bluuebunny/crossref_metadata_2025 huggingface dataset:
PEPTIDES FROM FIBRINOGENAND FIBRONECTIN CHANGE THE CONFORMATIONOF PURIFIED PLATELET GLYCOPROTEIN IIb-IIIa
Authors: Parise L V , Steiner B , Nannizzi L , Phillips D A
Published: August 2018

Specific amino acid sequences in fibrinogen and fibronectin appear to mediate the binding of these ligands to the glycoprotein (GP) IIb-IIIacomplex in platelets. Thesesequences include LGGAKQAGDV from the y chain of fibrinogen, and RGD(S) from the a chain of fibrinogenand the cell-binding domain of fibronectin. Several recent reports suggest thatfibrinogen and/or peptides with these sequences cause clustering of GPIIb-IIIa on the platelet surface and Na+/H+ exchange in epinephrine-stimulated platelets. Thus, it is possible that occupancy of specific sites on GP Ilb-IIIa affects its conformation, initiating such events. In this study,we determined whether LGGAKQAGDV, RGDS, and related peptides affect the conformation of purified platelet GP IIb-IIIa. Conformational changes in GP IIb-IIIa were evaluated bychanges in proteolytic susceptibility and hydrodynamic properties. Thepurified GP IIb-IIIa complex was fund to be resistant to proteolysis bythrombin. However, pretreatment of GP IIb-IIIa with various peptidesincreased the susceptibility ofGP libα to thrombin-induced proteolysis,as quantitated onpolyacryfamide gels.The order of potency of these peptides was RGDS<LGGAKQAGDV < KGDS < RGES. This order of potency agrees with that for the abilityof these peptides to inhibit 125I-fibrinogen binding to platelets. The effect of the peptides on proteolysis was time-, temperature-, and concentration-dependent; RGDS Induced a half-maximal effect at ˜60μM. Evaluation of the hydrodynamic properties of GP IIb-IIIa showed that LGGAKQAGDV orRGDS, but not RGES, decreased thesedimentation coefficient of GP IIb-IIIa from 8.5S to 7.7 S or7.4, S,respectively. This changewas accompanied by an increase in theStoke’s radius from 73 A to 84 A. These results suggestthat LGGAKQAGDV andRGDS alterthe conformationof the purified GPIIb-IIIa heterodimer complex by causing it to unfold.This change in conformation may be related to changesin the distribution and function of GP IIb-IIIaon the platelet surface that occurwith occupancy ofligand binding sites.
ENHANCED ASSOCIATION OF FIBRINOGEN WITH ITS PLATELET RECEPTOR DUE TO SODIUM CITRATE INDICATION FOR ONE FIBRINOGEN RECEPTOR ONLY
Authors: Bowry S X
Published: August 2018

Washed platelet suspensions are almost always prepared from blood anticoagulated with sodium citrate. As citrate has been implicated to affect platelet function and as estimates of the number of fibrinogen (Fbg) bindingsites on platelets range from 4,700 -82,500, we examined the involvement of citrate on the platelet-fibrinogen interaction. The binding of 125-1-fibrinogen to washed platelets from citrated blood (PCB) and thosefrom gel-filtered non-anticoagulated blood (PNB) from the same donor showed linear Scatchard plots for PNB and curvilinear plots for PCB. Assuming the presence of two classes of bindingsites, a high_.affinity site (5,540±760 molecules/ platelets; Kd, 1.29 x 10-7 M) and low affinity site (32,070 α 6,520 molecules/platelet; Kd 1.02 x 10-6 M) were determined for PCB. However, PNB indicated a single class of binding sites with 16,480α 2,800 Fbg molecules/platelet.When blood from one donor was collected into 10 mM and 20 mM citrate, increased binding of Fbg was observed onplatelets exposed to 20 mM citrate. The effects of citrate appear to be on theplatelet Fbg receptor since non-specific binding was not affected by the citrate. As no 14-C-citric acid binding to platelets was observed, citrate may affect the receptors without binding to the platelets. The dependence of binding on the pHof thecitrate suggests ionic interactions between platelets and citrate. Different amounts of Fbg were bound when four different preparations of sodium citrate varying in concentration and pH were used. Ourdata suggest that acombination of thedirect effects of citrate on the Fbg bindingof platelets, together with the variable concentrations and pH of the different citrate preparations routinelyused toanticoagulate blood, may explain why some investigators obtain upwardly concave Scatchard plotswhile others obtain linear plots. Ourresultsindicate that the major reasonfor the disparities in the Fbg binding datais due to the effects of citrate on platelets.
LIPOPROTEIN BINDING TOHUMAN PLATELETS IS LOCATED AT GPIIb/IIIa COMPLEX
Authors: Koller E , Koller F
Published: August 2018

Human Platelets possess specific binding sites for low density lipoproteins (LDL) and high density lipoproteins(HDL)(1). Binding of both classes of plasma lipoproteins, though competitive, has been shown by several groups to facilitate platelet activation.Isolated washed platelets occasionally aggregate upon addition of high concentrations of LDL even in the absence of known platelet activators. The proteins responsible for this binding have been visualized by ligand blotting (2). Both types of ligand specifically bind to two glycoproteins with molecular weights of 135 and 115 kD, respectively. The conditions of binding to these two proteins, however, markedly differ from those known for other lipoprotein receptors.

Following extensive purification, these two species are still present at concentrations relative to each other that depend markedly on the conditions of purification. The purified, solubilized receptor was tested under various conditions, including in the absence and presence of calcium, after disulfide-reduction, and following chymotrypsin digestion. In parallel experiments, the same preparations were tested with respect to binding of fibrinogen, different lectins, and thealloantibody anti-PlAI . The results strongly support the assumption, that the two protein bands associated with lipoprotein binding are constituents of the GP-IIb/IIIa complex.

These first results may have greatimplications for our understanding ofthe mechanism by which lipoproteins facilitate platelet stimulation.
ABOLITION OF IN VIVO PLATELET THROMBUS FORMATION WITH MONOCLONAL ANTIBODIES TO THE PLATELET GPIIb/IIla RECEPTOR: CORRELATION WITH PLATELET AGGREGATION AND BLEEDING TIME
Authors: Coller B S , Folts J D , Smith S R , Scudder L E
Published: August 2018

We previously reported that 0.8 mg/kgof the F(ab’)2 fragment of antibody 7E3, directed at theplatelet GPIIb/IIIa receptor, can abolish periodic platelet thrombus formation on partially stenosed carotid arteries in monkeys (Mnks). The present study was designed to: 1) test another antibody to GPIIb/IIIa (10E5), 2)find the minimum effective dose, and 3) correlate this effect with changesin the template bleeding time (BT) and platelet aggregation (PA). Periodicplatelet thrombi were established in the carotid arteries of 7 anesthetized Mnks after mechanical stenosis (∽70%) and intimal damage. 4 Mnks were treated with 7E3. Mnks 1 and 2 were given 0.2 mg/kg, and this dose: abolished thrombus formation and prevented its return in response to epinephrine infusion and increased intimal damage; abolished PA in response to ADP (10 μM); and increased the BT from 8.5 to 16 min and from 5 to 11 min. Mnk 3 was given 0.1 mg/kg, and this dose abolished the thrombi, inhibited PA by ∽41% and increased the BT only to 10 minfrom 8 min. Mnk 4 was givenincremental doses of 7E3. After 0.1 mg/kg, thrombi were reduced but not abolished, PA was minimally inhibited and the BTwas unchanged (7.5 vs 8 min pre). Afteranother 0.1 mg/kg, thrombi were abolished but could be partially restoredwith extreme provocation, PA was abolished and BT remained 7.5min. Afteranother 0.2 mg/kg, thrombicould not berestored, PA was abolished and the BTincreased to 21 min. After a final0.2mg/kg, the BT increased to 33 min. 3 Mnks were treated with 10E5. Mnk1received 0.4 mg/kg: thrombi and PA werebothabolished, and the BT increased from 5.5 to 14.5 min.Mnk 2 received 0.2mg/kg: thrombi and PA were again abolished while the BT increased to8.5from4.5 min. Mnk 3 received 0.1 mg/kg, and this abolished thrombus formation,butinhibitedPA by only ∽50%and increasedthe BT minimally (7.5 to 8.5 min). Increasedoozing from the neckwounds wasonly observed in animals with significant BT prolongations.We conclude that ∽0.1-0.2 mg/kg of eitherantibody can abolish in vivo thrombusformation, and that it is not necessary to abolish PA or cause marked prolongation ofthe BT in order toabolishthrombus formation in this model.
DISTRIBUTION OF GLYCOPROTEINS (GP) lib AND Ilia AND THEIR COMPLEX ON ADHERENT/ACTIVATED PLATELETS
Authors: Lewis J C , Hantgan R R , Kieffer N , Nurden A , Breton-Gorius J
Published: August 2018

Redistribution of GP Ilbllla has recentlybeen demonstrated for platelets activatedin suspension in the presence of either fibrinogen or specific monoclonal antibodies, and it has been suggested that redistribution is important for normal platelet function. Reported here is the distribution of GPIIb an GPIIIa following adhesion, and event focal to the hemostatic process. Human platelets isolated by centrifugation from heparinized blood and washed in Hank’s Salts, pH 6.5 with and without EDTA (3%) were activated by adhesion to carbon-stabilized formvar grids. Subsequent to activation/adhesion, GP’s were localized by immunoelectron microscopy on whole-mount cells using colloidal gold particles of different sizes. Primary antibodies included mouse monoclonal antibodies C17 (anti IIIa) and PI 12 (anti IIb), rabbit polyclonal antibodies aIIb, aIIIa and antibodies HpL2 and IgGL directed against IIb and the complex. Upon activation with pseudopod extension the Gp’s were colocalized with combined immunochemistry (C17 & aIIb, P112 & aIIIa). Greatest density was observed along pseudopods and the elaborating hyalomere. In addition to colocalization,discrete pools of either IIb or IIIa were interspersed with the complexes over the hyalomere. The pools, ranging in diameter to 0.3 μM were accentuated with hyalomere development; corresponding with release, both the pools and the complexes migrated centripetally to the granulomere. Thisresulted in an absence of Gp over the hyalomere. During hyalomere Gp migration, the label at pseudopods and the cell margin remained intact. Both centripetal movement and pool sizeswere accentuated at 37°C. Immunodetection over the granulomere was minimal following release, but small pools of individual Gp’s were again observed randomly over the hyalomere. Both clustering and relocation of Gp occurred in the presence of EDTA. Verification of individual Gp clusters and IIbIIIa complex clusters was achieved by double labeling using an antibody against the complex in conjunction with antibodies againstlib or IIIa. The complex always colocalized with the individual Gp’s; however, in all combinations the individual Gp’s were alsoobserved as separate pools.
THE SOLE USE OF MAGNESIUM CHLORIDE FOR BLOOD ANTICOAGULATION: PRESERVATION OF PLATELET SURFACE-BOUND “PROTEINACEOUS HALO”!
Authors: Crawford N , Crook M , Dawes J , Gray C R W
Published: August 2018

European Patent No. 83901336 [C.R.W. GRAY & N. CRAWFORD] refers to the use of bivalent cations for blood anticoagulationand the preparation of plasma and cellular products. Both CaCl2 and MgCl2 are effective as sole anticoagulants although the concentration of Ca+2 to maintain blood fluid for >7 days exceeds the osmolarity limits for good preservation of cellular integrity. With MgCl2 ,however, [at final concentrations 25-30 mM and suitable blood/MgCl2 volume ratios] red cells show good preservation, granulocytes phagocytose well and platelets are discoid and respond to all conventional agonists [although higher than normal doses are required]. The concentrations of 6 thromboglobulin (βTG) in “MgCl2-plasma” aresubstantially lower than with Cacomplexing anticoagulants containing theophylline and PGE . Thrombospondin [TSP]another major granule protein, unlike 6TG, binds after release to the activated platelet surface in the presence of physiological [Ca+2+]1 RIA assays of TSP has revealed that “Mg platelets” have 3 times more surface bound TSP than “CPD-platelets”. Subpopulation profiling by continuousflow electrophoresis shows that formol-fixed platelets from Mg2 anticoagulated blood are substantial>ly less electronegative than "CPD platelets" similarly processed. We believe that plateletsfrom blood in which the extracellular[Ca2+] is undisturbed carrya surface-associatd “halo”ofnonspecific [electrostatically-associated] and Ca2+-linked adsorbed proteins. Released TSP is an example of the latter association. Since plasma 0TG levels are influenced by kidney clearancekinetics and other factors, the measurement of surface bound TSP on platelets from MgCl2-anticoagulatedbloodmay have clinical value as an indexof invivo release events and particularly in vascular disorders accompanied by renal damage where the findings with 0TGhave been equivocal(2)
EFFECTS OF THROMBOXANE RECEPTOR BLOCKER BM 13.505 ON THE DEVELOPMENT OF PROTEINURIA IN AUTOIMMUNE NZB/W MICE
Authors: Stemeier K , Mertin J , Pill J , Hartig F
Published: August 2018

Female F1 hybrid of New Zealand black and white mice(NZB/W) spontaneouslydevelop an autoimmune disease characterize by afatal immune complex glomerulonephritis.Theyare considered to be a relevant model of human systemic lupus erythematosus. We observeda doubling of the concentration of TXB2 in urine at the same time when onset of proteinuria was noticed. This suggests that TXA2 synthetized by mesangial and epithelial cells of the glomeruli as well as by some inflammatory cells and platelets might be an important mediator in the pathogenesis of thi auto immune-mediated glomerular disease. Weused BM 13.505 as an long-acting TX receptor blocker for evaluating the importance of TXA2 on the development of proteinuria and compared its effects with that of the immunsuppressive agent cyclophosphamide.NZB/W mice were distributed to vehicle-treated (V-)group 20 mg/kg BM 13.505 (BM-) group and 20 mg/kgcyclophosphamide (C-) group( = 13 -14).Daily dosing by gavage was startedat the age of12 weeks. Every fourth week theurinary concentrations of proteins were measured by th biuret method and TXB2by a RIA. An increasein TXB2 was seen in the V- and BM-group, while in the C-group TXB2 was lowered. At 36 weeks of age 8of 14 animals of the V-group were proteinuria positive (>100 mg/100 ml). The study was finished at 44 weeks because more than 2/3 of the animals of the V-group haddeveloped a proteinuria. Previously four animals died and in most of other the disease was faradvanced. In the BM-group no animal had diedor showed signs of illness. However seven ofthe animals had slightly elevated protein concentrations in urine and two moderate elevated values. In the C-group no proteinuria was detected. Histological examinations of thekidneys showed a correlation in individualanimals of the V-group between the duration and extent of proteinuria and changes in the morphology of the glomeruli. In the BM-treated animals slight to moderate protein deposits were detectable, while in cyclophosphamide-treated animals glomeruli were of normal structure. This study presents someevidence that TXA2 may be an important mediator in the pathogenesis of this immune-mediated renal disease. Manifestation of this disease is delayed by the administration of thespecific TX receptor blocker BM 13.505.
INHIBITION OF FACTOR Xa AND THROMBIN BY ANTITHROMBIN III AND HEPARIN DURING HUMAN PROTHROMBIN ACTIVATION
Authors: Schoen P , Hemker H C , Lindhoubt T
Published: August 2018

Prothrombin-catalyzed human prothrombin activation results in the generation of thrombin and meizothrombin- des FI (MDF1) as was demonstrated by an immunoblot technique. Theheparin-independent second order rate -.onstants of inhibition of both thrombin and MDF1were 3.7 x 105 M-1min-1, whereas the rate constant of inhibition of purified thrombin was 6.5 x 105 M-1min-1 . In the presence of heparin the decay of amidolytic thrombin activity appeared to be bi-exponential and could be modelled by a 4-parameter equation. Fitting the experimental data to this equation gave the pseudo-first-order rate constants of inhibition, as well asthe composition of the reaction with respectto the levels of thrombin and MDF1. The occurence of MDF1 rather than meizothrombin (MT)suggests that MT is very rapidly converted into prothrombin fragment 1 (FI) and MDF1, a "dead-end" product of prothrombin activation. At high prothrombin concentrations (1.0 μM) predominantly MDF1 and not thrombin is formed, indicating that MT itself and/or MDF1 cleaves at Argl56 - Serl57 to produce FI and MDF1. Interestingly, heparin is unable to stimulate the inhibition of MT and MDF1 by AT III. We could demonstrate that MT and MDF1 have a low affinity, if any, for heparin.

The kinetics of the heparin-dependent inhibition of prothrom-binase formed thrombin differ from those of purified thrombin added to the prothrombin activation reaction. A lower rate of inactivation of endogenous formed thrombin was observed and,moreover, could not be modelled as a simple random order bi-reactant enzyme-catalyzed reaction. Theseeffects might be caused by the presence of prothrombin activation fragments.Taking the factor Xa inactivation also into account, it appeared that the major effect of heparin onthe thrombin generation was to enhance the inhibition of thrombin rather thanthat of factor Xa. When unfractionated heparin (UFH; mean Mr = 14000, 168 USP units/mg) was compared with a synthetic pentasaccharide (PS; Mr = 1714, 4000 anti-Xa U/mg) on basis of equal anti-Xa U/ml, UFH shortened the half-life time of factor Xa 190-fold, whereas PS shortened the half-life time 17-fold.
A NEW FIBRIN-SPECIFIC ANTIBODY DISCRIMINATING BETWEEN FIBRIN AND FIBRINOGEN IS DIRECTED AGAINST THE SYNTHETIC PEPTIDE LEU-ILE-ASP-GLY-LYS-MET
Authors: Scheefers-Borchel U , Muller-Berghaus G
Published: August 2018

To determine soluble fibrin in blood of patients with coagulation disorders we produced monoclonal antibodies which distinct fibrin from fibrinogen and other blood constituents. Fibrin-specific monoclonal antibodies were obtained by immunizing mice with the synthetic hexapeptide Leu-Ile-Asp-Gly-Lys-Met which was covalently linked to KLH via its C-terminus. Several of the monoclonal antibodies which reacted with the hexapeptide also reacted with batroxobin-induced desAA-fibrin and thrombin-induced desAABB-fibrin, but not with fibrinogen. No reaction was observed with plasmin-induced fibrinogenolytic and fibrinolytic degradation products, respectively. The epitope recognized by these fibrin-specific antibodies is located on the αchain of fibrin and is not accessible for an antibody in native fibrinogen. One monoclonal antibody (B/H11) was used to quantify the amount of soluble fibrin in plasma of patients with a variety of coagulation disorders. This antibody could also be used to develop an ELISA based on two different fibrin-specific monoclonal antibodies. For this assay anti-fbn 17 (Scheefers- Borchel et al., Proc. Natl. Acad. Sci. USA 82: 7091, 1985) was coated onto ELISA plates. After adding plasma which contained soluble fibrin, the fibrin bound was detected by the second fibrin-specific antibody B/H^ to which biotin was covalently linked. The second antibody was probed by the addition of peroxydase conjugated streptavidin and the substrate ABTS for peroxydase. This test can be used to detect fibrin at concentrations as low as 70 ng/ml. With this assay system, it is possible to measure the amount of soluble fibrin present in plasma samples without the interference of fibrinogen which is associated with soluble fibrin.
THE N-DSK γ-CHAIN BINDS TO IMMUNOPRECIPITATED GP IIBIIIa
Authors: Thorsen L I , Hessel B , Brosstad F , Gogstad G , Solum N O
Published: August 2018

We have previously demonstrated binding of the CNBr-split N-terminal disulphide knot of the fibrinogen molecule (N-DSK) to blood platelets and to their immunoprecipitated fibrinogen receptor, the glycoprotein IIb-IIIa complex. To further investigate which part of the N-DSK molecule that is responsible for its binding to GP IIb-IIIa, this fragment was split into its separate Aα (1-51), Bβ (1-118) and γ- (1-78) chains and carboxymethylated. GP IIb-IIIa was immunoprecipitated by crossed immunoelectrophoresis (CIE) of Triton X-100 extracts of platelets against rabbit antibodies to whole platelet proteins. The CIE plates were incubated with 125-I-radiolabelled N-DSK chains, and investigated for binding by autoradiography. The γ-chain but not the Aβ or Bβchains demonstrated binding to the GP IIb-IIIa. These results demonstrate that the fibrinogen molecule contains a third sequence of amino acids which is capable of binding to the fibrinogen receptor of the blood platelets, in addition to the two previously reported sequences C-terminally in the Aα-and γ-chains. Fragment E derived from fibrinogen (E fg) does not interact with the fibrinogen receptor. Thus, the γ-chain interaction site can not be present in the E fg-γ-chain (1-53). However, fragment E derived from fibrin (E1) with the γ-chain of residues 1-62, does react. The residues of N-DSK and E1 which are involved in binding to GP IIb-IIIa therefore appear to be present in the γ-chain sequences 54-62.
BIOCHEMICAL EVIDENCE OF THROMBUS FORMATION IN PATIENTS NITH PERSISTENT UNSTABLE ANGINA
Authors: Hame C W , Lorenz R L , Kuooer H , Bleifeld N , Weber P C
Published: August 2018

Thromboxane released from activated platelets and prostacyclin of the vessel wall may act as potent antagonistic modulators of platelet aggregability and coronary vascular tone. Therefore we studied urinary excretion of their major metabolites, 2,3-dinor-throeboxane B2 (TX-M)and 2,3-dinor-6-keto prostaglandin F, (PGI-H), in 16 patients presenting Mith prolonged angina at rest. The 10 patients who did not improve under vigorous antianginal treatment within 48 hours exhibited higher TX-M excretion than 6 patients responding to therapy (2208 ± 1542 vs 609 ± 312 ng/g creatinine) p<0.01). Elevated TX-H excretion Mas also found in 4/8 patients Mith sustained post-infarction angina. Mhen 9 patients Mere re-studied in a stable phase after 11 ± 5 Months TX-H Mas consistently in the norial or high normal range. Excretion of PGI-M Mas not depressed in any patient but correlated Meakly Mith TX-M (r=0.408). Thus, enhanced TX-M excretion as index of platelet activation lay identify patients Mho are at increased risk of active throsbus formation and Mho could benefit lost froi anti-aggregatory treatment.
FIBRINOLYTIC ALTERATIONS AS RISK FACTOR IN PATIENTS WITH CORONARY HEART DISEASE
Authors: Aznar J , Estelles A , Tormo G , Espafia F , Torino V
Published: August 2018

It has been reported that young survivors of myocardial infarction (MI) have elevated plasminogen activator inhibitor (PAI) levels. We have studied several fibrinolytic parameters (euglobulin lysis time, fibrin-plate lysis, tissue plasminogen activator (t-PA) antigen, t-PA activity, PAI activity, plasminogen, az antiplasmin and FDP/fdp in 55 patients with coronary heart disease (CHD), before and after an exercise test. The patients were classified in 4 groups:A) Patients with unstable angina (n=5); B) Patients with stable angina and previous history of MI (n=13); C) Patients with stable angina without previous history of MI (n=ll) and D) Patients with MI about 3 weeks before this study (n=26). All the groups were similars in age and life habits. Patients suffering from dislipemia and diabetes were excluded from the study. In basal conditions, PAI activity (U/ml) was high in the 4 patient groups (A:2.5±2.8; B:5.2 ± 4.9; C:2.8 ± 2.6; D: 4.6 ± 4.6) ascompared to a group of 10 healthy volunteers (0.46±0.5). In
all the clinical groups there were a large number of patients (about 60%) whose PA inhibitor level was > 2 U/ml. t-PA antigen (ng/ml) was slightly elevated in all patient groups (A: 12.4+±4.6; B:12.4±5.6; C:12.5+4.0; D: 13.3+4.3) in comparison with control group (10.1+2.9). The release of t-PA antigen after the exercise test did not differ significantly from one group to another. However, this release was < 3 ng/ml in about 50% of patients in all clinical groups, as compared to the control group, in which the release of t-PA antigen was higher than 3 ng/ml in all the subjects. After the exercise PAI activity remained high in the patient groups. The increased level of t-PA inhibitor activity founded in the patients was partially inhibited by antiserum against PA inhibitor-1 but not by antiserum against PA inhibitor-2. The formation of a complex of about 115,000 daltons between the increased plasma PA-Inhibitor and purified single t-PA was observed by a zymographic fibrin technique. These findings demonstrate that CHD patients have a fibrinolytic hypofunction caused basically by an increase in t-PA inhibitor. This increase in PAI activity is more evident in patient with a previous history of MI.
THE ROLE OF FACTOR VII IN HAEMOSTASIS: A DETAILED MICROSCOPIC ANALYSIS OF THE MORPHOLOGY OF THE EVOLVING HAEMOSTATIC PLUG IN NORMAL AND VII DEFICIENT DOGS
Authors: Giles Alan R , Vendervelden Peter
Published: August 2018

The role of F.VII in haemostasis remains controversial, both in terms of the functional consequences of the deficiency state and the activation pathways to which it makes its principal contribution In vivo. We have developed a cuticle bleeding time (CBT) model in dogs and used this to investigate the functional consequence of both congenital and acquired F.VII deficiency (SD) (Blood 65:1197, 1985). There was no significant difference between the CBT of these animals when compared to controls. However, the CBT prolonged at a significantly lower Heparin level than that observed in controls. F.VIIa was also infused into F.VIII deficient and normal dogs and FPA measured as an indicator of thrombin generation. Significant change in FPA level occurred in the latter but not the former, suggesting that activation of F.IX rather than F.X was favoured. We have now performed detailed morphological studies of the evolving haemostatic plug (HP) in the injured cuticle of F.VII and normal animals by light (LM) and electron microscopy (EM). Quantification of the EM changes noted were performed by morphometric analysis. The tightness of the intravascular component of the HP was assessed by random measurement of intraplatelet distance. The degree of platelet activation was measured by comparing the area of the open canalicular system (OCS) in comparison to the total platelet area. The appearance of fibrin in the plug was also noted. Qualitative LM revealed little difference between the two sets of animals. The appearance of fibrin at the periphery of HP plug was delayed in SD and was reduced in quantity. However, by morphometry although the pattern was identical in both groups, there was a significant delay in the changes noted in SD. These results suggest that the extrinsic pathway may play an important role in triggering the intrinsic pathway, either by providing for activation of the cofactors V and VIII or pulse generation of F.IXa. This may play a critical role in haemostasis when the vessel injured is larger than those in the nail cuticle of the dog (50 - 150 μm) or when other components of haemostatic mechanism are compromised
A MODIFIED METHOD FOR PURIFICATION OF BOVINE FACTOR VII AND ITS PRIMARY STRUCTURE
Authors: Takeya H , Kawabata S , Miyata T , Morita T , Iwanaga S
Published: August 2018

A modified method for purification of factor VII from bovine plasma was developed. The isolation procedures consisted of four steps with the ordinary barium citrate adsorption (1), DEAE-Sepharose chromatography (2), twice chromatographies on a benzamidine-Sepharose column (3) and affinity chromatography on Affigel-10 coupled with staphylocoagulase produced by Staphylococcus aureus, strain 213 (4). This final step was very effective for the removal of prothrombin contained in the pooled fraction of factor VII. Thus, these procedures allowed higher recovery of factor VII than the earlier methods and about 7 mg of the material was obtained from 15 liters of plasma. The purified preparation gave a single band on SDS-PAGE in the absence and presence of 2-mercaptoethanol and the apparent Mr was estimated as 53,000.

To establish the whole primary structure at protein level, the isolated single chain factor VII was incubated with bovine factor Xlla and the resulting two chains-factor VII was reduced and S-pyridylethylated. The S-alkylated heavy and light chains were then separated on a TSK gel G3000SW followed by reversed phase HPLC on a Phenyl-5PWRP column. The amino acid sequence of the isolated light chain was determined by sequencing the peptides obtained from lysyl endopeptidase and α-chymotrypsin digests and from trypsin digest of the succinylated light chain. The light chain consisted of the total of 152 residues with 10 r-carboxyglutamic acids and contained one oligosaccharide chain at Asn-145. The light chain sequence was compared with that predicted from the cDNA sequence for human factor VII. It had 76 % sequence identity with the human's. The sequence of the factor Vll-heavy chain is being studied
THE ANTITHROMBOTIC EFFECT OF PALM OIL IS CORRELATED WITH ITS CONTENTS OF VITAMIN E
Authors: Hornstra E G , Hennissen A H , Kalafusz R , Tan D T S
Published: August 2018

Dietary saturated fatty acids are known to increase platelet aggregation and arterial thrombogenesis.We recently demonstrated, however, that palm oil, rich in saturated palmitic acid, has a distinct antithrombotic affect, which is associated with a decrease of the thromboxane-prostacyclin ratio in activated whole blood. To identify the antithrombotic component(s) of palm oil, seven palm oil fractions were prepared with comparable fatty acid compositions of the triglycerides but containing Various amounts of non-triglyceride material with different compositions.These fractions were fed to rats in amounts of 50 energy% for a period of 8 weeks, after which arterial thrombosis tendency was measured upon insertion of an aortic prosthesis, the aorta-loop. During loop insertion, 1 ml blood was collected in citrate for measuring platelet aggregation and ATP release in response to collagen, using the Chronolog whole blood lumi-aggregometer. Arterial thrombosis tendency was found to be negatively related to the total amount of non-triglyceride material in the various fractions (r = 0.78; p <0.05).No significant relationship was observed between arterial thrombus formation and the various sterols present in the non-triglyceride material.A significant negative correlation was found, however, with a-tocopherol (r = 0.86; p <.02). Collagen-induced platelet aggregation and ATP release in whole blood were not correlated to total amounts or α-tocopherol content of the non-triglyceride material.However, significant positive relationships were found between these platelet functions and the amountsof the various sterols (Campesterol: r = 0.70; P < 0.10 β-sitostero1 : r = 0.69; P <0.10. Cholesterol : r = 0.81; P < 0.05).

These findings demonstrate that effects of edible oils on platelet function and arterial thrombogenesisare not only mediated by the fatty acid compostion of the triglycerides, but can also be determined by 'minor components', present in the non-triglyceride part of the oils.
IMPAIRMENT OF PLATELET PHOSPHOINOSITIDE METABOLISM IN PRIMARY HYPERTENSION
Authors: Koutouzov S , Remmal A , Marche P , Meyer P
Published: August 2018

Blood platelets from hypertensive patients and spontaneously hypertensive rats (SHR) display multiple abnormalities when compared with cells from normotensive controls. The major features of the modified platelet profile are an enhanced rate of adhesion/aggregation in response to many stimuli, a greater sensitivity for thrombin and adrenaline to produce increases in cytoplasmic free Ca2+, and an exaggerated release reaction. Furthermore, the resting levels of cytosolic free Ca2+ ions are specifically and constantly increased. Since phosphoinositides are involved in the stimulus-response coupling mediated by intracellular Ca2+ mobilization, the metabolism of these lipids was investigated in platelets of SHR and compared with those of normotensive Wistar-Kyoto rats (WKY). Following 32P-labelling of quiescent platelets, labeled lipids were analyzed both in platelets at rest and after thrombin stimulation. In resting platelets, the 32P associated with each of the phosphoinositides and phosphatidic acid (PA) was similar in SHR and WKY indicating that both the pool size of the various lipids and their basal turnover did not differ between the two strains. By contrast, within the first seconds after thrombin stimulation (10-60 sec), the dose-response and time-course curves of agonist-induced increase in 32P-PA were markedly shifted to the left and reached higher equilibrium levels in SHR. Since thrombin-induced 32P-PA formation is held as the most sensitive index of phospholipase C activity, our results indicate that this enzyme displays hyperreactivity in SHR (vs WKY). It is therefore likely that in SHR, the enhanced physiological responses (serotonin secretion, aggregation) that we observed under the same experimental conditions may be related to an increased formation of Phospholipase C products (inosi-toltriphosphate and diacylglycerol) which are the two second messengers responsible for internal Ca2+ mobilization and activation of protein kinase C, respectively. Therefore, these data suggest that the hypersensitivity of Phospholipase C may be involved in the overall alteration of cell calcium handling and hence in the SHR platelet responses.
Thranbospondin Promotes Cell-and Platelet-Substratum Adhesion
Authors: Tuszynski George P , Rothman Vicki L , Murphy Andrew , Siegler Katherine , Smith Linda , Smith Sena , Karczewski Jerzy , Knudsen Karen A
Published: August 2018

Thrombospondin (TSP), isolated from human platelets, promotes the in vitro, calcium-specific adhesion of a variety of cells, including platelets, melanoma cells, muscle cells, endothelial cells, fibroblasts, and epithelial cells. The cell adhesion-promoting activity of TSP is species independent since human, bovine, pig, rat and mouse cells all adhered to TSP. Furthermore, the cell adhesion-promoting activity of TSP is specific and not due to a nonspecific protein effect or to contamination by fibronectin, vitronectin, or laminin. That is, neither bovine serum albumin nor TSP preparations treated with a monospecific anti-TSP antibody support cell adhesion. As analyzed by polyacrylamide-gel electrophoresis and specific antibody binding assays, the TSP preparations used in these studies contained no detectable fibronectin or laminin and less than 0.04% vitronectin. The cell surface receptor for TSP appears distinct frcm that of fibronectin since an antiserum that blocks cell adhesion to fibronectin has no effect on adhesion to TSP. In addition, The platelet cell surface receptor for TSP appears distinct, frcm that of fibrinogen since thrcmbasthenic platelets adhere to TSP as well as control platelets. Antibodies to the GPIIb-GPIIIa complex block platelet adhesion to fibrinogen but have no effect on adhesion to TSP. Initial characterization of the cell surface receptor for TSP shows it to be protein in nature since cells treated with trypsin fail to adhere to TSP. In summary, our results provide the first clear evidence that TSP specifically promotes cell-substratum adhesion of a variety of cell types independent of the animal species. Our preliminary evidence suggests that the cell-surface receptor(s) for TSP is protein and that it is distinct for the receptor for fibronectin and fibrinogen. Our data suggest that TSP may play a central role in normal adhesive events mediated by platelets and other cells, such as those involved in hemostasis and wound healing. In addition, TSP may be involved in pathological adhesive events mediated by platelets and tumor cells, such as those involved in cardiovascular disease and tumor cell metastasis.
DETECTION OF ACTIVATED PLATELETS WITH MONOCLONAL ANTIBODIES
Authors: Metzelaar M J , Nieuwenhuis H K , Sixma J J
Published: August 2018

Blood tests reflecting in-vivo activation of platelets are potentially useful in evaluating patients with thrombotic diseases. Recently monoclonal antibodies have been described that react preferentially with activated platelets. We prepared an IgG2b antibody, designated RUU-AP 2.28, that reacted with a 53.000 MW protein that is located in a special subclass of platelet granules in unstimulated platelets and that is exposed on the surface of activated platelets. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed in patients undergoing cardiopulmonary bypass and in patients with acute deep venous thrombosis. The percentage of RUU-AP 2.28 positive platelets in the circulation was 3,9 ± 2.7 (SD)% in the controls, (n = 20), 24.6 ± 13.5% in patients after cardiopulmonary surgery (n = 10) and 8.5% in patients with acute deep venous thrombosis (n = 2).

In order to detect also earlier stages of platelet activation, such as secretion-independent phenomena, we produced new monoclonal antibodies by fusing spleen cells from Balb/c mice, immunized with thrombin stimulated, paraformaldehyde fixed platelets, with Ag 8653 myeloma cells. As a screening assay we used an ELISA with freshly fixed platelets or fixed thrombin-activated platelets. We detected six monoclonal antibodies (RUU-AP 1-6) specific for thrombin-activated platelets. The results of the ELISA were confirmed by flow cytofluorometry.

None of the antibodies inhibited platelet aggregation induced by ADP, collagen or ristocetin. Ascites of IgGl antibody RUU-AP 3 reacted with normal thrombin-activated platelets but did not react with thrombin-activated platelets from a patient with Glanzmann’s disease. In addition antibody RUU-AP 3 reacted with normal platelets stimulated with 1 pM of ADP. These data suggest that antibody RUU-AP 3 detects a secretion-independent conformational change in the platelet membrane glycoprotein IIb-IIIa complex.
DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY
Authors: Shattil S J , Hoxie J A , Cunningham M , Abrahms C S , O’Brien J , Budzynski Z
Published: August 2018

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.
BINDING OF CALCIUM TO HUMAN AND BOVINE FACTOR X
Authors: Monroe D M , Deerfield D W , Olson D L , Stewart T N , Roberts H R , Hiskey R G , Pedersen L G
Published: August 2018

Human and bovine factor X contain 11 and 12 glutamyl residues respectively within the first forty amino terminal residues that are posttranslationally modified to y-carboxyglutamyl (Gla) residues. Calcium binding to these Gla residues and at other sites is critical for activity in factor X. We have measured calcium binding to human factor X by equilibrium dialysis for the first time. We have also re-examined calcium binding to bovine factor X in order to compare the two species. Factor X (10 μM) was incubated with 45Ca in 20 mM Tris (pH 7.5), 100 mM NaCl in a half cell separated by a 12-14000 molecular weight fast-equilib-rium disk membrane at 25°C for 24 hours. Four aliquots (100 μL each) were removed from each side of the cell and counted. Data were analyzed with a variety of models that allow for more than one class of binding site and for cooperativity among binding sites. Calcium binding to bovine factor X was best simulated by a model that assumes 1 very tight site, 3 cooperative tight sites, and 18 equivalent, non-interacting sites. Based on data from des(Gla)factor X, the first site is probably a high affinity non-Gla binding site. Our results differ from two previously published reports that indicated either 1 tight and 39 loose noncooperative sites (R.H. Yue & M.M. Gertler (1978) Thrombos. Haemostas. (Stuttg.) 40, 350) or 20 calcium binding sites with the first 4 being cooperative (M.J. Lindhout & H.C. Hemker (1978) Biochimica Biophysica Acta 533, 318). Our data on human factor X fit the same model as used for bovine factor X; however, coop-erativity is less in the 3 cooperative sites. Shown below are the first six thermodynamic equilibrium constants derived from a Scatchard analysis of binding data (values are M−1).

Both proteins demonstrate the same total number of binding sites and essentially the same value for the first, tight binding site. Bovine factor X exhibits cooperativity, whereas human factor X has reduced cooperativity.
POLYMORPHIC SITES IN FACTOR X GENE
Authors: Chelucci C , Hassan H J , Guerriero R , Leonardi A , Mattia G , Peschle C
Published: August 2018

The structure of factor X gene has been analyzed by Southern blot in 5 subjects with factor X deficiency.

Genomic DNA was digested with 8 different endonucleases and hybridized with a cDNA probe. The congenital deficiency observed in these patients is not apparently due to a major deletion or rearrangement. Since the gene locus is grossly intact, the disease presumably results from point mutation(s) not identified by the utilized endonucleases.

Our study was also focused on the presence of polymorphic site(s) in the factor X gene locus. Analysis of 50 normal subjects allowed to identify several polymorphic restriction sites after digestion with EcoRI, Hind III and Pvu II.

The restriction pattern obtained after Hind III digestion showed two bands of 7.3 and 6.0 Kb, while in two families an additional 7.6 Kb band was observed. Genomic DNA digested with EcoRI showed a 7.1 and 5.1 Kb fragments, and also a 6.6 Kb band with a 10% f requency.

After DNA digestion with Pvu II 5.6, 2.7 and ∼1.0 Kb bands were observed. In three unrelated subjects we observed an additional 3.0 Kb fragment, in two other subjects a 3.5 kb band. Interestingly hybridization with a 178 bp cDNA subclone allowed to map the polymorphic sites in a the 3’ region of the gene locus.
THE EFFECT OF PROCOAGULANT PHOSPHOLIPID VESICLES WITH NET POSITIVE CHARGE ON THE ACTIVITY OF PROTHROMBINASE
Authors: Rosing J , Speijer H , Govers-Riemslag J W P , Zwaal R F A
Published: August 2018

It is generally thought that procoagulant phospholipid surfaces that promote the activation of vitamin K-dependent coagulation factors should have a net negative charge in order to promote calcium-dependent binding of the enzymes (FVIIa, FIXa and FXa) and substrates (prothrombin and FX) of the coagulation factor-activating complexes. Two models have been proposed to explain calcium-mediated association of vitamin K-dependent proteins with phospholipid: a) an electrostatic model, in which a positively-charged protein-calcium complex is attracted by a negatively-charged phospholipid surface and b) a chelation model in which a coordination complex is formed between calcium ions, γ-carboxyglutamic acids of the proteins and negatively-charged membrane phospholipids. To study the effect of the electrostatic potential of phospholipid vesicles on their activity in the pro-thrombinase complex the net charge of vesicles was varied by introduction of varying amounts of positively-charged stearylamine in the membrane surface. Introduction of 0-15 mole% stearylamine in phospholipid vesicles that contained 5 mole% phosphatidylseri-ne (PS) hardly affected their activity in prothrombin activation. Electrophoretic analysis showed that vesicles with > 5 mole% stearylamine had a net positive charge. The procoagulant activity of vesicles that contained phosphatidic acid, phosphatidylglyce-rol, phosphatidylinositol or phosphatidyl-glactate (PLac) as acidic phospholipid was much more effected by incorporation of stearylamine. Amounts of stearylamine that compensated the negative charge of acidic phospholipid caused considerable inhibition of the activity of the latter vesicles in prothrombin activation. The comparison of vesicles containing PS and PLac as acidic phospholipid is of special interest. PS and PLac only differ by the presence of NH+
3-group in the serine moiety of PS. Thus, in spite of the fact that vesicles with PLac are more negatively charged than vesicles with PS, they are less procoagulant. Our results show that a) although procoagulant membranes have to contain acidic phospholipids there is no requirement for a net negative charge, b) the amino group of phosphatidylserine has an important function in the interaction of procoagulant membranes with vitamin K-dependent proteins and c) the chelation model can satisfactorily explain calcium-mediated lipid-protein association.