Koiti Titani

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.

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-TTP and USS has not been reported. We studied 3 unrelated USS patients (ST, SY, and KI) who presented with severe indirect neonatal hyperbilirubimenia. 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.

von Willebrand Factor—Cleaving Protease and Upshaw-Schulman Syndrome

Vascular endothelial cell (EC)-produced plasma von Willebrand factor (vWF) plays a critical role in primary hemostasis through its action of anchoring platelets onto the injured denuded subendothelial matrices under high shear stress. Unusually large vWF multimers (UL-vWFMs), present in plasma immediately after release from ECs, are most biologically active, but they are soon cleaved and degraded into smaller vWFMs by a specific plasma protease, termed vWF-cleaving protease (vWF-CPase), in normal circulation. Recent studies on the relationship between UL-vWFMs and vWF-CPase, together with its autoantibody (inhibitor) have brought about a clear discrimination between thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Furthermore, a congenital deficiency of this enzyme activity has been shown to cause Upshaw-Schulman syndrome, a complex constitutional bleeding diathesis. Successful purification of vWF-CPase revealed that this enzyme is composed of a single polypeptide with a molecular mass of approximately 190 kd, and its complementary DNA cloning unambiguously indicated that it is uniquely produced in the liver and its gene is located on chromosome 9q34. The messenger RNA of vWF-CPase had a span of 4.6 kb, and its enzyme was designated ADAMTS 13. The predicted complete amino acid sequence of this enzyme consisted of 1427 residues, including a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat (TSP1), a cysteine-rich domain, an ADAMTS spacer, 7 additional TSP1 repeats, and 2 CUB domains.

ABO blood group genotype and plasma von Willebrand factor in normal individuals

von Willebrand factor (vWF) is a multimeric plasma protein with ABO (H) blood group sugar chains. We investigated a total of 330 plasmas from normal individuals having various ABO genotypes, with special reference to vWF antigen and its platelet glycoprotein‐Ib‐related biological activities, termed ristocetin cofactor (RCof) and botrocetin cofactor (BCof). RCof reflects the biological activity of higher vWF multimers, while BCof reflects that of vWF of multimers of all sizes. Plasmas from normal individuals carrying one O gene (genotypes AO and BO) had slightly, but proportionally lower levels of vWF antigen, RCof, and BCof than those carrying no O gene (genotypes AA, AB, and BB). Normal plasmas from individuals carrying two O genes (genotype 00) showed much lower values for these parameters than the other plasmas, as previously reported. However, multimeric analysis of plasma vWF antigen revealed no differences among the different genotypes.

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.

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.

Increase of glial fibrillary acidic protein fragments in the spinal cord of motor neuron degeneration mutant mouse

We analyzed protein fractions extracted from the spinal cord of the motor neuron degeneration (Mnd) mouse, a mutant that exhibits progressive degeneration of lower spinal motor neurons, by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) after solubilization of the tissue with medium containing sodium dodecyl sulfate (SDS)-urea during growth of the animal, in comparison with those of age-matched controls (C57BL/6). Several protein spots were detected around a region of pI 5.6-6.0 and molecular mass of 35-50 kDa in Mnd spinal cord tissue on the two-dimensional PAGE separation profile with Coomassie brilliant blue staining, while only a few spots around the same region were found in the control spinal cord. These spots were all immunoreactive with an antibody against glial fibrillary acidic protein (GFAP), a cytoskeleton filamentous protein specific to astroglial cells. The protein spot with molecular mass of 50 kDa showed immunoreactivity with anti-GFAP antibody, had a blocked amino-terminus, and is assumed to be intact GFAP. Several protein spots with slightly smaller molecular masses of 35 to 48 kDa lacked the head domain of the GFAP molecule as a result of cleavage at the 29th and 56th residues from the amino terminus. In Mnd spinal cord tissue, the densities of the immunoreactive GFAP bands with smaller molecular masses increased with development, and became dominant at the time of the appearance of behavioral paralytic gait around 6 to 7 months of age. These results suggest that the increased GFAPs devoid of head domains are related to the degenerative loss of motor neurons in the Mnd spinal cord. Histopathological and GFAP immunohistochemical examination of Mnd spinal cord preparation demonstrated progressive degenerative loss of motor neurons, and considerable increases in number of GFAP-stained astrocytes in the ventral horn at 7 to 9 months of age. These processes of degenerative loss of motor neurons and proliferation of reactive astrocytes with increased levels of fragmented GFAP in the Mnd spinal cord during development seem to be characteristic and preceded the deterioration of motor activities in this animal model of amyotrophic lateral sclerosis.

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.

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 ≥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. Am. J. Hematol. 66:105–115, 2001.

Cell adhesive activity of two animal lectins through different recognition mechanisms.

Cell adhesive activity of two animal lectins. frog (Rama catesbeiana) S‐type 14K lectin and echinoidin (a C‐type lectin from sea urchin plasma), was studied with human rhabdomyosarcoma (RD) cells. RD cells attached to and spread on plastic plates coatce with each lectin. Cell adhesion by the frog lectin was completely inhibited by the addition of lactose or asialofetuin glycopeptide. Echinoidin‐induced cell adhesion was only inhibited by peptide GRGDS. Since echinoidin is known to contain an RGD‐sequence, our results clearly indicate that this sequence is active as the cell adhesive signal. These results suggest that some of the animal lectins may function as a cell adhesive molecule rather than using the carbohydrate‐recognition mechanism.