bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation Of Vascular Prostacyclin (PGI2) Synthesis
Authors: Coughlin S , Moskowitz M , Antoniades H N , Levine L
Published: July 2018

We have examined the possibility that substances released during platelet degranulation modify vascular PGI2 synthesis. PGI2 is a potent inhibitor of platelet function produced by vascular endothelial and smooth muscle cells. Regulation of PGI2 synthesis by blood vessels is not well understood. We report that a platelet- dependent factor in serum dramatically stimulates PGI2 synthesis by vascular endothelial and smooth muscle cells in culture. We further report that platelet-derived growth factor (PDGF), a releasable protein found in platelet alpha granules, stimulates PGI2 synthesis by the above cell types by over 100 fold. The concentration of PDGF required to elicit this effect is below that reported in human serum. The above mentioned serum factor is relatively heat stable, non-dialyzable, and cationic; preliminary studies indicate that anti-PDGF antiserum is capable of blocking stimulation of PGI2 synthesis by both PDGF and serum. These data suggest that the serum factor may indeed be PDGF. PDGF acts synergistically with other platelet granule constituents (serotonin, ATP) and with thrombin to stimulate PGI2 synthesis by vascular cells in culture. We thus postulate that platelet-released PDGF, in concert with other substances generated during clotting, acts to increase vessel wall PGI2 synthesis as part of a negative feedback mechanism controlling platelet aggregation. A defect in the ability of a blood vessel to increase PGI2 production in response to platelet degranulation, as may occur in atherosclerotic vessels, could perhaps contribute to the genesis of thromboembolic events.
Roles Of Thromboxane A2, Releasable ADP, And CAMP In Fluid Shear-Induced Aggregation Of Human Platelets
Authors: Moritz M W , Sutera S P , Joist J H
Published: July 2018

Fluid shear-induced platelet alterations may be responsible for both thromboembolic complications and platelet dysfunction associated with extracorporeal circulation. Previous work in our laboratory using a Couette rotational viscometer with controlled laminar flow conditions demonstrated that shear-induced platelet aggregation (SIPA) in citrated human platelet-rich plasma at 24°C is associated with the release of substances from both the dense and α-granules of platelets and is not inhibited by aspirin (ASA) induced complete suppression of thromboxane A2-synthesis. We studied SIPA in suspensions of washed human platelets in Tyrode albumin solution at 37°C to determine the importance of releasable ADP, thromboxane A2 synthesis and the level of intracellular cAMP in SIPA. SIPA in this system was found to require the presence of added Ca++ (but not Mg++ or fibrinogen) in the medium. ASA (100 μM) did not cause appreciable inhibition of SIPA. Creatine phosphate- creatine phosphoklnase (CP/CPK), an enzyme system that converts ADP to ATP, caused 40% inhibition of SIPA. With both ASA and CP/CPK present in the suspension, SIPA was identical to that obtained with CP/CPK alone. However, preincubation of the suspension with PGE1 (1 μM) and theophylline (100 μM) caused complete inhibition of SIPA. These findings indicate that SIPA 1n this system can occur by thromboxane A2- and ADP-independent pathway(s), which may be modulated by intracellular cAMP.
Studies On The Interaction Of Thrombin And Platelet Proteins By Crossed Immunoelectrophoresis Using Immobilized Thrombin In The Intermediate Gel
Authors: Hagen I , Brosstad F , Korsmo R , Solum N O
Published: July 2018

The first step in the thrombin-platelet interaction is the binding of thrombin to the platelet surface. In order to identify platelet proteins that interact with thrombin Triton extracts of platelets were examined by crossed immunoelectrophoresis (CIE) against poly-specific anti-platelet antibodies under conditions where the proteins had to pass a gel of thrombin coupled to Sepharose 4B. Proteins which bind to thrombin will stick to the immobilized enzyme and their corresponding immuno- precipitates will be absent or reduced in area compared to a control without thrombin. Four immunoprecipitates representing platelet factor 4, glycocalicin-related protein, factor XIII and a hitherto unidentified protein were absent or reduced in area, whereas the arc representing GP IIb and GP IIIa and the other immunoprecipitates were unaffected. The interaction between glycocalicin- related protein and thrombin seemed to be dependent on both the macromolecular binding site and the active serine site since this protein passed unaffected when either or these sites were bloched by NBS, heparin, TLCK or PMSF. Treatment of the Triton extract with immobilized thrombin followed by CIE indicated some proteolysis of factor XIII, whereas no effect was observed on the immunoprecipitates representing glycocalicin-related protein and platelet factor 4. It is concluded that crossed immunoelectrophoresis with immobilized thrombin in the intermediate gel provides a means of studying the interaction between thrombin and individual platelet proteins directely without prior purification.
Platelet Aggregation By Membrane Glycoprotein I
Authors: Watanabe K , Yamamoto M , Ando Y , Iri H , Furihata K , Yoshii Y , Handa M , Imai M , Sugiura K , Ikeda Y , Toyama K
Published: July 2018

It has been recently shown that platelet membrane components, particularly glycoproteins, have a lectin activity, thus mediating an aggregation of platelets. To obtain further evidences for a crucial role of glycoproteins in an aggregation mechanism,we have investigated the possibility that membrane glycoprotein can directly induce an aggregation of platelets. The membrane glycoproteins ( GP I, GP II and GP III ) were isolated from 3-4 mg of human platelet membranes using preparative electrophoresis on 5 % polyacrylamide gels with 0.1% SDS. Platelet aggregation by isolated GP I, GP II or GP III was examined under phase_contrast microscopy after the incubation of these peptides with platelet rich plasma at 37°C for 15 min.. Among glycoproteins tested, only GP I( 20 μg/ml < ) exerted an apparent platelet aggregation. No such aggregation was induced by either GP II or GP III even at concentration of 80 μg/ml. GP I isolated separately using the wheat germ agglutinin affinity column also produced a platelet aggregation. Aggregation curve recorded with an aggregometer showed a long lag phase ( 10 min. < ) followed by an irreversible aggregation. The GP I-induced platelet aggregation occured in a dose dependent manner. This aggregation was completely inhibited by the addition of aggregating inhibitors such as indomethacin ( 25 μM ), PGE1 ( 1 μM ), EDTA ( 0.5 mM ) and TMB-8 ( lmg/ml ). A significant amount of serotonin ( 27% ) and β-thromboglobulin ( 14.6% ) was released from platelets by GP I ( 100 μg/ml ). Treatment of GP I with either trypsin ( 50 μg/ml ) or chymotrypsin ( 40 μg/ml ) reduced the aggregating activity of this glycopeptides. The platelet aggregation by GP I was inhibited in the presense of 30 mM N-acetylneuraminic acid, arginin or L-lysine, but N-acetyl- ated amino sugars and neutral sugars were without effect. This GP I-induced platelet aggregation may be an important findings in elucidating platelet aggregation mechanism.
Isolation And Purification Of Collagen And α1 Receptor From Platelet Membrane
Authors: Chiang T M , Kang A H
Published: July 2018

We have previously demonstrated that chick skin type I collagen and the α1(I) chain mediate platelet aggregation. Aggregation is associated with specific binding of these substances by platelet membranes. We now describe the isolation and purification of the receptor. Platelet membranes were prepared as described previously and isolated membranes were solubilized in 0.5% Triton. The receptor was then purified by a combination of gel filtration, affinity chromatography on α1(I)-sepharose or type I collagen-sepharose and preparative polyacrylamide gel electrophoresis. The receptor activity was assayed either directly by a binding assay using (14C)-glycine-labeled α1(I) or indirectly by an adhesion inhibition assay on Sepharose 2B with (14C)-sero- tinin-labeled platelets.

The results show that the α1(I) receptor can be purified to a single band on SDS-gel electrophoresis with a recovery of 2.5%. Its activity is destroyed by preincubation with trypsin or pronase indicating it is a protein. The apparent molecular weight as estimated by gel filtration and SDS-gel electrophoresis is 95,000 daltons. The binding of (14C)- labeled α1(I) is specifically displaced by unlabeled α1(I), and the bound radioactivity can be removed by treatment with purified bacterial collagenase. The binding of (14C)- labeled α1(I) by the purified α1(I) receptor can also be inhibited by the receptor isolated from collagen-sepharose affinity chromatography. These data suggest that the α1(I) binding site is identical to the collagen binding site.
Salicylate Accumulating During Repeated Aspirin Administration Prevents The Inhibitory Effect Of Aspirin On Vascular Prostacyclin
Authors: Dejana E , Cerletti C , de Castellarnau C , Galletti F , de Gaetano G
Published: July 2018

In vitro studies have shown that salicylate (SA) effectively protects cyclooxygenase from aspirin (ASA) inhibition. The purpose of this study was to investigate in vivo this pharmacological interaction and to evaluate the possibility that SA accumulating during repeated ASA treatment interferes with the inhibitory effect of the unchanged drug on vascular prostacyclin(PGI2). Male CD-COBS rats were given i.v. saline or SA (25-100 mg/kg) followed after 15 min by ASA (2.5-10 mg/kg). The animals were killed 30 min after the last treatment. PGI2 activity was bioassayed on thoracic aorta rings and confirmed by RIA of 6-Keto-PGF1α. Blood SA levels were measured by fluorimetric assay. SA pretreatment showed dose-related competitive prevention of the inhibitory effect of ASA on vascular PGI2 which was almost complete at a dose ratio of 5:1. Further experiments were performed with single or repeated doses of ASA (200 mg/kg). This resulted in different blood SA levels after 24 hours when in all conditions vascular PGI2 recovered by more than 50% of basal value. At that moment further inhibition (60-90%) of PGI2 by a test dose of ASA (10 mg/kg) could only be obtained if blood SA levels were lower than 100 μg/ml. Above this level an inverse linear correlation (r = 0.87, p< 0.01) was found between blood SA levels and the inhibitory activity of ASA. In particular, almost no ASA activity could be detected at blood SA levels around 250 μg/ml.

These results show that SA and ASA interact in vivo at vascular levels. This interaction could have clinical implications in view of the rapid hydrolysis of ASA to SA and slow elimination of SA from blood. These observations add a new piece of information to the current debate on the optimal ASA treatment schedule for thrombosis prevention.
Dipyridamole Reverses The Inhibitory Effect Of Aspirin On Prostacyclin Synthesis
Authors: Jackson C A , Preston F E , Greaves M
Published: July 2018

The value of aspirin (ASA) as an antithrombotic agent is limited by the fact that it inhibits the production of prostacyclin (PGI2) by the vessel wall. However, there is evidence that the combination of ASA and dipyridamole (DP) has antithrombotic potential for patients with certain types of thrombovascular disease and there appears to be some synergism between the two drugs. To study the relationship between ASA and DP we have examined the effect of the two drugs, singly and in combination on thromboxane (TX) production and PGI2 synthesis by platelets and vessel walls respectively. Rings of venous tissue prepared from veins obtained during surgery were incubated in Ringers solution with ASA, DP or a combination of both. PGI2 synthesis was assessed by radioimmunoassay for 6-keto PGF1α, its stable metabolite on aliquots of supernatant from the incubation mixture during a thirty minute period. All control samples synthesized PGI2. 0.1mM ASA caused 4299 per cent inhibition of PGI2. DP at a concentration of 0.05mg/ml had no significant effect on PGI2 production compared with values obtained in controls. The combination of DP and ASA caused significantly less inhibition of PGI2 synthesis compared with almost complete inhibition observed with ASA alone.

In similar studies on platelet rich plasma, TXA2 synthesis, determined by radioimmunoassay for TXB2, was abolished by ASA 0.15mM, whereas DP 0.05mg/ml had minimal inhibitory action. However, at lower concentrations of ASA the inhibition of both aggregation and TXA2 synthesis was markedly potentiated by DP 0.05mg/ml.

These results provide an explanation for the reported synergism between ASA and DP.
Pattern Of Plasma Beta-Thromboglobulin, Platelet Factor4 And Factor VIII Complex In Monitoring Haemolytic Uremic Syndrome In Children
Authors: Cossu M M , Tantalo V , Paracchini M L , Mondonico P , Rossi E , Claris-Appiani A , Tentori F , Edefonti A
Published: July 2018

Seven children, 2-5 years old, with HUS, were investigated sequentially for plasma BTG, PF4/FVIII complex. Plasma BTG is reported both as plasma concentration and as BTG/Platelets/μl ratio. Recent data suggest that BTG and PF4 enhance microangiopathic involvment of HUS, by inhibition of prostacyclin availability from vessel walls (BTG) and by bind ing of sulphated GAGs on the endothelial cells (PF4).High values of FVIII R: Ag/FVIII:C ratio are an index of endothelial damage. Six children were treated with plasma and antiaggregating agents and one child was treated only with antiaggregating agents. A close relationship between FVIII R:Ag/FVIII:C ratio and BTG and PF4 levels was found. These laboratory findings were in agreement with the clinical course: high values of FVIII R:Ag/FVIII:C ratio, of BTG and PF4 levels were found in the acute phase of the disease and normal values during remission. All patients had a favourable course of the disease and have now normal renal function. We think that plasma infusion may normalize the platelets/vesseI wall interaction.
Fibrinogen Proteolysis And Platelet Activation In Myeloproliferative Disorders
Authors: Ireland H , Lane D A , Wolff S , Pegrum G D
Published: July 2018

We have studied 18 patients with myeloproliferative disorders to determine whether their abnormal platelet function and increased plasma β thromboglobulin (β TG) are associated with activated coagulation and fibrinolytic systems. Of these patients, 8 had polycythaemia rubra vera, 8 had myelofibrosis and 2 had thrombocythaemia. We measured plasma concentrations of the thrombin sensitive fragment fibrinopeptide A (FPA), the plasmin sensitive fibrinogen fragment B β1-42, and β TG by radioimmunoassay. We also studied platelet aggregation in response to ADP. Mean normal values (n = 20) for FpA, B β1-42 and βTG were 1.06, 1.59 and 0.80 pmol/ml respectively. The 18 patients showed minimal plasma thrombin and plasmin activities with a disproportionately large platelet release. Mean FpA, B β1-42 and β TG levels were 1.74, 3.31 and 3.12 pmol/ml, respectively. These patients exhibited increased, decreased and normal platelet aggregation but no specific defect was associated with any particular radioimmunoassay result. 5 of these patients, 4 of whom had increased aggregation, were treated with aspirin in sufficient doses to eliminate their secondary response to ADP. FPA and B β1-42 were not altered but β TG was reduced from 3.39 to 2.72 pmol/ml. We also studied 10 patients with idiopathic polycythaemia. These patients showed a normal platelet response to ADP. Their FPA, B β1-42 and β TG levels were similar to normal, 0.7, 1.93 and 0.97 pmol/ml respectively. We conclude that in myeloproliferative disorders (a) the increased plasma concentration of β TG is not associated with a particular platelet aggregation abnormality (b) in the majority of patients the increased β TG level is not a consequence of thrombin action (c) the raised FpA and B β1-42 plasma levels in the minority of patients cannot be attributed solely to increased red cell mass (d) much of the β TG release is probably independent of the cyclooxygenase pathway.
Platelets Provide A Source Of Thromboxane B2 (TXB2) During Endotoxemia
Authors: Stuart Marie J
Published: July 2018

Thromboxane B2 the stable end product of Thromboxane A2 plays a central role in endotoxin shock in the experimental animal (Cook et al, JCI 65: 227, 80). Plasma Thromboxane B2 is elevated following the infusion of endotoxin, and animals pretreated with a thromboxane synthetase inhibitor are protected from its deleterious effects. The tissue source of Thromboxane A2 during endotoxemia has not however been delineated. Since one of the end products of platelet Ara- chidonic Acid metabolism is Thromboxane B2, we studied the effects of endotoxin (E. Coli, 1 μg/ml) on platelet Arachidonic Acid release and conversion to Thromboxane B2. In paired experiments, in the presence of endotoxin, the addition of thrombin (0.5 U/ml) caused human platelets to release 29.1 ± 3.4% (1S.E.) of 14C Arachidonic Acid. This value was increased (p<0.02) when compared to Arachidonic Acid release in the absence of endotoxin (21.9 ± 3.6%). Similarly, Thromboxane B2 production was elevated (p <0.01) when platelets were preincubated with endotoxin prior to the addition of thrombin (6.1 ± 0.6), compared to Thromboxane B2 formed in its absence (3.4 ± 0.5). Endotoxin thus facilitates the release of Arachidonic Acid from platelet phospholipids, and enhances conversion of the released Arachidonic Acid to Thromboxane B2 i.e. one of the sources of Thromboxane B2 during endotoxemia is the platelet. Since Thromboxane A2 is a potent aggregator, this study also provides a mechanism for the thrombocytopenia observed in endotoxemia.
Platelet Secretion Defects In Patients With Minimal Brain Dysfunction
Authors: Rao A K , Koike K , Holmsen H , Mueller P
Published: July 2018

Twelve MBD patients (P) with mucocutaneous bleeding diathesis (ages: 10-58 years, 11 females) had normal bleeding time, platelet count, activated PTT, PT and F-VIII activity. The mean threshold concentrations of ADP, epinephrine, collagen and ristocetin to elicit secondary aggregation or >20% secretion of preabsorbed 14C-serotonin in the patients’ platelet rich plasma (citrate) were normal. The aggregation response of gel-filtered platelets to 8uM divalent cationophore A23187 was markedly reduced (P=20.9 ± 6.6%, normal (N) = 61.4 ± 5.7%; p<0.001). The platelet contents of ATP, ADP, low-affinity platelet factor-4 (LA-PF4), β-N-acetyl- glucosaminidase (β-N), β-glucuronidase (β-G), and α- mannosidase (α-M), were normal. However, secretion of ATP + ADP (P=31.1 ± 5.3%, N=51.5 ± 6.4%, p<0.05), β-N (P=6.1 ± 2.5%, N=29.8 ± 5.2%, p<0.001) and β-G (P=1.8 ± 1.1%, N=10.5 ± 2.6%, p<0.01) in response to centrifugation of unfixed A23187-treated platelets from patients was markedly impaired, while of LA-PF4 and α-M was normal. Secretion with 0.1 u/ml thrombin was impaired for ATP + ADP, β-N and α-M; secretion of LA-PF4 and β-G was normal. Liberation of pre-incorporated 14C-arachidonate from phospholipids by thrombin (0.05-5u/ml) and production of thromboxane B2 during blood clotting were normal. Thus, platelets from these MBD patients have normal stores of secretable constituents and normal arachidonate pathway, but markedly impaired mechanism for dense granule and acid hydrolase secretion and impaired aggregation response to A23187, suggesting that impairment is in pathway common to aggregation and secretion. Similarity of secretion in platelets and other tissues, raises the question whether a defect in neuronal secretion is part of pathophysiology of MBD.
Lupus-Like Anticoagulant In A Patient With Severe Factor VIII Deficiency
Authors: Weinger R S , Moake J L , Deykin D , Lopez A , Olson J D
Published: July 2018

A severe hemophiliac (<1% FVIII-AHG) was transfused with FVIII concentrate and had orthopedic surgery without complication. His in vivo FVIII-AHG survival was normal (T½= 8hrs), as was his fibrinogen level, prothrombin (PT) and thrombin times. His prolonged (>100 sec.) activated partial thromboplastin time was incompletely corrected (40 sec.; normal<36 sec.) by the in vitro addition of an equal volume of pooled normal plasma, and did not become further prolonged after ½ and 2 hr. incubations at 37°C. A specific inhibitor to FVIII-AHG was not present. However, a thromboplastin inhibition test using patient plasma and progressively higher dilutions of rabbit brain thromboplastin (whole phospholipoprotein membranes) in a PT test system was positive. When either normal gel-separated platelets (GSP) or a dilute suspension of inosithin was used as a source of phospholipid, and clotting then initiated by the simultaneous addition of calcium and Russell’s viper venom (to activate FX directly), patient plasma clotting times were similar to the clotting times of normal plasma and other severe FVIII-AHG deficient plasmas. In contrast, when both normal GSP and dilutions of tissue thromboplastin reagent were added to plasma, and clotting then initiated by recalcification, patient plasma clotting time was prolonged in comparison to the clotting times of normal plasma and other FVIII-AHG deficient plasmas. This “lupus-like” anticoagulant had no effect on in vivo hemostasis, even in our patient with severe hemophilia A. The anticoagulant differs from a lupus anticoagulant recently well characterized (Thiagarajan et al., J.Clin. Invest. 66;397,1980) in that it interacts better with phospholipoprotein membranes than with free phospholipids, and the in vitro defect is not corrected by the addition of normal platelets.
Activation Of Extravascular Coagulation In The Inflamed Joints In Rheumatoid Arthritis (RA)
Authors: van Ginkel C J W , Oh J I H , van Mourik J A , van Aken W G , Vreeken J
Published: July 2018

Deposition of fibrin-like material on the synovival membrane of inflamed joints is a prominent and persistent feature of RA. To evaluate the involvement of the classical coagulation cascade in the formation of this material, fibrinopeptide A (FPA) and coagulation factors were analyzed in synovial fluid from RA patients. FPA was 1100 ng/ml (range 300-3200; n=l6), compared to 200 ng/ml (60-300;n=13) in synovial fluid from patients with osteoarthritis. No significant change (p> 0.2; n=l4) in FPA immunoreactivity (using R2 anti-FPA serum from Dr Nossel beside the antiserum from our institute) was observed after thrombin treatment of the diffusate of dialyzed synovial fluids. This finding indicates that the high levels of FPA immunoreactivity in joint fluids of RA reflect FPA (Aα 1-16) instead of FPA-like fibrinogen fragments (which can be cleaved from fibrinogen by e.g. granulocytic proteases). In view of the finding that all coagulation factors including fibrinogen were sufficiently present (30-100%; n=l8) - except FV (<5%) - the accumulation of FPA suggests activation of extravascular coagulation in the inflamed joints in RA.

To investigate the mechanisms of this activation, normal plasma was incubated with human articular cartilage and activation of FXII was determined by measuring the generation of bradykinin (radioimmunoassay). Cartilage activated FXII whereas its activation capacity was strongly enhanced by pre-exposition to granulocytic enzymes (compare in vivo!). Among its constituents collagen (type II) activated FXII, but proteoglycans were only weak activators. Both substances were isolated from human cartilage, obtained from a normal knee joint at necropsy. With respect to the contribution of the extrinsic coagulation pathway it was shown that synovial fluids from RA patients stimulated the thromboplastin generation by normal human monocytes. Taken together, these findings suggest that both intrinsic and extrinsic coagulation pathways, presumably triggered by the inflammatory process itself, are involved in articular fibrin deposition.
Antithrombin Assay - A Study Of Reaction Conditions, Reagents And Kinetics
Authors: Friberger P , Knös M , Andersson M
Published: July 2018

Antithrombin (AT) in plasma diluted as much as 1:600 in the final incubation mixture reacts rapidly and essentially quantitatively with thrombin in the presence of 3 IU/ml of heparin using tris buffer pH 8.3 and I 0.15. Normal plasma levels of AT consume approximately 60% of human thrombin when 7 nmol/1 is added. The reaction is similar to a 2nd order reaction, k2=8.106 1/mol.sec and is almost complete after 3 min incubation. The excess amount of thrombin is then determined with the chromogenic substrate S-2238.

Data collected prove: 1. The toal amount of the AT cofactor activity is titrated. 2. Other plasma inhibitors do not interfere. 3. The quality and/or source of thrombin and heparin is of definite importance. 4. The procedure for preparing the standard curve and the selection of standard is critical. 5. Reagents with good stability can be selected.

The assay is performed in two steps but easy handling is obtained by the use of the same volume of each reagent and the selection of suitable incubation times - all in order to allow reproducible results also in a routine laboratory. This is important since there are small differences between normal and pathologic values.
Kinetics Of Thrombin, Antithrombin III And Heparin Interactions
Authors: Olson S T , Evans S A , Shore J D
Published: July 2018

The rates of thrombin inactivation by AT III were studied by stopped-flow fluorimetry using p-aminobenzami- dine as an active site probe. p-Aminobenzamidine binds to thrombin with a of KD of 35 μM and a 50-fold fluorescence enhancement. The bimolecular rate constant for the thrombin-AT III reaction was 8.7 × 103 M-1 s-1 at 25° C and pH 7.4, with no diminished amplitude or evidence for a limiting rate at AT III concentrations as high as 10-4 M. Using lower AT III concentrations, an identical bimolecular rate constant was obtained by discontinuous assay of residual thrombin activity. Inactivation of thrombin by antithrombin III complexed with stoichiometric high affinity heparin at concentrations between 2.5 to 20 μM resulted in biphasic reactions: a rapid phase complete in less than 10 milliseconds and a slow phase with a rate constant of 4-6 s-1 . Relative amplitudes and slow phase rate constants showed slight changes over this AT III-heparin concentration range. These results are consistent with weak initial binding of AT III to thrombin in the absence of heparin (KD> 10-4 M), with a major part of the heparin rate enhancement due to a substantial decrease in the KD for the initial binding step. Supported by Michigan Heart Association and USPHS grant F32 HL06171-01.
Determination Of Antithrombin III Heparin-Binding Capacity By High Performance Liquid Chromatography
Authors: Nunez H A
Published: July 2018

The heparin-binding capacity of purified human antithrombin III (AT) both native and after a variety of denaturat- ing conditions was measured using a high performance liquid chromatographic (HPLC) system.

An exclusion chromatography, TSK-3000, 0.75 (ID) × 50 cm column equilibrated with 0.1M NaCl in 0.02M potassium phosphate buffer pH 7.35, at a flow rate of 1 ml/min was used. Under these conditions two essentially resolved peaks were obtained at 10.5 to 14 min. The peak corresponding to the unbound AT, or native AT in the absence of heparin (H), is sharp; that of the AT-H complex is broad due, probably, to the size heterogeneity of the H used in the tests. Without H, aggregates of AT are also resolved by this technique. The areas under the curve of the protein peaks monitored spectrophotometrically in the absence and in the presence of an excess of H are used to quantitate these species. A titration of AT with increasing amounts of H indicates the concentration of H sufficient to bind all the H-binding AT present in a given AT containing sample.

The results of the HPLC method correlate well with those obtained by two-dimensional or crossed Immunoelectrophoresis (CIEP), a technique that can be used to estimate the H-binding capacity of AT. Thus, for example, in non- pasteurized, pasteurized and heated AT samples, for which the HPLC method indicated that 5, 25 and 80% of the AT had lost its H-binding capacity, the CIEP gave similar results.

The results indicate that the HPLC method can adventage- ously be used to determine quantitatively the H-binding capacity of AT and, if present, AT aggregates, thus appearing more informative than CIEP. The analysis time is less than 30 minutes and uses inexpensive reagents.
Fractionation Of Heparin By Human PF-4 Affinity Resin
Authors: Aiken Martha , Novoa Eduardo , Fareed Jawed , Walz Daniel A
Published: July 2018

Platelet factor 4 (PF-4) is a high affinity heparin binding protein, specifically released from the alpha granule upon platelet activation. We have coupled purified human PF-4 to Sepharose and utilized salt gradients to fractionate commercial porcine sodium heparin. Under these conditions, all of the applied heparin bound to the PF-4, uni ike heparin fractionation by antithrombin but similar to heparin fractionation by thrombin or protamine. Three heparin fractions can be distinguished, eluting at approximately 0.5, 0.7 and 0.85 M NaCl. When each fraction was evaluated for its specific activity, no differences were apparent between the starting product and these fractions for USP units. However, the three fractions were each capable of prolonging the activated PTT clotting time of citrated pooled monkey plasma. The final fraction, 0.85 M, gave a four-fold longer APTT time than non-heparinized control plasma. There was no resolution of the antithrombotic and anticoagulant heparin activities. Each of these fractions is capable of competing effectively with PF-4 and its purified proteoglycan (PG) carrier to cause dissociation of the natural PG-PF-4 complex. These systems indicate that PF-4 whether released singularly or in complex form, will readily bind to natural heparinlike compounds with a high affinity and will function as a competitive inhibitor of the anti thrombin of plasma. Prior resolution of heparin, by chromatography on thrombin or protamine resins does not result in the purification of a more highly active fraction than was obtained by PF-4 chromatography alone. Experiments are underway to determine whether such procedures yields heparin with significantly longer animal survival times.
Gyki 14,451, A Specific Inhibitor Of Thrombin:“In Vitro” And“In Vivo”Studies
Authors: Tremoli E , Maderna P , Colli S , Morazzoni G , Paoletti R
Published: July 2018

The effects of a synthetic tripeptide, Gyki 14,451(Boc-D Phe-Pro-Arg-H)have been studied in vitro on human platelet aggregation and arachidonic acid (AA) metabolism and in vivo on experimentally induced venous thrombosis in the rat.

1 μM Gyki 14,451 concentration selectively inhibits thrombin induced platelet aggregation as well as malondial- dehyde (MDA) and thromboxane B2 (TXB2) formation by platelet rich plasma (PRP) stimulated with thrombin. Far higher concentrations (400μM)of the peptide are required to exert an inhibitory effect when collagen, ADP and AA are used to stimulate platelets. No effect has been observed on the conversion of 14C AA to metabolites using unstimulated platelets. Kinetic studies of MDA production by platelets stimulated with thrombin and its inhibition by Gyki 14,451 (0.15,0.3,0.6 uM) suggest that the peptide interacts with thrombin by an apparently competitive mechanism.

4 mg/Kg of Gyki 14,451, intravenously injected in the rat caudal vein, completely inhibited the occurrence of venous thrombosis, obtained by vena cava ligature.

The oral administration of the peptide (50 mg/Kg by gastric intubation) failed to reduce the percentage of incidence of venous thrombosis (88% in controls versus 90% in treated rats) resulting only in a reduction of the thrombus weight. These data suggest that the anticoagulant peptide Gyki 14,451, given intravenously, possesses a consistent activity in the prevention of experimentally induced venous thrombosis.
Fluorescence Studies On The Mode Of Action Of Two Synthetic Thrombin Inhibitors, No. 205 And No. 805
Authors: Nagano S , Okamoto S , Ikezawa K , Mimura K , Matsuoka A , Hijikata A , Tamao Y
Published: July 2018

Among a novel series of synthetic thrombin-inhibitors belonging to arginine derivatives found by S. Okamoto et al. , No. 205, dansyl-arginine-piperidineamide, was reported by Mann and his associates to show the enhancement of fluorescence intensity when bound to thrombin. However, No. 205 was reported by S. Okamoto et al. to inhibit pseudocholinesterase with very low Ki also, while No. 805, (4R- methyl- [ methyl-tetrahydro-quinolinesulfonyl-arginyl- ] 2R- piperidinecarboxylic acid), showed practically no inhibitory action on pseudocholinesterase remaining very potent inhibitory action to thrombin with a Ki of 0.019 μM. Therefore, comparative studies of fluorescence variation with No. 205 and No. 805 were made using thrombin, pseudocholinesterase and truecholinesterase as the target enzymes and the following results were obtained. 1) The enhancement of fluorescence intensity of No. 205 with thrombin was corn- firmed but the same order enhancement of fluorescence intensity of No. 205 was also observed with pseudocholinesterase, but not with truecholinesterase (to this enzyme Ki of No. 205 was large), indicating that the enhancement is concomitant with the inhibitory binding of No. 205 with the enzymes. 2) The enhancement of fluorescence intensity of No. 205 with thrombin was clearly reversed by the addition of No. 805, indicating the replacement of No. 205 bound to the thrombin by No. 805. This supports the possibility of the fluorometric differentiation of thrombin from pseudocholinesterase, which allows us to discuss the histo- chemical application of synthetic thrombin inhibitors to demonstrate the local thrombin.
Interaction Of Protamine Sulfate With Thrombin
Authors: Cobel-Geard R , Penner J A , Hassouna H I
Published: July 2018

Protamine sulfate, a simple protein M.W. 4,600, is a known antiheparin agent which, in the absence of heparin, demonstrates an anticoagulant activity both in vitro and in vivo. Much work has been done to elucidate the interactions of heparin with thrombin and ATIII, however, little is known about the mechanism of protamine sulfate anticoagulant activity and its effect on thrombin.

In binding studies, radiolabeled thrombin was reacted with protamine for 1 hour and ATIII was then used as a probe to test the availability of the thrombin active site. ATIII/*thrombin complex was separated from free *thrombin on a Sephacryl S200 molecular sieve column. As a result of preincubating thrombin with protamine, ATIII was unable to bind thrombin. Functional assays for thrombin activity, were carried out in a system where time taken for formation of a thrombin catalyzed fibrin clot was the measure used for thrombin activity. Effect of protamine on thrombin activity when tested in this system demonstrated that with increasing molar concentrations of protamine, there is a corresponding decrease in thrombin activity. Maximum inhibition of activity occurred at a protamine concentration of 200 μM. This inhibition was concentration dependent, partial and reversible. In contrast the amydolysis of the synthetic substrate H-D-Phe-Pip-Arg-p-nitroanilide by thrombin was unaffected by protamine.

Results of these studies have confirmed the anticoagulant role of protamine sulfate and provided information on its interaction with purified thrombin. Since the drug did not totally inhibit functional activity of thrombin, we feel that it binds the enzyme, partially blocking the active site.
Sex-Related Differences In Aspirin Pharmacokinetics Following IV Injection In Rabbits
Authors: Buchanan M R , Rosenfeld J , Hirsh J
Published: July 2018

Clinical and experimental studies suggest that aspirin (ASA) is a useful antithrombotic agent; however, its efficacy seems limited to males. This may be due to differences in i) the effects of ASA on platelets and/or vessel wall or ii) the pharmacokinetics of ASA, between males and females.

We examined the possible sex-related differences in 1) enhanced arterial thrombosis 3 h after treatment with 0, 3, 10 or 100 mg/kg ASA and 2) the distribution (α) and clearance (β) of ASA between male and female rabbits. Enhanced arterial thrombosis was determined by measuring 51Cr-platelet accumulation onto injured carotid arteries. ASA-plasma levels were determined, using a GC/MS assay.

51Cr-platelet accumulation onto injured arteries was 0.67 ± 0.15 × 106 (males) and 0.52 ± 0.07 × 106/100 mm2 vessel wall (females; mean ± SEM, n=8). 3 mg/kg ASA had no effect. 10 and 100 mg/kg ASA significantly enhanced 5lcr- platelet accumulation to 1.94 ± 0.41 × 106 and 2.59 ± 0.93 × 106 in males, and to 1.65 ± 0.32 × 106 and 2.16 ± 0.73 × 106 in females (p<0.001). There were no sex-related differences.

ASA-plasma levels were determined in serial samples collected from 10 rabbits injected with 10 mg/kg ASA. One week later, the study was repeated in the same animals. The α and β T½’s were 3.3 ± 0.2 and 29.2 ± 3.0 (min) respectively in males after the 1st treatment. Similar T½’s were found after the 2nd treatment (3.4 ± 0.1, 23.3 ± 3.5 respectively). In females, the initial α and β T½’s were 3.7 ± 0.2 and 26.1 ± 3.5 respectively. The α T½ was similar (3.6 ± 0.1) whereas the β T½ was significantly decreased (14.8 ± 0.3, p<0.007) after the 2nd ASA treatment. This decrease in females was associated with an increase in the apparent volume of distribution, p<0.02

The significance of these findings is uncertain.
Sex-Associated Differences On The Effect Of Asa Or Flurbiprofen On Platelet Function Ex Vivo
Authors: Nishizawa E E , Molony B A , Meinzinger M M , Williams D J
Published: July 2018

It has been reported that although ASA may have some beneficial effects in males, no differences from placebo was detected in females in clinical trials on venous thrombosis, TIA and stroke. Since ASA inhibits platelet function and because the end-points measured in the above clinical studies may be related to platelet function, we measured bleeding time and platelet aggregation induced by collagen or arachidonic acid following oral administration of ASA (650 mg) or flurbiprofen (100 mg) in normal male and female volunteers (10 per group, total of 40 subjects) between the age of 50 and 70.

No sex-associated differences in response with either drug were observed in bleeding time or platelet function. Thus, we were unable to confirm that the observation of beneficial effect of ASA in males was due to its effect on platelets. However, more interestingly there was a statistically shorter bleeding time in males (p = 0.026) before administration of drug. It was also found that despite the normal response to collagen, PRP prepared from pre-drug blood samples from 6 individuals (15%) did not aggregate when stimulated with arachidonic acid at concentrations as high as 1 mM. These results suggest that, at least in some individuals, collagen-induced aggregation may proceed by a pathway independent of the arachidonic acid pathway.
Unique Degradation Of Fibrinogen By Western Diamondback Rattlesnake Venom Proteases
Authors: Pandya Bharat V , Budzynski Andrei Z , Rubin Ronald N , Olexa Stephanie A
Published: July 2018

The venom of western diamondback rattlesnake, Crotalus atrox, renders blood unclottable even after addition of thrombin. The phenomenon is due to a limited proteolytic degradation of fibrinogen. Purified fibrinogen (1 mg/ml) or human plasma incubated with crude venom (7.5 μg/ml) became unclottable. Fibrinogen in a purified system was affected at a faster rate than that in plasma suggesting that plasma protease inhibitors partially inhibited the proteolysis of fibrinogen. The degradation of plasma fibrinogen did not result from plasminogen activation since the venom did not activate purified human plasminogen as determined by an amidolytic assay and SDS polyacrylamide gel electrophoresis. The degradation of the fibrinogen molecule (Mr 340,000) proceeded steadily with a continuous cleavage of small peptides as demonstrated by SDS polyacrylamide gel electrophoresis. The bulk of the degraded fibrinogen was represented in gels as a single band with gradually increasing electrophoretic mobility as the incubation time progressed. The correlation of molecular weight and clottability demonstrated that the earliest unclottable derivative had Mr 285,000; its Aα and Bβ chains were degraded loosing peptides of Mr 20,000 and 6,500, respectively, but the γ chain appeared intact. The degradation pattern of fibrinogen in plasma was different than that of fibrinogen alone. An unclottable fibrinogen derivative isolated from plasma by precipitation with 2.2 M glycine had Mr 325,000; its Aα and γ chains appeared unaffected but the Bβ chain had lost a peptide of Mr 6,500. This product represents a novel unclottable derivative of fibrinogen with apparently intact Aα and γ chains, cleaved Bβ chain and high molecular weight. The cleavage of the Bβ chain would indicate that the intact Bβ chain may have an important role in the conversion of fibrinogen to fibrin clot.
DES-AA Fibrin Clearance In Healthy Human Subjects
Authors: Brosstad F , Harrbora T , Holm B , Godal H C , Kierulf P
Published: July 2018

Plasma clearance of I125 des-AA fibrin mcncmers was studied in healthy subjects (N=7). Labelled mcnaners (fm), made fran labelled fibrinogen (F) in 2,5 M urea, pH 7,4 exposed to insoluble reptilase (Brosstad et al., Thrarto .Res. 13, 1978) vere solubilized upon addition to test-subject plasma, and subsequently injected. At intervals, citrated plasma samples ware tested for: I. Total radioactivity (Tr). II. Radioprecipitability (Rp) with trichloroacetic acid. III. Radioclottability (Rc) with plasmin-free thrcmbin (pH 6,3 1h). IV. Radiodistribution after ultrasentrifuga-ticn (Airfug, Beckmann) and V. Radiodistributicn in SDS-PAGE gels after electrophoresis of harvested EDTA-plasma clots, solubilized in urea-SDS.

RESULTS: Prior to injection, plasma demonstrated: Rp = 100 %, Rc = 94 %, single peak ultracentrifuge pattern and 95 % of total radioactivity moving as F on SDS-gels. Almost identical results were obtained in all subjects. After 30 min., Tr had dropped to 50 % of Tr value at 5 min., being 10 % and 2 % after 420 min. and 48 h., correspondingly.
A parallell drop in Rp and Rc scarcely noticeable at 10 min., reached a mininun of 30 % at 360 min; the Airfug and SDS-gel patterns substantiating these findings. Due to non-linearity on semilog. plots, a definite half-life of des-AA fibrin cannot be stated from decay parameters (Tr) alone, but metabolic data as outlined above (Rp, Rc, Airfug, SDS-elpho) suggest a half-life of 30-60 min .. In five subjects, I131-des-AA fibrin and I125-fibrinogen was injected simultaneously. Clearance rates characteristic of that of the individual molecular species were obtained. The extremely rapid elimination of fibrin ccnpared to that of fibrinogen suggests specific receptors on RES- cells to be involved.
Plasma Fibronectin Levels: Effect Of Obtaining Blood For Assay VIA Indwelling Plastic Catheters
Authors: Snyder E , Barash P , Mosher D
Published: July 2018

Plasma fibronectin (PFN) concentrations are reported to be decreased in patients with sepsis and disseminated intravascular coagulation. In such critically ill patients vascular access is often limited, and the indwelling plastic catheters inserted for clinical monitoring are often used for obtaining blood samples. Despite slow continuous infusion of heparinized saline, these catheters often become coated with fibrin. Since PFN is known to be precipi table by heparin and to be bound to fibrin(ogen), levels of PFN obtained from blood drawn through such indwelling catheters may be artifactually depressed. To provide data, levels of PFN were determined from blood samples drawn simultaneously through the distal port of Swan-Ganz (110 cm length), plastic arterial (3.2 cm length) or central venous pressure(CVP 20.3 cm length) catheters, and compared with those levels obtained after a percutaneous venipuncture. All catheters were in situ for 24-48 hours prior to blood sampling. The arterial and Swan-Ganz lines were flushed with heparinized saline continuously (2 units/ml;3 ml/hour). Prior to sample collection, 5 ml of blood was aspirated through the catheter and discarded. Plasma was frozen at -80C until assay. PFN concentration was determined using an electroimmunoassay.

We conclude that PFN levels determined from blood drawn through any of the plastic catheters studied were equivalent to those levels obtained from a percutaneous venipuncture. In critically ill patients in whom vascular access is limited, PFN concentration may be reliably obtained from blood samples collected through indwelling catheters.
Determination Of Malondialdehyde In Normal And Diabetic Pregnancies
Authors: Rákóczi I , Geró Gy , Demeter J , Gáti I
Published: July 2018

It is known that platelet hyperaggregation observed in diabetic patients is, at least in part, due to an increased activity of the endoperoxide-thromboxane forming metabolic pathway. It was interesting to determine the platelet malondialdehyde /MDA/ production in normal and diabetic pregnancies. Following individuals have been studied: /I/ twenty-five healthy non-pregnant volunteers; /II/ thirty women in third trimester of non-complicated pregnancies; /III/ twenty two diabetic pregnant women without retinopathy; /IV/ fifteen diabetic pregnant women with retinopathy. Platelet MDA production following N-ethyl-maleimide induced aggregation was measured according to Stuart et al. The mean value of MDA production was similar in volunteers and normal pregnant women /SDM, 7.07±0.73 nmoles MDA per 109 platelets; 7.22±0.81/. The mean MDA production in diabetic women without retinopathy was slightly but nonsignificantly higher than that in normal pregnant women /7.57±1.02; p>0.05/. The corresponding value in diabetic women with retinopathy was significantly higher than the values in the other three groups /8.47±0.82; p<0.01/. These data suggest that the activation of prostaglandin synthetic pathway /measured by MDA/ is significantly increased in diabetic pregnancy complicated by retinopathy. The increase of platelet prostaglandin synthesis in diabetic pregnancy might play an important role in initiating and/or promoting the small-vessel complications of placenta.
Increased Platelet Aggregation and Decreased Sensitivity of Platelets to Synthesized Prostacyclin in Alloxan-Diabetic Rabbits
Authors: Yamada K , Yoda T , Goto Y , Hirata T , Serizawa K
Published: July 2018

Platelet aggregation and sensitivity of platelets to prostacyclin were examined with a view to clarify platelet function in alloxan-diabetic rabbits. Diabetic rabbits were obtained by intravenous injection of alloxan (100mg/ kg body weight) and range of plasma glucose in these diabetic rabbits was 350-500mg/100ml. Blood was collected from the central artery of the ear of normal and alloxan- diabetic rabbits. Platelet aggregation was measured turbidometrically as rate of light transmission at maximal aggregation to light transmission of platelet poor plasma by a NKK aggregometer (Tokyo, Japan). Platelet aggregation was induced by ADP with each final concentration of 0.5, 1.0, 2.0 and 6.0 μM. Sensitivity of platelets to synthesized prostacyclin (Ono Pharma. Co., Tokyo, Japan) was represented as prostacyclin concentration of fifty percent inhibition of ADP-induced platelet aggregation.

Platelet aggregation rate in normal and alloxan-diabetic rabbits was as follows; 8.4±1.0, 22.8±2.6%(0.5 μM), 22.3±1.5, 35.2±3.5%(1.0 μM), 37.1±1.7,50.0±2.6%(2.0 μM), 51.0±2.2, 66.7±7.0%(6.0 μM ADP)(Mean±S.E., P< 0.005). Prostacyclin concentration of fifty percent inhibition of ADP-induced platelet aggregation in normal and alloxan- diabetic rabbits was as follows; 42.37±1.68, 48.50±0.48 ng/ml (Mean±S.E., P< 0.01).

In conclusion, platelets of alloxan-diabetic rabbits presented a significant decrease in sensitivity to synthesized prostacyclin as compared to platelets from normal rabbits, with a significant increase in ADP-induced platelet aggregation.