean apparent Mr of the reduced complex was around 12,000 by gel filtration under denaturating conditions. The Mrs of the alpha and beta subunits, with an apparent ratio of 1/1, were 12,600 and 13,580 by SDS-PAGE respectively. The Mr 72,000 glycoprotein is thought to be an alpha 3 beta 3 complex. The urea dissociated glycoprotein (Mr 72,000) retained its platelet-stimulating activity. It is concluded that the Mr 300,000 form isolated at acidic pH under native conditions, and showing a rosette - like, ring-shaped structure in the electron microscope as well as the Mr 144,000 form isolated at physiological pH under native conditions and active on platelets were the tetrameric and dimeric states of the molecule respectively.

Convulxin-induced activation of intact and of thrombin-degranulated rabbit platelets: specific crossed desensitisation with collagen.

The aggregation of plasma-free rabbit platelets induced by convulxin (Cx), a glycoprotein extracted from the venom of Crotalus durissus cascavella was accompanied by the secretion of ATP and by the formation of thromboxane A2 (TxA2) and of 'platelet-activating factor' (PAF-acether). Thrombin-induced exhaustion of the pool of releasable ADP, or inactivation of cyclooxygenase with aspirin or with arachidonic acid failed to suppress Cx-induced activation. Electron microscopy studies showed that platelets exposed to Cx could be recovered without damage to the cytoplasmic membrane, whereas dense bodies were depleted. Convulxin-treated platelets aggregated in response to ADP, to arachidonic acid and to thrombin, but failed to aggregate in response to Cx itself as well as to collagen. Crossed desensitisation between Cx and collagen was also observed when platelets were exposed to Cx in the presence of prostaglandin E1, which prevented granule depletion, demonstrating that desensitisation was not due to the inability of Cx-treated platelets to secrete ADP in response to collagen. Formation of PAF-acether by thrombin-treated platelets was impaired when thrombin was used as a second stimulus but was maintained when Cx was used as such. The formation of TxA2 by Cx-treated platelets stimulated with arachidonic acid or with thrombin was preserved of only slightly reduced whereas these platelets failed to synthesize TxA2 when stimulated with Cx or with collagen, showing that crossed desensitization between Cx and collagen was not restricted to aggregation, but extended to stimulation of arachidonate metabolism as well. Convulxin is a powerful platelet-stimulating agent which operates through mechanisms which may involve PAF-acether, and which interacts with sites related with those of collagen at an unknown level.

I--isolation and electron microscope studies of a potent platelet-aggregating glycoprotein from the venom of Crotalus durissus cascavella.

A potent acid-soluble platelet-aggregating glycoprotein was purified from the venom of Crotalus durissus cascavella by molecular sieve chromatography on Sephadex G-75 and by adsorption onto Sepharose 4B gels at acidic pH. This new protein with an apparent Mr of 300,000 at acidic pH and containing a low amount of sugars is non-toxic for mice. Electron microscope studies showed that the platelet-aggregating glycoprotein appeared as regular rosettes of 150 A in diameter at acidic pH and underwent polymerization in rod-like particles in the presence of sodium chloride. This glycoprotein, probably hydrophobic, is dissociated into an active molecular form whose apparent Mr was 144,000; however, it is believed to still be a not totally dissociated molecule.

Degranulation of rabbit platelets with PAF-acether: a new procedure for unravelling the mode of action of platelet-activating substances.

Aggregation and secretion of ATP induced by thrombin, collagen, the snake venom component convulxin and platelet-activating factor (PAF-acether) were studied after the exposure of rabbit platelets to 1 microM of PAF-acether. This concentration, which is around 6 orders of magnitude above the concentration needed to induce full aggregation, was required to remove most of the releasable ATP from the platelets. The depleted platelets aggregated to PAF-acether, to thrombin and to convulxin under conditions where only very low amounts of ATP were secreted, confirming that these agents do not require the release of dense body components to trigger aggregation. Furthermore, when exposure to PAF-acether was associated to inactivation of platelet cyclooxygenase with aspirin, aggregation to thrombin persisted, validating the claim that thrombin induces aggregation by a third pathway unrelated to ADP and to thromboxane A2. Aggregation by collagen was markedly reduced by exposure of the platelets to PAF-acether or to aspirin; when both procedures were associated, aggregation was suppressed. Failure to desensitize the rabbit platelets to PAF-acether upon exposure to high amounts of it indicates the absence of irreversible membrane changes due to PAF-acether, and allows its use as a depleting procedure for the dense body materials, which does not affect platelet membrane components as is the case for thrombin.

Purification and preliminary structure of a potent platelet aggregating glycoprotein isolated from the venom of Crotalus durissus cascavella.

A very potent platelet-aggregating glycoprotein, convulxin, was purified from the venom of Crotalus durissus cascavella by gel filtration on Sephadex G75 and by adsorption to Sepharose 4B gel. The apparent molecular weights of the native protein were 78400 and 60000 daltons determined by SDS electrophoresis and by gel filtration under denaturating conditions, respectively. Under the same conditions, the apparent molecular weights of the reduced protein were 13000 and 12000 respectively. These discrepancies are due to the presence of a carbohydrate moiety in the molecule. Analysis for carbohydrates showed the presence of around 4,8% sugars. Convulxin is built up of closely similar subunits linked by disulfide brides and is devoid of free sulfhydryl groups.

Activation of guinea-pig platelets induced by convulxin, a substance extracted from the venom of Crotalus durissus cascavella.

Convulxin (Cx), a component of the venom of the snake Crotalus durissus cascavella, induced the concentration-dependent aggregation of guinea-pig platelets when used at and above 50 +/- 5 ng/ml, accompanied by the release of ATP and by the formation of thromboxanes (Tx). Platelet activation by Cx was not due to potential contaminants found in the crude snake venom, such as phospholipase A2 and clotting enzymes. Aspirin (50-100 microM) failed to interfere with the platelet effects of Cx, demonstrating independence from cyclo-oxygenase. In contrast, indomethacin (50 microM) displayed a distinct inhibitory activity on the effects of Cx, as compared to aspirin, and thus exerts cyclo-oxygenase-independent effects on platelet activation. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) inhibited aggregation by Cx used at concentrations below 6-8 times the threshold, but failed to interfere with higher amounts. Platelet aggregation by Cx was inhibited and reversed once established by EDTA (5mM) and by prostacyclin (0.1-1 microM). Cx-induced activation of platelets is thus Ca2+-dependent and liable to control by the adenylate cyclase-cyclic AMP system. Convulxin induced hypotension, bronchoconstriction and thrombocytopenia when injected i.v. to the anesthetised guinea pig at 0.3-3 microgram/kg. Aspirin and indomethacin (20 and 5 mg/kg respectively) mepyramine and methysergide (02. mg/kg) failed to interfere with these effects, but the combination of either aspirin or indomethacin with methysergide and mepyramine, suppressed the bronchial effects of Cx, leaving the hypotensive and thrombopenic effects unchanged. This synergism remains unexplained. Bronchoconstriction was platelet-dependent, being suppressed by platelet depletion with antiplatelet serum or by i.v. prostacyclin (1-10 microgram/kg).