bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Plasma Membrane Bound Pyridine Nucleotide Reductase from Human Platelets
Authors: Moroff Gary , McGill Manley , Kosow David P.
Published: May 2019

Platelet membranes isolated by the glycerol lysis technique contain a pyridine nucleotide dependent reductase which can reduce cytochrome c. This reductase is not inhibited by superoxide dismutase. The Km (apparent) for NADH, the preferred cofactor, is 1 μM. The sulfhydryl reagent, parachloromercuribenzoate sulfonate (PCMBS), inhibits but KCN does not. PCMBS inhibition is time dependent. Inhibition of the reductase is also seen with quinacrine HCl (atebrin) and salts (both monovalent and polyvalent). Cytochemical staining with nitroblue tetrazolium has also localized the reductase to the platelet membrane. ADP and collagen induced aggregation in citrated plasma is inhibited by quinacrine HCl while aggregation by the calcium ionophore A23 187 is not. It has recently been reported that aggregation of human platelets by collagen or thrombin cause an increase in the non-superoxide dependent reduction of cytochrome c or nitroblue tetrazolium (Marcus, A. J., et al., J. Clin. Invest. 59, 149, 1977). The quinacrine HCl inhibition studies suggest that a pyridine nucleotide dependent reductase may be involved in one of the steps which trigger aggregation.
Circulating Platelet Aggregates in Normal Pregnancy and Rpeeclampsia
Authors: Rákóczi I. , Tallián F. , Cseh I. , Gáti I.
Published: May 2019

Circulating platelet aggregates have been observed in various thromboembolic states. It is known that severe preeclamsia is associated with features of intravascular coagulation. To evaluate the role of platelets in this disorder we have determined circulating platelet aggregates in 10 patients with severe preeclampsia, in 30 patients in the third trimester of uncomplicated pregnancies and in 35 healthy nonpregnant volunteers. Platelet aggregate ratio /P.A.R./ was measured by a modification of a method described by Wu and Hoak, The mean P.A.R. of severe preeclamptic patients /0.732 ± 0,063 SEM/ was significantly lower than that of the uncomplicated pregnant women /0,860 ± 0,052 SEM/ and of the nonpregnant volunteers /0.880 ± 0,061 SEM/.

The results indicate that severe preeclamptic patients have increased levels of circulating platelet aggregates and platelet activation is a feature of preeclampsia.
Heparin - PF4 Pulsing as a Measure of Platelet - Endothelial Cell Reaction in Vivo
Authors: Pumphrey C.W. , Pepper D.S. , Dawes J.
Published: May 2019

iv, Heparin (1000 units) causes an increase in plasma, PF4 of as much as 30-fold, when treasured by radioiiumunoassay. The PF4 reaches a peak within 5 minutes after injection and Is subsequently cleared to a normal value within 40-80 minutes. The Peak elevation is proportional to the initial heparin dose. Subsequent iv, Heparin doses, within the next few hours, fail to elevate PF4. Control experiments strongly indicate that the source of the PF4 is the vascular endothelium rather than the platelets. Further experiments have shown that this PF4. reservoir half fills within 46 hours. We suggest that iv. heparin pulses can be used for studying, indirectly, platelet - endothelial cell interaction in vivo.
Interaction of Prothrombin with Phospholipids Monolayers in Presence of Ca**.
Authors: Lecompte M.F. , Miller I.R.
Published: May 2019

The activation rate of Prothrombin and its conversion into thrombin through a proteolytic degradation is enhanced by phospholipids in the presence of Ca++. In this work, we could elucidate stoechiomet rio and structural aspects of the interaction between prothrombin. phospholipids and Ca++. We studied the adsorption equilibria of Ca++ and of prothrombin un phospholipids monolayers by a new approach which comoines two complementary methods of surface measurement.

Electrochemical investigations on lipid monolayer interacting with prothrombin showed penetration of prothrombin in presence or not of Ca++ (by increasing the lipid layer capacitance). with a preferential orientation. The adsorption of Ca++ and prothrombin to the lipid layer was measured by counting the surface radioactivity of 45Ca and 3H-prothrombin. Synergetic attachment of Ca++ and prothrombin to the phospholipid water interface was observed and the number of Ca++ per attached prothromoin was determinen. The two methods give comparative results for the proposed ionic-hydrophobic interaction.
Bovine Factors X1 and X2: Activation Peptide Based Chromatographic Differences
Authors: Morita Takashi , Jackson Craig M.
Published: May 2019

Bovine Factor X is eluted in two forms (X1 and X2) from anion exchange chromatographic columns. These two forms have indistinguishable amino acid compositions, molecular weights and specific activities. The amino acid sequences containing the γ-carboxyglu-tamic acid residues have been shown to be identical in X1 and X2, (H. Morris, personal communication). An activation peptide is released from the N-terminal region of the heavy chain of Factor X by an activator from Russell’s viper venom. This peptide can be isolated after activation by gel filtration on Sephadex G-100 under nondenaturing conditions. The activation peptides from a mixture of Factors X1 and X2 were separated into two forms by an ion-exchange chromatography. The activation peptide AP1) which eluted first was shown to be derived from Factor X1 while the activation peptide (AP2) which eluted second was shown to be derived from X2 on basis of chromatographic separations carried out on Factors X1 and X2 separately. Factor Xa was eluted as a symmetrical single peak. On the basis of these and other data characterizing these products, we conclude that the difference between X1 and X2 are properties of the structures of the activation peptides. (Supported by a grant HL 12820 from the National Heart, Lung and Blood Institute. C.H.J. is an Established Investigator of the American Heart Association).
Levels of Platelet Factor 3 in Citrate Plasma and in Whole Blood as Determined by A Chromogenic Peptide Substrate Assay
Authors: Sandberg H. , Gellerbring A.-K. , Andersson L.-O.
Published: May 2019

A newly developed sensitive method for determination of platelet factor 3 (PF 3) using a chromogenic substrate (S 2238) (Sandberg and Andersson, Thromb. Res., in press) was used for comparison of the PF 3 levels in platelet-rich plasma (PRP) in citrate with the levels in PRP in the anticoagulant EDTA/citrate/PCE 1/theophylline. It was shown that in citrate PRP, release of PF 3 was started after about 20 minutes from blood sampling. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3. No release of PF 3 or β-tg could be detected within 3 hours from blood sampling in PRP in the EDTA/citrate/PCE 1/theophylline anticoagulant.

The PF 3 level in whole blood was measured by a method which was a modification of the method used for plasma samples. It was found that, using the same donor, the PF 3 level of plasma and blood in the EDTA/citrate/PCE 1/theophylline anticoagulant was essentially the same. The levels of PF 3 found in blood upon standing in citrate or in EDTA/citrate/PCE 1/theophy11ine anticoagulant were in accordance with the levels found in plasma. Experiments with whole blood without any anticoagulant showed that release of PF 3 begin to occur simultaneously with release of β-tg, about 5 minutes before clotting. Thus the data show that PF 3 and βtg are released in parallel.
Experience with Aspirin-Dipyridamole Prophylaxis in Sickle Cell Disease
Authors: Alkjaersig N. , Chaplin H. , Fletcher A.P.
Published: May 2019

Crisis in sickle cell disease (SSD) patients is associated with reproducible sequential hemostatic changes indicative of intravascular fibrin formation and marked disturbance of platelet economy. With crisis onset, plasma high molecular weight fibrin(ogen) complexes are elevated and there is a transient fall in platelets. Subsequently fibrinogen and platelet counts rise, reaching their peaks respectively one and two weeks after onset. Three SSD patients have been followed by weekly clinical and laboratory examinations for a two year control and a two year prophylactic treatment period (aspirin 650 mg, dipyridamole 50 mg b.d.). Patient symptomatic status was classified weekly as 1 (asymptomatic) through 4 (hospitalized). Increase in fibrinogen concentration was significantly correlated with patient disease status. Wiile on treatment, th patients were asymptomatic or mildly symptomatic for 85% of the time compared to 73% of the control time.

Analysis of the clinical status data (2 x n Chi square) showed a statistically significant difference in favor of the treatment (p < 0.01) and the time spent in hospital was significantly reduced during treatment (p < 0.02).
Specific Factor VIII Fragmentation in Commercial Factor VIII Concentrates
Authors: Montgomery R.R.
Published: May 2019

Factor VIII antigen (VIIIag) is felt to consist of a series of oligomers that demonstrate immunologic reactions of identity with each other in normal plasma. This study demonstrates a fragment of VIIIag (VIIIag FRAG) which is present in commercial Factor VIII concentrate (VIII CONC) but not detected in cryoprecipitate (CRYO).

Crossed immunoelectrophoresis of 10 bags of CRYO and 10 different VIII CONC (5 different brands) was carried out against specific antibody to VIIIag. As expected, the mobility of VIIIag was less anodic in CRYO than in VIII CONC, but a specific fragment of VIIIag (VIIIag FRAG) was detected in the VIII CONC. VIIIag FRAG is more anodic and demonstrates a reaction of partial identity with normal VIIIag. VIIIag FRAG is Immunologically distinct from our previously reported von Willebrand’s disease antigen II. None of the CRYO samples demonstrate VIIIag FRAG.

Thus proteolysis in the preparation of commercial VIII CONC has produced VIII antigen that is deficient in some of the normal Factor VIII antigenic determinants. VIIIag in CRYO does not demonstrate this alteration. This proteolysis of VIIIag may contribute to the failure of VIII CONC to correct the bleeding time in von Willebrand’s disease.
Is IMP Accumulation in Platelets a Good Indicator for Increased Cytoplasmic Ca++?
Authors: Mürer E.H. , Holmsen H. , Siojo E.
Published: May 2019

Reduction of [ATP] and accumulation of hypoxanthine occurs in platelets·when consumption exceeds resynthesis of ATP. The intermediate IMP accumulates transiently ([IMP]↑) to a very varying degree. Several findings indicate that [IMP] is controlled by cytoplasmic [Ca++]: (1) The transient nature of [IMP]↑ would follow the release and subsequent uptake of cytoplasmic Ca++, the latter resulting in a return to resting state. (2) Under certain conditions antimycin does not cause [IMP]↑, but in combination with fluoride, which itself may cause release of intracellular Ca++, [IMP]↑ is prolonged, suggesting that Ca++ uptake is energy-requiring. (3) Concomitant addition of antimycin, deoxyglucose and gluconolactone causes irreversible [IMP]↑, suggesting that a rapid fall in [ATP] causes leakage of Ca++-from intracellular sites. Irreversible (IMP]↑ is also seen with high [A23l87] when cytoplasmic [Ca++] is irreversibly enhanced. (4) Secretion induced by 2-propyl-3-dimethylamino-5,6-methylenedioxyindene is accompanied by [IMP]↑. Chlortetracycline inhibits secretion and abolishes [IMP]↑. (5) The conversion of IMP to inosine + hypoxanthine in platelet lysates is enhanced by EGTA + Mg++ and inhibited by Ca++. This indicates that Ca++ inhibits Mg++-dependents 5′-nucleotidase responsible for IMP↑ inosine, which may explain the proposed regulatory effect of cytoplasmic Ca++ on [IMP]↑ in platelets. Since no reliable direct method is known for monitoring Ca++-movements in platelets, [IMP]↑ may be a useful indicator for [Ca++] in platelet cytoplasm.
Solution Studio of Bovins Prothrombin Fragments Using high Resolution Nuclear Magnetic Resonance Spectroscopy
Authors: Esnouf M.P. , Israel E.A. , Pluck N.D. , Nilliams R.J.P.
Published: May 2019

High resolution nuclear magnetic resonance spectroscopy (NMR) is now a widely used technique for investigating the solution structures of proteins. In particular, proton NMR provides information on the entire molecular structure and is not confined to the study of a few amino acid side chains with specific properties. We have used this technique (at 270 MHz) to investigate the solution behaviour of bovine prothrombin fragment 1. Large variations in temperature and pH are seen to have little or no effect on its spectrum. Indeed, the spectra are all very similar to those expected for a “random coil” protein. The protein has little tertiary structure. Calcium binding has been monitored and small changes involving tryptophan intensity have been seen in agreement with the results of other physical methods. However, NMR shows there to be no significant change in tertiary structure in fragment 1 on binding calcium. Prothrombin fragment 2 contains the second Kringle region - a large amount of its sequence being homologous to that in fragment 1. Preliminary experiments have shown bovine fragment 2 to have some degree of folded structure but the temperature dependence and chemical modification studies suggest that this is not very extensive.
Immune Response to Human Fibrinopeptide B.
Authors: Wilner George D. , Thomas David W. , Nossel Hymie L.
Published: May 2019

The serologic and cellular immune response to human fibrinopeptide B (hFPB), a unique thrombin proteolytic product of the fibrinogen Bβ chain (Bβ1-14), was studied as a basis for developing immunologic probes to quantitate restricted sequential regions in the fibrinogen molecule. Outbred rabbits hyper immunized with hFPB-bovine albumin conjugates produced anti-hFPB antibodies, as measured by radioimmunoassay (RIA) using 125I-hFPB analogue as tracer. Immunoreactive sites seen by these sera were characterized in terms of displacement of bound radiolabeled tracer by synthetic homologues of the hFPB sequence. In 4 inmune rabbit sera, displacement of binding comparable on a molar basis to using hFPB as inhibitor was obtained with fragments Bβ3-14, 5-14 and 7-14, but not with fragment Bβ9-14. In an additional 3 sera, inhibition comparable to intact.hFPB occured only with fragments BB3-14 and 5-14. In 3 of the 7 sera tested, Arg14 seemed to contribute critically to hFPB antigenicity. To study the cellular immune responsiveness to hFPB, inbred strain 2 guinea pigs were irrmunized with unconjugated hFPB. No antibody was demonstrated by RIA up to 1 month following immunization. However, immuno guinea pig T-cells showed a dramatic hFPB-specific proliferative response, as determined by 3H-thymidine incorporation. These cells were also stimulated by fragments Bβ3-14, 5-14 and 7-14, but not by fragments Bβ9-14 and 1-13. It is concluded that dominant antigenic determinants for both species reside in the COOH-terminal region of hFPB.(Supported by grant AI-14226 from NIAID, USPHS, and grants HL-15486 and HL-22642 from NHLI, NIH.)
Effect of Platelet Suppressants on Experimental Arterial Thrombosis
Authors: Gomez H. , Berzins A. , Kwaan H.C.
Published: May 2019

A reproducible model of platelet rich thrombi was produced in canine arteries by a previously described technique of intravascular insertion of metal tubing. The effect of platelet suppressants including sulfinpyrazone and ticrynafen, a clofibrate analog, on the rate and nature of thrombus formation was studied. ADP and epinephrine Induced platelet aggregation of the animal’s platelet rich plasma was monitored during the experiment. Platelet aggregation was impaired after the intravenous administration of either sulfinpyrazone (30 mg/kg body weight) or ticrynafen (100 mg/kg body weight). The rate of thrombus formation was not significantly altered by sulfinpyrazone when compared to the control, but histochemically, the thrombus studied showed a greatly diminished platelet content. In contrast, ticrynafen was able to prevent a complete occlusion of the involved vascular segment and a weak thrombus composed mostly of fibrin was produced instead. This effect of ticrynafen was dose related with minimal changes observed at a dose of 75 mg/kg. The results suggest that this arterial thrombosis model is of value in evaluating antithrombotic agents. They also indicate a potential value of ticrynafen as a therapeutic agent which should be further studied.
Effects of PGI2 and its more Stable forms on the Aggregation and on the Camp Content of Human Platelets
Authors: Nemesánszky E. , Blaskó Gy. , Stadler I. , Sas G. , Pálos L.A.
Published: May 2019

The authors investigated the anti-aggregating properties of some relatively stable derivatives of PGI2 concurrently determining the intracellular cAMP content of platelets. The ethyl-ester derivative of PGI2 and the complexes of PGI2-ethyl ester with β-cyclodextrine proved to be mora stable than PGI2-sodium salt. Their stimulating effect on adenyl-cyclase correlated with the potent inhibitory effect on platelet aggregation as well. The enhancing effect of these compounds for the intracellular cAMP conten t have a prolonged dura tion time -las ting 300 minutes - compared with the only a few minutes’ effect of PGE1.

These stable forms of PGI2 might be clinically useful as highly effective anti-aggregating compounds.
Fibronectin: Its Blood Clearance and Interaction with Fibrin in vivo
Authors: Sherman Laurence A. , Lee Joan
Published: May 2019

Fibronectin (cold insoluble globulin, CIG) binds to fibrin in vitro by both H+ bonds and F.XIII crosslinkinq. Potential in vivo significance was studied in a rabbit model. 131I-CIG purified by usual methods was ≥ 40% denatured as evinced by rapid blood clearance and complex t½. In contrast, the remaining nondenatured 131I-CIG in animals given 3H-amino acids had the same blood t½ (70-75 hours) and C1(0.8) as did the 3H-CIG. The CIG t½ was greater than that found for fibrinogen (55-65 hours). A second series received 131I-fibrinogen and nondenatured 125I-CIG i.V. Subsequently, thromboplastin or thrombin (50 u/Kg/lhr) was injected and both 125I-CIG and 13I-fibrinogen levels dropped during injection to 50-70% of preinjection levels. When ancrod was injected instead, 131I-fibrinogen fell by >90%, but 125I-CIG levels did not change. When animals were initially dofibrinated with ancrod CIG levels remained unchanged during subsequent thrombin injection. The results indicate that CIG has a longer normal blood t½ than fibrinogen, but the body distributions were approximately the same. When fibrin is formed, CIG and fibrin complex in vivo, and are rapidly cleared from the blood. The abnormal fibrin formed by ancrod apparently does not bind to CIG in vivo, or else does so reversibly prior to clearance from the blood.
Binding of Thrombin to Fibrin
Authors: Róka L. , Rademacher F.G.
Published: May 2019

After clot formation using bovine thrombin and bovine fibrinogen, one can detect no longer the total amount of thrombin. The amount missing in the supernatant is trapped in the clot and can be removed in active state after lysing the clot. The capacity for trapping thrombin by a constant amount of fibrin depends on the structure of the fibrinpolymer. This can be shown by producing different kinds of clots by variation of pH or monomer concentration during polymerisation or using Arvinmonomer insted of Thrombinmonomer. The trapped thrombin shows no clotting activity but some activity with chromogenic substrats remains. To neutralize trapped thrombin about 5 times more Antithrombin III is necessary than for the same amount of free thrombin. The trapped thrombin diffuses out of the clot indicating the reversibility of the binding of thrombin to fribin.
Immunological Studies of Prothrombin in Newborns
Authors: Muntean W. , Petek W.
Published: May 2019

Prothrombin of newborns was studied by means of the quantitative immuno-electrophoresis (QIE) and the two dimensional immunoelectrophoresis (TDIE). In the TDIE the prothrombin showed one precipitate in alpha-2-beta-globulin position when a buffer containing calcium was used. Another precipitate in alpha-1-globulin position was frequently found in preterm and term newborns. After administering vitamin K to the newborns the precipitate in alpha-1-globulin position was no longer demonstrable. When a buffer without calcium was used, in all newborns only one precipitate in the alpha-1-globulin position was found. The QIE and the activity assay gave equal results in those infants who showed only one precipitate in the TDIE. In those newborns who showed two precipitates, the QIE gave higher results than measurement of the activity.

From this study no evidence of differences between the prothrombin of newborns and that of adults could be derived. Vitamin K deficiency as judged from the appearance of a second peak in alpha-1-globulin position in the TDIE was a frequent finding in newborns, even on the first day of life.
Relationship Between Plasma Concentrations of Fibronectin α-Lipoprotein and Plasminogen
Authors: Boss N. , Bruder W. , Jilek F. , Bierner M. , Fleck E. , Hörmann H. , Koenig-Erich S. , Ruhenstroth-Bauer G.
Published: May 2019

In plasma samples of 24 patients with angiographically documented coronary artery disease the concentration of 12 proteins was determined by radial diffusion of monospecific antisera against gels containing various dilutions of plasma. The concentration of fibronectin, recently discussed to promote phagocytosis by cells of the reticulo - endothelial system, showed a good correlation (r = 0.541, 2P ≤ 0.01) with that of α-lipoprotein, a potentially antiatherogenic factor. In addition a significant correlation (r = 0.515, 2P ≤ 0.05) was found between the amounts of α-lipoprotein and plasminogen suggesting a possible relationship between various protective factors, which in some cases may promote regression of atherosclerosis.
Liver Devascularisation in Pig: Alterations of Coagulation & Ebrinolys
Authors: Morfini M. , Biliotti G. , Cinotti S. , Turchi A. , Marsili M. , Tonelli F. , Gross A. , Cordopatri F. , Lorenzi P. , Rossi Ferrini F.
Published: May 2019

All arteries leading to the liver were ligated and an end-to-side portacaval shunt was performed in twelve pigs. Before and 1,3,6 and 9 hrs after the laparatomy a blood sample was drawn by femoral artherial catheter. APTT, Prothrombin, Factor VIII AHF(one-stage method), platelet and red cells counts, fast-acting Antiplasmin by S-2251, AT III by S-2238 were performed on each sample. Prothrombin slowly but progressively decreased while APTT reduced. VIII AHF showed a little increase at first but the higher level, about the 160% of initial value, was recorded at the 9th hr.AT III increased during the 1st hr but, as Antiplasmin, slowly decreased to the 70% in the following, hours. The AT III and the first VIII AHF increase occurred during portal, vi na clumping, simultaneously to a very deep fall of blood pressure. The cases of portal system and an adrenergic stress may explain this finding. The survival of the pigs was quite costant, more than 24 hrs: this model of hepatitis failure has been chosen for application of articial devices to support liver function.
Coronary Flow Responses Following Stimulation of Lung Prostaglandin Release Before and after Embolization in a Rabbit Lung-Heart Model
Authors: Pedersen Kirstein A.
Published: May 2019

A new rabbit lung-heart preparation has been developed in order to evaluate the heart effects of the unstable PG’s (PGI2, TXA2) released by the lungs. The lungs were perfused (30 ml/min) and the effluent was used by the heart for anterograd constant pressure coronary perfusion. The coronary flow and the pulmonal perfusion pressure were recorded. Injections of arachidonic acid (AA, 10-100 μg) and the Ca+ +-ionophore A-23187 (50 μg) into the lungs caused a rapid increase in perfusion pressure and an increase in coronary flow. The first effect was inhibited by indomethacin (Indo) and N-butylimidazole (NBI, a TXA2-inhibitor), while the second effect was blocked only by Indo. The increase in coronary flow was not affected by selective administration of Indo through the left atrium. This effect was probably due to PGI2-release from the lungs. Embolization with 20-50 mg Sephadex S-25 microspheres caused an increase in the perfusion pressure, which was inhibited by Indo and NBI. Repeated injections of AA and A-23187 after embolization caused a paradoxical decrease in coronary flow, and this was blocked by both inhibitors. This may reflect a shift of AA metabolism from PGI2 to TXA2.
Interactions Between Blood Platelets and Vascular Endothelium in vitro Studies
Authors: Gimbrone M.A. , Curwen K.D. , Handin R. I.
Published: May 2019

Endothelial cells (EC) can actively influence the hemostatic response at sites of vascular injury through multiple mechanisms. For example, EC can degrade adenosine diphosphate, release plasminogen activator, and synthesize prostacyclin (PGI2), a potent inhibitor of platelet aggregation. We have examined whether PGI2 also might account for the normal lack of platelet adherence to the uninjured EC surface. In a monolayer adherence assay, radiolabeled human platelets in citrated plasma showed minimal interaction with primary cultures of human EC (<1 platelet adhering per cell). Platelets from aspirin-treated and untreated donors behaved similarly. However, aspirin pretreatment of EC consistently resulted in ~2-fold increases in platelet adherence which could be completely abolished by exogenous PGI2 (0.5–1.0 μg/ml). SV40-transformed human EC (SVHEC), which are deficient in PGI2 production compared to primary EC, showed 10-30 times more platelet adherence. Exogenous PGI2 produced a dose - related (.001-1.0 μg/ml) decrease in platelet adherence to SVHEC but did not result in the basal levels observed with normal EC monolayers. These data suggest that : 1) In addition to its effects on platelet aggregation, PGI2 can influence platelet endothelial cell interactions; 2) The increased platelet reactivity of transformed EC is associated with, but not completely attributable, to decreased PGI2 production; and 3) Factors other than PGI2 may play a role in the thromboresistance of normal vascular endothelium.
Molecular Events in the Activation of Bovine Factor V
Authors: Esmon C.T. , Gray S.F.
Published: May 2019

The conversion of bovine Factor V (FV) to Factor Va (FVa) by thrombin appears to proceed in two steps as shown below. FV(300,000) → thrombin Factor V intermediate (FVi) 2 sub-units; 220,000 & 100,000)→FVa (2 subunits; FVa-H.C., 100,000 & FVa-L.C., 73,000)+activation peptide/s. The 220,000 Mol. wt. subunit of FVi gives rise to the FVa-L.C. EDTA dissociates both FVi and FVa into subunits, Mn2+ facilitates reassociation. Even following dissociation with EDTA, 1 mole of Ca2+ remains bound to each of the FVa subunits.

Support of the above model of FV activation comes from an immunological characterization of the FV. Antibodies to FV cross react with FVa, FVi and both subunits of Fva. Antibodies to FVa cross react with FVi both subunits of FVi and both subunits of FVa. Antibodies to the FVa-L.C. cross react with FV, FVj and FVa, but do not with the FVa-H.L. Antibodies to the FVa-H.C. cross react with FV, the FVi, FVa but not with the FVa-L. C.

A protease from Russell’s viper venom activated FV by making a single proteolytic cleavage. This results in the formation of two subunits of Mol. wt., 250,000 & 73,000. The 73,000 Mol. wt. subunit is functionally and immunologically identical to the FVa-L.C. the 250,000 Mol. wt. subunit contains the antigenic determinants to the FVa-H.C. and can be converted into this subunit by incubation with thrombin. The appearance of two disdinct high Mol. wt. activation products from FV; one containing the FVa-L.C.; the other containing the FVa-H.C., is compatible with placing the activation peptide region between the two subunits of Fva.
Clinical Significance of Antithrombin
Authors: Sas G.
Published: May 2019

The clinical importance of antithrombin was directly proved by the description of the first hereditary antithrombin deficiency /HAD/ in 1965. Several other families have been studied since that time. Two main types of /HAD/ can be discerned. In one, both immunologic and fuctional methods reveal low level of antithrombin in the affected members. Some biochemical observations suggest the heterogenity even of this type of HAD. Oral anticoagulants seem effective in the treatment and prophylaxis of these patients. Thus, determination of antithrombin level should be performed in all young thrombotic patients especially. with prevalence of thromboembolism in the family. The second type of HAD, the congenital functional abnormality of antithrombin is very rare. Oestrogenic hormones can decrease the synthesis of antithrombin. This observation might be connected with •the thromboembolic complications of oral contraceptives. More data ar needed to evaluate the usefulness of monitoring the antithrombin level in the course of contraceptive treatment to avoid these complications. Decreased antithrombin levels and detection of antithrombin-complexes can provide useful information in the diagnosis of hypercoagulable states. Due to the methodologic development experienced in the last few years, one can expect a rapid rise of clinical investigations bringing about a better knowledge of clinical significance.
Platelet Secretion in Storage Pool Deficiency(SPD): Response to Thrombin and A23187
Authors: Lages B. , Holmsen H. , Danglemaier C. , Weiss H.J.
Published: May 2019

SPD platelets contain reduced amounts of the dense granular components serotonin, ATP, ADP, pyrophosphate, and calcium (Ca) and normal levels of lysosomal acid hydrolases (AH). We have examined the response of gel-filtered SPD platelets to thrombin(T) and ionophore A23187, substances which induce both dense granular and lysosomal secretion. SPD platelerel eased significant but less than normal amounts of Cainresponse to T and A23187. Ca secretion relative to ATP/ADP secretion was much greater in SPD platelets than in normal and platelets from the most severely dense granular deficient Hermansky-Pudlak patients released Cain the complete absence of ATP/ADe release. In agreement with previous studies, T-induced AH secretion was impaired in SPD platelets at low levels of T(0.04 units. ml) but normal at higher level s(0.3 units/ml). In response to A23187 AH secretion as wel as aggregation in platelet-rich plasma was markedly impaired in SPD platelets, and everally was not corrected at higher A23l 87 concentr ations. No differences were observed between SPD and normal platelets in metabolic ATP utilization in response to T or A23187. These results (1) suggest that secretabl e Camay bederived, in part, from sources other than the densegranules, and (2) further define the lysosomal secretion defect previously reported in SPD platelets.
The Combined Effect of Dipyridamole and Acetyl Salicylic Acid on Platelet Function and Bleeding Time : Evaluation of Dose for the Prophylaxis of Thrombosis
Authors: Penny A.F. , Crow M. J. , Rajah S. M. , Kester R.C.
Published: May 2019

The interaction of Dipyridamole (DPM) and Acetyl Salicylic Acid (ASA) on volunteers was investigated. ASA was given in single doses (multiples of 60mg) at 2 week intervals. DPM (multiples of 25 mg) was given tds for 2 days with a single dose of DPM and ASA on the morning of day 3. Bleeding time and platelet functions were performed 2 hours after each dose. ASA 300mg maximally inhibited aggregation, adhesion and PF4 release. Lower doses of ASA gave significant reductions in collagen aggregation at 120 mg, adhesion at 180mg and PF4 at 240mg. DPM 25mg tds produced reductions in collagen aggregation, adhesion and PF4. Maximal inhibition of platelet function without alteration in bleeding time was achieved by 50mg tds DPM + 180mg ASA or 75mg tds DPM + 120mg ASA. Bleeding time was normal with ASA 120mg, 180mg; prolonged at 300mg and 1000mg and tending to be normalised by 2g. Addition of DPM 75mg tds at each dose of ASA made no alteration in bleeding time. There was a cumulative effect of ASA on bleeding time. Combined DPM mg tds and low dose ASA present a balanced anti thrombotic regimen probably both inactivating thromboxane A2 production and enhancing prostacyclin activity. This dose combination is worthy of evaluation as an additional group, in trials.
Platelet Activation : Role of Exogenous and Endogenous Phospholipases
Authors: Perret B. , Mauco G. , Simon M.F. , Chap H. , Douste-Blazy L.
Published: May 2019

Phosoholipase A2 from bee venom induces aggregation of human platelets, provided that phospholipid hydrolysis is enabled by simultaneous incubation with sphingomyelinase C. Inhibition of the platelet response by indomethacin indicates that aggregation is due to arachidonic acid release. On another hand, this model allows to describe an asymmetrie distribution of arachidonic acid, whose only 6% is located in the outer leaflet of the plasma membrane.

During platelet aggregation by phospholipase C, the diacylglycerol and its hydrolysis product 2-acyl-glycerol are phosphorylated into phosphatide and lysophosphatidic acids, respectively. As the same kinds of changes occur in the presence of thrombin, a unifying hypothesis for platelet activation is proposed, involving the stimulation of an endogenous phospholipase C, whose some properties will be reported (neutral optimal pH, Ca-requlrement, phosphatidylinositol specificity and cytosol-localization). This model can be related to the recent finding that phosphatide acid behaves as a calcium-ionophore (Gerrard, J.M. et al., Prostaglandins Med., 1978, 1, 387) and provides an alternative pathway for arachidonic acid mobilization.
The Human Complement System in Thrombin-Mediated Platelet Function; Uptake of C5-9 Complexes
Authors: Polley M. J. , Nachmon R.L.
Published: May 2019

Thrombin-mediated platelet function is enhanced by the presence of C3, C5, C6, C7, C8 and C9 of human complement. The interaction of thrombin with its receptor on the platelet membrane initiates activation of complement on the platelet surface. Trypsin mediated platelet function is not enhanced by the addition of complement, probably since trypsin has no receptor on the platelet surface so activation of complement is triggered in the fluid phase and not on the platelet surface. Activation of complement by thrombin led to production of C5-9 complexes on the platelet surface. These complexes were eluted from the platelet membrane and were identified physicochemically and morphologically.

The mechanism of the complement induced enhancement of platelet function is not clear, however It is mediated via the arachidonic acid transformation pathway since this activity wos Inhibited by known inhibitors of cycla-oxygenase namely aspirin and indomethacin.
Prekallikrein Activator in Human Albumin Prepared by Cold-Ethanol Precipitation Methods and by ION Exchange Chromatography
Authors: Horowitz M.S. , Horowitz B.
Published: May 2019

Hypotensive reactions associated with the administration of albumin-containing plasma fractions have been attributed by Alving et al. to the presence of Hageman-factor fragments or prekallikrein activator (PKA). Using a radiochemical assay which measures kallikrein activation, we have investigated the PKA content of plasma fractions prepared according to the cold-ethanol method 6 of Cohn et al., several modified cold-ethanol procedures, and the ion-exchange chromatography system of Curling et al. Cohn fraction IV-4 contains both PKA and a precursor from which PKA activity can be generated by kaolin treatment, while Cohn Fraction V is free of both of these entities. Modification of Cohn method 6 by combining the IV-1 and IV-4 precipitation steps results in less efficient removal of PKA, especially when strong acids and bases replace acetate buffers for pH control. Prekal1ikrein activator activity accompanies albumin throughout the ion-exchange purification procedure and is present in the final albumin product prepared by this method. These results explain why PKA occurs frequently in the derivative known as plasma protein fraction and rarely in albumin which has been made according to Cohn method 6.
Influence of the Amount of Platelet Bound IgG (Antibody) on Platelet Survival and Site of Sequestration in Autoimmune Thrombocytopenia
Authors: Kernoff L.M. , Blake K.C.H. , Shackleton D.
Published: May 2019

This study aims to determine if increasing amounts of antiplatelet antibody result in progressive shortening of platelet survival and in proportionately greater hepatic sequestration. Antiplatelet antibody was measured by a quantitative antiglobulin technique using complement mediated lysis of IgG sensitised sheep RBC as an indicator system (1). Platelet survival was measured with autologous or ABO Rh compatible isologous platelets labelled with Cr-51 according to ICSH specifications (2). The survival data was fitted to linear exponential, multiple exponential and gamma functions on a Univac 1106 computer, incorporating a X2 measure of goodness-of-fit. Appropriately collimated detectors over liver and spleen were used to measure the relative uptake of labelled platelets which was expressed as the liver:spleen ratio at 48hr. after injection of the dose.

In 15 untreated patients with ITP, platelet antibody was 21-213ng IgG/106 platelets (normal 1.8 - 14.8, n=47). Survival data was best described by 2 exponentials. No correlation was observed between platelet antibody levels and the 2 exponential estimate of platelet meanlifespan. A highly significant correlation (p<0.001) was obtained between platelet antibody and the liver:spleen ratio demonstrating that greater amounts of antibody are associated with progressively greater hepatic sequestration.

(1) Dixon et al, New Engl.J. Med.292; 230. (2) Blood (1977) 50; 1137.

Supported by the Nellie Atkinson Bequest.
Life Style and Haemostatic Variables in Families of a Working Population
Authors: Duckert F. , Marbet G. , Bondeli S. , Isliker H. , Bruppacher R. , Seiler W. , Stähelin H.B.
Published: May 2019

Clinical and pathological data indicate that the haemostatic system is of importance in atherosclerosis. We have tried to relate established risk factors and life style to haemostatic variables in an epidemiological study on employees of a drug company with spouse and children. The families participated on a voluntary basis. The complete set of data could be collected for 94 fathers (F) median age 46, 92 mothers (M) 43y., 89 sons (S) 15y., 89 daughters (D) 15y. PTT was slightly higher in children (32” ±3.3 (D) vs 30 ± 2.6 (M) as was antithrombin III (AT III) concentration (113±13% (D) vs 105 ± 11 (M)) the differences beeing similar in F and S. Euglobulin lytic activity was found to be independent of age and sex (128±31 mm2 (D) vs 133 ± 26 (F)). PTT showed a highly significant negative correlation to age (α<0.0001), body mass index (BMI,α< 0.0001) and alcohol (α< 0.002). AT III correlated negativly with alcohol (α<0.025). Activation of fibrinolysis by venous stasis correlated with physical activity at work (α<0.02). No correlation was observed with cigarette smoking.
Platelet-Vessel Wall Interactions in von Willebrand’s Disease
Authors: Barnhart M.I. , Wilkins R.M. , Lusher J.M.
Published: May 2019

Adhesion of von willehrand (vW) platelets (P) from 12 patients was tested in an ex vivo human umbilical vein perfusion model. Experiments (42) employed twelve umbilical cords from Caesarian sections and P (fetal, adult and vW) either washed (with apyrase protection) or used as P rich plasma or whole blood. FVIII antigen (FVIII:ag), Ristocetin co-factor (RCF) and FVIII procoagulant were measured in blood and umbilical vein effluents. Either hypoxia or epinephrine pretreatment of vein released FVIII:ag and RCF into perfusates. Binding of a marker (latex linked anti-human FVIII:ag) demonstrated that FVIII:ag became exposed at endothelial surfaces. Scanning electron microscopy displayed vWP-vessel interactions. Although vWP adhered to injured vein wall, both qualitative and quantitative differences existed which related to the plasma RCF level. The vWP were less adhesive to exposed subendothelium than were control fetal or adult P. The vWP had less surface activity, spreading and fewer pseudopods. Vein pretreatment with FVIII antibody partly blocked P adhesion. Perfusion of cryoprecipitate with vWP improved their adhesion, activation and aggregation. These observations further establish the model's utility and validity for studies aimed at discovering the nature and extent of the vascular defect in various P disorders. The model seems especially well suited for testing impacts of P and/or endothelial cell reactive agents on platelet-vessel wall interactions.
The Isolation and Characterization of Platelet Fibringgen
Authors: Zimmermann R.E
Published: May 2019

Since platelets contribute essentially to the blood clotting mechanism the question arises as to what extent their physiological role is expressed by a specified type of fibrinogen.

Platelets are harvested , washed exhaustively and homogenated by freezing and thawing. After gradient centrifugation of the homogenate fibrinogen was isolated by controlled precipitation steps. According to biochemical analysis platelet
fibrinogen is of a unique polypeptid-composition, differs in its carbohydrate content and reveals a different kinetic of fibrin formation.
Purification of Platelet Glycocalicin and Glycoprotein I by Affinity Techniques
Authors: Moroi M. , Dubay E. , Jamieson G.A.
Published: May 2019

Glycocalicin and glycoprotein I are similar, or identical, proteins which appear to differ in their degree of affinity for the platelet surface and which mediate the interaction of thrombin and Factor VIII with platelets. Glycocalicin has now been purified from the soluble fraction of platelet homogenate by a two step procedure involving affinity chromatography on WGA-Sepharose and elution with borate-glucosamine, followed by affinity chromatography on thrombin-Sepharose and elution with heparin or 1 M sodium chloride. Glycoprotein I was prepared from a Triton extract of platelet membranes by a similar series of steps, but in this case two fractions were obtained requiring elution from WGA-Sepharose by borate-glucosamine alone or in the presence of deoxycholate. However, the borate-glucosamine eluate separated again into two fractions on rechromatography on WGA-Sepharose. The final product was less soluble than glycocalicin itself. Cyanogen bromide fragmentation of glycocalicin gave one Pas-positive band, a major band and three minor CBB bands. These studies suggest that glycocalicin and glycoprotein I may show functional and structural dissimilarities. (Supported, in part, by USPHS Grants Nos. HL14697 and HL20971.)
Effect of Thromboplastins and Instrumentation on the Prothrombin Time Test: Implications for National Standards
Authors: Evatt B.L. , Triplett D. , Koepke J.
Published: May 2019

The independent effects that different thromboplastins and types of methods have on prothrombin time were studied by analyzing data from a 1977 proficiency testing survey of 2735 laboratories. Each laboratory received three plasma samples with prothrombin times of approximately 12, 16 and 24 seconds which represented normal, partially anticoagulated, and fully anticoagulated plasma respectively. Combinations of 12 different instruments and 12 different thromboplastins were used by the laboratories in evaluating the samples. As determined by a two-way analysis of variance, both the type of thromboplastin and the type of instrument have similar effects upon the prothrombin time. When the mean prothrombin time corrected for independent effects of thromboplastin and method are plotted against the overall mean prothrombin times of each of the three types of plasma samples, the linear relationship between the two is obvious. These relationships can used to convert to a common scale a prothrombin time obtained with any thromboplastin method combination and provides a means for standardizing the prothrombin time test. Retrospective, analysis of data obtained from individual proficiency testing surveys in 1976, 1977, and 1978 suggests that the relationships remained constant during those years. These data suggest that the relationship of any thromboplastin to a thromboplastin standard should be determined independently of the method effect.
Measurement of Native and Degraded Forms of Plasminogen in Human Plasma
Authors: Scully M.F. , Kakkar V.V.
Published: May 2019

A method has been devised to measure the levels of native and degraded forms of plasminogen either alone or in mixtures using the known differences in the rate of activation as measured using the chromogenic substrate, S2251. Preliminary studies using the purified zymogens ascertained the conditions under which maximum differences in the rate of activation are observed. Blood is taken into citrate and trasylol (final concentration 1000K IU/ml of blood to prevent in-vitro degradation of plasminogen) and plasma prepared. Plasminogen is precipitated at 13% Na2SO4 and washed 5 times with 17% NA2SO4 to remove trasylol and antiplasmins. The fractions are reconstituted in saline and the rate of activation by urokinase measured in the presence and absence of 6-amino-hexanoic acid (final concentration 2.5mM), the ratio of these activities being linearly related to the percentage of degraded plasminogen in the mixture. The observed rate is not dependent an the concentration of plasminogen in the mixture. The behaviour of degraded and native plasminogen by this procedure was shown to be the same as that of the purified zymogens by the assay of plasma samples in which the original plasminogen was replaced by purified degraded and native plasminogen although the recovery of native plasminogen was less than the degraded forms. Various patient plasma samples have been assayed by this method
The Stabilizing Effect of Trasylol on Platelet Membranes
Authors: Reuter H.D.
Published: May 2019

The proteinase inhibitor TrasylolR (2000 U/ml) in vitro inhibits aggregation of human platelets induced in citrated plasma by 2 and 4 μM/ml adrenaline significantly (p < 0.01) by 61.3±14.0% and 61.8±15.1 % (mean + S. D.), respectively. Aggregation induced by 4µM/ml ADP was inhibited significantly (p < 0.01) by 24.6±7.6% while aggregation induced by 2 µg/ml collagen was inhibited by only 7.2±5.0% (n.s.). In adrenaline- and ADP-induced aggregation, first phase was inhibited slightly while there was strong inhibition of the second phase which indicates release reaction. The varying extent of inhibition obtained by the aggregation inducers adrenaline, ADP, and collagen may reflect the varying degree of replacement of aggregation inducers from the platelet membrane by the competitive inhibitor Trasylol. The inhibition of retraction (x = 66.6%) also reflects inhibition of the release of platelet constituents. Trasylol because of its cationic properties can bind to the negatively charged platelets thus influencing the membrane-bound enzyme adenylate cyclase which catalizes the formation of cyclic AMP. Cyclic AMP results in activating a platelet protein kinase which in turn phosphorylates a membrane protein thus stabilizing the platelet membrane.