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Upon stimulation of cells with platelet-derived growth factor (PDGF), phospholipase C-gamma1 (PLC-gamma1) binds to the tyrosine-phosphorylated PDGF receptor through one or both of its Src homology 2 (SH2) domains, is phosphorylated by the receptor kinase, and is thereby activated to hydrolyze phosphatidylinositol 4, 5-bisphosphate. Association of PLC-gamma1 with the insoluble subcellular fraction is also enhanced in PDGF-stimulated cells. The individual roles of the two SH2 domains of PLC-gamma1 in mediating the interaction between the enzyme and the PDGF receptor have now been investigated by functionally disabling each domain. A critical Arg residue in each SH2 domain was mutated to Ala. Both wild-type and mutant PLC-gamma1 proteins were transiently expressed in a PLC-gamma1-deficient fibroblast cell line, and these transfected cells were stimulated with PDGF. The mutant protein in which the COOH-terminal SH2 domain was disabled bound to the PDGF receptor. Accordingly, it was phosphorylated by the receptor, catalyzed the production of inositol phosphates, and mobilized intracellular calcium to extents similar to (but slightly less than) those observed with the wild-type enzyme. In contrast, the mutant in which the NH(2)-terminal SH2 domain was impaired did not bind to the PDGF receptor and consequently was neither phosphorylated nor activated. These results suggest that the NH(2)-terminal SH2 domain, but not the COOH-terminal SH2 domain, of PLC-gamma1 is required for PDGF-induced activation of PLC-gamma1. Functional impairment of the SH2 domains did not affect the PDGF-induced redistribution of PLC-gamma1, suggesting that recruitment of PLC-gamma1 to the particulate fraction does not involve the SH2 domains.

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-gamma in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (approximately 700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-gamma1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-gamma activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-gamma1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-gamma activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation.

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P2 with low affinity. But the PI(4,5)P2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P2. A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.

A 150-kDa protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) was previously purified from rat brain. The sequences of peptides derived from the purified PLD inhibitor now identify it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. Further characterization of the enzymatic properties of synaptojanin now shows that it hydrolyzes only the 5-phosphate from inositol 1,4,5-trisphosphate (I(1,4,5)P3) and that it does not catalyze the dephosphorylation of either I(1,3,4)P3 or inositol 1, 4-bisphosphate. However, synaptojanin hydrolyzes both the 4- and 5-phosphates of PI(4,5)P2 and the 4-phosphate of phosphatidylinositol 4-phosphate, converting both compounds to phosphatidylinositol. Magnesium is required for the hydrolysis of I(1,4,5)P3, but not for that of phosphoinositides, by synaptojanin. The inhibition of PLD by synaptojanin is attributable to its ability to hydrolyze PI(4,5)P2. Synaptojanin did not inhibit PLD in the absence of PI(4,5)P2, and the extent of PLD inhibition was related to the extent of PI(4,5)P2 hydrolysis in substrate vesicles. It has been proposed that the biosynthesis of PI(4,5)P2 and the activation of PLD by ADP-ribosylation factor constitute a positive loop to increase rapidly the concentrations of PI(4,5)P2 and phosphatidic acid (PA) during membrane vesiculation. The PA thus produced, probably together with PI(4,5)P2, facilitates vesicle coat assembly. The hydrolysis of PI(4,5)P2, and consequent inhibition of PLD, by synaptojanin might therefore constitute a mechanism to halt the positive loop connecting PI(4,5)P2 and PA during the endocytotic cycle of synaptic vesicles and serve as a signal for uncoating.

We recently showed that not only Ca2+ ions but also Mg2+ ions play a crucial role in stabilizing the native conformation of coagulation factor IX. We here report that Mg2+ ions at physiological concentrations greatly augment the biological activities of factor IX. In clotting assays with dialyzed plasma, addition of Mg2+ ions enhanced the apparent coagulant activity of factor IXa, while that of factor Xa was scarcely affected. Activation of factor X by factor IXa in the presence of factor VIIIa, phospholipids, and Ca2+ ions was accelerated by Mg2+ ions. It appeared that the cation increased the affinity between factor IXa and factor VIIIa, thereby increasing the apparent catalytic efficacy of the enzyme. We also evaluated the effect of Mg2+ ions in the coagulation pathway initiated by tissue factor and found that activation of factor IX by factor VIIa*tissue factor was accelerated by the cation. Consequently, clotting of normal plasma induced by factor VIIa*tissue factor was shortened by the cation, while no such effect was observed in plasma deficient in factor IX or VIII. These results indicate that the previously unrecognized plasma component, Mg2+ ions, plays crucial roles in blood coagulation and, moreover, that contributions of factors IX and VIII in the coagulation cascade have been seriously underestimated in previous investigations.

A collagen-binding glycoprotein was isolated from human platelets using affinity chromatography of immobilized collagen. Based upon characterizations of this protein we confirmed that it was identical to the propolypeptide of von Willebrand factor (pp-vWF), which is also called von Willebrand antigen II. The characteristics we have investigated are molecular weight, existence of carbohydrate chains, and the NH2-terminal amino acid sequence. pp-vWF has strong affinity to collagen and inhibits collagen-induced aggregation of human platelets at a concentration as low as 2 µg/ml even in the presence of plasma. This inhibitory effect is specific for collagen-induced aggregation since it does not inhibit aggregation of platelets induced by other agonists such as ADP, arachidonic acid, platelet-activating factor, ionophore A23187, and ristocetin. As pp-vWF is quickly released from platelets upon activation by various agonists, it is possible that pp-vWF functions as a repressor for excess platelet aggregation induced by collagen and constitutes a negative feed-back mechanism. Considering the fact that mature vWF supports platelet adhesion to subendothelium, present observations suggest that the propeptide portion and the mature protein could have opposing effects on hemostasis.

The venom of the viper Echis carinatus contains a metalloprotease, ecarin, that is a potent prothrombin activator. We here show that the venom is also rich in another prothrombin activator, which does not belong to any known category of prothrombin activators. The novel enzyme, designated carinactivase-1 (CA-1), consists of two subunits held together non-covalently but very tightly. One subunit is a 62-kDa polypeptide that has metalloprotease activity and is homologous to the single-chain enzyme ecarin; the other subunit of 25 kDa consists of two disulfide-linked polypeptides of 17 and 14 kDa, and this subunit resembles the anticoagulant in the habu snake venom, IX/X-bp, that specifically binds the Gla domains of coagulation factors IX and X in a Ca2+-dependent fashion. The activation of prothrombin by CA-1 requires Ca2+ ions at millimolar concentrations and in the absence of Ca2+ ions this enzyme is virtually inactive. By contrast, activation by ecarin is completely independent of Ca2+ ions. CA-1, unlike ecarin, does not activate prothrombin derivatives, in which binding of Ca2+ ions has been perturbed, namely prethrombin-1 and acarboxyprothrombin. Furthermore, the isolated catalytic subunit, although its activity is greatly reduced as compared to that of the holoenzyme, no longer requires Ca2+ ions for the activation of prothrombin. Reconstitution with the non-catalytic 25-kDa subunit restores high level activity and the dependence on Ca2+ ions. Finally, prothrombin activation by CA-1 is inhibited by prothrombin fragment 1, and the isolated non-catalytic subunit is capable of binding fragment 1 in the presence of Ca2+ ions. From these observations, we postulate the following unique mechanism for the activation of prothrombin by CA-1. The enzyme primarily recognizes the Ca2+-bound conformation of the Gla domain in prothrombin via the 25-kDa regulatory subunit, and the subsequent conversion of prothrombin to active thrombin is catalyzed by the 62-kDa catalytic subunit.

The indispensable role of Ca2+ ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation factors has been definitively established but the participation of Mg2+ ions, another alkaline-earth metal that is present abundantly in blood plasma, in such a process is not yet understood. We show here that the Ca(2+)-stabilized conformation of coagulation factor IX undergoes a further conformational change upon binding of Mg2+ ions using three independent structural probes. The probes we used were (i) IX/X-bp, a snake venom anticoagulant that recognizes the Gla domains in coagulation factors IX and X, (ii) conformation-specific polyclonal antibodies against bovine factor IX, and (iii) monoclonal antibodies against the Gla domain of human factor IX. The binding of all these probes had an absolute requirement for Ca2+ ions, and Mg2+ ions alone were ineffective. However, when added together with Ca2+ ions, Mg2+ ions at physiological concentrations greatly augmented the binding of these probes to factor IX; the required concentration of Ca2+ ions was much reduced, and the affinity of each probe for factor IX was increased even in the presence of an excess of Ca2+ ions. These results suggest the presence of a Mg(2+)-specific binding site that does not interact with Ca2+ ions in factor IX. Furthermore, Mg2+ ions potentiated the susceptibility of factor IX to activation by factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca2+ concentration was reduced by Mg2+ ions, and the rate of conversion to factor IXa was increased by Mg2+ ions in the presence of an excess of Ca2+ ions. At a saturating concentration of Ca2+ ions (5 mM), addition of 1 mM Mg2+ reduced the apparent Km value for factor IX from 0.31 to 0.18 microM, and in the presence of a physiological concentration of Ca2+ ions (1 mM), the reduction in Km by Mg2+ ions was far more striking (from 0.91 to 0.24 microM). The apparent Vmax values were hardly affected by Mg2+ ions. Our present data reveal a hitherto novel physiological role of the Mg2+ ions in plasma. Not only Ca2+ ions but also Mg2+ ions are important regulators of the stabilization of the native conformation of factor IX as well as of its efficient activation.

Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 × 10−8Mfor the high affinity site and 5.5 × 10−7Mfor the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.