bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation And Properties Of Intermediates Involved In Thrombin-Catalyzed Conversion Of Bovine Factor V To Factor Va
Authors: Nesheim M E , Mann K G
Published: July 2018

Bovine Factor V was incubated briefly with bovine β- thrombin, causing partial conversion to Factor Va. QAE- cellulose chromatography in Ca++ of the partially activated material yielded 3 fractions. Electrophoresis in DodSO4 indicated primarily starting material (Mr=330,000) in the first fraction; two peptides of Mr=150,000 and 200,000 in the second; and two peptides of Mr=200,000 and 90,000 in the third. The first of these latter fractions was active only after further incubation with thrombin, while the other was active without further thrombin catalyzed activation. After thrombin treatment both fractions had specific activities equal to that of fully activated, unfractionated Factor V. Incubation of either of these fractions with EDTA caused complete loss of activity and allowed isolation of the constituent polypeptides by further chromatography on QAE- cellulose in EDTA. The NH2-termini of the 150,000 and 90.000 apparent Mr peptides had sequences identical to that of Factor V. The 200,000 apparent Mr peptides of the two fractions obtained by chromatography in Ca++ had NH2- terminal sequences identical to each other but different from that of Factor V. The summed amino acid compositions of the Mr=200,000 and Mr=150,000 were equivalent to that of Factor V. Electrophoretic analysis indicated that thrombin converts the Mr=150,000 peptide to endproducts of Mr=90,000 and 71,000, and the Mr=200,000 peptide to endproducts of 83.000 and 31,000 apparent Mr. Recalcification of the individual isolated intermediates, followed by incubation with thrombin did not yield cofactor activity. Recombina- ion of the recalcified intermediates followed by incubation with thrombin, however, generated samples with activities approaching that of unfractionated, activated Factor V. These data indicate that the intermediates of Mr=150,000 and Mr=200,000 arise by a single cleavage of Factor V, and that Factor Va comprises peptides derived from both of these intermediates.
Studies On The Interaction Between Factor Va And Factor Xa
Authors: Lindhout M , Govers-Riemslag J , Rosing J , Hemker H
Published: July 2018

Thrombin activated bovine factor V is composed of two polypeptide chains with molecular weights 94,000 and 80,000. The two polypeptide chains are complexed via Ca2+ions.

Factor Va enhances the rate of thrombin formation by drastically increasing the Vmax of the prothrombin activation. We have undertaken a study of the interactions of factor Va with the different components of the prothrombinase complex (e.g. factor Xa and prothrombin), in order to get more insight in the mode of action of factor Va.

Our kinetic experiments in solution show that the functional enzyme in the prothrombinase complex is a equimolar complex of factor Va and factor Xa. The dissociationconstant, as determined over a wide range of prothrombin concentrations, has a value of 3×10-9M.

For the stimulating effect of factor Va on the prothrombin activation by factor Xa in solution, the presence of Ca2+ions is required. The dissociationconstant of the Va-Xa complex was found to be independent of the Ca2+ concentration. In order to reveal whether an interaction between Ca2+ and γ- carboxyglutamic acid residues is responsible for the observed Ca2+ requirement, identical experiments were carried out with decarboxyfactor Xa and decarboxyprothrombin.

The isolated polypeptide chains of factor Va have, in the presence or absence of factor Va, no effect on the kinetic parameters of the prothrombin activation. This let us conclude that there is no interaction between factor Xa and the separate polypeptide chains of factor Va.

The affinity of factor Xa for negatively charged phospholipid or stimulated bloodplatelets is greatly enhanced by the presence of factor Va. Our Kd value measured for the Xa-Va complex in combination with reported dissociationconstants of factor Xa-phospholipid and Factor Va-phospholipid complexes give a quantitative explanation for the above mentioned effect of factor Va on the binding of factor Xa to phospholipid membranes.
The Evaluation Of Platelet Suppressing Drugs In Patients With Unstable Angina
Authors: Gent M , Cairns J , Sackett D , Singer J
Published: July 2018

A double-blind, randomized multi-hospital study is in progress to investigate, among patients with unstable angina, the efficacy of aspirin (1300 mg daily) and sulfinpyrazone (800 mg daily), both singly and in combination, in reducing mortality, both overall and cause-specific; myocardial infarction; recurrence of unstable angina; and the frequency of aorto-coronary bypass surgery. It is proposed to enroll approximately 600 patients from six coronary care units and to follow each patient for up to two years.

An outline of the study design and execution will be presented and some of the specific methodological problems associated with this study will be discussed. In particular, attention will be drawn to the difficulty of recruiting patients to this type of trial (8000 patients screened to produce the first 200 patients actually entered into the study) and to the confounding created by other interventions among these patients, including both medication and coronary bypass surgery. An interim assessment of efficacy will not be presented but some overall descriptive data relating to patient characteristics, protocol adherance, and outcome measures will be summarized.
Intermittent Calf Compression: A Randomized Trial In Elective Hip Replacement
Authors: Gallus A , Darby T
Published: July 2018

We have evaluated venous thrombosis prevention with intermittent calf compression in 78 patients aged over 50 years having elective hip replacement. 38 patients were randomly allotted to intermittent calf compression with a “BOC-Roberts Venous Flow Stimulator”, begun at the start of surgery and continued for 7 days, while 40 patients had no prophylaxis. Age, weight, length of surgery, and other risk factors were similar in the two groups. All patients had ascending venography of the operated leg on the seventh day, or bilateral venography if routine 125I fibrinogen legscanning and impedance plethysmography suggested thrombosis on the unoperated side.

Venography showed thrombosis in 12/38 patients treated with intermittent calf compression (32%) and 21/40 untreated patients (53%). The difference is not statistically significant, but further analysis showed some interesting treatment effects: (a) calf vein thrombosis was found in 13% of treated and 35% of untreated patients (p <0.05), (b) femoral or popliteal vein thrombosis developed in 24% of treated and 38% of untreated patients, while isolated femoral vein thrombosis (without calf vein thrombosis) developed in 18% of treated and untreated patients, but (c) the femoral vein thrombi were smaller in treated patients - 8% of treated and 30% of untreated patients had femoral vein thrombi with an estimated length above 5 cm (p <0.05).

This suggests that intermittent calf compression prevents calf vein thrombosis and prevents extension of femoral vein thrombi after elective hip replacement, although it cannot prevent the initiation of femoral vein thrombi which is presumably due largely to local vein trauma. This limited effect may be clinically valuable, allowing delay in starting anticoagulant prophylaxis until this is safe.
Circulating Human Platelet Membrane Microparticles
Authors: George J N , Reimann T A , Thoi L L , Morgan R K
Published: July 2018

“Platelet dust” providing coagulant activity has been postulated to exist in human plasma. We studied human subjects to identify platelet membrane microparticles (MP) by immunologic methods in cell-free plasma. Blood was drawn into a syringe containing ACD-PGE1 and centrifuged at 30,000 g⋅min to obtain plasma with no identifiable platelets. The plasma concentration of platelet factor 4 was normal (9.4 ng/ml, n = 14), demonstrating no in vitro platelet activation. MP were isolated from diluted plasma by centrifugation at 7 × 105 g⋅min, the pellet was washed once, and solubilized in Triton X-100. Antibody from rabbits immunized with whole platelets did not cross-react with red cells or white cells. Using tandem crossed immunoelectrophoresis with MP and whole platelets, platelet antigens were identified in the MP. A specific antibody to platelet membrane glycoprotein II-III was prepared by adsorption of the anti-whole platelet antibody with cryoprecipitate and throm- basthenic platelets and used to quantify MP by rocket immunoelectrophoresis. Normal plasma MP concentration was 4.37 ± .71 (SE) μg/ml (n = 20). MP concentration was greater in serum (65.2 ± 13.2 μg/ml, p < .001) demonstrating platelet microparticle formation during coagulation. In a patient with thrombocytosis (1.76 × 106 platelets/μl) plasma MP were normal (7.5 μg/ml) but serum MP were increased (185 μg/ml). Plasma MP were assayed in 11 normal subjects before and after aspirin (640 mg/day × 7 days) and MP decreased in 9 (mean values: 4.45 μg/ml pre- and 1.92 μg/ml post-aspirin, paired t test: p <.02). Therefore platelet membrane fragmentation occurs during normal circulation and is inhibited by aspirin.
Abnormalities In Platelet Arachidonic Acid Metabolism In Chronic Idiopathic Thrombocytopenic Purpura
Authors: Stuart M J , Kelton J G , Allen J B
Published: July 2018

Patients with chronic idiopathic thrombocytopenic purpura (CITP) have been described to have bleeding times (B.Ts) that were shorter than would be predicted by their platelet counts. This phenomenon was explained by the presence in CITP of a young platelet population with increased hemostatic competence (NEJM 287:155, ’72). In contradistinction, we have observed patients with CITP to have a bleeding tendency at platelet counts >75,000/cu mm. We therefore evaluated B.Ts and platelet arachidonic acid (AA) metabolism in 7 patients with CITP who demonstrated increased amounts of platelet associated IgG (PAIgG >3fg per platelet) and compared them to 20 healthy controls. 3/7 patients with CITP and platelet counts of >75,000/cu mm demonstrated marked prolongations in their B.Ts. (10’, 12’ and 14’, normal <7’). Marked abnormalities in the metabolism of AA through the cyclo-oxygenase (Thromboxane B2 and HHT) and lipoxygenase (HETE) pathways were also observed in patients with CITP. Platelets in CITP synthesized less amounts (p <0.005) of Thromboxane B2 (10.3 ± 3.1%) in comparison to controls (22.9 ± 1.8). Values for HHT were decreased (23.7 ± 4.9 vs 39.7 ± 1.9; p<0.005), while HETE production was increased (59.5 ± 7.8 vs 30.7 ± 1.8; p<0.001). No correlation was observed between PAIgG and platelet Thromboxane B2 formation. However, an inverse correlation (r=0.81, p<0.05 was observed between the B.T. and platelet Thromboxane B2 formation in patients with chronic ITP. We have demonstrated platelet dysfunction and impaired Thromboxane B2 formation in CITP. This association should be investigated in the individual patient, since the bleeding tendency in these patients is exacerbated by the superimposed impairment in platelet function.
Variable Results With Plasma Exchange In Thrombotic Thrombocytopenic Purpura
Authors: Simon Toby L
Published: July 2018

The clinical efficacy of plasma exchange with fresh frozen single donor plasma in thrombotic thrombocytopenic purpura (TTP) was evaluated. Six patients with the classical findings of TTP, had plasmapheresis using the Haemonetics Model 30. Treatments were initiated daily. Two to three liters total exchanges were done; 1-1½ liters fresh frozen plasma were infused with each exchange. In five of the six cases, therapy was begun immediately after diagnosis. Steroids and antiplatelet agents were also used. Five of the patients were comatose when treatment was begun; the sixth had neurological symptoms of memory loss and aphasia with severe hemolytic anemia and thrombocytopenia. Circulating immune complex measurements were performed in all patients prior to treatment using the Clq and Raji cell assays.

Three patients (2 in coma) responded to therapy. The two comatose patients required more than four daily treatments before beginning to respond (followed by four and six alternate day treatments, respectively). The third patient went into remission after four daily treatments. Three patients in coma died, two deteriorating after the first plasma exchange. The third, treated after a delay due to severe cardiac complications, subsequently died.

The three responding patients were younger than the three who died. The two in coma who responded to therapy had a longer course of symptoms (suggesting disease onset prior to onset of coma) than the three who died. Circulating immune complexes by the Clq assay were negative in all six patients. The Raji cell assay was positive in one patient.

Even severely ill comatose patients with TTP respond to plasma exchange, but aggressive, persistent therapy may be required before response is seen. More fulminant disease with rapid onset may fail to respond. Circulating immune complexes do not appear to mediate the disease process.
Interaction Of Tritiated-Quinine With Human Platelets In The Presence Of A Quinine-Dependent Antibody
Authors: Christie D J , Aster R H
Published: July 2018

In drug-induced immune thrombocytopenia, it has not been established with certainty whether drug binds first to the platelet membrane or to antibody to promote platelet-antibody binding. The association of the drug, quinine, and a quinine dependent antibody (obtained from a patient with drug-induced thrombocytopenia) with human platelets was studied using a tritiated derivative of quinine prepared by catalytic hydrogen exchange. Tritiated quinine binding to platelets was measured in the presence of the antibody or normal human serum (NHS). Platelets were stirred with the appropriate reagents at room temperature for 20 minutes and washed. Bound tritiated quinine was measured by scintillation techniques.

Two types of drug binding sites, antibody-dependent and antibody-independent, were demonstrated on platelets. In the presence of NHS, fewer than 20,000 molecules of drug became tightly bound to the membrane. In the presence of excess antibody, approximately 200,000 molecules of quinine became membrane-bound. Antibody did not bind to platelets in the absence of drug. These findings appear to provide the first demonstration of antibody-dependent binding of quinine to platelets and suggest that interaction of drug and antibody in the soluble phase precedes the binding of either constituent to the platelets (“innocent bystander mechanism”). The use of radioactive drugs appears to provide a useful tool for the investigation of the molecular basis of drug- induced immune disorders.
Reconstitution Of Liposomes Bearing Platelet Membrane Glycoproteins. A Functional Study With Von Willebrand Factor
Authors: Sié P , Gillois M , Chap H J , Boneu B , Biermé R , Douste-Blazy L
Published: July 2018

Functional studies on isolated membrane glycoproteins (GP) are impaired by their poor solubility in the absence of detergent. The major glycoprotein of the platelet plasma membrane (GP lb) does not escape to the rule since its soluble derivative (glycocalicin or GP Is) does not exactly share the properties of the integral GP.

This study aimed to develop a membrane model where the GP Ib was solubilized from fresh human platelet membranes by desoxycholate, isolated using wheat-germ-agglutinin (WGA)- affinity chromatography and finally reincorporated into phospholipid vesicles by removal of the detergent. As shown by electron micrography, the method yielded individual, large - sized, single bilayered vesicles : WGA reversibly agglutinated theses vesicles arguing for the incorporation of the GP in the wall of the vesicles and the accessibility of the oligosacharidic moiety. Additionally, a rapid and convenient method for assessing agglutination was developped using thin -layer chromatography on agarose gel.

Since the GP Ib is the putative receptor for von Willebrand factor (vWF) we studied the effect of vWF on these vesicles. vWF was purified from cryoprecipitate by gel exclusion chromatography followed by an ion-exchange chromatography on DEAE-Sephacel. vWF, vWF plus ristocetin but not ristocetin alone induced a strong specific agglutination of the vesicles. This ristocetin-independent effect markedly differs from the natural behaviour of intact platelets and we speculate that the present model might help to analyse the factors which, at the platelet surface, refrain the plasmatic vWF from directly interacting with the exposed GP Ib.
Effect Of The Multimeric Structure Of The Factor VIII/Von Willebrand Factor Protein On Binding To Human Platelets
Authors: Gralnick H , Williams S , Morisato D J
Published: July 2018

We have studied the characteristics of binding of intact factor VIII/von Willebrand factor (f. VIII/vWf) protein and 2-mercaptoethanol (2ME) treated f. VIII/vWf protein to human platelets. The purified f. VIII/vWf was radiolabelled with tritiated 3H potassium borohydride; 4.5 × 103 molecules of the intact radiolabelled material bound per platelet. Of these some molecules bound with a high affinity/low capacity (Kd 0.21 nM and 1.5 × 103 molecules) and another with a low affinity/high capacity (Kd 2.5 nM and 3 × 103 molecules). When the material was reduced with 2ME at 0.01%, it bound with an intermediate affinity of 1.6 nM with a capacity of 4.0 × 103 and a low affinity binding of 12.5 nM and a capacity of 4.0 × 103 . The 0.1% 2ME-treated material revealed only low affinity binding with a Kd of 15 nM and the number of molecules bound 13 × 103. Studies of competitive inhibition of the intact f. VIII/vWf binding to human platelets by the reduced materials revealed that the smallest f. VIII/vWf protein (i.e., 0.1% 2ME) was the least effective while the 0.01% 2ME material was intermediate between the 0.1% and the intact material. The differences noted in the ability to displace the intact material as well as in binding to the human platelet were paralleled by decreases in the vWf activity of these proteins.

These studies aid in our understanding of the binding of the f. VIII/vWf to platelets in that the binding sites on platelets may be homogeneous while the ligand is heterogenous. These studies reinforce the structure/function relationships of f. VIII/vWf proteins which have been defined using ristocetin-induced platelet aggregation (i.e., the minimum molecular size of the f. VIII/vWf protein and the penultimate galactose residues on the carbohydrate side chain). We conclude that these defects of the f. VIII/vWf protein also interfere with the protein binding to its platelet receptor and that f. VIII/vWf binding to platelets is an important primary step in hemostasis.
Fibrinogen Bern I: A Hereditary Fibrinogen Variant With Defective Conformational Stabilization By Calcium Ions
Authors: Rupp C , Kuyas C , Häberli A , Furlan M , Beck E A
Published: July 2018

Inherited hypodysfibrinogenemia (fibrinogen Bern I) was found in four members (two generations) of a family with no haemorrhagic or thrombotic history. Fibrin aggregation curves (350 nm, 37°C) with patient plasma or purified fibrinogen Bern I, after addition of thrombin, were normal at high calcium concentrations (5mM) but delayed at lower calcium concentrations (≤0.lmM). The release of fibrinopeptide A was normal. Whereas the polypeptide chains of fibrinogen Bern I were indistinguishable from normal fibrinogen by SDS-gel-electrophoresis, an abnormal γ-chain with a decreased negative charge was found by isoelectric focussing.

Plasmic degradation o| normal fibrinogen, in the presence of calcium (≥ImM), results in only one terminal D fragment which is stabilized by calcium against further degradation of γ-chains. In contrast, degradation of fibrinogen Bern I, under the same conditions, yielded at least two additional smaller D fragments. In conclusion, fibrinogen Bern I is characterized by defective calcium binding in the D domain of the γ-chain.
The Role Of Glycoprotein V (GPV) In Thrombin Activation Of Human Platelets
Authors: Fujimura K , Maehama S , Kuramoto A
Published: July 2018

The analysis of platelet membrane glycoproteins and platelet functions was conducted to disclose the role of GPI and V in the thrombin activation of platelet. Our previous study proved that native and HNB thrombin hydrolyzed GPV(M. W.8-9 × 104) selectively and released new glycoprotein fragment (M.W. 6.2-6.8 × 104 ) of GPV, resulting in the development of 14C-5HT release reaction and platelet MDA production. But DIP thrombin could not induce these phenomena.

Membrane surface proteins of intact platelets were labeled with Na[3H]BH4 by neuraminidase and galactose oxidase method and analyzed by fluorography after SDS-PAGE.

The high molecular weight glycoproteins, GPI, GPIII and GPV were diminished by trypsin treatment in correlation with the concentration and incubation time. In correspond to the diminution of these membrane glycoproteins, platelet release reaction was increased .Chymotrypsin treatment in various concentrations, release reaction and MDA production were not induced in spite of long incubation times. But the ristocetin aggregation was decreased in Chymotrypsin treated platelets whose membrane glycoproteins did not change significantly. The Chymotrypsin treated platelets whose GPI was modified functionally, showed normal release reaction and MDA production by thrombin stimulation. On the other hand, the thrombin treated platelets in low concentration previously whose GPV was hydrolyzed partially, demonstrated little release reaction and MDA production by thrombin or trypsin stimulation. From these results, the GPV was hydrolyzed specifically by thrombin and nonspecifically by trypsin but was not hydrolyzed by Chymotrypsin. It was concluded that the thrombin binds to the GPI and hydrolyzed GPV specifically, and hydrolysis of GPV might act as a signal to induce the platelet release reaction and prostaglandin metabolism.
Ristocetin Interactions With Human Platelet Membranes
Authors: Takahashi M , McMahon P , Feldman J , Treffinger J
Published: July 2018

Ristocetin was coupled to CH-Sepharose-4B by coupling with l-ethyl-3-(3 dimethyl-aminopropyl) carbodiimide. The resulting adduct was treated with 1M hydroxylamine pH7.0, for 4 hours at 22°C to remove carbodiimide complexes of the phenolic hydroxyl groups of ristocetin. This ristocetin affinity column was found to strongly bind acetyl-D-alayl- D-alanine, an analogue of cell wall mucopeptides which is an inhibitor of ristocetin induced platelet aggregation. This bound acetyl-D-alanyl-D-alanine was removed by passing vancomycin, a structurally related antiobiotic, through the column. When Triton-XlOO solubilized human platelet membranes were applied to the column, only a small percentage of membrane proteins adhered (ca.<l%). This column was eluted with 50mM acetyl-D-alanyl-D-alanine which released bound membrane proteins. SDS gel electrophoresis of the eluted proteins revealed two bands which stained with PAS reagent. One PAS positive band was in a position corresponding to glycoprotein (GP-Ib). The binding observed both for acetyl-D-alanyl-D-alanine and the platelet membrane derived proteins points to functional specificity attributable to affinity binding to the immobilized ristocetin and gives further evidence for participation of glycoproteins in ristocetin mediated platelet aggregation.
Fibrinolysis And Coumarin Therapy: A Possible Relationship
Authors: Bauer J , Bachmann F
Published: July 2018

It has been reported that a good proportion of patients suffering from idiopathic deep vein thrombosis (IDVT) exhibit a defective release of plasminogen activator after venous stasis (Isacscn & Nilsson, Acta Chir Scand 138:313-319, 1972).

We have studied the 3 known activation pathways of physiological fibrinolysis in 36 patients with IDVT and 21 controls. The activity of the Hageman factor and contact phase Independant Intrinsic Fibrinolytic pathway (HIIF) was measured in the euglobulin fraction of a plasma obtained in fasting subjects after a 30 min rest period at 9 a.m. Activity of the Hageman factor Dependant Intrinsic Fibrinolytic pathway (HDIF) was measured after activation of the plasma by dextran sulfate. HDIF activities were expressed as A (after-before) dextran sulfate activation. Venous stasis induces the release of vascular activator. The latter triggers off the Extrinsic Fibrinolytic pathway (EF). Its activity was expressed as the Δ (euglobulin activity after venous stasis - HIIF activity).

26/36 patients (72%) with IDVT showed impaired fibrinolysis of at least one pathway. 24 (67%) exhibited a defective HIIF: in 14 (38%), this was an isolated defect, in 10 (28%), impaired HIIF was associated with defective EF. An isolated defect of EF occurred in only 2 patients. HDIF was normal in all patients. 20 of 24 antiooagulated patients, but only 4 of 12 nonanticoagulated patients had impaired HIIF, either alone or in combination (X2=9; p < 0,005).

These results strongly suggested an association of impaired HIIF and oral anticoagulation. When HIIF, HDIF and EF were measured before and 10 to 14 days after acenocouma- rol treatment in normal volunteers, there was marked decrease of HIIF activity, suggesting that an as yet non- identified vitamin K dependant factor participates in the HIIF pathway.
The Effect Of Exercise And Oral Contraceptive Agents On Hemostatic And Fibrinolytic Mechanisms In Trained Athletes
Authors: Huisveld I A , Hospers J E H , Bouma B N , Zevenbergen J L , Bernink M J E , Erich W B M
Published: July 2018

Exercise increases hemostatic and fibrinolytic activity in humans. The same has been reported for oral contraceptive agents (OCA). We studied the combined effects of strenuous exercise and OCA in twenty highly trained female athletes. Ten of the subjects had been using the pill (30 meg ethinylestradiol) for more than a year (users). The other ten females not using OCA served as a control (non users). All subjects were exercised to exhaustion which was attained within 10-16 min. by means of an increasing workload test on a bicycle ergometer. Tests were performed during morning hours and only in the first half of the therapeutic or menstrual cycle. Hemostatic and fibrinolytic components were determined in pre and postexercise plasma samples. Changes in plasma volume that occurred with exercise were calculated and postexercise results were corrected for these changes. Procoagulant activity of Factors XII, XI and VII were determined using partial thromboplastin time assays. HMWK, α2-macroglobulin and Ci-INH were quantified by an immunodiffusion technique using monospecific antisera. Synthetic chromogenic peptide substrates were used for the determination of plasminogen, prekallikrein,α2 antiplasmin and AT III amidolytic activities. Comparison or resting values showed an increase in F XII and plasminogen concurrent with a decrease in Cl-INH level in the group of users. The combination of exercise and OCA induced a decrease of all plasma components except F VII in the users. Postexercise decreases in F XII, HMWK, prekallikrein and they two inhibitors α2-macroglobulin and α2-antiplasmin were seen in the non users. Results indicate that hemostatic and fibrinolytic changes due to strenuous exercise are potentiated by OCA in trained females. The relevance of corrections for changes in plasma volume is demonstrated.
Plasminogen Activation In Normal Individuals And Patients With Thrombotic Tendency
Authors: Schulman S , Holmgren A
Published: July 2018

Following the principles described by Wohl, Summaria and Robbins we have studied the kinetics of human plasminogen activation by streptokinase. Plasma plasminogen activation kinetic parameters were measured in the following way: Citrated plasma was mixed with a Tris/NaCl buffer, pH 7.4, and increasing amounts of streptokinase were added. After incubation at 37°C for 5 min, synthetic substrate, H-D-val- -L-leu-L-lys-p-nitroanilide was added and initial plasmin rates were recorded. We also determined the Michaelis-Man- ten parameters for the plasmin of the plasma samples following maximal activation of the plasminogen with streptokinase. This was done by incubating citrated plasma (diluted 1:200) with streptokinase (5000 U/ml) for 10 min at 37°C.

In the normal individuals the zymogen.streptokinase complex gave a mean catalytic efficiency, kcat/KM, of 0.15 μM-1S-1. For the streptokinase activator species the average catalytic efficiency, kplg/Kplg, was 26 μM-1min-1, Iμ(95%) = {8.4; 44.5} μM-lmin-1.

So far we have studied eight patients with a history of thrombosis. With the exception of one patient, all showed values within the range mentioned above. The deviating patient had a significantly lower concentration of plasminogen, 6.7 mg/dl, as measured by immunodiffusion, and a significantly higher kplg/Kplg-value, 54 μM-1min-1, but a similar kcat/KM-value, 0.2 μM-1S-1. The lower plasminogen concentration is thus matched by an apparent increase of streptokinase activation. We do not know if the observed effect is caused by a mutation in the enzyme. An explanation for the low concentration of plasminogen could be chronic fibrinolysis of thrombi.

We will investigate this further and also screen more patients with a history of thrombosis for possible variants with lowered catalytic efficiency of zymogen activation.
Inhibitory Activity Of Heparin And Of A Heparin Side Fraction (Org.10172) On The Activation Of Thrombin In Human Plasma
Authors: Jonker R , van Houdenhoven F , van Dedem G
Published: July 2018

The dynamics of thrombin generation and -inactivation in human plasma was studied in the absence and presence of heparin and of Org 10172, with the purpose of defining (a) mechanism(s) whereby anticoagulant action is accomplished.

Plasma was activated with a standard stimulus of either thromboplastin and Ca++ or by kaolin-cephalin and Ca++ ; subsamples were taken at regular time intervals and assayed for amidolytic-(S 2238) and antithrombin-3 (AT3) activities. Similar experiments were done in the presence of heparin and of Org 10172.

With heparin, 2 domains could be distinguished: a) a domain (at heparin concentrations below 1 U/ml) where heparin accelerates the inactivation of thrombin and b) a domain (at heparin concentrations above 1 U/ml) where no measurable free thrombin is generated and no measurable drop in AT3 levels could be detected.

With Org 10172 the most pronounced effect was a dose- dependent delay in the onset of free thrombin generation and a weakly dose-dependent reduction in the free thrombin peak levels.

In AT3-free plasma, thrombin generation after a standard stimulus was very rapid and the final constant level of am- idolytic activity was higher than in normal plasma. Heparin concentrations of 2 - 4 U/ml changed this pattern to a gradual increase of amidolytic activity to its final level.

Org 10172 showed the same pattern as heparin and also delayed the onset of free thrombin generation in a dose- dependent manner.

It is concluded that a) the anticoagulant effect of heparin in plasma, activated by a standard stimulus is due to at least 2 mechanisms, one of which is probably independent of AT3, and b) Org 10172 exerts its effect independently of AT3, possibly by inhibiting one of the positive feedback loops in the coagulation sequence.
The Influence Of Heparin, Phospholipid And Platelets On The Thrombin Or Factor Xa Inactivation By Antithrombin III
Authors: Ofosu F , Blajchman M , Cerskus A , Hirsh J
Published: July 2018

It has been known for many years that the effects of heparin are antagonized by platelets. This effect has generally been attributed to the release of platelet factor 4 (PF4). Heparin has recently been shown to disrupt the interactions of vitamin K-dependent clotting factors with phospholipid. This is due in part to the binding of heparin to phospholipid. The present investigations therefore explored the possible effects of phospholipid (cephalin) and degranulated platelets on the interaction of thrombin or factor Xa with antithrombin-III in the presence of heparin. These interactions were determined by the measurement of the second order rate constants for the inactivation of purified factor Xa or thrombin by purified antithrombin-III. The rates of inactivation of thrombin or factor Xa by antithrombin-III increased with increasing concentrations of heparin. Cephalin reduced the magnitude of the heparin effect on factor Xa but had no additional effect on thrombin inactivation. Relative to cephalin, gel filtered platelets reduced further the ability of heparin to enhance the inactivation of factor Xa by antithrombin-III. When the platelets were depleted of PF4 by degranulation with thrombin (2u/ml), the effects of platelets on the protease- antithrombin-III-heparin reactions approached those obtained with cephalin. However, no detectable effect on the inactivation of factor Xa or thrombin by antithrombin- III was demonstrable by gel filtered platelets, degranulated platelets, or cephalin in the absence of heparin. These results indicate that in addition to the contribution made by the release of PF4 by platelets, the calcium dependent binding of factor Xa to cephalin and platelet phospholipid is capable of protecting factor Xa from inactivation by antithrombin-III and heparin.
Isolation And Characterization Of The Vitamin K Dependent Domain Of Human Prothrombin
Authors: Dodé C , Thiesce A , Labie D , Elion J
Published: July 2018

Chymotryptic cleavage of human prothrombin (hPt) produces two fragments of 68000 and 5000 MW respectively. These two species were separated by Ba citrate adsorption and Sephadex G100 chromatography. The 68000 MW species corresponds to hPt (des 1-44) and has lost all the vitamin K dependent properties of hPt : adsorption on Ba citrate, Ca+2 and phospholipid dependent stimulation of the activation by FXa, presence of strong Ca+2 binding sites (as shown by dialysis equilibrium) and Ca+2 induced fluorescence quenshing. The 5000 MW species corresponds to the N-terminal portion of hPt (residues 1-41). It contains the 10 Gla residues and the 17-22 disulfide bond. This peptide is quantitatively adsorbed on Ba citrate. Activation of hPt in a mixture containing FXa, Ca+2 and phospholipid is drastically inhibited by the addition of peptide 1-41 (≃ 50 % inhibition for a 1/1 peptide/Pt ratio, ≃ 7 % for a 40/1 ratio).

Ca+2 produces a quenshing of the intrinsic fluorescence of peptide 1-41 (λ emission = 355 nm). This quenshing plateausat 40 % of the initial fluorescence at 3 mM Ca+2. The plot of the fluorescence quenshing versus Ca+2 concentration however is not cooperative. Mn+2 and Mg+2 also induced fluorescence quenshing but to a lesser extent. Hence peptide 1-44 represents a functionnal domain in itself interacting with Ca+2 and phospholipid. It contains only one Trp (residue 41), showing directly the involvment of this residue in the Ca+2 induced fluorescence quenshing observed for Pt fragment (FI) and Pt. This isolated vitamin K dependent domain therefore retains many of the vitamin K dependent properties of FI or Pt, but shows differences in the Ca+2 induced conformational change in that it is no longer a cooperative process.
Preferential Binding Of Prothrombin Fragments On Monolayers Containing Phosphatidylserine
Authors: Lecompte M F , Miller I R
Published: July 2018

Phospholipids from platelet membranes play a crucial role in the last step of the blood coagulation cascade, as a catalytic surface for the conversion of prothrombin into thrombin by the prothrombinase complex. It provides a favora ble configuration of the required factors ( factor Xa, prothrombin, factor Va ), concentrated on it’s surface. The maximal biological activity is observed with mixtures of phospholipids containing phosphatidylserine, in presence of Ca++.

In order to understand at the molecular level, which part of prothrombin interact with phospholipids, we studied the correlation between the number of prothrombin molecules or fragments of it adsorbed ( by counting the surface radioactivity of 3H-protein ) and their ability to penetrate into the phospholipid monolayer ( measured by the increase of the lipid layer capacitance ), by two recent complementary methods of direct surface measurement ( radioactivity and electrochemistry ), in function of Ca++ concentration and phospholipid composition.

We could detect parallely the number of cystine residues ( by electrochemical reduction ) of the penetrating parts of the adsorbed protein.lt occurs that adsorbed protein don’t penetrate. The number of S-S groups reduced per adsorbed protein molecule can be deduced and its variation indicate different orientation of the molecules on the phospholipid surface. Indeed, it is the case at different Ca++ concentration,for prothrombin and fragment 1, which contain γ-carboxyglutamic groups.

We could also observe that penetration is specific of phospholipid composition for fragment 2 but not for thrombin and is not specific for both in function of the Ca++ concentration.

Results indicate that the ratio of penetration to the total number of adsorbed prothrombin molecules and fragment 1 is important for an optimal catalysis.
Calcification At The Blood-Material Interface. A Study Employing X-Ray Diffraction And Other Methods In Various Pumps
Authors: Murray J D , Harasaki H , Gibbons D F , Kiraly R J , Nose Y
Published: July 2018

Blood pumps assisting the natural heart have been tested successfully in calves for up to 9 months. The aim of current research is for an assist device to last 2 years or longer for clinical application. To achieve this, the problem of calcification on the blood contacting surface of the pump must be solved, or deterioration of the pump and a source of emboli could result.

Scanning electron microscopy(SEM),energy dispersion x-ray analysis(EDAX).thickness mapping,and histology have been used to characterize the pseudoneointima(PNI),or cellular layer.depositied on the diphargm of the assist devices. Calcification in the cellular components in the flexing area of the diaphragm has been seen in 22/47 left ventricular assist devices implanted 12-227 days,and in 19/26 par- athoracic ventricular assist devices implanted 5-160 days. Hydroxyapatite,the final crystalline form of calcium phosphate in calcification,was detected using x-ray diffraction, which is the best means of determining crystal structure.

In the rough, dacron fibril flocked surface of the PVAD, hydroxyapatite was first observed after 13 days,and in the smooth.biolized gelatin surface of the LVAD,hydroxyapatite was detected after 43 days. Relating to the pathogenesis of calcification, RBC and platelets were embedded separately in gelatin and reacted with calf serum in an in vitro experiment to determine the nidus for calcification. TEM,histology and x-ray diffraction were performed on the embedded material after 3,7,14,21,and 28 days. Hydroxyapatite was seen in 1 week in the RBC and after 3 weeks in platelets.

The in vitro data support the in vivo evidence that calcification is dystrophic in nature.occurring in the degraded RBC and platelets contained in the PNI. It is also indicated that increased stress at the flexing region,thicker PNI,and high serum phosphorus level exacerbate calcification.
Assessment Of Thromboresistant Materials By 125I-Fibrinogen Scanning In Animals And Man
Authors: Lewis J L , Sweeney J , Houlihan M J , Baldini L , Salzman E W
Published: July 2018

Further development of artificial organs requires improved methods to evaluate potentially thromboresistant materials, for which there is at present no simple method in man. We now report that thrombi associated with intravenous cannulae can be detected using 125I-fibrinogen and a portable gamma counter. FEP/Teflon cannulae were inserted percutaneously in 9 dogs receiving EACA and 1 sheep, and four sites over each cannula and over a control vein were counted before and at 30 to 45 minute intervals for up to 6 hours after cannula insertion. Animals were heparinized before sacrifice. Results were confirmed by autopsy. Nine of ten thrombi related to 12 cannulae were detected by a significant rise in counts (p<0.05, 3 way ANOVA). There were no false positives; the single false negative was related to a filmy thrombus with low gamma activity when counted directly. The smallest thrombus detected weighed 1 mg, representing fibrin arising from 500 μl of blood.

Thirty-nine patients were studied. Radioactivity.over an IV catheter introduced after administration of 125I- fibrinogen was compared with independent blinded clinical appraisal by a trained nurse. Significant progressive rise in radioactivity over the catheter tip or within 2 cm downstream developed in 13 of 14 forearms with 16 or 18 gage cannulae, all of whom subsequently developed rubor, tenderness, palpable thrombus, or plugging of the catheter. The rise in 125I counts preceded the clinical signs by 12 to 36 hours. 22 gage cannulae produced clinical signs of phlebitis in only 42% of cases but increased 1251 counts in 92%. Phlebograms were confirmatory.

125I-fibrinogen external counting is a simple, safe, and sensitive technique to detect thrombosis induced by intravenous cannulae in man and should be suitable for the comparison of potentially thromboresistant materials.
Human Platelet Aggregation In Laminar Flow: Stimulus And Shear Rate Dependence, With Effects Of Acetylsalicylic Acid
Authors: Yung W , Frojmovic M M
Published: July 2018

The shear rate (G) dependence of aggregate formation and stability of platelets in human citrated platelet-rich plasma (PRP) in laminar flow was studied for G=0-150 sec-1, a range encountered in many flow conditions, by injecting adenosine diphosphate (ADP) into PRP within a cone-in-plate device at 37°C. Macroaggregation was assessed by visual observation and light transmission (%T), as routinely done in aggregometry, while actual percent aggregation (PA) was determined by microscopy of sub-samples from the decrease in single platelet concentration. Control PA ≲ 8% (n=8), independent of G. Low [ADP] ≃ 0.75-1.5 μM, causing reversible aggregation in the aggregometer, gave shape change, and stable visible thrombus formation with maximal PA μ 25% at low G ≲ 30 sec-1; the apparent extent of aggregation (Δ%T) passed through a maximum at G μ 30 sec-1, with aggregate disruption occurring at G > 30 sec-1. For high ADP ≳ 5 μM, causing stable maximal aggregation in the aggregometer, the rate of aggregation increased continuously with G, with PA 90% and visible thrombi stable for G=15-150 sec-1. Aspirin (200 μM) (n=8) or indomethacin (10 μM) (n=2) - treated platelets activated with high ADP ≳ 5-7.5 μM, exhibited the same stable aggregation at low G (≲ 30 sec-1) as found for untreated platelets; higher G ≲ 30 sec-1 yielded only reversible aggregation as seen in the aggregometer, with PA only partially inhibited. It seems that (1) at high [ADP] (≳ 5 μM), increasing G normally increases the rate of platelet aggregation without causing deaggregation, and (2) for [ADP] up to - 7.5 μM, in the absence of endoperoxide synthetase activation, shear stresses (τ) of only ≃ 1-3 dynes cm-2 prevent the formation of stable large thrombi though still allowing appreciable PA; sufficiently high ADP (10 μM) overcomes these τ destabilizing effects.
Effects Of Short Time High Shear Exposure Upon Platelet Function And The Coagulation System Under Heparin Anticoagulation
Authors: Wurzinger L J , Opitz R , Blasberg P , Bialonski K , Schmid-Schönbein H
Published: July 2018

The fact that high shear activates and damages platelets has been suspected to be a major cause of thromboembolism in artificial internal organs (AIO) or in arterial stenosis. In AIO wall shear stresses well above 50 Nm-2 have been computed to which blood cells are exposed for times in the order of milliseconds (ms). Unfortunately, the studies on this subject employing defined flow conditions operate with exposure times higher than 10 seconds. The pupose of the present study was to elucidate the effects of high shear exposure for ms upon platelet function (ADP induced platelet aggregation (PA), platelet procoagulant activity {PF- 3)) under heparin anticoagulation, which is also used in AIO.

To apply shear rates ranging from 50 - 220 Nm-2 to heparinized PRP for defined exposure times between 7 - 700 ms a flow through Couette-viscometer was employed. Platelet factor 3 (PF-3) availability was estimated by using a modified Stypven time technique. Lactic dehydrogenase (LDH) liberation was taken as a measure for platelet destruction. All steps of the experimental procedure were carried out at 37°C.

From our data we conclude that, in the presence of physiological calcium levels (heparin anticoagulation) shear stresses and exposure times that certainly occur in AIO are able to activate platelets and procoagulant potential of blood.
Direct Evidence For The Contractility Of Endothelial Cells
Authors: Clerck F De , Brabander M De
Published: July 2018

Dog aorta endothelial cells, grown in culture, were resuspended in human citrated (Na3 . citrate . 2H2O, 0.313 %) platelet-free plasma (PFP) at cell densities between 2 and 0.5 × 106/ml. Fractions (60 % PFP) were coagulated at 37°C with thrombin, 2 N.I.H. U/ml, in the presence of barbital- HC1 buffer pH 7.35 (40 %) and CaCl2 5 × 10-4 M. Endothelial cells stimulated with thrombin induced clot retraction, the onset and degree of which depended upon cell density, cellular integrity, homogeneous cell distribution and availability of extracellular Ca2+. Contraction was completely inhibited by EGTA (8 mM), by papaverine (10-3 M)-induced phosphodiesterase inhibition and by VK 774 (10-4 M)- induced inhibition of glycolysis. It was partly reduced by nocodazole (I μg/ml)-induced microtubuli disruption, by cytochalasin B (1 μg/ml) and by verapamil (10-1 M)-induced intracellular Ca2+-blockade, but was unaffected by PGEl (3.3 μg/ml) and by the cycloxygenase inhibitor suprofen (10-1 M). E.M. examinations revealed pseudopod formation and interaction of the cells with fibrin strands. The contractile capacity of endothelial cells may contribute to the regulation of blood flow distribution in the microcirculation.
Thrombotect: An Optimized Sample Collection System For The Inhibition Of In Vitro Platelet Release
Authors: Workman E F , Clark D M
Published: July 2018

Platelet activation occurs in a variety of thrombotic disease states and may be monitored via measurement of Platelet Factor 4 (PF4) levels in plasma. In order for PF4 levels to reflect in vivo activation, care must be taken to eliminate in vitro α -granule release during the collection and preparation of blood specimens. Thrombotect is an evacuated blood collection tube containing an anticoagulant (EDTA) and two platelet inhibitors (2-chloroadenosine and procaine-HCl). This system is effective in preventing in vitro release under a variety of centrifugation conditions. Subsequent to a 30 minute icing step, whole blood samples may be centrifuged at room temperature and one of three centrifugation speeds - 2500 × g for 20 minutes,1500 × g for 30 minutes, and 1000 × g for 40 minutes. Plasma specimens from normal individuals were assayed after being prepared under the above conditions. PF4 levels were as follows: 2500 × g, *2.79 ± 1.00 (73); 1500 × g, 3.84 ±1.78 (20); 1000 × g, 3.35 ± 1.65 (20). Variations in the temperature of centrifugation had little effect on the ability of Thrombotect to inhibit PF4 release, whereas other commonly used blood collection cocktails became progressively less effective with increasing temperature.

*(Mean PF4 in ng/ml ± 1 S.D., “n” values in parenthesis). Results similar to those obtained for EDTA/Theophylline were obtained when EDTA was used in conjunction with adenosine and/or aspirin. The Thrombotect system has been employed in an examination of PF4 levels in a variety of thrombotic disease states. It also provides an excellent collection system for use with other “platelet-release” assays.