Kohji Kasahara

Venom from southern copperhead snake (Agkistrodon contortrix contortrix). II. A unique phospholipase A2 that induces platelet aggregation.

A platelet aggregation factor was purified from the venom of southern copperhead snake (Agkistrodon contortrix contortrix) by DEAE-cellulose ion-exchange chromatography, precipitation with ammonium sulfate, affinity chromatography using bovine serum albumin as ligand, and gel filtration on Cellulofine GCL-2000. It had molecular weights of 11,000 and 14,000, as determined by gel filtration chromatography and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), respectively. It consists of a single polypeptide, and was identified as a phospholipase A2. It was quite resistant to heat and various denaturing reagents including urea and SDS. It lost both phospholipase A2 activity and platelet aggregating activity upon modification of histidine residue(s) with p-bromophenacyl bromide. Its specificity towards the beta-position of phospholipid in esterolytic reaction was confirmed by gas-liquid chromatography using a pure synthetic phosphatidylcholine. Platelet aggregation by this phospholipase A2 was completely inhibited by prostacyclin, but was little inhibited by aspirin which indicates almost no direct participation of released arachidonic acid in the aggregation mechanism.

Plasma albumin is essential for collagen-induced platelet aggregation

We found that platelets must have albumin on the surface to respond to collagen and aggregate. Albumin, however, was not absolutely necessary for ADP-, platelet activating factor-, serotonin- or thrombin-induced aggregation, while fibrinogen was required for ADP- or serotonin-induced aggregation. Immunofluorescent microscopy revealed that albumin was retained on gel-filtrated platelets but not on washed platelets. Albumin was not required for platelet adhesion to immobilized collagen. Without albumin thromboxane formation upon collagen-stimulation was diminished. These data suggest that albumin is essential in some step(s) that results in production of thromboxane A2.

”A“ subunit of factor XIII is present on bovine platelet membrane and mediates collagen-induced platelet activation

The Fab fragment of a polyclonal antibody against platelet factor XIII inhibited the collagen-induced platelet aggregation in a dose-dependent manner. This inhibitory effect was specific for collagen, and it had no effect on arachidonic acid-, ADP-, and serotonin-induced aggregations. This finding strengthens our notion that platelet factor XIII is involved in collagen-induced platelet aggregation. (Saito, Y., Imada, T., Takagi, J., Kikuchi, T. and Inada, Y. J. Biol. Chem. 261, 1355-1358, 1986). We have investigated membrane localization of bovine platelet factor XIII using immunological techniques. Immunofluorescent visualization revealed that the factor XIII was expressed on the surface of non-permeabilized bovine platelets, where we detected neither lactate dehydrogenase, a cytoplasmic enzyme marker, nor B subunit of factor XIII, which is present in plasma. Cell surface iodination and immunoprecipitation also confirmed that it existed on the surface of platelets.

Subunit b of factor XIII is present in bovine platelets

Distribution of B subunit of coagulation factor XIII(FXIII B) in bovine platelets was determined. Intracellular location of FXIII B in platelets was confirmed by fluorescent antibody technique. Positive staining of FXIII B was observed with Triton-permeabilized platelets but not with non-permeabilized platelets. Immunoblots of plasma and platelet lysate revealed that the antibody was specific for FXIII B and this antigen existed in the supernatant of sonication-solubilized platelets. The amount of FXIII B in platelets was determined to be 17.3 +/- 6.7 ng/10(8) platelets by radioimmunoassay. This is the first visual and quantitative demonstration of intracellular FXIII B.

Analysis of distribution of receptors among platelets by flow cytometry

We have investigated the binding of fluorescein isothiocyanate (FITC)-labeled fibrinogen to platelets using a fluorescence spectrophotometer and a flow cytometer. The amount of fibrinogen bound by stimulation of adenosine diphosphate (ADP) and 5-hydroxytryptamine (5HT) increased in a dose-dependent manner. Stimulation of one agonist was independent of that of another agonist. Fibrinogen could bind to platelets in response to ADP, regardless of whether they were desensitized by 5HT, and vice versa. It was found by flow cytometric analysis that individual platelets responded uniformly to both ADP and 5HT, indicating that individual platelets have receptors for both ADP and 5HT on their surfaces.

Novel Bernard-Soulier syndrome variants caused by compound heterozygous mutations (case I) or a cytoplasmic tail truncation (case II) of GPIbα

A defective platelet glycoprotein (GP) Ib/IX/V complex [von Willebrand factor (VWF) receptor] results in Bernard-Soulier syndrome (BSS), which is characterized by macrothrombocytopenia and impaired ristocetin- and thrombin-induced platelet aggregation. We found 2 independent BSS-variant families: Case I [compound heterozygous mutations, p.Glu331X and a frame shift by a deletion at c.1444delA of GPIbα (GP1BA) terminating at a premature stop codon (p.Thr452ProfsX58)], and case II [homozygous nonsense mutation at c.1723C>T, p.Gln545X]. Case I platelets expressed no GPIbα, resulting in absence of ristocetin-induced platelet aggregation (RIPA) and 50% reduction in thrombin-induced aggregation with no shape change. The mothers platelets had 50% the expression level of A-type GPIbα (4-repeated VNTR: variable number of tandem repeats, p.[Thr145Met; Ser399_Pro411[4]]); the fathers platelets had the same expression level of C-type GPIbα (2-repeated VNTR, p.Ser399_Pro411dup) as the mothers platelets. The mothers RIPA was significantly higher than the fathers. Thrombin-induced aggregation was normal in both parents. Case II platelets expressed a GPIbα with an abnormal cytoplasmic tail, p.Gln545X-truncated GPIbα, which complexed with GPIX and GPV on the cell surface; its expression level of the complex was normal. Case II platelets had reversible RIPA, with no ATP release, and weak thrombin-induced aggregation without shape change. These results suggest that a signaling process through the GPIbα cytoplasmic tail required for full platelet activation is defective in BSS variant case II and a length polymorphism of GPIbα is associated with a modified level of RIPA heterozygous BSS case I.

Interaction between plasma factor XIII and collagen

Purified plasma Factor XIII efficiently bound to the immobilized collagen affinity column. In contrast, when plasma was applied to the column, as much as 80% of Factor XIII was not trapped. It was shown that the difficulty of binding of Factor XIII in plasma to collagen is not due to the covering of collagen with some plasma proteins. We then assumed that Factor XIII formed a complex in plasma with a certain protein(s), which would prevent Factor XIII from binding to collagen. Using an immobilized Factor XIII affinity column, we have indeed obtained one protein from plasma with apparent Mr of 260,000.

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

Blood Coagulation Factor XIII: A Multifunctional Transglutaminase

Factor XIII is a pro-enzyme of plasma transglutaminase consisting of two enzymatic A subunits and two non-catalytic B subunits, and platelet transglutaminase consisting of two enzymatic A subunits. FXIII plays a critical role in the generation of a stable hemostatic plug, wound healing, maintaining pregnancy, angiogenesis, apoptosis and bacterial immobilization. FXIII catalyzes intermolecular cross-linking reactions between fibrin monomers and α2-antiplasmin. These reactions increase the mechanical strength of the fibrin clot and its resistance to proteolytic degradation. Congenital FXIII deficiency is a rare autosomal recessive disorder, most cases of which are caused by defects in the FXIII-A gene, leading to a bleeding tendency. An autoimmune hemophilia-like disease is caused by anti-FXIII antibodies. Platelet surface FXIII-A2 is involved in fibrin translocation to lipid rafts and outside-in signaling, leading to clot retraction. FXIII-A2-mediated protein cross-linking is associated with assembly of the extracellular matrix on a variety of cell surfaces in physiological events such as differentiation.