bluuebunny/crossref_metadata_2025 huggingface dataset:
A Morphological Study Of Clot Contraction During The Development Of Isometric Tension
Authors: Gerrard j M , White J G , Cohen I
Published: July 2018

Under isometric conditions, and in the presence of micromolar Ca2+, clots of normal platelet-rich plasma develop tension at an initial rate of 0.1 to 0.2 g min-l cm-2 (initial cross-sectional area). When fixed under tension, after attaining a force of 1.6 g cm-2, electron microscopy reveals that platelets extend long pseudopods along the fibrin strands which are oriented in cable-like fashion in the direction of the isometric tension. Cross-sections of the pseudopods show microtubules in most. The extension of pseudopods paralleled the development of tension and was dependent on the quality of the platelets as well as on Factor XIIIa, catalyzed cross-linking of fibrin fibers. Normal platelets suspended in factor XIII deficient plasma failed to develop tension or long pseudopods but did so on addition of pure factor XIII. Pseudopod extension and development of tension were inhibited by either 10-4M vincristine which disrupted platelet microtubules or by 20 ug/ ml cytochalasin B which interfered with platelet microfilament function. When platelet numbers were decreased, tension development decreased approximately linearly. The decrease in platelet number was not compensated for by an increased length of platelet pseudopods suggesting that platelet fibrin interaction is essential for tension, while platelet-platelet contact is not. We suggest that clot contraction requires the extension of pseudopods along cross- linked fibrin strands, followed by their contraction orienting the fibrin fibers in the direction of tension and compacting the fibrin network into platelet-fibrin clumps.
Effects Of Hemostatic Agents On Platelet Aggregation
Authors: Sawyer P N , Schwann G , Stanczewski B , Jones M T
Published: July 2018

Several new collagen derived hemostatic agents have been introduced into the surgical armamentarium. The presumption is they produce platelet aggregation, ADP release and blood clotting through the intrinsic system.

Studies of interreaction of these new hemostatic agents with both blood and platelets suggests that the type of reaction with whole blood is not known or due to primary platelet activation. Avitene interreacts in part by platelet aggregation but also by interreacting with other components in blood including fibrinogen.

The available evidence with a new dissolving collagenous hemostat, “Superstat”™ indicates probable activation of the extrinsic coagulation system, directly converting prothrombin to thrombin, and fibrinogen to fibrin. Thrombin time ratios (experimental vs. control) are Superstat 1% - 0.81 ± 0.16/0.93 ± 0.06, Superstat 2% - 0.84 ± 0.11/0.93 ± 0.06, Superstat 3% - 0.61 ± 0.15/0.71 ± 0.02. Comparative studies have been completed on several other agents, among them, Kollagen Haemo. Vlies - 0.66 ± 0.1/0.71 ± 0.02, Gelfoam - 0.75 ± 0.15/0.71 ± 0.02, Surgicel - 0.73 ± 0.4/ 0.71 ± 0.02, Avitene - 0.75 ± 0.3/0.71 ± 0.02, Collatamp - 0.73 ± 0.4/0.71 ± 0.02. Derived data from thrombin times, clotting times and, when possible, platelet aggregation displays widely different rates of thrombus formation. We believe the enzymatic mechanisms for these reactions are currently only partially known.

The fact that clinically useful hemostatic agents each appear to produce hemostasis by several routes, and do not all use the same route, is of extreme interest in basic thrombosis studies as well as in clinical surgery.
Control Of Factor IXa Production By The Formation Of A Factor XIa-Factor IXa Complex
Authors: Jonan A , Slattery C W
Published: July 2018

The activation of factor IX represents a transition step between the “contact phase” and the “phospholipid-dependent phase” of intrinsic blood coagulation. A study was made to see if any of the constituents of either of these phases would influence this activation. Trypsin-activated factor XIa and factor IX were preincubated with various combinations of kaolin, high molecular weight kininogen, factor XII and human brain cephalin before being added to factor IX deficient plasma to determine the clotting time. Of these, only cephalin in the preincubation mixture apparently increased factor IXa activity. Measurements of the rate of activity generation with and without cephalin and with early and late addition of cephalin indicated that cephalin caused a five-fold increase in the maximum obtainable factor IXa activity. Late addition started at lower activity and gradually increased to the maximum. Factor IXa, isolated from factor XIa by means of an immobilized factor Xl-antibody column, responded to cephalin addition by becoming more active and to factor XIa addition by becoming less active. Centrifugation of cephalin-protein mixtures showed that neither factor XIa nor factor IX but only factor IXa was bound to the phospholipid. These data suggest that: 1) the activation of factor IX by factor XIa does not occur on a phospholipid surface; 2) once cleaved, the factor IXa that is produced remains bound to the active site of factor XIa, preventing further factor IX activation until a phospholipid surface is available for the reaction sequence to continue; 3) phospholipid competes with factor XIa for the bound factor IXa and these, in a time-dependent interaction, then form an active complex. This mechanism would prevent the production and dissemination of large amounts of factor IXa until required for further clotting.
A Simple Method For Preparation Of Nonthrombogenic Prothrombin Complex
Authors: Wickerhauser M
Published: July 2018

Prothrombin complex concentrate (PCC) when infused into patients may produce thrombotic episodes, presumably due to the presence of some thrombogenic material. In order to prepare a safer PCC we have investigated whether the addition of purified antithrombin III (AT III) during an early stage of the isolation process would be effective in reducing thrombogenicity as assessed by the Wessler stasis model. By our method the cryosupernatant of plasma is adsorbed with DEAE-Sephadex (1.5 g/L), the unadsorbed fraction removed, the DEAE-cake washed with 0.7-1.0 plasma volume of citrate saline, followed by elution with 0.1 plasma volume of 0.03 M citrate containing 2 M NaCl and 0.25 AT III unit/ml, at pH 6.8. The eluted PCC is then concentrated and desalted by ultrafiltration, filtered, dispensed and lyophilized.

In a bench-scale experiment starting from 6 L cryosupernatant, a PCC was obtained at 43% recovery and 50-fold purification over plasma based on Factor IX activity. The recoveries of Factors II, VII and X were 44, 23 and 64% respectively. The concentrate was free of detectable thrombin and plasmin and contained about 1 unit of AT III (determined immunologically) per 25 units of Factor IX. In the Wessler stasis model this PCC was nonthrombogenic at 100 Factor IX units per kilo body weight of the rabbit. Higher doses were not tried. A control PCC prepared without the addition of AT III to the DEAE-eluting buffer was nonthrombogenic at 50, but thrombogenic at 75 Factor IX units per kilo body weight of the rabbit. Subsequent incubation with AT III did not eliminate this thrombogenicity.

These results suggest that AT III can be effective in preventing thrombogenicity in PCC but not in abolishing it if already developed.
The Role Of Factor VIII: C In Fibrinogen - Fibrin Conversion
Authors: Frost T J , Lau R S
Published: July 2018

The cascade mechanism for the coagulation pathway has prevailed since its original proposal, and subsequent observations have supported the role of thrombin on coagulation reactions other than in Fibrinogen - Fibrin conversion. In very small quantities, thrombin has been reported as increasing the activity of Factor VIII: C. We report here, observations which support a reverse proposal, namely that Factor VIII: C increases activity of thrombin in Fibrinogen - Fibrin conversion.

Thrombin clotting times were performed employing human Fibrinogen solutions, fresh citrated normal platelet - poor human plasma and fresh citrated platelet - poor plasma from severely affected Hemophilia A patients, in whom Factor VIII : C assays were less than 1% in a one stage, activated partial thromboplastin time assay. A platelet aggregometer was used to assess the thrombin time, and the rate of clot formation (fibrin polymerization) was indicated by optical density change in the form of a curve, the tangent of the maximal slope of curve being equated with this clot formation rate.

Results indicate that with very small concentrations of thrombin, Factor VIII: C enhances the rate of fibrin clot formation and that Factor VIII: C restores the abnormal rate of clot formation observed in severe Hemophilia A; the test system was known to be calcium-free, which ensured that activation of Factor X did not ensue. The concentration of Fibrinogen remained constant in all experiments. The observed phenomena are confirmed when employing purified fibrinogen solutions, using decreasing amounts of Factor VIII: C.
Qualititative Structure Activity Relationships In Serveral New Types Of Oral Anticoagulants
Authors: Rehse k , Bienfait R , Emisch U , Kapp W , Schinkel W , Tenczer J
Published: July 2018

Various 1, 2-benzoxathiine-2, 2-dioxides (1), 2,3,4,5-tetra- hydrobenzothiepine-3, 5-dione-l,1-dioxides (2), 4-hydroxy-2- pyrones (3) 2, 4, 6-(1H,3H,5H)-pyrimidinetriones(4), tetronic acids (5) and 1, 2-oxathiolane-4-one-2, 2-dioxides (6) have been synthezised to examine structure activity relationships in the field of oral anticoagulants. The compounds are substituted by 4-chlorocinnamoyl, 1-phenylpropyl or l-(4-chlorophenyl)-3-oxo-butyl residues (R)in a suitable position. After single oral administration to rats (25-300 mg•kg-1) prothrombin levels (PT) of less than 25 percent are achieved within 10-48 hours with compounds of type 1-3 and 5. Compound of type 4 and 6 were inactive. The results show that not only 4-hydroxycoumarines (7) and 1,3-indanediones (8) exhibit indirekt anticoagulant activity. The lactone moiety of 7 can be replaced by a sultone moiety. The benzene ring needs not to be fused to the heterocyclic part of the molecule (see 3). The heterocyclic part may be a simple five memberea lactone. The effects of 8 can be extended to seven membered and even heterocyclic ring systems. Furthermore seme 2 showed platelet aggregation inhibiting activities (Born, IC50 = 5 • 10-4M). Some 5 showed direct anticoagulant activity (PT, PTT, in vitro).
Changes Of Plasma Thromboxane B22 And Cyclic Nucleotides At Exercise Test In Patients Of Coronary Heart Disease
Authors: Numano F , Yajima M , Hosaka T , Nishiyama K , Shimokado K , Numano F
Published: July 2018

To search the response of vascular wall at exercise, submaximal treadmill test was performed in 30 patients with the complaint of chest pain and plasma thromboxane B2(TXB2), cyclic AMP(cAMP) and cyclic GMP(cGMP) levels were measured before, immediately and 30 minutes after the test by radioimmunoassay. Using Hollenberg’s treadmill exercise score modified by us. 15 patients were judged as “positive” (P- group) and others as “negative” (N-group). Plasma levels of TXB2 in P-group were 262.4 ± 30.9 pg/ml, a statistically high level as compared with 186.7 ± 17.5 in N-group. In N-group no changes of plasma TXB2 levels were recognized after exercise. However, in P-group this level revealed a statistically significant decrease immediately after exercise and restored to the previous level 30 minutes after exercise. Although there were high levels of both cAMP and cGMP in N-group (cAMP: 11.9 ± 1.0 pmoles/ml; cGMP: 3.1 ± 0.5), as compared with these in P-group (10.9 ± 1.5, 2.6 ± 0.5), the significance was not statistical. However, in N-group levels of cAMP and cGMP increased significantly immediately after exercise and restored to the levels before test. On the other hand, no significant changes of these levels were found in P-group. These data may suggest that the poor response of vascular wall for haemodynamic change exists in patients of coronary heart disease which breaks the exquisite balance of the interrelationship between platelets and vascular wall.
Platelet Activation In Coronary System Of Patients With Coronary Artery Stenosis (CAS)
Authors: Zebe H , Andrassy K , Koderisch J , Tschöpe W , Kübler W , Ritz E
Published: July 2018

Platelets and platelet mediator substances are thought to play a role in the genesis of atheroma and spasm of coronary arteries. In the present study, indices of platelet activation (PA) were studied in the basal state and under conditions of exercise [Pacing (P) ; Handgrip (HG)] in patients with CAS and controls (CO). Pat, and methods; 22 pat. (14 with CAS > 75% in angiography; 8 with normal coronary arteries). No premedication, heparin or contrast media; Judkins technique; simultaneous sampling in the sinus coronarius (CS) and in the aorta (A) (basal, 5 min. P, 3 min. HG). Measurements: Platelet counts (PC), Beta-thromboglobulin (B-TG), platelet factor 4 (PF 4) with RIA, thromboxane B2 (RIA), ADP/collagen induced platelet aggregation (MA). Results : In the basal state no difference between A and CS was demonstrable for CO and CAS with respect to any of the above parameters. Both after P and during HG, a significant (p < 0.05) increase of B-TG was found in CAS; however, a significant increase was found in CO. In contrast to literature, no change of PC was observed. However, under P and HG a significant decrease of collagen and ADP-induced platelet aggregation (p< 0.05) was observed in CAS but not in CO. In some, but not all, CAS-patients, elevated TXB2 was demonstrable in A and CS blood. Conclusion: The present study demonstrates PA in the coronary system of pat. with CAS. Reduced aggregability but no change of PC may be due to subthreshold activation in proximity to the stenosis. This study (which avoided the use of heparin and contrast agents) also shows that PA, as measured by local sampling, is neither a sensitive nor a specific indicator of CAS.
Platelet Hypersensitivity In Acute Malaria Infection (Plasmodium Falciparum) In Man
Authors: Essien E M , Ebhota M I
Published: July 2018

We had earlier reported altered ADP-induced platelet aggregation in man during acute malaria infection. The present study sought to determine (i) whether the changes suggested platelet hypersensitivity to ADP and (ii) whether such changes occurred in vivo or in vitro.

The aggregation response of platelets (as citrated PRP) to addition of ADP from thirty patients with acute malaria infection has been compared with that of 29 control i.e., non-infected subjects. The age range of the subjects in both groups varied from 2 to 70 years. These tests were performed before the patients took any drugs. With addition of l.0μM ADP to 1 ml of PRP, the mean aggregation amplitude (as % light transmission) obtained from 8 patients, 39.8±27.1% was significantly greater than that from 9 control subjects (5.2±6.7%; t = 3.51; P < 0.005). With higher ADP concentrations (2.4 - 5.0μM) similar response in 22 subjects (mean 89.1±14.9%) was also significantly greater than that in 20 controls (77.8±16.5%; t = 12.45; P < 0.02). These results suggest that during acute malaria infection in man, circulating platelets become hypersensitive to ADP in vitro. No instances of spontaneous aggregation were however observed in the patients.

βTG was determined in 7 patients and 6 controls. The mean plasma βTG in the patients (208.3±15.6 ng/ml) was significantly higher than that in controls (59.2±15.7 ng/ml; t = 13.44; P <0.001). These latter results suggest that the platelets were probably activated in vivo to release the βTG. They further suggest that the hypersensitive changes noted earlier also probably occurred in vivo.

It is suggested that acute malaria (P.falciparum) infection in man is probably another clinical condition associated with platelet hypersensitivity.
Possible Differences In The Mode Of Action Of Ditazole And Non-Steroidal Anti-Inflammatory Drugs As Potential Antithrombotic Agents
Authors: Sim A K , McCraw A P , Caprino L , Antonetti F , Morelli L
Published: July 2018

Ditazole (4,5-diphenyl-2-diethanolamino-oxazole), a weak anti-inflammatory drug, has been shown to be a potent inhibitor of platelet aggregation, adhesiveness and bleeding time. Acetylsalicylic acid (ASA), dipyridamole and a combination of these two drugs induced a platelet shape change which was much shorter lasting than their effect on platelet aggregation. Conversely, similar doses of ditazole induced a potent shape change but no effect on aggregation. Ditazole has now been shown to reversibly antagonise thromboxane A2 (TXA2)-induced contraction of rabbit aortic strips at an optimal concentration of 25 μm in the perfusate. Separately, over a dose range of 50-400 mg/kg/p.o., TXA2 production was inhibited between 39% and 85% in spontaneously clotted rabbit blood. In addition, we have shown that TXA2 formation following arachidonic acid-induced aggregation of platelet-rich plasma (PRP) is similarly inhibited. Ditazole however did not inhibit prostacyclin (PGI2) production in rabbit aortic rings following oral drug administration over a dose range of 50-400 mg/kg. At 1000 and 2000 mg/kg PGI2 production was inhibited by 23% and 41% respectively. TXA2 and PGI2 levels were measured by radioimmunoassay of their stable derivatives TXB2 and 6-keto-PGF1α. It is suggested that the mode of action of ditazole may be more specific than the cyclo-oxygenase/PG-synthetase blocking activity of most other non-steroidal anti-inflammatory drugs.
Biological Nature Of Platelet Inhibitors From Allium Cepa, Allium Sativum And Auricularia Polytrica
Authors: Makheja Amar N , Low Chow Eng , Bailey J
Published: July 2018

Several lines of evidence indicate that extracts of onion (Allium cepa), garlic (Allium sativum) or Chinese black tree fungus (Auricularia polytrica) inhibit platelet aggregation both in-vitro and in-vivo.

A systematic study showed that aqueous extracts of these vegetables produced a dose-dependent inhibition of ADP, arachidonic acid (AA) or collagen-induced platelet aggregation. Onion and garlic juices were extracted sequentially into petroleum ether and diethyl ether. The anti-platelet activity in the aqueous phases was completely inactivated by purified adenosine deaminase. The activity in the petroleum ether layer was further characterised by its effects on the metabolism of 14C-AA by platelets. This oily fraction of onion and garlic suppressed thromboxane synthesis completely and induced a redistribution in the products of the lipoxygenase pathway. Adenosine and allicin (found in the ether layer) inhibited aggregation without affecting AA metabolism. Preparative GC and GC/MS studies showed that the activity of the oily fraction was due principally to paraffinic polysulfides (PPS) of which dimethyl trisulfide was one of the most active. Authentic samples of dimethyl trisulfide duplicated the effects of onion and garlic oils on both aggregation and AA metabolism.
Concentrations Of cAMP Phosphodiesterase Inhibitors That Block The ADP-Induced Shape Change Of Rabbit Platelets Do Not Increase Platelet cAMP
Authors: Lam S C T , Guccione M A , Packham M A , Mustard J F
Published: July 2018

When the cyclic AMP (cAMP) concentration in platelets is increased, platelet functions are inhibited. It is generally assumed that inhibitors of cAMP phosphodiesterase inhibit platelet reactions by increasing cAMP, but this is not established. We have studied the effects of cAMP phosphodiesterase inhibitors on ADP-induced platelet shape change, nucleoside diphosphokinase (NDK) activity, and cAMP concentrations. Papaverine (0.08 mM), dipyridamole (0.2 mM), caffeine (10 mM), or theophylline (8 mM) prevented the shape change of washed rabbit platelets induced by 1 μM ADP. We showed previously that PGEi, which increases cAMP, inhibits platelet NDK activity. Therefore we investigated the effects of cAMP phosphodiesterase inhibitors on platelet NDK activity and cAMP. At concentrations that prevented ADP-induced shape change, papaverine and dipyridamole had no effect on the formation of 14C-ATP from 14C-ADP by washed rabbit platelets. The methylxanthines partially inhibited NDK activity of washed rabbit platelets and of isolated membranes, probably due to the structural similarity between the adenine ring of ADP and these substances. However, it seems unlikely that these substances exert their inhibitory effects through interference with platelet NDK. None of these phosphodiesterase inhibitors increased platelet cAMP above basal levels, measured both by a protein binding assay and by prelabeling the platelet adenine nucleotides by incubation with 14C-adenine. When adenylate cyclase was stimulated with PGE1 (1.2 μM), the cAMP concentration was increased from 7.8 to 27.2 pmol/108 platelets, and in the presence of phosphodiesterase inhibitors,the cAMP concentration was greater than 50 pmol/108 platelets at 90 sec. Since the phosphodiesterase inhibitors by themselves had no detectable effect on cAMP at concentrations that inhibit ADP-induced shape change, it seems likely that they act through other mechanisms. Whether or not they all act in the same way remains to be determined.
Phosphorylation Of Platelet Proteins In Response To Activation By Phorbol 12-Myristate 13-Acetate
Authors: Carroll Roger C , Gerrard Jonathan M
Published: July 2018

We have investigated the 32P-1abelling of platelet proteins in response to 5uM to l0uM phorbol 12-myristate 13- acetate (PMA) which triggers pseudopod formation and aggregation but an atypical release without granule centralization by a contractile gel. Total platelet protein samples resolved on polyacrylamide-sodium dodecyl sulfate gels showed greater than 3 fold increases sustained over a 15 minute time course in the 32p-abelling of 260,000; 40,000; and 20,000 apparent molecular weight peptides. While similar increases in 32p-labelling are observed with other activators, such as thrombin, arachidonate, and A23187, peak phosphorylation routinely occurs between 30 to 60 seconds followed by an aggregation dependent dephosphorylation to less than 50% of peak levels between 3 to 5 minutes. The cytoskeletal cores which remain after 1% Triton X-100 extraction of platelets activated by typical stimuli contain mostly actin, myosin, and actin-binding protein. The presence in this cytoskeletal core most of the 32p-label associated with the 260,000 and 20,000 molecular weight peptides suggests that these phosphopeptide are the 260,000 molecular weight actin binding protein, and the 20,000 molecular weight myosin light chain subunits. Cytoskeletal cores prepared from PMA activated platelets still contain greater than 90% of the 32p-labelled 260,000 molecular weight peptide but less than 20% of the 32p-labelled 20,000 molecular weight peptide, most of which is found in the solubilized fraction. These results suggest that the lack of granule centralization by a contractile gel is due to a disruption of actin-myosin interaction even though the myosin light chain is phosphorylated. This effect seems to be specific in that actin-binding protein - actin interaction believed to be responsible for pseudopod formation is still present in PMA activated platelets.
Fluoride, An Internally Acting Platelet Activator, Causes Phosphorylation Of Platelet Phosphatidic Acid And 20K And 47K Proteins
Authors: Mürer E H , Siojo E , Daniel J L
Published: July 2018

The effects of fluoride, which is transported into platelets in order to induce secretion, are compared with known effects of thrombin, which acts via external sites. Thus, the changes related to transmission of signal through the platelet membrane will not be common to the two activators, only those changes which are subsequent to the internal triggering of platelet activation. Human platelets were prepared by collection in EDTA and washing in saline-EDTA or by gel filtration of citrated platelet-rich plasma. The two methods gave similar results. Platelets prelabeled in plasma with 32P and them separated were incubated at 37°C with 10 mM fluoride at pH 7.4, and samples removed at intervals. (1) The protein was precipitated with HC104, then solubilized by sonication with SDS buffer and the protein bands separated by acrylamide slab gel electrophoresis. The 20K and 47K bands showed 100 to 200% increase in label, with maximum at 8 min incubation (50% secretion) and a great increase seen already at 3 min incubation, where little secretion is observed. (2) Samples were extracted with chloroform-methanol, evaporated to dryness under N2, redissolved in chloroform and applied on thinlayer silica gels on aluminum plates. Two different systems for separating phosphatidic acid (PA) were used. No significant increase in 32P radioactivity was seen in PA the first 3 min. The label at 20 min was 3x that at 8 min. Thus the labeling related to contractile events, a late step in secretion, precedes the labeling of PA, suggesting that the major part of this labeling is not related to the initial phase of platelet activation.
Stroke Prevention - Introductory Remarks
Authors: Barnett H J M
Published: July 2018

A mass of experimental evidence indicates the benefit of platelet anti-aggregant therapy and suggests benefit to patients with thrombo-embolic disease. There are a number of varieties of threatening stroke with platelet-induced thrombogenesis related to ischemic events. The rationale for attempting to prevent stroke,with safe platelet anti- aggregants for which laboratory and animal studies are available,is sound. Several stroke-prevention trials have been conducted and it is appropriate to review the results and take stock of outstanding problems.

The most obvious disappointment is that a substantial number of failures occur. Those with advanced disease and of the female sex lack responsiveness. It is an interesting speculation in that the failure of arteriosclerotic arterial walls to produce prostacyclin might be related to the poor response in advanced disease. The evidence that acetyl- salicylic-esterase is less active in females may be part of the explanation for the sex differential. The better prognosis enjoyed by women suffering from threatened stroke may play a role in explaining some of this therapeutic difference.

Aspirin dosage may require some adjusting-either down or up. In favor of the former is the anti-thrombotic effect of 165 mg per day of ASA in dialysis patients. In favor of the latter is the benefit in women of a dose approaching 3 gm per day in preventing postoperative venous thrombosis.

Drugs other than aspirin have failed yet to establish efficacy in threatened stroke. Sulfinpyrazone and dipyridamole alone have both been tested. Theoretical reasons exist for considering that ASA and persantine may prove synergistic but the evidence is based on in vitro observation. Trials are under way and conclusions from the studies of platelet physiology and biochemistry respecting their combined advantage is premature. “The great tragedy of Science - the slaying of a beautiful hypothesis by an ugly fact”, the words of Thomas Hurley, may apply here.
Molecular Biology Of Factor Viii Coagulant Protein
Authors: Fass D N , Knutson G J
Published: July 2018

Several laboratories report factor VIII coagulant (VIII: C) from human, bovine and porcine sources interact with Willebrand factor (WF). Bovine coagulant may be S-S bonded to WF: the human and porcine VIII:C may reversibly bind to WF. This interaction has been used by various laboratories to help purify the VIII:C. The VIII:C from all 3 species are activated by Ila and inactivated in EDTA. Our work has involved the porcine proteins bound by WF and anti-VIII:C antibodies. Human antibodies to human VIII:C bind porcine plasma proteins which on SDS-PAGE band at apparent MW 160K, 130K and 80K. These data are consistent with the human VIII:C (Switzer and McKee (JCI 60:819, 1977)). Their human material appears to contain S-S linked chains, porcine chains separate in SDS alone. Thrombin treatment results in loss of high molecular weight proteins and an appearance of activation/deactivation products at MW = 80K and 73K and lower. Two murine monoclonal anti-porcine VIII:C antibodies partially inactivate VIII:C while a third doesn’t. This latter antibody, when immobilized, removes activity from solutions of VIII:C dissociated from WF but not from solutions of undissociated VIII:C. The VIII:C immobilized through this antibody is still active, can be removed by organic solvent in active form, and contains the same protein chains as those bound to the polyclonal human anti- VIII:C. The monoclonal antibody VIII:C complex is inactivated and releases the 130K + 160K chains in EDTA. The 80K MW chain can be removed separately. Based on relative staining intensity in SDS gels, the porcine VIII:C at this stage of purity may be heterogeneous, (130K + 80K and 160K + 80K), with the chains associated through Ca++. The 80K MW chain, may bind the VIII:C to porcine WF. The site of this interaction does not seem to be critical to the measurement of VIII:C activity in vitro.
Von Willebrand Factor In Platelet Interaction With Subendothelium
Authors: Baumgartner H R , Tschopp T B , Meyer D , Turitto V T , Weiss H J
Published: July 2018

Studies using citrated blood. A defect in adhesion of platelets from VWD patients to rabbit aorta subendothelium was first demonstrated in 1974 using our annular perfusion chamber. Later on we showed that the magnitude of the defect increased with the blood shear rate. Addition of void-volume fractions of cryoprecipitate chromatographed on Bio-gel A-15m to blood of patients corrected the adhesion defect. By treatment of normal human blood with homologous and heterologous antibodies to human FVIII/VWF a shear rate dependent adhesion defect was experimentally induced. Studies with reconstituted human blood by Sixma’s group confirmed the involvement of FVIII/VWF in platelet adhesion to subendothelium. Treatment of rabbit aorta subendothelium with antibodies to rabbit FVIII complex gave different results: an antiserum raised in goat inhibited adhesion, whereas an antiserum raised in guinea pig had no effect. The latter antiserum did, however, inhibit adhesion when added to rabbit blood. It is conceivable that the two antisera are predominantly directed towards different epitopes on FVIII/VWF.

Studies avoiding anticoagulation. In these studies blood is directly drawn from a vein through the annular chamber at a controlled flow rate. In addition to a shear rate dependent inhibition of platelet adhesion to subendothelium a significant inhibition of maximum thrombus heights, thrombus growth and/or stability was observed at shear rates which were not associated with an adhesion defect. It is not yet clear whether this additional, defect is due to FVIII/VWF and/or VIII: C deficiency.
The Molecular Structure Of Fibrinogen And The Architecture Of The Fibrin Clot: A Model From Electron Microscopy
Authors: Weisel J W , Phillips G N , Stauffacher C V , Ford E , Cohen C
Published: July 2018

The shape of the fibrinogen molecule and aspects of its packing to form fibrin have been derived from a study of electron microscope images of crystals and microcrystals. Using coordinated X-ray diffraction and electron microscopy, the length of the molecule was eastblished as 450Å. The molecules bond end-to-end to form filaments whose packing could be determined for several crystalline forms. A particularly useful aggregate in establishing the shape of the molecule was a “segment“ form of modified human fibrinogen that reveals direct information about the mass distribution. Computer simulations were carried out to refine the size and location of the globular domains. A single molecular model was derived to account for the substructure and packing in a variety of arrays seen in the electron microscope. The molecule can be seen in more detail than the tri-nodular Hall-Slayter model, and is made up of seven globular domains connected by rod-like regions.

The molecular architecture of the fibrin clot has been a puzzle for several decades. A simple, half-staggered arrangement of trinodular molecules can account for the 225 Å repeat, but not the detailed features of the band pattern of the negatively stained fibrin in the electron microscope. We can now simulate the fibrin band pattern using the heptad model and a packing arrangement derived from one of the microcrystalline forms. The molecular arrangement in fibrin is complex: the 450 Å long molecules are half staggered and bonded end-to-end; they are also bent and tilted slightly. Further aspects of the structure and packing will be available from the X-ray crystallographic analysis of the hydrated molecule now in progress.
A Direct, Plasmin Independent Assay For Plasminogen Activation
Authors: Mussoni Luciana , Lawrence Dan , Loskutoff David
Published: July 2018

We have modified the direct, 125I-plasminogen cleavage assay for plasminogen activator (PA) and employed it to compare urokinase (UK), tissue activator (TA), and PAs produced by cultured bovine aortic endothelial cells. The assay is based on conversion of single chain plasminogen to two chain plasmin as revealed by polyacrylamide gel electrophoresis in the presence of SDS andβ-mercapto- ethanol. Inclusion of Triton X-100, albumin and trasylol in the reaction mixture reduced the adsorptive and hydrolytic loss of reactants, and increased the linearity and sensitivity of the assay. Under these conditions, plasmin formation was linear for at least 6 hrs, dose- dependent over a 20-fold range of UK concentrations, and at least 100-fold more sensitive (0.01 units/ml) than previously reported direct assays for UK. In preliminary experiments, we determined a Km value of 10yM for UK and plasminogen. Activation of plasminogen by TA was minimal in the absence of fibrin, and independent of the concentration of activator. However, in the presence of fibrin, (1) the initial rate of activation increased dramatically, (2) 100-1000 fold less TA was required, and (3) activation was proportional to the concentration of both TA and fibrin. Surprisingly, activation by UK and cellular PAs was partially inhibited by fibrin. Epsilon amino caproic acid (EACA; 0.5-100mM) stimulated activation by UK both in the presence and absence of fibrin by 30-40%. In contrast,
EACA (0.1-100mM) inhibited TA activity in the presence of fibrin by over 90%. However, in the absence of fibrin, TA was inhibited by only 50%, even at high EACA concentrations (lOOmM). These results indicate that cleavage of 1251- plasminogen can be employed as a direct, sensitive and quantitative assay for various PAs, and offers a new approach for studying plasminogen activation and agents that stimulate or inhibit it.
Relative Affinity Of Vascular And Tissue Piasminogen Activator To Fibrin And Fibrinogen
Authors: Kruithof E K O , Bachroann F
Published: July 2018

Vascular and tissue plasminogen activator (VA and TA) induce lysis of preformed fibrin at concentrations which do not cause fibrinogenolysis. This phenomenon can be explained, in part, by the strong binding of VA and TA to fibrin. We have compared binding of TA to fibrinogen and to fibrin in various systems. Cn fibrinogen- and on fibrin-sepharose columns, the elution profile of TA was identical.

In further experiments, we determined if fibrinogen decreases the binding of TA to fibrin. To a solution of TA, we added 2 µM of fibrin monomer and varying concentrations of fibrinogen (0-6 µM). In the absence of Ca++, addition of 6 µM of fibrinogen resulted in 30% inhibition of binding of TA to fibrin (expected if affinities were identical: 75%). When citrated plasma containing VA (collected after i.v. injection of DDAVP into volunteers) was clotted by the addition of thrombin, 1,3 U activator per ml of plasma could be extracted from the clot with 0,017 M acetic acid. When fibrin monomer (final concentration 2 µM) was added to the plasma and allowed to clot, about 0,9 U of VA per ml of plasma could be extracted from the fibrin. As the plasma contained 5,4 µM fibrinogen, the 30 percent inhibition of binding of VA to fibrin is similar to that of TA to fibrin by fibrinogen.

The latter results demonstrate that the affinity of TA and VA to fibrin is at least five times higher than that to fibrinogen. The lack of difference in binding of TA to fibrin- and fibrinogen-sepharose indicates that the coupling of fibrinogen and/or fibrin to sepharose modifies their affinity characteristics for TA, or that fibrin polymerization itself increases the potential to bind TA.
The Effect Of Fibrinolysis In The Early Stage Of Acute Myocardial Infarction
Authors: Genth K , Frank J , Schaefer J , Korten V , Heene D
Published: July 2018

The influence of streptokinase (SK) on myocardial infarct size and left ventricular function after acute myocardial infarction was investigated. 21 patients with myocardial infarction received SK (SK-group), 27 patients underwent conventional therapy (C-group). In both groups therapy started within 8 hours after onset of chest pain. In the SK-group initially 250 000 IU were administered intravenously, followed by a maintenance dose of 100 000 IU/h, lasting 15 hours. Blood samples at 8 hours intervals were collected for 3 days for serial CPK-analysis to calculate infarct size (I=∫f(t)×dt×K×bw). M-mode echocardiography was taken before start of t her a py and after 15, 24, 48 and 72 hours. AOP and heart rate were recorded continuously. Infarct size was 47±12g in the SK-group and 84±25g in the C-group (p<0.05). The average time to peak blood CPK-activity was 24 hours in the SK-group and 40 hours in the C-group. Peak CPK-level was significantly higher (p<0.5) in the SK-group (841±160U/l) than in the C-group (532±13 8 U / l ) . In 16 patients of the SK-group short periods of ventricular tachycardia were recorded during the period of fibrinolysis. Before therapy all patients showed abnormal motion of the posterior left ventricular wall and/or the interventricular septum, detected by echocardiography. 14 patients showed after fibrinolysis an improved or normalized motion.

The results indicate that early fibrinolysis may reopen the occluded coronary artery. Reperfusion of the ischemic perfusion area may salvage jeo pardized myocardium, therefore infarct size was reduced and ventricular function improved.
Effect Of Intracoronary And Systemic Thrombolysis With Streptokinase In Patients With Acute Myocardial Infarction And Unstable Angina Pectoris. On Coagulation Tests And Plasminogen
Authors: Kösteriag H , Neuhaus K L , Kasten U , Schrader J , Artmann U , Rentrop P
Published: July 2018

Streptokinase, aplicated by intracoronary infusion in pat. with acute myocardial infarction has proven to be successfull in recanalisation of occluded coronary arteries. The good clinical, angiographic, chemical and EKG results suggests that jeopardized myocardium was salvaged by acute recanalisation. Till now, we infused Streptokinase (about 2000 U/min) in 78 pat. into the ischemia related occluded coronary artery. In this presentation we intend to demonstrate the results of these pat. and of a study, done before starting intracoronary Streptokinase infusion to be safe for bleeding complication. Neither after the infusion of 50 000 U.(n = 24) of 100 000 U. (n = 15) nor in 20 pat. who received SK equal to their ASTK-titres plus 50 000 U. SK whe found severe alterations of the blood-coagulation system. Only in the last group there was a small decrease of Fibrinogen of about 100 mg% and of Plasminogen 4 mg%. In none of the 78 pat., treated by intracoronary SK aplication, we resulted bleeding complication and the mean values of blood coagulation test remained within the normal range.
On the other hand, we infused SK by veins and controlled the thrombolytic effect by coronarangiography. In 5 of 6 pat. we succeded in recanalisation of occluded coronary arteries within 45 Used very high dosages of SK, (about 2 Mill U) there was only a small decrease of 210 mg of Fibrinogen and no bleeding complications.
Fibrinolytic Therapy Of Subclavian-Axillary Vein Thrombosis With Urokinase
Authors: Zimmermann R , Mörl H , Harenberg J , Janssen E , Kuhn H M , Wahl P
Published: July 2018

Acute vein thrombosis of the upper extremity occurs in consequence of trauma,stress,effort or spontaneously. The management was often performed conservatively,including anticoagulation and elevation of the involved arm. But the incidence of chronic residual edema,pain or disability in 6895 % of the cases suggests that active therapeutic procedures should be considered.

In 18 cases with recent and older thrombosis of the suclavian and axillary veins fibrinolytic therapy was performed with urokinase in combination with heparin. Urokinase and heparin therapy was adjusted to attain an anticoagulant effect not exceeding that aimed at usually by heparin alone.7 of 11 patients with recently developed (8 days or less) thrombosis showed a complete recanalization of the venous system.2 cases demonstrated a partial success. In 2 patients no phlebogra- phic alteration could be stated. These 2 patients had received urokinase at the lowest maintenance dosage of only 970 and 1000 IU/kg/h,respectively. In addition 7 patients with thromboses more than 10 days old showed no alteration of the thrombotic occlusions. Relevant side effects were not observed.

The therapeutic results unequivocally depended on the thrombus age,the dosage of urokinase applied and the duration of the fibrinolytic therapy. Under treatment with an median,initial maintenance dosage of urokinase 1300-2000 IU/kg/h in combination with heparin excellant therapeutic results could be attained. Our data suggest that the here presented therapeutic procedure has to be considered in serious cases of recently developed subclavian-axillary vein thrombosis.
A Monoclonal Anti Human Factor Ix Produced By A Mouse Hybridoma
Authors: Bertina R M , van der Linden I K , Muller H P , Derks J , Klein-Breteler E
Published: July 2018

Spleen cells of a Balb-c mouse immunized with purified human FIX were fused with mouse myeloma cells (MOPC-21 derivative]. Among the fusion products one hybridoma was found producing an inhibitor of factor IX procoagulant activity. After sub- clonation (5 x) a stable hybridoma was obtained. In vitro cell culture of the hybridoma cells gives anti-FIX titers (Bethesda units] of about 0.8 units/ml. Injection of the hybridoma cells in pristane pretreated mice results in antiFIX titers of 600-1000 units/ml ascites. Analysis of the produced immunoglobulin demonstrated the presence of one main band after iso-electric focussing, which contained heavy chains both of IgG1 and IgG2B subclass.

Monoclonal anti-factor IX was isolated from ascites liquid using affinity chromatography on protein-A-Sepharose. Using the purified antibody a radioimmunoassay was developed for the epitope on factor IX, against which this antibody is directed. The epitope is present both on FIX and activated FIXa; however, the affinity of the antibody for binding to FIXa is at least 10 times less. The antibody has no significant affinity for binding to the isolated heavy and light chain of FIXa.

About 27 different genetic variants of factor IX [from haemophilia B patients were tested for the presence of this epitope. All FIX-variants possessed the epitope. At least 2 variants demonstrated a reduced affinity for binding to the antibody.
Antisera To Human Factor VIII Raised In Balb/C Mice. The Production Of Monoclonal Antibodies To Factor VIII
Authors: Joshua D E , Exner T , Kronenberg H , Rickard K A
Published: July 2018

The aim of this series of experiments is to raise antisera to human Factor VIII in Balb/C mice, study the properties of these antisera, and use the spleen cells from such hyperimmune mice to raise a panel of monoclonal antibodies directed against the varying antigenic determinants of the human factor VIII complex. Antibody activity against factor VIII antigen was determined by a microtitre solid phase radioimmune assay. This involved exposing test antisera to human factor VIII-coated PVC wells, and detection of bound mouse antibody by subsequent exposure to 125I labelled rabbit antimouse immunoglobulin. Anticoagulant assays and ristocetin cofactor inhibition assays were based on standard methods adapted to microtitre plates. Balb/C mice were immunized with gel filtered factor VIII complex and boosted immediately prior to fusion. Fusion was performed with the parental line P3-NSI/l-Ag-l in polyethylene glycol (PEG). Compared to rabbits Balb/C mice produced considerably higher anticoagulant activity. Pooled rabbit anti-factor VIII coagulant activity was 1.2 Oxford units/ml whereas the anticoagulant activity of 6 mice ranged between 30-185 Oxford units/ml. Incubation of mouse antisera with human plasma samples and subsequent assay of residual mouse anti-VIII antigen demonstrated that normal and haemophiliac plasma markedly inhibited binding of the antisera whereas plasma samples from patients with Von Willebrand's disease did not. This system has been optimized as a microtitre radioimmune assay for human factor VIII antigen. There is good correlation with the Laurell “rocket” technique (r=0.86; n=14). A number of monoclonal antibodies which bind to the factor VIII complex have been produced. They have no anticoagulant activity nor activity against ristocetin cofactor. They recognize antigens present in plasma fractions after DEAE Sephadex A-50 separation which differ from those fractions recognized by the heterologous Balb/C antisera.
The Use Of A Cell-Free Synthesis System For The Study Of Factor FVIII
Authors: Heath P R , Malia R G , Tomlinson S , Preston F E
Published: July 2018

There is good evidence that FVIIIR:Ag is synthesized by endothelial cells but the site of synthesis of FVIII:C and the precise biochemical relationship between the various components of the factor VIII complex remains obscure. In order to study the biosynthesis of porcine factor VIII we have established a cell-free synthesis system based on a wheatgerm lysate wherein exogenous messenger R.N.A’s extracted from cultured porcine endothelial cells are used to direct protein synthesis in vitro. Preliminary biosynthetic labelling experiments confirmed that, using our recently described IRMA assay, the cultured cells synthesized and secreted FVIIIR:Ag. mRNA was extracted from lg cultured porcine endothelial cells by homogenizing the tissue in a low-salt buffer followed by treatment of the homogenate with proteinase K to degrade unwanted protein and ethanol to precipitate total nucleic acid. The mRNA was then separated on an oligo (dT) cellulose column. Confirmation that the mRNA was highly active in directing protein synthesis was shown by the incorporation of 35S-methionine into labelled products. Amongst the synthesized products is material which co-elates with FVIIIR:Ag as shown by radioactive and activity peaks. Further detailed studies are being undertaken to confirm immunological identity of this material with porcine factor VIII. The advantages of this technique to the study of the molecular characteristics of factor VIII and other clotting factors will be discussed.