Stimulation of human platelets with concanavalin A involves phospholipase C activation.

In response to concanavalin A, cytoplasmic calcium movement was observed in human platelets, both in the presence of 1 mM Ca2+ or 1 mM EGTA in the medium. Concanavalin A also caused the activation of inositide turnover and the production of inositol phosphates, suggesting that activation of phospholipase C occurs. The mechanism by which concanavalin A stimulates phospholipase C does not depend on GTP-binding transducers, because it was not inhibited by GDP beta S, while experiments performed in the presence of cytochalasin B suggested a role for membrane glycoprotein IIb-IIIa-cytoskeleton interaction in this process. Ca(2+)-proteases and Na+/H+ antiport also seemed to be related to concanavalin A-induced phospholipase C activation, as suggested by experiments performed in the presence of leupeptin and amiloride.     

Experimental acute Babesia caballi infections: II. Response of platelets and fibrinogen

Ponies were acutely infected with Babesia caballi by inoculation with infected red blood cells (RBCs) containing 1 × 10⁸ and 1 × 10⁹ piroplasms. A series of blood samples taken before and after inoculation were analyzed for platelets and fibrinogen, and the results compared with similar analyses made on challenged, premunized ponies and on equids inoculated with uninfected RBCs. In acutely infected animals there were immediate decreases in platelet counts that persisted at least through Day 18 after inoculation (AI). Concomitantly, plasma fibrinogen levels rose, reaching peak values between Days 6–17. Clot retractions in vitro were impaired in these ponies during Days 9–16. No large diminutions in platelet counts or elevations of fibrinogen levels were observed in the challenged, premunized ponies or the group transfused with uninfected RBCs. In fact, the effect of challenge was to maintain or increase platelet counts. Our results plainly indicate that B. caballi can elicit alterations in clotting factor levels in its hosts during acute infections.     

Plasma kallikrein is activated on dermatan sulfate and cleaves factor H

When human plasma is applied to a dermatan sulfate column, amidase activity is detected in the bound fraction and complement factor H is cleaved [A. Saito, H. Munakata, Factor H is a dermatan sulfate-binding protein: identification of a dermatan sulfate—mediated protease that cleaves factor H, J. Biochem. 137 (2005) 225-233]. Here, the amidase-active fraction was purified by sequential gel filtration and hydroxyapatite chromatography, and the amidase-active protein was identified to be plasma kallikrein by mass spectrometry. The activation of plasma kallikrein was further investigated by Western blotting using plasma deficient in prekallikrein or coagulation factor Xll. The dermatan sulfate column-bound fraction of the prekallikrein- and factor Xll-deficient plasmas did not show any amidase activity and factor H remained intact. Addition of kallikrein, but not activated factor Xll, to factor H purified from plasma resulted in cleavage of factor H. Thus, dermatan sulfate induces contact activation and activates kallikrein-mediated cleavage of FH.     

Missense mutations in the coagulation factor XII (Hageman factor) gene in hereditary angioedema with normal C1 inhibitor

Hereditary angioedema is characterized by recurrent skin swelling, abdominal pain attacks, and potentially life-threatening upper airway obstruction. The two classic types are both caused by mutations within the complement C1 inhibitor gene. A recently described new type does not show a deficiency of C1 inhibitor and affects almost exclusively women. We screened twenty unrelated index patients with this new type of hereditary angioedema for mutations in the coagulation factor XII gene. Two different missense mutations were identified in exactly the same position within exon 9 of the F12 gene. 'Mutation 1' (1032C-->A), encountered in five patients, predicts a threonine-to-lysine substitution (Thr309Lys). 'Mutation 2' (1032C-->G), observed in one patient, results in a threonine-to-arginine substitution (Thr309Arg). The predicted structural and functional impact of the mutations, their absence in 145 healthy controls, and their co-segregation with the phenotype in five families provide strong support that they cause disease.     

Probing single-stranded DNA and its biomolecular interactions through direct catalytic activation of factor XII, a protease of the blood coagulation cascade

Triggered self-activation of factor XII, a blood coagulation protease, was utilized for the amplified visual detection of ss-DNA targets in a non-sequence specific way. Factor XII holds potential as a low-affinity and therefore non-interfering probe for DNA secondary structure and for the screening of protein binding to ss-DNA. The observation that ss-DNA also accelerates coagulation of human blood plasma is relevant to the emerging field of aptamer therapeutics.     

Interactions between heparin and factor Xa inhibition of prothrombin activation

The effects of heparin on prothrombin activation have been examined. Heparin was found to inhibit the rate of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, heparin had no effect on the rate of prothrombin activation. In contrast, heparin was found to increase the rate of activation of prethrombin-1 and prethrombin-2. Initial velocity studies indicated that heparin blocks lipid stimulation of prothrombin activation. In accord with this, binding studies demonstrated that heparin could displace Factor Xa, and in separate experiments, prothrombin, from phospholipid vesicles.     

Mode of action of 2-(diethylamino)-7-ethoxychromone on human platelets.

The in vitro effect of 2-(diethylamino)-7-ethoxychromone (RC39XVIII) on human platelet aggregation induced by several agonists and on thromboxane B2 formation, granule release and intracellular cAMP elevation has been studied. The chromosome-derivative exerts a dose-dependent inhibitory effect on aggregation produced by U46619, arachidonic acid, thrombin, collagen and ADP. RC39XVIII inhibits aggregation, TxB2 formation and granule release in parallel. Moreover the drug potentiates cAMP accumulation induced by iloprost and forskolin. The drug also inhibits soluble cAMP phosphodiesterase in a dose-dependent manner. No effect on adenylate cyclase activity measured in platelet membranes was evident.     

Loading solution prevents activation damage of human platelets before lyophilization

The current study aims to optimize the compositions of platelet activation-inhibitors for a loading solution of lyophilizing protectants and to establish a series of perfect pretreatment methods for platelet lyophilization. The optimal combination of six kinds of inhibitors and loading solutions of lyophilizing protectants, including prostaglandin E1 (PGE1), adenosine, l-arginine, phyticacid, bivalirudin, and cilostazol, was analyzed using the orthogonal experimental design. The values of the expression rates of p-selectin (CD62p) and platelet membrane glyeoprotein (PAC-1), as well as of platelet and mean platelet volume (MPV), were selected as indices of platelet activation. The values of CD62p and Pac-1 induced by thrombin were determined as indices of platelet reactivity. The maximal aggregation and slide platelet aggregation test (SPAT) induced by the inducer were calculated as indices of the aggregation function of platelets. Level I of the loading condition factor had no adverse action on MPV, CD62p, PAC-1, SPAT, and the maximum platelet aggregation rate. Level II of factors PGE1, l-arginine, phycicacid sodium, and Bivalirudin could inhibit the activation of platelets and enable them to retain their function. The results show that the optimal solution compounding was the third group. The loading solution, which includes plasma, 1 μM prostaglandin E1, 5 mM l-arginine, 0.5 mM phyticacid, and 0.5 μM bivalirudin, could prevent the activation damage of platelets before lyophilization.     

On the degradation of heparin by human blood platelets.

Human blood platelets are able to degrade heparin from different tissues and species. The main degradation product is an oligosaccharide. Low molecular weight components such as inorganic sulfate or monosaccharides, i.e. products released by exoenzymes are not detected. The in vitro degradation of heparin by the crude enzyme is observed at pHs below 6.5 with an optimum temperature around 37 degrees C. The presence of sulfate in the substrate structure is shown to be essential for the enzyme activity. Since the oligosaccharides formed have only 10 per cent of the anticoagulant activity of the heparins tested, it is conceivable that the platelet enzyme may play an important role in the inactivation of some of the biological properties of heparin.     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Generation of megakaryocytes and platelets from human subcutaneous adipose tissues

Recent advances in regenerative medicine have created a broad spectrum of stem cell research. Among them, tissue stem cell regulations are important issues to clarify the molecular mechanism of differentiation. Adipose tissues have been shown to contain abundant preadipocytes, which are multipotent to differentiate into cells including adipocytes, chondrocytes, and osteoblasts. In this study, we have first shown that megakaryocytes and platelets can be generated from adipocyte precursor cells. Human adipocyte precursor cells were cultured in conditioned media for 12 days to differentiate adipocytes, followed by 12 days of culture in media containing thrombopoietin. The ultrastructures of adipocyte precursor cell- and bone marrow CD34-positive cell-derived megakaryocytes and platelets were similar. In addition, adipocyte precursor cell-derived platelets exhibited surface expression of P-selectin and bound fibrinogen upon stimulation with platelet agonists, suggesting that these platelets were functional. This is the first demonstration that human subcutaneous adipocyte precursor cells can generate megakaryocyte and functional platelets in an in vitro culture system.     

Arg-129 plays a specific role in the confirmation of antithrombin and in the enhancement of factor Xa inhibition by the pentasaccharode sequence of heparin

Small amounts of a variant antithrombin (AT) bearing an Arg-129 to Gln mutation were purified from plasma by means of affinity chromatography on insolubilized herapin at very low ionic strength. As a control, two variant antithrombins, one bearing on Pro-41 to Leu mutation and the other an Arg-47 to His mutation, were purified in the same way. The biochemical characterization of the variants and the kinetic study of thrombin and activated factor X (F Xa) inhibition in the presence of heparin and heparin derivatives suggest that Arg-129 plays a specific role in AT conformation and F Xa inhibition enhancement. Indeed, the purified variant adopted the locked conformation described ,for AT submitted to mild denaturing conditions (Carrell, R.W., Evans, D.Li and Stein, P.E. (1991) Nature 353, 576–578) and resembling the latent form of plasminogen activator inhibitor (PAI) (Mottonen J., Strand, A., Symersky, J., Sweet, R.M., Danley, D.E., Geohegan, K.F., Gerard, R.D. and Goldsmith, E.J. (1992) Nature 355, 270–273). Moreover, the mutant AT was partially reactivated by heparin for thrombin inhibition, but did not respond to the specific pentasaccharide domain of heparin for F Xa inhibition.     

Inhibition of the glycolytic pathway by methylglyoxal in human platelets

The incubation of human platelets with methylglyoxal and glucose produces a rapid transformation of the ketoaldehyde to D-lactate by the glyoxalase system and a partial reduction in GSH. Glucose utilization is affected at the level of the glycolytic pathway. No effect of the ketoaldehyde on glycogenolysis and glucose oxidation through the hexose monophosphate shunt was demonstrated. Phosphofructokinase, fructose 1,6 diphosphate (F1, 6DP) aldolase, glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate mutase were mostly inhibited by methylglyoxal. A decrease in lactate and pyruvate formation and an accumulation of some glycolytic intermediates (fructose 1,6 diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate) was observed. Moreover methylglyoxal induced a fall in the metabolic ATP concentration. Since methylglyoxal is an intermediate of the glycolytic bypass system from dihydroxyacetone phosphate to D-lactate, it may be assumed that ketoaldehyde exerts a regulating effect on triose metabolism.     

Interaction of terbium with human platelets

The effect of terbium on platelets has been studied by aggregation experiments and by fluorescence measurements. TbCl3 does not substitute for CaCl2 in the aggregation of platelets induced by ADP, but it may even inhibit, probably by a competition mechanism. It was impossible to observe a sensitized emission of Tb3+ in the presence of platelets. Instead the lanthanide, like Ca2+, significantly increases the aggregation of platelets induced by A23187. The fluorescence yield of this compound is greater in the presence of platelets than in buffer alone. Energy transfer appears to take place from the aromatic amino acids of the platelet membrane to the bound ionophore.     

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.     

N-terminal flanking region of A1 domain in von Willebrand factor stabilizes structure of A1A2A3 complex and modulates platelet activation under shear stress

von Willebrand factor (vWF) mediates platelet adhesion and thrombus formation via its interaction with the platelet receptor glycoprotein (GP)Ibα. We have analyzed two A1A2A3 tri-domain proteins to demonstrate that the amino acid sequence, Gln(1238)-Glu(1260), in the N-terminal flanking region of the A1 domain, together with the association between the A domains, modulates vWF-GPIbα binding and platelet activation under shear stress. Using circular dichroism spectroscopy and differential scanning calorimetry, we have described that sequence Gln(1238)-Glu(1260) stabilizes the structural conformation of the A1A2A3 tri-domain complex. The structural stabilization imparted by this particular region inhibits the binding capacity of the tri-domain protein for GPIbα. Deletion of this region causes a conformational change in the A1 domain that increases binding to GPIbα. Only the truncated protein was capable of effectively blocking ristocetin-induced platelet agglutination. To determine the capacity of activating platelets via the interaction with GPIbα, whole blood was incubated with the N-terminal region truncated or intact tri-A domain protein prior to perfusion over a fibrin(ogen)-coated surface. At a high shear rate of 1,500 s(-1), platelets from blood containing the truncated protein rapidly bound, covering >90% of the fibrin(ogen) surface area, whereas the intact tri-A domain protein induced platelets to bind <10%. The results obtained in this study ascertain the relevant role of the structural association between the N-terminal flanking region of the A1 domain (amino acids Gln(1238)-Glu(1260)) and the A1A2A3 domain complex in preventing vWF to bind spontaneously to GPIbα in solution under high shear forces.     

Activated protein C enhances human keratinocyte barrier integrity via sequential activation of epidermal growth factor receptor and Tie2

Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability.     

The interference of plasmic degradation products of human crosslinked fibrin with clot formation

The role of plasmic degradation products of human crosslinked fibrin on polymerization of fibrin monomer and clot formation was studied. Both reactions were inhibited by Fragment DD, which formed a complex with fibrin monomer in a molar ratio 1 : 1. The rate of polymerization was slightly increased by Fragment E but it was not affected by (DD)E complex and Fragment A. Approximately the same amount of fibrin was formed in the presence and absence of Fragments A, E and the complex. It was concluded that of the degradation products of crosslinked fibrin, only Fragment DD is a potent anticoagulant at physiologic pH. The (DD)E complex is inert and Fragments A and E have only marginal effects.     

Characterization of lipid rafts in human platelets using nuclear magnetic resonance: A pilot study

Lipid microdomains (‘lipid rafts’) are plasma membrane subregions, enriched in cholesterol and glycosphingolipids, which participate dynamically in cell signaling and molecular trafficking operations. One strategy for the study of the physicochemical properties of lipid rafts in model membrane systems has been the use of nuclear magnetic resonance (NMR), but until now this spectroscopic method has not been considered a clinically relevant tool. We performed a proof-of-concept study to test the feasibility of using NMR to study lipid rafts in human tissues. Platelets were selected as a cost-effective and minimally invasive model system in which lipid rafts have previously been studied using other approaches. Platelets were isolated from plasma of medication-free adult research participants (n=13) and lysed with homogenization and sonication. Lipid-enriched fractions were obtained using a discontinuous sucrose gradient. Association of lipid fractions with GM1 ganglioside was tested using HRP-conjugated cholera toxin B subunit dot blot assays. ¹H high resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectra obtained with single-pulse Bloch decay experiments yielded spectral linewidths and intensities as a function of temperature. Rates of lipid lateral diffusion that reported on raft size were measured with a two-dimensional stimulated echo longitudinal encode-decode NMR experiment. We found that lipid fractions at 10–35% sucrose density associated with GM1 ganglioside, a marker for lipid rafts. NMR spectra of the membrane phospholipids featured a prominent ‘centerband’ peak associated with the hydrocarbon chain methylene resonance at 1.3 ppm; the linewidth (full width at half-maximum intensity) of this ‘centerband’ peak, together with the ratio of intensities between the centerband and ‘spinning sideband’ peaks, agreed well with values reported previously for lipid rafts in model membranes. Decreasing temperature produced decreases in the 1.3 ppm peak intensity and a discontinuity at ~18 °C, for which the simplest explanation is a phase transition from L_d to L_o phases indicative of raft formation. Rates of lateral diffusion of the acyl chain lipid signal at 1.3 ppm, a quantitative measure of microdomain size, were consistent with lipid molecules organized in rafts. These results show that HRMAS NMR can characterize lipid microdomains in human platelets, a methodological advance that could be extended to other tissues in which membrane biochemistry may have physiological and pathophysiological relevance.     

人凝血酶原复合物生产过程中凝血因子活化分析

目的通过比较以组分Ⅲ沉淀和血浆为原料制备人凝血酶原复合物(Prothrombin complex concentrates,PCC)过程中凝血因子活化情况,为选择最适PCC制备原料提供数据支持。方法分别对以组分Ⅲ沉淀和血浆为原料制备PCC过程中中间品的活化的凝血因子活性和人凝血酶活性两个项目进行检定,分析凝血因子的活化情况。观察以组分Ⅲ沉淀为原料制备PCC过程中添加肝素能否抑制PCC中凝血因子的活化。结果以组分Ⅲ沉淀为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均不合格。以组分Ⅲ沉淀为原料制备PCC生产过程中添加肝素后,PCC中间品的活化的凝血因子活性和人凝血酶活性均不合格。以血浆为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均合格。结论组分Ⅲ沉淀为原料制备PCC会增加凝血因子活化的风险,新鲜冰冻血浆可作为制备PCC的原料。