Neurodegenerative diseases such as Alzheimer's, Parkinson's and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrP(res), a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrP(sen)) into PrP(res) (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrP(res) (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrP(res)-containing aggregates, PrP(res) was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17-27-nm (300-600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of < or =5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14-28 PrP molecules, are the most efficient initiators of TSE disease.

Platelet-and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from ϳ40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, plateletderived FVa contained only a fraction (ϳ10 -15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser 692 catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr 402 with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser 692 remained unmodified. N-terminal sequencing and MALDI-TOF analyses of plateletderived FV/Va peptides identified the presence of a fulllength heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr 1543 rather than Arg 1545 accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.Factor Va (FVa), 1 a heterodimeric protein composed of heavy chain (105 kDa) and light chain (74 kDa) subunits, is formed by limited proteolysis of factor V (FV) (1). In normal hemostasis, FVa functions as a non-enzymatic cofactor of the prothrombinase complex, which consists of a 1:1 stoichiometric and Ca 2ϩ -dependent complex of the serine protease factor Xa and FVa, bound to the membrane of appropriately activated platelets, and catalyzes the proteolytic conversion of prothrombin to thrombin (2). When incorporated into the prothrombinase complex, the catalytic activity of factor Xa is increased by approximately 5 orders of magnitude, and FVa contributes substantially to this increase (3). Removal of FVa from the prothrombinase complex results in a 10,000-fold decrease in the rate of thrombin generation (3), the physiologic effect of which is demonstrated in the bleeding diatheses expressed by FV-deficient individuals (4 -8)Factor V circulates in two pools in whole blood. The majority (75-80%) is found in the plasma as an inactive, singl...

Inhibition of the accumulation of protease-resistant prion protein (PrP-res) is a prime strategy in the development of potential transmissible spongiform encephalopathy (TSE) therapeutics. Here we show that curcumin (diferoylmethane), a major component of the spice turmeric, potently inhibits PrP-res accumulation in scrapie agent-infected neuroblastoma cells (50% inhibitory concentration, ϳ10 nM) and partially inhibits the cell-free conversion of PrP to PrP-res. In vivo studies showed that dietary administration of curcumin had no significant effect on the onset of scrapie in hamsters. Nonetheless, other studies have shown that curcumin is nontoxic and can penetrate the brain, properties that give curcumin advantages over inhibitors previously identified as potential prophylactic and/or therapeutic anti-TSE compounds.Transmissible spongiform encephalopathies (TSE) or prion diseases are untreatable, fatal neurodegenerative diseases that include bovine spongiform encephalopathy, chronic wasting disease, scrapie, and Creutzfeldt-Jakob disease. A central event in TSE diseases is the conversion of the normal, protease-sensitive isoform of prion protein (PrP-sen or PrP C ) to an abnormal, protease-resistant form, PrP-res or PrP Sc . Numerous compounds have been identified as inhibitors of PrPres formation in scrapie agent-infected murine neuroblastoma (ScNB) cells (1-3, 5). The most potent of these inhibitors can also protect rodents against scrapie if they are administered near the time of infection (7,8,10,11,14). Unfortunately, none of these compounds are known to be both safe and effective for use in humans and animals (8, 10, 11). One therapeutic weakness of most of these compounds is likely an inability to penetrate the brain where most of the PrP-res accumulates and TSE pathogenesis occurs.Curcumin, the major component of the spice turmeric and the yellow pigment in curry powder, has several properties that make it of interest as a possible anti-TSE drug. First, its structure resembles Congo red, the most potent of the small-molecule PrP-res inhibitors that have been assayed in ScNB cells (Fig. 1) in that both are potentially planar compounds that have two aromatic rings or ring systems with conjugated linkers. Structure-activity studies have provided evidence that the potential for coplanarity of the rings and linker is important for the inhibitory potency of Congo red (6). Second, unlike Congo red, curcumin is uncharged and is thought to have at least limited bioavailability to the brain after consumption. Indeed, recent studies with a rat model of Alzheimer's disease reported that dietary curcumin reduces ␤-peptide deposition in the brain as well as associated neuropathology and cognitive deficits (9, 12). Third, curcumin has antioxidant activity, a factor that may be important given that oxidative damage is a feature in TSE neuropathogenesis (13). Fourth, humans consume curcumin in large amounts with no apparent toxicity. Toxicology studies have indicated that rodents can tolerate for a long period up to 5%...

To cite this article: Gould WR, Simioni P, Silveira JR, Tormene D, Kalafatis M, Tracy PB. Megakaryocytes endocytose and subsequently modify human factor V in vivo to form the entire pool of a unique platelet-derived cofactor. J Thromb Haemost 2005; 3: 450-6.See also Hoffman M. One more way that mice and men are different. This issue, pp 448-9.Summary. Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential cofactor in thrombin production catalyzed by the prothrombinase complex. Plasma-derived factor V (FV) is synthesized in the liver. The source(s) of the platelet-derived cofactor remains in question. We identified a patient homozygous for the FV Leiden mutation, who received a liver transplant from a homozygous wildtype FV donor. Eighteen days post-transplant, phenotypic analysis of the patient's platelet-derived FV indicated that the platelets were acquiring wild-type FV, consistent with the temporal differentiation of megakaryocytes and subsequent platelet production. Nine months post-transplant, the plateletderived FV pool consisted entirely of wild-type FV. Consequently, megakaryocyte endocytosis of plasma-derived FV must account for the entire platelet-derived pool, because blood-borne platelets cannot bind or endocytose FV. Subsequent to this endocytic process, the patient's platelet-derived FV was cleaved to a partially active cofactor, and rendered resistant to phosphorylation catalyzed by a platelet-associated kinase, and hence less susceptible to activated protein C-catalyzed inactivation. These data provide the first in vivo demonstration of an endocytosed plasma protein undergoing intracellular modifications that alter its function. This process results in the sequestration of active FVa within the platelet compartment, poised for immediate action subsequent to release from platelets at a site of injury.

Effective hemostasis relies on the timely formation of-thrombin via prothrombi-nase, a Ca 2-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrom-bin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when pro-thrombin is processed on the activated platelet surface, the cleavage of prothrom-bin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin-or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating pre-thrombin-2, with-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma-or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC 50 0.3M), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagu-lant activity expressed by activated plate-lets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin. (Blood. 2011;117(5): 1710-1718) Introduction The activation of prothrombin to-thrombin is a critical step in the response to vascular injury. The generation of-thrombin is achieved through the action of prothrombinase, which is composed of the serine protease factor Xa and its nonenzymatic cofactor, factor Va, assembled on an appropriate membrane surface in the presence of Ca 2 ions. 1 In the physiologic setting, this surface is provided by the activated platelet. 2 Relative to the activity of factor Xa alone, incorporation of factor Xa into prothrombinase accelerates the rate of prothrombin cleavage by 5 orders of magnitude, 3 and both factor Va and the membrane surface are critical in this rate amplification, as removal of either component results in a substantial decrease in the rate of prothrombin cleavage. 3 Indeed, deficiencies or disorders of any component of this complex result in severe bleeding diatheses. 4-6 Prothrombin, the substrate for prothrombinase, consists of 4 domains: fragment 1, fragment 2, and the A and B chains of-thrombin (Figure 1). 1 Prothrombin is proteolytically activated to-thrombin by cleavage on the C-terminal side of 2 specific residues: Arg271 (located between fragment 2 and the A chain) and Arg320 (located between the A and B chains). Initial cleavage at Arg271 results in the generation of prethrombin-2, an inactive intermediate, and the release of fragment 1.2 (F1.2). Subsequent cleavage at Arg320 converts prethrombin-2 to-thrombin. 7 Alternatively , cleavage may occur first at Arg320, leading to the generation of the enzymatically active interme...

Decades after the prion protein was implicated in transmissible spongiform encephalopathies, the structure of its toxic isoform and its mechanism of toxicity remain unknown. By gathering available experimental data, albeit low resolution, a few pieces of the prion puzzle can be put in place. Currently, there are two fundamentally different models of a prion protofibril. One has its building blocks derived from a molecular dynamics simulation of the prion protein under amyloidogenic conditions, termed the spiral model. The other model was constructed by threading a portion of the prion sequence through a beta-helical structure from the Protein Data Bank. Here we compare and contrast these models with respect to all of the available experimental information, including electron micrographs, symmetries, secondary structure, oligomerization interfaces, enzymatic digestion, epitope exposure, and disaggregation profiles. Much of this information was not available when the two models were introduced. Overall, we find that the spiral model is consistent with all of the experimental results. In contrast, it is difficult to reconcile several of the experimental observables with the beta-helix model. While the experimental constraints are of low resolution, in bringing together the previously disconnected experiments, we have developed a clearer picture of prion aggregates. Both the improved characterization of prion aggregates and the existing atomic models can be used to devise further experiments to better elucidate the misfolding pathway and the structure of prion protofibrils.

No validated treatments exist for transmissible spongiform encephalopathies (TSEs or prion diseases) in humans or livestock. The search for TSE therapeutics is complicated by persistent uncertainties about the nature of mammalian prions and their pathogenic mechanisms. In pursuit of anti-TSE drugs, we and others have focused primarily on blocking conversion of normal prion protein, PrP(C), to the TSE-associated isoform, PrP(Sc). Recently developed high-throughput screens have hastened the identification of new inhibitors with strong in vivo anti-TSE activities such as porphyrins, phthalocyanines, and phosphorthioated oligonucleotides. New routes of administration have enhanced beneficial effects against established brain infections. Several different classes of TSE inhibitors share structural similarities, compete for the same site(s) on PrP(C), and induce the clustering and internalization of PrP(C) from the cell surface. These activities may represent a common mechanism of action for these anti-TSE compounds.

The M(r) = 94,000 heavy chain of bovine factor Va contains 10 cysteine residues which are distributed in the 2 A domains which make up this portion of the factor V molecule. The A1 domain contains four cysteines while the A2 domain contains six cysteines. The locations of disulfide bridges and free cysteines in bovine factor Va heavy chain were analyzed using iodo[14C]acetamide-labeled factor Va heavy chain digested with trypsin, plasmin, V-8 protease, and cyanogen bromide. Following HPLC separation of the resulting peptides, free cysteines were identified by the incorporation of radioactivity while disulfide-containing peptides were detected using an SBD-F fluorometric assay after reduction. All cysteine-containing peptides were analyzed by amino acid sequence analysis. The four cysteines in the A1 domain are associated with two disulfide bonds, Cys139-Cys165 and Cys220-Cys301. One disulfide bond was explicitly identified in the A2 domain; Cys471-Cys497, and a free cysteine was found in the A2 domain at Cys538. Significant difficulties were encountered in preparing identifiable or soluble peptides which would permit the explicit identification of the three remaining cysteines in the A2 domain. On the basis of homology, it is likely that Cys589 is a free SH while a disulfide bridge exists between Cys579 and Cys660. Thus, three major disulfide bonding patterns, characterized as "alpha", "beta", and "gamma" loops, are found in factor V. Each A domain contains a 26 residue "alpha loop at positions 139-165, 471-497, and 1684-1710. The A1 and A2 domains each contain 81 amino acid residue "beta" loops at 220-301 and 579-660.(ABSTRACT TRUNCATED AT 250 WORDS)