bluuebunny/crossref_metadata_2025 huggingface dataset:
CHARACTERIZATION OF THE FIBRINOGEN BINDING SITES USING MONOCLONAL ANTIBODIES TO HOMAN PLATELET MEMBRANE GLYCOPROTEINS IIb/IIIa*
Authors: Ruan C , Du X , Wan H , Hu X , Xi X , Li P
Published: August 2018

Two new murine monoclonal antibodies, SZ-21 and SZ-22, (both IgG1 subclass), were produced by the hybridoma technique using washed human platelets as the immunogen. Both SZ-21 and SZ-22 reanted specifically with normal platelets and megakaryocytes but not with Glanzmann's thrombasthenic platelets which lack the membrane glycoprotein(GP)IIb/IIIa complex. Platelets from 10 normal donors bound 64,500±20,300(x+SD) SZ-21 molecules/platelet with KD4.4±1.5nM and 61,000±19,900 SZ-22 molecules/platelet with KD18.8±6.7nM respectively.Affinity chromatography confirmedthat SZ-21 and SZ-22 reacted with theGPIIb/IIIa complex. SZ-21 inhibited the platelet aggregation and secretioninduced by collagen, arachidonic acidand thrombin,and the second wave of aggregation in response to ADP, adrenaline and ristocetin. The fibrinogen binding to human platelets induced by ADP, arachidonic acid and PAF was also inhibited by SZ-21. But SZ-22 hado effects on platelet aggregation andfibrinogen binding. Western blot analyses indicated that SZ-21reanted with GPIIIa and that SZ-22 bound to GPIIb. When the protein was reduced by 2-mercaptoethand, SZ-22 reacted with the enchain of GPIIb, but SZ-21 lost its ability of binding to GPIIIa, suggested that the antigenic determinants of SZ-21 depended on the integrity ofthe intra αchain disulfide bond of GPIIIa. On chymotrypsin treated platelets,the epitope for SZ-21wasidentified on a 66 KD membrane-bound fragment of GPIIIa. The appearanceofthe 66 KD fragment was related withthechymotrypsin-mediated fibrinogen binding and aggregation. However the prolonged treatment with chymotrypsin reduced the platelet aggregation, which coincided with the appearance of the 66KD fragment, a product of thefurther hydrolysis of the 66KD fragment.These results suggested that the domainbetween the 66KD and 60KD fragments of GPIIIa might be essential for the maintenance of the fibrinogen binding sites.

* Supported by the Science fund of the Chinese Academy of Sciences ( project No.82-172 ) and the National Science fund of China ( project No.3860713 ).
PLATELETS, ENDOTHELIUM AND VASOSPASM
Authors: Vanhoutte Paul M
Published: August 2018

The endothelium can secrete both relaxing and contracting substances. One of the most powerful stimuli to the release of the former are thrombin and aggregating platelets. This contributes to the protective role of the endothelium against inappropriate intraluminal platelet aggregation and coagulation in blood vessels with an intact intima. Thrombin-induced, endothelium-dependent relaxations have been obtained in isolated arteries of different species, including humans. Endothelium-dependent relaxations can be evoked by autologous platelets in isolated blood vessels of the dog, pig and rat; they can be obtained in canine coronary arteries with human platelets. The major platelet-products involved in these endothelium-dependent relaxations are 5-hydroxytryptamine (serotonin) and the adenine nucleotides. Although platelet-activating factor (PAF) can evoke endothelium-dependent relaxation it only does so at concentrations much higher than those occurring under physiological conditions; since the relaxations are not prevented by PAF-antagonists, they are non-specific in nature.The receptor mediating the endothelium-dependent relaxations to serotonin released from the aggregating platelets can be subtyped as a S1~(5HT1) serotonergic receptor;those mediating the response to the adenine nucleotides as P2y-purinergic receptors. In the absence of the endothelium aggregating platelets cause contractions of vascular smooth muscle; these are mediated by a mixture of S1-like and S2~serotoner-gic receptors in coronary arteriesof the dog, and by S2-serotonergic receptors in those of the pig. Thus, in the porcine coronary artery, the S2-serotonergic antagonist ketanserin markedly enhances the platelet-induced endothelium-dependent relaxation. After previous (four weeks) injury, the regenerated endothelium of the porcine coronary artery loses the ability to respond to serotonin,and is unable to prevent the constrictionsevoked by aggregating platelets. The endothelium-dependent relaxations of porcine coronary arteries evoked by aggregating platelets are potentiated by chronic treatmentof the donor animals with cod liver oil. These studies emphasize the protective roleof the endothelial cells against the vasoconstriction (vasospasm) induced by aggregating platelets. This role is depressed after previous injury, and can be facilitatedby dietary adj ustments.
CARBOHYDRATE COMPOSITION AND LECTIN BINDING AFFINITIES OF HUMAN PLACENTAL TISSUE FACTOR
Authors: Scheefers H , Kobus A , Geyer R
Published: August 2018

Tissue factor (TF) is a widely distibuted membrane glycoprotein and the most potent trigger of bloodcoagulation. It serves as an essential cofactor for the activation of Factor IX and X by Factor Vll/VIIa.TF is a lipoprotein composed of a phospholipid portion and a glycosylated apoprotein (apo-TF). The procoagulant activity of bovine brain TF is inhibited bythe lectin Con A indicating that the carbohydrates of TF might play a functional role in its interactionwith Factor Vll/VIIa.In the present study apo-TF was purified from human placenta by repeated SDS-PAGE to a purity of 95%. The carbohydrates of apo-TF wereanalyzed by capillary gas- liquid-chromatography andmass-fragmentography. This analysis revealed that apo-TF contains about 16% (w/w) carbohydrate consistingof 50.4 mole% N-acetylglucosamine, 22.2 mole% mannose, 21.0 mole% galactose, 3.2 mole% fucose and 3.2 mole% N-acetylgalactosamine. Further information on the structure of the carbohydrate moieties of the apoTF was achieved by determining the binding affinities of the apo-TF to ten different lectins. For this purpose a semiquantitative spot lectino sorbent assaywas developed. This assay is based on the detection of peroxidase-labeled lectins after being bound to the carbohydrate moieties of apo-TF adsorbed onto a nitrocellulose membrane. Human placental apo-TF showed the strongest affinity to wheat germ agglutinin which specifically binds to N-acetylglucosamine and sialic acid residues.In contrast to bovine brain apo-TF, human placental apo-TF only weakly interacted with Con A, which is known to recognize mannosyl residues in mannose-rich, hybrid- and biantennary glycans,but not in tri- or tetraantennary oligosaccharides of the complex type. From the carbohydrate constituent analysis and from the lectin binding studies it can be concluded that human placental apo-TF carriesabout four N-linked higher branched oligosaccharide chains.
MOLECULAR CLONING OF HUMAN TISSUE FACTOR cDNA
Authors: Edgington T S , Morrissey J H , Fakhrai H
Published: August 2018

Tissue factor (TF), the cell-surface receptor and allo-steric activator for factor Vll/VIIa, is important in hemostasis andinflammation. The TF apoprotein was purifiedfrom human brain using factor Vll-affinitychromatography and SDS gel electrophoresis,and was found to consist of a 47 kDa heavy chain plus a 12.5 kDa light chain. Approximately one-third of the heavy chain amino acid sequence was determined for four regions by microsequencing the intact protein and peptides derived from V8-protease digestion. A λgtll cDNA library, made from mRNA derived from the human fibroblastic cell line WI38,was screened with (a) affinity-purified rabbit antibodies to human tissue factor, and (b) a 45-mer oligonucleotide probe based on TF heavy chain amino acid sequence. Five overlapping cDNA clones were identifiedand sequenced which confirmed all four partial TF amino acid sequences. Together these clones span the entire heavy chain coding sequence as well as 5" and 3" nontranslated regions. The N-terminusof the TF heavy chain is preceded by an unusually long signal peptide which appears to be cleaved at alternative sites two amino acids apart. This results in two variants of TF heavy chains which differ slightly in length and amino-terminal sequence.The deduced protein sequence shows no major homology to known protein sequences. However,a relatively uncommon tripeptide sequence, Trp-Lys-Ser (WKS), appears three times in the TF heavy chain. This tripeptidesequence also occurs in HMW kininogen, factor VIII,von Willebrand"s factor andant ithrombin-III. Limited sequence similarity is observed in flanking sequences,andthis may indicate a possible functional domain for the recognition of members ofthe vitamin K-dependent serine protease famil.Supported by NIH grants HL-16411 andCA-41085.
EFFECT OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) ON BACTERIAL ENDOCARDITIS
Authors: Buiting A G M , Thomson J , Emeis J J , Mattie H , Brommer E J P , Van Furth R
Published: August 2018

In bacterial endocarditis the causing microorganisms are located in a fibrin-platelet matrix, making them less accessible to host-defence mechanisms and antibiotic therapy. Trombolytic treatment could break down the fibrin of these endocardial vegetations and thus eliminate the focus of infection. This approach was studied in vitroand in vivo using recombinant t-PA (rt-PA,Wellcome Biotech) having a fibrinolytic activity comparable with melanoma t-PA. The following results were obtained.

1.Incubation of Streptococcus sanguis infected plasma clots in the presence of t-PA resulted in lysis of the clots as evidenced by a significant decrease in the weight of the clots and by an increase in the number of streptococci in the medium.No effect of t-PA was found on the antimicrobial action of penicillin G (PenG)onthe streptococci in the non-lysed part ofthe clots.

2. Vegetations isolated from the heart of rabbits with a S. sanguis endocarditis,incubated in medium with t-PA were also lysed. This resulted in a rise in the mediumof both the number of bacteria and the concentration of fibrin degradation products.

3.Treatment of rabbits with a S. sanguis endocarditis using a combination of rt-PA, given as bolus injections at one hour intervals (4x0.5 or 4x1 mg per kg/day), andPenG decreased the weight of the endocardial vegetations significantly compared toa control group treated with PenGonly (43.3 and 81.8 mg respectively). No additional decrease in weight was obtained with repeated administration of t-PA at daily intervals up to 2 days. The significant decrease of the number of streptococci per gram of vegetation as a result of the treatmentwith PenG was not influenced by t-PA. Treatment with 4x1 mg t-PA per kg caused a small decrease (about 30%) in the plasma concentration of plasminogen, fibrinogen and aα2~antiplasmin.However, no bleeding complications were observed.

In conclusion rt-PA can influence the treatment of bacterial endocarditis in rabbits by decreasing the size of the vegetations but not by influencing antimicrobial action on the bacteria in the non-lysed part of the vegetation.
EPINEPHRINE INDUCED POTENTIATION OF ARACHIDONATE AGGREGATION IN OUIN 2 LOADED PLATELETS IS NOT MEDIATED BY ELEVATION OF CYTOSOLIC CALCIUM
Authors: Rao G H R , Gerrard J M , White J G
Published: August 2018

Previous studies have demonstrated that chelation of ionized cytosolic calcium by Ouin 2 leads to a refractory state in platelets.However, epinephrine (Epi) induced membranemodulation restored the sensitivity of Ouin 2loaded platelets to the action of agonists.Further studies with Ouin 2 and Fura 2 suggested that Epi induced recovery of sensitivity by refractory platelets to aggregation by arachidonate does not require elevation of cytosolic calcium. To further delineate the role of calcium in membrane modulation, we followed phosphoinositol metabolism and myosinlight chain phosphorylation using radiolabeled platelets. The total amount of PI metabolites generated after exposure to Epi, AA or Epi + AA were significantly less than that formed after 0.1 μ thrombin stimulation. Ouin 2 at 40 μM concentration had no inhibitory effect on PI hydrolysis. However, at this concentration it effectively blocked AA induced aggregation. Although Epi treatment restored the sensitivity of Ouin 2 loaded platelets to the action of AA, it did not enhance the formation of increased quantitites of PI metabolites. Similar to earlierstudies, Ouin 2 loading effectively blocked phosphorylation of myosin light chain (20 K).Although a combination of Epi + AA restoredsome phosphorylation of 20 K protein in Ouin2 loaded platelets, the degree of phosphorylation was significantly less than that achieved in control stimulated platelets. Resultsof these studies suggest that Epi induced restoration of sensitivity to refractory, Ouin2 loaded platelets is not mediated by 1) significant elevation of cytosolic calcium, 2) enhanced production of PI metabolites, 3) increased phosphorylation of 20 K protein. Epiinduced membrane modulation is a novel, independent mechanism capable of restoring sensitivity to agonists in platelets with compromised function.
EFFECT OF RECOMBINANT TISSUE TYPE PLASMINOGEN ACTIVATOR(rt-Pa)ON THE PREVENTION OF INTRAABDOMINAL ADHESION FORMATION
Authors: Mohler M , HollenBach S , Nguyen T , Reger V , Hothkiss A
Published: August 2018

The formation of intraperitoneal adhesions, the pathological adherence of organs and tissue surfaces, is the leading cause of postoperative intestinal obstruction following abdominal surgery, as well as the major contributing factor to infertility after reconstructive tubal surgery. At present, there is no generally accepted therapy for the prevention of adhesion formation. Since fibrin has been shown to be the physiological basis of adhesion formation, the current study was undertaken to determine if an application of exogenous rt-PA to the site of peritoneal injury would prevent adhesion formation. New Zealand white rabbits (3 kg) were anesthetized and midline laparotomies performed. A nine cm area of the peritoneal wall was surgically removed and resutured in place, creating an ischemic patch of peritoneal tissue. A proximal area of the cecum (75cm2 ) was abraided with dry gauze until punctate bleeding occurred. Approximately 2.5 grams of an ointment containing 0, 0.16, 0.31, or 0.63 mg rt-PA was applied to the ischemic peritoneal tissue and the abraided cecum. After seven days the rabbits were euthanized and the adhesions scored. In the rabbits which received placebo ointment (n=6) the cecum was adhered to the entire area of the ischemic patch. In the rabbits which received the ointment containing 0.63 mg of rt-PA, five had no adhesions and one rabbit had a very minor adhesion. The high dose of rt-PA was equally effective in preventing reformation of adhesions after surgical lysis. No evidence of systemic fibrinogen degradation or abnormal wound healing were evident in this model.

In summary, these studies demonstrate that intraperitoneal administration of rt-PA is effective in preventing initial adhesion formation as well as the reformation of adhesions after surgical trauma or injury.
ON THE MECHANISM OF THE IN VIVO SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) AND SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA)
Authors: Stassen J M , Collen D
Published: August 2018

t-PA and scu-PA, in molar ratios between 1:4 and 4:1 do not act synergically in vitro (Thromb. Haemost. 56,35,1986) but display marked synergism in a rabbit model (Circulation 74, 838, 1986) and in man (Am. Heart J. 112, 1083, 1986). To investigate the mechanism of in vivo synergism in the rabbit model (J. Clin. Invest. 71, 368, 1983), t-PA and scu-PA were infused 1) simultaneously over 4 hrs, 2) t-PA over 1 hr, then 15 min later scu-PA over 2 hrs and 3) scu-PA over 1 hr, then 15 min later t-PA over 2 hrs.

Significant synergism on thrombolysis is observed when t-PA and scu-PA are infused simultaneously or when t-PA is followed by scu-PA but not when scu-PA is followed by t-PA. These results suggest that low dose t-PA induces some plasminogen activation, sufficient to partially degrade fibrin, exposing COOH-terminal lysines with high affinity for plasminogen (Eur. J. Biochem. 140, 513, 1984). scu-PA might then activate surface-bound Glu-pla-minogen more efficiently.

Sequential therapy with t-PA (or any other agent which "predigests" the thrombus), followed by scu-PA might constitute an alternative to simultaneous infusion of synergistic thrombolytic agents.
RELEASE OF ENDOTHELIUM DERIVED RELAXING FACTOR (EDRF), PROSTACYCLIN (PGI2) AND ANTI-PLATELET ACTIVITY BY RABBIT AORTIC ENDOTHELIUM
Authors: Bult H , Jordaens F , Herman A G , Hove C Van , Verbeuren T J
Published: August 2018

Endothelium may release EDRF and PGI2; the former causes relaxation of arterial smooth muscle cells, whereas PGI2 suppresses phagocyte and platelet activation and may relax some arteries. The aim of the present experiments was to investigate whether EDRF from rabbit aorta affects platelet reactivity. To this end the isolated thoracic aorta was perfused with a Krebs' solution (3 ml/min), andthe perfusate was continuously super fused over a de-endothe-lialized ring of the abdominal aorta, contracted withnoradrenaline in the presence of atropine, to monitor EDRF. Aliquots (200 μl) ofeffluent were also added to 200 (il prewarmedautologous citrate platelet rich plasma, which was immediately stimulated with ADP or U46619, a thromboxane (TX) A2 mimetic. Both agonists induce aggregation independently of TXA2 formation. Finally, the PGI2 content of the effluent was assessed by specific radioimmunoassay of 6-oxo-PGF1alpha(ir-6oxo). Infusion of 0.3,1 and 3 μM acetylcholine (Ach) through the aorta for 7.5 min caused a dose-dependent relaxation of the abdominal ring (33 to 100 % at 3 (μM Ach), indicating intraluminal EDRF release. Maximum relaxation was reached in 3 min and maintained. Release of platelet inhibitory activity was variable. At 3 μM Ach, anti-platelet activity(more than 10 % suppression of aggregation)was present in the effluent of 5 (of 7) aorta's for ADP-, and in effluent of 6 (of 7) aorta's for U46617-induced aggregation. U46619 was more sensitive to the anti-platelet activity, a finding consistent withauthentic PGI2. When a clear anti-platelet activity was monitored, its release was abolished by indo-methacin, which affected neither EDRF release nor platelet aggregation. Baseline release of ir-6oxo was low and variable, but dose-dependently stimulated by Ach, reaching a maximum for each concentration in the 2nd to 3rd minute. In contrast to EDRF, ir-6oxo release was not maintained during the subsequent 4 min and tapered off. Formation of ir-6oxo and anti-aggregatingeffect were positively correlated, but therewas no association between EDRF and ir-6oxo or anti-platelet activity. In conclusion, rabbit aortic endothelium could release EDRF and PGI2, but the present set-up did not reveal an anti-platelet effect of EDRF.
THE EFFECT OF TWO CHOLESTEROL-LOWERING AGENTS ON PLATELETS IN PATIENTS WITH HYPERCHOLESTEROLEMIA
Authors: Norϕ A , Smimonsen T , Ytre-Arene K , Lyngmo B , Svensson B
Published: August 2018

Twenty-one subjects with type Ila hyperlipoproteinemia, receiving dietary treatment were given Synvinolin (MK-733), a HMG-CoA reductase inhibitor, 40 mg or Cholestyramin (Questran) 24 g daily for a period of 12 weeks.Serum lipids, platelet cholesterol, phospholipids and fatty acid composition and platelet function were measured before and after the intake of the two drugs.

Both drugs reduced serum total cholesterol with approximately 50%. No significant changes were observed in platelet lipid concentrations or in the primary bleeding time. Collagen induced aggregation and thromboxane (TXA2) production were reduced, whereas thrombin induced aggregation and TXA2 production were unaffected. This study shows that both a cholesterol synthetase inhibitor and Cholestyramin reduce the total serum cholesterol concentration and also reduce platelet aggregation and thromboxane synthesis without changing the platelet cholesterol content or the cholesterol/phospholipid ratio. The effect on serum lipids and platelet function may indicate a beneficial effect of both drugs on arterial disease in patients with hypercholesterolemia.
PROPERTIES OF PHOSPHATIDYLINOSITOL KINASE IN HUMAN PLATELETS
Authors: Suga K , Uemura Y , Tsuijinaka T , Sakon M , Kambayashi J , Mori T
Published: August 2018

We have reported the specific 32P-labelling in phosphatidyl-inositol-4-monophosphate(PIP) of intact platelets upon addition of the agents which elevate intracellular cAMP (Thrombos.Res.44, 155,1986).This event may be catalyzed by the action of Pl-kinase, the properties of which has not been elucidatedyet.Thereby, attempts were made to assay and to characterize PI-kinase of human platelets.Fresh lysed platelets prelabelled with 32P in cold Tris-HCl buffer containing 2mM EGTA were incubated at 37 C in the presence of MgCl2 for designated times and the phospholipids were extracted and analyzed by thin layer chromatography.32P-labelling in PIP was gradually increased in consort with the decreased labelling in PI-4,5-bisphosphate.As the changes in the labelling was not affected by the presence of apyrase and as the radioactive inositol trisphosphate was not detected,it was suggested that the changes is due to the action of phoshomono-esterase rather than PI-kinase or phospholipase C.When 32P-ATP was added to non-labelled lysed platelets upon incubation, 32P was labelled only into PIP and the amount was markedly increased until 5min. after incubation.Since the labelling was strongly inhibited by apyrase,it likely reflects the activity of Pl-kinase. The activity of PI-kinase thus measured required Mg2+ strictly for the activity and the maximal activity was obtained in the presence of 30mM Mg2+ .In contrast,it was markedly inhibited in the presence of Ca2+ (as low as 2mM Ca2+ in the presence of 2mM EGTA),which was compatible.with our previous findings with intact platelets. The activity of A-kinase was not inhibited by a low concentration of Ca2+ .Furthermore,the activity was inhibited by cAMP or dbcAMP in a dose related manner and no enhancement of the activity Was obtained by the addition of catalytic subunit of A-kinase,though a significant reduction in the activity was observed in the presence of inhibitor protein to A-kinase. From these observations,the following conclusions were obtained; l)The activity of Pl-kinase in lysed platelets may be determined by pulse labelling with 32P-ATP. 2)It requires Mg2+ absolutely and is inhibited by a very low concentration of Ca2+. 3)Pl-kinase is activated by A-kinase but the activated enzyme is inhibited by cAMP, suggesting the presence of feedback mechanism.
HAEMOSTATIC CHANGES INDUCED BY TWO LOW DOSE TRIPHASIC ORAL CONTRACEPTIVES
Authors: Machin S J , Mackie I J , Walshe K , Gillmer M D
Published: August 2018

The haemostatic system was investigated in 26 women taking cyclically administered triphasic combined oral contraceptives for the first time during their first six cycles. Fourteen women received Logynon (mean dose 32.4μg ethinyloestradiol, 92pg progestagen) and 12 received SHD 415G (Schering) which contains a mean dosage of 32.4μg ethinyloestradiol and 78pg gestodene, a recently developed progesterone. The Logynon group showed a significant increase (p<0.005) in fibrinogen (pre-mean 284.4 g/1; after 1 cycle 347.3 g/1, after 6 cycles 318.6 g/1) , factor VII (65.8 u/1 to 73.9 u/1 to 83.2 u/1), factor XII (1.74 u/1 to 2.41 u/1, to 2.25 u/1), plasminogen (100.9 u/1 to 135.1 u/1 to 126.3 u/1); decrease in ATIII (115.9 u/1 to 103.1 u/1 to 93.4 u/1) but no significant change in factor X (98.4 u/1 to 108.9 u/1 to 102.4) or protein C (0.85 u/1 to 0.88 u/1 to 0.94 u/1) activity. The SHD 415G group showed similar changes with an increase in fibrinogen (247.9 g/1 to 330.8 g/1 to 373 .1 g/1), factor VII (63.1 u/1 to 73.1 u/1 to 90.3 u/1, factor X (98.3 u/1 to 112.0 u/1 to 124.4 u/1), factor XII (1.46 u/1, to 1.93 u/1, to 2.03 u/1), plasminogen (110.8 u/1 to 125.4 u/1 to 136.7 u/1); decrease in ATIII (113.1 u/1 to 96.3 u/1 to 89.7 u/1), but no change in protein C (0.84 u/1 to - 0.78 u/1 to 0.85 u/1) activity. These changes were apparent after the first cycle of therapy and the differences were maintained over the six cycle period. There was no increase in protein C activity despite changes in the other vitamin K dependent proteins factors VII and X. Both low oestrogen dose triphasic pills caused similar prothrombotic changes which were not modified by the new progesterone, gestodene.
BIOLOGICAL CHARACTERIZATION OF AMINO-TERMINAL EXON DELETIONS OF T-PA
Authors: Larsen Glenn R , Henson Kim , Blue Yitzak
Published: August 2018

The secreted form of t-pa is proposed to be a mosaic protein which contains 4 different domain elements based on amino acid homologies with fibronectin finger elements, epidermal growth factor, kringle structures, and the active site of serine proteases. Of the 12 exons which encode these domains only the finger and epidermal growth factor are encoded separately by single exons. To investigate whether a single exon can encode a functional element or domain within a protein, the following precise exon alterations were made by loop-out mutagenesis techniques which deleted either the fibronectin finger, epidermal growth factor, or combination finger/growth factor domain(s). These mutant proteins were expressed in mammalian cells and characterized with respect to affinity for fibrin, fibrinolytic and fibrinogenolytic potential. All mutants demonstrated significantly lower affinity for fibrin with respect to the wild-type protein. We estimate the KD of these mutants for fibrin to be at least 100-fold higher than the wild-type form which we determined to be approximately 0.3 uM. Each mutant retained characteristic activator stimulation by fibrin and were also shown to have the same approximate specific activity as the wild-type form. These mutants were further evaluated in citrated human plasma [125-I]-fibrin clot lysis assays over a range of activator concentrations and shown to behave similarly to wild-type t-pa at therapeutic thrombolytic concentrations. At some lower concentrations, however, reduced fibrinolysis was observed for the mutant forms relative to wild-type. All mutants were evaluated for their fibrinogenolytic potential and demonstrated no significant decrease in coagulable fibrinogen over a five hour period. This was in dramatic contrast to an equivalent activator concentration of urokinase which showed a precipitous decline in coaguable fibrinogen in the first hour.
STUDIES OF PLATELET AND LEUKOCYTE ATTACHMENT TO WHOLE HUMAN ARTERY SEGMENTS
Authors: Schorer Anna E , Johnson Debra J , White James G
Published: August 2018

Activation of endothelial cells (EC) by inflammatory stimuli can result in heightened procoagulant properties towards coagulation proteins. These alterations can be readily studied in existing tissue culture models which lack a component of flow (shear). It is generally believed that thrombosis in the venous (low shear) regions of the vasculature are relatively dependent upon the coagulation proteins, whereas clots in the arterial system (high shear) are more dependent upon platelet plugs. Adhesion of the platelets to vessel wall elements under controlled conditions of flow have also been addressed in a number of models, such as the "Baumgartner chamber". In this model, blood or platelets are perfused over denuded, everted vessel segments, usually rabbit aortae. We have modified this model to study attachment of platelets to arterial segments of human origin which retain an intact layer of native EC. Arteries are aseptically removed from umbilical cords, trimmed, washed, and gently everted. The segments are placed in Medium 199 with 20% calf serum and standard growth supplements. After 4-10 days, these segments are placed on rods and perfused with whole citra-ted blood (800 sec−1). Such segments permit attachment of single platelets, but not platelet aggregates. Identical segments from which EC are mecnanically removed promote attachment of large (>5 cell) platelet thrombi. Endothelialized segments which are pre-treated (16 hours) with thrombin or phorbol myristate acetate show reduced attachment of platelets. In selected regions, clusters of leukocytes are attached to these segments, either upon or beneath the still- confluent layer of EC. Artery segments treated with bacterial endotoxin (1 mcg/ml) for 90 minutes, by contrast, show enhanced attachment of platelets, both as a monolayer of platelets and as small (2-5 cell) aggregates. This novel application of the Baumgartner assay demonstrates that platelet and leukocyte attachment to confluent EC may occur under conditions of moderate shear, and may be regulated in part by EC, which in turn may be modulated by exogenous mediators.
EFFECT OF ASPIRIN AND ASPIRIN COMBINED WITH DIPYRIDAMOLE IN EARLY DIABETIC RETINOPATHY
Authors: Samama M , Baudoin C E
Published: August 2018

In a double blind randomised controlled clinical trial the effect of antiplatelet agents (aspirin 330 mg × 3 × day) or in combination with dipyridamole (75 mg 3 × day) versus placebo, was tested in 475 patients with early diabetic retinopathy. Patients were follewed fourmonthly for 3 years. Ophtalmological examinations were carried out initially and at yearly intervals. The assessment of retinopathy was based on changes in the number of microaneurysms (MA) present in the macular field as seen on fluorescein angiograms over a period of three years. Forty one patients did not complete the study. Among the others at least three readable initial and yearly angiograms were available on 420 patients who had a 3 year follow up (266 insulin treated and 154 non insulin treated). The results are based on these patients.

It is concluded that either Aspirin alone or in conjunction with dipyridamole significantly slows down the progression of MA evolution in early diabetic retinopathy. Because of the very small, although significant changes, the clinical relevance of these drugs has not been established.
Effects of intermittent dosage of ASA on the increased in vivo formation of thromboxane during acute myocardial infarction
Authors: Vesterqvist O , Rasmanis G , Henriksson P , Edhag O , Gréen K
Published: August 2018

We have earlier demonstrated considerable increase of the in vivo formation of thromboxane (Tx) in connection with myocardial infarction (AMI). The in vivo formation of prostacyklin (PGI) was also increased, in some cases very much so, with a maximum after the CK peak. In normals ASA reduces the in vivo Tx synthesis to about 15% for several days while the PGI synthesis is only inhibited for 2-3 hrs. The in vivo synthesis of those prostanoids was measured by gas chromatographic mass spectrometric quantitation of the major urinary metabolites. In a serie of twenty patients with AMI we measured the in vivo synthesis of Tx and PGI during seven consecutive days. Ten patients received 0.5 g ASA every third day. In the nontreated group the Tx synthesis slowly decreased to about 70%, in the treated group to about 25% on day 3 as compared to the synthesis at admission. There was no difference in the in vivo synthesis of PGI between the two groups.

These data demonstrate that in most individuals with AMI it is possible to inhibit Tx synthesis considerably with intermittent dosages of 0.5g ASA while the PGI synthesis is essentially maintained intact. Therefore this regimen should be more beneficial to the patient than daily doses of any magnitude.
PLATELETS OF A PATIENT LACKING GLYCOPROTEINS lib AND Ilia AGGREGATE TO HIGH CONCENTRATIONS OF THROMBIN OR COLLAGEN
Authors: McGregor J L , McGregor L , Hans M , Sayegh A , Trzeeiak M C , Dechavanne M
Published: August 2018

The aim of this study was to investigate the platelets of a patient having bleeding episodes that began in infancy. The patient’s platelets in citrated-PRP did not aggregate when stimulated with ADP (5 and 10 uM), collagen (2.5 ug/ml), or sodium arachidonate (1 uM). However, washed patient platelets, in the presence of 2mM calcium, aggregated and secreted when stimulated with high concentrations of thrombin (0.36, 0.72 and lU/ml) or collagen (2, 4, 10 ug/ml). Monoclonal antibodies (Mab) LYP18 (directed against the IIb-IIIa glycoprotein complex) and LYP8 (anti-thrombospondin) inhibited thrombin and collagen induced aggregation of control but not the patient platelets. Patient thrombin -stimulated platelets did not bind 125I-labelled fibrinogen (40 to 320 ug/ml). Moreover, stimulating the washed patient's platelets with ADP (10-100 uM), in the presence of fibrinogen (2mg/ml), did not result in aggregation. Binding studies using Mab 125I-LYP2 (directed against the IIb-IIIa glycoprotein complex) showed the absence of the complex on the patient's platelets. The absence of the IIb-IIIa complex on the patient's platelets was also observed using crossed immunoelectro -phoresis and Mab 125I-LYP2 or 125I-LYP18. Individual glycoproteins (lib or Ilia) were not detected on silver stained two-dimensional (non-reduced/reduced) SDS-PAGE. Moreover, Western blots of |he patients platelets used in combination with anti-PLA or anti-LEK polyclonal antibodies failed to detect the presence of these two glycoproteins. These results indicate that this patient has Glanzmann's thrombasthenia or a variant of this disease. Moreover, this study shows that platelets lacking the IIb-IIIa glycoprotein complex can aggregate in responseto collagen or thrombin in the presence of physiological concentrations of calcium.
A NEW BLEEDING TENDENCY DUE TO HERITABLE HYPER α2-MACROGLOBULIN-EMIA (α2-MACROGLOBULIN-AKITA)
Authors: Endo Y , Iwamoto K , Mamiya S , Niitsu H , Itoh T , Miura A
Published: August 2018

A heritable elevation in α2-macroglobulin (α2M) was identified in a 9-year-old girl with bleeding tendency and prolonged APTT to 49.1 sec (normal 27-38) and recalcification time to 438 sec (<180). Plasma α2M level in her family tree i.e. sister, mother, maternal grand mother, father and paternal grand mother was 406, 380, 352, 339, 166 and 236 mg/dl (140-285), respectively. Thus it is thought to be an autosomal dominant disease, but other relatives show no apparent clinical syndrome. The significance of a possible causal association between elevated α2M and orolonged APTT was showed.

The activity of α2M of the patient determined as trypsin-protein esterase was 351 mg/dl (197%), also. The α2M of the patient showed normal mobility to anti-α2M plasma with acceralated anodal mobility by crossed immunoelectrophoresis and normal mobility by immuno-fixation electrophoresis. In addition, by the analysis with SDS-polyacrylamidegel electrophoresis we demonstrated no qualitative abnormality in α2M of the patient. The new familial abnormality is tentatively designated "α2-Macroglobulin -Akita".
INACTIVATION OF HEPARIN COFACTOR II (HC II) BY POLYMORPHONUCLEAR LEUKOCYTES (PMNL)
Authors: Sié P , Dupouy D , Dol F , Boneu B
Published: August 2018

Several authors have shown that antithrombin III (AT III) was catalytically inactivated by neutrophil elastase, an observation relevant to pathophysiological processes in the vicinity of inflammatory sites. The aim of this study was to investigate whether HC II, another natural thrombin inhibitor, is also inactivated by PMNL.

A rapid loss of HC II activity occured upon incubation with fresh human PMNL stimulated by phorbol myristate acetate (Tl/2 1 uM HC II, 0.35 108 cells/mm : -2 min) or with PMNL extracts prepared by nitrogen cavitation. Antithrombin (dermatan sulfate cofactor) and antichymotrypsin activities of HC II were lost at the same rate. Resting PMNL were ineffective. Inactivation was prevented by several serine-protease inhibitors but was Ca++ /Mg++ independent. Inactivation coincided with the formation of a 54 KD peptide after a first non-inactivating degradation into a 62 KD peptide (native HC II : 76 KD). These reaction products are reminiscent of those described upon incubation with proteinase I from Echis carinatus venom. HC II was inactivated more rapidly than AT III (T 1/2 of AT III in the same conditions -15 min). However, heparin (1-10 ug/ml) strongly accelerated the rate of AT III inactivation and slightly protected HC II, thus reversing the order of inactivation. Dermatan sulfate had no effect on this process.

In conclusion, this study shows ; l)both AT III and HC II are rapidly inactivated by PMNL enzymes, thus favoring locally thrombin-mediated processes ; 2) heparin increases AT III degradation by PMNL, a possible route of catabolism operating in patients with low AT III levels during heparin treatment.
INACTIVATION OF FACTOR Va BY PIASMIN
Authors: Omar M N , Lee C D , Mann K G
Published: August 2018

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The action of plasmin resulted in a rapid loss of the ability of Factor Va to serve as a cofactor to Factor Xa , as judged by clotting assays and . direct assays of prothrombin activation using the fluorophore, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA) . The rate of Factor Va inactivation catalyzed by plasmin was markedly enhanced by the addition of phospholipid vesicles (PCPS), suggesting that the action of plasmin on Factor Va may be a membrane bound phenomena. Both Factor Xa and prothrombin were capable of protecting Factor Va from inactivation by plasmin. SDS-PAGE was utilized to correlate plasmin catalyzed proteolysis of Factor Va with the concomitant loss of activity. Data obtained with Factor Va and the isolated chains of the cofactor indicated that the light chain (E) was cleaved by plasmin to yield products similar to those obtained with Factor Xa and Activated Protein C (APC). The heavy chain (D) was found to be degraded by plasmin to produce proteolytic fragments distinct from those produced by Factor Xa and APC. The action of plasmin on single chain Factor V was notable for an initial, transient increase in total Factor V activity, followed by subsequent loss of activity, indicating the transient formation of active intermediates. SDS-PAGE analysis revealed the degradation of Factor V by plasmin to final, inactive products via several transient, higher molecular weight intermediates. These findings may be of some significance in pathophysiologic states in which systemic fibrinolysis may occur, possibly contributing to the depletion of clotting factors. The identification of such ongoing processes may eventually be facilitated by the observation that the degradation of Factor Va by plasmin leads to end products which may be unique to this interaction.

(Supported by NIH Grants HL-35058 and HL 34575)
INTRA-ARTERIAL THROMBOLYTIC THERAPY IN ACUTE MIDDLE CEREBRAL ARTERY STROKE
Authors: Del Zoppo G J , Otis S M , Zyroff J , Hacke W , Zeumer H , Harker L A
Published: August 2018

18 patients presenting with acute carotid territory stroke, secondary to angiographically demonstrated occlusion of the middle cerebral artery (MCA), have been treated within 8 hours of the onset of acute symptoms by local intra-arterial infusion of urokinase or streptokinase. All patients were screened by baseline CT cerebral scan to exclude intracerebral hemorrhage as a cause of the acute stroke. 14 patients demonstrated complete, 2 partial, and 2 no recanalization (reopening) of the previously occluded artery following a 1 to 2 hour infusion of the fibrinolytic agent.

10 of the 14 patients displaying complete recanalization had complete neurological recovery or improvement with residual neurological deficits, while the 2 patients who did not display recanalization did not improve clinically. No clinical improvement was observed in the absence of recanalization.

Hemorrhagic transformation of cerebral ischemic areas may be classified as hemorrhagic infarction (minimal hemorrhage, no clinical deterioraton) and parenchymatous hemorrhage (mass effect, clinical deterioration). Minor infarction-related hemorrhages without detectable neurological sequelae (hemorrhagic infarctions) were found by CT scan in 4 patients; all displayed complete recanalization; and all hemorrhagic infarctions resolved.

This uncontrolled prospective clinical experience suggests that early local infusion of thrombolytic agents in selected patients may be efficacious and safe.
VINCULIN ISOFORMS IN HUMAN BLOOD PLATELETS
Authors: Asijee G M , Bruin T , Sturk A , ten Cate J E , Muszbek L
Published: August 2018

In several cells vinculin has been implicated in the interaction between the cytoskeleton and the outer ceil membrane. We have recently demonstrated that vinculin becomes associated with the Triton X-100 insoluble cytoskeleton of blood platelets upon thrombin-induced activation (Asijee et al, Exp Cell Res, 1987, in press). In the present study we demonstrate by SDS-PAGE and immunoblotting that vinculin is also present in the membrane skeleton of both non-activated and activated human blood platelets. The membrane skeleton was prepared by the method of Fox (J Clin Invest, 1985: 76, 1673), and platelets were stimulated 5 min at 37 °c with 0.1 U/ml thrombin. The association was specifically inhibited by DNase I-induced depolymerization of the actin filaments.

Protein analysis by the O’Farrell technique (first dimension IEF, second dimension SDS-PAGE) and subsequent immunoblotting demonstrated purified vinculin to consist of 4 major isoforms (pI 6.8 - 7.2). These isoforms differed in subcellular distribution. Upon thrombin-induced platelet activation, cytoskeletal vinculin consisted of the 2 most acidic isoforms, and cytoplasmic vinculin of 2 more basic isoforms. The membrane skeleton-associated vinculin contained all 4 isoforms.

We conclude that: 1. vinculin is a component of the membrane skeleton of both non-activated and activated human blood platelets, 2. similar to chicken gizzard smooth muscle cells, human blood platelet vinculin consists of several isoforms with differing subcellular distribution.
A STUDY OF HUMAN PLATELET SUBPOPULATION HETEROGENEITY IN FRACTIONS SEPARATED BY CONTINUOUS FLOW ELECTROPHORESIS (CFE)
Authors: Crook M , Crawford N
Published: August 2018

Earlier density gradient studies showed that the circulating platelet pool has substantial heterogeneity with respect to size, buoyant density and metabolic and functional properties. The interpretation of this heterogeneity is, however, controversial; some workers attributing it to in vivo ageing processes, whilst others consider it relates more closely to the megakaryocyte ploidy classes at the time of platelet production. Such considerations are important for further understanding of disease states where the heterogeneity profile may be distorted by the presence of megathrombocytes or other atypical platelet forms due to changes in the thrombopoeitic equilibrium (e.g. Idiopathic or drug-induced thrombocytopaenias, splenomegaly and leukaemia etc). We describe a new procedure for investigating platelet heterogeneity using a continuous flow method to isolate subpopulations on the basis of differences in surface membrane electrokinetic properties. To maintain the cells quiescent during the isolation and CFE they have been pre-treated with taxol, a microtubule stabilising agent. Taxol maintains platelet discoidicity, yet the cells show full functional responses to conventional agonists. The heterogeneity profile emerging from the CFE chamber extends over 15-20 fractions and spans a range of electrophoretic mobility (measured with a Laser Doppler Analytical apparatus) between 0.83-0.93 μmetres/sec/V. cm. Pooled fractions from the profile show differences in total and surface neuraminidase-labile sialic acid which correlate well with their electrokinetic properties. Interestingly, DTNB-titratable -SH groups in surface membrane proteins show an inverse relationship to platelet electronegativity as do phosphate-labile phosphate groups. There are significant differences in platelet size (Coulter volume) with the larger platelets being more electronegative in terms of unit charge density than the smaller ones. This novel procedure for studying platelet subpopulations on the basis of surface electrokinetic properties may also have value in profiling platelets from patients with specific platelet defects, during extracorporeal circuitry, transplant rejection and in vessel wall diseases.
INHIBITOR OF THE FACTOR VIIA-TISSUE FACTOR COMPLEX IS REDUCED IN PATIENTS WITH DISSEMINATED INTRAVASCULAR COAGULATION BUT NOT IN PATIENTS WITH SEVERE HEPATOCELLULAR DISEASE
Authors: Bajaj M S , Rana S V , Wysolmerski R B , Bajaj S P
Published: August 2018

Recently, inhibition of factor VIIa-tissue factor activity by a plasma component(s) which requires factor Xa has been described. In this communication, we have developed a specific radiometric assay (which utilizes 3H-factor IX and is sensitive to <1% of plasma level) for this inhibitor and have measured its activity in various disease states. Strikingly, the levels of this inhibitor were found to be normal in patients with advanced chronic hepatocellular disease but low in patients with disseminated intravascular coagulation (DIC). When endotoxin was used to induce DIC in rabbits, the levels of this inhibitor fell by 30 to 90%. Human umbilical vein endothelial cells (HUVE), bovine pulmonary artery endothelial cells, and a human hepatoma cell line (HepG2) all synthesized and secreted this inhibitor whereas a promyelocytic cell line (HL-60) did not and a monocytic cell line (U937) appears to synthesize only small amounts. When ammonium sulfate fractionated human plasma, and serum-free conditioned media from both HUVE and HepG2 cells were electrophoresed on sodium dodecyl sulfate acrylamide gels, two activity peaks corresponding to Mr ≃45,000 and Mr =33,000 were eluted in each case. These observations suggest that (a) the inhibitor is consumed in DIC and that (b) endothelial cells (or other cells) synthesize sufficient amounts of this inhibitor in vivo to compensate for any decreased production by liver cells. Furthermore, the inhibitor levels were found to be normal in patients on chronic warfarin therapy suggesting that the inhibitor is not a vitamin K-dependent protein.
RECOMBINANT HIRUDIN INHIBITS EXPERIMENTAL VENOUS THROMBOSIS INDUCED BY INJECTION OF TISSUE FACTOR AND STASIS
Authors: Freund M , Cazenave J-P , Wiesel M-L , Roitsch C , Riehl-Bellon N , Loison G , Lemoine Y E , Brown S , Courtney M
Published: August 2018

Hirudin (HIR), a polypeptide of 65 aminoacids, is the most potent natural inhibitor of coagulation by forming rapidly a very stable and specific non covalent 1:1 complex with α-thrombin, independent of antithrombin III. Although natural HIR has in vivo anticoagulant and antithrombotic properties, its limited availability for large scale purification has prevented further clinical testing and potential use; this can now be solved by recombinant DNA technology. We have previously reported the cloning and expression of a cDNA encoding one variant (called HV-2) of Hirudo medicinalis HIR (Proc. Natl. Acad. Sci. USA. 1986, 83, 1084-1088). The main factors responsible for venous thrombosis are stasis and thrombin generation secondary to tissue factor liberation from vascular cells and monocytes by injury, endotoxin, interleukin-1 or cachectin and the subsequent activation and circulation of activated clotting factors. We have studied the antithrombotic properties of recombinant HIR, HV-2, in a rat experiemental model of venous thrombosis. HV-2 was expressed in yeast, extracted from culture supernatant and purified by HPLC. Pure HV-2 had an isoleucine NH2-terminus and a specific activity of 13000 ATU/mg.30 male Wistar rats (225-300g) were anesthetized with pentobarbital. At time t (0 min) an i.v. (penis) injection of 0.4 ml of saline or HV-2 (2000 to 8000 ATU/kg) was given, followed at t (5min) by 25 mg/kg tissue factor (Thromboplastin C, Dade) i.v. ; 10 s later stasis of the exposed vena cava between 2 sutures 0.7 cm apart and at t (15 min) removal, blotting, fixation and weighing of the thrombus. Linear regression analysis showed a correlation (r=0.99) between the dose of HV-2 and thrombus weight and a calculated IC50 = 3000 ATU/kg. Total inhibition of thrombus formation was seen after injection of 6000 ATU/kg HV-2 and lasted up to 15 min of circulation, HV-2 being completely eliminated from blood in 60 min and accumulated in the kidneys as shown by gamma imaging with 131I-HV-2. In conclusion, the recombinant HIR HV-2 is a potent immediate antithrombin which inhibits venous thrombosis induced by tissue factor and stasis.