cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^

Cholesterol enhances phospholipid-dependent activated protein C anticoagulant activity

The influence of cholesterol on activated protein C (APC) anticoagulant activity in plasma and on factor Va inactivation was investigated. Anticoagulant and procoagulant activities of phosphatidylcholine/phosphatidylserine (PC/PS) vesicles containing cholesterol were assessed in the presence and absence of APC using factor Xa-1-stage clotting and factor Va inactivation assays. Cholesterol at approximate physiological membrane levels (30%) in PC/PS (60%/10% w/w) vesicles prolonged the factor Xa-1-stage clotting time dose-dependently in the presence of APC but not in the absence of APC. APC-mediated cleavage of purified recombinant factor Va variants that were modified at specific APC cleavage sites (Q306/Q679-factor Va; Q506/Q679-factor Va) was studied to define the effects of cholesterol on APC cleavage at R506 and R306. When compared to control PC/PS vesicles, cholesterol in PC/PS vesicles enhanced factor Va inactivation and the rate of APC cleavage at both R506 and R306. Cholesterol also enhanced APC cleavage rates at R306 in the presence of the APC cofactor, protein S. In summary, APC anticoagulant activity in plasma and factor Va inactivation as a result of cleavages at R506 and R306 by APC is markedly enhanced by cholesterol in phospholipid vesicles. These results suggest that cholesterol in a membrane surface may selectively enhance APC activities. © 2005 International Society on Thrombosis and Haemostasis.

Etude d'un anticoagulant oral (le rivaroxaban) sur les paramètres hémostatiques de chiens en santé

Chez le chien, les thromboses représentent une complication majeure de nombreuses conditions qui sont revues dans ce manuscrit. L’arsenal thérapeutique actuel présente certaines limites: des effets anticoagulants variables d’un patient à l’autre, des hémorragies et une administration par voie sous-cutanée pour l’héparine. Le rivaroxaban est un nouvel anticoagulant oral approuvé pour la prévention et le traitement des thromboses chez l’humain. C’est un inhibiteur direct du facteur Xa. La présente étude a pour objectif d’évaluer les effets hémostatiques du rivaroxaban chez des chiens en santé, en utilisant les tests de coagulation suivants: temps de prothrombine (PT), temps partiel de thromboplastine (aPTT), activité anti-facteur X, génération de thrombine (GT) et thromboélastographie (TEG®). Tout d’abord, l’effet anticoagulant du rivaroxaban a été évalué in vitro : le plasma citraté pauvre en plaquettes provenant de 20 Beagle en santé a été aliquoté et enrichi avec des solutions de rivaroxaban à des concentrations de 0 à 1000 mg/L d’anticoagulant. Une prolongation concentration-dépendante de tous les tests de coagulation a été notée. Les concentrations de 0.024 et 0.053 mg/L diminuent respectivement de 50% la vitesse de propagation de la GT et la densité optique de l’activité anti-facteur X. Ces derniers tests sont les plus sensibles et précis pour détecter l’effet anticoagulant du rivaroxaban. Ensuite, 24 Beagle en santé ont été répartis aléatoirement en 3 groupes (n=8). Chaque groupe a reçu par voie orale un placebo, ou 20 mg de rivaroxaban une ou deux fois à 8h d’intervalle. Quinze échantillons sanguins ont été prélevés pour chaque chien sur 30 heures. Pour tous les tests de coagulation excepté la TEG®, une différence significative a été notée dans les résultats entre les groupes traités et le groupe placebo (p<0.0001). La durée de l’effet anticoagulant du rivaroxaban était de 7.9-18.7h dans le groupe traité une fois; et de 17.5-26.8h dans le groupe traité deux fois. Le pic d’action de l’effet anticoagulant était d’environ 2h. Seul le paramètre R de la TEG® était significativement affecté dans les groupes traités. En conclusion, le rivaroxaban exerce un effet anticoagulant chez le chien à la dose de 2 mg/kg. Une administration biquotidienne semble appropriée pour un effet de 24h.

Use of the pentasaccharide fondaparinux as an anticoagulant during haemodialysis

No data about the use of the pentasaccharide fondaparinux, a highly selective indirect inhibitor of factor Xa, in patients treated with haemodialysis are available. Therefore, we investigated the pharmacokinetics and -dynamics of fondaparinux in 12 patients during haemodialysis. The anti-Xa activity (expressed as fondaparinux equivalent) was monitored, a semiquantitative clotting scale (SQCS) ranging from 0 (no visible traces of coagula) to 3 (complete clotting of the dialysis circuit) was applied, and the digital compression time necessary to achieve haemostasis at the puncture site was determined. After an initial period, when the regular heparin dose was replaced once weekly by fondaparinux, 0.05 mg/kg, the pentasaccharide was administered for nine consecutive haemodialysis sessions. Peak anti-Xa activity increased from 0.61 +/- 0.14 microg/l after the first dose to 0.89 +/- 0.24 microg/l after dose 9 (P < 0.001), whereas predialysis anti-Xa activity steadily rose to 0.32 +/- 0.09 microg/l (P < 0.001). A sufficient but slightly less effective anticoagulation with a mean SQCS of 1.19 +/- 0.71 (n = 121) was obtained by fondaparinux as compared with 0.65 +/- 0.58 (n = 60, P < 0.005) by 4,825 +/- 1,703 U of unfractionated heparin. Mean digital compression time rose slightly during fondaparinux from 23.7 +/- 7.4 minutes to 24.8 +/- 7.5 minutes (P < 0.05) and, more important, six of the 12 patients reported minor bleeding problems during the interdialytic interval. Thus, fondaparinux can be used to prevent circuit clotting during haemodialysis; however, accumulation results in an interdialytic increase of anti-Xa activity. Therefore, fondaparinux should be reserved for patients requiring systemic anticoagulation on the days off dialysis.

Anticoagulant repertoire of the hookworm Ancylostoma caninum.

Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

Mesure de la génération de thrombine et son application pour la surveillance pharmacothérapeutique de l'héparine de faible poids moléculaire chez le chien

Chez le chien, la daltéparine est un anticoagulant utilisé pour la prévention et le traitement de la thrombose. La surveillance thérapeutique de la daltéparine par l’activité anti-facteur Xa (FXa) n’est pas un test fonctionnel. Cette étude avait pour but d’étudier l’emploi de la génération de thrombine (GT) pour évaluer les effets in vitro de la daltéparine sur du plasma canin, ainsi que pour détecter les effets pharmacodynamiques de la daltéparine administrée chez des chiens sains. Premièrement, les paramètres normaux de la GT ont été établis à partir du plasma de 25 beagles et 11 chiens sains de clients. Ensuite, des pools de plasma canin fortifié avec de la daltéparine, à dose croissante, ont été analysés selon la GT, l’activité anti-FXa et selon le temps de thromboplastine partielle activée (aPTT). Finalement, 24 beagles sains répartis au hasard dans 4 groupes on reçu soit une dose sous-cutanée (SC) de 50U/kg, 100U/kg ou 150U/kg de daltéparine ou un placebo. Du plasma pauvre en plaquettes (PPP) a été récolté pendant 24 heures et analysé selon la GT, l’anti-FXa et l’aPPT. In vitro, la daltéparine a démontré un effet anticoagulant sur la GT qui était concentration-dépendant. Les tests de GT et anti-FXa étaient plus sensibles aux effets de la daltéparine que l’aPPT. L’étude pharmacodynamique a démontré que le temps, la dose ainsi qu’une interaction temps*dose avaient un effet significatif sur les paramètres de GT et anti-FXa. La GT peut mesurer les effets pharmacodynamiques de la daltéparine à des doses variées chez des chiens sains.

TICK INNATE IMMUNITY

Ticks are blood feeding parasites transmitting a wide variety of pathogens to their vertebrate hosts. The vector competence of ticks is tightly linked with their immune system. Despite its importance, our knowledge of tick innate immunity is still inadequate and the limited number of sufficiently characterized immune molecules and cellular reactions are dispersed across numerous tick species. The phagocytosis of microbes by tick hemocytes seems to be coupled with a primitive complement-like system, which possibly involves self/nonself recognition by fibrinogen-related lectins and the action of thioester-containing proteins. Ticks do not seem to possess a pro-phenoloxidase system leading to melanization and also coagulation of tick hemolymph has not been experimentally proven. They are capable of defending themselves against microbial infection with a variety of antimicrobial peptides comprising lysozymes, defensins and molecules not found in other invertebrates. Virtually nothing is known about the signaling cascades involved in the regulation of tick antimicrobial immune responses. Midgut immunity is apparently the decisive factor of tick vector competence. The gut content is a hostile environment for ingested microbes, which is mainly due to the antimicrobial activity of hemoglobin fragments generated by the digestion of the host blood as well as other antimicrobial peptides. Reactive oxygen species possibly also play an important role in the tick-pathogen interaction. The recent release of the Ixodes scapularis genome and the feasibility of RNA interference in ticks promise imminent and substantial progress in tick innate immunity research.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-Å resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin β-turn of the second inner loop pivots at Val64 and Asp70 by 60°. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 β-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle. © 1993 American Chemical Society.

Polymorphisms in antithrombin and in tissue factor pathway inhibitor genes are associated with recurrent pregnancy loss

Recurrent pregnancy loss (RPL) is a multifactorial condition. The effect of antithrombin (SERPINC1), protein C (PROC), thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI) single nucleotide polymorphisms (SNPs) on the risk of RPL is thus far unknown. Our objective was to determine the association of SNPs in the above mentioned genes with RPL. We included 117 non-pregnant women with three or more consecutive losses prior to 20 weeks of pregnancy without a previous history of carrying a fetus to viability, and 264 healthy fertile non-pregnant women who had at least two term deliveries and no known pregnancy losses. The PROC (rs1799809 and rs1799808), SERPINC1 (rs2227589), THBD (rs1042579) and TFPI (rs10931292, rs8176592 and rs10153820) SNPs were analysed by Real Time PCR. Genotype frequencies for PROC 2418A > G, PROC 2405C > T, THBD 1418C > T, TFPI (T-33C and TFPI C-399T) SNPs were similar in cases and controls. The carriers of SERPINC1 786A allele (GA + AA genotypes) had an increased risk for RPL (odds ratio [OR]: 1.77, 95% confidence interval [CI]: 1.05-3.00, p=0.034) while women carrying the TFPI-287C allele (TC + CC genotypes) had a protection effect on having RPL (OR: 0.46, 95% CI: 0.26 - 0.83, p=0.009). The TCC haplotype for TFPI T-33C/TFPI T-287C/TFPI C-399T SNPs was less frequent in cases (5.7%) than in controls (11.6%) (OR: 0.45, 95% CI: 0.23 - 0.90, p=0.025). In conclusion, our data indicate that SERPINC1 786G > A variant increases the risk for RPL, while TFPI T-287C variant is protective; however, further studies are required to confirm our findings.

Evaluierung der Prothrombinase-induzierten Gerinnungszeit zum Monitoring der Antikoagulation mit niedermolekularen Heparinen und Heparinoiden

Da der gemeinsame Endpunkt vieler Erkrankungen ein thrombotisches Ereignis ist, spielt eine effiziente Antikoagulation eine zentrale Rolle in der heutigen Medizin. Jedoch sind die gängigen Tests zum Monitoring der Gerinnungshemmung nicht ideal. Neue Tests, wie z.B. die Prothrombinase-induzierte Gerinnungszeit (PiCT), sollen die bestehenden Nachteile ausräumen und somit eine bessere Therapieüberwachung gewährleisten.rnDie vorliegende Arbeit untersucht dieses neue Verfahren beim Monitoring von Heparinen und Heparinoiden im Vergleich zu zwei gängigen anti-Faktor Xa-Aktivität(aXa)-Tests bei Patienten mit unterschiedlichem Thromboserisiko (z.B. Schwangere, Adipöse, Niereninsuffiziente).rnDazu wurde bei 175 Proben von 102 Patienten (53 männlich, 49 weiblich, medianes Alter 64 Jahre) die gerinnungshemmende Aktivität des verabreichten Medikaments (UFH, Enoxaparin, Dalteparin oder Danaparoid) mit zwei chromogenen Assays und dem PiCT-Test gemessen.rnHierbei ließen sich insgesamt Werte von 0 bis 1,36 IE/ml aXa feststellen. Im Vergleich lieferten die aXa-Tests höhere Messwerte als PiCT bei allen Medikamenten außer UFH. Während die Ergebnisse der chromogenen Verfahren insgesamt sehr stark korrelierten (r = 0,86), war die Übereinstimmung mit den PiCT-Ergebnissen schwach bis deutlich (r = 0,40 bzw. r = 0,66). Die Faktoren Schwangerschaft, Adipositas und Niereninsuffizienz zeigten auf die Gesamtergebnisse keinen nennenswerten Einfluss. rnZusammenfassend kann gesagt werden, dass PiCT dennoch ein brauchbarer Test zum Monitoring der Antikoagulation sein könnte. Um den Nutzen des Tests v.a. in Bezug auf seine Aussagekraft bei Komplikationen zu beweisen, sind aber weitere klinische Studien notwendig.rn

Surface expression and functional characterization of alpha-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin

Factor V (FV) present in platelet alpha-granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, alpha-granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% +/- 4.7% of the total population and is referred to as convulxin and thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187-activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of alpha-granule FV and the hemostatic effectiveness of young platelets. (Blood. 2000;95:1694-1702)