Crystal structure of bitiscetin, a von Willebrand factor-dependent platelet aggregation inducer.

Bitiscetin, a C-type lectin-like protein isolated from the venom of the snake Bitis arientans, promotes the interactions between plasma von Willebrand factor (VWF) and platelet membrane glycoprotein Ib (GPIb) to induce platelet aggregation. We report here the crystal structure of bitiscetin at 2.0 A resolution. The overall fold is similar to those of coagulation factor IX/X-binding protein (IX/X-bp) and flavocetin-A (a GPIb-binding protein), although these three proteins are functionally distinct from one another. The characteristic property determining target recognition is explained mainly by the differences in the surface potential on the central concave surface. A negatively charged patch on the surface of bitiscetin is a candidate for the site of binding to the positively charged surface of the VWF A1 domain, as shown in the case of another platelet aggregation inducer, botrocetin. However, a positively charged patch near the central concave surface is unique for bitiscetin and suggests that it is the binding site for the negatively charged surface of the VWF A3 domain. Thus, the interactions accounting for VWF activation by bitiscetin possibly involve both the A1 and A3 domains of VWF, indicating a specific mechanism of VWF activation by bitiscetin.

Inhibition of human platelet aggregation by L-amino acid oxidase purified from Naja naja kaouthia venom.

L-Amino acid oxidase (LAO) widely exists in snake venoms. Purification of LAO from the Naja naja kaouthia (monocellate cobra) venom has been reported (Tan and Swaminathan, 1992), but its structural characterization and physiological function remained to be determined. The function of snake venom LAOs in hemostasis, especially their effect on platelet aggregation, has been controversial. We determined the N-terminal amino acid sequence of the N. n. kaouthia LAO named K-LAO to be DDRRSPLEECFQQNDYEEFLEIAKNGLKKTxNPKHVXxV (38 residues). The protein data base search revealed that the enzyme had high similarities with other snake venom LAOs. Further, platelet aggregation studies revealed that K-LAO functionally did not induce platelet aggregation in a platelet-rich plasma system, but that it inhibited platelet aggregation induced by agonists such as ADP, collagen and ristocetin in a dose-dependent manner. K-LAO diminished platelet aggregation more intensely under low than high shear stress. This inhibitory activity of K-LAO on either ristocetin-induced or shear-induced platelet aggregation was quenched by addition of catalase. These results indicate that K-LAO functions as an inhibitor to platelet aggregation through the formation of hydrogen peroxide. The enzyme may contribute to the development of a severe hematological disorder due to cobra envenomation.

Upshaw-Schulman syndrome revisited: a concept of congenital thrombotic thrombocytopenic purpura.

Upshaw-Schulman syndrome (USS) is a congenital bleeding disorder characterized by repeated episodes of thrombocytopenia and microangiopathic hemolytic anemia that respond to infusions of fresh frozen plasma. Inheritance of USS has been thought to be autosomal recessive, because 2 siblings in the same family are often affected but their parents are asymptomatic. Recently, chronic relapsing thrombotic thrombocytopenic purpura (CR-TTP), reported almost exclusively in adults, was shown to be caused by inherited or acquired deficiency in the activity of a plasma von Willebrand factor-cleaving protease (vWF-CPase). The pathogenesis of USS is unknown, and a relationship between CR-YEP and USS has not been reported. We studied 3 unrelated USS patients (ST, SY, and KI) who presented with severe indirect neonatal hyperbilirubinemia. All 3 patients had undetectable vWF-CPase activity, and the inhibitors to vWF-CPase were all negative. In their parents with no clinical symptoms, vWF-CPase activities as a percentage of control samples (mother/father) were 17/20 for ST, 60/45 for SY, and 36/5.6 for KI. Thus, USS and vWF-CPase activity appear to be coinherited as autosomal recessive traits. Transfusion of fresh frozen plasma in 2 patients (ST and SY) resulted in the expected maximal increment of approximately 7% to 8% in vWF-CPase activity at 1 to 4 hours, but the levels became less than 3% within 2 days. After this decrease, platelet counts increased, plateaued in the normal range at 10 to 12 days, and declined thereafter. Thus, the 2 to 3 weeks of therapeutic benefit from plasma infusions will be discussed in relation to the intravascular lifetime of vWF-CPase.

Enhanced low shear stress induced platelet aggregation by Shiga-like toxin 1 purified from Escherichia coli O157.

The effect of Shiga-like toxin 1 (Stx1) produced by Escherichia coli O157 on platelets was studied with an argon laser light-assisted shear-induced platelet aggregometer and with binding assays. Stx1 markedly enhanced the platelet aggregation under low shear stress but did not affect it under high shear stress. Minimal concentration of Stx1 required for the enhancement was 0.25 ng/ml, and almost maximal enhancement was observed at a final concentration of > or =2.5 ng/ml. This enhanced platelet aggregation disappeared after leukocyte depletion from normal platelet-rich plasma with a specific filter. In contrast, a standard platelet aggregometer was unable to detect this enhanced platelet aggregation in either the presence or the absence of ADP. 125I-labeled purified Stx1 did not specifically bind to normal washed platelets depleted of leukocytes, and thin-layer chromatographic analysis of glycolipids extracted from normal platelet lysates also confirmed that leukocyte-depleted normal platelets lack Stx1-specific receptor globotriaosylceramide (Gb3). Supernatant from the monocyte suspension stimulated with Stx1 exhibited the enhanced low shear stress induced platelet aggregation, but that from the polymorphonuclear cell suspension did not. Several cytokines produced from monocytes reproduced this event in vitro. Further, plasmas from six out of seven patients with hemolytic uremic syndrome (HUS) had activity similar to the purified Stx1. This activity was almost totally impaired after treatment of HUS plasmas with Gb3 in accord with reduction of plasma Stx1 levels. Taken together, these results indicate that platelets lack Gb3, and Stx1 appears to modulate platelet aggregation in an indirect fashion, presumably by the release of cytokines or chemical compounds from the target tissues.

Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation.

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.

Comparative study of blood group-recognizing lectins toward ABO blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides.

Binding specificities of ABO blood group-recognizing lectins toward blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides were studied by lectin-blotting analysis, enzyme linked immunosorbent assay and lectin-conjugated agarose column chromatography. Human serum albumin conjugated with A- and B-trisaccharides was clearly recognized by Helix pomatia (HPA), Phaseolus lunatus, Dolichos biflorus agglutinins, and Griffonia simplicifolia I agglutinin B(4), respectively. Almost the same results were obtained for human group A and B ovarian cyst and A-active hog gastric mucins, but Glycine max agglutinin only reacted to the group A hog mucin. When human plasma von Willebrand factor (vWF), having Asn-linked blood group antigens, was tested, HPA was highly sensitive to blood group A antigen on the vWF. Ulex europaeus agglutinin I (UEA-I) preferentially bound to the vWF from blood group O plasma. Within the GalNAc-recognizing lectins examined, a biantennary complex-type oligosaccharide having the blood group A structure retarded on an HPA-agarose column, and the affinity was diminished after digestion with alpha-N-acetylgalactosaminidase. This product bound to UEA-I agarose column. These results indicate that HPA and UEA-I are most sensitive for detection of glycoproteins possessing small amounts of blood group A and H antigens and also useful for fractionation of complex-type oligosaccharides with blood group A and H antigens, respectively.

Complete amino acid sequence of kaouthiagin, a novel cobra venom metalloproteinase with two disintegrin-like sequences.

The primary structure of kaouthiagin, a metalloproteinase from the venom of the cobra snake Naja kaouthia which specifically cleaves human von Willebrand factor (VWF), was determined by amino acid sequencing. Kaouthiagin is composed of 401 amino acid residues and one Asn-linked sugar chain. The sequence is highly similar to those of high-molecular mass snake venom metalloproteinases from viperid and crotalid venoms comprised of metalloproteinase, disintegrin-like, and Cys-rich domains. The metalloproteinase domain had a zinc-binding motif (HEXXHXXGXXH), which is highly conserved in the metzincin family. Kaouthiagin had an HDCD sequence in the disintegrin-like domain and uniquely had an RGD sequence in the Cys-rich domain. Metalloproteinase-inactivated kaouthiagin had no effect on VWF-induced platelet aggregation but still had an inhibitory effect on the collagen-induced platelet aggregation with an IC(50) of 0.2 microM, suggesting the presence of disintegrin-like activity in kaouthiagin. To examine the effects of these HDCD and RGD sequences, we prepared synthetic peptides cyclized by an S-S linkage. Both the synthetic cyclized peptides from the disintegrin-like domain and from the Cys-rich domain) had an inhibitory effect on collagen-induced platelet aggregation with IC(50) values of approximately 90 and approximately 4.5 microM, respectively. The linear peptide (RAAKHDCDLPELC) and the cyclized peptide had little effect on collagen-induced platelet aggregation. These results suggest that kaouthiagin not only inhibits VWF-induced platelet aggregation by cleaving VWF but also disturbs the agonist-induced platelet aggregation by both the disintegrin-like domain and the RGD sequence in the Cys-rich domain. Furthermore, our results imply that the corresponding part of the Cys-rich domain in other snake venom metalloproteinases also has a synergistic disturbing effect on platelet aggregation, serving as a second disintegrin-like domain. This is the first report of an elapid venom metalloproteinase with two disintegrin-like sequences.

Plasma of patients with Upshaw-Schulman syndrome, a congenital deficiency of von Willebrand factor-cleaving protease activity, enhances the aggregation of normal platelets under high shear stress.

Upshaw-Schulman syndrome (USS) is an autosomal recessive disorder characterized by repeated episodes of chronic thrombocytopenia and microangiopathic haemolytic anaemia (MAHA) that responds dramatically to infusions of fresh frozen plasma (FFP). Recent studies have provided consistent evidence that USS is a congenital deficiency of plasma von Willebrand factor-cleaving protease (VWF-CPase) activity and, therefore, unusually large VWF multimers (UL-VWFMs) are present in the plasma. However, the molecular mechanism of the clinical symptoms of USS is not well understood. We studied the relationship between UL-VWFMs and thrombocytopenia in two USS patients by analysing platelet aggregation using a mixture of the patient's plasma and normal washed platelets under high shear stress. Our results clearly showed a remarkably enhanced high shear stress-induced platelet aggregation (H-SIPA) by the patient's plasma. At 24 h after FFP infusion (approximately equal to 10 ml/kg body weight), the enhanced H-SIPA became almost completely normalized but, 2 d later, it began to return to the preinfusion level. These results were in accordance with the change in VWFM patterns. The specific effects of enhanced H-SIPA on VWF, platelet glycoprotein Ib and endogenous ADP released from platelets upon stimulation were confirmed using reagents that specifically inhibit their respective functions. Our present results clearly indicate that thrombocytopenia in USS patients is caused by a combination of the presence of UL-VWFMs, platelets and high shear stress generated in the microcirculation.

The binding of myristoylated N-terminal nonapeptide from neuro-specific protein CAP-23/NAP-22 to calmodulin does not induce the globular structure observed for the calmodulin-nonmyristylated peptide complex.

CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is Nalpha-myristoylated and interacts with calmodulin (CaM) in the presence of Ca2+ ions. Takasaki et al. (1999, J Biol Chem 274:11848-11853) have recently found that the myristoylated N-terminal nonapeptide of CAP-23/NAP-22 (mC/N9) binds to Ca2+ -bound CaM (Ca2+/CaM). In the present study, small-angle X-ray scattering was used to investigate structural changes of Ca2+/CaM induced by its binding to mC/N9 in solution. The binding of one mC/N9 molecule induced an insignificant structural change in Ca2+/CaM. The 1:1 complex appeared to retain the extended conformation much like that of Ca2+/CaM in isolation. However, it could be seen that the binding of two mC/N9 molecules induced a drastic structural change in Ca2+/CaM, followed by a slight structural change by the binding of more than two but less than four mC/N9 molecules. Under the saturated condition (the molar ratio of 1:4), the radius of gyration (Rg) for the Ca2+/CaM-mC/N9 complex was 19.8 +/- 0.3 A. This value was significantly smaller than that of Ca2+/CaM (21.9 +/- 0.3 A), which adopted a dumbbell structure and was conversely 2-3 A larger than those of the complexes of Ca2+/CaM with the nonmyristoylated target peptides of myosin light chain kinase or CaM kinase II, which adopted a compact globular structure. The pair distance distribution function had no shoulder peak at around 40 A, which was mainly due to the dumbbell structure. These results suggest that Ca2+/CaM interacts with Nalpha-myristoylated CAP-23/NAP-22 differently than it does with other nonmyristoylated target proteins. The N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins suggests that the protein myristoylation plays important roles not only in the binding of CAP-23/NAP-22 to Ca2+/CaM, but also in the protein-protein interactions related to other myristoylated proteins.

Glutamine synthetase, hemoglobin alpha-chain, and macrophage migration inhibitory factor binding to amyloid beta-protein: their identification in rat brain by a novel affinity chromatography and in Alzheimer's disease brain by immunoprecipitation.

Proteins binding to amyloid beta-protein (Abeta) may modulate the accumulation of Abeta in Alzheimer's disease (AD) brain. We developed a monomeric Abeta column for isolation of the proteins binding to Abeta from rat brain. By amino acid sequence analysis and immunoreactivity with specific antibodies, we identified three new Abeta-binding proteins, glutamine synthetase, hemoglobin alpha-chain, and macrophage migration inhibitory factor as well as serum albumin, beta-tubulin, and glyceraldehyde-3-phosphate dehydrogenase already identified as proteins bound to amyloid beta-protein precursor. In addition, the retained fraction contained both apolipoprotein E and alpha(1)-antichymotrypsin already known as Abeta binding proteins. Furthermore, we detected the complexes of these new binding proteins with Abeta in a soluble fraction of the cerebral cortex of AD brain by immunoprecipitation. Our results suggest that these binding proteins also associate with Abeta, leading to the clearance or the accumulation of Abeta and the neuronal cell damage in human brain.

Activation of protein-tyrosine kinase pathways in human platelets stimulated with the A1 domain of von Willebrand factor.

The binding of multimeric von Willebrand Factor (vWF) to its specific receptor on platelets, glycoprotein (GP)Ib, is a critical event, allowing platelet activation and subsequent thrombus formation in the vessels. In this study, the effects of the monomeric A1 domain, which contains the GPIb-binding site of the vWF molecule, on platelet activation were examined. The binding of the A1 domain to GPIb resulted in Syk activation and association with Src, as is the case with intact vWF. However, the A1 domain, in contrast to vWF, did not induce platelet cytoskeletal association of tyrosine kinases, Src and Lyn. When platelet functional responses, such as aggregation and intracellular Ca2+ mobilization, were monitored, the A1 domain failed to induce the responses by itself and blocked the responses induced by the multimeric vWF molecule. These results suggested that the A1 domain triggers at least some of tyrosine kinase-related signals via GPIb and may be a partial agonist as well as a competitive antagonist for the vWF-GPIb interaction.

Snake venom proteases affecting hemostasis and thrombosis.

The structure and function of snake venom proteases are briefly reviewed by putting the focus on their effects on hemostasis and thrombosis and comparing with their mammalian counterparts. Up to date, more than 150 different proteases have been isolated and about one third of them structurally characterized. Those proteases are classified into serine proteases and metalloproteinases. A number of the serine proteases show fibrin(ogen)olytic (thrombin-like) activities, which are not susceptible to hirudin or heparin and perhaps to most endogenous serine protease inhibitors, and form abnormal fibrin clots. Some of them have kininogenase (kallikrein-like) activity releasing hypotensive bradykinin. A few venom serine proteases specifically activate coagulation factor V, protein C, plasminogen or platelets. The venom metalloproteinases, belonging to the metzincin family, generally show fibrin(ogen)olytic and extracellular matrix-degrading (hemorrhagic) activities. A few venom metalloproteinases show a unique substrate specificity toward coagulation factor X, platelet membrane receptors or von Willebrand factor. A number of the metalloproteinases have chimeric structures composed of several domains such as proteinase, disintegrin-like, Cys-rich and lectin-like domains. The disintegrin-like domain seems to facilitate the action of those metalloproteinases by interacting with platelet receptors. A more detailed analysis of snake venom proteases should find their usefulness for the medical and pharmacological applications in the field of thrombosis and hemostasis.

Copper-dependent formation of disulfide-linked dimer of S100B protein.

Previous cell biological studies demonstrated that S100B protein enhances neurite extension of cortical neurons and stimulates proliferation of glial cells. Although these activities of the protein are ascribed to its disulfide-linked dimeric form, there have been no indications as to how the dimer is formed in vivo. We have found by an in vitro study that it is produced by copper-dependent oxidation of noncovalent S100B dimer. The disulfide-linked dimer markedly stimulated nitric oxide production in a microglial cell line, BV2. Interestingly, the disulfide-linked dimer formation was found to be prevented by ascorbic acid. The copper-dependent formation of the dimer may not happen in vivo under normal conditions; however, under pathological conditions where copper is likely to be released from tissues and catalyze autoxidation of ascorbic acid, the dimer formation may proceed, resulting in the stimulated production of nitric oxide that would induce toxic signaling pathways.

Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1-loop fragments.

Under high shear stress, the binding of von Willebrand factor (VWF) A1-loop domain to platelet glycoprotein (GP) Ib alpha occurs as the earliest event in thrombus formation. Therefore recombinant VWF A1-loop fragments could be of therapeutic use in blocking this interaction as competing ligands. We have prepared three homodimeric VWF A1-loop fragments [315 kD Fr III (a homodimer of amino acid [aa] residues 1-1365 of the subunit), 220 kD Fr (a homodimer of aa residues 1-708 of the subunit), and 116 kD Fr (a homodimer of aa residues 449-728 of the subunit) and two monomeric fragments [39/34 kD Fr (a monomer of aa residues 480/481-718 of the subunit] and His-rVWF465-728 (a monomer of aa residues 465-728 of the subunit)], and assessed their potency as inhibitors of botrocetin-induced VWF binding to GPIb alpha and high shear stress induced platelet aggregation mediated by intact VWE All these fragments completely inhibited botrocetin-induced VWF binding to GPIb alpha at a final concentration of 40-200 microM. The homodimeric A1-loop fragments also totally inhibited high shear stress induced platelet aggregation at a final concentration of 0.45-2.0 microM in the following order: 315 kD Fr > or = 220 kD Fr >> 116 kD Fr. In contrast, the monomeric A1-loop fragments were only partial inhibitors of high shear stress induced platelet aggregation even at a final concentration of 20 microM, and their IC50s were 13-39 times higher than those of the homodimers. These results indicate that the homodimeric structure of the A1-loop fragment is important for optimal molecular interaction with GPIb alpha under high shear stress.

ABO blood group antigens on human plasma von Willebrand factor after ABO-mismatched bone marrow transplantation.

von Willebrand factor (vWF) is synthesized exclusively by endothelial cells and megakaryocytes, and stored in the intracellular granules or constitutively secreted into plasma. ABO blood group antigens are covalently associated with asparagine-linked sugar chains of plasma vWF. The effect of ABO-mismatched bone marrow transplantation (BMT) or blood stem cell transplantation (BSCT) on the expression of ABO blood group antigens on the vWF was examined to obtain information on the origin of these antigens. In ABO-mismatched (HLA-matched) groups, 8 cases of BMT and 4 cases of BSCT were examined. In all cases, the ABO blood groups on red blood cells were gradually converted to the donor's type within 80 to 90 days after the transplantation. The blood group antigens on the vWF were consistent with the recipient's blood group for the period monitored by enzyme-linked immunosorbent assay (ELISA). When vWF was isolated from normal platelets and examined for the blood group antigens using ELISA or immunoblotting, it showed few antigens. However, vWF extracted from veins expressed blood group antigens. These findings indicate that platelet (megakaryocyte)-derived vWF does not contain blood group antigens and that these antigens may be specifically associated with vWF synthesized in endothelial cells and secreted into plasma. Furthermore, it is possible that the persistence of the recipient's blood group antigens on plasma glycoproteins such as vWF, independent of the donor-derived erythrocytes, after ABO-mismatched stem cell transplantation, may influence the immunological system in the production of anti-blood group antibodies resulting in the establishment of immunological tolerance in the recipient plasma.

The cDNA cloning of human placental ecto-ATP diphosphohydrolases I and II.

The cDNA clones of two isoforms (enzymes I and II) of human placental ecto-ATP diphosphohydrolases have been isolated based on the N-terminal amino acid (aa) sequence of the immunopurified 82 kDa protein and characterized. The cDNA clone encoding enzyme I consists of 2081 nucleotides and the predicted enzyme I consists of 517 aa residues. Enzyme I has a 5'-UTR and an N-terminal 11 aa sequence that differ from CD39, but the rest of the sequence is the same as CD39. The hydropathy plot indicated that enzyme I has two hydrophobic regions near the N- and C-termini of the molecule. In contrast, enzyme II consists of 1814 nucleotides and the predicted protein consists of 306 aa residues. The sequence of 1-1018 nucleotides of enzyme II is identical to that of enzyme I, but the 1019-1814 nucleotide sequence is different from both enzyme I and CD39. The hydropathy plot indicated that enzyme II has one hydrophobic region near the N-terminus, suggesting that enzyme II is also anchored to the cell membrane. It is, however, likely that some of enzyme II exists as a soluble form in plasma, possibly after proteolytic processing.

Endogenous substrates of sphingosine-dependent kinases (SDKs) are chaperone proteins: heat shock proteins, glucose-regulated proteins, protein disulfide isomerase, and calreticulin.

Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly, substrates of all these SDKs so far identified are molecular chaperones or adapters capable of binding to enzymes and key molecules involved in signal transduction, maintaining tertiary structure of bioactive molecules, or maintaining cellular homeostasis in response to environmental stress. Thus, the essential role of Sph and DMS is to activate molecular chaperones, thereby providing a link to the mechanism by which SDK activity regulates cellular homeostasis and signal transduction.

Placental ecto-ATP diphosphohydrolase: its structural feature distinct from CD39, localization and inhibition on shear-induced platelet aggregation.

Human placental ecto-ATP diphosphohydrolase (ATPDase), an 82 kDa single-chain glycoprotein, was purified to high specific activity using a specific murine monoclonal antibody MK33 (IgG1-kappa). Structurally, protein-based analysis showed this enzyme to be almost identical to that of CD39 lymphoid cell activation antigen deduced by cDNA sequencing (Maliszewski CR, et al, J Immunol 1994; 153:3574); but differing in the NH2-terminal amino acid sequence, suggesting that placental ecto-ATPDase is most likely an isoform of CD39 generated by alternative splicing of the pre-mRNA. Functionally, placental ecto-ATPDase totally inhibits the secondary platelet aggregation induced by agonists at a final concentration (f.c.) of 1 microgram/ml. The purified enzyme (1 microgram/ml, final), pre-incubated with washed platelets prior to alpha-thrombin stimulation, completely inhibits the activation of platelet glycoprotein (GP) IIb/IIIa, thereby blocking the binding of fibrinogen or von Willebrand factor to platelets. Further, under different shear stresses, the enzyme modulates platelet aggregation differently. Low shear stress-induced platelet aggregation is blocked by this enzyme in a dose-dependent manner and is totally blocked at f.c. 0.5 microgram/ml. Under high shear stress, however, this protein at a f.c. of 0.5 microgram/ml mediates almost complete disaggregation of platelets without affecting the initial aggregation. Using immunohistochemical analysis, this enzyme was observed to be localized at the syncytiotrophoblasts of placental microvilli and the endothelial cells (ECs) of the umbilical vein obtained at full-term normal delivery, but scarcely at the ECs of the umbilical artery.

Purification and characterization of kaouthiagin, a von Willebrand factor-binding and -cleaving metalloproteinase from Naha kaouthia cobra venom.

A von Willebrand factor (vWF)-binding and -cleaving metalloproteinase, termed "kaouthiagin", was purified from the venom of cobra snake Naja kaouthia. Kaouthiagin is a monomer with a molecular mass of about 46 kDa and 51 kDa under non-reducing and reducing conditions, respectively, and the N-terminal amino acid sequence is homologous to high molecular mass snake venom metalloproteinases. Kaouthiagin bound to vWF in a divalent ion-independent manner, but the reduced kaouthiagin failed to interact with vWF, suggesting that the protein conformation maintained by intrachain-disulfide linkages of the molecule is essential for the binding to vWF. Neither botrocetin nor bitiscetin, vWF-binding modulators from another snake venom, interfered with the binding between kaouthiagin and vWF, but a monoclonal antibody VW92-3 specific to the N-terminal region of vWF (residues 1-910) inhibited the binding. Without affecting platelet GPIb/IX and GPIIb/IIIa, kaouthiagin specifically cleaved vWF between residues Pro-708 and Asp-709 in a divalent ion-dependent manner to diminish the multimeric structure of vWF in plasma, resulting in the loss of ristocetin-induced platelet aggregability and the collagen-binding activity of vWF. These results indicate that kaouthiagin is a unique metalloproteinase which specifically binds to and cleaves vWF at a specific site and that it will be a useful tool for functional dissection of vWF.

Changes in the specificity of antibodies against steroid antigens by introduction of mutations into complementarity-determining regions of the V(H) domain.

An attempt was made to change the specificity of antibodies (Abs) by introduction of mutations. A monoclonal Ab specific for 17alpha-hydroxyprogesterone (17-OHP) was used as the starting Ab. On the basis of a model that was generated by a computer-driven model-building system, we constructed a phage-display library of Abs in which 16 residues were mutated in three complementarity-determining regions of the heavy chain that appeared to form the steroid-binding pocket. We screened the library with 17-OHP and cortisol that had been conjugated with bovine serum albumin, and we isolated many clones that had retained 17-OHP-binding ability as well as clones with the newly developed ability to bind cortisol in addition to 17-OHP. We compared the amino acid sequences between 17-OHP-specific and cortisol-binding Abs, and then constructed several additional Abs. Our results indicated that a change in specificity could be achieved by changing only a single, critical amino acid residue. Models of the 17-OHP-and cortisol-binding pockets formed by the mutated Abs could explain these observations.